U.S. patent application number 12/069084 was filed with the patent office on 2009-02-12 for compositions and methods for drought tolerance.
This patent application is currently assigned to Michigan State University. Invention is credited to Suleiman Bughrara.
Application Number | 20090044287 12/069084 |
Document ID | / |
Family ID | 39682318 |
Filed Date | 2009-02-12 |
United States Patent
Application |
20090044287 |
Kind Code |
A1 |
Bughrara; Suleiman |
February 12, 2009 |
Compositions and methods for drought tolerance
Abstract
The present inventions relate to compositions and methods for
providing drought resistant grass plants comprising Festuca mairei
plant germplasm. Specifically, the inventions relate to providing
compositions and methods for introgressing Festuca mairei germplasm
and/or specific Festuca mairei genes into grass plants, such as
Lolium perenne plants. Further, the invention relates to methods of
grass plant breeding comprising genetic markers for identifying the
preferred Festuca mairei germplasm introgressed into grass plants,
and providing commercially desirable drought resistant cultivars of
grass plants.
Inventors: |
Bughrara; Suleiman; (East
Lansing, MI) |
Correspondence
Address: |
Medlen & Carroll, LLP;Sutie 350
101 Howard Street
San Francisco
CA
94105
US
|
Assignee: |
Michigan State University
Lansing
MI
|
Family ID: |
39682318 |
Appl. No.: |
12/069084 |
Filed: |
February 6, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60899837 |
Feb 6, 2007 |
|
|
|
Current U.S.
Class: |
800/260 ;
435/320.1; 530/350; 536/23.1; 800/278 |
Current CPC
Class: |
C12N 15/8273
20130101 |
Class at
Publication: |
800/260 ;
536/23.1; 530/350; 435/320.1; 800/278 |
International
Class: |
C12N 15/11 20060101
C12N015/11; C07K 14/00 20060101 C07K014/00; C12N 15/82 20060101
C12N015/82; A01H 1/02 20060101 A01H001/02 |
Goverment Interests
[0002] The present application was funded in part with government
support under grant numbers MICL01975, 1230-21000-035-OOD, and
1230-21000-045-OOD United States Department of Agriculture. The
government may have certain rights in this invention.
Claims
1-18. (canceled)
19. An isolated nucleic acid sequence comprising a sequence
selected from the group consisting of SEQ ID NOs: 1-39, 92-216, and
sequences at least 90% identical thereto.
20. The nucleic acid sequence of claim 19, wherein said sequence is
selected from the group consisting of SEQ ID NOs: 1-39 and
sequences at least 90% identical thereto.
21. The nucleic acid sequence of claim 19, wherein said nucleic
acid sequence comprises a sequence selected from the group
consisting of SEQ ID NOs: 1-4, 8-10, 12-19, 25-26, 29-30, 32-37,
and sequences at least 90% identical thereto.
22. The nucleic acid sequence of claim 21, wherein said nucleic
acid sequence encodes a polypeptide comprising a sequence selected
from the group consisting of SEQ ID NOs:40-91 and sequences at
least 90% identical thereto.
23. A polypeptide encoded by the nucleic acid sequence of claim
19.
24. The polypeptide of claim 23, wherein said polypeptide sequence
comprises a sequence selected from the group consisting of SEQ ID
NOs:40-91, and sequences at least 90% identical thereto.
25. A vector comprising the nucleic acid sequence of claim 19.
26. The vector of claim 25, wherein said nucleic acid sequence
comprises a sequence selected from the group consisting of SEQ ID
NOs: 1-39 and sequences at least 90% identical thereto.
27. The vector of claim 25, wherein said nucleic acid sequence
comprises a sequence selected from the group consisting of SEQ ID
NOs: 1-4, 8-10, 12-19, 25-26, 29-30, and 32-37, and sequences at
least 90% identical thereto.
28. The vector of claim 25, wherein said nucleic acid sequence
encodes a polypeptide comprising a sequence selected from the group
consisting of SEQ ID NOs:40-91 and sequences at least 90% identical
thereto.
29. A method for producing a grass plant comprising: a) providing;
i) a grass plant, and ii) the nucleic acid sequence of claim 19;
and b) introducing said nucleic acid sequence into said grass plant
to produce a progeny grass plant.
30. The method of claim 29, wherein said nucleic acid sequence
comprises a sequence selected from the group consisting of SEQ ID
NOs:1-4, 8-10, 12-19, 25-26, 29-30, 32-37, and sequences at least
90% identical thereto.
31. The method of claim 29, wherein said nucleic acid sequence
encodes a polypeptide comprising a sequence selected from the group
consisting of SEQ ID NOs:40-91, and sequences at least 90%
identical thereto.
32. The method of claim 29, wherein said introducing is by breeding
or transfecting.
33. The method of claim 29, wherein said grass plant is selected
from the group consisting of a turfgrass plant, forage grass plant,
ornamental grass plant, ground cover grass plant, and transgenic
grass plant.
34. The method of claim 29, wherein said grass plant is a ryegrass
plant selected from the group consisting of Lolium perenne plant
and Lolium perenne hybrid plant.
35. The method of claim 29, wherein said grass plant is a ryegrass
plant selected from the group consisting of Citation II plant and
Calypso plant.
36. The method of claim 29, wherein said grass plant is a hybrid
plant selected from the group consisting of (a) Festuca mairei
plant.times.grass plant, (b) Festuca mairei plant.times.Lolium
perenne plant, and (c) hybrid of Festuca mairei plant.times.Lolium
perenne plant.
37. The method of claim 29, wherein said grass plant is selected
from the group consisting of (s) F.sub.1 hybrid plant, (b)
4.times.F.sub.1 hybrid plant, (c) 3.times.F.sub.1 hybrid plant, and
(d) backcross progeny plant.
38. The method of claim 37, wherein said F.sub.1 hybrid plant is
selected from the group consisting of (a) Festuca mairei
plant.times.Lolium perenne plant, (b) hybrid of Festuca
maireiplant.times.Lolium perenne plant, (c) Festuca mairei
plant.times.Calypso plant, and (d) Festuca mairei
plant.times.Citation II plant.
Description
[0001] This application claims priority to co-pending U.S.
provisional patent application Ser. No. 60/899,837, filed Feb. 6,
2007, which is herein incorporated by reference in its
entirety.
FIELD OF THE INVENTION
[0003] The present inventions relate to compositions and methods
for providing drought resistant plants comprising Festuca mairei
plant germplasm. Specifically, the inventions relate to providing
compositions and methods for introgressing Festuca mairei germplasm
and/or specific Festuca mairei genes into grass plants, such as
Lolium perenne plants. Further, the invention relates to methods of
plant breeding comprising genetic markers for identifying the
preferred Festuca mairei germplasm introgressed into plants, and
providing commercially desirable drought resistant cultivars of
plants.
BACKGROUND OF THE INVENTION
[0004] Perennial ryegrass (Lolium perenne L.) (Lp) is a cool-season
grass (2n=2x=14, LL) that is in wide use as turfgrass and forage
grass due to superior quality and rapid establishment of plants.
However, lack of drought tolerance makes Lp less persistent and
reduces economical advantages of its use during hot and dry summers
or dry environments.
[0005] One approach for improvement of drought tolerance in
perennial ryegrass is introgression of alien genomes from other
drought tolerant genera, such as fescue plants (Rie and Mondart
1985, Ames: Iowa State Univ. Press, 1985:241-246; herein
incorporated by reference). Thus, plant breeders and geneticists
have attempted for several decades to genetically combine perennial
ryegrass (L. perenne L.) with fescue (Festuca spp.) to create novel
forage grasses containing both high forage quality and good drought
tolerance.
[0006] However, a major barrier in Festuca.times.Lolium breeding
programs for providing commercial cultivars of L. perenne plants
comprising the desirable traits of Festuca is the difficulty in
selecting hybrids with the desired alien chromatin or chromosome
addition-substitution. Further, Festuca germplasm is rapidly loss
from subsequent breeding lines when attempting to retain the
drought resistant Festuca germplasm and the desired agronomic
traits of Lolium plants.
[0007] Therefore, despite exhaustive efforts, especially in light
of the potential enormous economic and environmental significance
of success, there is still a need for identification and
characterization of plant genes that would confer drought tolerance
to Lolium plants. Further, there remains a need for providing
increased drought tolerance in L perenne L. ryegrass plants.
SUMMARY OF THE INVENTION
[0008] The present inventions relate to compositions and methods
for providing drought resistant plants (e.g., grass plants)
comprising Festuca mairei plant germplasm. Specifically, the
inventions relate to providing compositions and methods for
introgressing Festuca mairei germplasm and/or specific Festuca
mairei genes into plants (e.g., grass plants), such as Lolium
perenne plants. Further, the invention relates to methods of grass
plant breeding comprising genetic markers for identifying the
preferred Festuca mairei germplasm introgressed into plants (e.g.,
grass plants), and providing commercially desirable drought
resistant cultivars of plants (e.g., grass plants).
[0009] The present invention relates to compositions and methods
for identifying preferred plant germplasm associated with
successful adaptation to drought conditions, such as Festuca mairei
germplasm, including but not limited to individual nucleic acid
sequence associated with drought resistance.
[0010] The present invention also relates to the field of plant
breeding, specifically to methods of grass plant breeding
comprising introgression of preferred drought resistant germplasm
into plants. The breeding methods further comprise identifying and
using nucleic acid sequence tic markers for identifying germplasm
associated with drought resistance in breeding populations. The
grass plant breeding methods include but are not limited to natural
breeding, artificial breeding, selective breeding involving DNA
molecular marker analysis for germplasm associated with drought
tolerance and other desired agronomic traits, transgenics, and
commercial breeding. Further, the invention relates to new drought
resistant plants (e.g., grass plants) comprising preferred
germplasm, including but not limited to populations, cultivars,
varieties, lines and methods of breeding the same for commercial
use.
[0011] The invention further relates to new plants (e.g., grass
plants) that are resistant to drought, and in particular plants
(e.g., grass plants) comprising germplasm that was identified
during onset or adaptation to drought stress, methods of breeding
drought resistant plants, and the resulting new drought resistant
grass plant varieties, lines and cultivars developed through
traditional plant breeding methods that provide for successful
commercialization of the drought resistant germplasm. The present
invention is not limited to providing any particular grass plant
variety, line, and cultivar having drought resistance activities.
The present invention also provides breeding methods comprising DNA
marker analysis for identifying Festuca mairei plant nucleic acid
sequence in plants with increased tolerance for drought stress,
including but not limited to Festuca mairei and Lolium perenne
grass plants.
[0012] The invention provides an isolated nucleic acid sequence
comprising one or more of SEQ ID NOs:1-39, and 93-216. In one
embodiment, the nucleic acid sequence encodes a polypeptide
comprising one or more of SEQ ID NOs:40-91. In another embodiment,
the invention provides a polypeptide encoded by, and a vector
comprising, a nucleotide sequence that comprises one or more of SEQ
ID NOs:1-39, and 93-216. In one embodiment, the cell is a plant
cell.
[0013] The invention also provides a cell and/or plant comprising a
heterologous nucleotide sequence that comprises one or more of SEQ
ID NOs:1-39, and 93-216. In one embodiment, the plant has increased
drought tolerance than a control plant lacking the heterologous
nucleotide sequence. In a particular embodiment, the plant is a
grass plant, such as a perennial ryegrass plant, as exemplified by
Lolium perenne species and hybrid plants thereof. In one
embodiment, the hybrid plant is selected from the group consisting
of Festuca mairei.times.Lolium perenne species and hybrids
thereof.
[0014] The invention further provides a plant seed comprising a
heterologous nucleotide sequence that comprises one or more of SEQ
ID NOs:1-39, and 93-216. In one embodiment, the plant seed is
produced by a plant containing one or more of SEQ ID NOs:1-39, and
93-216.
[0015] The invention additionally provides a method for producing
transgenic plant cells, comprising: a) providing: i) plant cells
from a first plant, and ii) a nucleic acid sequence comprising one
or more of SEQ ID NOs:1-39, and 93-216, and b) transfecting the
nucleic acid sequence into the plant cells to produce a transgenic
plant cell. Optionally, the method further comprises c)
regenerating a transgenic plant from the transgenic plant cell,
whereby expression of the nucleic acid sequence in the transgenic
plant results in higher drought tolerance of the transgenic plant
than of the first plant. In an alternative embodiment, the first
plant is a grass plant, such as ryegrass, cereal grass, forage
grass, turf grass, ornamental grass, pasture grass, hay grass,
cover grass, and cereal grass. For example, the cereal grass is
selected from wheat, corn, rice, rye, oats, barley, and millet. In
another example, the grass plant is selected from Lolium
temulentum, creeping bent grass, colonial bent grass, tall fescue,
orchardgrass, Brachypodium distachyon, bromegrass, Bermuda grass,
zoysiagrass, Festuca arundinacea, and Lolium multiflorum. In a
further embodiment, the grass plant is a ryegrass plant, such as
Lolium perenne ryegrass plant.
[0016] The invention also provides a method for detecting the
presence of a drought tolerance nucleotide sequence in a cell,
comprising: a) providing genomic DNA isolated from a cell, and b)
detecting the presence in the genomic DNA of one or more sequence
selected from SEQ ID NOs:1-39, and 93-216, thereby detecting the
presence of a drought tolerance nucleotide sequence in the
cell.
[0017] The invention also provides a method for detecting the
presence of a drought tolerance nucleotide sequence in a cell,
comprising: a) providing genomic DNA isolated from a cell, and b)
hybridizing the genomic DNA with one or more primer sequence
selected from SEQ ID NO:219-227, 230-246, 249-265. In one
embodiment, detecting comprises hybridization under stringent
conditions. In another embodiment, the primer sequence is selected
from SEQ ID NO:219-227, 230-246, and 249-265, such as for AFLP. In
a further embodiment, the primer sequence is selected from SEQ ID
NO:266-306, such as for RAPD.
[0018] Accordingly, in some embodiments, the present invention
provides a nucleic acid sequence, wherein said nucleic acid
sequence comprises a sequence selected from the group consisting of
SEQ ID NOs: 1-39 (FIG. 7), and 93-216 (FIG. 9). In other
embodiments, the nucleic acid sequence comprises a sequence at
least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identical to a
sequence selected from the group consisting of SEQ ID NOs: 1-39,
and 93-216. In other embodiments, the nucleic acid sequence is at
least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identical to a
sequence selected from the group consisting of SEQ ID NOs: 1-39,
and 93-216. In other embodiments, the present invention provides
nucleotide sequences at least 90% identical to a sequence selected
from the group consisting of SEQ ID NOs: 1-39, and 93-216. In one
embodiment, the nucleic acid sequence comprises a sequence selected
from the group consisting of SEQ ID NOs: 1-39. In one embodiment,
the nucleic acid sequence is a sequence selected from the group
consisting of SEQ ID NOs: 1-39. In other embodiments, the present
invention provides nucleotide sequences at least 90% identical to a
sequence selected from the group consisting of SEQ ID NOs:1-39. In
one embodiment, the nucleic acid sequence comprises a sequence
selected from the group consisting of SEQ ID NOs:93-216. In one
embodiment, the nucleic acid sequence is a sequence selected from
the group consisting of SEQ ID NOs: 93-216. In other embodiments,
the present invention provides nucleotide sequences at least 90%
identical to a sequence selected from the group consisting of SEQ
ID NOs: 93-216. In one embodiment, the nucleic acid sequence
comprises a sequence selected from the group consisting of SEQ ID
NOs:1-4, 8-10, 12-19, 25-26, 29-30, and 32-37. In one embodiment,
the nucleic acid sequence is selected from the group consisting of
SEQ ID NOs:1-4, 8-10, 12-19, 25-26, 29-30, and 32-37. In one
embodiment, the nucleic acid sequence encodes a polypeptide
comprising a sequence selected from the group consisting of SEQ ID
NOs:40-91 (FIG. 8). In other embodiments, the polypeptide comprises
a sequence at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%
identical to a sequence selected from the group consisting of SEQ
ID NOs:40-91. In other embodiments, the polypeptide comprises a
sequence at least 90% identical to a sequence selected from the
group consisting of SEQ ID NOs:40-91. In one embodiment, the
expression of said nucleic acid sequence is altered during drought
stress treatment of a Festuca mairei plant as compared to a control
Festuca mairei plant. The present invention is not limited to any
particular type of altered expression of a nucleic acid sequence.
Indeed, a variety of altered nucleic acid sequence expression is
contemplated, including, but not limited to up-regulation,
down-regulation, silencing, up-regulation then down-regulation,
transient regulation, and differential regulation. In one
embodiment, the nucleic acid sequence is up-regulated during
drought stress treatment of a Festuca mairei plant as compared to a
control Festuca mairei plant. The present invention is not limited
to any particular function category of up-regulated nucleic acid
sequence. Indeed, a variety of function categories of up-regulated
nucleic acid sequences are contemplated, including, but not limited
to subcellular localization, transcription, defense, metabolism,
transport, et cetera. The present invention is not limited to any
particular sequence of up-regulated nucleic acid sequence. Indeed,
a variety of sequences of up-regulated nucleic acid sequences are
contemplated, including, but not limited to a nucleic acid sequence
comprising a sequence selected from the group consisting of SEQ ID
NOs: 94, 95, 99, 102, 105, 109, 113, 114, 116, 119, 122, 124, 126,
134, 140, 156, 157, 182, 192, 203, 209, and 215. In one embodiment,
the nucleic acid sequence is selected from the group consisting of
SEQ ID NOs: 94, 95, 99, 102, 105, 109, 113, 114, 116, 119, 122,
124, 126, 134, 140, 156, 157, 182, 192, 203, 209, and 215. In one
embodiment, the nucleic acid sequence is down-regulated during
drought stress treatment of a Festuca mairei plant as compared to a
control Festuca mairei plant. The present invention is not limited
to any particular function category of a down-regulated nucleic
acid sequence. Indeed, a variety of function categories of
down-regulated nucleic acid sequences are contemplated, including,
but not limited to subcellular localization, metabolism, transport,
energy, et cetera. The present invention is not limited to any
particular sequence of a down-regulated nucleic acid sequence.
Indeed, a variety of down-regulated nucleic acid sequences are
contemplated, including, but not limited to a nucleic acid sequence
comprising a sequence selected from the group consisting of SEQ ID
NOs: 96, 103, 104, 106, 108, 110, 111, 112, 162, 172, 181, 200,
202, 204, and 208. In one embodiment, the nucleic acid sequence is
selected from the group consisting of SEQ ID NOs: 96, 103, 104,
106, 108, 110, 111, 112, 162, 172, 181, 200, 202, 204, and 208. In
one embodiment, the nucleic acid sequence is up-regulated then
down-regulated during drought stress treatment of a Festuca mairei
plant as compared to a control Festuca mairei plant. The present
invention is not limited to any particular function category of
up-regulated then down-regulated nucleic acid sequence. Indeed, a
variety of function categories of up-regulated then down-regulated
nucleic acid sequences are contemplated, including, but not limited
to subcellular localization, energy, cell type differentiation,
protein fate, et cetera. The present invention is not limited to
any particular sequence of up-regulated then down-regulated nucleic
acid sequence. Indeed, a variety of sequences of up-regulated then
down-regulated nucleic acid sequences are contemplated, including,
but not limited to a nucleic acid sequence comprising a sequence
selected from the group consisting of SEQ ID NOs: 101, 118, 120,
141, and 185. In one embodiment, the nucleic acid sequence is
selected from the group consisting of SEQ ID NOs: 101, 118, 120,
141, and 185. In one embodiment, the nucleic acid sequence
comprises a transiently expressed fragment. In one embodiment, the
nucleic acid sequence is transiently regulated during drought
stress treatment of a Festuca mairei plant as compared to a control
Festuca mairei plant. The present invention is not limited to any
particular function category of transiently regulated nucleic acid
sequence. Indeed, a variety of function categories of transiently
regulated nucleic acid sequences are contemplated, including, but
not limited to nucleic acid sequences associated with transport,
subcellular localization, encoding a protein with a binding
function, defense, metabolism, interaction with the cellular
environment, cell type differentiation, et cetera. The present
invention is not limited to any particular sequence of transiently
regulated nucleic acid sequence. Indeed, a variety of sequences of
transiently regulated nucleic acid sequences are contemplated,
including, but not limited to a nucleic acid sequence comprising a
sequence selected from the group consisting of SEQ ID NOs: 93, 151,
153, and 179. In one embodiment, the nucleic acid sequence is
expressed in a drought stressed Festuca mairei plant. In one
embodiment, the nucleic acid sequence is a differentially expressed
fragment. In one embodiment, the nucleic acid sequence is silenced
in a drought stressed Festuca mairei plant. In one embodiment, the
altered nucleic acid sequence provides drought tolerance in a
Festuca mairei plant as compared to a control Festuca mairei plant.
In one embodiment, the altered nucleic acid sequence provides
increased drought tolerance in a Festuca mairei plant as compared
to a control Festuca mairei plant.
[0019] The present invention provides a polypeptide, wherein said
polypeptide is encoded by a nucleic acid sequence comprising a
sequence selected from the group consisting of SEQ ID NOs: 1-39,
and 93-216. In one embodiment, the polypeptide increases drought
tolerance in a grass plant.
[0020] The present invention provides a polypeptide, wherein said
polypeptide comprises an amino acid sequence selected from the
group consisting of SEQ ID NOs: 40-91. In other embodiments, the
polypeptide comprises a sequence at least 50%, 60%, 70%, 80%, 90%,
95%, 98%, or 99% identical to a sequence selected from the group
consisting of SEQ ID NOs: 40-91. In other embodiments, the
polypeptide sequence is at least 50%, 60%, 70%, 80%, 90%, 95%, 98%,
or 99% identical to a sequence selected from the group consisting
of SEQ ID NOs: 40-91. In other embodiments, the present invention
provides a polypeptide comprising a sequence at least 90% identical
to a sequence selected from the group consisting of SEQ ID NOs:
40-91.
[0021] The present invention provides a vector construct comprising
at least one nucleic acid sequence from a Festuca mairei plant,
wherein said nucleic acid sequence comprises a sequence selected
from the group consisting of SEQ ID NOs: 1-39 (FIG. 7), and 93-216
(FIG. 9). In other embodiments, the present invention provides a
nucleotide sequence at least 90% identical to a sequence selected
from the group consisting of SEQ ID NOs: 1-39, and 93-216. In one
embodiment, the nucleic acid sequence comprises a sequence selected
from the group consisting of SEQ ID NOs: 1-39. In one embodiment,
the nucleic acid sequence comprises a sequence selected from the
group consisting of SEQ ID NOs:1-4, 8-10, 12-19, 25-26, 29-30, and
32-37. In one embodiment, the nucleic acid sequence encodes a
polypeptide comprising a sequence selected from the group
consisting of SEQ ID NOs:40-91 (FIG. 8). In one embodiment, the
nucleic acid sequence is operably linked to an exogenous promoter.
The present invention is not limited to any particular type of
promoter. Indeed, the use of a variety of promoters is
contemplated. In some embodiments, the promoter is a eukaryotic
promoter. In some embodiments, the promoter is a constitutive
promoter or an inducible promoter. In some embodiments, the
eukaryotic promoter is active in a plant. The present invention is
not limited to any particular type of vector construct. Indeed, the
use of a variety of vector constructs is contemplated. In some
embodiments, the vector is a eukaryotic vector. In other
embodiments, said eukaryotic vector is a plant vector. In some
embodiments, the vector is a binary vector. In some embodiments,
the vector is an expression vector. In other embodiments, said
vector plant vector comprises a T-DNA vector. In other embodiments,
said vector is a prokaryotic vector.
[0022] The present invention provides a cultivar of a grass plant
comprising at least one heterologous nucleic acid sequence from a
Festuca mairei plant genome, wherein said nucleic acid sequence
comprises a sequence selected from the group consisting of SEQ ID
NOs: 1-39, and 93-216. In other embodiments, the present invention
provides a nucleotide sequence at least 90% identical to a sequence
selected from the group consisting of SEQ ID NOs: 1-39, and 93-216.
In one embodiment, the nucleic acid sequence comprises a sequence
selected from the group consisting of SEQ ID NOs: 1-39. In one
embodiment, the nucleic acid sequence comprises a sequence selected
from the group consisting of SEQ ID NOs: 1-39. In one embodiment,
the nucleic acid sequence comprises a sequence selected from the
group consisting of SEQ ID NOs: 1-4, 8-10, 12-19, 25-26, 29-30, and
32-37. In one embodiment, the nucleic acid sequence encodes a
polypeptide comprising a sequence selected from the group
consisting of SEQ ID NOs:40-91. In one embodiment, the grass plant
includes, but not limited to, a ryegrass, bluegrass, Bermuda grass,
and zoysiagrass plant. In one embodiment, the grass plant is a
perennial ryegrass plant. In one embodiment, the grass plant is
selected from the group consisting of Lolium perenne species and
hybrids thereof. In one embodiment, the grass plant is a hybrid
plant is selected from the group of hybrid plants consisting of
Festuca mairei.times.Lolium perenne species and hybrids thereof. In
one embodiment, the grass plant is a Festuca mairei
plant.times.Calypso plant or Festuca mairei plant.times.Citation II
plant. In one embodiment, the grass plant includes, but is not
limited to, a G15 plant or a G30a plant. In one embodiment, the
grass plant includes, but is not limited to, a grass plant derived
from a G15 or a G30a plant. In one embodiment, the nucleic acid
sequence is meiotically stable. In one embodiment, the cultivar is
drought resistant. In one embodiment, the cultivar is tolerant to
drought stress. In one embodiment, the grass plant remains turgid
during drought conditions. In one embodiment, the grass plant is an
agronomically desirable plant. In one embodiment, the grass plant
is a commercially desirable plant. In one embodiment, the invention
provides a seed of the cultivar.
[0023] The invention also provides a seed of a perennial ryegrass
Lolium perenne L. variety selected from the group consisting of
G11a, G14, and G16, as well as a plant produced by growing these
seeds. In one embodiment, the plant is capable of expressing all
the physiological and morphological characteristics, such as
drought tolerance characteristics, of the perennial ryegrass Lolium
perenne L. plant variety selected from the group consisting of
G11a, G14, and G16. The invention also contemplates any part of the
plant, such as pollen, ovule, tissue, seed, cell, and
germplasm.
[0024] The invention also provides a first generation (F.sub.1)
hybrid perennial ryegrass Lolium perenne L. seed having two
parents, wherein one or both parents is a plant described above.
The invention also provides the seed of the aforementioned plant,
wherein one parent of the seed is perennial ryegrass Lolium perenne
L. variety G1, and the second parent of the seed is selected from
perennial ryegrass Lolium perenne L. variety G14 and variety G16.
In one embodiment, one parent of the seed is perennial ryegrass
Lolium perenne L. variety G14, and the second parent of the seed is
selected from perennial ryegrass Lolium perenne L. variety G1 and
variety G16. In another embodiment, one parent of the seed is
perennial ryegrass Lolium perenne L. variety G16, and the second
parent of the seed is selected from perennial ryegrass Lolium
perenne L. variety G1 and variety G14. The invention also provides
a first generation (F.sub.1) hybrid perennial ryegrass Lolium
perenne L. plant produced by growing the any of the above
seeds.
[0025] Further provided is a method of producing perennial ryegrass
Lolium perenne L. seed, comprising: a) providing: i) a first plant
of perennial ryegrass Lolium perenne L. variety, and ii) a second
plant of perennial ryegrass Lolium perenne L. variety, wherein one
or both of the first and second plants is the plant described
above, b) crossing the first plant with the second plant to produce
a progeny plant, and c) growing the progeny plant to produce
perennial ryegrass Lolium perenne L. seed. In one embodiment, the
first plant is Lolium perenne L. variety G1, and the second plant
is selected from Lolium perenne L. variety G14 and variety G16. In
another embodiment, the first plant is Lolium perenne L. variety
G14, and the second plant is selected from Lolium perenne L.
variety G1 and variety G16. In an alternative embodiment, the first
plant is Lolium perenne L. variety G16, and the second plant is
selected from Lolium perenne L. variety G1 and variety G14. In
another embodiment, the produced seed is an F.sub.1 hybrid
perennial ryegrass Lolium perenne L. seed. Alternatively, the
produced seed is an F.sub.2 hybrid perennial ryegrass Lolium
perenne L. seed.
[0026] Also provided is a method of producing a perennial ryegrass
Lolium perenne L. plant, comprising: a) providing: i) a first plant
as described above, ii) a second perennial ryegrass Lolium perenne
L. plant, and iii) a third perennial ryegrass Lolium perenne L.
plant, b) crossing the first plant with the second plant to produce
a first progeny plant, and c) crossing the first progeny plant with
itself or with the third perennial ryegrass Lolium perenne L. plant
to produce a second progeny plant. Optionally, the method further
comprises d) crossing the second progeny plant with itself or with
a fourth perennial ryegrass Lolium perenne L. plant to produce a
third progeny plant.
[0027] The invention also provides a method for producing perennial
ryegrass Lolium perenne L. seeds, comprising: a) providing: i) a
first parent Lolium perenne L. plant, and ii) a second parent
Festuca mairei plant, b) crossing the first parent plant with the
second parent plant to produce a progeny plant, and c) growing the
progeny plant to produce perennial ryegrass Lolium perenne L. seed
that is capable of producing a progeny Lolium perenne L. plant that
has increased drought tolerance compared to the first parent plant.
Optionally, the method also comprises d) growing the seed to
produce a progeny Lolium perenne L. plant that has increased
drought tolerance compared to the first parent plant. In one
embodiment, the progeny plant comprises one or more genomic
sequences from the second parent plant, wherein expression of one
or more of the genomic sequences in the progeny plant results in
increased drought tolerance of the progeny plant compared to the
first parent plant. In a further embodiment, the progeny plant is
selected from the group consisting of F.sub.1 hybrid plant,
4.times.F.sub.1 hybrid plans, 3.times.F.sub.1 hybrid plant, and
backcross progeny plant. In yet another embodiment, the F.sub.1
hybrid plant is a Festuca mairei.times.Lolium perenne plant, or a
hybrid thereof. In a further embodiment, the F.sub.1 hybrid plant
is a Festuca mairei plant.times.Calypso plant, or a Festuca mairei
plant.times.Citation II plant. In another alternative, the
backcross progeny plant is selected from G11a, G14, and G16. The
invention also contemplates a seed produced by the above methods
and a plant produced by growing the seed.
[0028] The present invention provides a cultivar of a grass plant
grown from a seed deposited under American Type Culture Collection
(ATCC) No. ______, comprising at least one heterologous nucleic
acid sequence from a Festuca mairei plant. In one embodiment, the
nucleic acid sequence comprises a sequence selected from the group
consisting of SEQ ID NOs:1-39 (FIG. 7), and 93-216 (FIG. 9). In one
embodiment, the nucleic acid sequence comprises a sequence selected
from the group consisting of SEQ ID NOs: 1-39. In one embodiment,
the nucleic acid sequence comprises a sequence selected from the
group consisting of SEQ ID NOs:1-4, 8-10, 12-19, 25-26, 29-30, and
32-37.
[0029] The present invention provides a seed of a drought resistant
grass plant cultivar, wherein said drought resistant grass plant
comprises at least one heterologous nucleic acid sequence selected
from the group consisting of SEQ ID NOs: 1-39, and 93-216, wherein
a representative sample of seed of said cultivar was deposited
under ATCC Accession No. ______.
[0030] The present invention provides a plant, comprising the
morphological and physiological properties of a grass plant grown
from a seed deposited under American Type Culture Collection (ATCC)
No. ______.
[0031] The present invention provides a method of providing a
cultivar of a grass plant comprising: a) providing; i) a grass
plant, and ii) a nucleic acid sequence, wherein said nucleic acid
sequence comprises a sequence selected from the group consisting of
SEQ ID NOs: 1-39, and 93-216, and sequences at least 90% identical
thereto; and b) introgressing said nucleic acid sequence into said
grass plant under conditions to produce a progeny grass plant. In
other embodiments, the nucleic acid sequence comprises a sequence
at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identical to a
sequence selected from the group consisting of SEQ ID NOs: 1-39,
and 93-216. In other embodiments, the nucleic acid sequence is at
least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identical to a
sequence selected from the group consisting of SEQ ID NOs: 1-39,
and 93-216. In other embodiments, the present invention provides
nucleotide sequences at least 90% identical to a sequence selected
from the group consisting of SEQ ID NOs: 1-39, and 93-216. In one
embodiment, the nucleic acid sequence comprises a sequence selected
from the group consisting of SEQ ID NOs: 1-39. In one embodiment,
the nucleic acid sequence comprises a sequence selected from the
group consisting of SEQ ID NOs:1-4, 8-10, 12-19, 25-26, 29-30, and
32-37. In one embodiment, the nucleic acid sequence encodes a
polypeptide comprising a sequence selected from the group
consisting of SEQ ID NOs:40-91 (FIG. 8). In one embodiment, the
introgressing is breeding or transfecting. The present invention is
not limited to any particular type of introgression, including but
not limited to natural breeding, artificial breeding, breeding
using molecular marker selection, commercial breeding, and
transgenics. In one embodiment, the introgressed nucleic acid
sequence is meiotically stable. The present invention is not
limited to any particular source of Festuca mairei nucleic acid
sequence. Indeed, a variety of sources are contemplated including
but not limited to a hybrid plant, Lolium perenne hybrid plant,
F.sub.1 hybrid plant, 4.times.F.sub.1 hybrid plant, 3.times.F.sub.1
hybrid plant, backcross progeny plant, plant parts, and synthetic
nucleic acid sequence. In one embodiment, the F.sub.1 hybrid plant
is selected from the group of hybrid plants consisting of Festuca
mairei.times.Lolium perenne species and hybrids thereof. In one
embodiment, the F.sub.1 hybrid plant is a Festuca mairei
plant.times.Calypso plant. In one embodiment, the F.sub.1 hybrid
plant is a Festuca mairei plant.times.Citation II plant. In one
embodiment, the backcross progeny plant is selected from but not
limited to the group of G15, G30a, G6, G11b, G14, G27b, G30b, G11a,
and G14 plants. In one embodiment, the Festuca mairei nucleic acid
sequence is provided by a G15 plant or progeny thereof. In one
embodiment, the Festuca mairei nucleic acid sequence is provided by
a G30a plant or progeny thereof. In one embodiment, the Festuca
mairei nucleic acid sequence is provided by a plant that derives
from a G15 or a G30a plant. In one embodiment, the Festuca mairei
nucleic acid sequence is provided by a plant part. The present
invention is not limited to any particular plant part. Indeed, a
variety of plant parts are contemplated including but not limited
to a pollen grain, ovule, tissue, seed, and a cell. In one
embodiment, the Festuca mairei nucleic acid sequence is provided by
a synthetic Festuca mairei nucleic acid sequence. In one
embodiment, the Festuca mairei nucleic acid sequence is provided by
a transgenic nucleic acid sequence. The present invention is not
limited to any particular grass plant. Indeed, a variety of grass
plants are contemplated for breeding purposes including but not
limited to turfgrass plants, forage grass plants, ornamental grass
plants, ground cover grass plants, transgenic grass plants and
elite grass plants. In one embodiment, the plant is selected from
the group including but not limited to ryegrass, bluegrass, Bermuda
grass, zoysiagrass plants and cool season grass plants. In one
embodiment, an elite grass plant comprises at least one agronomic
trait desirable in the progeny grass plant. The present invention
is not limited to any particular agronomic trait. Indeed, a variety
of agronomic traits are contemplated, including but not limited to
drought resistance, heat resistance, microbe resistance, insect
resistance, particular color, particular height, particular type of
root development, forage quality, and the like. In one embodiment,
the ryegrass plant is a Lolium perenne plant or hybrid plant
thereof. In one embodiment, the ryegrass plant is a Citation II or
a Calypso plant. In one embodiment, the grass plant is a hybrid
grass plant. The present invention is not limited to any particular
hybrid grass plant. Indeed, a variety of hybrid grass plants are
contemplated for breeding purpose, including but not limited to
Lolium perenne species plant hybrids and Festuca mairei plant
hybrids. In one embodiment, the types of hybrid grass plants
include but are not limited to a Festuca mairei plant.times.a grass
plant, a Festuca mairei plant.times.Lolium perenne species plant or
hybrid plant thereof. In one embodiment, the types of hybrid grass
plants include but are not limited to F.sub.1 hybrid plants,
4.times.F.sub.1 hybrid plants, 3.times.F.sub.1 hybrid plant, and
backcross progeny plants.
[0032] In one embodiment, the F.sub.1 hybrid plant is selected from
the group of hybrid plants consisting of Festuca
mairei.times.Lolium perenne species and hybrids thereof. In one
embodiment, the F.sub.1 hybrid plant is a Festuca mairei
plant.times.Calypso plant. In one embodiment, the F.sub.1 hybrid
plant is a Festuca mairei plant.times.Citation II plant. In one
embodiment, the backcross progeny plant is selected from the group
consisting of G15, G30a, G6, G11b, G14, G27b, G30b, G11a, and G14
plants. In a preferred embodiment, the backcross progeny plant is a
G15 or a G30a plant. In one embodiment, the ryegrass plant derives
from a G15 plant or a G30a plant. In one embodiment, a molecular
marker is provided. In one embodiment, the method further comprises
a molecular marker and using said molecular marker for identifying
at least one Festuca mairei nucleic acid sequence in a grass plant
or a progeny grass plant.
[0033] The present invention is not limited to any particular
molecular marker. Indeed, a variety of molecular markers are
contemplated, including but not limited to a simple sequence repeat
(SSR), microsatellite marker, random amplified polymorphic DNA
(RAPD), cDNA-amplified fragment length polymorphism, Festuca mairei
nucleic acid, and Festuca mairei polypeptide. In one embodiment,
the cDNA-amplified fragment length polymorphism (AFLP) marker is
provided by a linker or primer nucleic acid sequence selected from
the group consisting of SEQ ID NOs:217-246 and 247-265. In one
embodiment, the random amplified polymorphic DNA (RAPD) marker is
provided by a primer sequence selected from the group consisting of
SEQ ID NOs:266-306. In one embodiment, the Festuca mairei nucleic
acid marker comprises a sequence selected from the group consisting
of SEQ ID NOs: 1-39, and 93-216. In other embodiments, the nucleic
acid sequence marker comprises a sequence at least 50%, 60%, 70%,
80%, 90%, 95%, 98%, or 99% identical to a sequence selected from
the group consisting of SEQ ID NOs: 1-39, and 93-216. In other
embodiments, the nucleic acid sequence marker is at least 50%, 60%,
70%, 80%, 90%, 95%, 98%, or 99% identical to a sequence selected
from the group consisting of SEQ ID NOs: 1-39, and 93-216. In other
embodiments, the present invention provides nucleotide sequence
markers at least 90% identical to a sequence selected from the
group consisting of SEQ ID NOs: 1-39, and 93-216. In one
embodiment, the nucleic acid sequence marker comprises a sequence
selected from the group consisting of SEQ ID NOs: 1-39. In one
embodiment, the nucleic acid sequence marker is a sequence selected
from the group consisting of SEQ ID NOs: 1-39. In other
embodiments, the present invention provides nucleotide sequence
markers at least 90% identical to a sequence selected from the
group consisting of SEQ ID NOs: 1-39. In one embodiment, the
nucleic acid sequence marker comprises a sequence selected from the
group consisting of SEQ ID NOs:93-216. In one embodiment, the
nucleic acid sequence marker is a sequence selected from the group
consisting of SEQ ID NOs: 93-216. In other embodiments, the present
invention provides nucleotide sequence markers at least 90%
identical to a sequence selected from the group consisting of SEQ
ID NOs: 93-216. In one embodiment, the polypeptide marker comprises
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 40-91. In one embodiment, the polypeptide marker is an amino
acid sequence selected from the group consisting of SEQ ID NOs:
40-91. In other embodiments, the polypeptide marker comprises a
sequence at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%
identical to a sequence selected from the group consisting of SEQ
ID NOs: 40-91. In other embodiments, the polypeptide marker is at
least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identical to a
sequence selected from the group consisting of SEQ ID NOs: 40-91.
In other embodiments, the present invention provides a polypeptide
marker at least 90% identical to a sequence selected from the group
consisting of SEQ ID NOs: 40-91. In one embodiment, the identifying
is an assay selected from the group consisting of a simple sequence
repeats (SSR), microsatellite marker, random amplified polymorphic
DNA (RAPD), cDNA-amplified fragment length polymqrphism,
microarray, macroarray, Northern, Southern, and Western. In one
embodiment, the progeny plant is drought resistant. The present
invention is not limited to any particular drought resistant
progeny plant. Indeed, a variety of drought resistant progeny
plants are contemplated, including but not limited to a drought
resistant progeny plant comprising an agronomically desirable
trait, a progeny plant that is tolerant to drought stress, a
progeny plant that remains turgid during drought stress, a progeny
plant that demonstrates increased resistance to drought stress as
compared to the grass plant, a progeny plant that is an
agronomically desirable plant, a progeny plant that is a
commercially desirable plant, and a progeny plant that is a
commercially desirable cultivar.
DESCRIPTION OF THE FIGURES
[0034] FIG. 1 shows an exemplary relationship between leaf water
content and soil moisture content of Festuca mairei compared with
Festuca arundinacea tall fescue cultivars (such as Falcon and
Borolex).
[0035] FIG. 2 shows an exemplary chart of relative leaf water
content of Festuca mairei during an extreme drought stress
period.
[0036] FIG. 3 shows an exemplary comparison of cDNA-AFLP profiles
of Festuca mairei plants under specific environmental growth
conditions: a: cDNA-AFLP (NspI-CC/TaqI-TC) profile on treatment
control plants. Transcript derived fragments (TEFs) were
constitutively expressed at the same level. b: cDNA-AFLP
(NspI-TC/TaqI-TG) profile on stress treated plants: A, up-regulated
DEF from day 0 to day 4; B, constitutively expressed fragment from
day 0 to day 5; C, down-regulated DEF from day 0 to day 5; D,
transiently expressed DEF on day 4. Stressed leaf samples of day 6,
7 and 8 were did not yield sufficient mRNA for evaluation, likely
due to the dry and highly fibrous leaf condition. Therefore, DEFs
were scored based on the first six lanes of the cDNA-AFLP profile
of stress treated plants while the last three lanes were used as
reference.
[0037] FIG. 4 shows an exemplary distribution of the patterns of
differentially expressed fragments (DEFs) revealed by cDNA-AFLP
during drought stress treatment in F. mairei.
[0038] FIG. 5 shows an exemplary portion of a hybridized
macroarray. The differentially expressed fragments (DEFs) from
cDNA-AFLP were arrayed in duplicate on nylon membranes A and B.
Membranes were separately hybridized to treatment control (A) and 5
days stress treated (B) cDNA probes, respectively. Spots in the
squares indicated the housekeeping controls used for normalization
between arrays before comparing gene expression). Spots in the
circles indicated the negative controls used to eliminate the
background effect. Spots in the up-triangles are examples of
up-regulated DEFs. Spots in the down-triangles are examples of
down-regulated DEFs.
[0039] FIG. 6 shows an exemplary comparison of functional
categories between up-regulated and down-regulated differentially
expressed fragments (DEFs) during drought stress treatment in
Festuca mairei. Each DEF was searched against the GenBank plant
protein database by BLASTX. The functional category was assigned
based on function classification criteria on the website of the
Munich Information Center for Protein Sequences (MIPS) (website
address at mips.gsf.de).
[0040] FIG. 7 shows exemplary preferred Fm nucleotide sequences
(SEQ ID NOs: 1-39).
[0041] FIG. 8 shows exemplary translated Fm sequences (SEQ ID
NOs:40-91).
[0042] FIG. 9 shows an exemplary preferred Fm nucleotide sequences
(SEQ ID NOs:93-216).
[0043] FIG. 10 shows an exemplary schematic for cross-breeding
scheme for introgression of Festuca mairei germplasm into Lolium
perenne plants.
[0044] FIG. 11 shows exemplary Festuca mairei/Lolium perenne genome
ratios of backcross progenies from Festuca mairei and perennial
ryegrass assessed by using SSR and RAPD markers.
[0045] FIG. 12 shows an exemplary variation of soil water content
(a), leaf water potential (b), leaf elongation (c), and leaf water
content (d) among genotypes of Atlas fescue, perennial ryegrass,
and their progeny in control (filled dots) and stressed (opened
dots) plants during the drought stress period.
[0046] FIG. 13 shows an exemplary plot of eigenvectors. LE, leaf
elongation; .PSI.w, leaf water potential; WC, leaf water content.
The numbers after LE, .PSI.w, and WC represent the week number
during the drought stress period.
[0047] FIG. 14 shows an exemplary principle component analysis of 9
genotypes for both control and stressed plants: a: Three genotype
components accounted for 33.4%, 16.4%, and 11.2% of the total
variation; and b: Three genotype components accounted for 30.5%,
16.2%, and 11.5% of the total variation.
[0048] FIG. 15 shows an exemplary grouping (classification) of
hybrid lines and cultivar plants of the present inventions.
[0049] FIG. 16 shows an exemplary soil water content (a), leaf
water potential decrease (b), leaf elongation decrease (c), and
leaf water content decrease (d) of the four classified groups
during the drought stress period. Error bars indicate standard
errors. *, ** shows significantly different means among the three
groups at certain weeks at P.ltoreq.0.05 and 0.01 respectively.
[0050] FIG. 17 shows an exemplary relationship between leaf
elongation decrease and soil water content.
[0051] FIG. 18 shows an exemplary relationship between leaf water
content decrease and soil water content.
[0052] FIG. 19 shows an exemplary relationship between leaf water
potential decrease and soil water content.
[0053] FIG. 20 shows an exemplary comparison of root length (a) and
biomass (b) between control (hydrated) and drought-stressed (water
deprived) plants. Bars indicate standard errors. Data for group
with the same letter (capitalized letter for comparison among the
groups in control plants and small letter for comparison among
groups in stressed plants) are no significant differences among
groups at P.ltoreq.0.05. ns, * and ** indicate no significant,
significantly different means between control and stressed plants
within group at P.ltoreq.0.05 and .ltoreq.0.01, respectively.
DEFINITIONS
[0054] To facilitate an understanding of the present invention, a
number of terms and phrases are defined below:
[0055] The use of the article "a" or "an" is intended to include
one or more.
[0056] The use of terms defined in the singular are intended to
include those terms defined in the plural and vice versa.
[0057] The term "drought resistant" or "drought tolerant" in
reference to a plant refers in general to a plant that is living
and/or thriving in surroundings having persistently low water
availability, such as low soil moisture content, for example, a
Festuca mairei drought tolerant plant growing in the Atlas
mountains or a plant of the present invention that continues to
live and/or thrive following the lowering of soil moisture content
and/or a under conditions of persistently low water availability.
On the other hand, a "drought susceptible" plant refers to a plant
that loses normal leaf color and/or increases leaf firing and/or
wilts during onset or persistent of low water availability.
[0058] The terms "drought resistance" or "drought tolerance" or
"drought stress resistance" or "drought stress tolerance" refer in
general to the response of a plant to a change in water
availability, naturally or artificially. An example of a natural
change in water availability is a seasonal difference in
availability of environmentally derived water. An example of an
artificial change of water availability is the lowering of the
amount of irrigation water, such that the soil moisture content is
lowered. Lowering water availability may be short-term or long-term
in time.
[0059] As used herein, "tolerant" in relation to "drought stress"
and equivalent terms also refers to a plant that is living and/or
thriving during time periods of low water availability.
[0060] The terms "altered environmental tolerance" and "altering
environmental tolerance" in reference to a plant refer to any
changes in a plant's ability to tolerate an environmental abiotic
stress. The terms "altered abiotic stress" and "altering abiotic
stress" refer to any changes in abiotic tolerance such as an
increased tolerance to an abiotic stress, such as onset or
persistence of "dry conditions" or "drought," heat, cold, "high
saline" or "salt."
[0061] The terms "altered drought tolerance" and "altering drought
tolerance" refer to any changes in drought and tolerance and
changes in environmental factors such as lower rainfall and lower
soil moisture content. An "altered drought tolerance phenotype"
refers to detectable change in the ability of a modified plant to
withstand low-water conditions compared to the similar, but
non-modified plant. In general, improved (increased) drought
tolerance phenotypes (i.e., ability to a plant to survive in
low-water conditions that would normally be deleterious to a plant)
are of interest.
[0062] For the purposes of the present invention, an "increasing"
or "increased" or "enhanced" tolerance refers to a higher level of
tolerance of a modified plant or plant part over a control plant or
plant part, such as a wild-type control plant or plant part or a
nonmodified control or plant part, such as when comparing a plant
or leaf from a Fm:Lp hybrid grass plant of the present invention to
a closely related cultivar of Lp plant or a Lp leaf from an Lp
cultivar. Examples include increasing expression of a gene
associated with increasing drought tolerance or maintaining turgor
under low water conditions, increasing water content of plants
under drought conditions, increasing the capability of a plant to
continue living and/or growing under environmental conditions such
as extreme dryness, such as depriving plants of irrigation water,
see, EXAMPLES.
[0063] As used herein, the term "drought adaptation" in reference
to a plant refers to an adaptation response of a plant during
lowering or lowered environmental water access.
[0064] As used herein, the term "environmental water access" refers
to natural soil moisture content or artificially controlling water
access to a plant.
[0065] As used herein, the terms "turgid" or "not wilted" or
"hydrated" applied to a plant cell, a plant tissue, a plant part
and a plant refer to a firm condition of the plant cell, plant
tissue, plant part or plant when it is filled with water. A turgid
condition of a plant or plant part refers to the opposite condition
of wilted plant or plant part. "Turgor" refers to a condition of a
cell or tissue or plant such as a turgid cell or turgid tissue or
turgid plant that is opposite of a dehydrated cell or dehydrated
tissue or dehydrated plant.
[0066] As used herein, the term "wilt" refers to a symptom or a
disease characterized by a loss of turgidity in a plant (for
example, vascular wilt).
[0067] As used herein, the term "wilting" refers to a symptom
characterized by loss of turgor or dehydration which results in
drooping of leaves and/or stems and/or flowers, such as
demonstrated when a physiological damaging decrease in turgidity
causes wilting.
[0068] As used herein, the term "plant" is used in it broadest
sense. It includes, but is not limited to, any species of grass
(e.g. ryegrass and turfgrass), ornamental or decorative, crop or
cereal, fodder or forage, fruit or vegetable, fruit plant or
vegetable plant, herb plant, woody plant, flower plant or tree. It
is not meant to limit a plant to any particular structure. It also
refers to a unicellular plant (e.g. microalga) and a plurality of
plant cells that are largely differentiated into a colony (e.g.
volvox) or a structure that is present at any stage of a plant's
development. Such structures include, but are not limited to, a
seed, a tiller, a sprig, a stolen, a plug, a rhizome, a shoot, a
stem, a leaf, a flower petal, a fruit, et cetera.
[0069] Thus, plants that may be useful in the invention's methods
include, without limitation, any plant that is capable of being
transformed by a nucleic acid sequence using any method. In one
embodiment, the plant has an agronomic, horticultural, ornamental,
economic, and/or commercial value. Exemplary plants include acacia,
alfalfa, aneth, apple, apricot, artichoke, arugula, asparagus,
avocado, banana, barley, beans, beet, blackberry, blueberry,
broccoli, brussels sprouts, cabbage, canola, cantaloupe, carrot,
cassaya, castorbean, cauliflower, celery, cherry, chicory,
cilantro, citrus, clementines, clover, coconut, coffee, corn,
cotton, cucumber, Douglas fir, eggplant, endive, escarole,
eucalyptus, fennel, figs, garlic, gourd, grape, grapefruit, honey
dew, jicama, kiwifruit, lettuce, leeks, lemon, lime, Loblolly pine,
linseed, mango, melon, mushroom, nectarine, nut, oat, oil palm, oil
seed rape, okra, olive, onion, orange, an ornamental plant, palm,
papaya, parsley, parsnip, pea, peach, peanut, pear, pepper,
persimmon, pine, pineapple, plantain, plum, pomegranate, poplar,
potato, pumpkin, quince, radiata pine, radicchio, radish, rapeseed,
raspberry, rice, rye, sorghum, Southern pine, soybean, spinach,
squash, strawberry, sugarbeet, sugarcane, sunflower, sweet potato,
sweetgum, tangerine, tea, tobacco, tomato, triticale, turf, turnip,
a vine, watermelon, wheat, yams, and zucchini.
[0070] "Plant cell" is the structural and physiological unit of
plants, consisting of a protoplast and the cell wall. The term
includes any composition that contains plant cells such as, without
limitation, a plant, plantlet, seed, tissue, organ, callus,
protocorm-like body, suspension culture, protoplasts, and the like.
"Plant tissue" is a group of plant cells organized into a
structural and functional unit. "Plant organ" is a collection of
tissues that performs a particular function or set of functions in
a plant's body. The leaf, stem, and root are exemplary organs found
in many plants. Organs are composed of tissues. "Plant callus" is a
cluster of undifferentiated plant cells that have the capacity to
regenerate a whole plant. "Plant cell suspension culture" refers to
plant cells in liquid medium.
[0071] The term "xerophytic plant" refers to a plant adapted for
life with a limited supply of water, as opposed to a "hydrophytic
plant" or "hydrophyte" in reference to a plant adapted to live in
water or in waterlogged soil and a "mesophytic plant" or
"mesophyte" in reference to a land plant growing in surroundings
having an average supply of water, for example, a moderate amount
of moisture, neither too dry nor too wet.
[0072] The term "xerophyte" refers to a plant adapted to living in
a dry arid habitat, such as a desert plant.
[0073] The term "xeromorphic" refers to a plant with morphological
and physiological characters that tolerate persistently low water
availability, such as succulent plant.
[0074] As used herein, the terms "crop" and "crop plant" are used
herein its broadest sense. The term includes, but is not limited
to, any species of plant or alga edible by humans or used as a feed
for animals or fish or marine animals, or consumed by humans, or
used by humans (natural pesticides), or viewed by humans (flowers)
or any plant or alga used in industry or commerce or education,
lawns, engineering, and agricultural uses. Indeed, a variety of
crop plants are contemplated, including but not limited to
ryegrass, such as and annual ryegrass and perennial ryegrass, turf
grass, forage grass, corn, wheat, rice, barley, sorgham, sunflower,
herbs and trees. The term includes, but is not limited to any
species of plant used as a feed for animals or birds, or fish, or
reptiles, or marine animals. In some embodiments of the present
invention, transgenic plants are crop plants.
[0075] As used herein, the term "grass" in reference to a plant
refers to any plant of the Poaceae family or Gramineae family,
Cyperaceae family (sedges), and Juncaceae family (rushes). A grass
may comprise a hollow, a segmented, and a round stem, bladelike
leaf, and extensively branching fibrous root systems, such as any
of grasses of the genus Festuca species or any of the Lolium
species. A grass plant may be a cereal grass, a forage grass, a
turf grass, an ornamental grass, pasture grass, a hay grass, a
cover grass, and a cereal grass, such as wheat, corn, rice, rye,
oats, barley, and millet. A grass plant may provide forage for
grazing animals, shelter for wildlife, stabilization of soils, such
as fire-burned soil, bare soil, etc., construction materials,
furniture, utensils, and food for humans. Some grass species are
grown as garden ornamentals, cultivated as turf for lawns and
recreational areas, or used as cover plants for erosion
control.
[0076] The term "ground cover" refers to a use of a plant to fill
in areas of land (e.g. sunny area, shaded area, and the like).
[0077] As used herein, "Festuca mairei" or "Fm" or "Atlas Fescue"
refers to an ornamental grass plant of the Festuca family as
described herein.
[0078] As used herein, "ryegrass" in general refers to a perennial
ryegrass "Lolium perenne L. ssp. Perenne" or "English ryegrass" or
"crested ryegrass." Ryegrass may also refer to an annual ryegrass
"Lolium perenne ssp. multiflorum" also referred to as "Italian
ryegrass," and any intermediate species of ryegrass.
[0079] As used herein, "common ryegrass" or "Lolium species" or
"Loliurn spp." refers to a commercial mixture of ryegrass species,
such as a mixture of annual ryegrass species, but may also contain
a substantial percentage of a perennial ryegrass species and
annual-perennial hybrid species of ryegrass, also referred to as
"intermediate ryegrass."
[0080] As used herein, "annual ryegrass" in general refers to a
"Lolium multiflorum" plant that lives for one year or less.
[0081] As used herein, "perennial ryegrass" in general refers to a
"Lolium perenne" plant that lives for more than two years.
[0082] As used herein, "intermediate ryegrass" in general refers to
a hybrid "Lolium hybridum" plant that developed by crossing annual
and perennial ryegrass.
[0083] The term "haploid" or "n" refers to an organism or cell with
no more than one set of chromosomes.
[0084] The term "polyploidy" refers to a condition of a cell or
organisms containing more than two homologous sets of chromosomes.
For example, polyploid types are termed according to the number of
chromosome sets in the nucleus: three sets refers to a triploid
(3n), four sets refers to a tetraploid (4n), five sets refers to a
pentaploid (5n), six sets refers to a hexaploid (6n, such as a
Sequoia sempervirens), et cetera.
[0085] The term "diploid" in reference to a plant refers to a plant
with 2 sets of chromosomes (for e.g. the majority of wild-type
grass plants).
[0086] The term "triploid" in reference to a plant refers to a
plant with 3 sets of chromosomes.
[0087] The term "tetraploid plant" refers to a plant that has 4
sets of chromosomes per cell. As used herein, the term "tetraploid
grasses" refers to grasses that have 4 sets of chromosomes per cell
(e.g. tetraploid varieties of grasses such as ryegrass, red clover,
lotus, etc.).
[0088] The term "allopolyploids" refers to a polyploidy with
chromosomes derived from different species, for example, triticale
has six chromosome sets, four from wheat (Triticum turgidum) and
two from rye (Secale cereale).
[0089] The term "amphidiploid" or "allopolyploid" refers to a
polyploid formed from the union of two separate chromosome sets and
their subsequent doubling, such as an organism produced by
hybridization of two species followed by chromosome doubling. An
allotetraploid may appear to be a normal diploid.
[0090] The term "autopolyploids" or "autotetraploidy" refers to a
polyploidy with chromosomes derived from a single species, such as
an autopolyploid can arise from a spontaneous, naturally-occurring
genome doubling (for example, potatoes), and as a further example,
bananas and apples can be triploid autopolyploids.
[0091] The term "plant tissue" includes differentiated and
undifferentiated tissues of plants including those present in
roots, shoots, leaves, pollen, seeds and tumors, as well as cells
in culture (e.g., single cells, protoplasts, embryos, callus,
etc.). Plant tissue may be in planta, in organ culture, tissue
culture, or cell culture.
[0092] As used herein, the term "plant part" as used herein refers
to a plant structure or a plant tissue, for example, pollen, an
ovule, a tissue, a pod, a seed, a leaf and a cell. Plant parts may
comprise one or more of a tiller, plug, rhizome, sprig, stolen,
meristem, crown, and the like.
[0093] For the purposes of the present inventions, the term
"hybrid" in reference to a seed or plant is produced as the result
of artificially controlled cross-pollination as opposed to a
"non-hybrid" seed produced as the result of natural pollination,
such as in a "hybrid seed" or "hybrid plant" produced by
introgression methods of the present invention, for example,
selective breeding or transgene insertion.
[0094] As used herein, the terms "F-generation" and "filial
generation" refers to any of the consecutive generations of cells,
tissues or organisms, such as a plant, after a biparental cross.
The generation resulting from a mating of the a biparental cross
(i.e. parents) is the first filial generation (designated as "F1"
and "F.sub.1") in reference to both a seed and a plant of that
generation, while that resulting from crossing of F1 individuals is
the second filial generation (designated as "F2" or "F.sub.2") in
reference to both a seed and a plant of that generation, et
cetera.
[0095] As used herein, "progeny" refers to a product of any cross
between two plants, such as a backcross or outcross, where progeny
may trace a pedigree back to the original cross. As used herein, a
second plant is derived from a first plant if the second plant's
pedigree includes the first plant.
[0096] As used herein, the terms "introgress" and "introgressing"
refer to incorporating a genetic substance, such as germplasm,
loci, allele, gene, DNA, and the like for introducing a trait into
an organism, such as a plant, a plant cell, a yeast cell, and the
like, for example, incorporating drought resistant transgenic
material and/or transgenes into a previously drought susceptible
plant variety. Introgression may refer to one of several types of
breeding methods for a incorporating a genetic trait, such as
drought resistance, provided by expression of a heterologous gene
or silencing of an endogenous gene.
[0097] The term "variety" refers to a biological classification for
an intraspecific group or population, that can be distinguished
from the rest of the species by any characteristic (for example
morphological, physiological, cytological, etc.). A variety may
originate in the wild but can also be produced through selected
breeding (for example, see, cultivar).
[0098] The terms "cultivar," "cultivated variety," and "cv" refer
to a group of cultivated plants distinguished by any characteristic
(for example morphological, physiological, cytological, etc.) that
when reproduced sexually or asexually, retain their distinguishing
features to produce a cultivated variety. In reference to a
ryegrass, a cultivar may be a diploid or a tetraploid cultivar,
such as "Big Daddy" (1995) and "Jumbo" (2000), released by the
University of Florida IFAS (Institute of Food and Agricultural
Sciences) Ryegrass Breeding Program.
[0099] The term "elite cultivar" refers to a commercial or breeding
stock cultivar. In reference to a ryegrass, an elite ryegrass
cultivar includes but is not limited to "Citation II," "Calypso,"
"Florida 80" (1982), "Surrey" (1989), Big Daddy, "Stampede" (1998),
"Natchez" (1999), "Fantastic" (1999), "Florlina" (1999), Jumbo,
"Passeral Plus" (2000), "King" (2000) and "Graze-N-Gro" (2000)
released by the University of Florida IFAS (Institute of Food and
Agricultural Sciences) Ryegrass Breeding Program. An elite ryegrass
cultivar in reference to ryegrass plants of the present inventions
may also refer to a preferred breeding stock such as hybrid
ryegrass plants comprising Fm germplasm as described herein.
[0100] The term "host cell" refers to any cell capable of
replicating and/or transcribing and/or translating a heterologous
gene. Thus, a "host cell" refers to any eukaryotic or prokaryotic
cell (e.g., plant cells, algal cells such as C. reinhardtii,
bacterial cells such as yeast cells, E. coli, insect cells, etc.),
whether located in vitro or in vivo. For example, a host cell may
be located in a transgenic plant, or located in a plant part or
part of a plant tissue or in cell culture.
[0101] As used herein, the term plant cell "compartments" or
"organelles" is used in its broadest sense. As used herein, the
term includes but is not limited to, the endoplasmic reticulum,
Golgi apparatus, trans Golgi network, plastids, sarcoplasmic
reticulum, glyoxysomes, mitochondrial, chloroplast, thylakoid
membranes and nuclear membranes, and the like.
[0102] The terms "leaf" and "leaves" refer to a usually flat, green
structure of a plant where photosynthesis and transpiration take
place and that grow attached to a stem or branch.
[0103] The terms "cotyledon," "true leaf," and "seed leaf" refer to
any one of the first leaves to appear after germination (there may
be one, such as a monocotyledon, two, such a dicotyledoen or more)
and the foliar portion of the embryo as found in the seed. The term
"hypocotyl" refers to a part of the stem of an embryo or young
seedling below the cotyledons.
[0104] As used herein, "aerial" and "aerial parts of Arabidopsis
plants" refer to any plant part that is above water in aquatic
plants or any part of a terrestrial plant part found above ground
level.
[0105] The terms "radicle" and "radicles" refer to rootlets
emerging from the sides and base of the stem and the end of a plant
embryo which gives rise to the first root. A radicle may also
comprise a "rhizoid" which refers to a cellular outgrowth of a
plant that usually aids in anchoring to the surface and increasing
surface area to acquire water or nutrients.
[0106] The term "lemma" refers to the lower of the two bracts
enclosing the flower in the spikelet of grasses.
[0107] The term "bract" refers to a leaf from the axil of which a
flower arises.
[0108] The term "axil" refers to the angle between a branch or leaf
and the stem from which it grows.
[0109] The term "inflorescence" refers to a flowering part of a
plant.
The term "meristem" refers to undifferentiated tissue from which
new cells are formed, e.g., the tips of roots or stems; the growing
tip. The term "meristem cloning" refers to artificial propagation
of a plant using cells taken from the meristem of a parent plant
and yielding genetically identical offspring.
[0110] The term "stem" refers to a main ascending axis of a
plant.
[0111] The term "seed" refers to a ripened ovule, consisting of the
embryo and a casing.
[0112] The term "propagation" refers to the process of producing
new plants, for example, asexual reproduction or sexual
reproduction.
[0113] The terms "vegetative propagation" and "asexual
reproduction" refer to the ability of plants to reproduce without
sexual reproduction, by producing new plants from existing
vegetative structures that are clones, i.e., plants that are
identical in all attributes to the mother plant and to one another.
In other words, vegetative propagation involves using parts of an
original plant to make new plants. Plants derived from vegetative
propagation may be produced by means such as tissue culture, the
division of a plant clump, rooting of root or stem or leaf
cuttings, or cutting of mature crowns or involving the rooting or
grafting of pieces of one plant onto other pieces of other
plants.
[0114] The terms "calli" and "callus" refer to a tough, often
hairy, swelling at the base or insertion of the lemma.
[0115] The term "tiller" refers to a portion of a plant where a
lateral stem (or shoot), usually erect, develops from the central
crown, often used for propagation of grass plants. Also refers to
the branch or shoot that originates at a basal node.
[0116] The terms "stolen" and "runner" refer to an elongated
horizontal stem (or shoot) that grows above the soil or just under
the soil surface that roots at nodes and can form new plants. The
term "stoloniferous" in reference to a plant, refers to spreading
or growing by means of stolons.
[0117] The term "rhizome" refers to a specialized slender or
swollen stem with branching close to the soil surface that can
produce a root, a stem, a leaf and a flower, along its length and
at its apex.
[0118] The term "sprig" refers to a small part of a plant
comprising a short piece of the stolon or rhizome, roots and
leaves, but not soil, (e.g. stolon, used for propagation). The term
"plug" refers to a small piece of sod usually two or more inches
wide comprising 2 to 3 inches of soil and grass roots.
[0119] The term "sod" refers to any one of a plug, square of grass,
and strip of grass, with adhering soil that are used in vegetative
planting, for example, top few centimeters of soil permeated by and
held together with grass roots or grass-legume roots.
[0120] The term "sodformer" refers to grass that propagates by seed
and vegetatively by rhizomes and/or stolons to form a sod.
[0121] As used herein, the term "trait" refers to an observable
and/measurable characteristics of an organism, such as a trait of a
plant, for example, resistance to low soil moisture, tolerance to
an herbicide, an agronomic trait, and the like.
[0122] As used herein, the terms "agronomic trait" or
"agronomically desirable trait "or" agronomically significant
trait" refers to any selected trait that increases the commercial
or economic or utility value of a plant and/or preferred plant
part, for example a preferred level of drought resistance or
drought tolerance, water resistance, cold weather resistance, hot
weather resistance, growth in a particular hardiness zone, yield,
nutritional content, protein content, fiber content, root
properties, root spreading, oil content, seed protein content, seed
size, seed color, seed coat thickness, seed sugar content, seed
free amino acid content, seed germination rate, seed texture, seed
fiber content, food-grade quality, hilum color, seed yield, and
color of a plant part.
[0123] As used herein, the term "modified plant" refers to plant
modified by man. A modified plant may be plant derived from
artificial breeding, such as artificial pollination, or transgenic
production, such as Agrobacterium-mediated, electroporation, etc.
to insert a heterologous gene or a combination of artificial
breeding and transgenic methods.
[0124] As used herein, the terms "modified" or "altered" regarding
a plant trait, refer to a change in the phenotype of a modified
plant relative to the similar non-modified plant, such as
introgressing a plant trait into a plant lacking that trait, for
example increasing drought tolerance in a plant. An "improvement"
is a feature that may enhance the utility of a plant species or
variety by providing the plant with a unique and/or novel
quality.
[0125] The terms "ABA" and "abscisic acid" refer to molecules that
induce "ABA-responsive proteins" comprising "abscisic acid
responsive elements" and "ABA responsive elements" that refer to
DNA regions of in the promoter region that bind to ABA of genes
that respond to ABA mediated environmental stress.
[0126] The term "abiotic stress" refers to a nonliving
environmental factors such as drought, salt, cold, excessive heat,
high winds, etc., that can have harmful effects upon plants. For
the purposes of the present invention, examples of abiotic stress
specifically include drought and salt factors.
[0127] The terms "eukaryotic" and "eukaryote" are used in its
broadest sense. It includes, but is not limited to, any organisms
containing membrane bound nuclei and membrane bound organelles.
Examples of eukaryotes include but are not limited to plants,
yeast, animals, alga, diatoms, and fungi.
[0128] The terms "prokaryote" and "prokaryotic" are used in its
broadest sense. It includes, but is not limited to, any organisms
without a distinct nucleus. Examples of prokaryotes include but are
not limited to bacteria, blue-green algae, archaebacteria,
actinomycetes and mycoplasma. In some embodiments, a host cell is
any microorganism.
[0129] As used herein the term "microorganism" refers to
microscopic organisms and taxonomically related macroscopic
organisms within the categories of yeast, algae, bacteria, and
fungi (including lichens).
[0130] The term "Agrobacterium" refers to a soil-borne,
Gram-negative, rod-shaped phytopathogenic bacterium that causes
crown gall. Agrobacterium is a representative genus of a
soil-borne, Gram-negative, rod-shaped phytopathogenic bacterium
family Rhizobiaceae. Its species are responsible for plant tumors
such as crown gall and hairy root disease. In the dedifferentiated
tissue characteristic of the tumors, amino acid derivatives known
as opines are produced and catabolized. The bacterial genes
responsible for expression of opines are a convenient source of
control elements for chimeric expression cassettes. Agrobacterium
tumefaciens causes crown gall disease by transferring some of its
DNA to the plant host. The transferred DNA (T-DNA) is stably
integrated into the plant genome, where its expression leads to the
synthesis of plant hormones and thus to the tumorous growth of the
cells. A putative macromolecular complex forms in the process of
T-DNA transfer out of the bacterial cell into the plant cell. The
term "Agrobacterium" includes, but is not limited to, the strains
Agrobacterium tumefaciens (which typically causes crown gall in
infected plants), and Agrobacterium rhizogens (which causes hairy
root disease in infected host plants). Infection of a plant cell
with Agrobacterium generally results in the production of opines
(e.g., nopaline, agropine, octopine etc.) by the infected cell.
Thus, Agrobacterium strains which cause production of nopaline
(e.g., strain GV3101, LBA4301, C58, A208, etc.) are referred to as
"nopaline-type" Agrobacteria; Agrobacterium strains which cause
production of octopine (e.g., strain LBA4404, Ach5, B6, etc.) are
referred to as "octopine-type" Agrobacteria; and Agrobacterium
strains which cause production of agropine (e.g., strain EHA105,
EHA101, A281, etc.) are referred to as "agropine-type"
Agrobacteria.
[0131] The term "transgene" refers to a foreign gene that is placed
into an organism or host cell by the process of transfection. The
term "foreign gene" or heterologous gene refers to any nucleic acid
(e.g., gene sequence) that is introduced into the genome of an
organism or tissue of an organism or a host cell by experimental
manipulations, such as those described herein, and may include gene
sequences found in that organism so long as the introduced gene
does not reside in the same location, as does the naturally
occurring gene.
[0132] The terms "transgenic" when used in reference to a plant or
leaf or fruit or seed or plant part, for example a "transgenic
plant," "transgenic leaf," "transgenic fruit," "transgenic seed,"
and a "transgenic host cell," refer to a plant or leaf or fruit or
seed or part or cell that contains at least one heterologous or
foreign gene in one or more of its cells. The term "transgenic
plant material" refers broadly to a plant, a plant structure, a
plant tissue, a plant seed or a plant cell that contains at least
one heterologous gene in one or more of its cells. The term
"portion" when used in reference to a protein (as in "a portion of
a given protein") refers to fragments of that protein. The
fragments may range in size from four amino acid residues to the
entire amino sequence minus one amino acid.
[0133] The terms "transformants" and "transformed cells" include
the primary transformed cell and cultures derived from that cell
without regard to the number of transfers. Resulting progeny may
not be precisely identical in DNA content, due to deliberate or
inadvertent mutations. Mutant progeny that have the same
functionality as screened for in the originally transformed cell
are included in the definition of transformants.
[0134] The terms "tissue culture" and "micropropagation" refer to a
form of asexual propagation undertaken in specialized laboratories,
in which clones of plants are produced from small cell clusters
from very small plant parts (e.g. buds, nodes, leaf segments, root
segments, etc.), grown aseptically (free from any microorganism) in
a container where the environment and nutrition can be
controlled.
[0135] The term "gene" refers to a nucleic acid (e.g., DNA or RNA)
sequence that comprises coding sequences necessary for the
production of an RNA, or a polypeptide or its precursor (e.g.,
proinsulin). A functional polypeptide can be encoded by a
full-length coding sequence or by any portion of the coding
sequence as long as the desired activity or functional properties
(e.g., enzymatic activity, ligand binding, signal transduction,
etc.) of the polypeptide are retained. The term "portion" when used
in reference to a gene refers to fragments of that gene. The
fragments may range in size from a few nucleotides to the entire
gene sequence minus one nucleotide. The term "a nucleotide
comprising at least a portion of a gene" may comprise fragments of
the gene or the entire gene. The term "cDNA" refers to a nucleotide
copy of the "messenger RNA" or "mRNA" for a gene. In some
embodiments, cDNA is derived from the mRNA. In some embodiments,
cDNA is derived from genomic sequences. In some embodiments, cDNA
is derived from EST sequences. In some embodiments, cDNA is derived
from assembling portions of coding regions extracted from a variety
of BACs, contigs, Scaffolds and the like.
[0136] The term "gene" encompasses the coding regions of a
structural gene and includes sequences located adjacent to the
coding region on both the 5' and 3' ends for a distance of about 1
kb on either end such that the gene corresponds to the length of
the full-length mRNA. The sequences which are located 5' of the
coding region and which are present on the mRNA are referred to as
5' non-translated sequences. The sequences which are located 3' or
downstream of the coding region and which are present on the mRNA
are referred to as 3' non-translated sequences.
[0137] The term "gene" encompasses both cDNA and genomic forms of a
gene. A genomic form or clone of a gene contains the coding region
termed "exon" or "expressed regions" or "expressed sequences"
interrupted with non-coding sequences termed "introns" or
"intervening regions" or "intervening sequences." Introns are
segments of a gene that are transcribed into nuclear RNA (hnRNA);
introns may contain regulatory elements such as enhancers. Introns
are removed or "spliced out" from the nuclear or primary
transcript; introns therefore are absent in the messenger RNA
(mRNA) transcript. The mRNA functions during translation to specify
the sequence or order of amino acids in a nascent polypeptide.
[0138] In addition to containing introns, genomic forms of a gene
may also include sequences located on both the 5' and 3' end of the
sequences that are present on the RNA transcript. These sequences
are referred to as "flanking" sequences or regions (these flanking
sequences are located 5' or 3' to the non-translated sequences
present on the mRNA transcript). The 5' flanking region may contain
regulatory sequences such as promoters and enhancers that control
or influence the transcription of the gene. The 3' flanking region
may contain sequences that direct the termination of transcription,
posttranscriptional cleavage and polyadenylation.
[0139] The term "derived" in reference to a gene, such as a "gene
derived from a Festuca mairei plant" refers to a gene comprising a
nucleic acid sequence present in Festuca mairei germplasm.
[0140] The term "germplasm" refers to any genetic material of
plants, animals or other organisms containing functional units of
heredity. As used herein, germplasm may refer to any hereditary
material, such as a nucleotide sequence, gene, linkage group, QTL,
chromosome, and groups of chromosomes. As used herein, the term
"germplasm" in reference to "drought resistant germplasm" and
"drought resistance germplasm" refers to and encompasses hereditary
material associated with resistance to drought conditions, such as
Festuca mairei nucleic acid sequences, linkage groups, chromosomes,
quantitative trait loci (QTLs) and the like.
[0141] The term "drought tolerance nucleotide sequence" means a
nucleotide sequence (e.g., regulatory sequence, structural gene
sequence, etc.) that alters (i.e., increases or decreases) drought
tolerance.
[0142] The term "meiotic stability" or "meiotically stable" in
reference to genetic material of a plant, such as a gene, linkage
group or chromosome, refers to the retention of parental genetic
material in a daughter cell following meiotic division. For the
purposes of the present inventions, meiotic stability refers to the
passage of desired Festuca mairei germplasm from parental plants to
progeny plants produced by sexual reproduction.
[0143] The terms "allele" and "alleles" refer to each version of a
gene for a same locus that has more than one sequence. For example,
there are multiple alleles for eye color at the same locus. The
terms "recessive," "recessive gene," and "recessive phenotype"
refer to an allele that has a phenotype when two alleles for a
certain locus are the same as in "homozygous" or as in "homozygote"
and then partially or fully loses that phenotype when paired with a
more dominant allele as when two alleles for a certain locus are
different as in "heterozygous" or in "heterozygote." The terms
"dominant," "dominant allele," and "dominant phenotype" refer to an
allele that has an effect to suppress the expression of the other
allele in a heterozygous (having one dominant allele and one
recessive allele) condition.
[0144] The term "heterologous" when used in reference to a gene or
nucleic acid refers to a gene that was manipulated in some way. For
example, a heterologous gene includes a gene from one species
introduced into another species. A heterologous gene also includes
a gene native to an organism that was altered in some way (e.g.,
mutated, added in multiple copies, linked to a non-native promoter
or enhancer sequence, etc.). Heterologous genes may comprise plant
gene sequences that comprise cDNA forms of a plant gene; the cDNA
sequences may be expressed in either a sense (to produce mRNA) or
anti-sense orientation (to produce an anti-sense RNA transcript
that is complementary to the mRNA transcript). Heterologous genes
are distinguished from endogenous plant genes in that the
heterologous gene sequences are typically joined to nucleotide
sequences comprising regulatory elements such as promoters that are
not found naturally associated with the gene for the protein
encoded by the heterologous gene or with plant gene sequences in
the chromosome, or are associated with portions of the chromosome
not found in nature (e.g., genes expressed in loci where the gene
is not normally expressed).
[0145] The terms "nucleic acid sequence," "nucleotide sequence of
interest" or "nucleic acid sequence of interest" refer to any
nucleotide sequence (e.g., RNA or DNA), the manipulation of which
may be deemed desirable for any reason (e.g., treat disease, confer
improved qualities, etc.), by one of ordinary skill in the art.
Such nucleotide sequences include, but are not limited to, coding
sequences of structural genes (e.g., reporter genes, selection
marker genes, oncogenes, drug resistance genes, growth factors,
etc.), and non-coding regulatory sequences which do not encode an
mRNA or protein product (e.g., promoter sequence, polyadenylation
sequence, termination sequence, enhancer sequence, etc.). The term
"up-regulated" refers to a gene or protein whose expression is
increased over a control or before exposure to a condition, such as
low soil moisture.
[0146] The term "down-regulated" refers to a gene or protein whose
expression is decreased over a control or before exposure to a
condition, such as low soil moisture.
[0147] The term "up-regulated then down-regulated" or
"up-then-down-regulated" refers to a gene or protein whose
expression is increased over a control or before exposure to a
condition, such as low soil moisture, then decreases in expression
over time.
[0148] The term "differentially expressed" or "differentially
regulated" or "DEF" refers to a gene or nucleic acid or protein
whose expression is different from that of a control.
[0149] The term "transiently expressed" or "transiently regulated"
or "TEF" refers to a gene or protein whose expression is
temporarily expressed.
[0150] As used herein, the term "wild-type" when made in reference
to a gene refers to a functional gene common throughout an outbred
population. As used herein, the term "wild-type" when made in
reference to a gene product refers to a functional gene product
common throughout an outbred population. A functional wild-type
gene is that which is most frequently observed in a population and
is thus arbitrarily designated the "normal" or "wild-type" form of
the gene.
[0151] As used herein, the term "modified" or "mutant" when made in
reference to a gene or to a gene product refers, respectively, to a
gene or to a gene product which displays modifications in sequence
and/or functional properties (i.e., altered characteristics) when
compared to the wild-type gene or gene product. Thus, the terms
"variant" and "mutant" when used in reference to a nucleotide
sequence refer to an nucleic acid sequence that differs by one or
more nucleotides from another, usually related nucleotide acid
sequence. A "variation" is a difference between two different
nucleotide sequences; typically, one sequence is a reference
sequence.
[0152] The terms "variant" and "mutant" when used in reference to a
polypeptide refer to an amino acid sequence that differs by one or
more amino acids from another, usually related polypeptide. The
variant may have "conservative" changes, wherein a substituted
amino acid has similar structural or chemical properties. One type
of conservative amino acid substitution refers to the
interchangeability of residues having similar side chains. For
example, a group of amino acids having aliphatic side chains is
glycine, alanine, valine, leucine, and isoleucine; a group of amino
acids having aliphatic-hydroxyl side chains is serine and
threonine; a group of amino acids having amide-containing side
chains is asparagine and glutamine; a group of amino acids having
aromatic side chains is phenylalanine, tyrosine, and tryptophan; a
group of amino acids having basic side chains is lysine, arginine,
and histidine; and a group of amino acids having sulfur-containing
side chains is cysteine and methionine. Preferred conservative
amino acids substitution groups are: valine-leucine-isoleucine,
phenylalanine-tyrosine, lysine-arginine, alanine-valine, and
asparagine-glutamine. More rarely, a variant may have
"non-conservative" changes (e.g., replacement of a glycine with a
tryptophan). Similar minor variations may also include amino acid
deletions or insertions (i.e., additions), or both. Guidance in
determining which and how many amino acid residues may be
substituted, inserted or deleted without abolishing biological
activity may be found using computer programs well known in the
art, for example, DNAStar software. Variants can be tested in
functional assays. Preferred variants have less than 10%, and
preferably less than 5%, and still more preferably less than 2%
changes (whether substitutions, deletions, etc.). Thus, nucleotide
sequences of the present invention can be engineered in order to
introduce or alter a preferred Fm coding sequence for a variety of
reasons, including but not limited to initiating the production of
environmental stress tolerance; alterations that modify the
cloning, processing and/or expression of the gene product (such
alterations include inserting new restriction sites and changing
codon preference), as well as varying the protein function activity
(such changes include but are not limited to differing binding
kinetics to nucleic acid and/or protein or protein complexes or
nucleic acid/protein complexes, differing binding inhibitor
affinities or effectiveness, differing reaction kinetics).
[0153] The term "structural" when used in reference to a gene or to
a nucleotide or nucleic acid sequence refers to a gene and/or A
nucleotide or nucleic acid sequence whose ultimate expression
product is a protein (such as an enzyme or a structural protein),
an rRNA, an sRNA, a tRNA, and the like.
[0154] The term "oligonucleotide" refers to a molecule comprised of
two or more deoxyribonucleotides or ribonucleotides, preferably
more than three, and usually more than ten. The exact size will
depend on many factors, which in turn depends on the ultimate
function or use of the oligonucleotide. The oligonucleotide may be
generated in any manner, including chemical synthesis, DNA
replication, reverse transcription, or a combination thereof.
[0155] The term "polynucleotide" refers to refers to a molecule
comprised of several deoxyribonucleotides or ribonucleotides, and
is used interchangeably with oligonucleotide. Typically,
oligonucleotide refers to shorter lengths, and polynucleotide
refers to longer lengths, of nucleic acid sequences.
[0156] The term "an oligonucleotide (or polypeptide) having a
nucleotide sequence encoding a gene" or "a nucleic acid sequence
encoding" a specified polypeptide refers to a nucleic acid sequence
comprising the coding region of a gene or in other words the
nucleic acid sequence which encodes a gene product. The coding
region may be present in a cDNA, genomic DNA or RNA form. When
present in a DNA form, the oligonucleotide may be single-stranded
(i.e., the sense strand) or double-stranded. Suitable control
elements such as enhancers/promoters, splice junctions,
polyadenylation signals, etc., may be placed in close proximity to
the coding region of the gene. When needed to permit proper
initiation of transcription and/or correct processing of the
primary RNA transcript. Alternatively, the coding region utilized
in the expression vectors of the present invention may contain
endogenous enhancers, exogenous promoters, splice junctions,
intervening sequences, polyadenylation signals, etc., or a
combination of both endogenous and exogenous control elements.
[0157] As used herein, the term "exogenous promoter" refers to a
promoter in operable combination with a coding region wherein the
promoter is not the promoter naturally associated with the coding
region in the genome of an organism. The promoter which is
naturally associated or linked to a coding region in the genome is
referred to as the "endogenous promoter" for that coding
region.
[0158] The terms "complementary" and "complementarity" refer to
polynucleotides (i.e., a sequence of nucleotides) related by the
base-pairing rules. For example, for the sequence "A-G-T," is
complementary to the sequence "T-C-A." Complementarity may be
"partial," in which only some of the nucleic acids' bases are
matched according to the base pairing rules. Or, there may be
"complete" or "total" complementarity between the nucleic acids.
The degree of complementarity between nucleic acid strands has
significant effects on the efficiency and strength of hybridization
between nucleic acid strands. This is of particular importance in
amplification reactions, as well as detection methods that depend
upon binding between nucleic acids.
[0159] The terms "EST" and "expressed sequence tag" refer to a
unique stretch of DNA within a coding region of a gene;
approximately 200 to 600 base pairs in length. The term
"recombinant" when made in reference to a nucleic acid molecule
refers to a nucleic acid molecule that is comprised of segments of
nucleic acid joined together by means of molecular biological
techniques. The term "recombinant" when made in reference to a
protein or a polypeptide refers to a protein molecule that is
expressed using a recombinant nucleic acid molecule.
[0160] The terms "protein," "polypeptide," "peptide," "encoded
product," and "amino acid sequence" are used interchangeably to
refer to compounds comprising amino acids joined via peptide bonds
and a "protein" encoded by a gene is not limited to the amino acid
sequence encoded by the gene, but includes post-translational
modifications of the protein. Where the term "amino acid sequence"
is recited herein to refer to an amino acid sequence of a protein
molecule, the term "amino acid sequence" and like terms, such as
"polypeptide" or "protein" are not meant to limit the amino acid
sequence to the complete, native amino acid sequence associated
with the recited protein molecule. Furthermore, an "amino acid
sequence" can be deduced from the nucleic acid sequence encoding
the protein. The deduced amino acid sequence from a coding nucleic
acid sequence includes sequences which are derived from the deduced
amino acid sequence and modified by post-translational processing,
where modifications include but not limited to glycosylation,
hydroxylations, phosphorylations, and amino acid deletions,
substitutions, and additions. Thus, an amino acid sequence
comprising a deduced amino acid sequence is understood to include
post-translational modifications of the encoded and deduced amino
acid sequence. The term "X" may represent any amino acid.
[0161] The terms "homolog," "homologue," "homologous," and
"homology" when used in reference to amino acid sequence or nucleic
acid sequence or a protein or a polypeptide refers to a degree of
sequence identity to a given sequence, or to a degree of similarity
between conserved regions, or to a degree of similarity between
three-dimensional structures or to a degree of similarity between
the active site, or to a degree of similarity between the mechanism
of action, or to a degree of similarity between functions. In some
embodiments, a homologue has a greater than 30% sequence identity
to a given sequence. In some embodiments, a homologue has a greater
than 40% sequence identity to a given sequence. In some
embodiments, a homologue has a greater than 60% sequence identity
to a given sequence. In some embodiments, a homologue has a greater
than 70% sequence identity to a given sequence. In some
embodiments, a homologue has a greater than 90% sequence identity
to a given sequence. In some embodiments, a homologue has a greater
than 95% sequence identity to a given sequence. In some
embodiments, homology is determined by comparing internal conserved
sequences to a given sequence. In some embodiments, homology is
determined by comparing designated conserved functional and/or
structural regions, a low complexity region or a transmembrane
region. In some embodiments, homology is determined by comparing
designated conserved "motif" regions. In some embodiments, means of
determining homology are shown in the Examples.
[0162] The term "homology" when used in relation to nucleic acids
or proteins refers to a degree of identity. There may be partial
homology or complete homology. The following terms are used to
describe the sequence relationships between two or more
polynucleotides and between two or more polypeptides: "identity,"
"percentage identity," "identical," "reference sequence," "sequence
identity," "percentage of sequence identity," and "substantial
identity." "Sequence identity" refers to a measure of relatedness
between two or more nucleic acids or proteins, and is described as
a given as a percentage "of homology" with reference to the total
comparison length. A "reference sequence" is a defined sequence
used as a basis for a sequence comparison; a reference sequence may
be a subset of a larger sequence, for example, the sequence that
forms an active site of a protein or a segment of a full-length
cDNA sequence or may comprise a complete gene sequence. Since two
polynucleotides or polypeptides may each (1) comprise a sequence
(i.e., a portion of the complete polynucleotide sequence) that is
similar between the two polynucleotides, and (2) may further
comprise a sequence that is divergent between the two
polynucleotides, sequence comparisons between two (or more)
pqlynucleotides are typically performed by comparing sequences of
the two polynucleotides over a "comparison window" to identify and
compare local regions of sequence similarity. A "comparison
window," as used herein, refers to a conceptual segment of in
internal region of a polypeptide. In one embodiment, a comparison
window is at least 77 amino acids long. In another embodiment, a
comparison window is at least 84 amino acids long. In another
embodiment, conserved regions of proteins are comparison windows.
In a further embodiment, an amino acid sequence for a conserved
transmembrane domain is 24 amino acids. Calculations of
identity-may be performed by algorithms contained within computer
programs such as the ClustalX algorithm (Thompson, et al. Nucleic
Acids Res. 24, 4876-4882 (1997)); herein incorporated by
reference); MEGA2 (version 2.1) (Kumar, et al. Bioinformatics 17,
1244-1245 (2001); herein incorporated by reference); "GAP"
(Genetics Computer Group, Madison, Wis.), "ALIGN" (DNAStar,
Madison, Wis.), BLAST (National Center for Biotechnology
Information; NCBI as described at
http://www.ncbi.nlm.nih.g-ov/BLAST/blast_help.shtml) and MultAlin
(Multiple sequence alignment) program (Corpet, Nucl. Acids Res., 16
(22), 10881-10890 (1988); herein incorporated by reference, at
http://prodes.toulouse.inra.fr/multalin/multalin.html), all of
which are herein incorporated by reference).
[0163] For comparisons of nucleic acids, 20 contiguous nucleotide
positions wherein a polynucleotide sequence may be compared to a
reference sequence of at least 20 contiguous nucleotides and
wherein the portion of the polynucleotide sequence in the
comparison window may comprise additions or deletions (i.e., gaps)
of 20 percent or less as compared to the reference sequence (which
does not comprise additions or deletions) for optimal alignment of
the two sequences. Optimal alignment of sequences for aligning a
comparison window may be conducted by the local homology algorithm
of Smith and Waterman (Smith and Waterman, Adv. Appl. Math. 2:482
(1981)) by the homology alignment algorithm of Needleman and Wunsch
(Needleman and Wunsch, J. Mol. Biol. 48:443 (1970); herein
incorporated by reference), by the search for similarity method of
Pearson and Lipman (Pearson and Lipman, Proc. Natl. Acad. Sci.
(U.S.A.) 85:2444 (1988); herein incorporated by reference), by
computerized implementations of these algorithms (GAP, BESTFIT,
FASTA, and TFASTA in the Wisconsin Genetics Software Package
Release 7.0, Genetics Computer Group, 575 Science Dr., Madison,
Wis.; herein incorporated by reference), or by inspection, and the
best alignment (i.e., resulting in the highest percentage of
homology over the comparison window) generated by the various
methods is selected.
[0164] The term "sequence identity" means that two polynucleotide
or two polypeptide sequences are identical (i.e., on a
nucleotide-by-nucleotide basis or amino acid basis) over the window
of comparison. The term "percentage of sequence identity" is
calculated by comparing two optimally aligned sequences over the
window of comparison, determining the number of positions at which
the identical nucleic acid base (e.g., A, T, C, G, U, or I) or
amino acid, in which often conserved amino acids are taken into
account, occurs in both sequences to yield the number of matched
positions, dividing the number of matched positions by the total
number of positions in the window of comparison (i.e., the window
size), and multiplying the result by 100 to yield the percentage of
sequence identity. The terms "substantial identity" as used herein
denotes a characteristic of a polynucleotide sequence, wherein the
polynucleotide comprises a sequence that has at least 85 percent
sequence identity, preferably at least 90 to 95 percent sequence
identity, more usually at least 99 percent sequence identity as
compared to a reference sequence over a comparison window of at
least 20 nucleotide positions, frequently over a window of at least
25-50 nucleotides, wherein the percentage of sequence identity is
calculated by comparing the reference sequence to the
polynucleotide sequence which may include deletions or additions
which total 20 percent or less of the reference sequence over the
window of comparison. The reference sequence may be a subset of a
larger sequence.
[0165] The term "ortholog" refers to a gene in different species
that evolved from a common ancestral gene by speciation. In some
embodiments, orthologs retain the same function. The term "paralog"
refers to genes related by duplication within a genome. In some
embodiments, paralogs evolve new functions. In further embodiments,
a new function of a paralog is related to the original
function.
[0166] The term "partially homologous nucleic acid sequence" refers
to a sequence that at least partially inhibits (or competes with) a
completely complementary sequence from hybridizing to a target
nucleic acid and is referred to using the functional term
"substantially homologous." The inhibition of hybridization of the
completely complementary sequence to the target sequence may be
examined using a hybridization assay (Southern or Northern blot,
solution hybridization and the like) under conditions of low
stringency. A substantially homologous sequence or probe will
compete for and inhibit the binding (i.e., the hybridization) of a
sequence that is completely complementary to a target under
conditions of low stringency. This is not to say that conditions of
low stringency are such that non-specific binding is permitted; low
stringency conditions require that the binding of two sequences to
one another be a specific (i.e., selective) interaction. The
absence of non-specific binding may be tested by the use of a
second target which lacks even a partial-degree of identity (e.g.,
less than about 30% identity); in the absence of non-specific
binding the probe will not hybridize to the second non-identical
target.
[0167] The term "substantially homologous" when used in reference
to a double-stranded nucleic acid sequence such as a cDNA or
genomic clone refers to any probe that can hybridize to either or
both strands of the double-stranded nucleic acid sequence under
conditions of low to high stringency as described above.
[0168] The term "substantially homologous" when used in reference
to a single-stranded nucleic acid sequence refers to any probe that
can hybridize (i.e., it is the complement of) the single-stranded
nucleic acid sequence under conditions of low to high stringency as
described above.
[0169] The term "hybridization" refers to the pairing of
complementary nucleic acids. Hybridization and the strength of
hybridization (i.e., the strength of the association between the
nucleic acids) is impacted by such factors as the degree of
complementary between the nucleic acids, stringency of the
conditions involved, the T.sub.m of the formed hybrid, and the G:C
ratio within the nucleic acids. A single molecule that contains
pairing of complementary nucleic acids within its structure is said
to be "self-hybridized."
[0170] The term "T.sub.m" refers to the "melting temperature" of a
nucleic acid. Melting temperature T.sub.m is the midpoint of the
temperature range over which nucleic acids are denatured (e.g.
DNA:DNA, DNA:RNA and RNA:RNA, etc.). Methods for calculating the
T.sub.m of nucleic acids are well known in the art (see, for e.g.,
Sambrook, et al. Molecular Cloning: A Laboratory Manual, 2nd ed.,
Cold Spring Harbor Laboratory Press, New York (1989) pp. 9.50-51,
11.48-49 and 11.2-11.3; herein incorporated by reference).
[0171] The term "stringency" refers to the conditions of
temperature, ionic strength, and the presence of other compounds
such as organic solvents, under which nucleic acid hybridizations
are conducted. With "high stringency" conditions, nucleic acid base
pairing will occur only between nucleic acid fragments that have a
high frequency of complementary base sequences. Thus, conditions of
"low" stringency are often required with nucleic acids that are
derived from organisms that are genetically diverse, as the
frequency of complementary sequences is usually less.
[0172] "Low stringency conditions" when used in reference to
nucleic acid hybridization comprise conditions equivalent to
binding or hybridization at 42.degree. C. in a solution consisting
of 5.times.SSPE (43.8 g/l NaCl, 6.9 g/l NaH.sub.2PO.sub.4H.sub.2O
and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS,
5.times.Denhardt's reagent (50.times.Denhardt's contains per 500
ml:05 g Ficoll (Type 400, Pharmacia):05 g BSA (Fraction V; Sigma))
and 100 .mu.g/ml denatured salmon sperm DNA followed by washing in
a solution comprising 5.times.SSPE, 0.1% SDS at 42.degree. C. when
a probe of about 500 nucleotides in length is employed.
[0173] "Medium stringency conditions" when used in reference to
nucleic acid hybridization comprise conditions equivalent to
binding or hybridization at 42.degree. C. in a solution consisting
of 5.times.SSPE (43.8 g/l NaCl, 6.9 g/l NaH.sub.2PO.sub.4H.sub.2O
and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,
5.times.Denhardt's reagent and 100 .mu.g/ml denatured salmon sperm
DNA followed by washing in a solution comprising 1.0.times.SSPE,
1.0% SDS at 42.degree. C. when a probe of about 500 nucleotides in
length is employed.
[0174] "High stringency conditions" when used in reference to
nucleic acid hybridization comprise conditions equivalent to
binding or hybridization at 42.degree. C. in a solution consisting
of 5.times.SSPE (43.8 g/l NaCl, 6.9 g/l NaH.sub.2PO.sub.4H.sub.2O
and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,
5.times.Denhardt's reagent and 100 .mu.g/ml denatured salmon sperm
DNA followed by washing in a solution comprising 0.1.times.SSPE,
1.0% SDS at 42.degree. C. when a probe of about 500 nucleotides in
length is employed. It is well known that numerous equivalent
conditions may be employed to comprise low stringency conditions;
factors such as the length and nature (DNA, RNA, base composition)
of the probe and nature of the target (DNA, RNA, base composition,
present in solution or immobilized, etc.) and the concentration of
the salts and other components (e.g., the presence or absence of
formamide, dextran sulfate, polyethylene glycol) are considered and
the hybridization solution may be varied to generate conditions of
low stringency hybridization different from, but equivalent to, the
above listed conditions. In addition, the art knows conditions that
promote hybridization under conditions of high stringency (e.g.,
increasing the temperature of the hybridization and/or wash steps,
the use of formamide in the hybridization solution, etc.).
[0175] As used herein, the term "polymerase chain reaction" and
"PCR" refers to the method of K. B. Mullis (U.S. Pat. Nos.
4,683,195; 4,683,202; and 4,965,188; herein incorporated by
reference), which describe a method for increasing the
concentration of a segment of a target sequence in a mixture of
genomic DNA without cloning or purification. This process for
amplifying the target sequence consists of introducing an excess of
two oligonucleotide primers to the DNA mixture containing the
desired target sequence, followed by a precise sequence of thermal
cycling in the presence of a DNA polymerase. The two primers are
complementary to their respective strands of the double stranded
target sequence. To effect amplification, the mixture is denatured
and the primers then annealed to their complementary sequences
within the target molecule. Following annealing, the primers are
extended with a polymerase so as to form a new pair of
complementary strands. The steps of denaturation, primer annealing
and polymerase extension can be repeated many times (i.e.,
denaturation, annealing and extension constitute one "cycle"; there
can be numerous "cycles") to obtain a high concentration of an
amplified segment of the desired target sequence. The length of the
amplified segment of the desired target sequence is determined by
the relative positions of the primers with respect to each other,
and therefore, this length is a controllable parameter. By virtue
of the repeating aspect of the process, the method is referred to
as the "polymerase chain reaction" (hereinafter "PCR"). Because the
desired amplified segments of the target sequence become the
predominant sequences (in terms of concentration) in the mixture,
they are said to be "PCR amplified".
[0176] The term "reverse-transcriptase" or "RT-PCR" refers to a
type of PCR where the starting material is mRNA. The starting mRNA
is enzymatically converted to complementary DNA or "cDNA" using a
reverse transcriptase enzyme. The cDNA is then used as a "template"
for a "PCR" reaction.
[0177] "Amplification" is a special case of nucleic acid
replication involving template specificity. It is to be contrasted
with non-specific template replication (i.e., replication that is
template-dependent but not dependent on a specific template).
Template specificity is here distinguished from fidelity of
replication (i.e., synthesis of the proper polynucleotide sequence)
and nucleotide (ribo- or deoxyribo-) specificity. Template
specificity is frequently described in terms of "target"
specificity. Target sequences are "targets" in the sense that they
are sought to be sorted out from other nucleic acid. Amplification
techniques were designed primarily for this sorting out.
[0178] As used herein, the term "primer" refers to an
oligonucleotide, whether occurring naturally as in a purified
restriction digest or produced synthetically, which is capable of
acting as a point of initiation of synthesis when placed under
conditions in which synthesis of a primer extension product which
is complementary to a nucleic acid strand is induced, (i.e., in the
presence of nucleotides and an inducing agent such as DNA
polymerase and at a suitable temperature and pH). The primer is
preferably single stranded for maximum efficiency in amplification,
but may alternatively be double stranded. When double stranded, the
primer is first treated to separate its strands before being used
to prepare extension products. Preferably, the primer is an
oligodeoxyribonucleotide. The primer must be sufficiently long to
prime the synthesis of extension products in the presence of the
inducing agent. The exact lengths of the primers will depend on
many factors, including temperature, source of primer and the use
of the method.
[0179] As used herein, the terms "PCR product," "PCR fragment," and
"amplification product" refer to the resultant mixture of compounds
after two or more cycles of the PCR steps of denaturation,
annealing and extension are complete. These terms encompass the
case where there was amplification of one or more segments of one
or more target sequences. Template specificity is achieved in most
amplification techniques by the choice of enzyme. Amplification
enzymes are enzymes that, under conditions they are used, will
process only specific sequences of nucleic acid in a heterogeneous
mixture of nucleic acid. For example, in the case of Q replicase,
MDV-1 RNA is the specific template for the replicase (see, for
e.g., Kacian et al. Proc. Natl. Acad. Sci. USA, 69:3038-3042
(1972); herein incorporated by reference). Other nucleic acids will
not be replicated by this amplification enzyme. Similarly, in the
case of T7 RNA polymerase, this amplification enzyme has a
stringent specificity for its own promoters (see, for e.g.,
Chamberlin et al. (1970) Nature, 228:227; herein incorporated by
reference). In the case of T4 DNA ligase, the enzyme will not
ligate the two oligonucleotides or polynucleotides, where there is
a mismatch between the oligonucleotide or polynucleotide substrate
and the template at the ligation junction (Wu and Wallace,
Genomics, 4:560 (1989); herein incorporated by reference). Finally,
Taq and Pfu polymerases, by virtue of their ability to function at
high temperature, are found to display high specificity for the
sequences bounded and thus defined by the primers; the high
temperature results in thermodynamic conditions that favor primer
hybridization with the target sequences and not hybridization with
non-target sequences (H. A. Erlich (ed.), PCR Technology, Stockton
Press (1989); herein incorporated by reference).
[0180] The term "amplifiable nucleic acid" refers to nucleic acids
that may be amplified by any amplification method. It is
contemplated that "amplifiable nucleic acid" will usually comprise
"sample template."
[0181] The term "sample template" refers to nucleic acid
originating from a sample that is analyzed for the presence of
"target" (defined below). In contrast, "background template" is
used-in reference to nucleic acid other than sample template that
may or may not be present in a sample. Background template is most
often inadvertent. It may be the result of carryover, or it may be
due to the presence of nucleic acid contaminants sought to be
purified away from the sample. For example, nucleic acids from
organisms other than those to be detected may be present as
background in a test sample.
[0182] The term "primer" refers to an oligonucleotide, whether
occurring naturally as in a purified restriction digest or produced
synthetically, which is capable of acting as a point of initiation
of synthesis when placed under conditions in which synthesis of a
primer extension product which is complementary to a nucleic acid
strand is induced, (i.e., in the presence of nucleotides and an
inducing agent such as DNA polymerase and at a suitable temperature
and pH). The primer is preferably single stranded for maximum
efficiency in amplification, but may alternatively be double
stranded. When double stranded, the primer is first treated to
separate its strands before being used to prepare extension
products. Preferably, the primer is an oligodeoxyribonucleotide.
The primer must be sufficiently long to prime the synthesis of
extension products in the presence of the inducing agent. The exact
lengths of the primers will depend on many factors, including
temperature, source of primer and the use of the method.
[0183] The term "linker" refers to a synthetic double-stranded
oligonucleotide that carries the sequence for one or more
restriction endonuclease sites.
[0184] The term "linker fragment" refers to a short synthetic
duplex oligonucleotide containing the target site for a restriction
enzyme that may be ligated to the end of a DNA fragment prepared by
cleavage with some other restriction enzyme during reconstruction
of recombinant DNA.
[0185] The term "expression" when used in reference to a nucleic
acid sequence, such as a gene, refers to the process of converting
genetic information encoded in a gene into RNA (e.g., mRNA, rRNA,
tRNA, or snRNA) through "transcription" of the gene (i.e., via the
enzymatic action of an RNA polymerase), and into protein where
applicable (as when a gene encodes a protein), through
"translation" of mRNA. Gene expression can be regulated at many
stages in the process. "Up-regulation" or "activation" refers to
regulation that increases the production of gene expression
products (i.e., RNA or protein), while "down-regulation" or
"repression" refers to regulation that decrease production.
Molecules (e.g., transcription factors) that are involved in
up-regulation or down-regulation are often called "activators" and
"repressors," respectively.
[0186] The term "vector" in reference to nucleic acid sequences
refers to nucleic acid molecules that transfer DNA segment(s).
Transfer can be into a cell, cell to cell, et cetera. A vector may
also refer to a "binary vector" or a "superbinary vector." The term
"vehicle" is sometimes used interchangeably with "vector."
[0187] The terms "expression vector" or "expression cassette" refer
to a recombinant DNA molecule containing a desired coding sequence
and appropriate nucleic acid sequences necessary for the expression
of the operably linked coding sequence in a particular host
organism. Nucleic acid sequences necessary for expression in
prokaryotes usually include a promoter, an operator (optional), and
a ribosome binding site, often along with other sequences.
Eukaryotic cells are known to utilize promoters, enhancers, and
termination and polyadenylation signals. The term "expression
vector" when used in reference to a construct refers to an
expression vector construct comprising, for example, a heterologous
DNA encoding a gene of interest and the various regulatory elements
that facilitate the production of the particular protein of
interest in the target cells. In certain embodiments of the present
invention, a nucleic acid sequence of the present invention within
an expression vector is operatively linked to an appropriate
expression control sequence(s) (promoter) to direct mRNA
synthesis.
[0188] The terms "in operable combination," "in operable order,"
and "operably linked" refer to the linkage of nucleic acid
sequences in such a manner that a nucleic acid molecule capable of
directing the transcription of a given gene and/or the synthesis of
a desired protein molecule is produced. The term also refers to the
linkage of amino acid sequences in such a manner so that a
functional protein is produced.
[0189] The term "regulatory element" refers to a genetic element
that controls some aspect of the expression of nucleic acid
sequences. For example, a promoter is a regulatory element that
facilitates the initiation of transcription of an operably linked
coding region. Other regulatory elements are splicing signals,
polyadenylation signals, termination signals, and the like.
[0190] Transcriptional control signals in eukaryotes comprise
"promoter" and "enhancer" elements. Promoters and enhancers consist
of short arrays of DNA sequences that interact specifically with
cellular proteins involved in transcription (see, for e.g.,
Maniatis, et al. (1987) Science 236:1237; herein incorporated by
reference). Promoter and enhancer elements were isolated from a
variety of eukaryotic sources including genes in yeast, insect,
mammalian and plant cells. Promoter and enhancer elements have also
been isolated from viruses and analogous control elements, such as
promoters, are also found in prokaryotes. The selection of a
particular promoter and enhancer depends on the cell type used to
express the protein of interest. Some eukaryotic promoters and
enhancers have a broad host range while others are functional in a
limited subset of cell types (for review, see Maniatis, et al.
(1987), supra; herein incorporated by reference).
[0191] The terms "promoter element," "promoter," or "promoter
sequence" refer to a DNA sequence that is located at the 5' end
(i.e. precedes) of the coding region of a DNA polymer. The location
of the majority of promoters known to occur in nature precedes the
transcribed region. The promoter functions as a switch, activating
the expression of a gene. When the gene is activated, it is said to
be transcribed, or participating in transcription. Transcription
involves the synthesis of mRNA from the gene. The promoter,
therefore, serves as a transcriptional regulatory element and also
provides a site for initiation of transcription of the gene into
mRNA.
[0192] The term "regulatory region" refers to a gene's 5'
transcribed but untranslated regions, located immediately
downstream from the promoter and ending just prior to the
translational start of the gene.
[0193] The term "promoter region" refers to the region immediately
upstream of the coding region of a DNA polymer, and is typically
between about 500 bp and 4 kb in length, and is preferably about 1
to 1.5 kb in length. Promoters may be tissue specific or cell
specific. The term "tissue specific" as it applies to a promoter
refers to a promoter that is capable of directing selective
expression of a nucleotide sequence of interest to a specific type
of tissue (e.g., seeds) in the relative absence of expression of
the same nucleotide sequence of interest in a different type of
tissue (e.g., leaves). Tissue specificity of a promoter may be
evaluated by, for example, operably linking a reporter gene and/or
A reporter gene expressing a reporter molecule, to the promoter
sequence to generate a reporter construct, introducing the reporter
construct into the genome of a plant such that the reporter
construct is integrated into every tissue of the resulting
transgenic plant, and detecting the expression of the reporter gene
(e.g., detecting mRNA, protein, or the activity of a protein
encoded by the reporter gene) in different tissues of the
transgenic plant. The detection of a greater level of expression of
the reporter gene in one or more tissues relative to the level of
expression of the reporter gene in other tissues shows that the
promoter is specific for the tissues in which greater levels of
expression are detected.
[0194] Promoters may be "constitutive" or "inducible." The term
"constitutive" when made in reference to a promoter means that the
promoter is capable of directing transcription of an operably
linked nucleic acid sequence in the absence of a stimulus (e.g.,
heat shock, chemicals, light, etc.). Typically, constitutive
promoters are capable of directing expression of a transgene in
substantially any cell and any tissue. Exemplary constitutive plant
promoters include, but are not limited to Cauliflower Mosaic Virus
(CaMV SD; see, for example, U.S. Pat. No. 5,352,605, incorporated
herein by reference), mannopine synthase, octopine synthase (ocs),
superpromoter (see for example, WO 95/14098; herein incorporated by
reference), ubi3 promoters (see, for example, Garbarino and
Belknap, Plant Mol. Biol. 24:119-127 (1994); herein incorporated by
reference) other constitutive promoters suitable for use in the
present invention are a rice actin promoter and a maize ubiquitin
promoter, a maize alcohol dehydrogenase gene (Adh-1) promoter, rice
or maize tubulin (Tub A, B or C) promoters; and the alfalfa His 3
promoter. Such promoters were used successfully to direct the
expression of heterologous nucleic acid sequences in transformed
plant tissue (see, for example, United States Patent Application
No. 20060212973; herein incorporated by reference).
[0195] In contrast, an "inducible" promoter is one that is capable
of directing a level of transcription of an operably linked nucleic
acid sequence in the presence of a stimulus (e.g., heat shock,
chemicals, light, etc.) that is different from the level of
transcription of the operably linked nucleic acid sequence in the
absence of the stimulus.
[0196] The term "regulatory element" refers to a genetic element
that controls some aspect of the expression of nucleic acid
sequence(s). For example, a promoter is a regulatory element that
facilitates the initiation of transcription of an operably linked
coding region. Other regulatory elements are splicing signals,
polyadenylation signals, termination signals, and the like.
[0197] The enhancer and/or promoter may be "endogenous" or
"exogenous" or "heterologous." An "endogenous" enhancer or promoter
is one that is naturally linked with a given gene in the genome. An
"exogenous" or "heterologous" enhancer or promoter is one that is
placed in juxtaposition to a gene by means of genetic manipulation
(i.e., molecular biological techniques) such that transcription of
the gene is directed by the linked enhancer or promoter. For
example, an endogenous promoter in operable combination with a
first gene can be isolated, removed, and placed in operable
combination with a second gene, therein making it a "heterologous
promoter" in operable combination with the second gene. A variety
of such combinations are contemplated (e.g., the first and second
genes can be from the same species, or from different species).
[0198] The term "naturally linked" or "naturally located" when used
in reference to the relative positions of nucleic acid sequences
means that the nucleic acid sequences exist in nature in the
relative positions.
[0199] The presence of "splicing signals" on an expression vector
often results in higher levels of expression of the recombinant
transcript in eukaryotic host cells. Splicing signals mediate the
removal of introns from the primary RNA transcript and consist of a
splice donor and acceptor site (Sambrook, et al. Molecular Cloning:
A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press,
New York (1989) pp. 16.7-16.8; herein incorporated by reference). A
commonly used splice donor and acceptor site is the splice junction
from the 16S RNA of SV40.
[0200] Efficient expression of recombinant DNA sequences in
eukaryotic cells requires expression of signals directing the
efficient termination and polyadenylation of the resulting
transcript. Transcription termination signals are generally found
downstream of the polyadenylation signal and are a few hundred
nucleotides in length. The term "poly(A) site" or "poly(A)
sequence" as used herein denotes a DNA sequence that directs both
the termination and polyadenylation of the nascent RNA transcript.
Efficient polyadenylation of the recombinant transcript is
desirable, as transcripts lacking a poly(A) tail are unstable and
are rapidly degraded. The poly(A) signal utilized in an expression
vector may be "heterologous" or "endogenous." An endogenous poly(A)
signal is one that is found naturally at the 3' end of the coding
region of a given gene in the genome. A heterologous poly(A) signal
is one which was isolated from one gene and positioned 3' to
another gene. A commonly used heterologous poly(A) signal is the
SV40 poly(A) signal. The SV40 poly(A) signal is contained on a 237
bp BamHI/BclI restriction fragment and directs both termination and
polyadenylation (Sambrook, supra, at 16.6-16.7).
[0201] The term "transfection" refers to the introduction of
foreign DNA into cells. Transfection may be accomplished by a
variety of means known to the art including calcium phosphate-DNA
co-precipitation, DEAE-dextran-mediated transfection,
polybrene-mediated transfection, glass beads, electroporation,
microinjection, liposome fusion, lipofection, protoplast fusion,
viral infection, biolistics (i.e., particle bombardment) and the
like.
[0202] The terms "stable transfection" and "stably transfected"
refer to the introduction and integration of foreign DNA into the
genome of the transfected cell. The term "stable transfectant"
refers to a cell that has stably integrated foreign DNA into the
genomic DNA.
[0203] The terms "transient transfection" and "transiently
transfected" refer to the introduction of foreign DNA into a cell
where the foreign DNA fails to integrate into the genome of the
transfected cell. The foreign DNA persists in the nucleus of the
transfected cell for several days. During this time the foreign DNA
is subject to the regulatory controls that govern the expression of
endogenous genes in the chromosomes. The term "transient
transfectant" refers to cells that have taken up foreign DNA but
have failed to integrate this DNA.
[0204] The term "calcium phosphate co-precipitation" refers to a
technique for the introduction of nucleic acids into a cell. The
uptake of nucleic acids by cells is enhanced when the nucleic acid
is presented as a calcium phosphate-nucleic acid co-precipitate.
The original technique of Graham and van der Eb in Virol., 52:456
(1973); herein incorporated by reference, was modified by several
groups to optimize conditions for particular types of cells. The
art is well aware of these numerous modifications.
[0205] The terms "infecting" and "infection" when used with a
bacterium refer to co-incubation of a target biological sample,
(e.g., cell, tissue, etc.) with the bacterium under conditions such
that nucleic acid sequences contained within the bacterium are
introduced into one or more cells of the target biological
sample.
[0206] The terms "bombarding, "bombardment, and "biolistic
bombardment" refer to the process of accelerating particles towards
a target biological sample (e.g., cell, tissue, etc.) to effect
wounding of the cell membrane of a cell in the target biological
sample and/or entry of the particles into the target biological
sample. Methods for biolistic bombardment are known in the art
(e.g., U.S. Pat. No. 5,584,807; herein incorporated by reference),
and are commercially available (e.g. the helium gas-driven
microprojectile accelerator (PDS-1000/He, BioRad)).
[0207] The term "microwounding" when made in reference to plant
tissue refers to the introduction of microscopic wounds in that
tissue. Microwounding may be achieved by, for example, particle
bombardment as described herein.
[0208] The term "selectable marker" refers to a gene which encodes
an enzyme having an activity that confers resistance to an
antibiotic or drug upon the cell in which the selectable marker is
expressed, or which confers expression of a trait which can be
detected (e.g., luminescence or fluorescence). Selectable markers
may be "positive" or "negative." Examples of positive selectable
markers include the neomycin phosphotrasferase (NPTII) gene that
confers resistance to G418 and to kanamycin, and the bacterial
hygromycin phosphotransferase gene (hyg), which confers resistance
to the antibiotic hygromycin. Negative selectable markers encode an
enzymatic activity whose expression is cytotoxic to the cell when
grown in an appropriate selective medium. For example, the HSV-tk
gene is commonly used as a negative selectable marker. Expression
of the HSV-tk gene in cells grown in the presence of gancyclovir or
acyclovir is cytotoxic; thus, growth of cells in selective medium
containing gancyclovir or acyclovir selects against cells capable
of expressing a functional HSV TK enzyme.
[0209] The term "reporter gene" refers to a gene encoding a protein
that may be assayed. Examples of reporter genes include, but are
not limited to, luciferase (See, e.g., deWet et al. Mol. Cell.
Biol. 7:725 (1987) and U.S. Pat. Nos. 6,074,859; 5,976,796;
5,674,713; and 5,618,682; all of which are herein incorporated by
reference in their entirety), green fluorescent protein (e.g.,
GenBank Accession Number U43284; GFP variants commercially
available from CLONTECH Laboratories, Palo Alto, Calif.; herein
incorporated by reference), chloramphenicol acetyltransferase,
.beta.-galactosidase (lacZ gene), alkaline phosphatase, and horse
radish peroxidase.
[0210] The term "probe" refers to an oligonucleotide (i.e., a
sequence of nucleotides), whether occurring naturally as in a
purified restriction digest or produced synthetically,
recombinantly or by PCR amplification, that is capable of
hybridizing to another oligonucleotide of interest. A probe may be
single-stranded or double-stranded. Probes are useful in the
detection, identification and isolation of particular gene
sequences. It is contemplated that any probe used in the present
invention will be labeled with any reporter molecule," so that is
detectable in any detection system, including, but not limited to
enzyme (e.g., ELISA, as well as enzyme-based histochemical assays),
fluorescent, radioactive, and luminescent systems. It is not
intended that the present invention be limited to any particular
detection system or label.
[0211] The term "antisense" refers to a deoxyribonucleotide
sequence whose sequence of deoxyribonucleotide residues is in
reverse 5' to 3' orientation in relation to the sequence of
deoxyribonucleotide residues in a sense strand of a DNA duplex. A
"sense strand" of a DNA duplex refers to a strand in a DNA duplex
that is transcribed by a cell in its natural state into a "sense
mRNA." Thus an "antisense" sequence is a sequence having the same
sequence as the non-coding strand in a DNA duplex. The term
"antisense RNA" refers to a RNA transcript that is complementary to
all or part of a target primary transcript or mRNA and that blocks
the expression of a target gene by interfering with the processing,
transport and/or translation of its primary transcript or mRNA. The
complementarity of an antisense RNA may be with any part of the
specific gene transcript, i.e., at the 5' non-coding sequence, 3'
non-coding sequence, introns, or the coding sequence. In addition,
as used herein, antisense RNA may contain regions of ribozyme
sequences that increase the efficacy of antisense RNA to block gene
expression. "Ribozyme" refers to a catalytic RNA and includes
sequence-specific endoribonucleases. "Antisense inhibition" refers
to the production of antisense RNA transcripts capable of
preventing the expression of the target protein.
[0212] The term "RNA interference" or "RNAi" refers to the
silencing or decreasing of gene expression by siRNAs. It is the
process of sequence-specific, post-transcriptional gene silencing
in animals and plants, initiated by siRNA that is homologous in its
duplex region to the sequence of the silenced gene. The gene may be
endogenous or exogenous to the organism, present integrated into a
chromosome or present in a transfection vector that is not
integrated into the genome. The expression of the gene is either
completely or partially inhibited. RNAi may also be considered to
inhibit the function of a target RNA; the function of the target
RNA may be complete or partial. In both plants and animals, RNAi is
mediated by RNA-induced silencing complex (RISC), a
sequence-specific, multicomponent nuclease that destroys messenger
RNAs homologous to the silencing trigger. RISC is known to contain
short RNAs (approximately 22 nucleotides) derived from the
double-stranded RNA trigger, although the protein components of
this activity are unknown. However, the 22-nucleotide RNA sequences
are homologous to the target gene that is being suppressed. Thus,
the 22-nucleotide sequences appear to serve as guide sequences to
instruct a multicomponent nuclease, RISC, to destroy the specific
mRNAs. Carthew (2001) has reported (Curr. Opin. Cell Biol.
13(2):244-248; herein incorporated by reference) that eukaryotes
silence gene expression in the presence of dsRNA homologous to the
silenced gene. Biochemical reactions that recapitulate this
phenomenon generate RNA fragments of 21 to 23 nucleotides from the
double-stranded RNA. These stably associate with an RNA
endonuclease, and probably serve as a discriminator to select
mRNAs. Once selected, mRNAs are cleaved at sites 21 to 23
nucleotides apart.
[0213] The term "siRNAs" refers to short interfering RNAs. In some
embodiments, siRNAs comprise a duplex, or double-stranded region,
of about 18-25 nucleotides long; often siRNAs contain from about
two to four unpaired nucleotides at the 3' end of each strand. At
least one strand of the duplex or double-stranded region of a siRNA
is substantially homologous to or substantially complementary to a
target RNA molecule. The strand complementary to a target RNA
molecule is the "antisense strand" the strand homologous to the
target RNA molecule is the "sense strand," and is also
complementary to the siRNA antisense strand. siRNAs may also
contain additional sequences; non-limiting examples of such
sequences include linking sequences, or loops, as well as stem and
other folded structures. siRNAs appear to function as key
intermediaries in triggering RNA interference in invertebrates and
in vertebrates, and in triggering sequence-specific RNA degradation
during posttranscriptional gene silencing in plants.
[0214] The terms "hpRNA" and "hairpin RNA" refer to
self-complementary RNA that forms hairpin loops and functions to
silence genes (e.g. Wesley et al. (2001) The Plant Journal
27(6):581-590; herein incorporated by reference). The term "ihpRNA"
refers to intron-spliced hpRNA that functions to silence genes.
[0215] The term "target RNA molecule" refers to an RNA molecule to
which at least one strand of the short double-stranded region of a
siRNA is homologous or complementary. Typically, when such homology
or complementary is about 100%, the siRNA is able to silence or
inhibit expression of the target RNA molecule. Although it is
believed that processed mRNA is a target of siRNA, the present
invention is not limited to any particular hypothesis, and such
hypotheses are not necessary to practice the present invention.
Thus, it is contemplated that other RNA molecules may also be
targets of siRNA. Such targets include unprocessed mRNA, ribosomal
RNA, and viral RNA genomes.
[0216] The terms "posttranscriptional gene silencing" and "PTGS"
refer to silencing of gene expression in plants after
transcription, and appears to involve the specific degradation of
mRNAs synthesized from gene repeats. The term "cosuppression"
refers to silencing of endogenous genes by heterologous genes that
share sequence identity with endogenous genes.
[0217] The term "coexpression" refers to the expression of a
foreign gene that has substantial homology to an endogenous gene
resulting in the suppression of expression of both the foreign and
the endogenous gene. As used herein, the term "altered levels"
refers to the production of gene product(s) in transgenic organisms
in amounts or proportions that differ from that of normal or
non-transformed organisms.
[0218] The term "overexpression" generally refers to the production
of a gene product in transgenic organisms that exceeds levels of
production in normal or non-transformed organisms.
[0219] The terms "overexpression" and "overexpressing" and
grammatical equivalents are specifically used in reference to
levels of mRNA to indicate a level of expression approximately
3-fold higher than that typically observed in a given tissue in a
control or non-transgenic animal. Levels of mRNA are measured using
any of a number of techniques known to those skilled in the art
including, but not limited to Northern blot analysis. Appropriate
controls are included on the Northern blot to control for
differences in the amount of RNA loaded from each tissue analyzed
(e.g., the amount of 28S rRNA, an abundant RNA transcript present
at essentially the same amount in all tissues, present in each
sample can be used as a means of normalizing or standardizing the
RAD50 mRNA-specific signal observed on Northern blots).
[0220] The terms "Southern blot analysis" and "Southern blot" and
"Southern" refer to the analysis of DNA on agarose or acrylamide
gels in which DNA is separated or fragmented according to size
followed by transfer of the DNA from the gel to a solid support,
such as nitrocellulose or a nylon membrane. The immobilized DNA is
then exposed to a labeled probe to detect DNA species complementary
to the probe used. The DNA may be cleaved with restriction enzymes
prior to electrophoresis. Following electrophoresis, the DNA may be
partially depurinated and denatured prior to or during transfer to
the solid support. Southern blots are a standard tool of molecular
biologists (Sambrook, et al. Molecular Cloning: A Laboratory
Manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York
(1989) pp. 9.31-9.58; herein incorporated by reference).
[0221] The term "Northern blot analysis," "Northern blot," and
"Northern" refer to the analysis of RNA by electrophoresis of RNA
on agarose gels to fractionate the RNA according to size followed
by transfer of the RNA from the gel to a solid support, such as
nitrocellulose or a nylon membrane. The immobilized RNA is then
probed with a labeled probe to detect RNA species complementary to
the probe used. Northern blots are a standard tool of molecular
biologists (Sambrook, et al. (1989) supra, pp 7.39-7.52; herein
incorporated by reference).
[0222] The term "isolated" when used in relation to a nucleic acid
or polypeptide, as in "an isolated oligonucleotide" refers to a
nucleic acid sequence that is identified and separated from at
least one contaminant nucleic acid with which it is ordinarily
associated in its natural source. Isolated nucleic acid is present
in a form or setting that is different from that in which it is
found in nature. In contrast, non-isolated nucleic acids, such as
DNA and RNA, are found in the state they exist in nature. For
example, a given DNA sequence (e.g., a gene) is found on the host
cell chromosome in proximity to neighboring genes; RNA sequences,
such as a specific mRNA sequence encoding a specific protein, are
found in the cell as a mixture with numerous other mRNAs that
encode a multitude of proteins. However, isolated nucleic acid
encoding a particular protein includes, by way of example, such
nucleic acid in cells ordinarily expressing the protein, where the
nucleic acid is in a chromosomal location different from that of
natural cells, or is otherwise flanked by a different nucleic acid
sequence than that found in nature. The isolated nucleic acid or
oligonucleotide may be present in single-stranded or
double-stranded form. When an isolated nucleic acid or
oligonucleotide is to be utilized to express a protein, the
oligonucleotide will contain at a minimum the sense or coding
strand (i.e., the oligonucleotide may single-stranded), but may
contain both the sense and anti-sense strands (i.e., the
oligonucleotide may be double-stranded).
[0223] The term "purified" refers to molecules, either nucleic or
amino acid sequences that are removed from their natural
environment isolated or separated. An "isolated nucleic acid
sequence" is therefore a purified nucleic acid sequence.
"Substantially purified" molecules are at least 60% free,
preferably at least 75% free, and more preferably at least 90% free
from other components with which they are naturally associated. As
used herein, the terms "purified" and "to purify" also refer to the
removal of contaminants from a sample. The removal of contaminating
proteins results in an increase in the percent of polypeptide of
interest in the sample. In another example, recombinant
polypeptides are expressed in plant, bacterial, yeast, or mammalian
host cells and the polypeptides are purified by the removal of host
cell proteins; the percent of recombinant polypeptides is therein
increased in the sample.
[0224] The term "accession" when used herein associated with
sequences of genes and proteins refers to a gene or group of
similar genes or proteins from these genes or proteins received
from a single source at a single time. The term "accession number"
when used herein refers to a unique identifier for protein and gene
sequences and is assigned when an accession is entered into a
database (for example GenBank at NCBI, European Molecular Biology
Laboratory (EMBL) and the like).
[0225] The term "accession" when used herein associated with
sources of plants refers to a plant or group of similar plants or
group of seeds from these plants received from a single source at a
single time. The term "accession number" when used herein
associated with sources of plants, such as the number following PI,
refers to a unique identifier for each accession and is assigned
when an accession is entered into a plant collection. As used
herein "PI" used before an accession number indicates the identity
of the genebank or national system that in this case refers to an
accession cataloged within the USA system where the term "PI"
refers to "plant introductions."
[0226] The term "sample" is used in its broadest sense. In one
sense it can refer to a plant cell or tissue, such as a leaf. In
another sense, it is meant to include a specimen or culture
obtained from any source, as well as biological and environmental
samples. Biological samples may be obtained from plants or animals
and encompass fluids, solids, tissues, and gases. Environmental
samples include environmental material such as surface matter,
soil, water, salt, and industrial samples. These examples are not
to be construed as limiting the sample types applicable to the
present invention.
[0227] The term "eigenvector" refers to a "vector" which, when
acted on by a particular linear transformation, produces a scalar
multiple of the original vector. The scalar in question refers to
an eigenvalue corresponding to a particular eigenvector. A "vector"
in reference to mathematical calculations, such as eigenvector
calculations, refers to an "element of a vector space" which can
include many mathematical entities.
[0228] As used herein, the terms "marker" and "DNA marker" and
"molecular marker" in reference to a "selectable marker" refers to
a physiological or morphological trait which may be determined as
marker for its own selection or for selection of other traits
closely linked to that marker, for example, a gene or trait that
associates with drought resistance, such as a marker, such as a DNA
marker including but not limited to simple sequence repeat (SSR),
single nucleotide polymorphism analysis (SNP), random amplified
polymorphic DNA analysis (RAPID), amplified fragment length
polymorphism analysis (AFLP), and the like that will link phenotype
information, such as drought resistance and agronomic traits to a
linkage or QTL locus, to provide a genomic map, for example a
fingerprint map, and chromosome location and/or map.
[0229] As used herein, the term "linkage group" or "LG" refers to a
group of two or more genetically or physically mapped loci with
observed linkage to a trait, for example, one or more of a SSR,
SNP, AFLP, and RAPD marker of the present invention that may map to
drought resistant germplasm.
[0230] The term "functional category" refers to a classification of
cellular function as defined and assigned by criteria on the
website of the Munich Information Center for Protein Sequences
(MIPS) (website address at mips.gsf.de). Functional categories
include but are not limited to metabolism energy, biogenesis of
cellular components, subcellular localization, transport,
transcription, signal transduction, interaction with the cellular
environment, protein synthesis, protein with binding function,
defense, development, cell fate, cell cycle and DNA processing,
protein fate, cell type differentiation, and protein activity
regulation.
[0231] The term "metabolic process" refers to a process that causes
a chemical change in a living organism, including anabolism and
catabolism. A metabolic process may transform a small molecule,
alter a macromolecular process such as DNA repair, alter DNA
replication, alter protein synthesis or alter protein
degradation.
[0232] Unless defined otherwise in reference to the level of
molecules and/or phenomena, the terms "increase," "elevate,"
"raise," and grammatical equivalents (including "higher,"
"greater," etc.) when in reference to the level of any molecule
(e.g., nucleotide sequence, amino acid sequence, etc.), and/or
phenomenon (e.g., drought tolerance, leaf elongation, root biomass,
leaf color, etc.) in a first sample relative to a second sample,
mean that the quantity of the molecule and/or phenomenon in the
first sample is higher than in the second sample by any amount that
is statistically significant using any art-accepted statistical
method of analysis. In one embodiment, the quantity of the molecule
and/or phenomenon in the first sample is at least 10% greater than,
at least 25% greater than, at least 50% greater than, at least 75%
greater than, and/or at least 90% greater than the quantity of the
same molecule and/or phenomenon in a second sample.
[0233] Unless defined otherwise in reference to the level of
molecules and/or phenomena, the terms "reduce," "inhibit,"
"diminish," "suppress," "decrease," and grammatical equivalents
(including "lower," "smaller," etc.) when in reference to the level
of any molecule (e.g., nucleotide sequence, amino acid sequence,
etc.), and/or phenomenon (e.g., drought tolerance, leaf elongation,
root biomass, leaf color, etc.) in a first sample relative to a
second sample, mean that the quantity of molecule and/or phenomenon
in the first sample is lower than in the second sample by any
amount that is statistically significant using any art-accepted
statistical method of analysis. In one embodiment, the quantity of
molecule and/or phenomenon in the first sample is at least 10%
lower than, at least 25% lower than, at least 50% lower than, at
least 75% lower than, and/or at least 90% lower than the quantity
of the same molecule and/or phenomenon in a second sample.
GENERAL DESCRIPTION OF THE INVENTION
[0234] The present inventions relate to compositions and methods
for providing drought resistant plants (e.g., grass plants)
comprising Festuca mairei plant germplasm. Specifically, the
inventions relate to providing compositions and methods for
introgressing Festuca mairei germplasm and/or specific Festuca
mairei genes into plants (e.g., grass plants), such as Lolium
perenne plants. Further, the invention relates to methods of grass
plant breeding comprising genetic markers for identifying the
preferred Festuca mairei germplasm introgressed into plants (e.g.,
grass plants), and providing commercially desirable drought
resistant cultivars of plants.
[0235] Environmental abiotic stresses such as drought, high
salinity, and extreme temperatures can severely impair plant growth
and performance. Thus, the response of plant to these abiotic
stresses were the focus of study for decades at physiological and
genetics levels (Levitt, J. 1980 Academic Press, NY; Quarrie et
al., 1994 Theor. Appl. Genet. 89:794-800; all of which are herein
incorporated by reference) and recently at molecular and genomics
levels (Seki et al., 2001 Plant Cell. 13: 61-72; Ozturk et al.,
2002 Plant Mol. Biol. 48: 55 1-573; all of which are herein
incorporated by reference). Among these stresses, drought or water
deficit is the most severe and complex limiting factor on plant
growth and crop production (Seki et al., 2002 Plant J.
30:279-292).
[0236] The Michigan Agricultural Statistics Service (MASS) showed
that over 1.9 million acres of turfgrass were maintained throughout
the state during 2002-2003. Over 84% of this acreage was in the
residential sector, while 96,000 acres were intensively managed by
golf course superintendents. In addition, 1.8 million acres were
used as forage grasses. The contribution of the turf grass industry
to Michigan's state economy exceeded $1.8 billion annually and
created jobs for over 30,000 full-time employees.
[0237] Thus, it is contemplated that drought tolerant cultivars
will save the turf grass industry millions of dollars by reducing
the irrigation rate, energy input, and more importantly conserving
water resources, as such resources become limited throughout the
USA and the world. In particular, drought resistant turf grass will
especially be cost efficient when grown in regions experiencing
increased drought conditions, such as when cities and
municipalities declare water emergencies causing restrictions in
the watering of landscapes.
I. Perennial Ryegrass (Lolium perenne (L.)) Plants, Annual Ryegrass
Plants (Lolium multiflorum) and Hybrids with Festuca Plants:
[0238] Perennial ryegrass (L. perenne L.) (Lp) is a cool-season
turfgrass grass (2n=2x=14, LL) that is widely used as turf and
forage with superior quality and rapid establishment of plants,
qualities lacking in Festuca plants. However, lack of drought
tolerance makes L. perenne less persistent during hot and dry
summers or dry environments unlike drought tolerant Festuca plants.
One approach for improvement of drought tolerance in perennial
ryegrass is introgression of alien genomes from other drought
tolerant genera, such as Festuca (Riewe et al., 1985, In Heath et
al. eds. pp. 241-246; herein incorporated by reference).
Intergeneric hybridization followed by backcrossing or by
chromosome doubling produced alien chromosome addition,
substitution, or translocation in progeny plants (Sharma et al.,
1995, Genome 38:406-413; herein incorporated by reference).
[0239] Thus, intergeneric hybridization between Lolium and Festuca
was used for developing novel allopolyploids for combining
desirable agronomic attributes from two important turf grass and
forage grass species. Amphiploids (LLG.sub.1G.sub.1G.sub.2G.sub.2,
2n=6x=42) between diploid ryegrass (L. multiflorum, LL, 2n=2x=14)
and tetraploid tall fescue (F. arundinacea var. glaucescens gauct.
[=F. arundinacea subsp. fenas (Lag.) Arcang.]
(G.sub.1G.sub.1G.sub.2G.sub.2, 2n=4x=28) were synthesized to
improve palatability of tall fescue by introducing the L genome
from annual ryegrass (Cao et al. 1994, Boiss. Sci. Agric. Sin.
27:69-76; herein incorporated by reference). Amphiploids (2n=8x=56)
between annual ryegrass and hexaploid tall fescue (F. arundinacea,
2n 6.times.42) were produced to transfer the nutritive quality of
annual ryegrass into tall fescue while retaining the adaptive
qualities of tall fescue (Buckner et al. 1985, Crop Sci.
25:757-761; Buckner, 1965, Crop Sci. 5:395-397; all of which are
herein incorporated by reference).
[0240] Amphidiploids between ryegrasses (L. multiflorum or L.
perenne) and meadow fescue (F. pratensis Huds.) were selected for
better agronomic performance as forage grass (Zwierzykowski et al.
1998, J. of Hered. 89:324-328; herein incorporated by reference)
and some are widely used in grasslands, such as `Elmet` and `Prior`
(Jauhar 1993, In Cytogenetics of the Festuca-Lolium complex:
Relevance to breeding. Springer-Verlag, Berlin; herein incorporated
by reference). Eleven cultivars were developed from these Lolium
spp..times.F. pratensis hybrids and seven cultivars from L.
multiflorum.times.F. arundinacea (Zwierzykowski, 2004, In: Yamada
et al. (eds) Development of a novel grass with environmental stress
and high forage quality through intergeneric hybridization between
Lolium and Festuca, NARO, Tsukuba, pp: 17-29; herein incorporated
by reference). The amphiploids and tetraploid hybrids derived from
these ryegrass and fescue hybrids were subjected to extensive
cytological analyses and were determined to be good breeding
material for improving forage quality of tall fescue (Cao et al.
2003, Crop Sci. 43:1659-1662; Zwierzykowski et al., 2006, Theor.
Appl. Genet. 113:539-547; all of which are herein incorporated by
reference).
[0241] Further, several genes were identified in Festuca
arundinacea L. germplasm, including dehydrin and a cytosolic-heat
shock protein (HSC 70), that associated with a response to drought
stress in two tall fescue (Festuca arundinacea L.) cultivars,
`Southeast` and `Rebel Jr.` (Jiang and Huang, 2002, Crop Sci.
42(1):202-207; herein incorporated by reference).
II. Festuca mairei Plants:
[0242] Festuca mairei St. Yves (Fm) is a tetraploid plant
(2n=4x=28, M, M.sub.1M.sub.2M.sub.2) species, commonly known as
Atlas Fescue, that grows in the Moroccan Atlas mountains.
[0243] The M genome in Festuca is associated with a xerophytic
adaptation allowing the plant to survive long summers under drought
stress (Marlatt et al., 1997, Neotyphodium/Grass Interactions,
Baxon and Hill, Plenum Press, NY; herein incorporated by
reference). Fm plants shares it's M.sub.1M.sub.2 genomes with F.
arundinacea var. atlantigena
(GiG.sub.1G.sub.2G.sub.2M.sub.1M.sub.1M.sub.2M.sub.2) a species
that also grows near and in the Atlas Mountain ranges of northwest
Africa but with a broader environmental range including lowland
areas. F. mairei tolerates high temperature and drought stress
(Borill et al. 1971, Cytologia 36:1-14; herein incorporated by
reference) in addition to a high photosynthetic rate (Randall et
al., 1985, NY pp:409-418; herein incorporated by reference).
[0244] As described herein, unexpected results were obtained when
Festuca mairei plants demonstrated a greater capability to
withstand drought stress treatment than other species of Festuca,
including commercial cultivars of F. arundinacea, see, FIG. 1).
[0245] Thus, it is contemplated that breeding programs using
Festuca mairei germplasm, in particular providing germplasm
associated with drought tolerance to grass plants, had the
potential to provide superior hybrid drought tolerant grass
cultivars. Furthermore, it is contemplated that F. mairei's genome
contains unique Festuca genetic material and/or expresses a unique
combination of genes providing for conserved and highly developed
systems for growing under severe drought conditions.
[0246] Moreover, additional unexpected results were obtained after
evaluation of the hybrid plants developed through plant breeding
methods of the present inventions, see, for example, FIG. 14b.
These unexpected results demonstrate that a F. mairei.times.L.
perenne `Calypso` hybrid plant and backcrossed plants (lines G15
and G30a) of the present invention show superior levels of drought
resistance when compared to parental and F.sub.1 F. mairei.times.L.
perenne hybrid plants. In other words, the F. mairei.times.L.
perenne hybrid backcrossed plants of the present inventions
demonstrated unexpected "hybrid vigor" also known as "hybrid
superiority," "heterosis" and "transgression" for resisting drought
stress. These unexpected results were obtained using PCA-based
evaluation methods for drought tolerance of the present inventions,
as described herein.
[0247] Therefore, the inventor used this polyploidy monocot
species, F. mairei as a model plant system for providing a genetic
study of drought tolerance for providing drought tolerant hybrid
grass plants and for comparing drought tolerance mechanisms to
other grass species. Further, in some embodiments, this genetic
information is used to provide novel grass varieties demonstrating
strong vegetative growth and selected agronomic performance, such
as for providing novel grass plant cultivars showing a combined
enhanced drought resistance with desired characteristics of
ornamental grasses, or turf grasses or forage grasses.
III. Genetics of Drought Tolerance:
[0248] A. Molecular Regulation:
[0249] Drought stress triggers a wide range of plant responses
manifested by changes from growth rates, physiological, and
metabolic processes to altered gene expression. A stress response
is initiated when a plant recognizes stress at the cellular level,
which then activates signal transduction pathways to transmit the
information within individual cells and throughout the plants.
Ultimately, changes in gene expression will occur and are
integrated into plant adaptive response to modify growth and
development.
[0250] Genes involved in many different pathways expressed in
response to drought stress in Arabidopsis plants, were extensively
studied as a model plant that tolerates moderate water deficit.
Several hundred Arabidopsis genes were shown to be differentially
expressed in response to dehydration, as evidenced by transcript
profiling (Bockel et al., 1998 J. Plant Physiol. 152: 158-166;
herein incorporated by reference). These differentially expressed
genes (DEFs) were assigned to diverse metabolic pathways. For
example, genes encoding enzymes involved in sugar metabolism and
biosynthesis of other compounds acting as compatible solutes were
found up-regulated in response to drought (Bohnert et al., 1995
Plant Cell 7: 1099-10111; herein incorporated by reference). The
ion and water channel proteins are likely to be important in
regulating water flux, which were supported by isolation of channel
protein genes from pea (Pisum sativum) in response to water deficit
(Guerrero et al., 1990 Plant Mol. Biol. 15: 11-26; herein
incorporated by reference).
[0251] Enzymes required for cell wall lignification processes are
increased in drought stressed tissue (Peleman et al., 1989 Plant
Cell 1: 81-93; herein incorporated by reference). Genes encoding
proteins similar to proteases and enzymes that detoxify active
oxygen species were induced by drought (Williams et al., 1994 Plant
Mol. Biol. 25: 259-270; Mittler et al., 1994 Plant J. 5: 397 405;
all of which are herein incorporated by reference). Although
precise function of these genes has not yet been demonstrated, five
main groups were summarized by Bartels and Salamini (Bartels et
al., 2001 Plant Physiol. 127: 1346-1353; herein incorporated by
reference) as genes encoding: (a) proteins with protective
properties; (b) membrane proteins involved in transport processes;
(c) enzymes related to carbohydrate metabolism; (d) regulatory
molecules, such as transcription factors, kinases, or other
putative signaling molecules; and (e) open reading frames that show
no homologies to known sequences.
[0252] The molecular complexity of the process during drought
stress response was illustrated by recent microarray experiments
(Seki et al., 2002 Plant J. 30:279-292; herein incorporated by
reference). These results indicate that a network of signal
transduction pathways allows the plant to adjust its metabolism to
the demands imposed by water deficit (Shinozaki et al., 2000 Curr.
Opin. Plant Biol. 3:217-223; Kirch et al., 2001 In Scheel et al.,
eds, Vol. II, 2nd ed. Academic press. NY; all of which are herein
incorporated by reference). The complex signal transduction cascade
is divided into three basic steps: (a) perception of stimulus; (b)
signal amplification and integration; and (c) response reaction in
the form of de novo gene expression (Ingram et al., 1996 Annu. Rev.
Plant Physiol. Plant Mol. Biol. 47: 377-403; herein incorporated by
reference).
[0253] Several experimental approaches were followed to identify
signaling molecules involved in signal transmission process and the
activation of gene expression in response to stress. The majority
of information was derived from promoter analyses and from
differential screening procedures. One molecule that was found as
central to dehydration-regulated gene expression is the plant
hormone abscisic acid (ABA). Endogenous ABA levels were reported to
increase as a result of water deficit in many physiological
studies, and therefore ABA is thought to be involved in the signal
transduction (Chandler et al., 1994 Annu. Rev. Plant Physiol. Plant
Mol. Biol. 45: 113-141.; Giraudat et al., 1994 Plant Mol. Biol. 26:
1557-1577; all of which are herein incorporated by reference).
Besides the ABA-mediated gene expression, the investigation of
drought-induced genes in A. thaliana also revealed ABA-independent
signal transduction pathways (Yamaguchi-Shinozaki et al., 1994
Plant Cell 6: 251-264; herein incorporated by reference). Both
ABA-dependent and -independent stress signaling first modifies
constitutively expressed transcription factors, leading to the
expression of early response transcriptional activators, which then
activate downstream stress tolerance effective genes (Zhu et al.,
2001, Trends Plant Sci. 6: 66-71; herein incorporated by
reference).
[0254] Therefore, even though a large number of drought-induced
genes were identified in a wide range of species, the molecular
basis for increasing drought tolerance remains unknown (Ingram et
al., 1996 Annu. Rev. Plant Physiol. Plant Mol. Biol. 47: 377-403;
herein incorporated by reference).
[0255] Thus, it is contemplated that the use of whole
genomic-related technology for identifying differential gene
expression in Fm provides the necessary tools to identify the key
genes that are altered during drought stress within large-scale
drought stress experiments and for providing grass plant breeding
goals.
[0256] B. Genomics:
[0257] Recently, within the rapidly expanding field of genomics,
the creation of a large-scale EST database from various plant
species, including the complete sequencing of Arabidopsis
(Arabidopsis genome initiative [AGI 2000, Nature 408: 796-815;
herein incorporated by reference) and rice genome (Yu et al., 2002,
Science 296:79-92; herein incorporated by reference) were made
public. This genomic information source provides a vast reference
database for evaluating the coordinated function and expression of
genes identified by using cDNA-AFLP in plants where few genes have
been identified or sequenced, such as in Festuca mairei plants
(see, Wang and Bughrara, 2005, Mol. Biotechnol. 29(3):211-20;
herein incorporated by reference).
[0258] Functional genomics of Festuca species lags far behind other
plant systems. Molecular genetic mechanisms controlling the
expression of drought tolerance in these species also was an
unknown field, even though a significant effort were invested into
the physiological mechanisms studies (Levitt, 1980, Academic Press,
NY; Youngner et al., 1985, In: Gibeault et al., eds., 37-43 Qian et
al., 1997, Crop Sci. 37:905-9 10; all of which are herein
incorporated by reference) and developing and evaluating drought
resistance in grass species (Aronson et al., 1987, Crop Sci. 27:
1261-1266; Fry et al., 1989, Crop Sci. 29: 1535-1541; all of which
are herein incorporated by reference).
[0259] It is contemplated that by relating gene regulation to
adaptive events occurring during stress using a variety of
techniques, such as cDNA-AFLP, provides genetic tools for gaining
information on differential gene expression during stress. cDNA
amplified fragment length polymorphism (cDNA-AFLP) is an extremely
efficient method for isolating differentially expressed fragments
(DEFs) or transcript derived fragments (TDFs) in a genome wide
scale (Bachem et al., 1996 Plant J. 9: 745-753; herein incorporated
by reference). cDNA-AFLP shows high reproducibility and
sensitivity, good correlation with northern blot analysis and low
set-up cost, even though it requires a comprehensive reference
database (Donson et al., 2002 Plant Mol. Biol. 48: 75-97; herein
incorporated by reference). Therefore, the inventor investigated
drought-induced gene expression of F. mairei plants in order to
facilitate germplasm introduction and gene manipulation for
increasing drought tolerance of plants within grass plant breeding
programs.
[0260] Unlike previous studies where one or two stress time-points
were compared with the control, including limited microarray
analysis, the inventor provides herein an analysis of more relevant
drought stress treatments, such as a nine serial time-point study
of the drought stress cycle, which covered the whole course of
dynamic changes of the plant adaptive response to drought stress.
These studies revealed four separate differential expression
patterns detected by the cDNA-AFLP analysis, even though two
expression patterns of transient and up-then-down expression
patterns were not abundant. The inventor further contemplates that
using a complete differential expression pattern that included
spatial and temporal regulation patterns would lead to the design
of a programmed control of a survivial response to drought, and
thus, will allow the artificial regulation of a stress response
mechanism at the gene level. Thus, the cDNA-AFLP technique provided
a means to provide genomic sequence information and functional
analysis but also served as a powerful tool for identification of
gene regulation and types of differential expression patterns
responsible for stress adaptation.
[0261] Also unlike previous studies where a large number of genes,
such as stress inducible or up-regulated transcripts and proteins,
were identified associated with stress responses, the inventor
additionally identified herein, down-regulated genes, transiently
expressed genes and other types of altered gene expression
patterns, such as up-then-down regulation. Thus, down-regulation or
other types of gene regulation are contemplated by the inventor to
play important roles in responding favorably to drought stress
and/or long-term stress tolerance, such as retaining green leaves
and turgid tissues.
[0262] C. Molecular Identification of Festuca Germplasm in Grass
Plants:
[0263] When combined in hybrid plants, Fm and Lp genomes show a
distant relationship according to a low level of homologous
chromosome pairing and hence less genetic recombination (Chen et
al., 1995 Crop Sci. 35:720-725; Humphreys et al., 1997 New Phytol.
137:55-60; all of which are herein incorporated by reference). When
Fm germplasm was identified in Lp hybrids, demonstrating that Fm
and Lp genomes could combine, Fm Lp hybrid plants were contemplated
to provide an effective means to produce hybrids of high agronomic
potential (Cao et al., 2000, Genome 43:398-403; herein incorporated
by reference). However, the Lolium.times.Festuca amphiploids were
found to be unstable and oftentimes shifted back towards one of the
parent types, particularly Lolium, Humphreys et al., 2003, Annals
of Applied Biololgy 143, 1-10; herein incorporated by
reference.
[0264] Therefore, in some embodiments, the present invention
contemplates using molecular markers for identifying F. mairei
germplasm associated with drought tolerance. In particular, the
inventor contemplates identifying meotically stable Fm germplasm on
a Lp plant background for choosing plants with increased drought
resistance for use in grass plant breeding programs.
[0265] Previously, within Fm.times.Lp hybrid plants, F. mairei
chromatin was identified in the L. perenne genetic background by
FISH and RFLP methods (see, for example, Chen and Sleper (1999)
Crop Science 39, 1676-1679; PaSakinskien and Jones, 2005, Cytogenet
Genome Res 109: 393-399; herein incorporated by reference). While
genomic in situ hybridization, (GISH) showed homologous chromosome
pairing between L and M genomes detected in hybrids from crosses
between Fm and Lp plants (Cao et al., 2000 Genome 43:398-403;
Morgan et al. (2001) Theoretical and Applied Genetics 103, 696-701;
all of which are herein incorporated by reference).
[0266] Compared to PCR-based molecular markers, the procedure of
FISH is more difficult, needs trained personnel, and is relatively
expensive. In addition, the amount of information obtained by the
FISH procedure is very limited due to fewer genome specific probes.
Thus PCR based markers such as simple sequence repeat (SSR) and
random amplified polymorphic DNA (RAPD) are important genetic
markers for plant genome analysis due to their genome wide
distribution, simple assay by PCR, and high levels of
polymorphism.
[0267] Additional Festuca markers contemplated for use in the
present inventions include markers used for comparative genome RFLP
mapping for meadow and tall fescue (Chen et al., 1995 Crop Sci.
35:720-725; herein incorporated by reference), SSR markers for tall
fescue (Festuca arundinacea Schreb.) (FA), that were used to
generate a large number of SSR markers through mining the FA
expressed sequence tag (EST) database, then applied in molecular
mapping, comparative genomics, and molecular plant breeding across
a wide range of turfgrass species (Saha et al., 2004, Theor. Appl.
Genet. 109:783-791; Saha, et al., 2005, Theor. Appl. Genet.,
110:323-336; all of which are herein incorporated by
reference).
[0268] The use of ryegrass markers are also contemplated in methods
of the present inventions whereas SSR markers are co-dominantly
inherited and were successfully isolated from perennial ryegrass,
which constitute a valuable resource of markers for the molecular
breeding of ryegrass (Kubik et al., 2001, Crop Sci. 45:1565-1571;
Jones et al., et al., 2001, Theor. Appl. Genet. 102:405-415; all of
which are herein incorporated by reference) with additional
ryegrass markers described in Warnke, et al., 2004, Theor. Appl.
Genet. 109:294-304; and Saha, et al., 2006, Theor Appl Genet.
113(8):1449-58; all of which are herein incorporated by
reference).
[0269] RAPD DNA markers are also contemplated for use in the
present inventions for routine fingerprinting of germplasm and
cultivars, because of the low cost and random marker distribution
throughout the genome. Even though RAPD markers are dominantly
inherited, they are useful for monitoring genome introgressions
from wild donor to cultivated species (Bemabdelmouna et al., 1999
Theor. Appl. Genet. 98:10-17; Siffelova et al., 1997 Biologia
Plantarum 40:183-192; all of which are herein incorporated by
reference).
[0270] Assessment of the genome introgression status in the progeny
from intergenic hybridization by using SSR and RAPD markers will be
useful in directing breeding programs to develop improved grass
cultivars. With the goal of transferring drought tolerance of Fm
into Lp, a population comprised of hybrids, amphidiploid, and
backcross progeny derived from intergeneric crosses between Fm and
Lp were generated Monitoring the Fm and Lp genome compositions in
these progeny using molecular markers will assist in identifying
individuals with desirable genome combinations to develop new
perennial ryegrass cultivars with improved drought tolerance. This
reference describes the use of molecular markers for tracing
genomic introgression between Lolium perenne and Festuca
mairei.times.L. perenne in the progeny of a backcross population
(Wang et al. (2003) Crop Science 43, 2154-2161; herein incorporated
by reference).
[0271] Further, desirable agronomic traits are also contemplated
for tracking with molecular markers. For example, genetic control
of herbage quality variation in perennial ryegrass (Lolium perenne
L.) was assessed through the use of the molecular marker-based
reference (QTL) genetic map of perennial ryegrass (Lolium perenne
L.). Restriction fragment length polymorphism (RFLP), amplified
fragment length polymorphism (AFLP) and genomic DNA-derived simple
sequence repeat-based (SSR) framework marker set was enhanced, with
RFLP loci corresponding to genes for key enzymes involved in lignin
biosynthesis and fructan metabolism. Quality traits such as crude
protein (CP) content, estimated in vivo dry matter digestibility
(IVVDMD), neutral detergent fiber content (NDF), estimated
metabolisable energy (EstME) and water soluble carbohydrate (WSC)
content were measured by near infrared reflectance spectroscopy
(NIRS) analysis of herbage harvests (see, Cogan et al., 2005, Theor
Appl Genet. II 0(2):364-80; herein incorporated by reference).
DETAILED DESCRIPTION OF THE INVENTION
[0272] In some embodiments, the present invention relates to
compositions and methods for providing drought resistant plants
(e.g., grass plants) comprising Festuca mairei plant germplasm.
Specifically, the invention relates to providing compositions and
methods for introgressing preferred Festuca mairei germplasm and/or
specific Festuca mairei genes into plants (e.g., grass plants),
such as Lolium perenne plants. Further, the invention relates to
methods of grass plant breeding comprising genetic markers for
identifying the preferred Festuca mairei germplasm introgressed
into plants, and providing commercially desirable drought resistant
cultivars of plants.
[0273] In some embodiments, the present invention further relates
to methods of breeding Lp hybrid plants comprising Fm preferred
germplasm further comprising compositions and methods for using DNA
markers for identifying the Fm germplasm including but not limited
to simple sequence repeat (SSR), single nucleotide polymorphism
analysis (SNP), random amplified polymorphic DNA (RAPD), amplified
fragment length polymorphism analysis (AFLP), DNA fingerprinting,
and the like, for identifying introgressed drought resistance
germplasm into a formerly drought-susceptible plant variety, for
example, certain elite ryegrass plant varieties.
[0274] In some embodiments, the present invention additionally
provides comparative methods for identifying superior drought
resistant plants.
[0275] In some embodiments the present invention relate to
compositions and methods utilizing genes derived from Festuca
mairei (Fm) plant genomes. Thus, in some embodiments, the present
invention provides compositions comprising Fm genes, Fm nucleic
acid sequences, Fm coding sequences, and Fm polypeptides, and in
particular to plants and expression vectors comprising any one of
SEQ ID NOs: 1-39 (FIG. 7), and 93-216 (FIG. 9), for encoding Fm
polypeptides (see, e.g., polypeptides comprising any one of SEQ ID
NOs:40-91, FIG. 8) and related genes associated with onset,
adaptation, and maintenance of drought resistance in plants, see,
for e.g., related genes in Tables 3-6. In some embodiments, the
nucleic acid sequences are at least 50%, 60%, 70%, 80%, 90%, 95%,
98%, or 99% identical to the aforementioned sequences.
[0276] In some embodiments, the present invention also provides
breeding methods for using Fm genes, Fm nucleic acid sequences, and
Fm polypeptides; such methods include but are not limited to use of
these genes to produce drought resistance plants (e.g., grass
plants) for providing breeding and commercial cultivars through
artificial breeding and/or transgenic plant production, to produce
Fm nucleic acid sequences and/or Fm protein in a plant, to increase
levels of Fm nucleic acid sequences and/or Fm protein in a plant,
to transiently express Fm nucleic acid sequences and/or Fm protein
in a plant, to decrease Fm nucleic acid sequences and/or Fm protein
in a plant, to silence Fm nucleic acid sequences and/or Fm protein
in a plant, to alter environmental stress tolerance of a plant, to
alter environmental stress plant phenotypes of a plant, and for
controlled production of drought tolerance in plants. It is not
meant to limit the present invention to alterations in Fm
expression. In some embodiments, an Fm nucleotide or Fm protein
alters Lp gene expression and/or Lp gene activity and/or Lp protein
expression and/or Lp protein activity. In some embodiments, Fm
genes and/or polypeptides are overexpressed in modified plants,
modified plant tissue, modified leaves, and modified host cells. It
may be desirable to integrate the nucleic acid sequence of interest
into a plant genome. Introduction of the nucleic acid sequence of
interest into a plant cell genome may be achieved by, for example,
by artificial breeding or heterologous recombination using
Agrobacterium-derived sequences, such as described herein.
[0277] In some embodiments, the present invention also provides
methods for inhibiting Fm nucleic acid sequences and/or Fm
polypeptides; such methods include but are not limited to use of
these genes in antisense constructs to produce transgenic plants,
to inhibit Fm protein in a plant, to decrease Fm protein in a
plant, to alter levels of endogenous Lp protein in a plant, to
alter environmental stress tolerance of a plant, to alter
environmental stress phenotypes of a plant, and for controlled
production of drought tolerance. Introduction of the inhibitory
nucleic acid sequence of interest, such as an Fm antisense nucleic
acid sequence, into a plant cell genome may be achieved by, for
example, heterologous recombination using Agrobacterium-derived
sequences.
[0278] In some embodiments, the invention provides an expression
vector, comprising a first nucleic acid sequence encoding a nucleic
acid product that interferes with the expression of a second
nucleic acid sequence comprising a sequence set forth in any one of
SEQ ID NOs:96, 103, 104, 106, 108, 110, 111, 112, 162, 172, 181,
200, 202, 204, 208, 101, 118, 120, 141, and 185. In some
embodiments, the second nucleic acid sequence encodes a polypeptide
comprising a sequence set forth in any one of SEQ ID NOs:40-91
(FIG. 8). In some embodiments, the nucleic acid or polypeptide
sequences are at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%
identical to the aforementioned sequences. The present invention is
not limited to any particular nucleic acid product that interferes
with the expression of a second nucleic acid sequence. Indeed a
variety of types of nucleic acid products are contemplated. In some
embodiments, said nucleic acid product that interferes is an
antisense sequence. In some embodiments, said nucleic acid product
that interferes is a dsRNA that mediates RNA interference. In some
embodiments, said nucleic acid product that interferes is a siRNA
sequence. In some embodiments, said nucleic acid product that
interferes is a hpRNA sequence. In some embodiments, the expression
vector silences the gene in a plant.
[0279] Alternatively, the responsiveness of a plant or plant cell
to a stress condition, such as water deprivation, or a return to an
irrigated condition, can be modulated by use of a suppressor
construct containing a dominant negative mutation for any of the
stress-regulated sequences described herein. In particular, a
suppressor construct would suppress a silenced Fm gene found to be
up-then-down-regulated during adaptation to low water conditions.
Expression of a suppressor construct containing a dominant mutant
mutation generates a mutant transcript that, when coexpressed with
the target transcript inhibits the action of the target transcript.
Methods for the design and use of dominant negative constructs are
well known (see, e.g., in Herskowitz, (1987) Nature 329:219-222;
Lagna and Hemmati-Brivanlou, (1998) Curr. Topics Devel. Biol.
36:75-98; all of which are herein incorporated by reference).
[0280] Thus, in some embodiments, the presentinvention provides
compositions comprising Fm and Fm related (homologous) genes,
nucleic acids sequences, and coding sequences, Fm polypeptides and
Fm homologous polypeptides, and in particular to expression vectors
providing Fm nucleic acid sequences and Fm genes encoding Fm
polypeptides, related genes and their encoded polypeptides,
associated with providing for drought tolerance and increasing
drought tolerance in a plant.
[0281] In some embodiments, the present invention also provides
methods for using Fm related genes, and Fm related polypeptides
(see, for examples, Tables 3-6); such methods include but are not
limited to use of these genes to produce new plants (e.g., grass
plants), to alter environmental stress tolerance in plants, to
alter plant phenotypes, and for controlled production of drought
resistance in plants. It may be desirable to target the nucleic
acid sequence of interest to a particular locus on the plant
genome. As such, the identification of Lp linkage groups comprising
Fm germplasm in grass plants with superior drought resistance may
find use in breeding methods of the present inventions, see, for
example, Table 10.
[0282] In some embodiments, Fm genes and polypeptides are
overexpressed in hybrid grass plants, in nontransgenic plants, in
transgenic plants, transgenic tissue, transgenic leaves, transgenic
seeds, transgenic host cells. In some embodiments, Fm polypeptides
are underexpressed in hybrid grass plants, in nontransgenic plants,
in transgenic plants, transgenic tissue, transgenic leaves,
transgenic seeds, transgenic host cells. In some embodiments, Fm
genes and polypeptides are overexpressed then underexpressed in
hybrid grass plants, in nontransgenic plants, in transgenic plants,
transgenic tissue, transgenic leaves, transgenic seeds, transgenic
host cells. In some embodiments, Fm genes and polypeptides are
transiently expressed in hybrid grass plants, in nontransgenic
plants, in transgenic plants, transgenic tissue, transgenic leaves,
transgenic seeds, transgenic host cells.
[0283] The present invention is not limited to any particular
mechanism of action of genes for providing drought tolerance.
Indeed, an understanding of the mechanism of action is not needed
to practice the present invention. The descriptions provided
herein, are provided merely to describe pathways involved in
regulating environmental stress, with an emphasis on controlling
drought tolerance, Fm gene expression, protein production or
controlling Fm protein activity. In some embodiments, the present
invention provides methods for identifying genes involved in
providing or controlling Fm gene activity, and of Fm mutants and
related Fm genes discovered through use of these methods. Further,
using the sequences and methods of the present invention,
additional genes, nucleic acid sequences, and amino acid sequences
related to regulating drought tolerance are identified and
contemplated for use in the methods of the present inventions. This
description also provides methods of identifying and characterizing
and using Fm genes and their encoded proteins. In addition, the
description provides specific, but not limiting, illustrative
examples of embodiments of the present invention.
[0284] In some embodiments, methods of the invention can be
performed with respect to identifying a functional pathway
involving any of the Fm stress-regulated genes, such as a sequence
set forth in any one of SEQ ID NOs: 1-39 (FIG. 7), and 93-216 (FIG.
9), and/or polypeptides as encoded by a polypeptide comprising a
sequence set forth in any one of SEQ ID NOs:40-91 (FIG. 8),
including for example, functions such as a stress-regulated
transcription factor, metabolism, energy, biogenesis of cellular
component, subcellular localization, transport, transcription,
signal transduction, interaction with the cellular environment,
protein synthesis, protein with binding function, defense,
development, cell fate, cell cycle and DNA processing, protein
fate, and cell type differentiation, see, for example, Tables 2-6.
In some embodiments, the nucleic acid sequences are at least 50%,
60%, 70%, 80%, 90%, 95%, 98%, or 99% identical to the
aforementioned sequences. Functions are defined by The Gene
Ontology project (G0) at the public website
www.geneontology.org/index.shtml. Pathways in which the disclosed
stress-regulated genes and/or polypeptides are involved are
contemplated to be identified herein, for example, a nucleotide
with a homolog, such as Arabidopsis thaliana gi|25090853|
identified as a homolog of Fm sequence SSBII-D09, involved in
electron transport, and Fm sequences SSBI-B09 identified a Hordeum
vulgare homolog gi|28866019|, farnesylated protein 3 involved in
metal ion transport, by searching UniProt (Universal Protein
Resource at www.pir.uniprot.org/) and the EBI (European Molecular
Biology Laboratory) Gene Ontology website (e.g. QuickGO at the
UniProt site) at European Bioinformatics Institute (2006) or the
Munich Information Center for Protein Sequences (MIPS) Arabidopsis
thaliana database (MATDB at mips.gsf.de/projects/plants).
[0285] In some embodiments, the present invention provides methods
of modulating the activity of a biological pathway, such as a
pathway identified herein, in a plant cell, wherein the pathway
involves a stress-regulated Fm gene and/or Fm polypeptide. As used
herein, reference to a pathway that "involves" a stress-regulated
polypeptide means that the polypeptide is required for normal
function of the pathway. For example, plant stress-regulated Fm
polypeptides as disclosed herein include those acting as
transcription factors or as protein binding elements or affecting
drought induced and/or mediated stress responses, which are well
known to be involved in metabolic pathways. As such, a method of
the invention provides a means to modulate a biological pathway
involving plant stress-regulated Fm polypeptides, for example, by
altering the expression of the Fm polypeptides in response to a
stress condition or in response to changes in soil moisture content
or irrigation water. Thus, a method of the invention can be
performed, for example, by introgressing an Fm polynucleotide
portion of a plant stress-regulated Fm gene into the plant cell,
therein modulating the activity of the biological pathway.
[0286] In some embodiments, the present invention also provides
methods of identifying a polynucleotide that modulates a stress
response in a plant cell. Such methods are contemplated to be
performed, for example, by contacting an array of probes
representative of a plant cell genome, such as an Fm genechip
array, and nucleic acid molecules expressed in plant cell exposed
to a particular stress; such as drought stress, detecting a nucleic
acid molecule that is expressed at a level different from a level
of expression in the absence of the stress; introducing the nucleic
acid molecule that is expressed differently into a plant cell; and
detecting a modulated response of the plant cell containing
introgressed nucleic acid molecule to a stress, therein identifying
a polynucleotide that modulates a stress response in a plant cell.
The contacting is under conditions that allow for selective
hybridization of a nucleic acid molecule with probe having
sufficient complementarity, for example, under stringent
hybridization conditions.
[0287] In some embodiments, the present invention also provides
methods of using a polynucleotide portion of a plant
stress-regulated gene, such as an Fm polynucleotide, to confer a
selective advantage on a plant cell. In some embodiments, such
methods are performed by introducing a plant stress-regulated
regulatory element into a plant cell, for example, a regulatory
element for a gene sequence described herein, wherein, upon
exposure of the plant cell to a stress condition to which the
regulatory element is responsive, a nucleotide sequence operatively
linked to the regulatory element is expressed, such as an
up-regulated Fm nucleotide as described herein. In another
embodiment, the stress-regulated element is operably linked to a
reporter molecule, such as luciferase, therein identifying a
drought stress regulatory element. The operatively linked
nucleotide sequence can be, for example, a transcription factor for
an Fm gene described herein, the expression of which induces the
further expression of polynucleotides involved in a stress
response, therein enhancing the response of a plant to the stress
condition for resisting drought. In another embodiment, a coding
sequence of a plant stress-regulated Fm gene as disclosed herein is
introgressed into the cell, therein providing the plant with a
selective advantage in response to a stress condition. In still
another embodiment, the method results in the knock-out of a plant
stress-regulated gene, such as a down-regulated Fm nucleotide, as
disclosed herein, in a first population of plants, therein
providing a selective advantage to a stress condition in the
knock-out population of plants.
[0288] In some embodiments, the present invention provides methods
of identifying an agent, such as a nucleic acid or polypeptide,
that modulates the activity of a stress-regulated regulatory
element of a gene. In some embodiments, methods are provided for
identifying an agent that alters the activity of a response to an
abiotic stress, such as drought stress, such that a composition
comprising an agent to be tested is contacted with a responsive
regulatory element, preferably an element associated with
regulating, e.g., a nucleic acid sequence comprising any one of a
sequence as set forth in SEQ ID NOs: 1-39 (FIG. 7), and 93-216
(FIG. 9), and determining the effect of the agent on the ability of
the regulatory sequence to regulate Fm or Lp gene transcription. In
some embodiments, the nucleic acid sequences are at least 50%, 60%,
70%, 80%, 90%, 95%, 98%, or 99% identical to the aforementioned
sequences. In further embodiments, the regulatory elements are
associated with particular stresses or combination of stresses such
as drought stress and heat stress. In one embodiment, the
regulatory element can be operatively linked to a heterologous
polynucleotide encoding a reporter molecule, and an agent that
modulates the activity of the stress-regulated regulatory element
can be identified by detecting a change in expression of the
reporter molecule due to contacting the regulatory element with the
agent. Such a method can be performed in vitro in a plant cell-free
system, or in a plant cell in culture or in a plant in situ. In
another embodiment, the agent is contacted with a transgenic plant
containing an introgressed plant stress-regulated regulatory
element, and an agent that modulates the activity of the regulatory
element is identified by detecting a phenotypic change in the
transgenic plant. The methods of the invention can be performed in
the presence or absence of the stress condition to which the
particularly regulatory element is responsive, in particular to
drought stress.
[0289] In some embodiments, the present invention provides
nucleotide probes useful for detecting an abiotic stress in a
plant, such as a drought stress response in plants, the probes
comprising a nucleotide sequence of at least 15, 25, 50 or 100
nucleotides that hybridizes under stringent, preferably highly
stringent, conditions to a sequence comprising a sequence set forth
in any one of SEQ ID NOs: 1-39, and 93-216. In some embodiments,
the nucleic acid sequences are at least 50%, 60%, 70%, 80%, 90%,
95%, 98%, or 99% identical to the aforementioned sequences. Also
provided are nucleotide probes comprising at least 15, 25, 50 or
100 nucleotides in length that hybridize under stringent,
preferably highly stringent conditions, to at least one gene
associated with a particular stress, for example drought stress a
sequence comprising a sequence set forth in any one of SEQ ID NOs:
1-39, and 93-216.
I. Festuca mairei (Fm) Plant Genes and Polypeptides:
[0290] The present invention provides plant Fm genes and proteins
including their homologues, orthologs, paralogs, variants and
mutants, all of which are identified in relation to Fm and/or Fm
genes and proteins of the present inventions. In some embodiments,
an isolated nucleic acid sequence comprising a sequence set forth
in any one of SEQ ID NOs: 1-39 (FIG. 7), and 93-216 (FIG. 9) is
provided.
[0291] In some embodiments, the nucleic acid sequences are at least
50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identical to the
aforementioned sequences. Some embodiments of the present
inventions provide polynucleotide sequences that comprise at least
one sequence set forth in any one of SEQ ID NOs: 1-39, and 93-216.
In some embodiments, a sequence encoding a Fm polypeptide comprises
a sequence set forth in any one of SEQ ID NOs:40-91.
[0292] Some embodiments of the present invention provide
polynucleotide sequences that do not encode polypeptides. In some
embodiments, the Fm polynucleotide encodes a polypeptide that shows
0 homology or results equivalent to "No significant similarity" to
known plant polypeptides in publicly accessible sequence databanks
using methods comprising publicly available search engines, such as
BLASTX at a NCBI website (www.ncbi.nlm.nih.gov/BLAST/Blast.cgi).
Other embodiments of the present invention provide polynucleotide
sequences encoding polypeptides comprising a sequence set forth in
any one of SEQ ID NOs:40-91. In yet further embodiments, the
present invention provides Fm polypeptide homologues; see, for
nonlimiting examples, Tables 3-6. In other embodiments, the Fm
polynucleotide encodes a polypeptide at least, 3.00E-33, 2.00E-44,
3.00E-51, 1.00E-108 (or more) identical to any of exemplary
polypeptides encoded by nucleotide sequences comprising SEQ ID NOs:
93-216, such as a polypeptide obtained by a BLASTX search. These
sequences include nucleotide sequences comprising Fm DNA sequences,
with or without genomic sequences.
II. Additional Genes and Polypeptides Associated with Onset and
Adaptation to Drought Tolerance:
[0293] The methods of the present invention are not limited to the
use of germplasm from Festuca plants. Indeed, the germplasm of a
variety of plants are contemplated for use in methods for altering
drought tolerance of any plant, including but not limited to
eukaryotes, such as Plantae and Protista, monocotyledon and
dicotyledon plants, and in particular, Commelimidae plants, such as
Festuca spp.; xeromorphic plants of Restionaceae; and the like. In
further embodiments, germplasm comprising homologes of Fm nucleic
acid sequences are provided. Such germplasm includes but is not
limited to Lolium perenne; Lolium rigidum (annual ryegrass; Wimmera
ryegrass); Poa pratensis (Kentucky Bluegrass); Oryza sativa (rice);
Triticum aestivum (wheat); Avena sativa (oats); Spinacia oleracea
(spinach); Apium graveolens (celery); and Flayeria trinervia
(clustered yellowtops); Brassica sp. (e.g., Arabidopsis thaliana,
B. napus, B. oleracea, etc.), Zea mays (corn), Hordeum vulgare
(barley), Mesembryanthemum crystallinum (common iceplant);
Gossypium barbadense (cotton); Chlamydomonas reinhardtii (green
alga) and the like.
[0294] Thus, the present invention provides nucleic acid sequences
comprising additional plant genes and polypeptides for use in
compositions and methods for altering drought tolerance in plants,
for example, GenBank Accessions Zea mays' AY588275 and
gi|46560602|, plant sequences as described in Tables 2-6, and the
like). Some embodiments of the present invention provide additional
polynucleotide sequences that are homologous to at least one of
exemplary Fm SEQ ID NOs: 1-39, and 93-216, such as Zea mays'
gi|46560602. As such, in some embodiments, the nucleic acid
sequences are at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%
identical to the aforementioned sequences. In some embodiments, the
additional Fm polynucleotides provide sequences that are at least,
3.00E-33, 2.00E-44, 3.00E-51, 1.00E-108 (or more) identical to any
of exemplary polypeptides encoded by nucleotide sequences
comprising SEQ ID NOs: 93-216. In some embodiments, the Fm
polynucleotides are at least, 3.00E-33, 2.00E-44, 3.00E-51,
1.00E-108 (or more) identical to any of exemplary polypeptides
encoded by nucleotide sequences comprising SEQ ID NOs: 1-39. Other
embodiments of the present invention provide homologes of
polynucleotide sequences encoding polypeptides that are homologous
to at least one of exemplary polypeptide comprising a sequence set
forth in any one of SEQ ID NOs:40-91.
[0295] A polynucleotide sequence of a stress-regulated gene as
disclosed herein is contemplated to be particularly useful for
performing the methods of the invention on a variety of plants,
including but not limited to plants described herein.
III. Alleles of Fm Genes:
[0296] In some embodiments of the present invention, alleles of Fm
gene sequences comprising a sequence set forth in any one of SEQ ID
NOs: 1-39, and 93-216 are provided. In some embodiments, the
alleles are at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%
identical to the aforementioned sequences. Any given gene may have
none, one or many allelic forms. Common mutational changes that
give rise to alleles are generally ascribed to deletions,
additions, or insertions, or substitutions of nucleic acids. Each
of these types of changes may occur alone, or in combination with
the others, and at the rate of one or more times in a given
sequence. Mutational changes in alleles also include
rearrangements, insertions, deletions, additions, or substitutions
in upstream regulatory regions.
[0297] In some embodiments, alleles result from a mutation, (i.e.,
a change in the nucleic acid sequence) and generally produce
altered mRNAs or polypeptides whose structure or function may or
may not be altered. In preferred embodiments, the invention
provides alleles resulting from a mutation for producing altered
mRNAs or polypeptides whose structure or function increase
tolerance to abiotic stress, such as drought stress.
[0298] In some embodiments of the present invention, the
pQlynucleotide sequence encoding a Fm nucleotide sequence is
extended utilizing the nucleotide sequences (e.g., SEQ ID NOs:
1-39, and 93-216) in various methods known in the art to extend
sequences, such as to obtain coding and noncoding gene sequences,
such as to detect upstream and/or downstream sequences such as
promoters and regulatory elements. For example, it is contemplated
that for some Fm, or related Fm sequences, the sequences upstream
and/or downstream are identified using RACE or Fm genomic
information. For other Fm sequences for which a similar sequence is
identified and a database is available, the sequences upstream or
downstream of the identified Fm genes can be identified. For other
Fm genes for which a public genomic database is not available, or
not complete, it is contemplated that polymerase chain reaction
(PCR) finds use in the present invention, such as Rapid
Amplification of cDNA Ends (RACE) assays and assays described
below.
[0299] In another embodiment, inverse PCR is used to amplify or
extend sequences using divergent primers based on a known region
(see, e.g., Triglia et al. (1988) Nucleic Acids Res., 16:8186;
herein incorporated by reference). In yet another embodiment of the
present invention, capture PCR (see, for e.g., Lagerstrom et al.
PCR Methods Applic., 1:111-19 (1991); herein incorporated by
reference) is used. In still other embodiments, walking PCR is
utilized. Walking PCR is a method for targeted gene walking that
permits retrieval of unknown sequence (see, for e.g., Parker et al.
Nucleic Acids Res., 19:3055-60 (1991); herein incorporated by
reference). The PROMOTERFINDER kit (Clontech) uses PCR, nested
primers and special libraries to "walk in" genomic DNA. This
process avoids the need to screen libraries and is useful in
finding intron/exon junctions. In yet other embodiments of the
present invention, add TAIL PCR is used as a preferred method for
obtaining flanking genomic regions, including regulatory regions
(see, for e.g., Liu and Whittier, (1995) Genomics, 25(3):674-81;
Liu et al. (1995) Plant J., (3):457-63; all of which are herein
incorporated by reference). Preferred libraries for screening for
full-length cDNAs include libraries that have been size-selected to
include larger cDNAs. Also, random primed libraries are preferred,
in that they contain more sequences that contain the 5' and
upstream gene regions. A randomly primed library may be
particularly useful in cases where an oligo d(T) library does not
yield full-length cDNA. Genomic Libraries are useful for obtaining
introns and extending 5' sequence.
IV. Variant Fm Genes and Polypeptides:
[0300] In some embodiments, the present invention provides isolated
variants of the disclosed nucleic acid sequences encoding Fm genes
and the polypeptides encoded therein; these variants include
mutants, fragments, fusion proteins, homologes, and functional
equivalents of genes and gene protein products.
[0301] Thus, nucleotide sequences of the present invention are
contemplated for engineering in order to introduce or alter a Fm
coding sequence for a variety of reasons, including but not limited
to initiating the production of abiotic stress tolerance, such as
drought stress; augmenting or increasing stress tolerance, such as
drought stress, alterations that modify the cloning, processing
and/or expression of the gene product (such alterations include
inserting new restriction sites and changing codon preference), as
well as varying the protein function activity (such changes include
but are not limited to differing binding kinetics to nucleic acid
and/or protein or protein complexes or nucleic acid/protein
complexes, differing binding inhibitor affinities or effectiveness,
differing reaction kinetics, varying subcellular localization, and
varying protein processing and/or stability).
[0302] A. Homologues: In some embodiments, the present invention
provides isolated variants of the disclosed nucleic acid sequence
encoding Fm nucleotides, or related plant genes, and the
polypeptides encoded therein; these variants include mutants,
fragments, fusion proteins or functional equivalents genes and
protein products.
[0303] Some homologues or variants of encoded Fm products are
contemplated to have an intracellular half-life dramatically
different than the corresponding wild-type protein. For example,
the altered protein is rendered either more stable or less stable
to proteolytic degradation or other cellular process that result in
destruction of, or otherwise inactivate the encoded Fm product.
Such homologues, and the genes that encode them, can be utilized to
alter the activity of the encoded Fm and/or Fm products by
modulating the half-life of the protein. For instance, a short
half-life can give rise to more transient biological effect, such
as to mimic the up then down regulated or transiently regulated
nucleotides, or to turn on genes for upregulation or turn off genes
for down regulation. Other homologues have characteristics that are
either similar to wild type Fm, or which differ in one or more
respects from wild-type Fm.
[0304] In some embodiments of the present invention, the amino acid
sequences for a population of Fm nucleic acid product homologues
are aligned, preferably to promote the highest homology possible.
Such a population of variants can include, for example, Fm gene
homologues from one or more species, such as rice, oats, maize,
barley, cotton, or Fm gene homologues from the same species, such
as Festuca, but which differ due to mutation. Amino acids that
appear at each position of the aligned sequences are selected to
create a degenerate set of combinatorial sequences.
[0305] In a preferred embodiment of the present invention, the
combinatorial Fm gene library is produced by way of a degenerate
library of genes encoding a library of polypeptides that each
include at least a portion of candidate encoded Fm-protein
sequences. For example, a mixture of synthetic oligonucleotides is
enzymatically ligated into gene sequences such that the degenerate
set of candidate Fm sequences are expressible as individual
polypeptides, or alternatively, as a set of larger fusion proteins
(e.g., for phage display) containing the set of Fm sequences
therein.
[0306] There are many ways by which the library of potential Fm
homologues can be generated from a degenerate oligonucleotide
sequence. In some embodiments, chemical synthesis of a degenerate
gene sequence is carried out in an automatic DNA synthesizer, and
the synthetic genes are ligated into an appropriate gene for
expression. The purpose of a degenerate set of genes is to provide,
in one mixture, all of the sequences encoding the desired set of
potential Fm sequences. The synthesis of degenerate
oligonucleotides is well known in the art (see, e.g., Narang,
(1983) Tetrahedron Lett. 39(1):3-22; Itakura et al. Recombinant
DNA, in Walton (ed.), Proceedings of the 3rd Cleveland Symposium on
Macromolecules, Elsevier, Amsterdam, pp 273-289 (1981); Itakura et
al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1977)
Science 198:1056; Ike et al. (1983) Nucl. Acid Res., 11:477; all of
which are herein incorporated by reference in their entirety. Such
techniques have been employed in the directed evolution of other
proteins (See e.g., Scott et al (1990) Science, 249:386-390;
Roberts et al. (1992) Proc. Natl. Acad. Sci. USA, 89:2429-2433;
Devlin et al (1990) Science, 249:404-406; Cwirla et al (1990) Proc.
Natl. Acad. Sci. USA, 87:6378-6382; in addition to U.S. Pat. Nos.
5,223,409, 5,198,346, and 5,096,815; all of which are herein
incorporated by reference in their entirety).
[0307] Functional homologues can be screened for by expressing the
homologues in an appropriate vector (described in more detail
below) in a plant cell and analyzing the level of drought tolerance
of a plant derived from the cell.
[0308] B. Mutants: In some embodiments, the present invention
provides nucleic acid sequences encoding mutant forms of Fm
proteins, (i.e., mutants), and the polypeptides encoded therein. In
some embodiments of the present invention, mutations in
up-regulated nucleic acid sequences of Fm genes are provided, (see,
Table 6). In some contemplated embodiments, mutations in
upregulated genes result in altered abiotic stress tolerance, such
as increased drought stress tolerance, and drought stress
phenotypes, such as maintaining leaf color and less firing compared
to control plants. In some embodiments of the present invention,
isolated nucleic acid sequences comprising downregulated genes are
provided, (see, Table 5). In some embodiments, mutations in
downregulated genes result in altered abiotic stress tolerance,
such as drought stress tolerance, and drought stress phenotypes,
such as maintaining leaf color and less firing compared to control
plants. In some embodiments, mutations in nucleic acid sequences
comprising upregulated-then-downregulated genes are provided, (see,
Table 6). In some contemplated embodiments, mutations in
upregulated-then-downregulated genes result in altered abiotic
stress tolerance, such as drought stress, and abiotic stress
tolerance, such as drought stress, phenotypes, such as maintaining
leaf color and less firing compared to control plants. In some
contemplated embodiments, mutations in isolated nucleic acid
sequences comprising transiently regulated genes are provided,
(see, Table 6). In some contemplated embodiments, mutations in
transiently regulated genes result in altered abiotic stress
tolerance, such as drought stress, and abiotic stress tolerance,
such as drought stress, phenotypes, such as maintaining leaf color
and less firing compared to control plants.
[0309] In preferred embodiments, mutants result from mutation of
the coding sequence, (i.e., a change in the nucleic acid sequence)
and generally produce altered mRNAs or polypeptides whose structure
or function may or may not be altered. Any given gene may have
none, one, or many variant forms. Common mutational changes that
give rise to variants are generally ascribed to deletions,
additions or substitutions of nucleic acids. Each of these types of
changes may occur alone, or in combination with the others, and at
the rate of one or more times in a given sequence.
[0310] Mutants of Fm genes can be generated by any suitable method
well known in the art, including but not limited to EMS (ethyl
methanesulfonate) induced mutagenesis, site-directed mutagenesis,
randomized "point" mutagenesis, and domain-swap mutagenesis or
fusion proteins in which portions of Fm cDNA are "swapped" with the
analogous portion of Fm encoding cDNAs (Back and Chappell, (1996)
PNAS 93: 6841-6845; herein incorporated by reference).
[0311] It is contemplated that is possible to modify the structure
of a peptide having an activity (e.g., such as a drought resistance
activity, such as activity for leaf elongation), for such purposes
as increasing synthetic activity or altering the affinity of the Fm
protein for a binding partner or a kinetic activity. Such modified
peptides are considered functional equivalents of peptides having
an activity of an Fm function as defined herein. A modified peptide
can be produced in which the nucleotide sequence encoding the
polypeptide has been altered, such as by substitution, deletion, or
addition. In some preferred embodiments of the present invention,
the alteration increases or decreases the effectiveness of the Fm
gene product to exhibit a phenotype caused by altered abiotic
stress tolerance production, such as drought stress. In other
words, construct "X" can be evaluated in order to determine whether
it is a member of the genus of modified or variant Fm genes of the
present invention as defined functionally, rather than
structurally. Accordingly, in some embodiments the present
invention provides nucleic acids comprising an Fm sequence that
complements the coding regions of any of SEQ ID NOs: 1-39, and
93-216, as well as the polypeptides encoded by such nucleic acids.
In some embodiments, the nucleic acid sequences are at least 50%,
60%, 70%, 80%, 90%, 95%, 98%, or 99% identical to the
aforementioned sequences.
[0312] Moreover, as described above, mutant forms of Fm proteins
are also contemplated as being equivalent to those peptides that
are modified as set forth in more detail herein. For example, it is
contemplated that isolated replacement of a leucine with an
isoleucine or valine, an aspartate with a glutamate, a threonine
with a serine, or a similar replacement of an amino acid with a
structurally related amino acid (i.e., conservative mutations) will
not have a major effect on the biological activity of the resulting
molecule.
[0313] Accordingly, some embodiments of the present invention
provide nucleic acids comprising sequences encoding variants of Fm
gene products disclosed herein containing conservative
replacements, as well as the proteins encoded by such nucleic
acids. Conservative replacements are those that take place within a
family of amino acids that are related in their side chains.
Genetically encoded amino acids can be divided into four families:
(1) acidic (aspartate, glutamate); (2) basic (lysine, arginine,
histidine); (3) nonpolar (alanine, valine, leucine, isoleucine,
proline, phenylalanine, methionine, tryptophan); and (4) uncharged
polar (glycine, asparagine, glutamine, cysteine, serine, threonine,
tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes
classified jointly as aromatic amino acids. In similar fashion, the
amino acid repertoire can be grouped as (1) acidic (aspartate,
glutamate); (2) basic (lysine, arginine, histidine), (3) aliphatic
(glycine, alanine, valine, leucine, isoleucine, serine, threonine),
with serine and threonine optionally be grouped separately as
aliphatic-hydroxyl; (4) aromatic (phenylalanine, tyrosine,
tryptophan); (5) amide (asparagine, glutamine); and (6)
sulfur-containing (cysteine and methionine) (e.g., Stryer ed.,
Biochemistry, pg. 17-21, 2nd Ed, WH Freeman and Co., 1981; herein
incorporated by reference). Whether a change in the amino acid
sequence of a peptide results in a functional homologue can be
readily determined by assessing the ability of the variant peptide
to function in a fashion similar to the wild-type protein. Peptides
having more than one replacement can readily be tested in the same
manner.
[0314] More rarely, a mutant includes "nonconservative" changes
(e.g., replacement of a glycine with a tryptophan). Analogous minor
variations can also include amino acid deletions or insertions, or
both. Guidance in determining which amino acid residues can be
substituted, inserted, or deleted without abolishing biological
activity can be found using computer programs (e.g., LASERGENE
software, DNASTAR Inc., Madison, Wis.; herein incorporated by
reference in its entirety). Accordingly, other embodiments of the
present invention provide nucleic acids comprising sequences
encoding variants of Fm gene products disclosed herein containing
non-conservative replacements where the biological activity of the
encoded protein is retained, as well as the proteins encoded by
such nucleic acids.
V. Plant Breeding Methods:
[0315] The present invention also relates to the field of plant
breeding, specifically to methods of grass plant breeding and the
resulting grass plant s and grass plant lines for commercial
distribution. The grass plant breeding methods include but are not
limited to natural breeding, artificial breeding, molecular marker
selection, commercial breeding, and transgenics. More particularly,
the invention relates to producing drought resistant plants (e.g.,
grass plants), populations, cultivars, varieties, lines and methods
of breeding the same, the methods further comprising DNA molecular
marker analysis.
[0316] Plants are crossed by either natural or artificial
techniques for introgressing germplasm from one plant into another
plant. Plants, in particular grass plants, are reproduced sexually
by seed (sexual reproduction) or via vegetative propagation
(asexually), for example, by cultivation of tillers which arise
from buds on culm nodes, rhizomes, and stolons. Natural pollination
occurs in fields through transfer of pollen to stamens by wind
(grasses) or insects (plants in general). Artificially directed
pollination can be effected either by controlling the types of
pollen that can blow onto the flowers (ovaries) or by pollinating
by hand. Grass plants are crossed mechanically using artificial
pollination techniques, for example, by collection of pollen in
pollination bags that are then tied over the flowers of the
receiving plants.
[0317] Backcross breeding was used to transfer germplasm by
introgressing a heritable trait into a desirable homozygous
cultivar or inbred line, called the recurrent parent. The source of
the trait to be transferred is called the donor parent (e.g., Fm
plants). The resulting plant, progeny plant, is expected to have
the attributes of the recurrent parent (e.g., cultivar) and the
desirable trait transferred from the donor parent. After ihe
initial cross, individuals possessing the phenotype of the donor
parent are selected and repeatedly crossed (backcrossed) to the
recurrent parent. The resulting progeny are expected to have the
attributes of the original recurrent parent (e.g., cultivar) and
the desirable trait transferred from the donor parent.
[0318] Near-isogenic lines (NIL) are created by numerous
backcrosses to produce an array of individuals that are nearly
identical in genetic composition except for the trait or genomic
region under interrogation that can be used in a mapping population
for identifying the genetics of a desirable trait.
[0319] The methods of the present invention are not limited to
altering drought tolerance in any particular plant. Indeed, a
variety of plant classifications, plants, and types of plant are
contemplated for use including but not limited to monocotyledon and
dicotyledon plants, Classes Liliopsida and Magnoliopsida of
flowering plants, and plants such as grasses, grains, vegetables,
flowers, herbs, ornamentals, plants for use as ground covers,
plants for use as water covers, plants for use in erosion control,
plants for use in slope stabilization, plants for use in shore
stabilization, bushes, and trees. For example, plant Liliopsida
plants include but are not limited to Commelimidae, further
including, for example, the grass family Poaceae (also known
Gramineae), the sedges Cyperaceae (such as papyrus), the bur-reeds
(Sparganiaceae); pipeworts (Eriocaulaceae), the Cattail Family or
perennial marsh plants (Typhaceae), the rushes (Juncaceae), the
yellow-eyed grass family (Xyridaceae; such as American marsh
plants), succulent herb family (Commelinaceae), Amaranthaceae,
Asteraceae, and the ginger family (Zingiberaceae or Zingiberidae),
such as Costus speciosus or `crepe` ginger. For another example,
Magnoliopsida plants include but are not limited to Gossypium
barbadense (cotton). Preferred forage and turf grass for use in the
methods of the invention include perennial ryegrass, intermediate
ryegrass, annual ryegrass, and tall fescue. In particular, the
following plants are contemplated for use in the present
inventions: wheat (Triticum), rice (Oryza spp.), corn (Zea Mays),
oats (Avena spp.), alfalfa (e.g., Medicago sativa, Medicago spp.,
etc.), rye (e.g., Secale cereale, spp., etc.), sorghum (e.g.,
Sorghum bicolor, Sorghum vulgare, etc.), millet (e.g., pearl millet
(Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail
millet (Setaria italica), finger millet (Eleusine coracana), etc.),
Melilotus, e.g., clover, Lotus, trefoil, lentil, hemp, buckwheat,
cotton (e.g., Gossypium barbadense, Gossypium hirsutum, etc.),
sugarcane (e.g., Saccharum spp., etc.), oats (e.g., Aveneae spp.,
such as Avena sativa), barley (Hordeum spp.), sugar cane (Saccharum
spp.); bead grass (Paspalum spp.), bluegrass (Poa spp.), bluestem
(Andropogon, Dichanthium spp.); fescue (Festuca spp.), gramma grass
(Bouteloua spp.), timothy (Phleum spp.,), wheatgrass (Agropyron,
Elytrigia, Elymus spp.), and zoysia (Zoysia spp.); little bluestem
(Schizachyrium spp.), Indian grass (Sorghastrum spp.), fountain
grass (Pennisetum spp.), hair grass (Deschampsia spp.), Miscanthus
spp., Andropogon spp., and Panicum virgatum.
[0320] A. Selection of Parents to Produce Superior Transgressive
Segregants:
[0321] The goal of plant breeding in general is to produce progeny
that exceed their parents in performance for one or more traits.
The inventor discovered grass lines, G15 and G30a that demonstrated
superior drought resistance when compared to groups of plants
segregating for drought resistant levels comprising the parental
plants. Thus the inventor contemplates the use of such superior
plants for identifying the DNA markers for germplasm that provides
drought resistance and use these superior plants as a basis for
selection of parents and progeny that comprise a similar complement
of desired Fm genes. For example, when examining the marker
genotypes of G15 and G30a (Table 8), it is apparent that these two
lines differ in genotype at least 7 out of 13 SSR loci tested
(Table 8). Therefore, in one embodiment, the inventor contemplates
crossing G15 and G30a and select inbred progeny plants that contain
the desired alleles within the 7 loci. The desired alleles will
contribute to drought tolerance performance in some additive and/or
epistatic fashion, and it will be likely that such progeny will be
agronomically superior to either parent.
[0322] 1. Selection of Superior Lines From Segregating
Populations:
[0323] DNA markers used to select breeding parents that will be
used in crosses are contemplated for use in screening progeny from
such crosses. For example, in a G15 and G30a cross, progeny plant
lines at various stages of inbreeding will be screened for the
favorable allele at least 7 loci that are segregating. The breeder
will select progeny that contain as many of the favorable alleles
as possible. The best possible transgressive segregant or "ideal
segregant" will be the one that contains the favorable alleles at
segregating loci (for example, Table 8).
[0324] 2. Selection of Superior Hybrids:
[0325] In addition to selection of parents that will produce
superior drought resistant recombinant inbred plants,
complementation at QTL's affecting desirable agronomic traits are
contemplated for use in predicting and providing superior hybrid
agronomic performance. The inventors' methods will be used to
identify selected agronomic traits in drought resistant plants, in
particular ryegrass plants (see, for example, Yamada et al., (2004)
Crop Sci. 44:925-935; Bert et al., (1999) Theor. Appl. Genet.
99:445-452; all of which are herein incorporated by reference).
[0326] 3. Selection for and Maintenance of Fm Germplasm:
[0327] In some embodiments, the inventor contemplates plants
comprising meoitically stable Fm germplasm. In some embodiments,
meotically stable germplasm remains stable during under green house
growing conditions. In some embodiments, meotically stable
germplasm remains stable during environment interactions under
field growing conditions. For example, in the case of a locus "A"
where allele "A1" is necessary for maximum drought resistance in
one type of environment and allele "A2" is necessary for maximum
drought resistance in another type of environment, for example, a
field of ryegrass grown in a greenhouse or a field of ryegrass
located in the United States Midwest or a field of ryegrass located
in the southern region of the United States. In such cases, a
population that is heterogeneous for these alleles also retains and
shows drought resistance trait stability over both types of
environments. The inventors' methods will allow the breeder to
select for and maintain such heterogeneity.
[0328] A method of confirming whether intra-line heterogeneity at a
specific locus will be beneficial for agronomic performance,
comprises: 1) identify lines that are heterogeneous for the locus
in question; 2) develop sub-populations of the line that are
homogeneous for one or the other allele based on selection with DNA
markers; 3) field test the original heterogeneous line along with
each of derived homogeneous lines over a number of defined
environments; and 4) determine whether the heterogeneous line will
perform, (such as provide an economical yield) greater than either
homogeneous line when averaged over test environments.
[0329] 4. Use of Genetic Markers for Linkage Groups and
Quantitative Trait Loci (QTLs) to select Superior Plants:
[0330] The inventor contemplates using linkage groups and/or QTL's
affecting agronomic performance for selecting superior breeding
plants. For example, a plant breeder would use a method to for
selecting a parental plant comprising: 1) identify parents that
will produce superior transgressive segregants; 2) ide ntify
superior lines from crosses that are segregating at a DNA marker or
loci; 3) identify parents that will produce superior hybrid
progeny; 4) identify heterogeneous lines to fix favorable alleles;
and 5) identify plants maintaining desirable heterogeneity.
[0331] Genetic linkage of marker molecules can be established by a
gene mapping model such as, without limitation, the flanking marker
model reported by Lander and Botstein, Genetics, 121:185-199
(1989), and the interval mapping, based on maximum likelihood
methods described by Lander and Botstein, Genetics, 121:185-199
(1989), and implemented in the software package MAPMAKER/QTL
(Lincoln and Lander, Mapping Genes Controlling Quantitative Traits
Using MAPMAKER/QTL, Whitehead Institute for Biomedical Research,
Massachusetts, (1990); herein incorporated by reference. Additional
software includes Qgene, Version 2.23 (1996), Department of Plant
Breeding and Biometry, 266 Emerson Hall, Cornell University,
Ithaca, N.Y.); herein incorporated by reference.
[0332] A maximum likelihood estimate (MLE) for the presence of a
marker is calculated, together with an MLE assuming no QTL effect,
to avoid false positives. A log.sub.10 of an odds ratio (LOD) is
then calculated as: LOD=log.sub.10 (MLE for the presence of a QTL
(MLE given no linked QTL).
[0333] The LOD score essentially indicates how much more likely the
data are to have arisen assuming the presence of a QTL than in its
absence. The LOD threshold value for avoiding a false positive with
a given confidence, say 95%, depends on the number of markers and
the length of the genome. Graphs indicating LOD thresholds are set
forth in Lander and Botstein, Genetics, 121:185-199 (1989); herein
incorporated by reference, and further described by Ars and
Moreno-Gonzlez, Plant Breeding, Hayward, Bosemark, Romagosa (eds.)
Chapman & Hall, London, pp. 314-331 (1993); herein incorporated
by reference.
[0334] Additional models can be used. Many modifications and
alternative approaches to interval mapping have been reported,
including the use of non-parametric methods (Kruglyak and Lander,
Genetics, 139:1421-1428 (1995); herein incorporated by reference).
Multiple regression methods or models can be also be used, in which
the trait is regressed on a large number of markers (Jansen,
Biometrics in Plant Breed, van Oijen, Jansen (eds.) Proceedings of
the Ninth Meeting of the Eucarpia Section Biometrics in Plant
Breeding, The Netherlands, pp. 116-124 (1994); Weber and Wricke,
Advances in Plant Breeding, Blackwell, Berlin, 16 (1994); all of
which are herein incorporated by reference). Procedures combining
interval mapping with regression analysis, whereby the phenotype is
regressed onto a single putative QTL at a given marker interval,
and at the same time onto a number of markers that serve as
`cofactors,` have been reported by Jansen and Stam, Genetics,
136:1447-1455 (1994); Zeng, Genetics, 136:1457-1468 (1994); all of
which are herein incorporated by reference. Generally, the use of
cofactors reduces the bias and sampling error of the estimated QTL
positions (Utz and Melchinger, Biometrics in Plant Breeding, van
Oijen, Jansen (eds.) Proceedings of the Ninth Meeting of the
Eucarpia Section Biometrics in Plant Breeding, The Netherlands, pp.
195-204 (1994); herein incorporated by reference, thereby improving
the precision and efficiency of QTL mapping (Zeng, Genetics,
136:1457-1468 (1994); herein incorporated by reference). These
models can be extended to multi-environment experiments to analyze
genotype-environment interactions (Jansen et al., (1995) Theo.
Appl. Genet. 91:33-37; herein incorporated by reference).
[0335] Backcross populations (e.g., generated from a cross between
a successful variety (recurrent parent) and another variety (donor
parent) carrying a trait not present in the former) can be utilized
as a mapping population. A series of backcrosses to the recurrent
parent can be made to recover most of its desirable traits. Thus a
population is created consisting of individuals nearly like the
recurrent parent but each individual carries varying amounts or
mosaic of genomic regions from the donor parent. Backcross
populations can be useful for mapping dominant markers if all loci
in the recurrent parent are homozygous and the donor and recurrent
parent have contrasting polymorphic marker alleles (Reiter et al.,
Proc. Natl. Acad. Sci. (U.S.A.) 89:1477-1481 (1992); herein
incorporated by reference). Backcross populations, however, are
more informative (at low marker saturation) when compared to RELs
as the distance between linked loci increases in RIL populations
(i.e., about 0.15% recombination). Increased recombination can be
beneficial for resolution of tight linkages.
[0336] B. Marker-Assisted Trait Selection and Plant Breeding
[0337] In one embodiment, the present invention provides a method
for marker-assisted selection. Marker-assisted selection involves
the selection of plants having desirable phenotypes based on the
presence of particular nucleotide sequences "markers," such as SSR,
RAPD, cDNA AFLP and the like, or for transformed plants, an
expressed product is a marker, such as GUS or GFP. The use of
markers allows plants to be selected by genotype for breeding
superior grass lines. The use of markers also allows plants to be
selected early in development, often before a desired phenotype
would normally be manifest. Because it allows for early selection,
marker-assisted selection decreases the amount of time need for
selection and thus allows more rapid genetic progress. Briefly,
marker-assisted selection involves obtaining nucleic acid from a
plant to be selected. The nucleic acid obtained is then identified,
or not, with probes that selectively hybridize under stringent,
preferably highly stringent, conditions to a nucleotide sequence or
sequences associated with the desired phenotype. In one embodiment,
the probes hybridize to any of the stress-responsive genes or
regulatory regions disclosed herein, for example, any one SEQ ID
NOs: 1-39, and 93-216 or homologs thereof as described above. The
presence of any hybridization products formed is detected, or not,
and plants are then selected on the presence or absence of the
desired hybridization products.
[0338] An additional aspect provides a method for marker-assisted
breeding to select plants having an altered resistance to abiotic
stress tolerance, such as drought stress tolerance, comprising
obtaining nucleic acid molecules from the plants to be selected;
contacting the nucleic acid molecules with one or more markers or
probes that selectively hybridize under stringent, preferably
highly stringent, conditions to a nucleic acid sequence selected
from any of sequences set forth in SEQ ID NOs:217-246 and 247-265.
Such that the detecting the hybridization of the one or more probes
to the nucleic acid sequences, for example using PCR amplification
of hybridized probes or Southern or Northern gels or
micro/macroarrays, wherein the presence of the hybridization
indicates the presence of a gene associated with altered resistance
to abiotic stress tolerance, such as drought tolerance; and
selecting plants on the basis of the presence or absence of such
hybridization. Marker-assisted selection can also be accomplished
using one or more probes which selectively hybridize under
stringent, preferably highly stringent conditions, to a nucleotide
sequence comprising a polynucleotide expressed in response
associated with a particular stress, for example, a nucleotide
sequence comprising any of sequences set forth in SEQ ID
NOs:217-246 and 247-265. In each case marker-assisted selection can
be accomplished using a probe or probes to a single sequence or
multiple sequences or as fusion sequences. In one embodiment,
marker assisted selection is by using a PCR primer or linker for
amplifying a marker or as a marker. If multiple sequences, such as
PCR primers, are used they can be used simultaneously or
sequentially. Nonlimiting examples of such primers are any of RAPD
markers as set forth in SEQ ID NOs: 266-306, any of AFLP markers,
such as primers or linkers as set forth in SEQ ID NOs:217-265, or
an Lp SSR primer, or a Festuca EST-SSR primer, and the like. The
use of such markers, primers, and linkers is provided in the
Examples.
[0339] C. Generation of Drought Tolerant Grass Plant Lines using
Transgenic Grass Plants:
[0340] The breeding methods of the present inventions further
comprise introgressing a heterologous transgene from a transgenic
plant. Thus in one embodiment, the inventor contemplates
traditional plant breeding methods comprising a transgenic
perennial ryegrass (Lolium perenne L.) plant for introgressing a
transgene into a drought resistant plant of the present inventions.
A purpose of such introgression is to provide a desired agronomic
trait. In one embodiment, the transgenic ryegrass plant is an
established plant. For one example, the use of an established plant
of a salt-tolerant transgenic perennial ryegrass (Lolium perenne
L.) is contemplated for providing increased salt tolerance in a
plant of the present invention provided by a rice vacuolar
Na[+]/H[+] antiporter transgene, OsNHX1 (Wu, et al., 2005, Plant
science, 169, (1):65-73). In another embodiment, the inventor
contemplates introgressing a heterologous transgene of a novel
transgenic plant of the present invention. For example, a
traditional breeding methods comprising a novel transgenic ryegrass
plant of the present inventions comprising a Fm gene, such as a
gene comprising a nucleic acid sequence set forth in any of SEQ ID
NOs: 1-39, and 93-216. In some embodiments, the nucleic acid
sequences are at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%
identical to the aforementioned sequences. Another purpose for
using transgenic ryegrass plants in breeding methods of the present
inventions, in addition to introgression of the transgene, is to
introgress germplasm for desired agronomic traits of the transgenic
plant into a drought resistant plant of the present inventions.
[0341] D. Regeneration of Plants:
[0342] Plants may be regenerated from plant cells and plant parts,
such as seeds, stems, and cultured plant tissue, using well-known
methods for regenerating whole plants. Specifically, after
selecting for a modified plant comprising Fm germplasm
demonstrating desired drought resistant traits or selecting a
transformed plant, such as a plant that expresses a heterologous Fm
gene encoding a Fm protein or variant thereof, a whole plant is
regenerated. It is known that many plants can be regenerated from
cultured cells or tissues, including but not limited to all major
species of grass, including but not limited to perennial ryegrass
(for example, Folling et al., 1995 Plant science. 108:229-239;
herein incorporated by reference), oats, barley (for example, Nobre
et al., 1995, Barley Genetics Newsletter 25:46-50; herein
incorporated by reference), rice (for example, Fujimura, et al.
(1985) Plant Tissue Culture Lett. 2:74-75; herein incorporated by
reference), Zea Mays (for example, Shillito et al. (1989)
Bio/Technology 7:581-587; herein incorporated by reference),
sugarcane, cotton, legumes, vegetables, and monocots.
[0343] Methods for regeneration of plants vary from species to
species of plants. In one embodiment, a suspension of transformed
protoplasts comprising copies of the heterologous gene is provided.
Plant regeneration from cultured protoplasts is described in Evans
et al. Handbook of Plant Cell Cultures, Vol. 1: (MacMillan
Publishing Co., New York, 1983); and Vasil (ed.), Cell Culture and
Somatic Cell Genetics of Plants, Acad. Press, Orlando, Vol. I,
1984, and Vol. III, 1986; herein incorporated by reference. A
protoplast suspension may be used to form embryogenic callus from
which shoots and roots may be induced to form plants.
[0344] Alternatively, embryo formation can be induced from a
protoplast suspension. These embryos can germinate and form mature
plants. The culture media of the protoplast suspention will
generally contain various amino acids and hormones, such as auxin
and cytokinins. Efficient regeneration will depend on the type of
cultures medium, or the genotype, or the history of the culture.
The reproducibility of regeneration depends on the control of these
variables.
VI. Evaluation of Stress Tolerance, Such as Drought Stress:
[0345] The modified plants and grass plant lines are tested for the
effects of the Fm gene or Fm transgene on abiotic stress tolerance,
such as a drought stress phenotype. For example, the parameters
evaluated for drought stress tolerance, are compared to those in
control (irrigated) plants, or unmodified plants and plant lines.
Parameters evaluated include measures of leaf elongation, leaf
water content, leaf water potential, root biomass, root length,
flowering, reproduction, and the like, in response to water
deprivation, including responses to additional abiotic stresses
such as high or low salt, changes in light exposure, heat, cold,
and the like. Differences in levels of capability to resist abiotic
stress, such as a level of response to drought stress, can be
expressed as a distance of leaf elongation, weight as in root
biomass, a unit of time, or in a particular tissue, such a leaf
color, or as a developmental state, such as stage of flowering; for
example, comparative levels of drought stress capabilities in
Festuca and grass plant hybrids was measured in leaves and roots,
see, Examples and Figures. These tests may be conducted both in the
greenhouse and in the field.
[0346] A further aspect provides a method for monitoring a
population of plants comprising providing at least one sentinel
plant comprising a recombinant polynucleotide, wherein the
polynucleotide comprises a stress responsive sequence comprising a
sequence set forth in any one of SEQ ID NOs: 1-39, and 93-216
operatively linked to a nucleotide sequence encoding a detectable
marker, for example, a fluorescent protein. In some embodiments,
the nucleic acid sequences are at least 50%, 60%, 70%, 80%, 90%,
95%, 98%, or 99% identical to the aforementioned sequences.
[0347] It should be recognized that one or more polynucleotides,
which are the same or different can be introgressed into a plant,
therein providing a means to obtain a genetically modified plant
containing multiple copies of a single transgenic sequence, or
containing two or more different transgenic sequences, either or
both of which can be present in multiple copies. Such transgenic
plants can be produced, for example, by simply selecting plants
having multiple copies of a single type of transgenic sequence; by
cotransfecting plant cells with two or more populations of
different transgenic sequences and identifying those containing the
two or more different transgenic sequences; or by crossbreeding
transgenic plants, each of which contains one or more desired
transgenic sequences, and identifying those progeny having the
desired sequences.
VII. Transgenic Plants, Seeds, and Plant Parts:
[0348] Plants of the present invention are transformed with at
least one heterologous gene encoding an Fm or Fm related gene, or
encoding a sequence designed to increase Fm or Fm related gene
expression, according to any procedure well known or developed in
the art. In some embodiments, the heterologous gene may introduce
Fm or Fm gene expression and protein activity of the expressed
protein. In some embodiments, expression of the heterologous gene
may decrease endogenous Fm or Fm expression. In some embodiments,
the heterologous gene may replace endogenous homologues of Fm or Fm
gene expression. It is contemplated that these heterologous genes,
or nucleic acid sequences of the present invention and of interest,
are utilized to increase the level of the polypeptide encoded by
heterologous genes, or to decrease the level of the protein encoded
by endogenous genes. It is contemplated that these heterologous
genes, or nucleic acid sequences of the present invention and of
interest, are utilized augment and/or increase the level of the
protein encoded by endogenous genes. It is also contemplated that
these heterologous genes, or nucleic acid sequences of the present
invention and of interest, are utilized to provide a polypeptide
encoded by heterologous genes.
[0349] Introduction of the nucleic acid sequence of interest into
the plant cell genome may be achieved by, for example, heterologous
recombination using Agrobacterium-derived sequences and other plant
transformation methods. Transgenic grass plant lines are
contemplated to be developed from transgenic plants by tissue
culture propagation. The presence of nucleic acid sequence of a
heterologous Fm gene and/or an encoded a Fm polypeptide or mutants
or variants thereof may be transferred from a transgenic plant to
related varieties by traditional plant breeding techniques.
Examples of transgenic lines are described herein. Transgenic lines
are contemplated for establishment from transgenic plants by tissue
culture propagation.
[0350] Methods for Agrobacterium transformation of grass plants are
exemplified by those disclosed herein (Example VII) and those used
for Lolium temulentum (Ge et al. (2007) Plant Cell Reports 26 (6):
783-789), creeping bent grass (Fu et al. (2007) Plant Cell Reports
26 (4): 467-477, Yu et al. (2000) Hereditas 133 (3): 229-233);
colonial bent grass (Chai et al. (2004) Plant Cell Tissue And Organ
Culture 77 (2): 165-171, Aswath et al. (2005) Plant Growth
Regulation 47 (2-3): 129-139), tall fescue (Zhao et al. (2007)
Plant Cell Reports 26 (9): 1521-1528; Wang et al. (2005) J. Plant
Physiol. 162 (1): 103-113), orchardgrass (Lee et al. (2006) Plant
Science 171 (3): 408-414), Brachypodium distachyon (Vogel et al.
(2006) Plant Cell Tissue And Organ Culture 84 (2): 199-211),
bromegrass (Bromus inermis) (Nakamura et al. (2006) Plant Cell
Tissue And Organ Culture 84 (3): 293-299), forage and turf grasses
(Wang et al. (2006) In Vitro Cellular & Developmental
Biology--Plant 42 (1): 1-18), Bermuda grass (Hu et al. (2005) Plant
Cell Tissue And Organ Culture 83 (1): 13-19), zoysiagrass (Toyama
et al. (2003) Molecules And Cells 16 (1): 19-27), and for Festuca
arundinacea and Lolium multiflorum (Bettany et al. (2003) Plant
Cell Reports 21 (5): 437-444). Methods for Agrobacterium
transformation of ryegrass are exemplified by Example VII herein,
as well as Wu et al. (2007) Russian Journal Of Plant Physiology 54
(4): 524-529, Bajaj et al. (2006) Plant Cell Reports 25 (7):
651-659, Wu et al. (2005) Plant Science 169 (1): 65-73, and
Vandermaas et al. (1994) Plant Molecular Biology 24 (2): 401-405.
Methods for Agrobacterium transformation of turfgrass are
exemplified by U.S. Pat. No. 7,057,090]
[0351] Transgenic lines over-expressing Fm genes of drought
resistant cultivars may be utilized for evaluation of drought
resistant activity. These transgenic lines are then contemplated
for evaluation of abiotic stress tolerance, such as drought stress
tolerance, and agronomic traits such as phenotype, color, pathogen
resistance and other desired agronomic traits.
[0352] A. Expression Cassettes:
[0353] The methods of the present invention contemplate the use of
at least one heterologous gene encoding a Fm gene and/or Fm related
gene, or encoding a sequence designed to decrease or increase a Fm
genes or Fm related gene expression, as described herein.
Heterologous genes include but are not limited to naturally
occurring coding sequences, as well variants encoding mutants,
variants, truncated proteins, and fusion proteins, as described
above. Heterologous genes may be used alone or in combination with
a selected agronomic trait (such as yield, etc.). Heterologous
genes intended for expression in plants are first assembled in
expression cassettes comprising a promoter. Methods which are well
known to or developed by those skilled in the art may be used to
construct expression vectors containing a heterologous gene and
appropriate transcriptional and translational control elements.
These methods include in vitro recombinant DNA techniques,
synthetic techniques, and in vivo genetic recombination. Exemplary
techniques are widely described in the art (see e.g., Sambrook et
al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring
Harbor Press, Plainview, N.Y., and Ausubel, F. M. et al. (1989)
Current Protocols in Molecular Biology, John Wiley & Sons, New
York, N.Y.; herein incorporated by reference).
[0354] In general, these vectors comprise a nucleic acid sequence
encoding a Fm gene and/or Fm related gene, or encoding a sequence
designed to decrease Fm gene and/or Fm related gene expression, (as
described herein) operably linked to a promoter and other
regulatory sequences (e.g., enhancers, polyadenylation signals,
etc.) required for expression in a plant.
[0355] Promoters include but are not limited to constitutive
promoters, tissue specific promoters, organ specific promoters,
developmentally specific promoters, inducible promoters and stress
response promoters. Examples of promoters include but are not
limited to: rice actinl promoter, maize ubiquitin promoter (for
example, Dalton et al., 1999, Plant Cell Reports, 18(9): 721-726),
fructosyltransferase (LpFT1) promoter (for example, Chalmers, et
al., 2005, Plant Biotechnology Journal 3(5):459, constitutive
promoter 35S of cauliflower mosaic virus; a wound-inducible
promoter from tomato, leucine amino peptidase ("LAP," see, e.g.,
Chao et al. (1999) Plant Physiol 120: 979-992; herein incorporated
by reference); a chemically-inducible promoter from tobacco,
Pathogenesis-Related 1 (PR1) (induced by salicylic acid and BTH
(benzothiadiazole-7-carbothioic acid S-methyl ester)); a tomato
proteinase inhibitor II promoter (PIN2) or LAP promoter (both
inducible with methyl jasmonate); a heat shock promoter (see, e.g.
U.S. Pat. No. 5,187,267; herein incorporated by reference); a
tetracycline-inducible promoter (see, e.g. U.S. Pat. No. 5,057,422;
herein incorporated by reference); and seed-specific promoters,
such as those for seed storage proteins (e.g., phaseolin, napin,
oleosin, and a promoter for soybean beta conglycin (see, e.g.,
Beachy et al. (1985) EMBO J. 4: 3047-3053; herein incorporated by
reference).
[0356] The expression cassettes may further comprise any sequences
required for expression of mRNA. Such sequences include, but are
not limited to transcription terminators, enhancers such as
introns, viral sequences, and sequences intended for the targeting
of the gene product to specific organelles and cell
compartments.
[0357] A variety of transcriptional terminators are available for
use in expression of sequences using the promoters of the present
invention. Transcriptional terminators are responsible for the
termination of transcription beyond the transcript and its correct
polyadenylation. Appropriate transcriptional terminators and those
which are known to function in plants include, but are not limited
to, the CaMV 35S terminator, the tml terminator, the pea rbcS E9
terminator, and the nopaline and octopine synthase terminator (see,
for examples, Odell et al. (1985) Nature 313:810; Rosenberg et al.
(1987) Gene, 56:125; Guerineau et al. (1991) Mol. Gen. Genet.
262:141; Proudfoot (1991) Cell 64:671); Sanfacon et al. (1991)
Genes Dev., 5:141; Mogen et al. (1990) Plant Cell, 2:1261; Munroe
et al. (1990) Gene 91:151; Ballas et al. (1989) Nucleic Acids Res.
17:7891; Joshi et al. (1987) Nucleic Acid Res., 15:9627, all of
which are incorporated herein by reference in their entirety).
[0358] In addition, in some embodiments, constructs for expression
of the gene of interest include one or more of sequences found to
enhance gene expression from within the transcriptional unit. These
sequences can be used in conjunction with the nucleic acid sequence
of interest to increase expression in plants. Various intron
sequences have been shown to enhance expression, particularly in
monocotyledonous cells. For example, the introns of the maize Adhl
gene have been found to significantly enhance the expression of the
wild-type gene under its cognate promoter when introgressed into
maize cells (see, e.g., Callis et al. (1987) Genes Develop. 1:1183;
herein incorporated by reference). Intron sequences have been
routinely incorporated into plant transformation vectors, typically
within the non-translated leader.
[0359] In some embodiments of the present invention, the construct
for expression of the nucleic acid sequence of interest also
includes a regulator such as a nuclear localization signal (see,
e.g., Kalderon et al. (1984) Cell 39:499; Lassner et al. (1991)
Plant Molecular Biology 17:229; all of which are herein
incorporated by reference), a plant translational consensus
sequence (see, e.g., Joshi (1987) Nucleic Acids Research 15:6643;
all of which are herein incorporated by reference), an intron (see,
e.g., Luehrsen and Walbot (1991) Mol. Gen. Genet. 225:81; all of
which are herein incorporated by reference), and the like, operably
linked to the nucleic acid sequence encoding a Fm gene.
[0360] In preparing the construct comprising the nucleic acid
sequence encoding a Fm gene, or encoding a sequence designed to
decrease Fm gene expression, various DNA fragments can be
manipulated, so as to provide for the DNA sequences in the desired
orientation (e.g., sense or antisense) orientation and, as
appropriate, in the desired reading frame. For example, adapters or
linkers can be employed to join the DNA fragments or other
manipulations can be used to provide for convenient restriction
sites, removal of superfluous DNA, removal of restriction sites, or
the like. For this purpose, in vitro mutagenesis, primer repair,
restriction, annealing, resection, ligation, or the like is
preferably employed, where insertions, deletions or substitutions
(e.g., transitions and transversions) are involved.
[0361] Numerous transformation vectors are available for plant
transformation. The selection of a vector for use will depend upon
the preferred transformation technique and the target species for
transformation. For certain target species, different antibiotic or
herbicide selection markers are preferred. Selection markers used
routinely in transformation include the nptII gene which confers
resistance to kanamycin and related antibiotics (see, e.g., Messing
and Vierra, (1982) Gene 19: 259; Bevan et al. (1983) Nature
304:184; all of which are incorporated herein by reference), the
bar gene which confers resistance to the herbicide phosphinothricin
(see, e.g., White et al. (1990) Nucl Acids Res. 18:1062; Spencer et
al. (1990) Theor. Appl. Genet. 79:625; all of which are
incorporated herein by reference), the hph gene which confers
resistance to the antibiotic hygromycin (see, e.g., Blochlinger and
Diggelmann (1984) Mol. Cell. Biol. 4:2929; herein incorporated by
reference), and the dhfr gene, which confers resistance to
methotrexate (see, e.g., Bourouis et al. EMBO J., 2:1099 (1983);
herein incorporated by reference).
[0362] In some preferred embodiments, the Ti (T-DNA) plasmid vector
is adapted for use in an Agrobacterium mediated transfection
process (see e.g., U.S. Pat. Nos. 5,981,839; 6,051,757; 5,981,840;
5,824,877; and 4,940,838; all of which are herein incorporated by
reference in their entirety). Construction of recombinant Ti and Ri
plasmids in general follows methods typically used with the more
common vectors, such as pBR322. Additional use can be made of
accessory genetic elements sometimes found with the native plasmids
and sometimes constructed from foreign sequences. These may include
but are not limited to structural genes for antibiotic resistance
as selection genes.
[0363] There are two systems of recombinant Ti and Ri plasmid
vector systems now in use. The first system is called the
"cointegrate" system. In this system, the shuttle vector containing
the gene of interest is inserted by genetic recombination into a
non-oncogenic Ti plasmid that contains both the cis-acting and
trans-acting elements required for plant transformation as, for
example, in the pMLJ1 shuttle vector and the non-oncogenic Ti
plasmid pGV3850. The use of T-DNA as a flanking region in a
construct for integration into a Ti- or Ri-plasmid has been
described in EPO No. 116,718 and PCT Application Nos. WO 84/02913,
02919 and 0292; all of which are herein incorporated by reference
in their entirety). See, for further examples, Herrera-Estrella
(1983) Nature 303:209-213; Fraley et al. (1983) Proc. Natl. Acad.
Sci, USA 80:4803-4807; Horsch et al. (1984) Science 223:496-498;
and DeBlock et al. (1984) EMBO J. 3:1681-1689, all of which are
herein incorporated by reference).
[0364] The second system is called the "binary" system or "binary
vector" in which two plasmids are used; the gene of interest is
inserted into a shuttle vector containing the cis-acting elements
required for plant transformation. The other necessary functions
are provided in trans by the non-oncogenic Ti plasmid as
exemplified by the pBIN19 shuttle vector and the non-oncogenic Ti
plasmid PAL4404. In some embodiments of the invention, the nucleic
acid sequence of interest is targeted to a particular locus on the
plant genome. Site-directed integration of the nucleic acid
sequence of interest into the plant cell genome may be achieved by,
for example, homologous recombination using Agrobacterium-derived
sequences. Generally, plant cells are incubated with a strain of
Agrobacterium which contains a targeting vector in which sequences
that are homologous to a DNA sequence inside the target locus are
flanked by Agrobacterium transfer-DNA (T-DNA) sequences, as
previously described (see, e.g. U.S. Pat. No. 5,501,967; herein
incorporated by reference). One of skill in the art knows that
homologous recombination may be achieved using targeting vectors
that contain sequences that are homologous to any part of the
targeted plant gene, whether belonging to the regulatory elements
of the gene, or the coding regions of the gene. Homologous
recombination may be achieved at any region of a plant gene so long
as the nucleic acid sequence of regions flanking the site to be
targeted is known.
[0365] Agrobacterium tumefaciens is a common soil bacterium that
causes crown gall disease by transferring some of its DNA to the
plant host. The transferred DNA (T-DNA) is stably integrated into
the plant genome, where its expression leads to the synthesis of
plant hormones and thus to the tumorous growth of the cells. In yet
other embodiments, the nucleic acids such as those disclosed herein
is utilized to construct vectors derived from plant (+) RNA viruses
(e.g., brome mosaic virus, tobacco mosaic virus, alfalfa mosaic
virus, cucumber mosaic virus, tomato mosaic virus, and combinations
and hybrids thereof). Generally, the inserted heterologous
polynucleotide can be expressed from these vectors as a fusion
protein (e.g., coat protein fusion protein) or from its own
subgenomic promoter or other promoter. Methods for the construction
and use of such viruses are described in U.S. Pat. Nos. 5,846,795;
5,500,360; 5,173,410; 5,965,794; 5,977,438; and 5,866,785, all of
which are incorporated herein by reference.
[0366] B. Vectors for Expressing a Fm and/or a Fm Gene
[0367] The nucleic acid sequences of the present invention may be
employed for producing polypeptides by recombinant techniques.
Thus, for example, the Fm nucleic acid sequence may be included in
any one of a variety of expression vectors for expressing a
polypeptide. In some embodiments of the present invention, vectors
include, but are not limited to, chromosomal, nonchromosomal and
synthetic DNA sequences (for example, derivatives of SV40,
bacterial plasmids, phage DNA; baculovirus, yeast plasmids, vectors
derived from combinations of plasmids and phage DNA, and viral DNA
such as vaccinia, adenovirus, fowl pox virus, and pseudorabies). It
is contemplated that any vector may be used as long as it is
replicable and viable in the host plant cell or microbe.
[0368] In particular, some embodiments of the present invention
provide recombinant constructs comprising one or more of the
nucleic sequences as broadly described above (for example, SEQ ID
NOs: 1-39, and 93-216 and sequences that are at least 50%, 60%,
70%, 80%, 90%, 95%, 98%, or 99% identical to the aforementioned
sequences.). In some embodiments of the present invention, the
constructs comprise a vector, such as a plasmid or viral vector,
into which a nucleic acid sequence of the invention has been
inserted, in a forward or reverse orientation. In preferred
embodiments of the present invention, the appropriate nucleic acid
sequence is inserted into the vector using any of a variety of
procedures. In general, the nucleic acid sequence is inserted into
an appropriate restriction endonuclease site(s) by procedures known
in the art.
[0369] Large numbers of suitable vectors are known to those of
skill in the art, and are commercially available. Such vectors
include, but are not limited to, the following vectors: 1)
Bacterial--pQE70, pQE60, pQE-9 (Qiagen), pBS, pD10, phagescript,
psiX174, pbluescript SK, pBSKS, pNH8A, pNH16a, pNH18A, pNH46A
(Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5
(Pharmacia); and 2) Eukaryotic--pWLNEO, pSV2CAT, pOG44, PXT1, pSG
(Stratagene), pSVK3, pBPV, pMSG, and pSVL (Pharmacia). Any other
plasmid or vector may be used as long as they are replicable and
viable in the host. In some preferred embodiments of the present
invention, plant expression vectors comprise an origin of
replication, a suitable promoter and enhancer, and also any
necessary ribosome binding sites, polyadenylation sites, splice
donor and acceptor sites, transcriptional termination sequences,
and 5' flanking nontranscribed sequences. In some embodiments, DNA
sequences derived from the SV40 splice, and polyadenylation sites
may be used to provide the required nontranscribed genetic
elements.
[0370] In some embodiments of the present invention, a heterologous
nucleic acid sequence of interest is introgressed directly into a
plant. One vector useful for direct gene transfer techniques in
combination with selection by the herbicide Basta (or
phosphinothricin) is a modified version of the plasmid pCIB246,
with a CaMV 35S promoter in operational fusion to the E. coli GUS
gerie and the CaMV 35S transcriptional terminator (Intl.
Publication No. WO 93/07278; herein incorporated by reference).
[0371] C. Generating Transgenic Plants: Transformation
Techniques
[0372] Introduction of the nucleic acid sequence of interest into
the plant cell genome may be achieved by, for example, heterologous
recombination using Agrobacterium-derived sequences and other plant
transformation methods. Examples of such transgenic plants are
provided in an over-expressed transgene in ryegrass plants (see,
for example, Hisano, et al., 2004, Plant science, 167, (4):861-868;
herein incoroproated by reference), and ryegrass transformed with
an antisense construct for providing a down-regulated (silenced)
gene, (Bhalla, et al., 1999, PNAS, 96(20):11676-11680; herein
incoroproated by reference). Transgenic grass plant lines are
contemplated to be developed from transgenic plants by tissue
culture propagation. The presence of nucleic acid sequence of a
heterologous Fm gene and/or an encoded a Fm polypeptide or mutants
or variants thereof may be transferred from a transgenic plant to
related varieties by traditional plant breeding techniques.
Examples of transgenic lines are described herein. Transgenic lines
are contemplated for establishment from transgenic plants by tissue
culture propagation.
[0373] Transgenic lines over-expressing Fm genes of drought
resistant cultivars may be utilized for evaluation of drought
resistant activity. These transgenic lines are then contemplated
for evaluation of abiotic stress tolerance, such as drought stress
tolerance, and agronomic traits such as phenotype, color, pathogen
resistance and other desired agronomic traits. A nucleic acid
sequence encoding a Fm gene operatively linked to an appropriate
promoter and inserted into a suitable vector for the particular
transformation technique utilized (e.g., one of the vectors
described herein), the recombinant DNA is introgressed into the
plant cell in a number of art-recognized ways. Those skilled in the
art will appreciate that the choice of method might depend on the
type of plant targeted for transformation. In some embodiments, the
vector is maintained episomally. In some embodiments, the vector is
integrated into the genome. In one embodiment, a method of the
present invention is performed by introducing a polynucleotide
portion of a plant stress-regulated gene into the plant. A
polynucleotide can be introgressed into a cell by a variety of
methods well known to those of ordinary skill in the art. For
example, the polynucleotide can be introgressed into a plant cell
using a direct gene transfer method such as electroporation or
microprojectile mediated transformation, or using Agrobacterium
mediated transformation. Non-limiting examples of methods for the
introduction of polynucleotides into plants are provided in greater
detail herein.
[0374] In addition to direct transformation, in some embodiments,
the vectors comprising a nucleic acid sequence encoding a Fm gene
are transferred using Agrobacterium-mediated transformation (see,
e.g., Hinchee et al. (1988) Biotechnology, 6:915; Ishida et al.
(1996) Nature Biotechnology 14:745, all of which are herein
incorporated by reference). Introduction of the nucleic acid
sequence of interest into the plant cell genome may be achieved by,
for example, heterologous recombination using Agrobacterium-derived
sequences and other plant transformation methods. Transgenic grass
plant lines are contemplated to be developed from transgenic plants
by tissue culture propagation. The presence of nucleic acid
sequence of a heterologous Fm gene and/or an encoded a Fm
polypeptide or mutants or variants thereof may be transferred from
a transgenic plant to related varieties by traditional plant
breeding techniques. Examples of transgenic lines are described
herein. Transgenic lines are contemplated for establishment from
transgenic plants by tissue culture propagation.
[0375] Transgenic lines over-expressing Fm genes of drought
resistant cultivars may be utilized for evaluation of drought
resistant activity. These transgenic lines are then contemplated
for evaluation of abiotic stress tolerance, such as drought stress
tolerance, and agronomic traits such as phenotype, color, pathogen
resistance and other desired agronomic traits. Agrobacterium is a
representative genus of the gram-negative family Rhizobiaceae. Its
species are responsible for plant tumors such as crown gall and
hairy root disease. In the dedifferentiated tissue characteristic
of the tumors, amino acid derivatives known as opines are produced
and catabolized. The bacterial genes responsible for expression of
opines are a convenient source of control elements for chimeric
expression cassettes. Heterologous genetic sequences (e.g., nucleic
acid sequences operatively linked to a promoter of the present
invention) can be introgressed into appropriate plant cells, by
means of the Ti plasmid of Agrobacterium tumefaciens. The Ti
plasmid is transmitted to plant cells on infection by Agrobacterium
and is stably integrated into the plant genome (Schell (1987)
Science, 237: 1176; herein incorporated by reference). Species
which are susceptible infection by Agrobacterium may be transformed
in vitro.
[0376] In particular, examples of methods for transformation
techniques for overexpressing nucleic acids include but are not
limited to providing transgenic forage plants are described in U.S.
Patent Appln. Pub. Nos. 20020019997A1; 20020023279A1; and U.S. Pat.
No. 5,985,666; all of which are herein incorporated by reference;
alfalfa (Galili et al. (2000) Transgenic Res 9, 137-144; Trieu et
al. (2000) Plant Journal 22, 531-541; all of which are herein
incorporated by reference); fescue (Wang et al. (2000) Plant Cell
Rep 20, 213-219; herein incorporated by reference); and an herb
(Niu et al. (2000) Plant Cell Rep 19, 304-310; herein incorporated
by reference).
[0377] In some embodiments, direct transformation into the plastid
genome is used to introduce the vector into the plant cell (See
e.g., U.S. Pat. Nos. 5,451,513; 5,545,817; 5,545,818; PCT
application WO 95/16783, all of which are incorporated herein by
reference). The basic technique for chloroplast transformation
involves introducing regions of cloned plastid DNA flanking a
selectable marker together with the nucleic acid encoding the RNA
sequences of interest into a suitable target tissue (e.g., using
biolistics or protoplast transformation with calcium chloride or
PEG). The 1 to 1.5 kb flanking regions, termed targeting sequences,
facilitate homologous recombination with the plastid genome and
thus allow the replacement or modification of specific regions of
the plastome. Initially, point mutations in the chloroplast 16S
rRNA and rpsl.sup.2 genes conferring resistance to spectinomycin
and/or streptomycin are utilized as selectable markers for
transformation (see, e.g., Svab et al. (1990) PNAS, 87:8526; Staub
and Maliga, (1992) Plant Cell, 4:39; all of which are herein
incorporated by reference). The presence of cloning sites between
these markers allowed creation of a plastid targeting vector
introduction of foreign DNA molecules (see, e.g., Staub and Maliga
(1993) EMBO J., 12:601; herein incorporated by reference).
Substantial increases in transformation frequency are obtained by
replacement of the recessive rRNA or r-protein antibiotic
resistance genes with a dominant selectable marker, the bacterial
aadA gene encoding the spectinomycin-detoxifying enzyme
aminoglycoside-3'-adenyltransferase (Svab and Maliga (1993) PNAS,
90:913; herein incorporated by reference). Other selectable markers
useful for plastid transformation are known in the art and
encompassed within the scope of the present invention. Plants
homoplasmic for plastid genomes containing the two nucleic acid
sequences separated by a promoter of the present invention are
obtained, and are preferentially capable of high expression of the
RNAi encoded by the DNA molecule.
[0378] In some embodiments, vectors useful in the practice of the
present invention are microinjected directly into plant cells by
use of micropipettes to mechanically transfer the recombinant DNA
(see, e.g., Crossway (1985) Mol. Gen. Genet, 202:179; herein
incorporated by reference). In still other embodiments, the vector
is transferred into the plant cell by using polyethylene glycol
(see, e.g., Krens et al. (1982) Nature, 296:72; Crossway et al.
(1986) BioTechniques, 4:320; all of which are herein incorporated
by reference); fusion of protoplasts with other entities, either
minicells, cells, lysosomes or other fusible lipid-surfaced bodies
(see, e.g., Fraley et al. (1982) Proc. Natl. Acad. Sci., USA,
79:1859; herein incorporated by reference); protoplast
transformation (see, e.g., EP 0 292 435; herein incorporated by
reference); direct gene transfer (see, e.g., Paszkowski et al.
(1984) EMBO J., 3:2717); Hayashimoto et al. (1990) Plant Physiol.
93:857; all of which are herein incorporated by reference).
[0379] In still further embodiments, the vector may also be
introgressed into the plant cells by electroporation (see, e.g.,
Fromm, et al. (1985) Pro. Natl Acad. Sci. USA 82:5824; Riggs et al.
(1986) Proc. Natl. Acad. Sci. USA 83:5602; all of which are herein
incorporated by reference). In this technique, plant protoplasts
are electroporated in the presence of plasmids containing the gene
construct. Electrical impulses of high field strength reversibly
permeabilize biomembranes allowing the introduction of the
plasmids. Electroporated plant protoplasts reform the cell wall,
divide, and form plant callus.
[0380] In yet other embodiments, a vector comprising nucleotides of
the present invention is introgressed through ballistic particle
acceleration using devices (e.g., such as those devices, available
from Agracetus, Inc., Madison, Wis. and Dupont, Inc., Wilmington,
Del.); (see, e.g., U.S. Pat. No. 4,945,050; and McCabe et al.
Biotechnology 6:923 (1988); all of which are herein incorporated by
reference). Other examples of transformation techniques of vectors
for introgression of drought resistant genes into plants are See,
for further examples, Sato et al. 2006 Grassland Science, 52(2):95
(ryegrass); Murray et al. Mol Gen Genet. (1992) 233(1-2):1-9
(ryegrass); Klein et al. Proc. Natl. Acad. Sci. USA, 85:4305 (1988)
(maize); Klein et al. Bio/Technology, 6:559 (1988) (maize); Klein
et al. Plant Physiol., 91:4404 (1988) (maize); Fromm et al.
Bio/Technology, 8:833 (1990); and Gordon-Kamm et al. Plant Cell,
2:603 (1990) (maize); Koziel et al. Biotechnology, 11:194 (1993)
(maize); Hill et al. Euphytica, 85:119 (1995) and Koziel et al.
Annals of the New York Academy of Sciences 792:164 (1996);
Shimamoto et al. Nature 338: 274 (1989) (rice); Christou et al.
Biotechnology, 9:957 (1991) (rice); Datta et al. Bio/Technology
8:736 (1990) (rice); European Application EP 0 332 581
(orchardgrass and other Poaceae); Vasil et al. Biotechnology, 11:
1553 (1993) (wheat); Weeks et al. Plant Physiol., 102: 1077 (1993)
(wheat); Wan et al. Plant Physiol. 104: 37 (1994) (barley); Jahne
et al. Theor. Appl. Genet. 89:525 (1994) (barley); Knudsen and
Muller, Planta, 185:330 (1991) (barley); Umbeck et al.
Bio/Technology 5: 263 (1987) (cotton); Casas et al. Proc. Natl.
Acad. Sci. USA 90:11212 (1993) (sorghum); Somers et al.
Bio/Technology 10:1589 (1992) (oat); Torbert et al. Plant Cell
Reports, 14:635 (1995) (pat); Weeks et al. Plant Physiol., 102:1077
(1993) (wheat); Chang et al. WO 94/13822 (wheat) and Nehra et al.
The Plant Journal, 5:285 (1994) (wheat); all of which are herein
incorporated by reference in their entirety.
[0381] In general, advances in understanding the drought tolerance
mechanism were obtained from studies on the mild drought tolerant
model species, Arabidopsis plants. However, those studies were not
sufficient to explain the adaptation of plants, such as Festuca
species, to severe drought stress, such as long-term severe drought
conditions. The inventor's research on the F. mairei plant and Fm
genome uncovered a large number of novel genes associated with
onset of drought stress and adaptation to drought, for example, low
soil moisture growing conditions. The combination of data from
studies on a genetic model F. mairei plant and on diverse plant
species should help us have a better understand of underlying
mechanism of thought tolerance in plant. Existence of variety of
drought responsive genes suggests complex processes of a plants
response to the stress. The genes are involved in thought stress
tolerance and stress responses. Although more work is necessary to
define gene functions and dissect the complex regulation of gene
expression, the genes isolated and characterized to data give us
many intriguing insights into the protective mechanisms that
determine desiccation tolerance.
EXPERIMENTAL
[0382] The following examples are provided in order to demonstrate
and further Illustrate certain preferred embodiments and aspects of
the present invention and are not to be construed as limiting the
scope thereof.
[0383] In the experimental disclosure which follows, the following
abbreviations apply: M (molar); mM (millimolar); .mu.M
(micromolar); nM (nanomolar); mol (moles); mmol (millimole);
.mu.mol (micromole); nmol (nanomole); gm (gram); mg (milligram);
.mu.g (microgram); pg (picogram); L (liter); ml (milliliter); .mu.l
(microliter); cm (centimeter); mm (millimeter); .mu.m (micrometer);
nm (nanometer); .degree. C. (degrees Centigrade or Celsius), d
(day), s (second), v (volt), h and hr (hour), and wk (week).
Example I
Materials and Methods
[0384] A. Experimental Procedures for Demonstrating the Superior
Drought Tolerance of F. mairei Plants Over Other Festuca Plants and
for Demonstrating Hybrid Vigor.
Plant Materials and Drought Treatments:
Twelve Weeks of Drought Treatment:
[0385] A comparison of four Festuca grasses, a selection of Atlas
fescue originally collected from Morocco and three commercial tall
fescue cultivars used as entries for turf and forage grass plants
to compare levels of drought resistance (soft-leaf tall fescue,
Festuca arundinacea Schreb., `Barolex, turf-type tall fescue,
Festuca arundinacea `Falcon II,` and tall fescue, Festuca
arundinacea, cultivar `Kentucky 31`. A single tiller of each entry
was used to vegetatively propagate a mature donor plant in the
greenhouse. From each plant, two vegetative tillers were
transplanted into each of six polyvinyl chloride (PVC) tubes (100
cm deep.times.34 cm diameter). The tubes were lined with a sleeve
of heavy duty plastic to facilitate moving the root system and soil
from the tube at the end of the experiment. Tubes were filled with
the same weight (11.8 kg) of a recommended substrate for athletic
fields consisting of 85% sand and 15% field soil. The transplanted
tillers were established for 15 weeks in the greenhouse during the
fall with natural lighting, regular irrigation, fertilizer and
trimming. Greenhouse temperature was 25.+-.3.degree. C., with an
average 13-hour photoperiod.
[0386] After trimming the plants to leave stubble of 7.5 cm for
equal size plants, a pre-conditioning drought was applied by
withholding water for 2 weeks. Plants were allowed to recover by
irrigation for one week before being trimmed again to 7.5 cm. Then,
three tubes of each entry were randomly allocated to the drought
treatment and the control. Drought stress was gradually imposed by
progressively applying decreasing amounts of water from 200 ml/day
for week one (up to 100% soil capacity in the tube), to 150 ml, 100
ml, and 50 ml/day for weeks 2, 3, and 4, respectively. Water was
not applied during the remainder of the 12-week drought period.
Control plants were irrigated regularly during this period. The PVC
tubes were re-randomized weekly during this drought period to
minimize effects of possible environmental gradients within the
greenhouse.
Soil Water Content (SWC) Measurement:
[0387] Polyvinyl chloride (PVC) tubes were weighed every week at
the same time (1:00 p.m.) to determine gravimetric soil water
content (SWC) from water loss. The mass of soil mixture was
measured for each tube at the beginning of the experiment, which
also ensured the same weight of substrate (11.8 kg) in each tube.
The moisture of the soil mixture was estimated by weighing 10
samples, first fresh and then after oven drying at 80.degree.
Celsius.
Leaf Water Content (LWC) Measurement:
[0388] An upper fully developed leaf of the drought stressed plants
was detached weekly (for long-term experiments, e.g. weeks) for
leaf water content (LWC) measurement or for shorter drought tests
as described herein. Control plants were sampled in weeks 3, 6, and
9 during the drought period or for shorter tests the same day as
the test leaf.
[0389] The fresh weight (FW) (weight of the leaf immediately after
detachment), turgor weight (TW) (weight of the leaf after soaking
in milliQ water for 24 hr at room temperature), and dry weight (DW)
(weight of the leaf after oven drying at 80.degree. C. for 24 hr)
of the leaf were measured. Relative leaf water content (LWC) was
calculated according to Slavik (1974, Direct methods of water
content determination. p. 121-156, In B. Slavik (ed.) Methods of
studying plant water relations. Springer-Verlag, Berlin; herein
incorporated by reference) and White et al., (1992, Crop Sci. 32:
25 1-2563; herein incorporated by reference) as LWC
(%)=(FW-DW)/(TW-DW).times.100.
Leaf Water Potential (.PSI.w) Measurement:
[0390] Plants were covered by a black plastic sheet in the evening
for one night to imitate a pre-dawn condition of closed stomata and
low respiration. The following morning, duplicate fully-emerged,
undamaged leaf blades in each tube were removed and immediately
subjected to leaf water potential (.PSI.w) measured immediately by
using a pressure chamber (Soil Moisture Equipment Corp., Santa
Barbara, Calif.). Measurements were conducted at 22-25.degree. C.
within 2 hours in the greenhouse. Data were eliminated when the
.PSI.w of the control was greater than -0.6 MPa.
Leaf Elongation (LE) Measurement:
[0391] After 15 weeks establishment, three tillers in each tube
were randomly chosen and labeled with wires of different color.
Length of the top two emerging leaves on each tiller were measured
from the tip of each lamina to the ligule of the next oldest leaf
(Norris and Thomas, 1982, Wales J. Agric. Sci. 99:547-553; herein
incorporated by reference) every week until leaf growth of
drought-stressed plant ceased.
Root Length (RL) and biomass (RM) Measurement:
[0392] At the end of the experiment, the heavy-duty plastic sleeve
containing the soil and root system was slid out of each tube. The
soil substrate was gently washed from the root system by flowing
water. Length of the root system (RL) was measured using a ruler.
Root biomass (RM) was weighed after blotting with a paper towel and
air-drying at room temperature for about 6 hr to remove surface
moisture.
Soil Water Content (SWC) Measurement:
[0393] The PVC tubes were weighed every week at the same time (1:00
p.m.) to determine gravimetric soil water content (SWC) from water
loss. The mass of soil mixture was measured for each tube at the
beginning of the experiment, which also ensured the same weight of
substrate (11.8 kg) in each tube. The moisture of the soil mixture
was estimated by weighing 10 samples, first fresh and then after
oven drying at 80.degree. Celsius.
Statistical Analyses:
[0394] The data of LE, LWC, SWC, and .PSI.w, were subjected to
analysis of variance (ANOVA), using repeated measurements in time
by SAS program (SAS Institute Inc. 2003). Comparisons were made
within the four entries by one-way ANOVA and between drought and
control treatments by the student-I test at each specified week.
Mean separations were performed by a least significant difference
(LSD) procedure where the F-value was significant at the 0.05
probability level. Data for RL and RM were subjected to one-way
ANOVA analysis to compare within the four entries and between
stressed and control plants. Relationships between parameters were
fitted to appropriate nonlinear regression models using Microsoft
Office Excel (Microsoft Co., 2002).
[0395] B. Experimental Procedures for Identification of F. mairei
(Fm) Germplasm Associated with Xerophytic Adaptation
[0396] See also, Wang, et al., 2005, Molecular Biotechnology,
29(3):211-220; herein incorporated by reference in its
entirety.
Plant Materials and Drought Treatment for Identifying Preferred Fm
Germplasm:
Nine Days of Drought Treatment:
[0397] Ten plants of a F. mairei clone (Fm1, originally collected
from Morocco) were transplanted into polyethylene pots (20.32 cm
diameter at the top, 15.24 cm diameter at the bottom, and 35.56 cm
height) filled with 90% sand and 10% silt and clay. The plants were
established (grown) for three months with regular irrigation and
fertilization in a uniform greenhouse environment condition. After
establishment (three months), five F. mairei plants were deprived
of water until they were severely stressed (dehydrated) and passed
the permanent wilting point (nine days).
[0398] The five control plants, referred to as a "treatment
control," were watered daily (irrigated) throughout the drought
stress period of the five drought stressed plants. During the
drought stress treatment, leaf samples from both the stressed and
the control F. mairei plants were collected at noon of each day to
eliminate the possible gene expression variation from occurring
during the day. Leaf samples were immediately frozen in liquid
nitrogen and stored in -80.degree. C. for subsequent RNA
isolation.
Relative Leaf Water Content Measurements:
[0399] As described above, a fully extended leaf of F. mairei plant
was detached during the drought stress treatment. Leaf water
content (LWC) was measured daily (see, FIG. 2).
RNA Isolation and cDNA Synthesis:
[0400] Total RNA was isolated with plant RNA purification reagent
(Invitrogen Life Technologies, Carlsbad, Calif.) and then
quantified using a spectrophotometer at a wavelength 260 nm.
Quality of the RNA was checked by running 2 .mu.g of the total RNA
on 1.2% agarose gel with 2.5% formaldehyde in 40 mM MOPS
(3-(N-morpholino) propane sulfonic acid) running buffer for 2.5
hours. Poly (A)+ RNA was isolated from the total RNA by using
PolyATract mRNA isolation systems III (Promega, Madison, Wis.).
Double-stranded (ds) cDNA was synthesized from Poly(A)+ RNA using
the Universal RiboClone cDNA Synthesis System (Promega) and
purified with an equal volume of Tris-EDTA (TE, 10 mM Tris-Cl, pH
7.5 1 mM EDTA):saturated phenol: chloroform: isoamyl alcohol
(25:24:1). The ds cDNA was quantified using Hoechst 33258
(bisbenzimide) dye on DyNA quant 200 fluorometer (Hoefer Pharmacia
Biotech, Inc., San Francisco, Calif.).
cDNA-AFLP Analysis:
[0401] The cDNA-AFLP procedure was conducted as described by Bachem
et al. (1998, Plant Mol. Biol. Rep. 16, 157-173; herein
incorporated by reference) with some modifications. Briefly, AFLP
is a method for genotyping individuals for a large number of loci
using a minimal number of PCR reactions. This method is based on
Vos et al. 1995 (Nucleic Acids Research 23: 4407-14; herein
incorporated by reference). In brief; 1. DNA is cut with
restriction enzymes and then linkers are ligated onto the cut ends.
Typically this involves a combination of two restriction enzymes: a
4 base cutter (such as MseI) and a 6 base cutter (such as EcoRI);
2. Pre-selective PCR is performed using primers that match the
linkers. These primers have a two base overhang. Selective PCR is
performed using primers with three base overhangs. For any given
pre-selective amplification, there were 16 possible selective
primer combinations. The EcoRI primer is labeled so that fragments
that contain an EcoRI site are exclusively detected. PCR products
are then analyzed by gel or capillary electrophoresis. Fragments
are combined and then analyzed for fingerprint similarity with
other samples.
[0402] Specifically, 30 nanograms of cDNA were digested with 5 U
NspI at 37.degree. C. for 2.5 hours, and then immediately digested
with 5 U of TaqI at 65.degree. C. for 2.5 hours followed by heat
inactivation at 80.degree. C. for 20 minutes. The two steps of
digestion were conducted in NEbuffer 2 (New England Biolabs,
Beverly, Mass.) in a total volume of 30 .mu.l. The digestion mix
(20 .mu.l) was ligated to 0.5 mM NspI adapter and 2.5 mM TaqI
adapter using 1 U of T4 ligase supplemented with T4 DNA ligase
buffer (Promega). Oligonucleotide (adapters, linkers, and primers)
used during the development of the present inventions, were
synthesized by MWG Biotech, Inc. (Charlotte, N.C.) (see, Tables 2
and 3 for sequences).
[0403] A PCR reaction solution (20 .mu.l) for pre-amplification
contained 1 .mu.l digestion mix, 0.5 .mu.M of each primer, 0.3 mM
dNTP mix, 1.5 mM Mg.sup.2+, and 0.5 U Taq polymerase (Promega). The
PCR reaction was conducted on a PTC-225 machine at: 72.degree. C.,
2 min; 94.degree. C., 1 min; 15 cycles of 94.degree. C., 30 sec;
56.degree. C., 30 sec; and 72.degree. C., 1 min; then followed by
10 min at 72.degree. C. for a final extension. For selective
amplification, the PCR solution included 1 .mu.l of 5.times.
diluted pre-amplification product, 0.4 .mu.M of each selective
primer, 1.5 mM Mg2+, 0.3 mM dNTP mix, and 0.4 U Taq polymerase in
15 .mu.l total reaction volume. The PCR reaction was performed
following the program: 10 cycles: 94.degree. C., 30 s; 65.degree.
C. (-0.7.degree. C./cycle), 30 sec; 72.degree. C., 1 min and 25
cycles: 94.degree. C., 30 sec; 56.degree. C., 30 sec; 72.degree.
C., 1 min; followed by a final extension step of 10 min at
72.degree. Celsius.
[0404] The selective PCR product (15 .mu.l) was denatured at
96.degree. C. for 6 min after adding 9 .mu.l of 98% formamide
loading buffer. The denatured PCR product (6 .mu.l) was loaded into
a 5% denatured polyacrylamide sequencing gel with 45.4% urea for
fractionation by electrophoresis at 90 W for 2.5 hours. The
fractionated fragments on the gel were then detected by using the
Silver Sequence DNA Sequencing System (Promega). The gel on the
back plate was allowed to dry overnight at room temperature for
scoring on a light box. For recovery of TDFs from the
polyacrylamide gel, silver staining had advantages over radioactive
fingerprints by being directly visualized and excised from the
gel.
TABLE-US-00001 TABLE 1A Sequences of the linkers and primers used
for cDNA-AFLP synthesized by MWG Biotech Inc. Restriction Lab SEQ
ID Enzyme Primers linkers No. No:XX Sequences (5'-3') NspI linker 1
217 GTAGACTGCGTTCCCATG NspI linker 2 218 GGAACGCAGTCTACGAG NspI
pre- 219 GTAGACTGCGTTCCCATG amplification primer NspI selective N1
220 GTAGACTGCGTTCCCATGTA amplification primer NspI selective N2 221
GTAGACTGCGTTCCCATGTT amplification primer NspI selective N3 222
GTAGACTGCGTTCCCATGTC amplification primer NspI selective N4 223
GTAGACTGCGTTCCCATGTG amplification primer NspI selective N5 224
GTAGACTGCGTTCCCATGCA amplification primer NspI selective N6 225
GTAGACTGCGTTCCCATGCT amplification primer NspI selective N7 226
GTAGACTGCGTTCCCATGCC amplification primer NspI selective N8 227
GTAGACTGCGTTCCCATGCG amplification primer TaqI linker 1 228
AAGTCCTGAGTAGCAC TaqI linker 2 229 CGTTCAGGACTCATC TaqI pre- 230
CACGATGAGTCCTGAACG amplification primer TaqI selective T1 231
CACGATGAGTCCTGAACGAAA amplification primer TaqI selective T2 232
CACGATGAGTCCTGAACGAAT amplification primer TaqI selective T3 233
CACGATGAGTCCTGAACGAAC amplification primer TaqI selective T4 234
CACGATGAGTCCTGAACGAAG amplification primer TaqI selective T5 235
CACGATGAGTCCTGAACGATA amplification primer TaqI selective T6 236
CACGATGAGTCCTGAACGATT amplification primer TaqI selective T7 237
CACGATGAGTCCTGAACGATC amplification primer TaqI selective T8 238
CACGATGAGTCCTGAACGATG amplification primer TaqI selective T9 239
CACGATGAGTCCTGAACGACA amplification primer TaqI selective T10 240
CACGATGAGTCCTGAACGACT amplification primer TaqI selective T11 241
CACGATGAGTCCTGAACGACC amplification primer TaqI selective T12 242
CACGATGAGTCCTGAACGACG amplification primer TaqI selective T13 243
CACGATGAGTCCTGAACGAGA amplification primer TaqI selective T14 244
CACGATGAGTCCTGAACGAGT amplification primer TaqI selective T15 245
CACGATGAGTCCTGAACGAGC amplification primer TaqI selective T16 246
CACGATGAGTCCTGAACGAGG amplification primer
TABLE-US-00002 TABLE 1B Sequences of the linkers and primers used
for cDNA-AFLP synthesized by MWG Biotech., Inc. SEQ ID Restriction
Primers/ Lab No: Enzyme linkers No. XX Sequence (5'-3') EcoRI,
linker 247 CTCGTAGACTGGGTACC EcoRI linker 2 248 AATTGGTACGCAGTCTAC
EcoRI pre- 249 CACTGCGTACCAATTC amplification primer EcoRI
selective E1 250 CACTGCGTACCAATTCAA amplification primer I EcoRI
selective E2 251 CACTGCGTACCAATTCAT amplification primer 2 EcoRI
selective E3 252 CACTGCGTACCAATTCAC amplification primer 3 EcoRI
selective E4 253 CACTGCGTACCAATTCAG amplification primer 4 EcoRI
selective E5 254 CACTGCGTACCAATTCTA amplification primer S EcoRI
selective E6 255 CACTGCGTACCAATTCTT amplification primer 6 EcoRI
selective E7 256 CACTGCGTACCAATTCTC amplification primer 7 EcoRI
selective E8 257 CACTGCGTACCAATTCTG amplification primer 8 EcoRI
selective E9 258 CACTGCGTACCAATTCCA amplification primer 9 EcoRI
selective E10 259 CACTGCGTACCAATTCCT amplification primer 10 EcoRI
selective E11 260 CACTGCGTACCAATTCCC amplification primer 11 EcoRI
selective E12 261 CACTGCGTACCAATTCCG amplification primer 12 EcoRI
selective E13 262 CACTGCGTACCAATTCGA amplification primer 13 EcoRI
selective E14 263 CACTGCGTACCAATTCGT amplification primer 14 EcoRI
selective E15 264 CACTGCGTACCAATTCGC amplification primer 15 EcoRI
selective E16 265 CACTGCGTACCAATTCGG amplification primer 16
Identification of Differentially Expressed Fragments (DEFs) and
Fragment Recovery from Polyacrylamide Gel:
[0405] For each primer combination, the final PCR products from a
series of days of drought stress were loaded in order into lanes
next to each other in the sequencing gel for comparison of band
density for bands of the same size. When the density of the bands
showed an increase from lane to lane gradually across the time
points, the bands were identified as up-regulated differentially
expressed fragments. When the band density showed a gradual
decrease over time, the bands were identified as down-regulated
DEFs (for example, see, Bachem et al., 1996, Plant J. 9:745-753;
herein incorporated by reference). When a band appeared at a
specific time point, those bands were identified as a transient
expressed DEF. A few bands were also identified as an up-then-down
regulated DEF, which meant the density of the bands increased at
first several lanes and then decreased at the last several
lanes.
[0406] The four types of DEFs were then excised from a
polyacrylamide gel with a sterile surgical blade. DNA was eluted by
soaking the excised gel in 50 .mu.l water for 12 h and was then
used as the template to re-amplify the DNA fragment using the same
PCR condition as used for selective amplification. The re-amplified
product was run on a 1% agarose gel in 1.times.TBE buffer for
confirmation of the target fragment and separation from possible
DNA contamination. DNA fragments of the target size were purified
from the agarose gel with a QIAquick gel extraction kit (QIAGEN,
Inc., Valencia, Calif.) and eluted in 50 .mu.l sterile water.
Macroarray Hybridization and Data Analysis:
[0407] Double stranded cDNA samples from control and stressed
plants at different time points were labeled with DIG-dUTP by using
a PCR DIG probe synthesis kit (Roche Applied Science, Penzberg,
Germany). The labeled product was purified with a high pure PCR
product purification kit (Roche).
[0408] Ten microliters of both the recovered DEF DNA samples and
control samples were denatured with 10 .mu.l denature solution (0.4
N sodium hydroxide, 0.01 M EDTA, pH=8.0) at 37.degree. C. for 15
minutes and then neutralized with 10 .mu.l 2 M ammonium acetate
(pH=7.0).
[0409] Controls included a negative control, which contained
sterile water but no DNA, and a housekeeping control, which
contained only DNA fragments with the same expression level
(constitutively expressed) throughout the control and the different
days of the stressed plant. Denatured solutions of the DEF and
controls, described above, were spotted onto a nylon membrane
(115.times.76 mm) (Nalge Nunc International, Naperville, Ill.) with
two replications using the Beckman BioMek.RTM. 2000 laboratory
automation workstation (see, example method, Dilks et al. 2003; J
Neurosci Methods. 123(1):47-54; herein incorporated by
reference).
[0410] The controls were spotted in different sections of the
membrane to compensate for variable background levels. Identical
nylon arrays were prepared serially and then subjected to separate
hybridization with labeled ds cDNA probes, including control and
stressed probes. The hybridization and washing were performed by
using DIG high prime DNA labeling and detection starter kit II
(Roche). The luminescent signal on the membrane was exposed to
Lumi-Film Chemiluminescent detection film (Roche).
[0411] The array image on the film was scanned and saved as an
individual ".TIFF" file and analyzed with BIORAD.RTM. Quantity One
Software 4.2.3 (Bio-Rad Laboratories, Hercules, Calif.). For each
array image, the spots were delimitated with the same size circles,
which could include 100% of the pixels in the spot. The average
volume (=pixel intensity.times.area of the circled spot) of
negative control spots on the array image was subtracted from the
volume of each of the other spots to eliminate background
effect.
[0412] The average volume of housekeeping control spots on the
array image was used to normalize the spots of unidentified DEFs
between array images. The ratio of the average volume of
housekeeping spots between images was applied as a scaling factor
for the volume of unknown DEF spots, which were compared to its
counterpart between membranes to confirm the differential
expression pattern.
DNA Fragment Sequencing and Sequence Analysis:
[0413] The DEFs were recovered from the polyacrylamide gel whose
differential expression patterns were confirmed with macroarray
analysis, cloned into pGEM-T easy vector (Promega), and transformed
into JM109 component cells (Promega) by heat shock. Plasmid DNA was
extracted from successful transformants using the Wizard plus SV
minipreps DNA purification system (Promega), and plasmid inserts
were sequenced using an ABI PRISM 3100 genetic analyzer (Applied
Biosystems, Foster City, Calif.) at The Genomics Technology Support
Facility (Michigan State University).
[0414] The sequences of the DEFs were searched against the AGI
(Arabidopsis Genome Initiative) protein database using BLASTX
(website address at http://www.arabidopsis.org/Blast/). Additional
analysis using BLASTX against the GenBank plant protein database
and TBLASTX against the GenBank plant dbEST were performed for DEFs
with zero matches or low similarity (E value greater than 1E-6) in
AGI protein database. A tool of the "Blast 2 sequences", which can
be found at: http://www.ncbi.nlm.nih.gov/blast/bl2seq/wblast2.cgi,
was used for sequence comparisons, for example, see, Table 3
below.
[0415] C. Breeding Methods for Providing Plants of the Present
Inventions.
[0416] 1. Plants Used in Plant Breeding Methods:
[0417] A Fm plant (Fm1) was chosen as a founder plant from a Fm
population collected in Morocco. This Fm population was adapted to
the hot and dry summers of Northwest Africa (Borill et al. 1971,
Cytologia 36:1-14; herein incorporated by reference). Another Fm
plant line, Fm2, was obtained from a plant introduction, PI 283313,
United States Department of Agriculture (USDA) germplasm database.
Two Lp plant lines from elite turfgrass cultivars `Citation II`
(Lp1) and `Calypso` (Lp2), respectively, were also used as founder
plants. The initial parental crosses were made by Chen (1996, Ph.D
dissertation, Univ. of Missouri-Columbia, Columbia, Mo.; herein
incorporated by reference) and the scheme for producing initial
backcross derivatives was presented by Chen and Sleper (1999, Crop
Sci. 39:1676-16793; herein incorporated by reference) (see, for
example, FIG. 10). Plants used during the development of the
present inventions, included, but were not limited to, parental
plants (Fm1, Lp1 and Lp2), two F.sub.1 hybrids: a 4.times.F.sub.1
(Fm1.times.Lp2) and a 3.times.F.sub.1 (Lp2.times.Fm2), 13 backcross
plants, and an amphidiploid (2n 6x=42) that was obtained from a
triploid F.sub.1 hybrid of Fm1.times.Lp1 by Chen (1996, Ph.D
dissertation, University of Missouri-Columbia; herein incorporated
by reference). Fm1 and Fm1 derived plants were used in developing
the majority of plants of the present inventions. See, exemplary
crossing schematic, FIG. 10.
[0418] 2. Identifying Intergeneric Hybridization of Festuca
Germplasm in Lolium Plants.
DNA Isolation:
[0419] Total plant genomic DNA samples were extracted from young
growing leaves. Plant cells were lysed using the extraction buffer
(0.1M Tris-HCl, 0.05M EDTA-Na, 0.25M NaCl, pH=8.0 and 0.04M dodecyl
sulfate (SDS)). Potassium acetate (5M) was used for
deproteinization and recovery of DNA. Nucleic acid was precipitated
by isopropanol followed by RNase treatment to degrade the RNA. The
DNA concentration was measured by spectrophotometer readings at 260
nm and the purity was determined by the ratio of the absorptions at
260 nm and 280 nm. DNA quality was checked by loading 100 ng DNA in
a 1% agarose gel followed by electrophoresis at 72 V for 2
hours.
RAPD Screening Protocol:
[0420] DNA samples from plants, in particular progeny plants, were
used as templates for RAPD analyses with these 41 polymorphic
primers. Forty-one decamer RAPD oligonucleotides (see, Table 7)
were synthesized using C and Y kits (Operon Technologies Inc.,
Alameda, Calif., and used in screening and detecting maximum
polymorphism between the Fm and Lp parents and a F.sub.1 hybrid.
(See, for examples, Charmet et al. 1997, Theor. Appl. Genet.
94:1038-1046; Siffelova et al. 1997, Biologia Plantarum 40:183-192;
all of which are herein incorporated by reference). A 25 .mu.l RAPD
reaction mixture contained 10 mM Tris-HCl (pH=8.3), 4 mM
MgCl.sub.2, 0.24 mM of each dNTP, 1.2 .mu.M of primers, 30 ng of
template DNA, and 1 U Taq DNA polymerase (Promega, Madison, Wis.).
Amplification conditions were as follows: 3 pre-amplification
cycles (94.degree. C. for 1 mm, 35.degree. C. for 1 mm and
72.degree. C. for 2 mm). After initiation of the reaction, 35
amplification cycles were conducted (94.degree. C. for 20 s,
40.degree. C. for 20 s, and 72.degree. C. for 2 mm). The last cycle
was followed by 5 mm at 72.degree. C. to ensure that primer
extension reactions proceeded to completion. RAPD profiles were
generated in 2% agarose gels with 0.003% ethidium bromide subjected
to electrophoresis at 72 V for 3.5 hours. A 1 Kb ladder was used to
mark the size of the fragments. RAPD images were obtained through
an Eagle Eye II still video system V3.2 (Stratagene, La Jolla,
Calif.).
SSR Screening Protocol:
[0421] Seventy-six tall fescue EST-SSR primer pairs (NFFA series)
developed at the Samuel Roberts Noble Foundation (Saha et al. 2004,
Theor. Appl. Genet. 109:783-791; herein incorporated by reference),
and 32 Lp SSR primer pairs developed from ryegrass (Kubik et al.
2001, Crop Sci. 45:1565-1571; Jones et al. 2001, Theor. Appl.
Genet. 102:405-415; all of which are herein incorporated by
reference) were tested on the Fm and Lp parents, then test plant
materials, for example, hybrid progeny. The primer combinations
that produced polymorphic bands between parents were utilized to
test plant materials.
[0422] An ethidium bromide detection protocol was used for ryegrass
and 19 tall fescue EST-SSR primer pairs and the silver staining
protocol was used for screening of the remaining primer pairs. In
the ethidium bromide detection protocol, 10 .mu.l PCR reaction
mixture contained 10 mM Tris-HCl (pH=8.3), 3 mM MgCl.sub.2, 0.25 mM
of each dNTP, 0.2 .mu.M of forward and reverse primers, 10 ng of
template DNA, and 1 U Taq polymerase (Gibco Invitrogene, Grand
Island, N.Y.). PCR amplification was conducted in a PTC-100
programmable thermal controller (MJ Research, Waltham, Mass.).
Amplification conditions were as follows: initial denaturation at
95.degree. C. for 5 min., 40 amplification cycles [95.degree. C.
for 50 s, 42-approximately 60.degree. C. (the optimum annealing
temperature for each primer pair) for 50 s, and 72.degree. C. for
90 s], and the final extension of the reaction at 72.degree. C. for
10 min. SSR profiles were generated by running PCR products in a 6%
non-denaturing polyacrylamide gel for 2.5 h at 350V. TBE buffer
with 0.002% ethidium bromide filled in the positive node tank was
prerun one hour for visualizing bands under UV light.
[0423] In a silver staining protocol, 20 ng of DNA was used as a
template for each PCR reaction. Ten ul PCR reactions consisted of
one U of AmpliTaq Gold.RTM. with GeneAmp PCR buffer II (Applied
Biosystems/Roche, Branchburg, N.J.), 3 mM MgCl.sub.2, 0.2 mM of
dNTPs, and 0.2 .mu.M of each primer. PCR amplification conditions
were same as in ethidium bromide detection protocol. PCR products
were resolved on 6% polyacrylamide denaturing gels (Gel Mix 6,
Invitrogen Life Technologies). Gels were silver stained using
Silver Sequence Kit (Promega, Madison, Wis.) for SSR band
detection.
Data Analysis:
[0424] Intense (dark) and repeatable bands in RAPD profiles were
scored as 0 and 1 for absence and presence, respectively. In SSR
profiles, the intense bands within the expected size range were
scored as 0 and 1 for absence and presence, respectively. The Fm
genome introgression levels in the progeny from SSR data equaled
the number of loci showing Fm alleles in the backcross individual
divided by total number of polymorphic SSR loci. Similarly, the Fm
genome introgression levels in the progeny from RAPD data were
calculated as follows: the number of Fm-specific markers present in
the backcross individual/total number of the Fm-specific markers.
Parental Fm/Lp genome specific band ratios (Fm/Lp genome ratio) of
the Fm-Lp hybrids and backcross progeny were calculated as the
ratio of the percentage of Fm-specific-bands to that of Lp-specific
bands with an assumption that all markers were randomly dispersed
in the whole genome. The correlation analysis of genome
introgression levels between SSR and RAPD data was conducted with
SAS system V8 (SAS Institute, Cary, N.C.). A SAS Proc Corr
procedure was run to obtain the correlation coefficient and the P
value. Dice coefficient (Dice et al., 1945 Ecology 26:297-302;
herein incorporated by reference) similarity matrices for both SSR
and RAPD data were calculated by running similarity for the
qualitative data module in numerical taxonomy and multivariate
analysis system (NTSYSpc version 2.1, Exeter software, Setauket,
New York; herein incorporated by reference).
[0425] D. Evaluating Progeny Plants and Identification of Superior
Drought Resistant Hybrid Plants.
Plants:
[0426] Nineteen genotypes of plants were evaluated that included
Atlas Fescue plants originally collected from Morocco; two
perennial ryegrass cultivars `Citation II` (Lp1) and `Calypso`
(Lp2); two F.sub.1 hybrids from crosses of Atlas fescue and
perennial ryegrass; 12 second-generation backcross progeny; an
amphidiploid generated from a triploid F.sub.1 hybrid by colchicine
treatment; and a tall fescue cultivar, Kentucky 31, which was used
as a drought tolerant control plant. The plants were bred using
methods described above.
[0427] A single tiller of each plant was propagated vegetatively in
the greenhouse to provide experimental plant material. From each
plant genotype, two tillers were transplanted into each of six PVC
tubes (100 cm deep.times.34 cm in diameter) that was filled with
11.8 kg of a mixed soil substrate, 85% sand and 15% field soil of
the type recommended for use in athletic fields. Prior to planting,
a heavy duty plastic sleeve was placed inside each tube, between
the inner surface and the substrate, to facilitate removing the
root system from the tube at the end of the experiment.
Transplanted tillers were established by growing for 15 weeks in
the greenhouse. Grass plants were clipped to leave a 7.5 cm stubble
every week and irrigated daily. The greenhouse temperature was
25.+-.3.degree. C., with an average photoperiod of 13 hours.
[0428] A pre-conditioning drought was applied by withholding water
for 2 weeks. Plants were then irrigated daily for one week and then
trimmed to the same height (around 8 cm). Three tubes of each
genotype were randomly allocated to the drought treatment; the
other three were allocated to the irrigated control.
[0429] Drought stress (treatment) was imposed by withholding water
progressively from plants in the drought treatment group by
supplying 200 ml (up to 100% soil capacity in the tube), 150 ml,
100 ml, and 50 ml water weekly in the first four weeks respectively
and then stopping irrigation completely during the remaining
12-week drought period. The plants in the control treatment were
irrigated regularly during this period. The PVC tubes were
re-randomized weekly during this drought period to minimize effects
of possible environmental gradients within the greenhouse
[0430] Measurements of physiological parameters are described
previously (see, Wang et al., (2003) Crop Sci. 43:2154-2161; herein
incorporated in its entirety by reference).
Eigenvector Diagrams Generated by Principle Component Analysis
(PCA):
[0431] Principle component analysis (PCA) was conducted by using
the numerical taxonomy and multivariate analysis system (NTSYSpc
version 2.1, Exeter Software, Setauket, N.Y.). A correlation matrix
was calculated from a standardized data matrix that included 14 wks
of leaf elongation, 12 wks of leaf water content, and 14 wks of
leaf water potential.
[0432] Eigenvalues and eigenvectors (the principal component axes)
of the matrix were computed using EIGEN module. Eigenvectors were
plotted using the Matrix plot module. The PROJ module was used to
project the genotypes onto the principal component axes and
displayed by the Mod3D plot module. Analysis of variance (ANOVA)
was conducted by Proc mixed model in the SAS program (SAS
Institute, Inc., 2003). Relationships between parameters were
fitted to appropriate nonlinear regression models using Microsoft
Office Excel (Microsoft Co., 2002).
Example II
Festuca mairei Plants Show Superior Drought Resistance Over Plants
from Other Festuca Species in Response to Drought Stress
Treatment
[0433] Turfgrass plant cultivars vary in drought resistance (White
et al., 1992, Crop Sci. 32: 25 1-256; Carrow, 1996, Crop Sci. 36:
687-694; all of which are herein incorporated by reference). Of the
grass plants, tall fescue is recognized for its exceptional drought
tolerance (Norris and Thomas, 1982, J. Agric. Sci. Camb. 98:
623-628; Fry and Butler, 1989, Crop Sci. 29:1536-1541; all of which
are herein incorporated by reference). Tall fescue originated from
North Africa and is now a popular turfgrass and forage grass plant
species that grows in cool and transition zone regions. In
particular, the tall fescue cultivars of F. arundinacea Schreb.
named `Kentucky 31` and `Falcon II` were previously identified as
having good drought tolerance (Huang and Gao, 1999, HortScience 34:
897-901; Huang, 2001, HortScience 36:148-152; all of which are
herein incorporated by reference). `Barolex` is a new tall fescue
forage-type cultivar and its comparative level of drought tolerance
was unknown. Atlas fescue species (Festuca mairei) was originally
found restricted to the Atlas Mountain ranges of northwest Africa.
There were no reports of relative drought tolerance capabilities of
Atlas fescue, although it is known for it's xerophytic adaptation
to survive long, dry summers in its Mediterranean climate (Marlatt
et al., 1997, Investigations on xerophytic Festuca species from
Morocco and their associated endophytes, In: Bacon, G. W.; Hill, N.
S. ed. Neotyphodium/grass interactions, New York, Plenum Pres, p.
73-75; herein incorporated by reference).
[0434] Morphological and physiological drought responses of these
Fescue plants were compared, in particular leaf elongation, leaf
water content, leaf water potential, root biomass and root length,
for determining the relative capability of Fescue plants to provide
germplasm associated with drought resistance for providing drought
tolerance to plants in plant breeding programs.
[0435] The inventor shows herein that when the drought resistance
of Festuca mairei (Fm) plants was compared to other species of
Festuca plants, the Fm plants retained normal levels of leaf water
content while the other species of Festuca rapidly dehydrated over
the 12 week drought period. These comparisons were made on plants
grown under greenhouse conditions and deprived of irrigation water.
Thus unlike the large percentage of leaf water loss in Falcon II
and Barolex under drought conditions, the Festuca mairei plants
retained leaf water content and remained green. See, for example,
FIG. 1. The results for the other parameters that demonstrate
higher levels of drought resistance in Atlas plants are described
below.
Soil Water Content and Leaf Water Potential:
[0436] At full water capacity, SWC was 9.33%, after which it
declined significantly (P<0.001) starting at week 2 of the
drought treatment (FIG. 1). The rate of soil water depletion was
similar among the grasses except for that of Atlas fescue, which
was slower. Specifically, SWC of Atlas fescue was significantly
higher than the other entries from week 4 to week 8, indicating
that it extracted less soil water.
[0437] The imposed drought stress had a significant effect on
.PSI.w, an indicator of plant stress, of the grasses studied. In
irrigated plants, .PSI.w was similar (P=0.086) among the grasses,
and remained relatively high across the 12-week period. In
contrast, .PSI.w of stressed plants showed significant differences
from the irrigated ones after 4 (Falcon II), (Kentucky 31), 6
(Barolex), and 8 (Atlas fescue) weeks. Further, .PSI.w of stressed
plants decreased differently among the four grasses. Atlas fescue
maintained .PSI.w at the level of control plants longer into the
drought period than did the three cultivars. Variation of .PSI.w
was highly dependent on SWC with a similar pattern for the four
grasses, i.e., .PSI.w gradually decreased but remained below -1 Mpa
as SWC decreased from 9.33 to about 2.8%. The results reflected
that soil water was readily available and kept sufficient for the
plants in the SWC range from 9.33 to 2.8%. The critical SWC of 2.8%
was basically in agreement with the threshold of SWC for initial
stomatal closure due to drought stress in tobacco (Nicotiana
tabacum L.) (Riga and Vartanian, 1999, Australian Journal of Plant
Physiology 26(3) 211-220; herein incorporated by reference). The
three cultivars showed a more rapid decrease in .PSI.w as the soil
dried below a SWC of 1.5-1.8%, whereas .PSI.w of Atlas fescue did
not decrease steeply until SWC was near 1%. This shows that Atlas
fescue is less sensitive to soil water deficits than the other
Fescue cultivars.
Leaf Elongation Rate:
[0438] Atlas fescue showed a lower LE than Barolex and Kentucky 31
in the first week. As the stress gradually increased there was a
negative effect on LE of all grasses (P<0.001) when compared
with the control. However, the mean LERs were similar for irrigated
Atlas fescue, Barolex, and Kentucky 31 across the 12-week period,
and significantly greater than that of Falcon II. These results
revealed that irrigated Falcon II grew relatively slower than other
grasses, and Atlas fescue initially had a low LE but it increased
in later weeks during the drought stress period.
[0439] Between weeks 8 and 10, LE of the irrigated plants was
greater than during the first 7 weeks of the drought treatment
period. At week 10, LE of irrigated plants of Kentucky 31 dropped
dramatically, when the plants started to bloom and vegetative
growth was switched to reproductive growth. In drought-treated
plants, the average LB for four grasses across the whole drought
stress period was not significantly different (P=0.5078). For the
three tall fescue cultivars, the LE of stressed plants started to
decrease to below the level of irrigated plants at week 5 or 6 of
the stress treatment, whereas for Atlas fescue, LE started to
decrease later, at week 7 of stress. The LE of drought-treated
plants in Barolex and Falcon II ceased after 9 weeks of treatment,
while in Atlas fescue and Kentucky-31 LB lasted longer, up to week
10.
[0440] The relation between LE and SWC was fitted to a second order
polynomial function. When SWC was near full soil capacity
(8-9.33%), the LE of Barolex and Kentucky 31 were higher than those
of Falcon II and Atlas fescue, indicating that Barolex and Kentucky
31 were growing faster at a high SWC. As the SWC was declining, LE
decreased differently among the four grasses. Falcon II and Atlas
fescue showed a relatively slow decreasing rate compared with
Barolex and Kentucky 31, because the slopes of trend line for
Falcon II and Atlas fescue were less steep, suggesting that the
growth of Falcon II and Atlas fescue was less sensitive to the
declining SWC.
[0441] The LE responded to the decreasing .PSI.w following a
polynomial function. As .PSI.w was declining and becoming more
negative, the LE decreased for all grasses, but at different rates.
The decrease in rate of LE of Atlas fescue and Falcon II was less
than that of Barolex and Kentucky 31 indicating that, on a relative
basis, LEs of Atlas fescue and Falcon II were less sensitive to the
increasing severity of drought stress.
Leaf Water Content:
[0442] Drought stress treatment had a significant (P<0.001)
effect on LWC of the grasses. The LWC of irrigated plants remained
constant at about 87.7% during the whole experimental period. In
plants subjected to drought, LWC decreased differently among the
four grasses. For the tall fescue cultivars, LWC of stressed plants
was at the level of irrigated plants during the first 3 or 4 weeks
of growth, whereas for Atlas fescue, LWC of stressed plants
maintained the same level as irrigated plants up to 8 weeks. The
LWC of Atlas fescue was significantly higher than that of the
cultivars between week 6 and week 9 in the drought stress
treatment. The results imply that Atlas fescue may accumulate or
conserve water in leaf tissue and maintain turgor as a stress
avoidance mechanism through adapted leaf and root morphology.
[0443] The relationship of LWC in response to SWC showed three
stages. When SWC was high (8-9.33%), LWC of all grasses was
maintained at a high level (between 80 and 90%). In the second
stage, as SWC decreased from 8% to about 4%, LWC showed a slightly
increasing trend, more so in Atlas fescue than the cultivars. In
the third stage, when the SWC was decreasing from 4% to near 0%,
the LWC decreased dramatically for all the grasses. It was notable,
however, that with SWC was decreasing from 6% to 2%, a medium
drought stress status, LWC of Atlas fescue remained higher than
that of the other grasses, and then decreased most rapidly.
[0444] The association of LWC with .PSI.w was described by a
polynomial function. As .PSI.w became more negative, specifically
between -1 and -2.5 MPa, the LWC of grasses declined, but at a much
slower rate for Atlas fescue than the cultivars, especially between
.PSI.w of -1.2 and -2.4 Mpa. This again suggested that Atlas fescue
had an adaptation ability to accumulate or conserve water in leaf
tissue under drought stress.
Root Length and Biomass:
[0445] The RL among grasses ranged from 115 to 132 cm and varied
significantly (P=0.034). Barolex had the longest root system, while
Kentucky 31 had the shortest. The RL of Falcon II was negatively
affected by the drought treatment, whereas there was no significant
difference in RL between irrigated and drought-treated plants of
Atlas fescue, Barolex, and Kentucky 31. No significant difference
was found in RM among the grasses when the irrigated and drought
stress treatments were avenged (P=0.072). However, the drought
treatment had a significant (P=0.003) effect on RM. Stressed plants
of Atlas fescue, Barolex, and Falcon had significantly less RM than
did their irrigated controls, but not for Kentucky 31). The results
suggested that control plants of Barolex and Atlas fescue with
longer roots might be more adaptive to drought stress than Kentucky
31. However, the RM of Kentucky 31 was not reduced by severe
drought stress suggesting that Kentucky 31 may tolerate the drought
stress through maintenance of viable roots capable of extracting
available water, even though it had a shorter root. In summary,
drought stress reduced LE, LWC, .PSI.w, root biomass, and root
length of the grasses. Thus Festucas species avoid drought stress
through changes in leaf and root morphology and through osmotic
adjustment to maintain sufficient turgor pressure in the growing
zone for leaf elongation.
[0446] Unlike annual plants that escape drought by maturing before
stress becomes severe, perennial grasses do not escape drought
completely by early flowering. Indeed, a few of the control plants
but no drought stressed plants flowered indicating that plant
maturity was delayed and/or reproductive growth was inhibited by
the imposed drought stress. Further, the four grasses maintained
leaf elongation until a very low SWC (1.2%), showing that active
growth rather than dormancy occurred during the drought stress
period. However, while the leaves of the grasses rolled initially,
as SWC decreased further, the leaf tip showed firing and lower
leaves became bleached. These symptoms show that these grasses
employ an escape strategy to reduce the transpiration surface area
and close stomata to limit plant water loss similar to tobacco
plants (Nicotiana tabacum L.) (Riga and Vartanian, 1999).
[0447] Additionally, in contrast to previous studies on drought
stress responses where the root system was chosen as a selection
trait in breeding programs to improve drought tolerance of fescue
(Torvert et al., 1990, Appl. Agric. Res. 5: 18 1-187; herein
incorporated by reference), the results described herein show the
benefits of using leaf water content measurements in breeding
methods of the present inventions. In particular, LE measured
weekly during the drought stress period was a major indicator of
the status of plant response to drought. Cell expansion was
acknowledged as the most sensitive trait in plants (Boyer, 1988,
Physiol. Plant. 73: 311-316; herein incorporated by reference) and
is reduced by drought before other physiological processes
(Wardlaw, 1969, Aust. J. Biol. Sci. 22: 1-16; herein incorporated
by reference). In the present studies LE of Atlas fescue and
Kentucky 31 declined significantly one week earlier than .PSI.w,
which was previously shown to be an effective measurement of the
maximum soil water potential available to roots (Tardieu and
Simonneau, 1998, J. Exp. Bot. 49: 419-432; herein incorporated by
reference). These results confirmed that LE is a sensitive
parameter for drought tolerance evaluation in plants. In addition,
it is difficult to make measurements of .PSI.w on severely
drought-stressed leaves, however LE is measured at any time and
water condition.
[0448] Further, the slower decrease in LE, LWC, and .PSI.w for
Atlas fescue during the drought-stress period demonstrated its
greater capability for drought tolerance and the value for
introgressing this characteristic into a plant breeding
program.
[0449] Therefore, Festuca mairei plants demonstrated a greater
capability to resist drought, in other words a higher level of
drought resistance, than the Festuca species and their
representative commercial plant cultivars that were previously used
for providing drought tolerant germplasm to grass plants, including
Lolium species.
Example III
Identifying Fm Germplasm in Festuca mairei Plants Associated with
Drought Stress
[0450] See also, for example, Wang et al., 2005, Molecular
Biotechnology, 29(3):21 1-220; herein incorporated by reference in
its entirety.
[0451] A. Plant Performance During Nine Days of Extreme Drought
Stress.
[0452] A complete drought response was completed in nine days. Of
the nine days, eight days were actual drought stress plus the day
before withholding irrigation water, was considered to be a whole
drought stress period because it covered the range of dynamic
changes of the plants responding to drought stress, as described
below.
[0453] During the nine days of treatment, control F. mairei plants
maintained green and survived throughout the stress treatment
period. The F. mairei plants under stress remained green during the
first three days after water deprivation. However, on the 4th day
after water deprivation the treated plants began discoloring and
firing, an indication of tissue injury due to stress. Specifically,
the RWC of leaves from the stressed plants decreased dramatically
from 83% to 26% between the 3rd and 5th day of drought stress (FIG.
2).
[0454] On the 8th day of drought stress, the leaves of the stressed
plants were completely fired with a RWC of 17%. Therefore, the 4th
day under these extreme stress conditions was critical for the
evaluation of information for identifying phenotypical and
physiological changes significant of water deprivation.
[0455] B. cDNA-AFLP Analysis.
[0456] The inventor noticed that in the majority of previous
drought tolerance studies, a large number of identified genes,
transcripts and proteins related to drought were induced by stress
(inducible) or up-regulated. However, there were few reported
down-regulated genes and no transient or other types of altered
genes reported that would contribute to drought adaptation.
Further, low numbers of time points during the onset and duration
of drought stress were reported, for example, one or two
time-points during stress periods were compared with the control,
especially by microarray analysis. Therefore, some genes, such as
transiently expressed genes, and up or down regulated genes, would
not be identified. However, because the plant response to stress is
a complicated procedure, down-regulation or other types of
regulation may also play important roles in drought response or
even drought tolerance.
[0457] The inventor found during the development of the present
inventions, that investigation of the systemic and dynamic changes
of gene expression on a daily basis during induction of stress
provided more complete information for understanding the molecular
mechanism of stress response. Thus in contrast to previous studies,
four different differential expression patterns were detected by
cDNA-AFLP analysis as described herein, even though the third and
fourth patterns, transient and up-then-down expression, was not
abundant. Further study on genes with these four differential
expression patterns, further including spatial and temporal
regulation patterns, should lead to a programmed control of the
drought stress response, and thus the inventor contemplates
regulating a stress response mechanism at the gene regulation
level. The cDNA-AFLP technique used by the inventor not only
provided an approach to generate genomic sequence information and
functional analysis but also served as a powerful tool for the
identification of genes with additional kinds of differential
expression patterns for a plant's stress response.
[0458] Further, in contrast to other studies, the inventor obtained
information during the development of the present inventions, which
covered the whole dynamic change of the plant responding to the
stress. This dynamic change was covered in the nine days (with
three later days as reference) during a drought stress treatment of
complete water depravation as described below.
[0459] C. A Functional Approach to Identify Stress Response
Genes:
[0460] Control Plants: DEFs induced by regular plant development
and any changes of greenhouse conditions during the drought stress
treatment were investigated by cDNA-AFLP analysis performed on the
control plants of F. mairei over nine days using four randomly
picked primer combinations (NspI-CC/TaqI-TC, NspI-TG/TaqI-GT,
NspI-TG/TaqI-CC, and NspI-TC/TaqI-AG, see, Table 1A). DEFs were not
detected across the nine days in control plants (FIG. 3a), which
suggested that both plant development and/or greenhouse conditions
did not affect gene expression in F. mairei plants during the
application of the drought stress treatment.
[0461] Drought Stressed Plants: cDNA-AFLP analysis was conducted
using all of the 128 primer combinations over nine days of the
stressed F. mairei plants and revealed 11,346 transcript derived
fragments (TDFs) with an average of 89 fragments obtained per
primer pair. The size of the observed fragments ranged from 50 to
1000 bp. Of these TDFs, 464 fragments (4.1%) were identified as
being differentially expressed across the nine days during the
drought stress treatment, indicating the gene expression had been
altered by the drought conditions (FIG. 3b).
[0462] The expression pattern of these DEFs included up-regulated
(138, 29.7%), down-regulated (252, 54.3%), transient-expressed (57,
12.3%), and up-then-down-regulated (17, 3.7%) (FIG. 4). Of these
464 DEFs, 434 (94%) fragments were recovered from acrylamide gel
and isolated as genes potentially related with plant response to
drought stress. Thus the inventor provided isolated nucleic acids
for SEQ ID NOs: 1-39, and 93-216.
[0463] Upon analysis, the majority of the identified proteins fell
into one or more statistical significant functional category, thus
each of those categories were counted in the analysis. In
comparison between the functional categories of the up-regulated
and the down-regulated DEFs (FIG. 6), the down-regulated genes were
primarily involved in metabolism and cellular biogenesis, such that
they were found to be nearly twice that of the up-regulated DEFs.
On the other hand, more than two times the amounts of the
up-regulated DEFs were involved in transcription, defense, cell
cycle and DNA processing compared to down-regulated DEFs.
[0464] These results generally indicated that during drought stress
there was a decrease in metabolic function and biogenesis of
cellular components during the plants degenerative drying (and
dying) process. Up-regulated genes were associated with the cell
cycle and DNA synthesis that would be involved with increasing
activity of growth particularly in specific guard cells that
function in stress defense. However, the transiently expressed DEFs
were primarily involved in subcellular localization, defense, and
heavy metal carriers for transport. The inventor contemplates that
the TEFs function to meet the temporary need for turning on and/or
regulating sets of genes that are expressed in stress defense,
transport and subcellular localization during a plant response to
drought stress. In contrast, the up-then-down regulated DEFs were
mostly involved in transport, subcellular localization, and energy.
Thus the inventor contemplated that genes necessary for
electron/hydrogen transport, subcellular localization, and
photosynthesis were first stimulated by a drought stress signal,
and then inactivated by the continued or severe stress conditions
leading to death. Thus the inventor contemplated that the plant
system appeared to save energy while at the same time providing for
new gene transcription, in particular transcription of genes
related to stress defense.
[0465] D. Macroarray Hybridization Analysis:
[0466] In addition to 13 positive and 13 negative nucleic acid
controls, 406 samples of 434 recovered DEFs were printed on a
membrane with two replications. Twenty-eight DEFs with small sizes
of less than 100 bp were not included in the macroarray analysis.
The dot intensity volume of two or three identical membrane arrays
hybridized with cDNA probes from different days respectively were
compared for confirmation of the differential expression pattern of
the 406 DEFs (as described herein). FIG. 5 shows an exemplary image
of a portion of the hybridized macroarray. The comparison results
revealed that 54 of 128 (42.2%) up-regulated, 97 of 210 (46.2%)
down-regulated, 14 of 51 (27.5%) transiently expressed, and 6 of 17
(35.3%) up-then-down regulated DEFs showed a consistent
differential expression pattern. In total, the expression pattern
of 171 (42.1%) of DEFs were confirmed. These 171 DEFs were cloned
as drought responsive gene fragments also referred to herein as
preferred Fm germplasm.
[0467] After comparing cDNA-AFLP results with microarray results,
42% of DEFs were considered to be consistently expressed by plants.
The inconsistency between the two techniques could be due to (1)
the subjective evaluation on the DEF in the cDNA-AFLP gel; or (2)
the different macroarray hybridization intensities and or
background between the membranes compared; or (3) possible cross
hybridization of closely related sequences in macroarray; and/or
(4) low expression genes in the probe for macroarray hybridization
(Miller et al., 2002, BioTechniques 32:620-625; herein incorporated
by reference). Therefore, the inventor excluded more than half
(58%) of the DEF from subsequent sequence analysis. Additionally, a
few of the drought inducible DEFs identified by cDNA-AFLP coupled
with macroarray as described herein, were previously reported as
stress-inducible genes in other species. These discoveries showed
that methods of the present inventions for the analysis system
functioned properly to discover stress-inducible genes of
plants.
[0468] E. DEF Sequence Analysis and Assignment of Functional
Category:
[0469] One hundred seventy-one DEFs were sequenced and an expected
size sequence was obtained for 166 of these sequences. Of the 166
sequences, three pairs of fragments showed above 98% similarity to
each other after sequence alignment. Therefore one of each pair was
excluded from further analysis, for 163 sequences. Of the 163
sequences, 92 were down-regulated, 50 were up-regulated, 15 were
transiently expressed, and 6 were up-then-down-regulated (for
example, Tables 3-6). One hundred twenty-four sequences were
deposited to the GenBank EST database with accession numbers of
DW248995 through DW249118 (SEQ ID NOs:93-216).
[0470] BLASTX analysis was conducted against the GenBank protein
database for SEQ ID NOs: 1-39, and 93-216. The results revealed
that 101 DEFs (62.0%) showed significant homology to protein
sequences in the database (E value less than 1E-6). The other 62
DEFs (38.0%) showed zero matches (no hits found) or no significant
homology (E value higher than 1E-6). When the entire GenBank EST
database was screened for the presence of sequences similar to the
62 DEFs (TBLASTX analysis), 23 DEFs showed statistically
significant degrees of similarity to the public available ESTs. The
remaining 39 DEFs were defined as novel sequences (SEQ ID NOs:
1-39), which are not identified in other organisms. Translation of
SEQ ID NOs: 1-39 into amino acid sequences SEQ ID NOs:40-91 (FIG.
8) was done via ExPASY (at website ca.expasy.org/tools/dna.html.
For several sequences, more than one predicted amino acid sequence
was identified according to different read frames, and a few were
not read completely through because of a stop codon.
[0471] The 39 novel DEFs included 13 down-regulated, 6 transiently
expressed, and 20 up-regulated sequences. Therefore, 40% of the 50
up-regulated, 15 transiently expressed DEFs respectively and 14.1%
of the 92 down-regulated DEFs were found to be novel in the genome
of F. mairei plants under drought stress treatment.
[0472] The predicted function for the 101 DEFs was subdivided into
17 functional categories while 4.8% of these DEFs were functionally
unknown, hypothetical or an unclassified protein based on function
classification criteria defined at the website of MIPS (Munich
Information Center for Protein Sequences) (http://mips.gsf.de)
(see, Table 2).
TABLE-US-00003 TABLE 2 Distribution of the differentially expressed
fragments (DEFs) during drought stress cycle in F. mairei by
functional categories Function Up-regulated Down- Transiently
Up-then down category In general (%) (%) regulated (%) expressed
(%) regulated (%) Metabolism 16.45 9.76 19.63 9.09 6.25 Energy 7.36
4.88 7.98 0.00 12.5 Biogenesis of 4.76 2.44 5.52 0.00 6.25 cellular
components Subcellular 22.94 17.07 24.54 27.27 18.75 localization
Transport 10.39 9.76 9.20 9.09 25 Transcription 5.19 12.20 4.29
0.00 0 Signal 2.60 0.00 3.07 0.00 6.25 transduction Interaction
3.90 4.88 3.68 9.09 0 with the cellular environment Protein 1.73
2.44 1.84 0.00 0 synthesis Protein with 5.63 4.88 4.91 18.18 6.25
binding function Defense 6.93 17.07 3.68 18.18 6.25 Development
2.16 2.44 2.45 0.00 0 Cell fate 1.30 2.44 1.23 0.00 0 Cell cycle
and 0.87 2.44 0.61 0.00 0 DNA processing Protein fate 2.16 0.00
2.45 0.00 6.25 Cell type 0.43 0.00 0.00 9.0 9 0 differentiation
Protein activity 0.43 0.00 0.61 0.0 0 0 regulation Function 4.76
7.32 4.29 0.00 6.25 unknown, hypothetical, and unclassified
protein
[0473] In Table 2 classification was performed for 101 DEFs with
strong statistical similarity to GenBank plant protein sequence (E
values lower than 1.00E-06) by BLASTX search. The functional
category was assigned based on function classification criteria in
the website of Munich Information Center for Protein Sequences
(MIPS) (http://mips.gsf de).
[0474] Comparing the functional categories between up-regulated and
down-regulated DFEs (FIG. 7 and Tables 3 and 4), the inventor found
that down-regulated genes involved in metabolism and cellular
biogenesis were nearly twice of the up-regulated. On the other
hand, more than two times of the percentage of up-regulated DEF was
involved in transcription, defense, and cell cycle and DNA
processing compared to down-regulated DEF. The results reflected
that during the drought stress generally more metabolic function
and biogenesis of cellular components in the plant were under
degenerative processes. The plant system seemed to save the energy
for new genes transcription and stress defense. More genes involved
in cell cycle and DNA synthesis that were up-regulated may suggest
the increasing activity of growth in some specific guard cell for
stress defense. The transiently expressed DEFs were basically
involved in subcellular localization, defense, or acted as heavy
metal carrier for transport reflecting the temporary needs for sets
of gene in defense, transport and subcellular localization during
plant response to drought. The up-then-down regulated DEFs were
primarily involved in transport, subcellular localization, and
energy indicating that some genes for electron-hydrogen transport,
subcellular localization, and photosynthesis were stimulated by
drought stress signal firstly and then inactivated by the continued
or severe stress.
[0475] F. Drought Inducible Genes:
[0476] Functional analyses of the stress-inducible genes are
important for manipulation of molecular mechanisms of stress
response and for stress tolerance improvement of crops. At least
200 drought inducible genes were reported in plants (Seki et al.,
2002, Plant J. 30:279-292, Ozturk et al., 2002, Plant Mol. Biol.
48: 55 1-573; all of which are herein incorporated by reference),
and some were transferred successfully into several crops to
improve the stress tolerance of plants (Bajaj et al., 1999,
Molecular Breeding 5: 493-503; herein incorporated by reference).
However, because of differences between various crop plant species
which are reflected in their germplasm, and the need for further
improvement in specific types of stress tolerance, such as drought
tolerance, the identification of additional drought-inducible genes
would provide improved drought resistant plants and provide a
larger picture of the genes involved in stress tolerance and
cis-acting promoter elements that function in drought specific gene
expression (Seki et al., 2001, Plant Cell. 13: 61-72; herein
incorporated by reference).
[0477] During the development of the present inventions, 50 drought
inducible gene fragments were identified from the drought adaptive
monocot plant, F. mairei. 22 (44%) had hits with significant
similarity in the protein database and were assigned functions.
Several of them have been reported as up-regulated by stress in
Arabidopsis such as zinc finger and MYB family transcription
factors, raffinose synthases and trehalose-6-phosphate synthase,
heat-shock protein, auxin-regulated protein, etc. (Seki et al.,
2002, Plant J. 30:279-292; herein incorporated by reference). The
remaining 28 (56%) either had hits with no significant similarity
in the protein database, or had significant hits in the EST
database with unknown function, or had no hits in either database
and were defined as novel drought-inducible gene fragments.
Functional analysis of such novel DEFs might be informative to
follow up in later experiments based on more natural drying plants
in the field.
TABLE-US-00004 TABLE 3 Relationship of DEFs to known sequences
provided by BLASTX searches SEQ ID plant protein nr Plant EST Size
NO: XX sequence BLASTX BLASTX TBLASTX kb 01 SSBI-A12 No hits found
No hits found No hits found 228 N5T8-100u-2 125 III 85 02 SSBI-A2
gi|15292985| No hits found Low 020 N8T1-200t 230 II 188 03 SSBI-A3
No hits found No hits found Low 053 N7T2-100u 165 II 122 04 SSBI-A6
gi|34903618| Low Low 104.5 N4T4-100u 170 132 05 SSBI-C1 No hits
found No hits found Low 002 N1T1-100u 135 III 101 06 SSBI-C3
gi|50905077| Low Low 056 N8T2-300d 385 II 328 07 SSBI-C8
gi|50908947| Low Low 155 N5T6-300d 310 II 275 08 SSBI-E1
gi|13568486| 3.00E-26 Low 006 N2T1-300t-2 330 II 291
gi|51459300|ref|XP_498769.1| 09 SSBI-E5 No hits found No hits found
Low 099 N4T4-100u 110 III 75 10 SSBI-F11 gi|45181461| No hits found
Low 211 N1T8-100u-3 110 III 77 11 SSBI-F3 No hits found Low Low 060
N8T2-100u-1 198 II 162 12 SSBI-F7 No hits found Low Low 133
N7T5-200t 220 II 186 13 SSBI-F8 No hits found No hits found Low 163
N8T6-100d-1 155 II 113 14 SSBI-G1 gi|18406088| No hits found Low
012 N4T1-100u 120 II 81 15 SSBI-G11 No hits found No hits found Low
222 N4T8-100u 175 II 138 16 SSBI-G2 No hits found No hits found Low
041 N5T2-100u-2 118 II 78 17 SSBI-G3 No hits found No hits found
Low 061 N8T2-100u-2 105 III 69 18 SSBI-G4 gi|50898902| Low Low 082
N6T3-200u 290 II 166 19 SSBI-G7 No hits found No hits found Low 134
N7T5-100d 155 II 116 20 SSBI-G8 gi|50939069| Low Low 165 N1T7-500t
570 II 545 21 SSBI-H11 gi|31431423| Low Low 227 N5T8-100u-1 198 II
165 22 SSBI-H9 gi|50905401| Low Low 182 N5T7-200d 250 II 211 23
SSBII-A10 gi|33285910| No hits found Low 428 N1T15-100u-2 168 II
126 24 SSBII-A9 No hits found No hits found Low 415 N5T14-100u-1
190 III 153 25 SSBII-B10 gi|50912857| Low Low 433 N1T15-50u 99 III
68 26 SSBII-B11 gi|34910006| Low Low 220 100d 135 II 97 27 SSBII-B3
gi|34902330| Low Low 310 N4T10-100t-2 185 II 147 28 SSBII-B7 No
hits found No hits found Low 388 N4T13-100d-3 160 III 119 29
SSBII-B9 No hits found No hits found Low 418 N6T14-100u-1 148 III
108 30 SSBII-C4 No hits found No hits found Low 339 N4T11-100u 145
III 101 31 SSBII-C5 gi|40538965| Low Low 360 N6T12-300d 340 I 299
32 SSBII-C6 gi|5853329| No hits found Low 375 N8T12-100d-1 199 II
169 33 SSBII-C9 No hits found Low Low 419 N6T14-100u-2 110 III 71
34 SSBII-D6 No hits found No hits found Low 377 N2T13-200d-1 240 II
204 35 SSBII-E10 No hits found No hits found No hits 455 N2T16-50u
80 III 23 found 36 SSBII-E8 gi|50908607| Low Low 409 N2T14-200t 208
II 177 37 SSBII-F9 No hits found No hits found Low 424 N7T14-100d
120 III 88 38 SSBII-G5 gi|34896478| No hits found Low 366
N6T12-100d 192 III 161 39 SSBII-H1 No hits found No hits found Low
285 N8T9-300d 380 II 335
[0478] The products of the stress-inducible genes can be classified
into two groups: (1) those that directly protect against
environmental stresses; and (2) those that regulate gene
expressions and signal transductions in stress response (Seki et
al., 2002, Plant J. 30:279-292; herein incorporated by reference).
The proteins in the first group have the ability to function in
stress tolerance. The raffinose synthases and trehalose-6-phosphate
synthase were osmoprotectant biosynthesis-related proteins for
adjusting the osmotic pressure under stress conditions. The
heat-shock proteins have been reported to be involved in protecting
macromolecules such as enzymes and lipids (Shinozaki et al., 1999,
In Molecular Responses to Cold, Drought, Heat and Salt Stress in
Higher Plants, pp. 11-28; herein incorporated by reference). The
fibrillin and fiber proteins might contribute to cell wall
structure modification. The type-1 pathogenesis-related protein is
considered to be a protein with antifungal activity (Antoniw et
al., 1980, J. Gen. Virol. 47: 79-87; herein incorporated by
reference) that may have multiple stress-related roles even though
the function is still unknown. Tonoplast intrinsic protein
(aquaporin) functions as a water channel to transport water through
plasma membrane and tonoplast to adjust the osmotic pressure under
stress conditions (Daniels et al., 1994, Plant Physiol. 106:
1325-1333; herein incorporated by reference). The transporters for
anion and zinc may function in adjustment of ion homeostasis. The
second group contains regulatory proteins involved in regulation of
signal transduction and gene expression in stress responses. The
zinc finger and MYB family transcription factors may function in
the regulation of stress-inducible gene expression. The peptide
chain release factor induced by drought stress reflected that the
post-transcriptional regulatory mechanism also affected the gene
expression. Ankyrin protein kinase is thought to be involved in
signal transduction and in further regulating the functional genes
under stress conditions. The auxin-regulated gene was identified as
drought-inducible suggesting a link between auxin and drought
stress signaling pathways. Some DEFs annotated to the same
functional genes were probably derived from the same gene or from
redundant homologous genes. However, the functions of the majority
of these genes are not known.
[0479] Some DEFs annotated to the same functional genes were
probably derived from the same gene or from redundant homologous
genes. Currently the functions of the majority of these genes are
not fully understood. Moreover, 56% of the drought inducible gene
fragments discovered during the development of the present
inventions, were still functionally unknown and remain to be
elucidated. Functional analysis of such set of DEFs might be
informative to follow up in later experiments based on more natural
drying plants in the field.
[0480] G. Predicted Function of Up-Regulated DEFs.
[0481] Twenty two DEFs up-regulated or induced by drought stress in
F. mairei had significant similarity with protein sequences in the
GenBank database (Table 4). The majority had the highest similar
hits in the monocot species such as rice (Oryza sativa), maize (Zea
mays), and wheat (Triticum aestivum). SSBII-H2, SSBI-B6, and
SSBI-C09 encoded enzymes, respectively, involved in biosynthesis of
purine nucleotide, raffinose, which has also been induced by water
stress in Cicer arietinum (Romo et al., 2001, Plant Physiol.
Biochem. 39: 1017-10263; herein incorporated by reference), and
trehalose, the most effective osmoprotectant sugar in terms of
minimum concentration required (Crowe et al., 1992, Annu. Rev.
Physiol. 54: 579 599; herein incorporated by reference). The
SSBI-D11 encoding of an enzyme for C-compound and carbohydrate
utilization has been identified as a transcript differentially
expressed in response to high salinity in the mangrove, Bruguiera
gymnorrhiza (Banzai et al., 2002, Plant Sci. 162: 499-505; herein
incorporated by reference).
[0482] The farnesylated protein encoded by SSBI-B9 has been found
in Hordeum vulgare to be a nuclear protein involved in stress
response and leaf senescence (Barth et al., 2004 Physiol. Plantarum
121: 282-293; herein incorporated by reference). SSBI-D4, SSBI-D9,
SSBII-A3, and SSBI-A4 encoded a fiber protein Fbl9, a
dehydration-responsive family protein, a type-1
pathogenesis-related protein, and a DNAJ heat shock N-terminal
domain-containing protein, respectively, which have been widely
studied in association with stress response. SSBI-E2 encoded a 34
kD fibrillin-like protein, the major constituent of
elastin-associated extracellular microfibrils, and has been
recently identified in a network of rice genes associated with
stress response (Cooper et al., 2003, Proc Natl Acad Sci USA.
4945-4950; herein incorporated by reference). SSBI-A5 encoded a
brown planthopper susceptibility protein and shared similarity with
the sequence of a rice gene induced in response to herbivore
grazing. Several DEFs encoded proteins involved in heavy metal ion
transport, electron/hydrogen transport, and membrane channel,
reflecting the plant actively adjusted the ion and water status for
homeostasis. The DEFs encoding proteins for transcription and
translation regulation were induced such as zinc finger protein,
MYB transcription factor, and peptide chain release factor
suggesting certain stress-responsive genes are activated by these
factors for positive stress defense.
TABLE-US-00005 TABLE 4 Function annotation and size of the
differentially expressed fragments (DEFs) up-regulated by drought
stress in F. mairei. SEQ ID DEF Top hit NO: XX sequence in GenBank
E-value* Description Organism Size (bp) 209 SSBII- gi|46560602|
2.00E-44 putative inosine-uridine preferring 245 H02 nucleoside
hydrolase [Zea mays] 102 SSBI-B06 gi|50540754| 7.00E-44 putative
raffinose synthase or seed 248 imbibition protein [Oryza sativa]
105 SSBI-B09 gi|28866019| 3.00E-40 farnesylated protein 3 [Hordeum
263 vulgare] 114 SSBI-C09 gi|50252610| 1.00E-33 putative
trehalose-6-phosphate 251 synthase/phosphatase [Oryza sativa] 94
SSBI-A04 gi|52353404| 1.00E-29 DNAJ heat shock N-terminal 194
domain-containing protein [Oryza sativa] 126 SSBI-E02 gi|50898740|
1.00E-25 putative chloroplast drought-induced 306 stress protein,
34 kD/fibrillin-like protein [Oryza sativa] 95 SSBI-A05
gi|33771376| 2.00E-25 putative brown planthopper 228 susceptibility
protein Hd002A [Oryza sativa] 113 SSBI-C07 gi|49328143| 2.00E-24
putative peptide chain release factor 172 subunit 1 (eRF1) [Oryza
sativa] 124 SSBI-D11 gi|34894800| 6.00E-19 putative
dihydrolipoamide 156 dehydrogenase precursor [Oryza sativa] 119
SSBI-D04 gi|38016525| 9.00E-19 fiber protein Fb19/universal stress
273 protein USP1-like protein [Gossypium barbadense] 109 SSBI-C02
gi|56784864| 8.00E-18 auxin-regulated protein-like [Oryza 310
sativa] 140 SSBI-F09 gi|53749298| 8.00E-16 putative
polyprotein/GAG-POL 166 precursor [Oryza sativa] 156 SSBII-
gi|3702665| 1.00E-13 type-1 pathogenesis-related protein 100 A03
[Triticum aestivum] 99 SSBI-B02 gi|50919725| 2.00E-13 Putative
anion transporter [Oryza 114 sativa] 122 SSBI-D09 gi|51469000|
3.00E-13 Ankyrin protein kinase- 103 like/dehydration-responsive
protein- like [Poa pratensis] 203 SSBII- gi|30420736| 4.00E-13 zinc
transporter [Oryza sativa] 108 G04 134 SSBI-E11 gi|168473| 9.00E-13
ferredoxin [Zea mays] 119 182 SSBII- gi|25090853| 2.00E-09
NADH-ubiquinone oxidoreductase18 77 D09 kDa subunit, mitochondrial
precursor [Arabidopsis thaliana] 215 SSBII- gi|4996646| 1.00E-08
Dof zinc finger protein [Oryza 185 H09 sativa] 116 SSBI-C11
gi|4996646| 1.00E-08 Dof zinc finger protein [Oryza 199 sativa] 157
SSBII- gi|32879770| 5.00E-07 tonoplast intrinsic protein [Oryza 70
A04 sativa] 192 SSBI-F02 gi|51572282| 5.00E-07 MYB transcription
factor [Triticum 80 aestivum] *E-value < 1.00E-06 means the
significant similarity with the protein sequences in the Genbank
database.
[0483] H. Drought Repressible Genes.
[0484] The analysis of drought-repressible genes is as important as
analysis of drought-inducible genes in understanding the molecular
mechanism of plant response to stress. During the development of
the present inventions, many photosynthesis-related genes were
found such as chlorophyll a/b binding protein,
ribulose-1,5-isphosphate carboxylase, high molecular mass early
light-inducible protein HV58, etc., all reflecting that
photosynthesis was inhibited by the water deficit. This can be due
to a reduction in light interception as leaf senesce, or to a
reduction of intercellular CO.sub.2 concentration as closure of
stomata (Bartels et al., 2001, Plant Physiol. 127: 1346-1353;
herein incorporated by reference). The benefit of the depressed
photosynthesis appears to be the switch toward another carbohydrate
utilization pathway, which leads to the production of valuable
stress tolerance molecules (Pattanagul et al., 1999, Plant Physiol.
121: 987-993; herein incorporated by reference). Bockel &
Bartels proposed that down-regulation of photosynthesis-related
genes possibly contributed to reduced photooxidative stress.
[0485] Lipoxygenase, glutamate synthase, malate dehydrogenase
up-regulated under drought in barley (Ozturk et al., 2002, Plant
Mol. Biol. 48:551-573; herein incorporated by reference) were
down-regulated in this study. Some clones have been up-regulated by
drought in Arabidopsis encoding protein products the same as
protein encoded by down-regulated clones, e.g. Cytochrome P450
protein and malate dehydrogenase have shown in both up-regulated
and down regulated groups (Seki et al., 2002, Plant J. 30:279-292;
herein incorporated by reference). This distinct behavior has also
been found in barley and rice (Kawasaki et al., 2001, Plant Cell
13: 889-906; herein incorporated by reference). Their role and
importance in tolerance or sensitivity is impossible to judge based
on the experiments alone under controlled environment conditions.
But these DEFs, at least, provide clues about the genes
differentially expressed with a reference database for comparison
later on with data from natural field drought conditions.
[0486] I. Predicted Function of Down-Regulated DEFs.
[0487] In total, 70 down-regulated DEFs showed significant
similarity to previously identified proteins (Table 5). A much
larger quantity of down-regulated DEFs than up-regulated were
isolated in F. mairei during drought stress indicating that the
plant was mainly under degenerative processes imposed by the
stress. Down-regulated genes were involved in a number of basic
metabolic or biosynthetic functions and systemic development or
growth, such as photosynthesis (light-inducible protein HV58,
SSBII-D7), respiration (chlorophyll A-B binding family protein,
SSBI-B10), amino acid metabolism (victorin binding protein,
SSBI-B12), oligopeptide synthesis (GTP-binding protein type A,
SSBII-C2), carbohydrate metabolism (UDP-glycosyltransferase 88B1,
SSBII-G7), tissue development (homeobox protein knotted-7,
SSBI-H3), DNA cell division (helicase, SSBI-G10) and so on. In
addition, some proteins for transport facilitation were
down-regulated, such as ADP-ribosylation factor for vesicular
transport (SSBII-H8), iron-phytosiderophore transporter protein for
aligopeptide transport (SSBII-B8), ferric reductase for electron
transport (SSBII-B2), cation diffusion facilitator for ion
transport (SSBII-C1.), and triose phosphate translocator for
c-compound transport (SSBI-E7). Moreover, several proteins involved
in transcription and signal transduction were also down-regulated
indicating some pathways for signaling and basic biosynthesis or
metabolism were turned down in the plant during drought stress.
Those proteins included cleavage and polyadenylation specificity
factor (SSBII-F10), homeobox gene knotted 7 (SSBI-H3), TATA-binding
protein associated factor (SSBII-F3), SEUSS transcriptional
co-regulator (repressor) (SSBI-B7), EREBPltranscription factor
(SSBI-C10), zinc finger protein (SSBII-E5), and
phosphatidylinositol-4-phosphate 5-kinase (SSBII-G2).
TABLE-US-00006 TABLE 5 Functional annotation and size of the
differentially expressed fragments (DEFs) down-regulated by drought
stress in F. mairei. SEQ ID Top hit in Size NO: XX DEF GenBank
E-value Description Organism (bp) 158 SSBII-A05 gi|710308|
1.00E-108 victorin binding protein/glycine 582 dehydrogenase P
protein [Avena sativa] 111 SSBI- gi|50940811| 2.00E-75 putative
non-phototropic hypocotyl 3 449 C05 (NPH3)/phototropic response
protein [Oryza sativa] 143 SSBI- gi|14861035| 2.00E-71
protoporphyrin IX Mg-chelatase 399 G05 subunit XANTHA-F [Hordeum
vulgare] 116 SSBII- gi|710308| 5.00E-71 victorin binding
protein/glycine 398 C11 dehydrogenase P protein [Avena sativa] 210
SSBI- gi|33333542| 1.00E-70 knotted 7/homeobox gene [Hordeum 460
H03 vulgare] 190 SSBII- gi|1181331| 9.00E-69 calnexin [Zea mays]
356 E09 186 SSBII- gi|861199| 1.00E-68 protoporphyrin IX
Mg-chelatase 398 E04 subunit precursor [Hordeum vulgare] 199 SSBII-
gi|50921909| 3.00E-65 OSJNBa0032B23.5/cleavage and 398 F10
polyadenylation specificity factor (CPSF) [Oryza sativa] 208 SSBII-
gi|13661772| 2.00E-64 putative cytochrome P450 [Lolium 357 G10
rigidum] 131 SSBI- gi|68599| 2.00E-59 glutamate-ammonia ligase
precursor, 390 E08 chloroplast - barley [Hordeum vulgare] 214
SSBII- gi|861205| 3.00E-55 ADP-ribosylation factor 309 H08
[Chlamydomonas reinhardtii] 184 SSBII- gi|6409335| 7.00E-54
ribulose-1,5-bisphosphate carboxylase 431 E02 small subunit [Avena
clauda] 138 SSBI- gi|50912639| 4.00E-51 putative serine-threonine
rich antigen 473 F05 [Oryza sativa] 206 SSBII- gi|18147771|
3.00E-49 cycloartenol synthase [Costus 345 G08 speciosus] 175
SSBII- gi|54290318| 1.00E-48 unknown protein/IWS1 C-terminus 318
D01 family protein [Arabidopsis thaliana] [Oryza sativa] 167 SSBII-
gi|40888826| 1.00E-46 iron-phytosiderophore transporter 338 B08
protein yellow stripe 1 [Oryza sativa] 192 SSBII- gi|21842139|
1.00E-43 cytochrome P450 monooxygenase 390 F02 CYP72A28 [Zea mays]
133 SSBI- gi|32251039| 3.00E-38 glyoxysomal malate dehydrogenase
238 E10 [Triticum aestivum] 193 SSBII- gi|49388196| 4.00E-37
putative TATA-binding protein 400 F03 associated factor (IID
(TFIID) component TAF4 family) [Oryza sativa] 198 SSBII-
gi|51091645| 2.00E-35 rab3-GAP regulatory domain-like 235 F08
[Oryza sativa] 213 SSBII- gi|50872458| 1.00E-34 putative c-type
cytochrome synthesis 223 H07 protein [Oryza sativa] 155 SSBII-
gi|4325354| 1.00E-34 contains similarity to retrovirus-related 223
A02 polyproteins and to CCHC zinc finger protein/gag-pol
polyprotein [Arabidopsis thaliana] 147 SSBI- gi|14334888| 1.00E-34
putative glycine 224 H01 hydroxymethyltransferase [Arabidopsis
thaliana] 189 SSBII- gi|2072727| 1.00E-34 ferredoxin-dependent
glutamate 232 E07 synthase [Oryza sativa] 103 SSBI- gi|18033922|
2.00E-34 SEUSS transcriptional co-regulator 286 B07 (Represser)
[Arabidopsis thaliana] 115 SSBI- gi|50726040| 3.00E-34 putative
transcription factor 280 C10 EREBP1/BTH-induced ERF transcriptional
factor 1 [Oryza sativa] 195 SSBII- gi|45357045| 5.00E-34 coatomer
alpha subunit [Hordeum 209 F05 vulgare] 162 SSBII- gi|2072727|
7.00E-31 ferredoxin-dependent glutamate 215 A11 synthase [Oryza
sativa] 166 SSBII- gi|2072727| 7.00E-31 ferredoxin-dependent
glutamate 215 B06 synthase [Oryza sativa] 194 SSBII- gi|20153218|
9.00E-31 putative sucrose: sucrose 1- 192 F04 fructosyltransferase
[Lolium perenne] 128 SSBI- gi|50948231| 1.00E-30 putative
cytochrome p450 (CYP78A9) 230 E04 [Oryza sativa] 130 SSBI-
gi|13195734| 2.00E-30 triose phosphate translocator [Triticum 201
E07 aestivum] 178 SSBII- gi|34922538| 1.00E-28 Lipoxygenase 2.3,
chloroplast 194 D04 precursor [Hordeum vulgare] 216 SSBII-
gi|21839| 2.00E-28 phosphoribulokinase; ribulose-5- 254 H10
phosphate kinase [Triticum aestivum] 205 SSBII- gi|54290956|
1.00E-27 putative UDP-glycosyltransferase 88B1 245 G07 [Oryza
sativa] 110 SSBI- gi|51451358| 3.00E-27 putative
o-methyltransferase ZRP4 206 C04 [Oryza sativa] 145 SSBI-
gi|50912885| 8.00E-26 putative Ribosomal RNA processing 285 G09
protein/S1 self-incompatibility locuslinked pollen G211 protein
[Oryza sativa] 196 SSBII- gi|13785467| 9.00E-26 phosphoenolpyruvate
carboxykinase 172 F06 (ATP-dependent) [Flaveria trinervia] 164
SSBII- gi|47169677| 2.00E-25 ferric reductase [Oryza sativa] 178
B02 210 SSBII- gi|39750999| 1.00E-24 unnamed protein product/Alpha-
232 H03 glucan water dikinase, chloroplast precursor
(Starch-related R1 protein) [Lolium perenne] 108 SSBI- gi|710308|
1.00E-23 victorin binding protein [Avena sativa] 166 B12 129 SSBI-
gi|50905199| 1.00E-23 cycloartenol synthase [Oryza sativa] 185 E06
112 SSBI- gi|32481061| 1.00E-23 Rubisco activase alpha form
precursor/ 233 C06 ribulose-bisphosphate carboxylase activase
[Deschampsia antarctica] 212 SSBII- gi|38347077| 9.00E-23
OSJNBa0006A01.18/unknown protein 161 H06 [Oryza sativa] 187 SSBII-
gi|50906397| 1.00E-22 zinc finger protein-like/GATA-1 zinc 231 E05
finger protein [Oryza sativa] 168 SSBII- gi|56784641| 4.00E-21
putative cation diffusion facilitator 9 167 C01 [Oryza sativa] 165
SSBII- gi|51964240| 3.00E-19 PREDICTED P0666E12.10 gene 278 B04
product [Oryza sativa] 202 SSBII- gi|48716905| 2.00E-18
apospory-associated protein C-like 129 G03 [Oryza sativa] 135 SSBI-
gi|710308| 2.00E-17 victorin binding protein [Avena sativa] 122 E12
207 SSBII- gi|50251471| 3.00E-17 unknown protein [Oryza sativa] 323
G09 154 SSBI- gi|50907447| 6.00E-17 unknown protein [Oryza sativa]
132 H08 169 SSBII- gi|50906979| 1.00E-15 putative GTP-binding
protein typA 134 C02 (tyrosine phosphorylated protein A)/
elongation factor family protein [Oryza sativa] 180 SSBII-
gi|119284| 1.00E-15 High molecular mass early light- 170 D07
inducible protein HV58, chloroplast precursor (ELIP) [Hordeum
vulgare] 197 SSBII- gi|121343| 2.00E-15 Glutamine synthetase shoot
isozyme, 109 F07 chloroplast precursor (Glutamate-- ammonia ligase)
[Oryza sativa] 121 SSBI- gi|53749331| 1.00E-13 putative
2-oxoglutarate/malate 128 D06 translocator [Oryza sativa] 170
SSBII- gi|119748| 7.00E-13 Fructose-1,6-bisphosphatase, cytosolic
121 C03 (D-fructose-1,6-bisphosphate 1- phosphohydrolase) (FBPase)
[Spinacia oleracea] 204 SSBI- gi|50905143| 7.00E-12 putative 50S
ribosomal protein L3 204 G06 [Oryza sativa] 188 SSBII- gi|11761654|
2.00E-11 peroxiredoxin/thioredoxin peroxidase 178 E06 CATP [Oryza
sativa] 196 SSBI- gi|32400293| 7.00E-11 hydroxyanthranilate 91 F06
hydroxycinnamoyltransferase 2 [Avena sativa] 173 SSBII-
gi|12060390| 2.00E-10 response regulator 7 [Zea mays] 148 C10 152
SSBI- gi|38345616| 4.00E-10 OSJNBb0003B01.8 (BAC 91 H06
clone)/unknown protein [Oryza sativa] 161 SSBII- gi|1769849|
3.00E-09 photosystem II type I chlorophyll a/b 166 A08 binding
protein [Apium graveolens] 181 SSBII- gi|15223823| 2.00E-08
armadillo/beta-catenin repeat family 204 D08 protein/unknown
protein [Arabidopsis thaliana] 96 SSBI- gi|82619| 6.00E-08
ribulose-bisphosphate carboxylase 212 A07 [Triticum aestivum] 201
SSBII- gi|50915986| 2.00E-07 putative phosphatidylinositol-4- 90
G02 phosphate 5-kinase [Oryza sativa] 104 SSBI- gi|31323256|
2.00E-07 photosystem II type I chlorophyll a/b 116 B08 binding
protein [Brassica oleracea] 146 SSBI- gi|50911901| 2.00E-07
putative DNA helicase/DNA-binding 82 G10 protein [Oryza sativa] 106
SSBI- gi|2196772| 4.00E-07 chlorophyll a/b-binding protein 116 B10
[Mesembryanthemum crystallinum] 172 SSBII- gi|50928287| 6.00E-07
OSJNBa0013K16.8/putative glutamate 113 C08 receptor [Oryza sativa]
200 SSBII- gi|133872| 2.00E-06 ribosomal protein S1, chloroplast
114 G01 30S precursor (CS1) [Spinacia oleracea] 'E-value <
1.00E-06 means the significant similarity with the protein
sequences in the Genbank database.
[0488] J. Predicted Function of Up-then-Down Regulated and
Transiently Expressed DEFs.
[0489] Five up-then-down regulated and four transiently expressed
DEFs were identified sharing significant similarity with proteins
in the public database (Table 6). The rieske Fe--S precursor
protein (SSBI-F10), a chlorophyll a/b-binding protein (SSBII-D5),
digalactosyldiacylglycerol synthase (SSBI-D3), and a disease
resistance protein (SSBI-D5) were up-regulated at the earlier
stress period and then turned down with the stress continuing,
indicating that these proteins may have a positive response to the
mild stress but were not retained during the severe stress. The
glutamine-dependent asparagine synthase, plasma membrane H+ ATPase,
small heat shock protein Hsp23.5, and type 2 metallothioneine were
temporally expressed at approximately day 4 stress suggesting the
transient regulation of these proteins might be critical for droug
ht stress response. Predicted function of up-then-down regulated
and transiently expressed DEF.
[0490] K. Transiently Expressed and Up-then-Down-Regulated.
[0491] DEFs including the novel ones should be analyzed with
breeding lines under more natural drought conditions to further
confirm the correlation of expression pattern of the transcripts
with drought tolerance. The particular functions of these DEFs need
to be studied by using knock-out mutants and transgenics, such as
over-expression, antisense suppression, and double-stranded RNA
interference (RNAi). It has been found that some genes induced by
drought stress have no effect on drought tolerance in transgenic
plants (Karakas et al., 1997, Plant cell and Environment. 20:609-6
16). Therefore, a challenge for future research is to distinguish
between gene products with a potential in osmoprotection and those
that are only involved in secondary reaction. The combination of
quantitative transcript profiles with an appropriate QTL analysis
could possibly lead to the identification of candidate genes for
agronomically valuable traits. Reverse genetic approach as well as
classical genetics will become more important to understand not
only functions of stress-inducible genes but also the complex
signaling process in environmental stress response.
TABLE-US-00007 TABLE 6 Functional annotation and size of the
differentially expressed fragments (DEFs) transiently expressed (T)
and up-then-down-regulated (UD) during drought stress in F. mairei.
Representative examples of Representative similarity examples of
SEQ ID hits in Description Organism Size NO: XX DEF GenBank E-value
Expression Pattern (bp) 179 SSBII- gi|53680379| 3.00E-51
glutamine-dependent T 290 D05 asparagine synthetase [Triticum
aestivum] 93 SSBI- gi|20302435 1.00E-33 plasma membrane H+ T 222
A10 ATPase [Oryza sativa] 153 SSBI- gi|4138869| 9.00E-16 small heat
shock T 128 H07 protein Hsp23.5 [Triticum aestivum] 151 SSBI-
gi|23954355| 8.00E-15 metallothioneine type2 T 287 H05 [Hordeum
vulgare] 141 SSBI- gi|32394644| 3.00E-33 putative Rieske Fe--S UD
259 F10 precursor protein [Triticum aestivum] 101 SSBI- gi|82682|
4.00E-31 chlorophyll a/b-binding UD 197 B05 protein precursor [Zea
mays] 118 SSBI- gi|50252668| 1.00E-21 putative UD 309 D03
digalactosyldiacylglycerol synthase [Oryza sativa] 120 SSBI-
gi|50943213| 5.00E-12 putative disease UD 231 D05 resistance
protein [Oryza sativa] 185 SSBII- gi|50912463| 8.00E-07 unknown
protein UD 84 E03 [Oryza sativa]
[0492] In summary, over one hundred DEFs identified from cDNA-AFLP
analysis were confirmed by macroarray hybridization analysis. Thus
the inventor showed that cDNA-AFLP technique coupled with
macroarray hybridization analysis was an efficient procedure in
detecting differentially expressed genes associated with responding
to drought stress. The DEFs provided herein are the first
transcripts derived from an Atlas fescue plants's response to
drought stress. The use of the methods of the present inventions
demonstrated the presence of a variety of drought responsive gene
responses showing a comprehensive molecular regulation level in
Atlas fescue plants that responded to drought stress.
[0493] Further, predicted functions of the sequences were
subdivided into 17 functional categories. Some DEFs discovered in
Atlas fescue are novel genes, which in combination with their
superior capability to resist drought effects showed that Atlas
fescue plants comprise novel compositions and mechanisms as a
defense against adverse effects of drought stress. The inventor
contemplated that the novel sequences provide a valuable resource
for future compositions and methods for use in specific types of
drought tolerant gene regulation in plants. During drought stress
treatment in Atlas fescue, increased metabolic function and
biogenesis of cellular components in the plant undergo a
degenerative process potentially causing the plant system to save
energy for new gene transcription and stress defense. The genes
isolated and characterized herein provide compositions and methods
for increasing protective mechanisms against desiccation tolerance
in plants. In particular for use in breeding programs of grass
plants for providing agronomically and/or economically desirable
grass plant cultivars.
Example IV
Breeding Methods for Providing Lp Plants COMPRISING Fm
Germplasm
[0494] An exemplary schematic for a breeding strategy for providing
drought resistant plants of the present inventions is shown in FIG.
10. In particular, 4.times.Fm1 plants were crossed with 2.times.Lp
plants to provide 4.times.F.sub.1 hybrid progeny plants for
subsequent breeding programs, including the plants provided herein.
Backcrosses were made in order to restore the desired agronomic
traits of the Lp grass plants while retaining drought resistant
germplasm from Fm plants. The introgression of germplasm was
tracked, (FIG. 11) by identifying ratios of Fm/Lp germplasm using
the RAPD and Lp SSR methods as described herein.
[0495] The capability of molecular markers, such as RAPD and SSR
markers, primers and linkers, to discriminate between Lolium and
Festuca DNA in hybrid and backcross plant progeny in combination
with locating these markers on a linkage map enabled the creation
of introgression maps. Thus combining genetic mapping and desired
physiological traits for identifying and using these methods in
breeding methods for providing drought tolerance in grass plants
(see, for example, Humphreys et al., 1997 New Phytol. 137:55-60;
Humphreys et al., 2005 Theor. Appl. Genet. 110:579-597; all of
which are herein incorporated by reference). Markers associated
with desired or undesirable trait components are contemplated for
application to assist in drought tolerant progeny selection and
decrease the time of the breeding process to provide desired plant
cultivars.
[0496] The inventor discovered that Fm-Lp plant progeny
successfully combined germplasm form both Fm and Lp plants and
further that progeny showed desirable agronomic traits in initial
greenhouse evaluation. Further, evaluation of Lp hybrid plants
using morphological criteria for demonstrating drought resistance
showed that certain hybrid plants showed superior levels of drought
resistance. These elite progeny, such as line G15 and G30a, are
contemplated as the basis of new cultivar release and for use in
more specific development of marker(s) associated with drought
tolerance.
Example V
Hybrid Superiority of Festuca mairei.times.Lolium perenne
Plants
[0497] This example is provided to show that the inventor
unexpectedly identified hybrid plants demonstrating superior
drought resistance traits using Principle Component Analysis (PCA)
and plots of the comparative eigenvectors comprising morphological
and physiological information from drought stressed plants.
[0498] Evaluation of eigenvector plots demonstrated that when grass
plants of the present inventions, including parental Fm, drought
tolerant Fescue species, and backcrossed Lp plants comprising Fm
germplasm, were grouped by their ability to resist drought stress,
several hybrid grass lines, specifically 2 backcrossed Lp plant
lines comprising Fm gennplasm, provided a group of plants that
showed a higher level of drought tolerance when compared to grouped
levels of drought resistance of parental grass plants, FIGS. 12-20,
specifically lines G15, G30a and a F.sub.1 hybrid of Atlas
fescue.times.Calypso (Lp cultivar).
[0499] A F.sub.1 hybrid from Atlas fescue.times.`Calypso` was used
as a female parent for backcrossing as described herein. The higher
drought tolerance of this F.sub.1 hybrid and the existence of a
drought-tolerant backcross progeny evidenced that this hybrid
contained drought tolerance genes from Atlas fescue.
Grouping of Plants Based Upon Drought Response Measurements:
[0500] First the irrigated and drought stressed plants fell into
two distinct groups by PCA based on leaf elongation, leaf water
content, and leaf water potential during the treatment period (FIG.
14a). One group included all of the control plants and the other
group included the drought stress treated plants, indicating the
successful treatment application and parameters' utility for such
grouping methods.
[0501] Genotypes of both irrigated control and non-irrigated plants
(19 plants) varied dramatically in response to drought treatment in
terms of soil water content, leaf elongation, leaf water content,
and leaf water potential (see, for example, FIG. 13). Forty drought
responsive variables, including 14 (week 1 to 14) variables of leaf
elongation, 14 variables of leaf water potential, and 12 variables
leaf water content, were used in PCA to classify the 19 plant
genotypes. The plot of the eigenvectors showed that leaf elongation
of week 3, 5, 6, and 8, and leaf water content at week 2 and 12
were relatively independent because they were relatively divergent
and separated by large angles in terms of closeness of angles from
the origin. The other variables were positively and closely
correlated to one or more variables by showing relative small
angles between or among them. For example, leaf elongation data of
weeks 1, 2, 10, 11, 12, 13, and 14, and leaf water potential data
of weeks 9, 10 11, 12, 13, and 14, and leaf water content data for
week 3 were positively and closely correlated showing relatively 8
small differences in angles. Leaf water potential data of weeks 7
and 8 were also highly correlated.
[0502] The PCA based on the plant responses to the drought
treatment revealed four groups of all genotypes. The first three
principal components accounted for 59% of variation. Group 1
included Kentucky 31, Atlas fescue founder plants, an F.sub.1
hybrid, an amphiploid, and five backcross progeny. Group 2 included
two perennial ryegrass parents and four backcross progeny. Group 3
contained an F.sub.1 hybrid and two backcross progeny. Group 4 had
only one backcross progeny, G24 (FIG. 15).
Morphological-Physiological Responses:
[0503] The soil water content of three groups declined steadily
during the 14-wk drought stress treatment (FIG. 16), but the effect
of drought duration, differed (P<0.05) among groups for weeks 5
to 13 as the soil dried faster for Group 3 than for Group 2 with
Group 1 being intermediate to the others. Leaf water content
decrease (FIGS. 16 and 18) tended to be inversely related to soil
water content during the treatment indicating the drought responses
of the three groups varied with drought treatment duration and was
reflected similarly by both soil and leaf water content. Even
though a difference among the three groups was noted in both soil
and leaf water content there was no significant difference among
leaf elongation rates (FIGS. 16 and 17). Leaf water potential,
among the three groups responded to the drought treatment similarly
except in weeks 6, 8, and 9 (FIGS. 16 and 19). Thus data were
evaluated at common soil water contents for group comparisons.
[0504] The regression analyses showed that the leaf elongation of
Group 2 decreased below the controls when the soil water content
was reduced to 3.8%, while leaf elongation of Groups 1 and 3
decreased until soil water content decrease to 2.5 and 1.98%,
respectively (FIG. 17). At the soil water content of 2%, the leaf
elongation of Group 3 had not begun to decrease, while Group 1 was
decreased about 0.21 cm/d and Group 2 decreased 0.44 cm/d. These
results suggest that genotypes in Group 2 were more sensitive to
drought stress in terms of leaf elongation, while Group 3 had the
most drought tolerance and Group 1 had moderate drought tolerance.
Leaf water potential showed no major differences among the three
groups as the soil water content declined (FIG. 19).
[0505] Group 4 was represented by only one genotype (G24). Leaf
water potential and especially leaf water content of Group 4,
compared with the other three groups, showed that G24 responded to
the 14-wk drought stress treatment in an aberrant way (FIG. 16).
This line was dropped from the majority of studies.
Soil Water Content Estimates of Water Depletion Rate:
[0506] Monitoring of the soil water content revealed a different
water depletion rate among the three groups (FIG. 16). Group 2
showed the lowest water depletion rate suggesting that the
genotypes in Group 2 consumed water slower, which was probably due
to a shallow root system and small plant size as they were
observed. Regardless of relative drought tolerance, the water
content and water potential may decline more rapidly for larger
versus smaller genotypes (Cregg, 2004, In: Fernandes et al., (ed.)
Pro Celsius. XXVI IHC-Nursery Crops, Can. Int. Dev. Agency; herein
incorporated by reference). Thus, interpretations were based on
common levels of soil water that were highly correlated with leaf
water content.
Unexpected Finding of Higher Tolerance to Drought in Hybrid
Plants:
[0507] Genetic differences in water use might affect the rate of
stress development, which can be reduced by growing the plants
together (Thomas, 1987, J. Exp. Bot. 38:115-125; herein
incorporated by reference) for an accessibility to the same soil
volume and soil water to control the uniformity of water stress
development (White et al., 1992, Crop Sci. 32: 25 1-256; herein
incorporated by reference). Leaf elongation, as a sensitive
parameter, was compared among the three groups at the same soil
water content (FIG. 17) and was an indicator of the order of
drought tolerance, Group 3>Group 1>Group 2. However, no
significant differences were found in leaf water potential and leaf
water content among the three groups. These results implied that
the three groups exhibited similarly in terms of leaf water content
and leaf water potential as the stress condition was becoming
severe, suggesting (1) the other two parameters were not sensitive
enough to significantly discriminate groups, or most likely (2) the
three groups applied a similar mechanism related to leaf water
status to respond to the drought stress treatment. Using leaf
elongation measurements Genotypes in Group 3 showed better drought
tolerance than Atlas fescue, the drought tolerant parent, which was
in Group 1. These results supported the discovery that Group 3
plants showed hybrid superiority.
Root System Development:
[0508] Drought stress significantly reduced the root biomass (FIG.
20) especially in Group 2, the drought sensitive group, by 51%, and
only 36% for drought tolerant Group 3. Reduction of root biomass
may be associated with root death and desiccation. Even though the
physiological factors influencing root death under drought stress
are not yet understood, considerable root death from drought stress
was reported in various plant species (Smucker et al., 1991, Ecol.
1:1-5; Stasovski and Peterson, 1991, Can. J. Bot. 69:1170-1178;
Huang and Nobel, 1992, J. Exp. Bot. 43:1441-1449; all of which are
herein incorporated by reference. Some species exhibit more
tolerance to drought with little root death (Kosola and Eissenstat,
1994, I Exp. Bot. 45:1639-1645; herein incorporated by reference).
In this study, a lesser amount of reduced root biomass in Group 3
further indicated that the genotypes in Group 3 had better drought
tolerance. Noticeably, even though root length of Group 2 was
enhanced, root biomass was significantly reduced by drought. The
results suggested that the Group 2 root system responded to drought
by producing a secondary thinner root with less biomass, and may be
subject to severe root death and loss of the stronger primary
root.
[0509] Large root systems help increase water uptake and can
increase water-use efficiency. Both the irrigated and non-irrigated
plants in Groups 1 and 3 had a significantly longer root system
(around 120 cm for both irrigated and non-irrigated plants) than
Group 2 (around 82 cm for irrigated and 102 cm for non-irrigated
plants) (FIG. 20). Root length responded differently to drought.
When comparing the non-irrigated to the irrigated plants in each
group, minimal change in root length was noted in Groups 1 and 3.
However, in Group 2 the root length of stressed plants was
significantly increased by 24%. Measurements of root biomass were
more variable, yet stressed plants of each group had less root mass
(P<0.05) for all groups (FIG. 20). The drought treatment
decreased root biomass by 48% for Group 1, 51% for Group 2, and 36%
for Group 3. When stressed, plants maintain or actually increase
root growth in length, but have thinner root with less biomass.
This response was most noticeable in Group 2.
[0510] The longer root system in Groups 1 and 3 (FIG. 20) suggested
these genotypes in Groups 1 and 3 are more capable to extract
available water, therefore are more tolerant to drought stress than
Group 2. In Group 2, the root of non-irrigated plants was
significantly longer than the irrigated plant, indicating that the
smaller root system of genotypes in Group 2 was triggered for a
secondary growth by drought stress. The root re-growth during
drought stress implied that the genotypes in Group 2 applied root
phenotypic adaptation to avoid drought stress. Enhanced root growth
during drought stress has been considered as an important
adaptation mechanism to improve efficiency of plant water uptake
(Gallardo et al., 1996, Plant Cell Environ. 19:1169-1178; herein
incorporated by reference). A reduction of root growth caused
merely by soil drying also results in low water uptake rates (Huang
and Gao, 2000, Crop Sci. 40:196-203; herein incorporated by
reference).
[0511] Root growth of genotypes of Groups 1 and 3 (FIG. 20) was not
enhanced. This might be due to a long root system that has occupied
much of the volume in the pot (100 cm high PVC tube). Kentucky 31 a
moderate drought tolerant plant and identified as a good drought
resistant cultivar (Huang and Gao, 1999, HortSci. 34:897-901;
herein incorporated by reference) was found to have a Group 1 level
of drought tolerance. IN a previous study, soil drying increased
root length of Kentucky 31 by 10% in the deep soil layer (Huang and
Gao, 2000, Crop Sci. 40:196-203; herein incorporated by reference).
The study of Huang and Gao (1999, 2000, supra) suggested that
Kentucky 31 showed a great potential for enhanced growth during
drought stress. Therefore, Groups 1 and 3 in this study might also
have potential to increase root length during drought, which may
have been limited by little available space for secondary growth in
the small one meter PVC tube.
[0512] Principle component analysis (PCA) involved a mathematical
procedure that transformed a set of correlated response variables
into a smaller set of uncorrelated variables (for example, Johnson
1998, In: Applied multivariate methods for data analysts.
Brooks/Cole Publishing Co. Pacific Grove, Calif.; herein
incorporated by reference). PCA analysis of the data obtained
during the course of the development of the present inventions
showed that a large portion of the response to drought provided
variables that were correlated or duplicated up to a certain point,
while some of the variables stood by themselves and had a greater
weight to evaluate the drought response of the genotypes. Of three
parameters measured, leaf elongation was more typical of drought
responses, because seven variables (during weeks 3 to 9) out of the
14 variables (during weeks 1 to 14) were relatively independent
from each other (see, for example, FIG. 11). Whereas, for leaf
water content, four (weeks 1, 2, 3, and 12) of 12 variables were
independent and for leaf water potential, three (weeks 1, 2, and 3)
of 14 variables were independent of each other. Leaf elongation is
mainly caused by turgor pressure of enlarging cells (Matyssek et
al., 1988, Plant Physio. 86:1163-1167; herein incorporated by
reference). Cell expansion, which directly contributes to the leaf
elongation, was reported as the most sensitive trait (Boyer, 1988,
Physiol. Plant. 73:311-316; herein incorporated by reference) and
is reduced by drought before any other physiological process
(Wardlaw, 1969, Aust. J. Biol. Sci. 22:1-16; herein incorporated by
reference). Additionally, results of this study confirmed the value
of using leaf elongation as a parameter for drought tolerance
evaluation in methods for identifying plant for breeding programs
described herein. Due to its sensitivity and high variability, thus
allowing greater distinctions and thus comparisons between plants
for drought resistance.
[0513] In summary, drought stress reduced leaf elongation, leaf
water content, and leaf water potential. Leaf elongation was a
sensitive and typical parameter for screening drought tolerant
plants during drought stress. The drought tolerance of Atlas fescue
was inherited in the progeny derived from intergeneric
hybridization between ryegrass and Atlas fescue. Some progeny
groups expressed higher levels of drought tolerance than groups
comprising comparable levels of drought tolerance of the parental
Atlas fescue plants.
Example VI
[0514] The use of molecular markers, primers, and linkers for
identifying plants for use in breeding programs.
[0515] This example is provided to demonstrate the capability of
SSR and RAPD markers to identify Fm germplasm within Lp hybrid
plants. RAPD and SSR markers, primers, and linkers were used to
overcome the problems in identifying specific genomic segments that
are found when using methods comprising fluorescence in situ
hybridization (FISH), which utilizes chromosome-specific DNA probes
and genomic in situ hybridization (GISH). Further, restriction
fragment length polymorphism (RFLP) based markers do not detect
alien DNA in a hybrid when a small portion of a chromosome or a few
chromosomes are transferred (Chen and Sleper, 1999, Crop Science
39:1676-1679; herein incorporated by reference).
[0516] A. The use of Festuca EST-SSR Markers for identifying Fm
Germplasm in plants for use in plant breeding methods of the
present inventions.
[0517] Simple sequence repeats (SSR) or microsatellite markers
developed from, Fescue and Lolium perenne L. and random amplified
polymorphic DNA (RAPD) markers of Fescue were used to assess
genomic introgression of Festuca mairei St. Yves (Fm) into L.
perenne (Lp) as briefly described in EXAMPLE I and below.
[0518] Seventy-six tall fescue EST-SSR primer pairs, including but
not limited to NFFA005; NFFA007; NFFA012; NFFA013; NFFA015;
NFFA017; NFFA019; NFFA021; NFFA022; NFFA024; NFFA029; NFFA031;
NFFA032; NFFA033; NFFA034; NFFA036; NFFA037; NFFA039; NFFA041;
NFFA047; NFFA048; NFFA050; NFFA052; NFFA057; NFFA059; NFFA061;
NFFA062; NFFA066; NFFA068; NFFA069; NFFA071; NFFA072; NFFA073;
NFFA074; NFFA075; NFFA076; NFFA077; NFFA084; NFFA087; NFFA090;
NFFA091; NFFA092; NFFA094; NFFA095; NFFA096; NFFA098; NFFA100;
NFFA103; NFFA108; NFFA109; NFFA113; NFFA114; NFFA120; NFFA123;
NFFA125; NFFA126; NFFA129; NFFA131; NFFA132; NFFA135; NFFA13;
NFFA140; NFFA142; NFFA143; NFFA146; NFFA147; NFFA149; NFFA150;
NFFA151; NFFA155; and NFFA157; and 32 Lp SSR primer pairs developed
from ryegrass were first tested on the Fm and Lp parents.
Preliminary screening showed that eight out of 32 ryegrass primer
pairs and 27 of 76 tall fescue EST-SSR primer pairs demonstrated
polymorphic bands (used as markers) between parental plants.
[0519] Specifically, amplification of ryegrass genomic SSRs and
tall fescue EST-SSRs in a preliminary screening panel showed 127
polymorphic bands scored from the 35 SSR primer pairs. The primer
combinations that produced polymorphic bands between parents were
then utilized to test plant materials. The 127 bands segregated
among the Fm-Lp progeny. More than half of the alleles of both
parents (Fm1 and Lp 1/Lp2) were combined in the 4.times.F.sub.1
hybrid (Fm1.times.Lp2) and the amphiploid derived from a
3.times.F.sub.1 (Fm 1.times.Lp1) crosses, indicating successful
wide crosses. In backcross plants, different levels of alleles of
both Fm and Lp parents were present in each individual plant which
demonstrated segregation of alleles from both parents during
backcrossing. Among the 127 bands, 23 (18%) were present in the
Fm-Lp progeny but not in the three parents (Fm1, Lp1, and Lp2). A
relative higher Fm1/Lp genome ratio showed that more Fm genome was
introgressed into the progeny than Lp. Progeny that showed an Fm/Lp
genome ratios above zero indicated that the Fm genome was
successfully introgressed into those individuals. However, the
ratios varied widely from 0.09 (014) to 1.95 (Fm1.times.Lp2)
indicating that the Fm genome had been retained in these progeny at
various extents.
[0520] Therefore, 27 EST-SSR primers pairs and 8 Lp SSR primers
demonstrated usefulness in analyzing progeny plants for
introgression of Fm germplasm.
[0521] B. The Use of RAPD Markers for identifying Fm Germplasm in
Plants for use in Plant Breeding Methods of the Present Inventions
(See, Wang et al., (2003) Crop Sci. 43:2154-2161; Herein
Incorporated by Reference in its Entirety).
[0522] Forty-one RAPD primers were chosen to detect genome
introgression of the backcross progeny. A total of 188
parent-specific markers were obtained. Ninety-two (49%) were
Fm-specific markers. The 13 backcross progenies showed a range of
introgression of Fm-specific markers (5.4-60.9%).
[0523] Two-hundred and twenty two polymorphic bands were generated
from 41 RAPD primers. The number of polymorphic bands scored for
each primer ranged from 1 to 11. Distribution of the 222 RAPD bands
among the parents was similar to that of the SSR markers.
Thirty-six bands (15.7%) were present in the progeny but not in the
three parents indicating the contribution of the Fm2 genome.
Ninety-six (41.9%) were Fm-specific and 87 (38.0%) were Lp-specific
bands including the Lp1- and Lp2-specific bands and the bands
common to both parents. Similar to the SSR results, a higher number
of common bands between Lp1 and Lp2 (41, 17.9%) suggested a
relatively close relationship between Lp1 and Lp2, and a lower
number of common bands between Fm and Lp (Lp, 4.5%; Lp2, 3.0%)
suggested a distant relationship between Fm and Lp.
[0524] RAPD results were consistent with SSR results as both
parent-specific bands were itherited in the F.sub.1 hybrids and
amphiploid, and various ratios of segregation occurred in backcross
progeny. Fm1 Lp genome ratios of these Fm-Lp progeny ranged from
0.08 (G11a) to 1.79 (Fm1.times.Lp2). This result confirmed that all
progeny retained the Fm genome at different levels. The correlation
coefficient (r=0.80) of the Fm/Lp genome ratios assessed by SSR and
RAPD markers was highly significant (P=0.0004), which reflected
upon the reliability of the two marker systems in assessing genome
introgression.
TABLE-US-00008 TABLE 7 The sequence of 41 RAPD primers and the
number of fragments amplified (see, Wang et al., (2003) Crop Sci.
43:2154-2161 herein incorpo- rated by reference in its entirety).
SEQ ID Number of polymorphic Primer NO:XX Sequence; 5'- 3'
fragments scored OPA-04 266 AATCGGGCTG 7 OPA-05 267 AGGGGTCTTG 4
OPA-07 268 GAAACGGGTG 6 OPA-08 269 GTGACGTAGG 6 OPA-20 270
GTTGCGATCC 4 OPB-12 271 CCTTGACGCA 9 OPC-01 272 TTCGAGCCAG 9 OPC-02
273 GTGAGGCGTC 5 OPC-04 274 CCGCATCTAC 2 OPC-05 275 GATGACCGCC 11
OPC-06 276 GAACGGACTC 3 OPC-07 277 GTCCCGACGA 5 OPC-08 278
TGGACCGGTG 11 OPC-09 279 CTCACCGTCC 9 OPC-10 280 TGTCTGGGTG 5
OPC-11 281 AAAGCTGCGG 7 OPC-13 282 AAGCCTCGTC 7 OPC-15 283
GACGGATCAG 7 OPC-16 284 CACACTCCAG 4 OPC-19 285 GTTGCCAGCC 3 OPC-20
286 ACTTCGCCAC 6 OPE-09 287 CTTCACCCGA 4 OPY-01 288 GTGGCATCTC 2
OPY-02 289 CATCGCCGCA 10 OPY-03 290 ACAGCCTGCT 6 OPY-05 291
GGCTGCGACA 7 OPY-06 292 AAGGCTCACC 5 OPY-07 293 AGAGCCGTCA 4 OPY-09
294 AGCAGCGCAC 1 OPY-10 295 CAAACGTGGG 4 OPY-13 296 GGGTCTCGGT 1
OPY-14 297 GGTCGATCTG 5 OPY-15 298 AGTCGCCCTT 3 OPY-16 299
GGGCCAATGT 4 OPY-17 300 GACGTGGTGA 6 OPY-18 301 GTGGAGTCAG 2 OPY-19
302 TGAGGGTCCC 5 OPY-20 303 AGCCGTGGAA 5 OPX-01 304 CTGGGCACGA 11
OPX-06 305 ACGCCAGAGG 4 OPX-13 306 ACGGGAGCAA 3 Total 222
[0525] RAPD results demonstrated that RAPD could be equally
effective and informative in monitoring the introgression of alien
DNA fragments, as compared to SSR markers. Random amplified
polymorphic DNA markers are also efficient in detecting genome
introgression because of its small tissue sample requirements,
rapid analysis, low cost, no previous knowledge of DNA sequence
requirement, and easy establishment. The potential for genome
labeling and chromosome tagging are contemplated for further
increases in sensitivity of detecting specific regions of DNA by
converting RAPD markers into sequence characterized amplified
regions (SCARs) (Paran and Michelmore, 1992, Theor. Appl. Genet. 8
5 985-993; herein incorporated by reference in its entirety).
[0526] C. The Use of Lp SSR Markers for Identifying Fm Germplasm in
Plants for Use in Plant Breeding Methods of the Present Inventions
(see, Wang et al., (2003) Crop Sci. 43:2154-2161; Herein
Incorporated by Reference in its Entirety).
[0527] Additionally, mapping Lp SSR markers in hybrid plants is
provided for use in methods for determining meiotic stability in
plants comprising Fm germplasm. Forty Lp SSR primer pairs were used
to detect polymorphism between the Lp and Fm parents. Of these, 19
primer pairs failed to produce a detectable amplification product.
Three primer pairs did not show distinguishable differences between
species with this method due to showing similarly sized amplified
products. Four SSR primers showed shared alleles between the
parents thus Fm was not fully distinguishable from Lp.
Amplification of the same size bands in both Fm and Lp parents
indicated a close relationship or certain homology between these
two genomes. One marker was unscorable in this test.
[0528] The remaining 13 markers covering seven linkage groups
(Table 8) fully discriminated Fm and Lp by amplifying completely
different sizes of SSR products from the parents. nine markers
detected that the Fm genome had introgressed in at least one place
of the genomes of the backcross individuals, because each of the
nine markers amplified the Fm-specific alleles in one or more
back-cross individuals. Out of the 13 backcross individuals
analyzed, 11 showed that the Fm genome fragments were introduced
and that the introgression levels ranged from 0 to 66.7%. This
demonstration showed significant DNA structural differentiation in
the genomes of the two species on the basis of these Lp SSR
markers. The 13 SSR markers were then used to test for Fm genome
introgression in additional progeny plant lines derived from
hybridization between Fm and Lp.
[0529] During the development of the present inventions, the Lp SSR
markers tested for a particular linkage group did not completely
detect simultaneous genome introgression into the individual
progeny. For example, at linkage group 7, three SSR primer pairs
(LPSSRK15F05, LPSSRK11E11, and LPSSK14F07) (Table 8) were used to
identify Fm germplasm, however only one primer (LPSSRK11E11)
revealed the Fm fragment in G8. Two primers (LPSSRK11E 11 and
LPSSRK14F07) showed a Fm allele in G15, but not all of the three
markers detected Fm alleles in G8 and G15. Such introgression
suggested that the mechanism of alien chromosome segments
transmission is due to genetic recombination through crossover
rather than substitution of whole or large segments of chromatin
from Fm. Otherwise, the SSR markers for the same linkage group
should have detected the Fm genome introgression in the same
individual at the same time.
[0530] During the development of the present inventions, it was
discovered that the introgression levels detected by Lp SSR and
RAPD markers were significantly correlated. In particular, the
introgression levels of the backcross progeny revealed by Lp SSR
and RAPD markers were significantly correlated at
P.ltoreq.0.0116.
TABLE-US-00009 TABLE 8 Lp SSR markers that revealed F. mairei
genome intro- gression into the backcross progeny (see, Wang et
al., (2003) Crop Sci. 43:2154-2161 herein incorpo- rated by
reference in its entirety). SSR markers (see, Jones et al., 2001,
Theoretical And Applied Lp Genetics 102 (2-3): linkage Expected
Progeny with 405-415). group alleles in F.sub.1 Fm alleles
LPSSRK03G05 3 G27a, G30b LPSSRK10B07 6 none none LPSSRK15F05 7
G27a, G27b LPSSRK12E06 2 none LPSSRK10F08 1 G8, G11b, G15, G24,
G26, G27a, G27b, G30a, G30b LPSSRH10G02 5 G15 LPSSRK08A09 3 G11b,
G15, G24, G27b, G30a LPSSRH02H05 6 none none LPSSRH03A08 2 none
LPSSRK02D08 4 G14, G16, G24, G26, G27a LPSSRK11E11 7 G8, G11b, G15,
G24, G30a, G30b LPSSRK14F07 7 G11b, G15, G27a, G27b LPSSRH02D10 2
and 6 G11b, G15, G24, (two G27a, G27b, G30a, loci) G30b
[0531] The ability of molecular markers to discriminate between
Lolium and Festuca DNA in hybrids and backcross progeny enables
introgression maps to be created when these markers were localized
on a linkage map. By combining the genetic mapping approach and
physiological complex trait dissection, it should be possible to
identify and localize the importance of trait components that
contribute to drought tolerance (Humphreys et al, 1997, New Phytol.
137:55-60; Humphreys et al., 2005, Theor. Appl. Genet. 110:579-597;
herein incorporated by reference). Markers associated with trait
components are contemplated for application to assist in drought
tolerant progeny selection and speed up the breeding process.
During the development of the present inventions, a number of Fm-Lp
progeny successfully combined both genomes from Fm and Lp and some
of those progeny showed desirable agronomic traits during initial
greenhouse evaluation. The results described herein demonstrate
that Fm-Lp progeny plants of the present invention are useful for
new grass plant cultivar release, plant breeding methods, and for
the development of marker(s) associated with drought tolerance.
[0532] D. Combining Lp SSR and RAPD Results for Evaluating Grass
Plant Progeny and Application of Fm-Lp Ratios in Grass Plant
Progeny for Use in Turfgrass Breeding.
[0533] Genome introgression levels of Fm and Lp determined by SSR
and RAPD ratios (FIG. 11) were favorably consistent with
morphological characteristics (FIGS. 14b and 15). These results
demonstrated that the two molecular techniques were efficient in
assessing alien genome introgression.
[0534] Specifically, backcross progeny plants tested using RAPD and
SSR markers showed different introgression levels of Fm DNA
fragments that were consistent with morphological characteristics.
For example, in G27a, which resembled Fm with rigid, long leaves as
compared to Lp with softer and shorter leaves, Fm DNA segments were
introgressed at five SSR loci out of the nine loci tested. Fm
genome introgression level was measured up to 66.7%. More than half
of 92 RAPD markers (introgression level at 60.9%) revealed Fm
chromatin introgressed in G27a. While in G11a, which resembled Lp
with soft and short leaves, none of the Fm genome was introgressed
at the nine SSR loci, and only five out of 92 RAPD markers (5.4%
introgression level) showed introgression of Fm chromatin.
Fm-Lp Genome Recombination in the 4.times.F.sub.1:
[0535] The partially fertile 4.times.F.sub.1 hybrid was found to be
useful in a backcross-breeding program to develop a diploid
perennial ryegrass, which was then tested for inheritance of
drought tolerance from Fm. Although 3.times.F.sub.1 hybrid plants
were sterile, fertility was largely restored through chromosome
doubling and therefore has potential use in developing new
cultivars.
[0536] With several generations of backcrossing and selection for
meiotic stability and turf quality, several drought tolerant
cultivars were developed. In particular, progeny G11a plants and
G14 plant lines recovered the majority of the Lp genome within one
generation of backcrossing to Lp. They were then tested for drought
tolerance and meiotic stability to evaluate the potential for a new
cultivar release. The drought resistance level of the line G14
grouped with the parental Atlas plants. The other backcross plants
required additioanl generations of backcrossing to Lp to recover
desired perennial ryegrass attributes.
[0537] An example of an application of SSR and RAPD analysis of
chromorsomal integration in hybrid plants is provided. In a
4.times.F.sub.1 hybrid plant derived from Fm1.times.Lp1, 84.3 and
90.6% of Fm-specific SSR and RAPD bands were inherited,
respectively. The inventor contemplated that at these loci, the
genotype of Fm, as an autotetraploid, is Aaaa, AAaa, AA.Aa or AAAA
while the Lp genotype is aa. As a result, the band ratios of
F.sub.1 are expected be 1:1 for Aaaa.times.aa, 5:1 for
AAaa.times.aa, 1:0 for AAAa.times.aa and 1:0 AAAA.times.aa. When
the four Fm genotypes among these loci showed the same ratio, then
on average, the F.sub.1 plant was estimated to comprise at least
83.2% Fm-specific bands.
[0538] The Chi-squared test (X.sup.2) test was used to test the
significance of consistency of observed Fm-specific bands presented
in the 4.times.F.sub.1 with the expected results. The analysis
revealed that, for both SSR and RAPD data, the observations were
consistent with the expectation (P=0.8 and P=0.05 for SSR and RAPD
data, respectively). Fm was considered an autotetraploid or at
least a partial allotetraploid because the genomes of M.sub.1 and
M.sub.2 are closely related and readily paired in the F.sub.1
hybrid plants of Fm.times.Lp (Chen et al. 1995, Crop Sci.
35:720-725; herein incorporated by reference).
[0539] The inventor's results supported the finding of
autotetraploidy in Fm plants. The genome of the other founder
parent, Lp1 was transferred into the 4.times.F.sub.1 by 75% and 53%
dominant alleles detected by SSR and RAPD, respectively. Lp1 was
transferred to the F.sub.1 through 2n pollen and the relatively
high rate of dominant alleles transferred suggested that the 2n
pollen were produced through first division restitution (FDR) (Chen
et al. 1997, Crop Sci. 462 37:76-80; herein incorporated by
reference). Fm-Lp genome recombination in progeny plants.
[0540] Improvement of any plant depends on the ability to
introgress the desirable genomes of source plants into cultivated
varieties (Prakash et al., 2002 Euphytica 124:265-271; herein
incorporated by reference). This strategy is largely facilitated by
precise monitoring of alien and cultivated genome combinations at
the molecular level. In backcross progeny detected in this study,
dominant alleles from both Fm and Lp parents were present in each
individual at various levels suggesting segregation of alleles of
both parents during backcrossing. The results were in agreement
with the finding that L chromosomes of Lp could pair with M.sub.1
and M.sub.2 chromosomes from Fm (Cao et al., 2000 Genome
43:398-403; herein incorporated by reference). The pairing of
homologous chromosomes would cause segregation of alleles of both
parents. Of the 27 polymorphic EST-SSR loci, 15 were mapped to
ryegrass linkage groups (LGs) (Wanke et al., 2004 Theor. Appl.
Genet. 109:294-304). Three groups of these marker loci were
uniquely mapped on both male and female maps (NFFA031 and NFFA075
on LG 1; NFFAO15, 036, and 048 on LG 6; NFFA019 and NFFA069 on LG
7).
[0541] To investigate the event of chromosome crossover between M
and L genomes, the co-segregation of markers on each of the three
LGs was assessed among the Fm-Lp hybrids and backcross individuals.
The results indicated that the linked markers were not co-inherited
into the hybrids or backcross individuals. The separation of the
linked markers demonstrated the crossover of homeologous
chromosomes and genome recombination between M and L in the progeny
from intergeneric hybridization. The mapped EST-SSR markers on
ryegrass LGs showed a great applicable value in assessment of the
homeologous chromosome crossover and genome recombination in the
intergeneric hybrid, which is usually tested by sophisticated
cytogenetic studies. In addition, the map location of EST-SSRs
derived from transcripts with known function would provide
functional genetic markers for direct characterization of the QTLs
for putatively correlated traits (Saha et al., 2005 Theor. Appl.
Genet. 110:323-336; herein incorporated by reference).
Example VII
Exemplary Protocol For Agrobacterium-Mediated Transformation of
Ryegrass
A. Media
[0542] The following media may be used in the protocols described
below. YEP solid medium: 10 g/L bacto-peptone, 10 g/L yeast
extract, 5 g/l NaCl, 15 g/l bacto-agar and Rifamycin (100 mg/L). 1
M acetosyringone stock: Dissolve 196.2 mg acetosyringone in 1 mL
DMSO; store at 4.degree. C. Add 0.01 ml (10 .mu.l) stock solution
to 100 mL medium for final concentration of 100 .mu.M. Inoculation
medium: 1/10 MS basal medium with Gamborg (B5) vitamins (Phytotech
Lab # M404), and 3% sucrose. 0.01% Silwet L-77 (Vac-in-Stuff): 0.01
ml (10 .mu.l) in 100 mL solution. Cefotaxime (250 mg/L): Dissolve 2
g Claforan in 8 ml sterile dH.sub.2O; store at 4.degree. C. Add 1
ml for 1 L after autoclave. PPT (20 mg/L): Dissolve 200 mg PPT in
10 mL. Aliquot 1 ml into 10 Eppendorf tubes (25 mg/ml each); store
at 4.degree. C. Add 100 .mu.l for 100 ml medium. Chlorophenol Red
(50 mg/l): Dissolve 500 mg clorophenol red in 10 ml. Aliquot 1 ml
into 10 tubes (50 mg/ml each); store at 4.degree. C. Add 100 .mu.l
for 100 ml medium.
B. Agrobacteria Preparation
[0543] On day 1, a fresh plate of A. tumefaciens strain EHA 105
(Rif.sup.r) is streaked from glycerol stock and incubated on
YEP+Rifamycin (selection) at 28.degree. C. On day 4, a loop of A.
tumefaciens strain EHA 105 (Rif.sup.r) is inoculated in 5 mL of
YEP+Rifamycin and incubated overnight at 175 rpm, 28.degree. C.
(starter culture). On day 5, one or 2 mL starter culture is added
to 100 mL of YEP+Rifamycin supplemented with mg/L (100 .mu.M)
acetosyringone and incubated overnight at 175 rpm, 28.degree. C. On
day 6, when OD.sub.600 is 0.8-1.0, the culture is centrifuged at
5000 rpm (9000 g) 10 min. Pellets are resuspended in Inoculation
Medium (MS-B5+3% sucrose) containing 100 .mu.M aceotsyringone
dissolved in DMSO. The final OD.sub.600 is adjusted to 0.2 for
inoculation.
C. Plant Callus Preparation
[0544] On day 6, one or two week-precultured mature seed-derived
embyrogenic cultures are prepared by transferring
microscopically-identified embyrogenic sectors to maintenance
medium on Whatman #1 paper filters. Embryogenic sectors range in
size from 2 to 3 mm and 0.1 g per filter. Plates are prepared right
before inoculation.
D. Inoculation
[0545] On day 6, 10 to 15 mL final inoculum is added to each plate
containing filters of callus tissue prepared the same day, making
sure to cover calli completely. Calli are loosened from the filter
paper via forceps. Plates are intermittently shaken manually for 10
min.
E. Co-Culture, Delay
[0546] On day 6, the inoculum is aspirated out using a sterile
pipette. Calli are transferred to another sterile Petri dish
containing a sterile 85 mm Whatman #1 filter. Inoculated calli,
approximately 20 pieces, are arranged in a 4-cm diameter circle. To
the center of the circle is added 200 .mu.L water using a
micropipette. Plates are placed in 22-24.degree. C. for 2 days in
the dark. On day 8, calli are transferred directly to delay
(maintenance/subculture) medium containing 250 mg/L cefotaxime, and
cultured 3 days in the dark at 22-24.degree. C.
F. Selection Culture
[0547] On day 11, calli are transferred to callusing/selection
medium (20 mg/L PPT) containing 250 mg/L cefotaxime. Plates are
maintained in the dark at 22-24.degree. C. On day 25, calli are
transferred to new selection medium (20 mg/L PPT) containing 250
mg/L cefotaxime. On day 39, calli are transferred to
callusing/selection medium (20 mg/L PPT) containing 250 mg/L
cefotaxime. On day 53, embryogenic sectors are transferred to
regeneration/selection medium (10 mg/L PPT) containing 100 mg/L
cefotaxime. One week in the dark and then move to illumination (16
h:8 h L/D) with covering with cheesecloth the first week. On day
85, sectors are transferred to rooting/selection medium (add 50
mg/L chlorophenol red) containing 100 mg/L cefotaxime. Chlorophenol
red will stay yellow if the shoots are transgenics, it will stay
purple if the shoots are negatives.
[0548] All publications and patents mentioned in the above
specification are herein incorporated by reference. Various
modifications and variations of the described method and system of
the invention will be apparent to those skilled in the art without
departing from the scope and spirit of the invention. Although the
invention has been described in connection with specific preferred
embodiments, it should be understood that the invention as claimed
should not be unduly limited to such specific embodiments. Indeed,
various modifications of the described modes for carrying out the
invention which are obvious to those skilled in the relevant fields
are intended to be within the scope of the following claims.
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 306 <210> SEQ ID NO 1 <211> LENGTH: 85 <212>
TYPE: DNA <213> ORGANISM: Festuca mairei <400>
SEQUENCE: 1 catgcatagg acccagtaat ggactgtaga gtaagttgtc ccgcgtgcga
cggcgtgtac 60 gcgtgttcgt gacacactga catcg 85 <210> SEQ ID NO
2 <211> LENGTH: 188 <212> TYPE: DNA <213>
ORGANISM: Festuca mairei <400> SEQUENCE: 2 catgcgctgt
tatttgccaa agctcgctgg ccattgtttt cccaccattg cccattcttg 60
gcacctcgca tcctgtcgtc actgagattg gaaagcgaaa tcagggccga ccgacggaca
120 cctccgacaa caacaacctc accaatcttg cacatcaagt cgtgacattc
aatagacgta 180 agctttcg 188 <210> SEQ ID NO 3 <211>
LENGTH: 122 <212> TYPE: DNA <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 3 catgccgtgc tacaggaggt agataatcag
ggtgcctaat tttggatggt gttttgtatg 60 caatatggcg tttgtgtgtt
agcatcacag attaatgagg gaatctctgg atgatttatt 120 cg 122 <210>
SEQ ID NO 4 <211> LENGTH: 132 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 4
catgcaccgg agataggcac tgcggaagcc ctgtaggaga cgccctgcct ccgaattggc
60 atcaacgcct ggaggtagaa catgagctgg cgttgatgaa gtgacaccaa
agctttccat 120 ggtcgccctt cg 132 <210> SEQ ID NO 5
<211> LENGTH: 101 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 5 catgtatcat gaaacaacgc
atggcagtgt tccttctttt taggttatag cttcactggc 60 ttcgctagct
ccaggtctcc aaaactcatc atcttttttc g 101 <210> SEQ ID NO 6
<211> LENGTH: 328 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 6 catgcgctct gacaaagggc
cgtggaagga ccctgaaatc ctaaaggtta atggttactc 60 tccgaagatt
ttggagttca tgcatgacct gtgggaaaat gtagtgcaga cttgccactg 120
aactgaagga agtagcagta ctactacagc taaactcctg agcacaggtc cattaacatt
180 ttcctggttt gattcttcag atggtgcaat gtggaatggg gagatgcgga
atgaacagct 240 cggaccttat atgcaggtat tatatggcct ttttcgccat
atttttctga acattgccgt 300 ctgatttcat acgcagaagc aagattcg 328
<210> SEQ ID NO 7 <211> LENGTH: 275 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 7
cgattccggt taaaggacgc tgggtgctgg gaaagcaggt cggcctcttt atggctgcct
60 gcgatggcca cgaaggcccc agcctgattc ctccccagat ttttggtctt
gctgtgctgg 120 tgtcatgtaa ctcatgtttg gatgccgttc aacaacttgg
tgaatgagca agttctgagc 180 tcaagtttcc atgtgtatga gtttccatgc
gtacgagtgt ctgttacggt tccgttatac 240 cggtatttat tgtctgtgtt
ggtttcttct gcatg 275 <210> SEQ ID NO 8 <211> LENGTH:
291 <212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 8 catgtttcag aactactcta tgaaaagcaa tgtgaaactc
tgggagttga acacaaacat 60 cacagagaag tttctgagaa tgcttctgtt
tagtttttat gtgaagatat tcccgtttcc 120 aaagacatct tcggagaggt
ccacatttcc acttgcagat tccacaaaaa gggagtttca 180 acactgctct
atccatagga gggttcaact ctgtgagttg aatgcaatca tcacagagaa 240
gtttcagaga aggcttctgt ctagatttta tgcgaagata tacccgtttc g 291
<210> SEQ ID NO 9 <211> LENGTH: 75 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 9
catgtgcaaa tctggcttgc acccgttgta tgcacacgtt gaatctatca cacacgatca
60 tcacgtgatg cttcg 75 <210> SEQ ID NO 10 <211> LENGTH:
77 <212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 10 cgatgatgcc gccgcaggag gcgtactcgc
agcaggggca gtcaatgcag cagtggtcgc 60 cgtcgtacct gtacatg 77
<210> SEQ ID NO 11 <211> LENGTH: 162 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 11
catgccaggt aatagccctg ttgaaacacc cagggcattg tgtacttgtt tatttttctt
60 tttgcttgta aatgctacag cccctcggat gaatcacaac caccgtggtt
gctatgtgtg 120 gtggtaatga ccagtctatt ggttgagccc agcataaatt cg 162
<210> SEQ ID NO 12 <211> LENGTH: 186 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 12
catgcccccc acaggtgttg tcggtggtac atttgtcgga gatgctaatc taactgtaac
60 agcgggtatt acaccgtcgt atatcctaca agagggtgat cttactgccg
actggactat 120 tacatttggg cagaacgatc aatttggtcg tcccgtccta
gacggagcta gttttagaat 180 ctatcg 186 <210> SEQ ID NO 13
<211> LENGTH: 113 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 13 cgattagatc gtgggaggaa
tgtctttttc caattttgga agggcttaat catattaccg 60 acccggcaat
attttcggat cggagggagt actgatcttt ttcaccgcgc atg 113 <210> SEQ
ID NO 14 <211> LENGTH: 81 <212> TYPE: DNA <213>
ORGANISM: Festuca mairei <400> SEQUENCE: 14 cgaaacggcg
cgcagtaaac tccttcgttt acgcgcaagt ggagaaaatg ggccgggcgc 60
accgattcct tcctccacat g 81 <210> SEQ ID NO 15 <211>
LENGTH: 138 <212> TYPE: DNA <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 15 catgtgtaaa ggtgcataca aatctgaacc
aagaattact tttaatgcat gaactgtgaa 60 catctaccgg atggtaggta
tcatttttcg tgctaagcgc aaatcttcgt aaaccatgta 120 gtcgttcagg actcatcg
138 <210> SEQ ID NO 16 <211> LENGTH: 78 <212>
TYPE: DNA <213> ORGANISM: Festuca mairei <400>
SEQUENCE: 16 catgcagagg attgaacgac actagtatat gggtgtcctc gtagtgtttt
cctttgcacg 60 tgggtgtcca cttattcg 78 <210> SEQ ID NO 17
<211> LENGTH: 69 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 17 cgaatagcaa atggccgagg
tccctgctgc atgaacctga tctgctgcga aatgagagct 60 gggcgcatg 69
<210> SEQ ID NO 18 <211> LENGTH: 166 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 18
cgatggcagc tagggcgcta ggcagcgcgc tacggcagct ggcagccagc aggctcaagg
60 tgcgtggacg ggctaatcag ggcgtgcggg cgtgcgttgc aagcaggagg
ccggaggctg 120 gcgttcaggc ggatgacgcg cagaaggctg ggccgaaagg tgcatg
166 <210> SEQ ID NO 19 <211> LENGTH: 116 <212>
TYPE: DNA <213> ORGANISM: Festuca mairei <400>
SEQUENCE: 19 catgcctagc tgtgcgcacc tgttcgccga tgagtacgcc accacgggca
accaaacaat 60 acccgtcacg ctgccgcgcc tatggcagcc atcggatggc
tattggggta ttatcg 116 <210> SEQ ID NO 20 <211> LENGTH:
545 <212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 20 catgtacact ggtataccac cggatacgta
cattgaatta agaacaccgc catcagacgc 60 aactacttta acagaactat
tgataccatc ggctgcccga acaccgtcat agcagaaatg 120 tccagtatgg
ttaccgtacg tgtcgccacc aataatacca ccgatataag cggacttaac 180
gttggcaccc aggtataccg cacaggcatt atctccggta tagtagttac tgttgttgta
240 gtctttaata tcggtaacga tcattacacg acgagcattt tcaagccaga
cgggattgcc 300 tacaaatgta gacatttgcc cgccagtcca actaatagag
ttcacagacc cattagtggc 360 ggtagaacct ttgtcactaa tagctcagta
gttattaccc tgacccaggg ctgaacaacc 420 cgtaaagtgg attgaagcta
cttgtccact tgtatcttca ttttcaaaac gaatacaagc 480 atcattagtg
ccgataccgc atggatcaaa tgtactgttg acaaaattca agtcttgaat 540 gatcg
545 <210> SEQ ID NO 21 <211> LENGTH: 165 <212>
TYPE: DNA <213> ORGANISM: Festuca mairei <400>
SEQUENCE: 21 catgcatgaa tgccacatga atgcaagaaa ggtaaaaagg gtctaggtgt
tactcttggg 60 atgttacacg tagtgtggtg cgaccatgaa cttggcgaag
gcctggcggc caagcacgca 120 gtggaacggg ctgtggaagc tgacgacctc
gttcaggact catcg 165 <210> SEQ ID NO 22 <211> LENGTH:
211 <212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 22 catgcatata tgcagacatg acacataaca
cagccgccac cggcgacatg gctgacagta 60 ctcatctagc tcatccgtac
atcggctata agtacatcgg ctataagcgg tagcataatt 120 acagttgtgt
agagaactgg tgagcactat cagtatgtac tatctactca ccagtagcta 180
gttcggttcg gctagagcgc cttacagatc g 211 <210> SEQ ID NO 23
<211> LENGTH: 126 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 23 catgtatgtg ttggacacca
tgtatagggg cggatgggcg gttatgtagg gatctcatat 60 cagctatgat
ctggttgctg ttccgtatct ttggatgacc accaggaggg gctcagcacc 120 cgctcg
126 <210> SEQ ID NO 24 <211> LENGTH: 153 <212>
TYPE: DNA <213> ORGANISM: Festuca mairei <400>
SEQUENCE: 24 cgagtcaact caaaggttaa tttttgctgg ccttgctgta gagaagctag
tgatgaaatt 60 aagcaaggta gcttgttgat taagttgtaa tcaagacagt
aactagtata ggtagcccca 120 cacactactt tgcaggttca gtttagatgc atg 153
<210> SEQ ID NO 25 <211> LENGTH: 68 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 25
catgtacaag tataacaccc accagatcgc ctcctctgct tcggatcagg agctcatgaa
60 agcgctcg 68 <210> SEQ ID NO 26 <211> LENGTH: 97
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 26 cgatggcatt ggcgttcatg tccaggaagg
caaggtgcca tctcagagag ctagcagacc 60 agcaaacggg aggagcccgt
cgccgagcta caacatg 97 <210> SEQ ID NO 27 <211> LENGTH:
147 <212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 27 catgtgtaca tttcctctgt actcctccct
actccgacgc tagactgaat cgctgatcac 60 atatatccac ggtcaaaaca
cttgcttcac tcttcctcct gcgcgtgaat ccgatggacc 120 ctgatgggga
ggcccttggg gaagtcg 147 <210> SEQ ID NO 28 <211> LENGTH:
119 <212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 28 catgtgctaa ataaaacatt ttggatatgc
tagtacaaat gtggtctgga tgctcgcata 60 tagaagcaag gtccataaga
gcgacaattg gaagatcaag ctagcattgt gtggtctcg 119 <210> SEQ ID
NO 29 <211> LENGTH: 108 <212> TYPE: DNA <213>
ORGANISM: Festuca mairei <400> SEQUENCE: 29 cgagttcccg
gccttctgat ccaatccaat ccaaggagtc gtgtcatgcc ttcctgattc 60
ctagctattc atttgctctt cccttacaga ataaactgtg ggagcatg 108
<210> SEQ ID NO 30 <211> LENGTH: 101 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 30
cgaccgtagg gctagtaata caattgtgtg gagccacttg gcttgtgagc atactataca
60 ctcccctaca catttatcat cccttactag aagtgcacat g 101 <210>
SEQ ID NO 31 <211> LENGTH: 299 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 31
catgctcacc acagggccaa ggaagaggcc aaagtgcccc tgaacgagtc caccctgctg
60 ctggatccat ccatccatct cctcctcaac ggacctacgg caccctgcct
aattgcctag 120 atgtgttctc gtgtagcttc cctctgctcc tgctagttag
tttttttttt tttgaacagg 180 ctagttagct agtgtgatgc gtattgtctg
ttggattcgc gtgctgtacg tgcctgaagc 240 tacgtatatg ttgtcgttgt
cagcttgtaa gagtaatgtt ctgctagcca ggatcgtcg 299 <210> SEQ ID
NO 32 <211> LENGTH: 169 <212> TYPE: DNA <213>
ORGANISM: Festuca mairei <400> SEQUENCE: 32 catgcgcaac
acggaaatta cacacatacg gagtataatt tacagataca atcaacactg 60
cgttcgtgcg aacatatatg tgaatttatc ggtggaacgc tcctcggaat cttgaaacga
120 tcaagcgccg gaaaccaccg ccgccgcagg ctgatcgccg ggaacgtcg 169
<210> SEQ ID NO 33 <211> LENGTH: 71 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 33
catgctactt tggggcctcc aacgcactct cgatctgctg gattagctga tcagcaggca
60 acgctcactc g 71 <210> SEQ ID NO 34 <211> LENGTH: 204
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 34 catgttttgg tttccagata attaacttgt
gccggagtac gaatacgtcg tcacccccac 60 ccaggatagg tcctagtatt
gataatctag ccggatgcaa tgcgctagtc gtatttaatt 120 agccactgtt
ctctcgttgt gctgcagaat tgtaaagatg tgtaagctgt agtgcacatg 180
gagcagcttc agtaccagtt ctcg 204 <210> SEQ ID NO 35 <211>
LENGTH: 177 <212> TYPE: DNA <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 35 catgttagcg taaaacacgt tcttgatcgg
cttgcgctta atcccgataa ggtctgcgtt 60 ttcctggtga gggtcatggc
cctctgtgta gaatcgcttg cccagttcgg gattcggcag 120 aaagtgagcg
aaacatcgca tctgagcggc cgcagcatcg tagccgacca gtactcg 177 <210>
SEQ ID NO 36 <211> LENGTH: 88 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 36
catgcccgtt ccaggcttcc agcagatctg ttgtcatcta ctcatctgtc agtgccgggt
60 gcgaaccaag gcctcccacc cgaactcg 88 <210> SEQ ID NO 37
<211> LENGTH: 161 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 37 catgctagga tgatgacgtt
ggcaacagcg tcgcgttaga gtggggccgg gcgcgtggac 60 tgccgttagt
aatgcgagct cgtacaacat ctacgagaag ctacccgtgg cgacgacgat 120
ggtcctcctc cgtggtcgtc acgatctcct ccaggtcgtc g 161 <210> SEQ
ID NO 38 <211> LENGTH: 335 <212> TYPE: DNA <213>
ORGANISM: Festuca mairei <400> SEQUENCE: 38 cgacaacgag
gcaacgtggc ttgatttgag aggaccaagc ctggtgtgct ggccaggtag 60
aagtgctact cgttttgctc actggtaagg cacgtcgccc agatattttt agctaatgcc
120 taagcggcgg gcggcaagat attttacaca gtttgagcgg ctagattttt
agctgacttg 180 ggaaccgacg ttgagcacct atatatagat agccttgccg
cttctgcggc tgctaacatc 240 agtagactgc aaatagagct ggacctacca
aacgagagtg agagagtaga gaaagagagc 300 gagagaaggg ccggtgaaga
tcattcatgc gcatg 335 <210> SEQ ID NO 39 <211> LENGTH:
23 <212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 39 cgagggaacg cgtctacaac atg 23 <210>
SEQ ID NO 40 <211> LENGTH: 28 <212> TYPE: PRT
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 40 Arg
Cys Gln Cys Val Thr Asn Thr Arg Thr Arg Arg Arg Thr Arg Asp 1 5 10
15 Asn Leu Leu Tyr Ser Pro Leu Leu Gly Pro Met His 20 25
<210> SEQ ID NO 41 <211> LENGTH: 28 <212> TYPE:
PRT <213> ORGANISM: Festuca mairei <400> SEQUENCE: 41
Asp Val Ser Val Ser Arg Thr Arg Val His Ala Val Ala Arg Gly Thr 1 5
10 15 Thr Tyr Ser Thr Val His Tyr Trp Val Leu Cys Met 20 25
<210> SEQ ID NO 42 <211> LENGTH: 27 <212> TYPE:
PRT <213> ORGANISM: Festuca mairei <400> SEQUENCE: 42
Met Ser Val Cys His Glu His Ala Tyr Thr Pro Ser His Ala Gly Gln 1 5
10 15 Leu Thr Leu Gln Ser Ile Thr Gly Ser Tyr Ala 20 25 <210>
SEQ ID NO 43 <211> LENGTH: 62 <212> TYPE: PRT
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 43 Cys
Ala Val Ile Cys Gln Ser Ser Leu Ala Ile Val Phe Pro Pro Leu 1 5 10
15 Pro Ile Leu Gly Thr Ser His Pro Val Val Thr Glu Ile Gly Lys Arg
20 25 30 Asn Gln Gly Arg Pro Thr Asp Thr Ser Asp Asn Asn Asn Leu
Thr Asn 35 40 45 Leu Ala His Gln Val Val Thr Phe Asn Arg Arg Lys
Leu Ser 50 55 60 <210> SEQ ID NO 44 <211> LENGTH: 62
<212> TYPE: PRT <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 44 Arg Lys Leu Thr Ser Ile Glu Cys His Asp
Leu Met Cys Lys Ile Gly 1 5 10 15 Glu Val Val Val Val Gly Gly Val
Arg Arg Ser Ala Leu Ile Ser Leu 20 25 30 Ser Asn Leu Ser Asp Asp
Arg Met Arg Gly Ala Lys Asn Gly Gln Trp 35 40 45 Trp Glu Asn Asn
Gly Gln Arg Ala Leu Ala Asn Asn Ser Ala 50 55 60 <210> SEQ ID
NO 45 <211> LENGTH: 44 <212> TYPE: PRT <213>
ORGANISM: Festuca mairei <400> SEQUENCE: 45 Arg Arg Ala Thr
Met Glu Ser Phe Gly Val Thr Ser Ser Thr Pro Ala 1 5 10 15 His Val
Leu Pro Pro Gly Val Asp Ala Asn Ser Glu Ala Gly Arg Leu 20 25 30
Leu Gln Gly Phe Arg Ser Ala Tyr Leu Arg Cys Met 35 40 <210>
SEQ ID NO 46 <211> LENGTH: 43 <212> TYPE: PRT
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 46 Glu
Gly Arg Pro Trp Lys Ala Leu Val Ser Leu His Gln Arg Gln Leu 1 5 10
15 Met Phe Tyr Leu Gln Ala Leu Met Pro Ile Arg Arg Gln Gly Val Ser
20 25 30 Tyr Arg Ala Ser Ala Val Pro Ile Ser Gly Ala 35 40
<210> SEQ ID NO 47 <211> LENGTH: 96 <212> TYPE:
PRT <213> ORGANISM: Festuca mairei <400> SEQUENCE: 47
Met Phe Gln Asn Tyr Ser Met Lys Ser Asn Val Lys Leu Trp Glu Leu 1 5
10 15 Asn Thr Asn Ile Thr Glu Lys Phe Leu Arg Met Leu Leu Phe Ser
Phe 20 25 30 Tyr Val Lys Ile Phe Pro Phe Pro Lys Thr Ser Ser Glu
Arg Ser Thr 35 40 45 Phe Pro Leu Ala Asp Ser Thr Lys Arg Glu Phe
Gln His Cys Ser Ile 50 55 60 His Arg Arg Val Gln Leu Cys Glu Leu
Asn Ala Ile Ile Thr Glu Lys 65 70 75 80 Phe Gln Arg Arg Leu Leu Ser
Arg Phe Tyr Ala Lys Ile Tyr Pro Phe 85 90 95 <210> SEQ ID NO
48 <211> LENGTH: 96 <212> TYPE: PRT <213>
ORGANISM: Festuca mairei <400> SEQUENCE: 48 Lys Arg Val Tyr
Leu Arg Ile Lys Ser Arg Gln Lys Pro Ser Leu Lys 1 5 10 15 Leu Leu
Cys Asp Asp Cys Ile Gln Leu Thr Glu Leu Asn Pro Pro Met 20 25 30
Asp Arg Ala Val Leu Lys Leu Pro Phe Cys Gly Ile Cys Lys Trp Lys 35
40 45 Cys Gly Pro Leu Arg Arg Cys Leu Trp Lys Arg Glu Tyr Leu His
Ile 50 55 60 Lys Thr Lys Gln Lys His Ser Gln Lys Leu Leu Cys Asp
Val Cys Val 65 70 75 80 Gln Leu Pro Glu Phe His Ile Ala Phe His Arg
Val Val Leu Lys His 85 90 95 <210> SEQ ID NO 49 <211>
LENGTH: 24 <212> TYPE: PRT <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 49 Met Cys Lys Ser Gly Leu His Pro Leu
Tyr Ala His Val Glu Ser Ile 1 5 10 15 Thr His Asp His His Val Met
Leu 20 <210> SEQ ID NO 50 <211> LENGTH: 24 <212>
TYPE: PRT <213> ORGANISM: Festuca mairei <400>
SEQUENCE: 50 Lys His His Val Met Ile Val Cys Asp Arg Phe Asn Val
Cys Ile Gln 1 5 10 15 Arg Val Gln Ala Arg Phe Ala His 20
<210> SEQ ID NO 51 <211> LENGTH: 25 <212> TYPE:
PRT <213> ORGANISM: Festuca mairei <400> SEQUENCE: 51
Asp Asp Ala Ala Ala Gly Gly Val Leu Ala Ala Gly Ala Val Asn Ala 1 5
10 15 Ala Val Val Ala Val Val Pro Val His 20 25 <210> SEQ ID
NO 52 <211> LENGTH: 25 <212> TYPE: PRT <213>
ORGANISM: Festuca mairei <400> SEQUENCE: 52 Met Met Pro Pro
Gln Glu Ala Tyr Ser Gln Gln Gly Gln Ser Met Gln 1 5 10 15 Gln Trp
Ser Pro Ser Tyr Leu Tyr Met 20 25 <210> SEQ ID NO 53
<211> LENGTH: 25 <212> TYPE: PRT <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 53 Met Tyr Arg Tyr Asp Gly Asp
His Cys Cys Ile Asp Cys Pro Cys Cys 1 5 10 15 Glu Tyr Ala Ser Cys
Gly Gly Ile Ile 20 25 <210> SEQ ID NO 54 <211> LENGTH:
25 <212> TYPE: PRT <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 54 Cys Thr Gly Thr Thr Ala Thr Thr Ala Ala
Leu Thr Ala Pro Ala Ala 1 5 10 15 Ser Thr Pro Pro Ala Ala Ala Ser
Ser 20 25 <210> SEQ ID NO 55 <211> LENGTH: 61
<212> TYPE: PRT <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 55 Met Pro Pro Thr Gly Val Val Gly Gly Thr
Phe Val Gly Asp Ala Asn 1 5 10 15 Leu Thr Val Thr Ala Gly Ile Thr
Pro Ser Tyr Ile Leu Gln Glu Gly 20 25 30 Asp Leu Thr Ala Asp Trp
Thr Ile Thr Phe Gly Gln Asn Asp Gln Phe 35 40 45 Gly Arg Pro Val
Leu Asp Gly Ala Ser Phe Arg Ile Tyr 50 55 60 <210> SEQ ID NO
56 <211> LENGTH: 37 <212> TYPE: PRT <213>
ORGANISM: Festuca mairei <400> SEQUENCE: 56 Arg Leu Asp Arg
Gly Arg Asn Val Phe Phe Gln Phe Trp Lys Gly Leu 1 5 10 15 Ile Ile
Leu Pro Thr Arg Gln Tyr Phe Arg Ile Gly Gly Ser Thr Asp 20 25 30
Leu Phe His Arg Ala 35 <210> SEQ ID NO 57 <211> LENGTH:
26 <212> TYPE: PRT <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 57 Glu Thr Ala Arg Ser Lys Leu Leu Arg Leu
Arg Ala Ser Gly Glu Asn 1 5 10 15 Gly Pro Gly Ala Pro Ile Pro Ser
Ser Thr 20 25 <210> SEQ ID NO 58 <211> LENGTH: 26
<212> TYPE: PRT <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 58 Lys Arg Arg Ala Val Asn Ser Phe Val Tyr
Ala Gln Val Glu Lys Met 1 5 10 15 Gly Arg Ala His Arg Phe Leu Pro
Pro His 20 25 <210> SEQ ID NO 59 <211> LENGTH: 27
<212> TYPE: PRT <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 59 His Val Glu Glu Gly Ile Gly Ala Pro Gly
Pro Phe Ser Pro Leu Ala 1 5 10 15 Arg Lys Arg Arg Ser Leu Leu Arg
Ala Val Ser 20 25 <210> SEQ ID NO 60 <211> LENGTH: 26
<212> TYPE: PRT <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 60 Met Trp Arg Lys Glu Ser Val Arg Pro Ala
His Phe Leu His Leu Arg 1 5 10 15 Val Asn Glu Gly Val Tyr Cys Ala
Pro Phe 20 25 <210> SEQ ID NO 61 <211> LENGTH: 45
<212> TYPE: PRT <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 61 Met Ser Pro Glu Arg Leu His Gly Leu Arg
Arg Phe Ala Leu Ser Thr 1 5 10 15 Lys Asn Asp Thr Tyr His Pro Val
Asp Val His Ser Ser Cys Ile Lys 20 25 30 Ser Asn Ser Trp Phe Arg
Phe Val Cys Thr Phe Thr His 35 40 45 <210> SEQ ID NO 62
<211> LENGTH: 25 <212> TYPE: PRT <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 62 Asn Lys Trp Thr Pro Thr Cys
Lys Gly Lys His Tyr Glu Asp Thr His 1 5 10 15 Ile Leu Val Ser Phe
Asn Pro Leu His 20 25 <210> SEQ ID NO 63 <211> LENGTH:
23 <212> TYPE: PRT <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 63 Arg Ile Ala Asn Gly Arg Gly Pro Cys Cys
Met Asn Leu Ile Cys Cys 1 5 10 15 Glu Met Arg Ala Gly Arg Met 20
<210> SEQ ID NO 64 <211> LENGTH: 22 <212> TYPE:
PRT <213> ORGANISM: Festuca mairei <400> SEQUENCE: 64
Asn Ser Lys Trp Pro Arg Ser Leu Leu His Glu Pro Asp Leu Leu Arg 1 5
10 15 Asn Glu Ser Trp Ala His 20 <210> SEQ ID NO 65
<211> LENGTH: 23 <212> TYPE: PRT <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 65 His Ala Pro Ser Ser His Phe
Ala Ala Asp Gln Val His Ala Ala Gly 1 5 10 15 Thr Ser Ala Ile Cys
Tyr Ser 20 <210> SEQ ID NO 66 <211> LENGTH: 22
<212> TYPE: PRT <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 66 Met Arg Pro Ala Leu Ile Ser Gln Gln Ile
Arg Phe Met Gln Gln Gly 1 5 10 15 Pro Arg Pro Phe Ala Ile 20
<210> SEQ ID NO 67 <211> LENGTH: 22 <212> TYPE:
PRT <213> ORGANISM: Festuca mairei <400> SEQUENCE: 67
Cys Ala Gln Leu Ser Phe Arg Ser Arg Ser Gly Ser Cys Ser Arg Asp 1 5
10 15 Leu Gly His Leu Leu Phe 20 <210> SEQ ID NO 68
<211> LENGTH: 54 <212> TYPE: PRT <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 68 Met Ala Ala Arg Ala Leu Gly
Ser Ala Leu Arg Gln Leu Ala Ala Ser 1 5 10 15 Arg Leu Lys Val Arg
Gly Arg Ala Asn Gln Gly Val Arg Ala Cys Val 20 25 30 Ala Ser Arg
Arg Pro Glu Ala Gly Val Gln Ala Asp Asp Ala Gln Lys 35 40 45 Ala
Gly Pro Lys Gly Ala 50 <210> SEQ ID NO 69 <211> LENGTH:
38 <212> TYPE: PRT <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 69 Met Pro Ser Cys Ala His Leu Phe Ala Asp
Glu Tyr Ala Thr Thr Gly 1 5 10 15 Asn Gln Thr Ile Pro Val Thr Leu
Pro Arg Leu Trp Gln Pro Ser Asp 20 25 30 Gly Tyr Trp Gly Ile Ile 35
<210> SEQ ID NO 70 <211> LENGTH: 38 <212> TYPE:
PRT <213> ORGANISM: Festuca mairei <400> SEQUENCE: 70
Cys Leu Ala Val Arg Thr Cys Ser Pro Met Ser Thr Pro Pro Arg Ala 1 5
10 15 Thr Lys Gln Tyr Pro Ser Arg Cys Arg Ala Tyr Gly Ser His Arg
Met 20 25 30 Ala Ile Gly Val Leu Ser 35 <210> SEQ ID NO 71
<211> LENGTH: 22 <212> TYPE: PRT <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 71 Met Tyr Lys Tyr Asn Thr His
Gln Ile Ala Ser Ser Ala Ser Asp Gln 1 5 10 15 Glu Leu Met Lys Ala
Leu 20 <210> SEQ ID NO 72 <211> LENGTH: 22 <212>
TYPE: PRT <213> ORGANISM: Festuca mairei <400>
SEQUENCE: 72 Glu Arg Phe His Glu Leu Leu Ile Arg Ser Arg Gly Gly
Asp Leu Val 1 5 10 15 Gly Val Ile Leu Val His 20 <210> SEQ ID
NO 73 <211> LENGTH: 32 <212> TYPE: PRT <213>
ORGANISM: Festuca mairei <400> SEQUENCE: 73 Asp Gly Ile Gly
Val His Val Gln Glu Gly Lys Val Pro Ser Gln Arg 1 5 10 15 Ala Ser
Arg Pro Ala Asn Gly Arg Ser Pro Ser Pro Ser Tyr Asn Met 20 25 30
<210> SEQ ID NO 74 <211> LENGTH: 31 <212> TYPE:
PRT <213> ORGANISM: Festuca mairei <400> SEQUENCE: 74
Met Ala Leu Ala Phe Met Ser Arg Lys Ala Arg Cys His Leu Arg Glu 1 5
10 15 Leu Ala Asp Gln Gln Thr Gly Gly Ala Arg Arg Arg Ala Thr Thr
20 25 30 <210> SEQ ID NO 75 <211> LENGTH: 32
<212> TYPE: PRT <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 75 His Val Val Ala Arg Arg Arg Ala Pro Pro
Val Cys Trp Ser Ala Ser 1 5 10 15 Ser Leu Arg Trp His Leu Ala Phe
Leu Asp Met Asn Ala Asn Ala Ile 20 25 30 <210> SEQ ID NO 76
<211> LENGTH: 35 <212> TYPE: PRT <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 76 Ser Ser Arg Pro Ser Asp Pro
Ile Gln Ser Lys Glu Ser Cys His Ala 1 5 10 15 Phe Leu Ile Pro Ser
Tyr Ser Phe Ala Leu Pro Leu Gln Asn Lys Leu 20 25 30 Trp Glu His 35
<210> SEQ ID NO 77 <211> LENGTH: 33 <212> TYPE:
PRT <213> ORGANISM: Festuca mairei <400> SEQUENCE: 77
Asp Arg Arg Ala Ser Asn Thr Ile Val Trp Ser His Leu Ala Cys Glu 1 5
10 15 His Thr Ile His Ser Pro Thr His Leu Ser Ser Leu Thr Arg Ser
Ala 20 25 30 His <210> SEQ ID NO 78 <211> LENGTH: 33
<212> TYPE: PRT <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 78 Thr Val Gly Leu Val Ile Gln Leu Cys Gly
Ala Thr Trp Leu Val Ser 1 5 10 15 Ile Leu Tyr Thr Pro Leu His Ile
Tyr His Pro Leu Leu Glu Val His 20 25 30 Met <210> SEQ ID NO
79 <211> LENGTH: 33 <212> TYPE: PRT <213>
ORGANISM: Festuca mairei <400> SEQUENCE: 79 Cys Ala Leu Leu
Val Arg Asp Asp Lys Cys Val Gly Glu Cys Ile Val 1 5 10 15 Cys Ser
Gln Ala Lys Trp Leu His Thr Ile Val Leu Leu Ala Leu Arg 20 25 30
Ser <210> SEQ ID NO 80 <211> LENGTH: 23 <212>
TYPE: PRT <213> ORGANISM: Festuca mairei <400>
SEQUENCE: 80 His Ala Thr Leu Gly Pro Pro Thr His Ser Arg Ser Ala
Gly Leu Ala 1 5 10 15 Asp Gln Gln Ala Thr Leu Thr 20 <210>
SEQ ID NO 81 <211> LENGTH: 7 <212> TYPE: PRT
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 81 Arg
Gly Asn Ala Ser Thr Thr 1 5 <210> SEQ ID NO 82 <211>
LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 82 Glu Gly Thr Arg Leu Gln His 1 5
<210> SEQ ID NO 83 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Festuca mairei <400> SEQUENCE: 83
Arg Glu Arg Val Tyr Asn Met 1 5 <210> SEQ ID NO 84
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 84 His Val Val Asp Ala Phe Pro
1 5 <210> SEQ ID NO 85 <211> LENGTH: 7 <212>
TYPE: PRT <213> ORGANISM: Festuca mairei <400>
SEQUENCE: 85 Cys Cys Arg Arg Val Pro Ser 1 5 <210> SEQ ID NO
86 <211> LENGTH: 59 <212> TYPE: PRT <213>
ORGANISM: Festuca mairei <400> SEQUENCE: 86 His Val Ser Val
Lys His Val Leu Asp Arg Leu Ala Leu Asn Pro Asp 1 5 10 15 Lys Val
Cys Val Phe Leu Val Arg Val Met Ala Leu Cys Val Glu Ser 20 25 30
Leu Ala Gln Phe Gly Ile Arg Gln Lys Val Ser Glu Thr Ser His Leu 35
40 45 Ser Gly Arg Ser Ile Val Ala Asp Gln Tyr Ser 50 55 <210>
SEQ ID NO 87 <211> LENGTH: 59 <212> TYPE: PRT
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 87 Arg
Val Leu Val Gly Tyr Asp Ala Ala Ala Ala Gln Met Arg Cys Phe 1 5 10
15 Ala His Phe Leu Pro Asn Pro Glu Leu Gly Lys Arg Phe Tyr Thr Glu
20 25 30 Gly His Asp Pro His Gln Glu Asn Ala Asp Leu Ile Gly Ile
Lys Arg 35 40 45 Lys Pro Ile Lys Asn Val Phe Tyr Ala Asn Met 50 55
<210> SEQ ID NO 88 <211> LENGTH: 58 <212> TYPE:
PRT <213> ORGANISM: Festuca mairei <400> SEQUENCE: 88
Glu Tyr Trp Ser Ala Thr Met Leu Arg Pro Leu Arg Cys Asp Val Ser 1 5
10 15 Leu Thr Phe Cys Arg Ile Pro Asn Trp Ala Ser Asp Ser Thr Gln
Arg 20 25 30 Ala Met Thr Leu Thr Arg Lys Thr Gln Thr Leu Ser Gly
Leu Ser Ala 35 40 45 Ser Arg Ser Arg Thr Cys Phe Thr Leu Thr 50 55
<210> SEQ ID NO 89 <211> LENGTH: 29 <212> TYPE:
PRT <213> ORGANISM: Festuca mairei <400> SEQUENCE: 89
His Ala Arg Ser Arg Leu Pro Ala Asp Leu Leu Ser Ser Thr His Leu 1 5
10 15 Ser Val Pro Gly Ala Asn Gln Gly Leu Pro Pro Glu Leu 20 25
<210> SEQ ID NO 90 <211> LENGTH: 29 <212> TYPE:
PRT <213> ORGANISM: Festuca mairei <400> SEQUENCE: 90
Met Pro Val Pro Gly Phe Gln Gln Ile Cys Cys His Leu Leu Ile Cys 1 5
10 15 Gln Cys Arg Val Arg Thr Lys Ala Ser His Pro Asn Ser 20 25
<210> SEQ ID NO 91 <211> LENGTH: 28 <212> TYPE:
PRT <213> ORGANISM: Festuca mairei <400> SEQUENCE: 91
Cys Pro Phe Gln Ala Ser Ser Arg Ser Val Val Ile Tyr Ser Ser Val 1 5
10 15 Ser Ala Gly Cys Glu Pro Arg Pro Pro Thr Arg Thr 20 25
<210> SEQ ID NO 92 <400> SEQUENCE: 92 000 <210>
SEQ ID NO 93 <211> LENGTH: 222 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 93
catgcacaga ggacactcca tgggttgcag ccaccggatg ccaagctgtt ccccgagaag
60 gcaggctaca acgagctgaa tcagatggct gaagaggcaa aacggagagc
tgaaattgca 120 aggctcaggg agcttcacac tctcaagggg cacgtagagt
cggttgtgaa gctgaagggc 180 ctggacattg acaccattca gcaatcttac
acagtgtgat cg 222 <210> SEQ ID NO 94 <211> LENGTH: 194
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 94 catgttcgtc aacgaggttt acacggttct
gaccgatccg gtgcagcgtg ccgtgtatga 60 tgagctccat ggctacgcag
caacggccgc caaccctttc tttaatgaca gtgcgcccaa 120 ggatcacgtc
tttgttgacg agtttacctg tataggatgc aagatttgtg ccaatgtgtg 180
ccccaatgtg ttcg 194 <210> SEQ ID NO 95 <211> LENGTH:
228 <212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 95 catgcgcctc taaatcttca gcatggcctc
caacgcgcga agtacgtcat cacgggtaac 60 tatacctatc acttggttgt
cctgatttac tattggtaat ctgtggatct tcttcttgag 120 catcagagct
gcggcatcgg tcactgttct atcacatgat agcgtgatcg ctggagaggt 180
catcacttgt gcaatctttg tccttgaccc atatgaagcc cttgttcg 228
<210> SEQ ID NO 96 <211> LENGTH: 212 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 96
catgttcctt ctacgttgat aggtacggtg catacacaca caattatatg tggaaataaa
60 agtaaaaccc ggaaagcgga gttgtcatca aaaactaaac caagagactc
catatggatt 120 cctagctcgc agcttatgcc ttgccggact cctcacagcc
tggtggcttg aaggcgatga 180 agctgacgca ctgcacctga cggatgttat cg 212
<210> SEQ ID NO 97 <211> LENGTH: 152 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 97
catgcgcttt acaataatca tgatgtatca gtagaaatcg gctcttgtac aaattattac
60 acgaatgaca gacgccacaa ggcgcgtaac gtggggtact ctttccaaaa
taggcgcagt 120 actttctagc atcgggtaat taatccttat cg 152 <210>
SEQ ID NO 98 <211> LENGTH: 378 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 98
catgtactac agagtatgcg attccagcct gtttccgaaa cctgccttac agaacagcac
60 atggaagttt atgtacctct tccaaatatc atctcctcaa actgaaccag
gcttgcctaa 120 tattccatat aacccatgat cctaccgtaa ttgctgactg
aaccaactag tatttccatc 180 ttacagcttg ccagcaaata cctgcagtaa
aacttttgtc tatctgcatc tgaagatcca 240 ggcctcccat gcaagtagtc
atcaaaattg tacccgagat cgtcagcagc tcactggtca 300 actgagttaa
ccagttttga caaaaacgaa ttccaattac gctctgtcca cattactgca 360
caatgcgctt ccttttcg 378 <210> SEQ ID NO 99 <211>
LENGTH: 114 <212> TYPE: DNA <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 99 catgcgcgtt ccaccagctc ccacagcgca
ttgggtatgc cagttctatt gaaaccatcc 60 accgttatga acatcccgca
gaagaagatc aacagcgagt aggatacctt ttcg 114 <210> SEQ ID NO 100
<211> LENGTH: 151 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 100 catgccctta aaccaccatt
aataatgcca ttattctcag caaaaacaaa cgcctgctct 60 tccaaccctc
acccgggcac aaaacataac aaatcctccg cctagacaga ctgtaagata 120
atgcaaaaaa aaaaggatag ttgacaattc g 151 <210> SEQ ID NO 101
<211> LENGTH: 197 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 101 cgaactcgcg gttcttggcg
aaggtctcgg ggtcggcgga cagcccagcg gtgtcccagc 60 catagtcgcc
ggggaactcg ccggtgaggt agctcggggg ctcgccggag agcgggccga 120
ggtagagcac gcggtcggag ccgtaccacg ggctgccgga cgcggccacc ttgggcttgc
180 cggccgtctt gcgcatg 197 <210> SEQ ID NO 102 <211>
LENGTH: 248 <212> TYPE: DNA <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 102 cgaagttgtg gttccccggc ttgtcgctga
cataaatcgg gcagccgccg atcgcccttg 60 cggcgccgtg gtactccgcc
gccgggtgca agctatgaaa catatcccag tcgggctgca 120 tgaactcgcc
gaggaagagg gtgttgtaag ccacggagga gatgtggatg gtatgcgacg 180
ccgggtcgtg cgggtagaag tcgtcggagg cgcgcacgac ggctgtctgc ctggcgctgt
240 agagcatg 248 <210> SEQ ID NO 103 <211> LENGTH: 286
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 103 catgtcctga gttgataaac tggttgggtt
ttgagcagca gcctggtatt tctgtacaac 60 cgcgccaagc tgactaacct
gcggtattat tgacctccgt ggaataagtt cctcatgacg 120 cctagcacag
aactcccaag atgcgatctt gaggtctgga ttaaaaatta tcctcagatg 180
cccctcacgt acgacacgca attgatcaaa gacactttct tgaattgctt ttgtatagtc
240 cagaacaatc tgaccagata cattcttgga ctcacgtggc atatcg 286
<210> SEQ ID NO 104 <211> LENGTH: 116 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 104
catgcggctc acaggcatgg ccgtagtgga tgccttactt gccgggaaca aagttggtgg
60 cgaacgccct tgcgttgttg ttgacagggt cagcgaggtg gtcggcgagg ttatcg
116 <210> SEQ ID NO 105 <211> LENGTH: 263 <212>
TYPE: DNA <213> ORGANISM: Festuca mairei <400>
SEQUENCE: 105 cgatcagcgg tgtgacgagc gtggccgtga acccgaagat
gagcagggtg acggtgacgg 60 ggtacgtgga gccgcgcaag gtgctggaga
aggttaagag cacggggaag gcggcagaga 120 tgtggccgta cgtgccctac
accatggcca cctaccccca cgtcggcggc gcctacgaca 180 agaaggcacc
ggcgggcttc atccggagcg cgccgcaggc catggccgcc cccggggcgc 240
cagaggtcca gtacatgaac atg 263 <210> SEQ ID NO 106 <211>
LENGTH: 116 <212> TYPE: DNA <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 106 cgatcacgtt gctgacccag tcaacaacaa
cgcatgggct ttcgccacca acttcgctcc 60 tggaagttaa atgagttagc
catccgtccg accaccggcc gggcgagata tgcatg 116 <210> SEQ ID NO
107 <211> LENGTH: 165 <212> TYPE: DNA <213>
ORGANISM: Festuca mairei <400> SEQUENCE: 107 catgcgcgga
gcacgcgtac aaatttacat ttcacaccca cacccttgca tatatacctc 60
tcgcacgcac acaggtatac catgcacagg acgacgatgc ttttggccta gtggaacttg
120 aggctggtga ggatgttgtt gttgacgggg tcggggaggt gatcg 165
<210> SEQ ID NO 108 <211> LENGTH: 166 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 108
catgctaaag attgggtgag ttaggtaggg gctgtcgcgc acaaggctgc taggaatgga
60 gcttgagact tcaggtgcaa tggattcagc tgtgaagccc actggctttc
caccagagaa 120 caccttgaac agctggtcaa catcctccaa ggtggtggtc tcatcg
166 <210> SEQ ID NO 109 <211> LENGTH: 310 <212>
TYPE: DNA <213> ORGANISM: Festuca mairei <400>
SEQUENCE: 109 cgaataagga agcattaaag tcaggctgaa ctccatgtgt
gcaatatatg gtttgcctag 60 tccagcgaaa tcaagttgta gcagatgttg
gcacttatgc ggttgtccta gagaagtaga 120 agaagcttag ataacgagtt
ctccggttag ctacactcct ctcagtcttg actgtgttct 180 tacaagagat
ggctgcagcg cgtcatagtg cccataaccg tcgtagagaa cacggactgg 240
attatccttg gcatactcct gaccatattc tgctatgatc ctggggccat cagacccctt
300 ggtgtacatg 310 <210> SEQ ID NO 110 <211> LENGTH:
206 <212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 110 cgaacaaggc ctgcatctct ctttgcttca
catctgacga ccctgctcca atcactatgt 60 ctatgattat cacctttccc
ccatcctctc ttgaaggaat agctttcttg cagttcttta 120 gtatcttgac
acactcttgg tcgccccagt catgcataac ccacttgagg aagacaacgt 180
ttgccggagg aacgctctca aacatg 206 <210> SEQ ID NO 111
<211> LENGTH: 449 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 111 cgaagaagag tacctgcacg
atgacacgca gagggagacg ctcattctga gcggcgtggg 60 tgcaagcctc
cagggagagt ttctggcagt ccattacacg gcaaagttct tccttctctg 120
actccgggag atggggatgc gccttcagat agatgtcaac agcacgataa agtccatcgt
180 ctattggccg agcataatct ggtatggcag cagccaaaga cttgaacttt
ggcaacttta 240 ggttggcatc tggcgcaact tcagctaggt agccgtcaat
caacttagca accatagtta 300 ccggcattag agatggagaa gctaatagtt
gcccatcgtc gccaaggcca ggggaagttc 360 caccagtttc ttgatccatt
gccaagaagt gtccaagaat cctatggacg caatccacat 420 cgtagagcgt
ttcatcagat tcagacatg 449 <210> SEQ ID NO 112 <211>
LENGTH: 233 <212> TYPE: DNA <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 112 catgcttgtc caggagcagg acaatgttaa
gcgtgtgcag cttgctgaca cttacatgag 60 ccaggcagct ctgggtgatg
ctaaccagga tgccacgaag actggttcct tctacggtta 120 gaacactctt
catacaccca ccatctctag ctgcatagga ggaggtaaag gagcacaaca 180
aagaactttg cctgtgccgg aaggttgtac cgaccgggaa gccaagaact tcg 233
<210> SEQ ID NO 113 <211> LENGTH: 206 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 113
cgaacaaggc ctgcatctct ctttgcttca catctgacga ccctgctcca atcactatgt
60 ctatgattat cacctttccc ccatcctctc ttgaaggaat agctttcttg
cagttcttta 120 gtatcttgac acactcttgg tcgccccagt catgcataac
ccacttgagg aagacaacgt 180 ttgccggagg aacgctctca aacatg 206
<210> SEQ ID NO 114 <211> LENGTH: 251 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 114
catgttgaac agcctttgcc gcgacaagaa caacatggtc ttgctcgcta gcacgaagac
60 tcgggcgatg ttaagcgaat ggttttcgcc atgtgagaac ctagggctgg
ctgctgagca 120 cggctatttc ctcaggctga gaggagatgc agagtgggag
acgtgcgctc ctgcgcctga 180 ctctggctgg aagcagattg tggagcctgt
gatgaaaacc tacacggaga caaccgacgg 240 gtcaacgatc g 251 <210>
SEQ ID NO 115 <211> LENGTH: 280 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 115
catgctatcc gatcagagca gcaactcatt tggctctacc gactttgggt gggatgatga
60 ggccatgaca ccggactaca catccgtctt cgttccaaat gctgccatgc
cagcatatgg 120 cgggcccgct tacctgcaag gcggagcgcc aaagaggatg
aggaacaatt tcggtgtagc 180 tgtgcttcct cagggaaatg atgcgccaca
agatgtctgt gcttttgacc atgagatgaa 240 gtattcactg ccttacgttg
agagtagctc agacggatcg 280 <210> SEQ ID NO 116 <211>
LENGTH: 199 <212> TYPE: DNA <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 116 catgtactta ccagctagct gttggtccgg
tcgtcgttaa gaagcaatta accacagctt 60 aattgaagtg atcgtgacga
gtaactaaac caaactaggg taggtagacg gacgggtccg 120 ggacgtccgt
ccagcagctc ccggcgttcc agtacgcggc cggcgacgcg tcgtccccga 180
gctcgttcag gactcatcg 199 <210> SEQ ID NO 117 <211>
LENGTH: 76 <212> TYPE: DNA <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 117 catgtgtggt ctctagagga acttgaacag
caggcctccg tacgtggcaa agaacggcgc 60 aaacacaagg gattcg 76
<210> SEQ ID NO 118 <211> LENGTH: 309 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 118
catgcgacag gtagtagtac aaaccaacag actacaagat tagtcaggac aacagctaca
60 gagcgtattc ctactatgta cacatatatg gcaccatcta tacgtagtag
taacttaatg 120 tgtgcaatgc atgtccacat caccagccat atacagggtg
ctgtacctgg ggaggcagca 180 ggcccatatc agcacggtgt tgttcatcgt
agtcacgtgt accaggaatc gcgcctgtag 240 ccaacctgag gacctcacta
gaggtcaggc aacgatgcgc aaatgccgat ccactgtcca 300 cgacattcg 309
<210> SEQ ID NO 119 <211> LENGTH: 273 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 119
catgtggtat ccagggtcca tttatccaca caaatgcaca atcggcaata catacgtaag
60 cacagactgg tcactgggtt cagcgatgaa tactgatcac tgggtttcaa
ggctggggca 120 tttgttcagt gcttgtgttt tggcttcttc acaatcatca
ccgtgcagtg cgcgtgatgg 180 gtgcagtaat cgctcacact tccaagaaca
gcccttttaa ttgctccata gccatggttg 240 cccacaacca acatctccgc
gtgatgccgt tcg 273 <210> SEQ ID NO 120 <211> LENGTH:
231 <212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 120 catgtggtgc tgtctctcca cgaagtgaag
gtgaagctga agccaaagcg cttttgacat 60 ggaagtctac tttgatgttc
tccgacgtca acggctcttc tccgctctcg tcatgatcac 120 cggccaactc
cctctgcaat tcttggtctg gcatcacgtg caacacggct ggccatatcg 180
tggagctcac ggttcccgga gctggtgtcg caggcacgct ggacgccttc g 231
<210> SEQ ID NO 121 <211> LENGTH: 128 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 121
cgcagggcaa actgcaagca gatgcaaagg atcctgagac agcactggcc ccaagcattg
60 tagagatctc tccagcacca agtttgtctc tgagtaactc aacactctgg
tcataggaag 120 cgagcatg 128 <210> SEQ ID NO 122 <211>
LENGTH: 103 <212> TYPE: DNA <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 122 catgttaagg cttatagcaa tgtgaacaag
tatctactta ctggtaggta cagaaacatc 60 atggacatga acgcaggctt
tgggggtttc gctgcagcga tcg 103 <210> SEQ ID NO 123 <211>
LENGTH: 256 <212> TYPE: DNA <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 123 catgctaagc gcactgtttc ataaatataa
tgttgtgcag acgatgataa atacagtaga 60 tgcaaccaga ggcgactggt
aacccagctt cattatccag ggaagtgggc gaacccttgg 120 tcctaaagca
gtcgctcact gcttaggaga gtgccaagga tcaatctgat ctcacaggag 180
atgcagaacc ggataagctc ttgctagggc ttctgctcac tgattttcca ggagaggccg
240 aagaaactgg ggatcg 256 <210> SEQ ID NO 124 <211>
LENGTH: 156 <212> TYPE: DNA <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 124 ccatgatgct gaagggatgg tgaatgttat
atctgagaag gaaactgaca gaatcctcgg 60 cgtacacatt atgtcccctg
gcgcgggaga gatcatccat gaggctgtgc ttgcgcttca 120 gtatggagct
tccagcgagg acattgcccg tacatg 156 <210> SEQ ID NO 125
<211> LENGTH: 120 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 125 cgacagcaac ggacagggtg
tacagcgggg ccaccttata gtcaactacc agttcgtcaa 60 ctgcggcgac
aacgagctgc tgctccagcg cgaagagaaa taagaagcta ccagtacatg 120
<210> SEQ ID NO 126 <211> LENGTH: 306 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 126
cgaatgagaa cgcggcatcc tccgtcgcca agaccatctc cggtcagccc ccactgaaga
60 taccgatcag gagcgacaac gccgggtcct ggctgctcac aacctacctt
gatgacgagc 120 ttagaatctc cagaggagat ggcagcagca tctttgtgct
gttcaaggaa gggagcactc 180 tcttaatata ggcttacgtg tatctcttct
cagagtagaa tttgggcgaa tccaatagat 240 agttgtggct atgtgtttgt
tttgttagcc cgtgcgttta tagttcgttc ttgtgtgttg 300 tgcatg 306
<210> SEQ ID NO 127 <211> LENGTH: 168 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 127
catgcgtaga attcttcgcg aagtaaacac gacatgatac gtacgtaaag catcacgtat
60 acgtagctaa tctcggttga ttctgtcctc gcaacctaca taaactggct
gcaaggacgc 120 ggtactagtt aatttcgcaa aaagtatatc ggccacgtgt acgattcg
168 <210> SEQ ID NO 128 <211> LENGTH: 230 <212>
TYPE: DNA <213> ORGANISM: Festuca mairei <400>
SEQUENCE: 128 cgaactcgtg ccgcagcgtg gcgacccaga acccgacggt
ggcgatggcg agcgacttgc 60 cggggcagct ccgcctgccc gacccgaacg
gcgccagcct gaggtctgag cccgttatgg 120 agaactcggc ggcgccggcg
tgatcccgcg acggtccggc gaggaaccgg tcaggcctga 180 actctgccgg
ctcggtccag acggccgggt cgtgcgttat ggcccacatg 230 <210> SEQ ID
NO 129 <211> LENGTH: 185 <212> TYPE: DNA <213>
ORGANISM: Festuca mairei <400> SEQUENCE: 129 cgaagggaag
agatagacaa atgtatccat aaagccgatg gcttcattca gagtattcaa 60
agaagtgacg gttcatggta cggctcctgg ggtgtttgtt tcacatatgg gacatggtat
120 gcagtgaggg gattagttgc cgctggaagg acattcaaga actgtcctgc
tatcaggaag 180 gcatg 185 <210> SEQ ID NO 130 <211>
LENGTH: 201 <212> TYPE: DNA <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 130 cgataattgc tcttatcgtc tgcataccac
cagcagttat tattgaaggt ccccaactta 60 tgcagtatgg attaaatgac
gcaattgcaa aagtaggtct gacaaagttt gtttcagacc 120 ttttcctggt
cggactgttc taccatctgt ataaccagct tgctacaaac acattggagc 180
gggtggcccc tctgacacat g 201 <210> SEQ ID NO 131 <211>
LENGTH: 390 <212> TYPE: DNA <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 131 catgcgtgaa gatggaggtt ttgaagtgat
taagaaagca atcctgaacc tttcacttcg 60 tcacgacttg cacataagtg
aatatggtga aggaaatgaa cggaggttga cagggttaca 120 tgagacagct
agcatatcag acttttcatg gggtgtagca aaccgtggtt gctctattcg 180
ggtggggcga gacactgagg caaaagggaa aggatacctg gaagaccgtc gtccggcctc
240 aaacatggac ccatacactg tgacggccct actggctgaa accacaattc
tctgggagcc 300 gacccttgaa gcagaggctc ttgctgccaa gaagctggcg
atgaacgtat gaaggactga 360 aaaggatgaa tttctgggga aaataaatcg 390
<210> SEQ ID NO 132 <211> LENGTH: 69 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 132
catgttcggc ggcggcaagt tcaagaagtg gaagtaatct gccagtagct ttccatagct
60 gatggatcg 69 <210> SEQ ID NO 133 <211> LENGTH: 238
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 133 catgcccgcc aacaaccgga acactgactt
ctctaggatc aactccaagc acttcagcca 60 caaaggtgtt agccctcgct
acatcaagag ttgtcactcc aaggagacgt ttggggcagt 120 aagttccagc
cctcttgaaa actttcgccg caatggggac agttgagttc acagggttgc 180
tgatcaaatt cataattgca ttagggcagc tcttggcaac gccctcacag attgatcg 238
<210> SEQ ID NO 134 <211> LENGTH: 119 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 134
catgtacacg caagcccccc taatacaggt cgccttcctt gtgggtgtgg atgatgcagt
60 cagacttggg gtatgagacg caggtcagca cgtagccttc ctcctgctgg ttgtcatcg
119 <210> SEQ ID NO 135 <211> LENGTH: 122 <212>
TYPE: DNA <213> ORGANISM: Festuca mairei <400>
SEQUENCE: 135 catgtaccac actgagcacg agcttctccg ttacctacac
aagttgcaaa ccaaggatct 60 ctcactgtgc cacagtatga ttcctcttgg
ttcttgcacc atgaaactaa atgctactgt 120 cg 122 <210> SEQ ID NO
136 <211> LENGTH: 153 <212> TYPE: DNA <213>
ORGANISM: Festuca mairei <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (11)..(11) <223> OTHER
INFORMATION: n is a, c, g, or t <400> SEQUENCE: 136
cgaaacgggg ngatttcttt ttctttttat ggaggaaaag aacattcaag tgaacaacat
60 cccagcagaa gatggggaga aagagagatg aataagaatt attccgatca
ggggaggaac 120 aaacaagctc cctttcttaa ttatgatgac atg 153 <210>
SEQ ID NO 137 <211> LENGTH: 80 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 137
cgaatcctgg ctgtgcaata ccccggaccg aatctattga cagatcatcc atccttggtt
60 tcttgggaga aggctgcatg 80 <210> SEQ ID NO 138 <211>
LENGTH: 473 <212> TYPE: DNA <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 138 catgcatcct taccagcttc ctcagttgtc
ctgttgctct ccaatgtaac aactgctgat 60 ttatcacctt cctccaaacc
tgcgacccct ttaatctcat tgctatgcca agactcagca 120 ttagcatcac
aagctaactg aggctgagaa ttttcagtct tcattttcac ccgccgacca 180
ttctgttcat gcttatcagc aagcacagga gatgaagatc tactcccagt gacagatggg
240 tcgtcaaatg agccaccact cacacttcta gcaggactac gacttggatt
tgaaaagcga 300 actatcaatc tatggctatt accattatca ggaggtgtat
cacctacttt ctcagacacc 360 attccaggtt gtgatgcttt ttcctggaat
gaggaccgat caagtgaagt agatcttccc 420 acggtagctt ctttctttac
cccagaacca agacgggcac tgtttgccct tcg 473 <210> SEQ ID NO 139
<211> LENGTH: 91 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 139 cgacttcacc gggggcatct
catcctaccc gctcctcgtc gcccaggtga cccacttcaa 60 gtgcggaggc
gtggccctcg gcataggcat g 91 <210> SEQ ID NO 140 <211>
LENGTH: 166 <212> TYPE: DNA <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 140 cgatccggtg aagcaatccc tccgccgctt
cgccgaagaa aggagaaaag ccattggtga 60 agagatagcc cggctgctcg
cagccggctt tatcatggaa gtgctgcccc cagactggtt 120 ggctaaccca
gtcctggtct tgaagaagaa tgacacctgg cacatg 166 <210> SEQ ID NO
141 <211> LENGTH: 259 <212> TYPE: DNA <213>
ORGANISM: Festuca mairei <400> SEQUENCE: 141 cgatcgccgt
ctccgcactg cagcagctac catggcgtcc accgcgctct ccaccgcctc 60
caaccctacc cagctctgca ggtccagagc ttcgccgtgc aagcccatca agggcctggg
120 catcggccgg gagcgcgtcc cgaggaacat cacatgcatg gccggcagca
tctccgccga 180 ccgcgtgccg gacatgagca agagggagac gatgaacctc
ctcctgctcg gcgccatctc 240 gctccccacc ttcggcatg 259 <210> SEQ
ID NO 142 <211> LENGTH: 276 <212> TYPE: DNA <213>
ORGANISM: Festuca mairei <400> SEQUENCE: 142 catgttctct
tttgcaggaa gttaggacag gagcgaagcc gaacgtcttt agcttgataa 60
aaaagatgaa gcaaaggaag acgccgcatc ctgagacggg gtccttgtgg gttaacgagc
120 aatccgggac ccagtgtgcg gcgtatgtct tgaagttcaa gcagaagcac
ggcgagagct 180 ccaacccaga ggccgaggat tttgacgttg aggttgcggt
gcttgcggga gaaggcatga 240 agcatggccg cctatggctt ggtgatgggt gtgtcg
276 <210> SEQ ID NO 143 <211> LENGTH: 399 <212>
TYPE: DNA <213> ORGANISM: Festuca mairei <400>
SEQUENCE: 143 cgaagaggtt attcgtgata aggaggccca gttcagcagc
cccaacctca atgttgttta 60 ccgcatgaat gtgcgggagt accaggcact
aaccccctat gcctccatgc tggaggagaa 120 ctggggcaag gcacctgggc
atctcaattc tgatggcgag aacctccttg tctatgggaa 180 gcagtatgga
aacatcttca tcggagtgca gcccactttt ggttatgaag gtgatcctat 240
gcggctcctg ttctcaaaat ctgccagccc tcaccatgga tttgcagcat actacaccta
300 tgttgagaag atcttcaagg cagatgctgt tctgcatttc ggcacacacg
gatcccttga 360 gttcatgccc gggaaacaag tcgggacgag tgatgcatg 399
<210> SEQ ID NO 144 <211> LENGTH: 204 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 144
catgtataca aaacctggac ctcagaatac aacacatcca gtaataaggt aaaaacaaat
60 taactcttaa caggatggaa aacatcatct atctagctct tggggatgtt
cttgccaacg 120 atcttggcag gtgtgatgcg gagaaggttc ccctgcttcc
caggaatggc tcccttgatc 180 atcacaactt taagatcgtt atcg 204
<210> SEQ ID NO 145 <211> LENGTH: 285 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 145
cgatcggact atcctcacca ccaattgcat gaataaccac cgggatgtcg ctcaggtagc
60 gtatcaccag gggcagagcc gctggggcaa catcagcttt cactacgtca
tcgctcagca 120 aaactgaatc ggcagcagcg ggaccgccag cctctattgc
tcctggttca tctttgtctt 180 cagataacat taactcggat gggttcacca
ccactgcgac ttcctcaatt gctatgctac 240 atttgaactg gcttcccctc
ctttgggcag gcttgttcct gcatg 285 <210> SEQ ID NO 146
<211> LENGTH: 82 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 146 catgccggcg tgcccttgta
atagccacat tcatgagcct gctgtcaccc atgaacccta 60 cagctcctag
agggtttgat cg 82 <210> SEQ ID NO 147 <211> LENGTH: 224
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 147 cgaaatcata cttaacctcc ttcccttgct
tgtttatttc tttcaacccc ttcctgaaaa 60 agatcatggc tccacgtggt
ccacggagtg acttgtgagt agtggtagta acaacatctg 120 catactcaaa
aggagatgga atgacaccag cagcaactag gccactgata tgtgccatgt 180
ctgcgagaag tattgccttc tgcttgttac agatcttccg catg 224 <210>
SEQ ID NO 148 <211> LENGTH: 120 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 148
cgaattccat aatgaaatat gttgtaattg ctcatgtgaa cgaatggaga acaggagacc
60 tccatgggcg gcccagaaat tcagcaatga cgcggacctg cgcttccgcc
tccaagcatg 120 <210> SEQ ID NO 149 <211> LENGTH: 460
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 149 catgtttgaa ggaaatgacg tgtcagatgg
tatgggtttc ggaatgctaa ccgagggtga 60 gagatccctg gttgagcgtg
taaggcaaga gctgaagcac gagcttaaac aggggtacag 120 agaaaagctt
gtggacatta gggaagagat acttcggaag cgaagagccg gaaagctccc 180
aggagacaca gcgtctactc tgaaagcctg gtggcaagct catgcaaaat ggccataccc
240 gactgaggag gacagggccc gcctggtgca ggaaacaggg ctgcaactga
agcagatcaa 300 caattggttc atcaaccaac gcaagcgcaa ctggcacagc
aaccccacct catcctcatc 360 agacaagagc aagagaaaaa gaaacaatgc
aggtgatggc aacgccgagc ggtcttggta 420 ggacatggtt ggagaagaac
acgcgtgtgt aaacagttcg 460 <210> SEQ ID NO 150 <211>
LENGTH: 177 <212> TYPE: DNA <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 150 ccaaccgctc ataccagcat gatgatttgg
aggccttgct gatgatatct ctccccccct 60 tcctgttcat ataggaagga
tttagtgtac ctattgcccg aatagtgtat ttctggtgca 120 cctgccggtt
ccctgggtac tggcttgaat atgtgaatac tgtgcatatg gggcatg 177 <210>
SEQ ID NO 151 <211> LENGTH: 287 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 151
catgcatgca gcctggggcg tacagattga caggcctatg tgtagctcag ccctcatcag
60 gtagcgatac atggtggtaa ttaagtagtg atgcaagcgg ccagatcata
gctcgttgac 120 tgatgatcta gcaggtgcag caggagcagc cacagctggt
gctgcagttg cacttgctgc 180 aggggcagcc gccgttctcc gcctccgcgg
ccatgtccat cccaccggcg ctcgccttgt 240 gggtggcggc ggcgacgagg
aagacgttgc cgttgccggc ggcttcg 287 <210> SEQ ID NO 152
<211> LENGTH: 91 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 152 cgataagaag gacagcgagg
aggccaagca ggcgctagac cagctgaagg agctcggctg 60 ggccaagcga
tggagctcgc agccctacat g 91 <210> SEQ ID NO 153 <211>
LENGTH: 128 <212> TYPE: DNA <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 153 catgccgggg ctggggaagg agcacgtcaa
ggtgtgggcg gagcagaaca gcctggtgat 60 caagggcgag ggcgagaagg
actccgagga ggagggcgtc gccgccccga ggtacagcgg 120 ccgtatcg 128
<210> SEQ ID NO 154 <211> LENGTH: 132 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 154
catgtagccg atctttgttc caagagatgg taaagctttg ctttcataga tgcacctata
60 tgacctcttc ccaggtggta gtcatcccag gcggcgacga ggtgttcagg
ggagaggccc 120 ttggcgcgat cg 132 <210> SEQ ID NO 155
<211> LENGTH: 223 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 155 catgcggcaa acaatcttcc
cactccttca agttcttctt gatcatggat ctcaacagtt 60 gtgacaatgt
tctattcacc acctcagttt gaccatcagt ttggggatga caagtagtgt 120
tgaaaagtag cttcgtcccc agctttctcc aaagcgtctt ccagaagtag ctcatgaact
180 tcacgtcacg atcagaaaca atagtcttcg ggactccatg tcg 223 <210>
SEQ ID NO 156 <211> LENGTH: 100 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 156
cgacttgcgc cacaccacct gcgtgtagtg cccgcacacc ttgccggcgt cgcaggtgtt
60 gctgctgagg tggtagttct tcttctcgtc cacccacatg 100 <210> SEQ
ID NO 157 <211> LENGTH: 70 <212> TYPE: DNA <213>
ORGANISM: Festuca mairei <400> SEQUENCE: 157 catgttagcc
ccgagcagga agcccacggc gagcggcccg atggtgccca cgtggcccct 60
cttggggtcg 70 <210> SEQ ID NO 158 <211> LENGTH: 582
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 158 catgttgttg gccagaactt ggcaccacgg
agccacgccg cagggaacgc ggcgtactcc 60 ctggagtatg gcttagtcca
tgcgtcactc atcaggagtt ggggtgggtg gggagcgccc 120 ttcaggacat
tgttgttcac atctgctacg ccattttcta cctgtgcaat ttcttccctg 180
atggaaataa gggcatcaca gaacctgtct agttcagcct tgctttcact ttcagtgggt
240 tcaatcataa gtgtgcctgg aacaggccat gacatggttg gtccatggaa
cccatagtcc 300 atcaagcgct tcgccacatc ctcaggctct ataccagcag
tcgccttgag ccctcttaaa 360 tcaatgatga attcatgggc aacagttcca
ttgactccac ggaaaagaac tgggtagtgt 420 ttctccagac gctttgccat
gtagtttgca ttcaagatcg caatctttga agcatcagtg 480 agtccctgag
accccatcat ggctatgtat gtataggaaa ttggaagaat caaagcagat 540
ccccatggag cagcagaaat ggaacccagg aggtcggttt tc 582 <210> SEQ
ID NO 159 <211> LENGTH: 130 <212> TYPE: DNA <213>
ORGANISM: Festuca mairei <400> SEQUENCE: 159 catgccgctg
tactgatcta ccggaagctg cagctggttg cagaacaagc tgctgctgga 60
tctcctcctc ttcatcccat tatacttgtt accagtagcg taatcacggt catctaactg
120 cggcacgtcg 130 <210> SEQ ID NO 160 <211> LENGTH:
122 <212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 160 catgtggcat caattttaca tcaacctgcc
gcagctgtcc tgatcatacg actaattagc 60 cggagaaggt ctggatgatg
gtgttgtgcc acgggtcaga caagtgctgg aacaggttct 120 cg 122 <210>
SEQ ID NO 161 <211> LENGTH: 166 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 161
catgcggcag catccatcag caatgaagtt gtcggccaag cacgcgcgcg cacgcccgcg
60 ctactgctag agagctgaca aagctcactt tccggggacg aagttggtgg
cgaaggccca 120 ggcgttgttg ttgacggggt cggcgaggtg gtcggcgagg ttctcg
166 <210> SEQ ID NO 162 <211> LENGTH: 178 <212>
TYPE: DNA <213> ORGANISM: Festuca mairei <400>
SEQUENCE: 162 cgacaaagaa cacggccggc gaccatggca gcagcggttt
tggaactgct atactttgaa 60 gtttgaacag cgccttgacc tcagatgctg
gtggaattag ctatttgcgt gccaaatgta 120 gcgggtaaaa aatagctgtg
gtggttccag gattgtgtat tcggtaccgt gccacatg 178 <210> SEQ ID NO
163 <211> LENGTH: 178 <212> TYPE: DNA <213>
ORGANISM: Festuca mairei <400> SEQUENCE: 163 catgcggtac
ctcccatcca tggggctgga agacacactg aacgggtgcc actgcaagaa 60
cgacagctcc cgcacttgaa caaagatgaa gctgagagca ctgtaccgga ggcttgctgg
120 ctttgaggag actagctcca cagttccgca ggggcggcag gcagcagaaa caatgtcg
178 <210> SEQ ID NO 164 <211> LENGTH: 278 <212>
TYPE: DNA <213> ORGANISM: Festuca mairei <400>
SEQUENCE: 164 catgtgtctc tttgtccaaa tcagtttctt gaagaggtgc
tttctcagtg gtcttgcttt 60 cttcacaaag ctgctgtggc gtcaccgttg
gatcaacagg taaggcagtg cagcttccat 120 ctttctctaa tttctcatct
gctgcatcgg tagcgacagg ttcttcatca gtgacagcag 180 cagcagaagc
ggatacatct tcagactctt gtttctcagc tggagctgcc tgcttgttct 240
tgccatgcat tgccttgtag gtggcgacgg cgtggtcg 278 <210> SEQ ID NO
165 <211> LENGTH: 278 <212> TYPE: DNA <213>
ORGANISM: Festuca mairei <400> SEQUENCE: 165 catgtgtctc
tttgtccaaa tcagtttctt gaagaggtgc tttctcagtg gtcttgcttt 60
cttcacaaag ctgctgtggc gtcaccgttg gatcaacagg taaggcagtg cagcttccat
120 ctttctctaa tttctcatct gctgcatcgg tagcgacagg ttcttcatca
gtgacagcag 180 cagcagaagc ggatacatct tcagactctt gtttctcagc
tggagctgcc tgcttgttct 240 tgccatgcat tgccttgtag gtggcgacgg cgtggtcg
278 <210> SEQ ID NO 166 <211> LENGTH: 215 <212>
TYPE: DNA <213> ORGANISM: Festuca mairei <400>
SEQUENCE: 166 cgacgtactt taatatccgt aaaggcctag acggctccct
agacaaggca attaatgctc 60 tttgtgaaga agctgacgct gctgtgcgga
gtggttctca acttctggtc ctttctgatc 120 gttctgaagc acttgaacca
acacggcctg ccatcccaat acttctagcc gttggtgcca 180 tccaccagca
tctgattcaa aatggcctcc gcatg 215 <210> SEQ ID NO 167
<211> LENGTH: 338 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 167 catgtaccaa caacgaggcc
tgcaacgacg ccattgtccc tgccagccca accggcaaag 60 acgaagagtc
ctatcttgcc atagttataa cccatgttga tatcggtaag ccctgtccca 120
taggaattgg cgaatccaag catgggggcg acgacatagg ctataaccac gtagtaccat
180 ttcacctgtc ggaacattat tggcgtggta accactgcaa cagcacttaa
caaggcatac 240 ccggtgtacg ccaaccaaga ggggatatgg cccttccgga
agatctcgtc gcgctgcaga 300 tcctcaagtg agaccgtatt gtccacatct ttcactcg
338 <210> SEQ ID NO 168 <211> LENGTH: 167 <212>
TYPE: DNA <213> ORGANISM: Festuca mairei <400>
SEQUENCE: 168 cgacacgctc actgatatct gatggaggtg aattcagctt
gacaaatgag caggaaaagt 60 gggttgtaga cattatgctc tcagtaacgc
tggtgaaact tgctctagct ttatattgcc 120 gcacattcac caatgaaatt
gtcaaggctt atgcgcagga tcacatg 167 <210> SEQ ID NO 169
<211> LENGTH: 134 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 169 catgcggagg gaaggatatg
aatttatgat tggacctcca aaggtcataa acaagagtgt 60 agatgggaag
ctactggagc cgtatgagat agctgctata gaggtaccag aggaatatat 120
gtgatcagct gtcg 134 <210> SEQ ID NO 170 <211> LENGTH:
121 <212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 170 cgactgcggc gtctccatcg gaacgatctt
tgggatttac atgatcaaga actttgacac 60 cgtgaccctt gaggaagtgc
cgctgcctgg gaaggacatg attgctgctg gatactgcat 120 g 121 <210>
SEQ ID NO 171 <211> LENGTH: 201 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 171
catgcatatg ctgcaaatgt ttcttcccac ctagtgtttc ttttttcctt ttaccccgca
60 attgaaccgt gcaaagctca aggctccgat catatatacg ccttcgtatc
tagcgacaag 120 agtgaatgag cgcggtaagc tgttatggaa tctccttggc
acgtctgatc aatgtacata 180 ctgacactcg catttgtctc g 201 <210>
SEQ ID NO 172 <211> LENGTH: 113 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 172
cgagtcgcag tcgcagacca acgatctgga gtcggacagt ctgcaggtgt acagcttctc
60 cgggctgttc ctcatctgcg gcgtggcgtg cgtgatcacc ctcgccatac atg 113
<210> SEQ ID NO 173 <211> LENGTH: 148 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 173
catgtcgttg gacggcagca ccatcaccac atggagagaa aggagccgtt ggacaatctg
60 tgcaggagct cgccgaggca caggatggcg tagaccagca ggatgaactt
gaagaggaac 120 gggaagtagt gcgagagccc ggcgctcg 148 <210> SEQ
ID NO 174 <211> LENGTH: 398 <212> TYPE: DNA <213>
ORGANISM: Festuca mairei <400> SEQUENCE: 174 catgcaccca
tttgccccta ttgatcaggc tgcaggctat catgaaatgt ttgacaactt 60
gggtgatctg ttgaacacga tcaccggttt tgattccttc tctctgcaac caaatgctgg
120 tgcttcagga gagtatgctg gactgatggt tattcgggcc caccacaggg
caagaggaga 180 ccatcaccga aatgtctgca tcattcctgt ctcggcacac
ggtacaaatc ctgcaagtgc 240 tgctatgtgt ggaatgaaga ttattactgt
cggaactgac tccaaaggta acattaacat 300 tgcggagttg aagaaagctg
ctgaagcaaa caaggacaac ctgtctgctc tgatggttac 360 ctatccttca
acccatggag tctatgaaga aagcatcg 398 <210> SEQ ID NO 175
<211> LENGTH: 318 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 175 cgacattatg cacaggcaga
ggaagctgaa gaggatgatg aaattgagcg gctctttagt 60 agtaagaaag
agaagaagaa tgatcggcca cgagcagata ttggtcttat cgttgagcag 120
ttcattgccg agtttgaagt agcgtctgaa gaagatgcaa acctaaatag gcaatccaaa
180 ccggccatta acaaacttat gaagcttcca ctgctcatag aggttctctc
aaagaagaat 240 ctccagcagg aattccttga tcatggaatt ctcactcttc
tgaaaaactg gcttgaacct 300 ttacctgatt gaagcatg 318 <210> SEQ
ID NO 176 <211> LENGTH: 86 <212> TYPE: DNA <213>
ORGANISM: Festuca mairei <400> SEQUENCE: 176 catgtacgtg
cttcgttgca tcttctgaac agcctcggtg acctccttac gtacgccaag 60
ccatcgcact gagctgagct cagtcg 86 <210> SEQ ID NO 177
<211> LENGTH: 116 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 177 catgcatatc tcgcccggcc
ggtggtcgga cggatggcta actcatttaa catccaggag 60 cgaagttggt
ggcgaaagcc catgcgttgt tgttgactgg gtcagcaacg cagtcg 116 <210>
SEQ ID NO 178 <211> LENGTH: 194 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 178
cgaccataaa cgccccgtga tcggcggcaa ggagcacccc taccctcgcc gatgccgcac
60 cggtcgccct aaaaccatca ttgactcaga gacggagaag aggagctcac
cagtgtatgt 120 gccacgtgac gagcagttct cggacattaa agggcagaca
ttcagcgcga cgacactgcg 180 gtctggattg catg 194 <210> SEQ ID NO
179 <211> LENGTH: 290 <212> TYPE: DNA <213>
ORGANISM: Festuca mairei <400> SEQUENCE: 179 cgacgaccta
aaggctcacg cagaatcaaa tgtgactgat aagatgatgt caaatgcaaa 60
gttcatctac ccacacaaca ccccgacaac aaaggaggca tactgttaca gaacgatctt
120 tgagaggttc ttcccccaga actcggcgat cctgacagtg ccaggtggac
caagcgtcgc 180 atgcagcacg gcgaaggcgg tagagtggga tgctcagtgg
tcggggaacc tggatccctc 240 agggagagca gcgcttggag tccatctctc
agcctatgaa caagagcatg 290 <210> SEQ ID NO 180 <211>
LENGTH: 170 <212> TYPE: DNA <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 180 catgcacgta cggggttcgt aacactactc
tagcttaatt aatctagacg ttgacaaagg 60 gcgtgccggt gatgaactcg
gtgacggcga gcgcgacgag tccgagcatg gcgaagcggc 120 cgttccagag
ctcggcgtcg gcgctccaga cgccgctgga cttgctctcg 170 <210> SEQ ID
NO 181 <211> LENGTH: 204 <212> TYPE: DNA <213>
ORGANISM: Festuca mairei <400> SEQUENCE: 181 catgttggtg
cttacaaata tggcagctgg ggacgaatta agcaaggaag ctgtaatgga 60
tgttattgtt cctcacagat cagatcgcat caagccatct tttgtggtca actttctgca
120 gagcaaggac gaacaattga gagttgcatc tttgtggtgc attcttaact
tagcttaccc 180 aaaaagtgat gcttcatcta ctcg 204 <210> SEQ ID NO
182 <211> LENGTH: 77 <212> TYPE: DNA <213>
ORGANISM: Festuca mairei <400> SEQUENCE: 182 catgcttggg
aataggtgtg tcatcttcaa tcacctcgca ttcgtagtca tcaggaacgc 60
cccagggatc ccactcg 77 <210> SEQ ID NO 183 <211> LENGTH:
114 <212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 183 catgctgtcg ttcaggactc atcgtgactg
tagactgcgt tcccatgctt tctcctccaa 60 agtgagttgc acatccttca
tctcacactg gactgatgcc atttccccgt gtcg 114 <210> SEQ ID NO 184
<211> LENGTH: 431 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 184 catgttcctt ctacgttgat
atgtacggtg catacacaaa caattatacg tggaactaaa 60 agtaaaaccc
ggaaagcgga gttgtcatca aaaactaaac caagagactc cacatggatt 120
cctagctcgc ggcttatgcc ttgccggact cctcacagcc tggtggcttg aaggcgatga
180 agctgacgca ctgcacctga cggatgttgt caaaaccgat gatgcggaca
taggcgtcag 240 ggtactcctt cttgacctcc tccacctcct tgaggacctg
ggtggcgtct gtgcagccga 300 acatgggcag cttccacatt gtccagtacc
tgccgtcgta gtatccggga gtgctgccgt 360 gctcacggaa gatgaagcca
accttgctga actccaggca gggaacccat ttggagcgga 420 tcaagaagtc g 431
<210> SEQ ID NO 185 <211> LENGTH: 84 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 185
catgcagtgg aatgtcttct gataaacgta ggggagaaca tgactgggga aggacttcgg
60 tggaagctat tttcttgcca gtcg 84 <210> SEQ ID NO 186
<211> LENGTH: 398 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 186 catgcttgcg cacatagttc
atctctactg gctcgtccag ctcagcaacc atctttactg 60 cccggtccag
cagattcatc tggttgatga aaagacctct gaacacgccc gagcagttga 120
cgacgacatc aaccctagga cgtccaagct cctcaatgct gacaggctcc acacggttga
180 cacggccaag gccatcagta accggctcca caccaagcat ccaaaacacc
tgggccaggg 240 actcgccgta ggtcttgatg ttgtcagtac cccacaagac
aagagcaatt gtctcaggat 300 acttgccacc attgtcagcc ttttgccgct
ccagcagacg ttccacaaca accttggcac 360 tcttcgtggc cgctgcggtc
gggattgact gcgggtcg 398 <210> SEQ ID NO 187 <211>
LENGTH: 231 <212> TYPE: DNA <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 187 cgacgtccag cgctctcttc tccttcttca
ccttgggctg cgtgatcaca gtggcatcac 60 ccggcccaga gaccgccttg
gcgccgacgt ctgtgcctgg agccgccgtg gaggccatca 120 tcgcccggcg
cgccttccgc tgcctgatgc cacaagcgtt gcaaagtgac ttggggccac 180
atggaccact cctccacaag ggggttttgg tggtgttgca gtcggagcat g 231
<210> SEQ ID NO 188 <211> LENGTH: 178 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 188
catgttcaaa ctagattgat acgacaccag gtccacatga tcactgcggt tccgaagagc
60 tgaagatcct acagtgcctt gaggatctcc tcagcaccag agattgtgaa
ctggccgccc 120 tcctcaatgt ttgcaacagt gaccttgagg tcatcagcaa
ggagagcaaa ccgtctcg 178 <210> SEQ ID NO 189 <211>
LENGTH: 232 <212> TYPE: DNA <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 189 cgagagtact ttgggcttca attttgggtc
attcaataga gaatctagtt caccttcatt 60 cagtacagga cttgatagag
taacctggtc agcattttca gggccaacct ccaagatgtt 120 gcctcgttta
ccaatattaa cttcaagaga catgactaaa ccttctcgga gtggatcaat 180
tgcagggttt gtaacctgtg caaatcgctg cttgaaataa tcaaagagca tg 232
<210> SEQ ID NO 190 <211> LENGTH: 356 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 190
catgccactt gcccttgtag gcagggttct gcttcgttgg cttcttccat tcaccacatc
60 caggtgcctc ttcgcacttg gggttgtcaa tcttcggtgc ctcccattca
ccatcctcct 120 catcatccca gtctttaggc ttagcagcct caggatcgtc
aatttcatca ggctcatcat 180 ccagccatcc ttctggcttg gtggcctcct
catccacaat ctctattggg gcatcctcat 240 cccagtcgtc aggcttagta
gcatctggat cagggatctt agctctctcg tcccagtcct 300 ctggcttctt
gtcgtcaggg tcaggaatcg tctttgatgg aataagtgct gactcg 356 <210>
SEQ ID NO 191 <211> LENGTH: 122 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 191
catgccttca tatctgcaac aactggtcag tagggcaatc acatcagaag ataaattcgt
60 tgatgatgtt ggtccctcca ggcaaacaat aagcaagggt gtcccatcag
acaacaatgt 120 cg 122 <210> SEQ ID NO 192 <211> LENGTH:
390 <212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 192 cgactccagc agcaacccga gaagatcgtc
gctcgttgct tcgccagctt ttaaggcgtt 60 ctctcttttg gtgatgatcc
tcttaggatc ctcccaatct ccgcagcaat ctgcttcatc 120 cttctgttgg
ctttggttgg caagaacagg taaccaggga tatgtatctt gttcatggcc 180
tgcatgacga gcaggatctg ctccccctga agctggaata tcctcacgcc ctcaaggtag
240 ctgctgccga atgcggcgcg ggagatgaca tcccctgtca ggttctgcat
atcaggccag 300 acatctacct cgcatggcag gtcaccggtg actaaacctt
cccatctgtg taccagctcc 360 gtgcaacatt cggcgaaagc cggcaacatg 390
<210> SEQ ID NO 193 <211> LENGTH: 400 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 193
catgctgtca aagtggcagc tgatggctga acaagctagg cagaagcgag caccagtaca
60 gaggccagca catggatctg gaaaaggttc ggcagaacag aatgaagctt
caaagaggag 120 ccattttgca gccttcggaa ctggaggcac gaaaaggcaa
ggcaagggtt cattcgctac 180 gcgtcactcg catgggccac aacgaactgt
ttccgtgaag gatgtaatct gcgtcctgga 240 gagggagcct cagatgacga
aatcacggct aatttatcgg ctgtacgagc gattgcctgg 300 agatttcacc
acagattagg ctgaattatg tagtgtaact tatagcgtgt aactgtttgt 360
tgatgcacag cccgtcgctc agactgacgt gttccagtcg 400 <210> SEQ ID
NO 194 <211> LENGTH: 192 <212> TYPE: DNA <213>
ORGANISM: Festuca mairei <400> SEQUENCE: 194 cgacccatgc
cgtgagcggg tcgcggaagt ccttgagccc gatcccgggt ggcgggaaga 60
ggatggggtt ggcggggtgc ttgatccagc tgcggaggag cgggtcggat gggtcggcgg
120 gcacggccag gcactggacc tgcgcgaagg tgtcagtgtt ccccgtgtcg
agcaggatca 180 cgcgcccgtc ag 192 <210> SEQ ID NO 195
<211> LENGTH: 209 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 195 catgctattg gagaacggtc
ccaacgaggc ccaggcaaag aaggcccgcc aggtcctgca 60 ggcctgcggc
gataggaaag acggctacca gctgaactac gacttcagga acccgttcgt 120
tgtgtgcggg gcgacctttg tcccgatcta ccgcgggcag aaggacgtct cctgccccta
180 ctgcacttcc cggttcgtgc cctccgtcg 209 <210> SEQ ID NO 196
<211> LENGTH: 172 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 196 cgagatagac gagcagacct
tcctgaccaa cagggagagg gcggtggact acctcaactc 60 cctggacaag
gtgttcgtga acgaccagtt cctcaactgg gacccggaga accgcatcaa 120
ggtgcgcatc atctccgcca gggcctacca ctcgctcttc atgcacaaca tg 172
<210> SEQ ID NO 197 <211> LENGTH: 109 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 197
cgagagaatc acggagcaag ctggtgtagt gctcactctt gacccaaaac caatccaggg
60 tgactggaat ggagctggct gccacacaaa ttacagcaca aagagcatg 109
<210> SEQ ID NO 198 <211> LENGTH: 235 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 198
cgagtcagcg ccactactgt gccattacag ttggagagga tgctgtcgtt tcagcatata
60 ggttatcaga agacagaggc aggtcattag ttggagcaat tttgtcaagg
ggtgtagctg 120 caacattttc aacaatatca tctttgtcca aaattctatg
gcggagtgaa ccatcaccaa 180 ctaagaagcc acggccaaaa cctcaatcct
ttgcaaaaac ttcacctctg acatg 235 <210> SEQ ID NO 199
<211> LENGTH: 398 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 199 cgaggttcgc gttttggagt
tggagaaacc tggtggacgt tgggagacaa ggtccactat 60 tccaatgcaa
ccatttgaaa atgctctgac tgtgcgcatt gttacattac ataacacaac 120
caccaaggaa aatgaaaccc tgatggccat cgggactgct tatgtccaag gagaggatgt
180 agctgctaga ggacgggtgc ttctgttctc tttcacgaaa agtgaaaatt
ctcaaaatct 240 ggtgacagaa gtctactcaa aagagagtaa aggtgctgta
tcagctgttg catcgcttca 300 aggtcatctt gtgatagctt ctggcccaaa
aatcacattg aacaaatggt ccggttctga 360 attgacagct gttgcattct
atgatgcccc tttgcatg 398 <210> SEQ ID NO 200 <211>
LENGTH: 114 <212> TYPE: DNA <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 200 catgcctcat ctataggcaa gaaggcagta
gcctttgact ggatgtccat aaatgcgcca 60 tttgaagtag tcatgaatac
tgttccttta atgagtgagc cgatttcagt gtcg 114 <210> SEQ ID NO 201
<211> LENGTH: 90 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 201 catgttcacg cctagctgga
tctgaaccct tactgtgcct ggaagcagga ggtctacttc 60 cccgaatcca
gcagcagatg atcgcagtcg 90 <210> SEQ ID NO 202 <211>
LENGTH: 129 <212> TYPE: DNA <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 202 catgcttcca tctgcaagtg aggattccac
aacactgcat ctgaccaatt tgtatttgag 60 atgacaattt tgtcacccaa
tccattgtcc agggtaagct cactcggtgc gccaaggtaa 120 acgcagtcg 129
<210> SEQ ID NO 203 <211> LENGTH: 108 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 203
catgccatgt gaccatcaca caggatgcgg atgctgatca gatgcttgac aaggtcattg
60 ggtacatcaa ggcagagtac aacatcagtc atgtgaccat tcaggtcg 108
<210> SEQ ID NO 204 <211> LENGTH: 119 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 204
cgagacactg gctacagtgc cctcaagctg ctcaaggcgc tactagcact ctctccatag
60 gtagatataa gatagctcgc cggccaatgg atcagtagct gtagttcttg acgaacatg
119 <210> SEQ ID NO 205 <211> LENGTH: 245 <212>
TYPE: DNA <213> ORGANISM: Festuca mairei <400>
SEQUENCE: 205 cgagaagtcc gggcacaggt tcctgtgggt gctgcgtgcg
cctcctgcct tcgctgcggc 60 cgccgctgaa ccggatgcgg cgctttctct
cctcccagag gggttcttgg cacggaccgc 120 agacaggggc ctcgtggtga
ccgcgtcctg ggtgccgcag gtggacgtgc ggcgtcacgc 180 ctccactggt
gccttcgtaa cgcactgcgg atggaactca acactggagg caggcgaccg 240 gcatg
245 <210> SEQ ID NO 206 <211> LENGTH: 345 <212>
TYPE: DNA <213> ORGANISM: Festuca mairei <400>
SEQUENCE: 206 cgagtcacga tgagtcctga acgagtgttt tcatcttcat
aatggatatg ttgcatggca 60 tttttcaaag ctttttccct caatttgctt
cccggccaat gcgacagaac tggtacaaca 120 aatttgttaa gagcagccca
tatgatatct tgtcccagtg gatgtggaca gtacagatcc 180 tccttagcac
attggttccg agccttgtcc caatcaacct catcataggg tactttatag 240
agctccttcc gtaagttcaa tacaactggt gtaattgggc caacaaacct ctttccatat
300 acgtaagaca tgggaaagta taccattcgg caatgggacc acatg 345
<210> SEQ ID NO 207 <211> LENGTH: 323 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 207
cgagttacaa ttatatgagt actactgacc agacctgtgt atactggaaa gaactgggtc
60 gctaagaggc tgctgagaaa gaattaccta caagccgtgg attacatggt
gaagattgct 120 tttatggaga gagaaaaaaa ggagaaaaat cagagatatg
tgtatgttat atgtactctc 180 agcaggggaa caacaaaaac gcagcctccc
tgtggatcct cctattctct accagtatga 240 tcttgtccag cttcgccttg
caccactgca gctgctcgct ggtgatcctc ggcatccgca 300 gcacccgcgc
caccagccgc atg 323 <210> SEQ ID NO 208 <211> LENGTH:
357 <212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 208 catgcacccg gagtggcaag agcaggcaag
aaaggaagtg ttgcaccact tcggaagaat 60 cacaccagac tttgagaact
agagtcggct gaagatagta acgatggttc tatatgaggt 120 tcttaggttg
tacccgccag caatctttgt taccagaaga acatacaaga caatggagct 180
tggtggcatc acatatccgg caggagtgaa ccttatgttg cccattctct ttatccacca
240 tgaccccaat atatggggaa aagatgcaag cgagttcaat ccacagaggt
ttgctgatgg 300 catctcaagt gccgtgaagc atccggctgc gttcttccca
tttggagggg gtcctcg 357 <210> SEQ ID NO 209 <211>
LENGTH: 245 <212> TYPE: DNA <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 209 cgactgtcca tgcgattgaa atcggtttgt
aacctgacca tggattttct gaattccact 60 tcttcaatcc atgatccatc
aaagtatgtc cggtgcaaat gccctgggtc tcaactctca 120 caacaccctt
cttaaatgtg aagtactcag gatgaactaa ggccgtaaag ctcacaggat 180
catggaggaa aatcccatgg aagccgtcag acttggtatg ccaatctctg tagaacttgc
240 acatg 245 <210> SEQ ID NO 210 <211> LENGTH: 232
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 210 catgccataa aggctcatat atcctactac
tctacaactt gagctgccta tacaaacgta 60 ttacatctgt ggtctagtct
ggactacgta gatcttccca tccttcacta ctccctcaac 120 gtcctgtggt
gacccataga gctcctcaat ggcatggcca gcccgtgcaa tgcttgagag 180
aattgagctt cggaatccag agtctgtgat gagagcgtcg gtcgtgtagt cg 232
<210> SEQ ID NO 211 <211> LENGTH: 258 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 211
catgcctgac atggcgaacc catccaaatt gcaaagagga tttgggaagt ttctgacaac
60 ctttcctgca aatatcttgg tagccacaat gctcaggggg cccaatattg
tttgattctt 120 caaagcacct gctaacagtt catgagactc caaggaacgc
tcagcttgct tgatcatctc 180 atctaaacat gctttctcct ccaaagtgag
ttgcacatcc ttcatctcac actggactga 240 tgccattttc ccgcgtcg 258
<210> SEQ ID NO 212 <211> LENGTH: 161 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 212
catgtgatcc aggcgccacc cttatgtcca ccttgtaacg aggaggaagg ctccgcatga
60 gcttcacacg taagcaaagg ccaataatag tcgccatact gcaatgctcc
actgttgggg 120 tgaaagtaac ccgcacatga ctaagatcat cgctgatctc g 161
<210> SEQ ID NO 213 <211> LENGTH: 223 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 213
cgagatctgc agtctattgt gttgtatgat caaaatggta agtttgtggg ggttcgtcgg
60 ccaagctcaa aactccccat tgaaatcaat ggtaatgaaa tactaattga
agacgctatt 120 ggcagtactg gtctggatct taagaccgat ccaggaattc
ctgtcgtgta tgctggattt 180 ggcgcgctca tgttgacgac ctgcattagc
tatctttcgc atg 223 <210> SEQ ID NO 214 <211> LENGTH:
309 <212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 214 catgtgctct ggatgtacca gtgtcgctgg
cgaagggaat gcaggccaag cttatcggtg 60 atttcagcag cgttcatggc
attaggaaga tcttgtttgt ttgcaaacac cagaagcaca 120 gcatcacgca
actcatcctc attgagcatt cggtgaagct catctctggc ctcaacaaca 180
cgctctctgt cgttgctgtc caccacaaaa ataaggccct gggtgttctg gaagtagtgc
240 ctccacaggg gcctgatctt gtcctgaccc ccgacatccc aaactgtgaa
actaatgttc 300 ttgtactcg 309 <210> SEQ ID NO 215 <211>
LENGTH: 185 <212> TYPE: DNA <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 215 catgtactta ccagctaggt gttggttcgg
tcgtcgttaa gaagcattta accacagctt 60 aattgaagtg atcgtgatga
gaaagtaagc caaactaggg taggtagacg gatggatccg 120 ggacgtccgt
ccagcagctc ccggcgttcc agtacgcggc cggcgacgcg tcgtcgccga 180 gctcg
185 <210> SEQ ID NO 216 <211> LENGTH: 254 <212>
TYPE: DNA <213> ORGANISM: Festuca mairei <400>
SEQUENCE: 216 ccagggtttg cacccaatgt acgacgaacg tgtgagagac
ctccttgatt tcagcatcta 60 cttagacatc agcaatgagg ttaagtttgc
atggaaaatt cagagagaca tggcagagcg 120 tgggcacagc cttgaaagca
tcaaggctag cattgaagcc aggaaaccaa attttgatgc 180 atttattcgt
agtgcctttt tgccatctga aaacaataat tgtttgccat aaacccaact 240
taacatgggg catg 254 <210> SEQ ID NO 217 <211> LENGTH:
18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
<400> SEQUENCE: 217 gtagactgcg ttcccatg 18 <210> SEQ ID
NO 218 <211> LENGTH: 17 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic <400> SEQUENCE: 218 ggaacgcagt
ctacgag 17 <210> SEQ ID NO 219 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
<400> SEQUENCE: 219 gtagactgcg ttcccatg 18 <210> SEQ ID
NO 220 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic <400> SEQUENCE: 220 gtagactgcg
ttcccatgta 20 <210> SEQ ID NO 221 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
<400> SEQUENCE: 221 gtagactgcg ttcccatgtt 20 <210> SEQ
ID NO 222 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic <400> SEQUENCE: 222 gtagactgcg
ttcccatgtc 20 <210> SEQ ID NO 223 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
<400> SEQUENCE: 223 gtagactgcg ttcccatgtg 20 <210> SEQ
ID NO 224 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic <400> SEQUENCE: 224 gtagactgcg
ttcccatgca 20 <210> SEQ ID NO 225 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
<400> SEQUENCE: 225 gtagactgcg ttcccatgct 20 <210> SEQ
ID NO 226 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic <400> SEQUENCE: 226 gtagactgcg
ttcccatgcc 20 <210> SEQ ID NO 227 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
<400> SEQUENCE: 227 gtagactgcg ttcccatgcg 20 <210> SEQ
ID NO 228 <211> LENGTH: 16 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic <400> SEQUENCE: 228 aagtcctgag
tagcac 16 <210> SEQ ID NO 229 <211> LENGTH: 15
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
<400> SEQUENCE: 229 cgttcaggac tcatc 15 <210> SEQ ID NO
230 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic <400> SEQUENCE: 230 cacgatgagt
cctgaacg 18 <210> SEQ ID NO 231 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
<400> SEQUENCE: 231 cacgatgagt cctgaacgaa a 21 <210>
SEQ ID NO 232 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 232
cacgatgagt cctgaacgaa t 21 <210> SEQ ID NO 233 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic <400> SEQUENCE: 233 cacgatgagt cctgaacgaa c 21
<210> SEQ ID NO 234 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 234
cacgatgagt cctgaacgaa g 21 <210> SEQ ID NO 235 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic <400> SEQUENCE: 235 cacgatgagt cctgaacgat a 21
<210> SEQ ID NO 236 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 236
cacgatgagt cctgaacgat t 21 <210> SEQ ID NO 237 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic <400> SEQUENCE: 237 cacgatgagt cctgaacgat c 21
<210> SEQ ID NO 238 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 238
cacgatgagt cctgaacgat g 21 <210> SEQ ID NO 239 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic <400> SEQUENCE: 239 cacgatgagt cctgaacgac a 21
<210> SEQ ID NO 240 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 240
cacgatgagt cctgaacgac t 21 <210> SEQ ID NO 241 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic <400> SEQUENCE: 241 cacgatgagt cctgaacgac c 21
<210> SEQ ID NO 242 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 242
cacgatgagt cctgaacgac g 21 <210> SEQ ID NO 243 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic <400> SEQUENCE: 243 cacgatgagt cctgaacgag a 21
<210> SEQ ID NO 244 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 244
cacgatgagt cctgaacgag t 21 <210> SEQ ID NO 245 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic <400> SEQUENCE: 245 cacgatgagt cctgaacgag c 21
<210> SEQ ID NO 246 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 246
cacgatgagt cctgaacgag g 21 <210> SEQ ID NO 247 <211>
LENGTH: 17 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic <400> SEQUENCE: 247 ctcgtagact gcgtacc 17
<210> SEQ ID NO 248 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 248
aattggtacg cagtctac 18 <210> SEQ ID NO 249 <211>
LENGTH: 16 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic <400> SEQUENCE: 249 cactgcgtac caattc 16
<210> SEQ ID NO 250 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 250
cactgcgtac caattcaa 18 <210> SEQ ID NO 251 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic <400> SEQUENCE: 251 cactgcgtac caattcat 18
<210> SEQ ID NO 252 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 252
cactgcgtac caattcac 18 <210> SEQ ID NO 253 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic <400> SEQUENCE: 253 cactgcgtac caattcag 18
<210> SEQ ID NO 254 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 254
cactgcgtac caattcta 18 <210> SEQ ID NO 255 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic <400> SEQUENCE: 255 cactgcgtac caattctt 18
<210> SEQ ID NO 256 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 256
cactgcgtac caattctc 18 <210> SEQ ID NO 257 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic <400> SEQUENCE: 257 cactgcgtac caattctg 18
<210> SEQ ID NO 258 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 258
cactgcgtac caattcca 18 <210> SEQ ID NO 259 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic <400> SEQUENCE: 259 cactgcgtac caattcct 18
<210> SEQ ID NO 260 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 260
cactgcgtac caattccc 18 <210> SEQ ID NO 261 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic <400> SEQUENCE: 261 cactgcgtac caattccg 18
<210> SEQ ID NO 262 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 262
cactgcgtac caattcga 18 <210> SEQ ID NO 263 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic <400> SEQUENCE: 263 cactgcgtac caattcgt 18
<210> SEQ ID NO 264 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 264
cactgcgtac caattcgc 18 <210> SEQ ID NO 265 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic <400> SEQUENCE: 265 cactgcgtac caattcgg 18
<210> SEQ ID NO 266 <211> LENGTH: 10 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 266
aatcgggctg 10 <210> SEQ ID NO 267 <211> LENGTH: 10
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 267 aggggtcttg 10 <210> SEQ ID NO 268
<211> LENGTH: 10 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 268 gaaacgggtg 10 <210>
SEQ ID NO 269 <211> LENGTH: 10 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 269
gtgacgtagg 10 <210> SEQ ID NO 270 <211> LENGTH: 10
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 270 gttgcgatcc 10 <210> SEQ ID NO 271
<211> LENGTH: 10 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 271 ccttgacgca 10 <210>
SEQ ID NO 272 <211> LENGTH: 10 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 272
ttcgagccag 10 <210> SEQ ID NO 273 <211> LENGTH: 10
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 273 gtgaggcgtc 10 <210> SEQ ID NO 274
<211> LENGTH: 10 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 274 ccgcatctac 10 <210>
SEQ ID NO 275 <211> LENGTH: 10 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 275
gatgaccgcc 10 <210> SEQ ID NO 276 <211> LENGTH: 10
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 276 gaacggactc 10 <210> SEQ ID NO 277
<211> LENGTH: 10 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 277 gtcccgacga 10 <210>
SEQ ID NO 278 <211> LENGTH: 10 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 278
tggaccggtg 10 <210> SEQ ID NO 279 <211> LENGTH: 10
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 279 ctcaccgtcc 10 <210> SEQ ID NO 280
<211> LENGTH: 10 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 280 tgtctgggtg 10 <210>
SEQ ID NO 281 <211> LENGTH: 10 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 281
aaagctgcgg 10 <210> SEQ ID NO 282 <211> LENGTH: 10
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 282 aagcctcgtc 10 <210> SEQ ID NO 283
<211> LENGTH: 10 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 283 gacggatcag 10 <210>
SEQ ID NO 284 <211> LENGTH: 10 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 284
cacactccag 10 <210> SEQ ID NO 285 <211> LENGTH: 10
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 285 gttgccagcc 10 <210> SEQ ID NO 286
<211> LENGTH: 10 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 286 acttcgccac 10 <210>
SEQ ID NO 287 <211> LENGTH: 10 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 287
cttcacccga 10 <210> SEQ ID NO 288 <211> LENGTH: 10
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 288 gtggcatctc 10 <210> SEQ ID NO 289
<211> LENGTH: 10 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 289 catcgccgca 10 <210>
SEQ ID NO 290 <211> LENGTH: 10 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 290
acagcctgct 10 <210> SEQ ID NO 291 <211> LENGTH: 10
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 291 ggctgcgaca 10 <210> SEQ ID NO 292
<211> LENGTH: 10 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 292 aaggctcacc 10 <210>
SEQ ID NO 293 <211> LENGTH: 10 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 293
agagccgtca 10 <210> SEQ ID NO 294 <211> LENGTH: 10
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 294 agcagcgcac 10 <210> SEQ ID NO 295
<211> LENGTH: 10 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 295 caaacgtggg 10 <210>
SEQ ID NO 296 <211> LENGTH: 10 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 296
gggtctcggt 10 <210> SEQ ID NO 297 <211> LENGTH: 10
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 297 ggtcgatctg 10 <210> SEQ ID NO 298
<211> LENGTH: 10 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 298 agtcgccctt 10 <210>
SEQ ID NO 299 <211> LENGTH: 10 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 299
gggccaatgt 10 <210> SEQ ID NO 300 <211> LENGTH: 10
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 300 gacgtggtga 10 <210> SEQ ID NO 301
<211> LENGTH: 10 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 301 gtggagtcag 10 <210>
SEQ ID NO 302 <211> LENGTH: 10 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 302
tgagggtccc 10 <210> SEQ ID NO 303 <211> LENGTH: 10
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 303 agccgtggaa 10 <210> SEQ ID NO 304
<211> LENGTH: 10 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 304 ctgggcacga 10 <210>
SEQ ID NO 305 <211> LENGTH: 10 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 305
acgccagagg 10 <210> SEQ ID NO 306 <211> LENGTH: 10
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 306 acgggagcaa 10
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 306
<210> SEQ ID NO 1 <211> LENGTH: 85 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 1
catgcatagg acccagtaat ggactgtaga gtaagttgtc ccgcgtgcga cggcgtgtac
60 gcgtgttcgt gacacactga catcg 85 <210> SEQ ID NO 2
<211> LENGTH: 188 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 2 catgcgctgt tatttgccaa
agctcgctgg ccattgtttt cccaccattg cccattcttg 60 gcacctcgca
tcctgtcgtc actgagattg gaaagcgaaa tcagggccga ccgacggaca 120
cctccgacaa caacaacctc accaatcttg cacatcaagt cgtgacattc aatagacgta
180 agctttcg 188 <210> SEQ ID NO 3 <211> LENGTH: 122
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 3 catgccgtgc tacaggaggt agataatcag ggtgcctaat
tttggatggt gttttgtatg 60 caatatggcg tttgtgtgtt agcatcacag
attaatgagg gaatctctgg atgatttatt 120 cg 122 <210> SEQ ID NO 4
<211> LENGTH: 132 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 4 catgcaccgg agataggcac
tgcggaagcc ctgtaggaga cgccctgcct ccgaattggc 60 atcaacgcct
ggaggtagaa catgagctgg cgttgatgaa gtgacaccaa agctttccat 120
ggtcgccctt cg 132 <210> SEQ ID NO 5 <211> LENGTH: 101
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 5 catgtatcat gaaacaacgc atggcagtgt tccttctttt
taggttatag cttcactggc 60 ttcgctagct ccaggtctcc aaaactcatc
atcttttttc g 101 <210> SEQ ID NO 6 <211> LENGTH: 328
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 6 catgcgctct gacaaagggc cgtggaagga ccctgaaatc
ctaaaggtta atggttactc 60 tccgaagatt ttggagttca tgcatgacct
gtgggaaaat gtagtgcaga cttgccactg 120 aactgaagga agtagcagta
ctactacagc taaactcctg agcacaggtc cattaacatt 180 ttcctggttt
gattcttcag atggtgcaat gtggaatggg gagatgcgga atgaacagct 240
cggaccttat atgcaggtat tatatggcct ttttcgccat atttttctga acattgccgt
300 ctgatttcat acgcagaagc aagattcg 328 <210> SEQ ID NO 7
<211> LENGTH: 275 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 7 cgattccggt taaaggacgc
tgggtgctgg gaaagcaggt cggcctcttt atggctgcct 60 gcgatggcca
cgaaggcccc agcctgattc ctccccagat ttttggtctt gctgtgctgg 120
tgtcatgtaa ctcatgtttg gatgccgttc aacaacttgg tgaatgagca agttctgagc
180 tcaagtttcc atgtgtatga gtttccatgc gtacgagtgt ctgttacggt
tccgttatac 240 cggtatttat tgtctgtgtt ggtttcttct gcatg 275
<210> SEQ ID NO 8 <211> LENGTH: 291 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 8
catgtttcag aactactcta tgaaaagcaa tgtgaaactc tgggagttga acacaaacat
60 cacagagaag tttctgagaa tgcttctgtt tagtttttat gtgaagatat
tcccgtttcc 120 aaagacatct tcggagaggt ccacatttcc acttgcagat
tccacaaaaa gggagtttca 180 acactgctct atccatagga gggttcaact
ctgtgagttg aatgcaatca tcacagagaa 240 gtttcagaga aggcttctgt
ctagatttta tgcgaagata tacccgtttc g 291 <210> SEQ ID NO 9
<211> LENGTH: 75 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 9 catgtgcaaa tctggcttgc
acccgttgta tgcacacgtt gaatctatca cacacgatca 60 tcacgtgatg cttcg 75
<210> SEQ ID NO 10 <211> LENGTH: 77 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 10
cgatgatgcc gccgcaggag gcgtactcgc agcaggggca gtcaatgcag cagtggtcgc
60 cgtcgtacct gtacatg 77 <210> SEQ ID NO 11 <211>
LENGTH: 162 <212> TYPE: DNA <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 11 catgccaggt aatagccctg ttgaaacacc
cagggcattg tgtacttgtt tatttttctt 60 tttgcttgta aatgctacag
cccctcggat gaatcacaac caccgtggtt gctatgtgtg 120 gtggtaatga
ccagtctatt ggttgagccc agcataaatt cg 162 <210> SEQ ID NO 12
<211> LENGTH: 186 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 12 catgcccccc acaggtgttg
tcggtggtac atttgtcgga gatgctaatc taactgtaac 60 agcgggtatt
acaccgtcgt atatcctaca agagggtgat cttactgccg actggactat 120
tacatttggg cagaacgatc aatttggtcg tcccgtccta gacggagcta gttttagaat
180 ctatcg 186 <210> SEQ ID NO 13 <211> LENGTH: 113
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 13 cgattagatc gtgggaggaa tgtctttttc
caattttgga agggcttaat catattaccg 60 acccggcaat attttcggat
cggagggagt actgatcttt ttcaccgcgc atg 113 <210> SEQ ID NO 14
<211> LENGTH: 81 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 14 cgaaacggcg cgcagtaaac
tccttcgttt acgcgcaagt ggagaaaatg ggccgggcgc 60 accgattcct
tcctccacat g 81 <210> SEQ ID NO 15 <211> LENGTH: 138
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 15 catgtgtaaa ggtgcataca aatctgaacc
aagaattact tttaatgcat gaactgtgaa 60 catctaccgg atggtaggta
tcatttttcg tgctaagcgc aaatcttcgt aaaccatgta 120 gtcgttcagg actcatcg
138 <210> SEQ ID NO 16 <211> LENGTH: 78 <212>
TYPE: DNA <213> ORGANISM: Festuca mairei <400>
SEQUENCE: 16 catgcagagg attgaacgac actagtatat gggtgtcctc gtagtgtttt
cctttgcacg 60 tgggtgtcca cttattcg 78 <210> SEQ ID NO 17
<211> LENGTH: 69 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 17 cgaatagcaa atggccgagg
tccctgctgc atgaacctga tctgctgcga aatgagagct 60 gggcgcatg 69
<210> SEQ ID NO 18 <211> LENGTH: 166 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 18 cgatggcagc tagggcgcta ggcagcgcgc
tacggcagct ggcagccagc aggctcaagg 60 tgcgtggacg ggctaatcag
ggcgtgcggg cgtgcgttgc aagcaggagg ccggaggctg 120 gcgttcaggc
ggatgacgcg cagaaggctg ggccgaaagg tgcatg 166 <210> SEQ ID NO
19 <211> LENGTH: 116 <212> TYPE: DNA <213>
ORGANISM: Festuca mairei <400> SEQUENCE: 19 catgcctagc
tgtgcgcacc tgttcgccga tgagtacgcc accacgggca accaaacaat 60
acccgtcacg ctgccgcgcc tatggcagcc atcggatggc tattggggta ttatcg 116
<210> SEQ ID NO 20 <211> LENGTH: 545 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 20
catgtacact ggtataccac cggatacgta cattgaatta agaacaccgc catcagacgc
60 aactacttta acagaactat tgataccatc ggctgcccga acaccgtcat
agcagaaatg 120 tccagtatgg ttaccgtacg tgtcgccacc aataatacca
ccgatataag cggacttaac 180 gttggcaccc aggtataccg cacaggcatt
atctccggta tagtagttac tgttgttgta 240 gtctttaata tcggtaacga
tcattacacg acgagcattt tcaagccaga cgggattgcc 300 tacaaatgta
gacatttgcc cgccagtcca actaatagag ttcacagacc cattagtggc 360
ggtagaacct ttgtcactaa tagctcagta gttattaccc tgacccaggg ctgaacaacc
420 cgtaaagtgg attgaagcta cttgtccact tgtatcttca ttttcaaaac
gaatacaagc 480 atcattagtg ccgataccgc atggatcaaa tgtactgttg
acaaaattca agtcttgaat 540 gatcg 545 <210> SEQ ID NO 21
<211> LENGTH: 165 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 21 catgcatgaa tgccacatga
atgcaagaaa ggtaaaaagg gtctaggtgt tactcttggg 60 atgttacacg
tagtgtggtg cgaccatgaa cttggcgaag gcctggcggc caagcacgca 120
gtggaacggg ctgtggaagc tgacgacctc gttcaggact catcg 165 <210>
SEQ ID NO 22 <211> LENGTH: 211 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 22
catgcatata tgcagacatg acacataaca cagccgccac cggcgacatg gctgacagta
60 ctcatctagc tcatccgtac atcggctata agtacatcgg ctataagcgg
tagcataatt 120 acagttgtgt agagaactgg tgagcactat cagtatgtac
tatctactca ccagtagcta 180 gttcggttcg gctagagcgc cttacagatc g 211
<210> SEQ ID NO 23 <211> LENGTH: 126 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 23
catgtatgtg ttggacacca tgtatagggg cggatgggcg gttatgtagg gatctcatat
60 cagctatgat ctggttgctg ttccgtatct ttggatgacc accaggaggg
gctcagcacc 120 cgctcg 126 <210> SEQ ID NO 24 <211>
LENGTH: 153 <212> TYPE: DNA <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 24 cgagtcaact caaaggttaa tttttgctgg
ccttgctgta gagaagctag tgatgaaatt 60 aagcaaggta gcttgttgat
taagttgtaa tcaagacagt aactagtata ggtagcccca 120 cacactactt
tgcaggttca gtttagatgc atg 153 <210> SEQ ID NO 25 <211>
LENGTH: 68 <212> TYPE: DNA <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 25 catgtacaag tataacaccc accagatcgc
ctcctctgct tcggatcagg agctcatgaa 60 agcgctcg 68 <210> SEQ ID
NO 26 <211> LENGTH: 97 <212> TYPE: DNA <213>
ORGANISM: Festuca mairei <400> SEQUENCE: 26 cgatggcatt
ggcgttcatg tccaggaagg caaggtgcca tctcagagag ctagcagacc 60
agcaaacggg aggagcccgt cgccgagcta caacatg 97 <210> SEQ ID NO
27 <211> LENGTH: 147 <212> TYPE: DNA <213>
ORGANISM: Festuca mairei <400> SEQUENCE: 27 catgtgtaca
tttcctctgt actcctccct actccgacgc tagactgaat cgctgatcac 60
atatatccac ggtcaaaaca cttgcttcac tcttcctcct gcgcgtgaat ccgatggacc
120 ctgatgggga ggcccttggg gaagtcg 147 <210> SEQ ID NO 28
<211> LENGTH: 119 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 28 catgtgctaa ataaaacatt
ttggatatgc tagtacaaat gtggtctgga tgctcgcata 60 tagaagcaag
gtccataaga gcgacaattg gaagatcaag ctagcattgt gtggtctcg 119
<210> SEQ ID NO 29 <211> LENGTH: 108 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 29
cgagttcccg gccttctgat ccaatccaat ccaaggagtc gtgtcatgcc ttcctgattc
60 ctagctattc atttgctctt cccttacaga ataaactgtg ggagcatg 108
<210> SEQ ID NO 30 <211> LENGTH: 101 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 30
cgaccgtagg gctagtaata caattgtgtg gagccacttg gcttgtgagc atactataca
60 ctcccctaca catttatcat cccttactag aagtgcacat g 101 <210>
SEQ ID NO 31 <211> LENGTH: 299 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 31
catgctcacc acagggccaa ggaagaggcc aaagtgcccc tgaacgagtc caccctgctg
60 ctggatccat ccatccatct cctcctcaac ggacctacgg caccctgcct
aattgcctag 120 atgtgttctc gtgtagcttc cctctgctcc tgctagttag
tttttttttt tttgaacagg 180 ctagttagct agtgtgatgc gtattgtctg
ttggattcgc gtgctgtacg tgcctgaagc 240 tacgtatatg ttgtcgttgt
cagcttgtaa gagtaatgtt ctgctagcca ggatcgtcg 299 <210> SEQ ID
NO 32 <211> LENGTH: 169 <212> TYPE: DNA <213>
ORGANISM: Festuca mairei <400> SEQUENCE: 32 catgcgcaac
acggaaatta cacacatacg gagtataatt tacagataca atcaacactg 60
cgttcgtgcg aacatatatg tgaatttatc ggtggaacgc tcctcggaat cttgaaacga
120 tcaagcgccg gaaaccaccg ccgccgcagg ctgatcgccg ggaacgtcg 169
<210> SEQ ID NO 33 <211> LENGTH: 71 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 33
catgctactt tggggcctcc aacgcactct cgatctgctg gattagctga tcagcaggca
60 acgctcactc g 71 <210> SEQ ID NO 34 <211> LENGTH: 204
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 34 catgttttgg tttccagata attaacttgt
gccggagtac gaatacgtcg tcacccccac 60 ccaggatagg tcctagtatt
gataatctag ccggatgcaa tgcgctagtc gtatttaatt 120 agccactgtt
ctctcgttgt gctgcagaat tgtaaagatg tgtaagctgt agtgcacatg 180
gagcagcttc agtaccagtt ctcg 204 <210> SEQ ID NO 35 <211>
LENGTH: 177 <212> TYPE: DNA <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 35 catgttagcg taaaacacgt tcttgatcgg
cttgcgctta atcccgataa ggtctgcgtt 60
ttcctggtga gggtcatggc cctctgtgta gaatcgcttg cccagttcgg gattcggcag
120 aaagtgagcg aaacatcgca tctgagcggc cgcagcatcg tagccgacca gtactcg
177 <210> SEQ ID NO 36 <211> LENGTH: 88 <212>
TYPE: DNA <213> ORGANISM: Festuca mairei <400>
SEQUENCE: 36 catgcccgtt ccaggcttcc agcagatctg ttgtcatcta ctcatctgtc
agtgccgggt 60 gcgaaccaag gcctcccacc cgaactcg 88 <210> SEQ ID
NO 37 <211> LENGTH: 161 <212> TYPE: DNA <213>
ORGANISM: Festuca mairei <400> SEQUENCE: 37 catgctagga
tgatgacgtt ggcaacagcg tcgcgttaga gtggggccgg gcgcgtggac 60
tgccgttagt aatgcgagct cgtacaacat ctacgagaag ctacccgtgg cgacgacgat
120 ggtcctcctc cgtggtcgtc acgatctcct ccaggtcgtc g 161 <210>
SEQ ID NO 38 <211> LENGTH: 335 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 38
cgacaacgag gcaacgtggc ttgatttgag aggaccaagc ctggtgtgct ggccaggtag
60 aagtgctact cgttttgctc actggtaagg cacgtcgccc agatattttt
agctaatgcc 120 taagcggcgg gcggcaagat attttacaca gtttgagcgg
ctagattttt agctgacttg 180 ggaaccgacg ttgagcacct atatatagat
agccttgccg cttctgcggc tgctaacatc 240 agtagactgc aaatagagct
ggacctacca aacgagagtg agagagtaga gaaagagagc 300 gagagaaggg
ccggtgaaga tcattcatgc gcatg 335 <210> SEQ ID NO 39
<211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 39 cgagggaacg cgtctacaac atg
23 <210> SEQ ID NO 40 <211> LENGTH: 28 <212>
TYPE: PRT <213> ORGANISM: Festuca mairei <400>
SEQUENCE: 40 Arg Cys Gln Cys Val Thr Asn Thr Arg Thr Arg Arg Arg
Thr Arg Asp 1 5 10 15 Asn Leu Leu Tyr Ser Pro Leu Leu Gly Pro Met
His 20 25 <210> SEQ ID NO 41 <211> LENGTH: 28
<212> TYPE: PRT <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 41 Asp Val Ser Val Ser Arg Thr Arg Val His
Ala Val Ala Arg Gly Thr 1 5 10 15 Thr Tyr Ser Thr Val His Tyr Trp
Val Leu Cys Met 20 25 <210> SEQ ID NO 42 <211> LENGTH:
27 <212> TYPE: PRT <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 42 Met Ser Val Cys His Glu His Ala Tyr Thr
Pro Ser His Ala Gly Gln 1 5 10 15 Leu Thr Leu Gln Ser Ile Thr Gly
Ser Tyr Ala 20 25 <210> SEQ ID NO 43 <211> LENGTH: 62
<212> TYPE: PRT <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 43 Cys Ala Val Ile Cys Gln Ser Ser Leu Ala
Ile Val Phe Pro Pro Leu 1 5 10 15 Pro Ile Leu Gly Thr Ser His Pro
Val Val Thr Glu Ile Gly Lys Arg 20 25 30 Asn Gln Gly Arg Pro Thr
Asp Thr Ser Asp Asn Asn Asn Leu Thr Asn 35 40 45 Leu Ala His Gln
Val Val Thr Phe Asn Arg Arg Lys Leu Ser 50 55 60 <210> SEQ ID
NO 44 <211> LENGTH: 62 <212> TYPE: PRT <213>
ORGANISM: Festuca mairei <400> SEQUENCE: 44 Arg Lys Leu Thr
Ser Ile Glu Cys His Asp Leu Met Cys Lys Ile Gly 1 5 10 15 Glu Val
Val Val Val Gly Gly Val Arg Arg Ser Ala Leu Ile Ser Leu 20 25 30
Ser Asn Leu Ser Asp Asp Arg Met Arg Gly Ala Lys Asn Gly Gln Trp 35
40 45 Trp Glu Asn Asn Gly Gln Arg Ala Leu Ala Asn Asn Ser Ala 50 55
60 <210> SEQ ID NO 45 <211> LENGTH: 44 <212>
TYPE: PRT <213> ORGANISM: Festuca mairei <400>
SEQUENCE: 45 Arg Arg Ala Thr Met Glu Ser Phe Gly Val Thr Ser Ser
Thr Pro Ala 1 5 10 15 His Val Leu Pro Pro Gly Val Asp Ala Asn Ser
Glu Ala Gly Arg Leu 20 25 30 Leu Gln Gly Phe Arg Ser Ala Tyr Leu
Arg Cys Met 35 40 <210> SEQ ID NO 46 <211> LENGTH: 43
<212> TYPE: PRT <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 46 Glu Gly Arg Pro Trp Lys Ala Leu Val Ser
Leu His Gln Arg Gln Leu 1 5 10 15 Met Phe Tyr Leu Gln Ala Leu Met
Pro Ile Arg Arg Gln Gly Val Ser 20 25 30 Tyr Arg Ala Ser Ala Val
Pro Ile Ser Gly Ala 35 40 <210> SEQ ID NO 47 <211>
LENGTH: 96 <212> TYPE: PRT <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 47 Met Phe Gln Asn Tyr Ser Met Lys Ser
Asn Val Lys Leu Trp Glu Leu 1 5 10 15 Asn Thr Asn Ile Thr Glu Lys
Phe Leu Arg Met Leu Leu Phe Ser Phe 20 25 30 Tyr Val Lys Ile Phe
Pro Phe Pro Lys Thr Ser Ser Glu Arg Ser Thr 35 40 45 Phe Pro Leu
Ala Asp Ser Thr Lys Arg Glu Phe Gln His Cys Ser Ile 50 55 60 His
Arg Arg Val Gln Leu Cys Glu Leu Asn Ala Ile Ile Thr Glu Lys 65 70
75 80 Phe Gln Arg Arg Leu Leu Ser Arg Phe Tyr Ala Lys Ile Tyr Pro
Phe 85 90 95 <210> SEQ ID NO 48 <211> LENGTH: 96
<212> TYPE: PRT <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 48 Lys Arg Val Tyr Leu Arg Ile Lys Ser Arg
Gln Lys Pro Ser Leu Lys 1 5 10 15 Leu Leu Cys Asp Asp Cys Ile Gln
Leu Thr Glu Leu Asn Pro Pro Met 20 25 30 Asp Arg Ala Val Leu Lys
Leu Pro Phe Cys Gly Ile Cys Lys Trp Lys 35 40 45 Cys Gly Pro Leu
Arg Arg Cys Leu Trp Lys Arg Glu Tyr Leu His Ile 50 55 60 Lys Thr
Lys Gln Lys His Ser Gln Lys Leu Leu Cys Asp Val Cys Val 65 70 75 80
Gln Leu Pro Glu Phe His Ile Ala Phe His Arg Val Val Leu Lys His 85
90 95 <210> SEQ ID NO 49 <211> LENGTH: 24 <212>
TYPE: PRT <213> ORGANISM: Festuca mairei <400>
SEQUENCE: 49 Met Cys Lys Ser Gly Leu His Pro Leu Tyr Ala His Val
Glu Ser Ile 1 5 10 15 Thr His Asp His His Val Met Leu 20
<210> SEQ ID NO 50 <211> LENGTH: 24 <212> TYPE:
PRT <213> ORGANISM: Festuca mairei <400> SEQUENCE: 50
Lys His His Val Met Ile Val Cys Asp Arg Phe Asn Val Cys Ile Gln
1 5 10 15 Arg Val Gln Ala Arg Phe Ala His 20 <210> SEQ ID NO
51 <211> LENGTH: 25 <212> TYPE: PRT <213>
ORGANISM: Festuca mairei <400> SEQUENCE: 51 Asp Asp Ala Ala
Ala Gly Gly Val Leu Ala Ala Gly Ala Val Asn Ala 1 5 10 15 Ala Val
Val Ala Val Val Pro Val His 20 25 <210> SEQ ID NO 52
<211> LENGTH: 25 <212> TYPE: PRT <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 52 Met Met Pro Pro Gln Glu Ala
Tyr Ser Gln Gln Gly Gln Ser Met Gln 1 5 10 15 Gln Trp Ser Pro Ser
Tyr Leu Tyr Met 20 25 <210> SEQ ID NO 53 <211> LENGTH:
25 <212> TYPE: PRT <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 53 Met Tyr Arg Tyr Asp Gly Asp His Cys Cys
Ile Asp Cys Pro Cys Cys 1 5 10 15 Glu Tyr Ala Ser Cys Gly Gly Ile
Ile 20 25 <210> SEQ ID NO 54 <211> LENGTH: 25
<212> TYPE: PRT <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 54 Cys Thr Gly Thr Thr Ala Thr Thr Ala Ala
Leu Thr Ala Pro Ala Ala 1 5 10 15 Ser Thr Pro Pro Ala Ala Ala Ser
Ser 20 25 <210> SEQ ID NO 55 <211> LENGTH: 61
<212> TYPE: PRT <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 55 Met Pro Pro Thr Gly Val Val Gly Gly Thr
Phe Val Gly Asp Ala Asn 1 5 10 15 Leu Thr Val Thr Ala Gly Ile Thr
Pro Ser Tyr Ile Leu Gln Glu Gly 20 25 30 Asp Leu Thr Ala Asp Trp
Thr Ile Thr Phe Gly Gln Asn Asp Gln Phe 35 40 45 Gly Arg Pro Val
Leu Asp Gly Ala Ser Phe Arg Ile Tyr 50 55 60 <210> SEQ ID NO
56 <211> LENGTH: 37 <212> TYPE: PRT <213>
ORGANISM: Festuca mairei <400> SEQUENCE: 56 Arg Leu Asp Arg
Gly Arg Asn Val Phe Phe Gln Phe Trp Lys Gly Leu 1 5 10 15 Ile Ile
Leu Pro Thr Arg Gln Tyr Phe Arg Ile Gly Gly Ser Thr Asp 20 25 30
Leu Phe His Arg Ala 35 <210> SEQ ID NO 57 <211> LENGTH:
26 <212> TYPE: PRT <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 57 Glu Thr Ala Arg Ser Lys Leu Leu Arg Leu
Arg Ala Ser Gly Glu Asn 1 5 10 15 Gly Pro Gly Ala Pro Ile Pro Ser
Ser Thr 20 25 <210> SEQ ID NO 58 <211> LENGTH: 26
<212> TYPE: PRT <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 58 Lys Arg Arg Ala Val Asn Ser Phe Val Tyr
Ala Gln Val Glu Lys Met 1 5 10 15 Gly Arg Ala His Arg Phe Leu Pro
Pro His 20 25 <210> SEQ ID NO 59 <211> LENGTH: 27
<212> TYPE: PRT <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 59 His Val Glu Glu Gly Ile Gly Ala Pro Gly
Pro Phe Ser Pro Leu Ala 1 5 10 15 Arg Lys Arg Arg Ser Leu Leu Arg
Ala Val Ser 20 25 <210> SEQ ID NO 60 <211> LENGTH: 26
<212> TYPE: PRT <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 60 Met Trp Arg Lys Glu Ser Val Arg Pro Ala
His Phe Leu His Leu Arg 1 5 10 15 Val Asn Glu Gly Val Tyr Cys Ala
Pro Phe 20 25 <210> SEQ ID NO 61 <211> LENGTH: 45
<212> TYPE: PRT <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 61 Met Ser Pro Glu Arg Leu His Gly Leu Arg
Arg Phe Ala Leu Ser Thr 1 5 10 15 Lys Asn Asp Thr Tyr His Pro Val
Asp Val His Ser Ser Cys Ile Lys 20 25 30 Ser Asn Ser Trp Phe Arg
Phe Val Cys Thr Phe Thr His 35 40 45 <210> SEQ ID NO 62
<211> LENGTH: 25 <212> TYPE: PRT <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 62 Asn Lys Trp Thr Pro Thr Cys
Lys Gly Lys His Tyr Glu Asp Thr His 1 5 10 15 Ile Leu Val Ser Phe
Asn Pro Leu His 20 25 <210> SEQ ID NO 63 <211> LENGTH:
23 <212> TYPE: PRT <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 63 Arg Ile Ala Asn Gly Arg Gly Pro Cys Cys
Met Asn Leu Ile Cys Cys 1 5 10 15 Glu Met Arg Ala Gly Arg Met 20
<210> SEQ ID NO 64 <211> LENGTH: 22 <212> TYPE:
PRT <213> ORGANISM: Festuca mairei <400> SEQUENCE: 64
Asn Ser Lys Trp Pro Arg Ser Leu Leu His Glu Pro Asp Leu Leu Arg 1 5
10 15 Asn Glu Ser Trp Ala His 20 <210> SEQ ID NO 65
<211> LENGTH: 23 <212> TYPE: PRT <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 65 His Ala Pro Ser Ser His Phe
Ala Ala Asp Gln Val His Ala Ala Gly 1 5 10 15 Thr Ser Ala Ile Cys
Tyr Ser 20 <210> SEQ ID NO 66 <211> LENGTH: 22
<212> TYPE: PRT <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 66 Met Arg Pro Ala Leu Ile Ser Gln Gln Ile
Arg Phe Met Gln Gln Gly 1 5 10 15 Pro Arg Pro Phe Ala Ile 20
<210> SEQ ID NO 67 <211> LENGTH: 22 <212> TYPE:
PRT <213> ORGANISM: Festuca mairei <400> SEQUENCE: 67
Cys Ala Gln Leu Ser Phe Arg Ser Arg Ser Gly Ser Cys Ser Arg Asp 1 5
10 15
Leu Gly His Leu Leu Phe 20 <210> SEQ ID NO 68 <211>
LENGTH: 54 <212> TYPE: PRT <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 68 Met Ala Ala Arg Ala Leu Gly Ser Ala
Leu Arg Gln Leu Ala Ala Ser 1 5 10 15 Arg Leu Lys Val Arg Gly Arg
Ala Asn Gln Gly Val Arg Ala Cys Val 20 25 30 Ala Ser Arg Arg Pro
Glu Ala Gly Val Gln Ala Asp Asp Ala Gln Lys 35 40 45 Ala Gly Pro
Lys Gly Ala 50 <210> SEQ ID NO 69 <211> LENGTH: 38
<212> TYPE: PRT <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 69 Met Pro Ser Cys Ala His Leu Phe Ala Asp
Glu Tyr Ala Thr Thr Gly 1 5 10 15 Asn Gln Thr Ile Pro Val Thr Leu
Pro Arg Leu Trp Gln Pro Ser Asp 20 25 30 Gly Tyr Trp Gly Ile Ile 35
<210> SEQ ID NO 70 <211> LENGTH: 38 <212> TYPE:
PRT <213> ORGANISM: Festuca mairei <400> SEQUENCE: 70
Cys Leu Ala Val Arg Thr Cys Ser Pro Met Ser Thr Pro Pro Arg Ala 1 5
10 15 Thr Lys Gln Tyr Pro Ser Arg Cys Arg Ala Tyr Gly Ser His Arg
Met 20 25 30 Ala Ile Gly Val Leu Ser 35 <210> SEQ ID NO 71
<211> LENGTH: 22 <212> TYPE: PRT <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 71 Met Tyr Lys Tyr Asn Thr His
Gln Ile Ala Ser Ser Ala Ser Asp Gln 1 5 10 15 Glu Leu Met Lys Ala
Leu 20 <210> SEQ ID NO 72 <211> LENGTH: 22 <212>
TYPE: PRT <213> ORGANISM: Festuca mairei <400>
SEQUENCE: 72 Glu Arg Phe His Glu Leu Leu Ile Arg Ser Arg Gly Gly
Asp Leu Val 1 5 10 15 Gly Val Ile Leu Val His 20 <210> SEQ ID
NO 73 <211> LENGTH: 32 <212> TYPE: PRT <213>
ORGANISM: Festuca mairei <400> SEQUENCE: 73 Asp Gly Ile Gly
Val His Val Gln Glu Gly Lys Val Pro Ser Gln Arg 1 5 10 15 Ala Ser
Arg Pro Ala Asn Gly Arg Ser Pro Ser Pro Ser Tyr Asn Met 20 25 30
<210> SEQ ID NO 74 <211> LENGTH: 31 <212> TYPE:
PRT <213> ORGANISM: Festuca mairei <400> SEQUENCE: 74
Met Ala Leu Ala Phe Met Ser Arg Lys Ala Arg Cys His Leu Arg Glu 1 5
10 15 Leu Ala Asp Gln Gln Thr Gly Gly Ala Arg Arg Arg Ala Thr Thr
20 25 30 <210> SEQ ID NO 75 <211> LENGTH: 32
<212> TYPE: PRT <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 75 His Val Val Ala Arg Arg Arg Ala Pro Pro
Val Cys Trp Ser Ala Ser 1 5 10 15 Ser Leu Arg Trp His Leu Ala Phe
Leu Asp Met Asn Ala Asn Ala Ile 20 25 30 <210> SEQ ID NO 76
<211> LENGTH: 35 <212> TYPE: PRT <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 76 Ser Ser Arg Pro Ser Asp Pro
Ile Gln Ser Lys Glu Ser Cys His Ala 1 5 10 15 Phe Leu Ile Pro Ser
Tyr Ser Phe Ala Leu Pro Leu Gln Asn Lys Leu 20 25 30 Trp Glu His 35
<210> SEQ ID NO 77 <211> LENGTH: 33 <212> TYPE:
PRT <213> ORGANISM: Festuca mairei <400> SEQUENCE: 77
Asp Arg Arg Ala Ser Asn Thr Ile Val Trp Ser His Leu Ala Cys Glu 1 5
10 15 His Thr Ile His Ser Pro Thr His Leu Ser Ser Leu Thr Arg Ser
Ala 20 25 30 His <210> SEQ ID NO 78 <211> LENGTH: 33
<212> TYPE: PRT <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 78 Thr Val Gly Leu Val Ile Gln Leu Cys Gly
Ala Thr Trp Leu Val Ser 1 5 10 15 Ile Leu Tyr Thr Pro Leu His Ile
Tyr His Pro Leu Leu Glu Val His 20 25 30 Met <210> SEQ ID NO
79 <211> LENGTH: 33 <212> TYPE: PRT <213>
ORGANISM: Festuca mairei <400> SEQUENCE: 79 Cys Ala Leu Leu
Val Arg Asp Asp Lys Cys Val Gly Glu Cys Ile Val 1 5 10 15 Cys Ser
Gln Ala Lys Trp Leu His Thr Ile Val Leu Leu Ala Leu Arg 20 25 30
Ser <210> SEQ ID NO 80 <211> LENGTH: 23 <212>
TYPE: PRT <213> ORGANISM: Festuca mairei <400>
SEQUENCE: 80 His Ala Thr Leu Gly Pro Pro Thr His Ser Arg Ser Ala
Gly Leu Ala 1 5 10 15 Asp Gln Gln Ala Thr Leu Thr 20 <210>
SEQ ID NO 81 <211> LENGTH: 7 <212> TYPE: PRT
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 81 Arg
Gly Asn Ala Ser Thr Thr 1 5 <210> SEQ ID NO 82 <211>
LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 82 Glu Gly Thr Arg Leu Gln His 1 5
<210> SEQ ID NO 83 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Festuca mairei <400> SEQUENCE: 83
Arg Glu Arg Val Tyr Asn Met 1 5 <210> SEQ ID NO 84
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 84 His Val Val Asp Ala Phe Pro
1 5
<210> SEQ ID NO 85 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Festuca mairei <400> SEQUENCE: 85
Cys Cys Arg Arg Val Pro Ser 1 5 <210> SEQ ID NO 86
<211> LENGTH: 59 <212> TYPE: PRT <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 86 His Val Ser Val Lys His Val
Leu Asp Arg Leu Ala Leu Asn Pro Asp 1 5 10 15 Lys Val Cys Val Phe
Leu Val Arg Val Met Ala Leu Cys Val Glu Ser 20 25 30 Leu Ala Gln
Phe Gly Ile Arg Gln Lys Val Ser Glu Thr Ser His Leu 35 40 45 Ser
Gly Arg Ser Ile Val Ala Asp Gln Tyr Ser 50 55 <210> SEQ ID NO
87 <211> LENGTH: 59 <212> TYPE: PRT <213>
ORGANISM: Festuca mairei <400> SEQUENCE: 87 Arg Val Leu Val
Gly Tyr Asp Ala Ala Ala Ala Gln Met Arg Cys Phe 1 5 10 15 Ala His
Phe Leu Pro Asn Pro Glu Leu Gly Lys Arg Phe Tyr Thr Glu 20 25 30
Gly His Asp Pro His Gln Glu Asn Ala Asp Leu Ile Gly Ile Lys Arg 35
40 45 Lys Pro Ile Lys Asn Val Phe Tyr Ala Asn Met 50 55 <210>
SEQ ID NO 88 <211> LENGTH: 58 <212> TYPE: PRT
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 88 Glu
Tyr Trp Ser Ala Thr Met Leu Arg Pro Leu Arg Cys Asp Val Ser 1 5 10
15 Leu Thr Phe Cys Arg Ile Pro Asn Trp Ala Ser Asp Ser Thr Gln Arg
20 25 30 Ala Met Thr Leu Thr Arg Lys Thr Gln Thr Leu Ser Gly Leu
Ser Ala 35 40 45 Ser Arg Ser Arg Thr Cys Phe Thr Leu Thr 50 55
<210> SEQ ID NO 89 <211> LENGTH: 29 <212> TYPE:
PRT <213> ORGANISM: Festuca mairei <400> SEQUENCE: 89
His Ala Arg Ser Arg Leu Pro Ala Asp Leu Leu Ser Ser Thr His Leu 1 5
10 15 Ser Val Pro Gly Ala Asn Gln Gly Leu Pro Pro Glu Leu 20 25
<210> SEQ ID NO 90 <211> LENGTH: 29 <212> TYPE:
PRT <213> ORGANISM: Festuca mairei <400> SEQUENCE: 90
Met Pro Val Pro Gly Phe Gln Gln Ile Cys Cys His Leu Leu Ile Cys 1 5
10 15 Gln Cys Arg Val Arg Thr Lys Ala Ser His Pro Asn Ser 20 25
<210> SEQ ID NO 91 <211> LENGTH: 28 <212> TYPE:
PRT <213> ORGANISM: Festuca mairei <400> SEQUENCE: 91
Cys Pro Phe Gln Ala Ser Ser Arg Ser Val Val Ile Tyr Ser Ser Val 1 5
10 15 Ser Ala Gly Cys Glu Pro Arg Pro Pro Thr Arg Thr 20 25
<210> SEQ ID NO 92 <400> SEQUENCE: 92 000 <210>
SEQ ID NO 93 <211> LENGTH: 222 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 93
catgcacaga ggacactcca tgggttgcag ccaccggatg ccaagctgtt ccccgagaag
60 gcaggctaca acgagctgaa tcagatggct gaagaggcaa aacggagagc
tgaaattgca 120 aggctcaggg agcttcacac tctcaagggg cacgtagagt
cggttgtgaa gctgaagggc 180 ctggacattg acaccattca gcaatcttac
acagtgtgat cg 222 <210> SEQ ID NO 94 <211> LENGTH: 194
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 94 catgttcgtc aacgaggttt acacggttct
gaccgatccg gtgcagcgtg ccgtgtatga 60 tgagctccat ggctacgcag
caacggccgc caaccctttc tttaatgaca gtgcgcccaa 120 ggatcacgtc
tttgttgacg agtttacctg tataggatgc aagatttgtg ccaatgtgtg 180
ccccaatgtg ttcg 194 <210> SEQ ID NO 95 <211> LENGTH:
228 <212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 95 catgcgcctc taaatcttca gcatggcctc
caacgcgcga agtacgtcat cacgggtaac 60 tatacctatc acttggttgt
cctgatttac tattggtaat ctgtggatct tcttcttgag 120 catcagagct
gcggcatcgg tcactgttct atcacatgat agcgtgatcg ctggagaggt 180
catcacttgt gcaatctttg tccttgaccc atatgaagcc cttgttcg 228
<210> SEQ ID NO 96 <211> LENGTH: 212 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 96
catgttcctt ctacgttgat aggtacggtg catacacaca caattatatg tggaaataaa
60 agtaaaaccc ggaaagcgga gttgtcatca aaaactaaac caagagactc
catatggatt 120 cctagctcgc agcttatgcc ttgccggact cctcacagcc
tggtggcttg aaggcgatga 180 agctgacgca ctgcacctga cggatgttat cg 212
<210> SEQ ID NO 97 <211> LENGTH: 152 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 97
catgcgcttt acaataatca tgatgtatca gtagaaatcg gctcttgtac aaattattac
60 acgaatgaca gacgccacaa ggcgcgtaac gtggggtact ctttccaaaa
taggcgcagt 120 actttctagc atcgggtaat taatccttat cg 152 <210>
SEQ ID NO 98 <211> LENGTH: 378 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 98
catgtactac agagtatgcg attccagcct gtttccgaaa cctgccttac agaacagcac
60 atggaagttt atgtacctct tccaaatatc atctcctcaa actgaaccag
gcttgcctaa 120 tattccatat aacccatgat cctaccgtaa ttgctgactg
aaccaactag tatttccatc 180 ttacagcttg ccagcaaata cctgcagtaa
aacttttgtc tatctgcatc tgaagatcca 240 ggcctcccat gcaagtagtc
atcaaaattg tacccgagat cgtcagcagc tcactggtca 300 actgagttaa
ccagttttga caaaaacgaa ttccaattac gctctgtcca cattactgca 360
caatgcgctt ccttttcg 378 <210> SEQ ID NO 99 <211>
LENGTH: 114 <212> TYPE: DNA <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 99 catgcgcgtt ccaccagctc ccacagcgca
ttgggtatgc cagttctatt gaaaccatcc 60 accgttatga acatcccgca
gaagaagatc aacagcgagt aggatacctt ttcg 114 <210> SEQ ID NO 100
<211> LENGTH: 151 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 100 catgccctta aaccaccatt
aataatgcca ttattctcag caaaaacaaa cgcctgctct 60 tccaaccctc
acccgggcac aaaacataac aaatcctccg cctagacaga ctgtaagata 120
atgcaaaaaa aaaaggatag ttgacaattc g 151 <210> SEQ ID NO 101
<211> LENGTH: 197 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei
<400> SEQUENCE: 101 cgaactcgcg gttcttggcg aaggtctcgg
ggtcggcgga cagcccagcg gtgtcccagc 60 catagtcgcc ggggaactcg
ccggtgaggt agctcggggg ctcgccggag agcgggccga 120 ggtagagcac
gcggtcggag ccgtaccacg ggctgccgga cgcggccacc ttgggcttgc 180
cggccgtctt gcgcatg 197 <210> SEQ ID NO 102 <211>
LENGTH: 248 <212> TYPE: DNA <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 102 cgaagttgtg gttccccggc ttgtcgctga
cataaatcgg gcagccgccg atcgcccttg 60 cggcgccgtg gtactccgcc
gccgggtgca agctatgaaa catatcccag tcgggctgca 120 tgaactcgcc
gaggaagagg gtgttgtaag ccacggagga gatgtggatg gtatgcgacg 180
ccgggtcgtg cgggtagaag tcgtcggagg cgcgcacgac ggctgtctgc ctggcgctgt
240 agagcatg 248 <210> SEQ ID NO 103 <211> LENGTH: 286
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 103 catgtcctga gttgataaac tggttgggtt
ttgagcagca gcctggtatt tctgtacaac 60 cgcgccaagc tgactaacct
gcggtattat tgacctccgt ggaataagtt cctcatgacg 120 cctagcacag
aactcccaag atgcgatctt gaggtctgga ttaaaaatta tcctcagatg 180
cccctcacgt acgacacgca attgatcaaa gacactttct tgaattgctt ttgtatagtc
240 cagaacaatc tgaccagata cattcttgga ctcacgtggc atatcg 286
<210> SEQ ID NO 104 <211> LENGTH: 116 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 104
catgcggctc acaggcatgg ccgtagtgga tgccttactt gccgggaaca aagttggtgg
60 cgaacgccct tgcgttgttg ttgacagggt cagcgaggtg gtcggcgagg ttatcg
116 <210> SEQ ID NO 105 <211> LENGTH: 263 <212>
TYPE: DNA <213> ORGANISM: Festuca mairei <400>
SEQUENCE: 105 cgatcagcgg tgtgacgagc gtggccgtga acccgaagat
gagcagggtg acggtgacgg 60 ggtacgtgga gccgcgcaag gtgctggaga
aggttaagag cacggggaag gcggcagaga 120 tgtggccgta cgtgccctac
accatggcca cctaccccca cgtcggcggc gcctacgaca 180 agaaggcacc
ggcgggcttc atccggagcg cgccgcaggc catggccgcc cccggggcgc 240
cagaggtcca gtacatgaac atg 263 <210> SEQ ID NO 106 <211>
LENGTH: 116 <212> TYPE: DNA <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 106 cgatcacgtt gctgacccag tcaacaacaa
cgcatgggct ttcgccacca acttcgctcc 60 tggaagttaa atgagttagc
catccgtccg accaccggcc gggcgagata tgcatg 116 <210> SEQ ID NO
107 <211> LENGTH: 165 <212> TYPE: DNA <213>
ORGANISM: Festuca mairei <400> SEQUENCE: 107 catgcgcgga
gcacgcgtac aaatttacat ttcacaccca cacccttgca tatatacctc 60
tcgcacgcac acaggtatac catgcacagg acgacgatgc ttttggccta gtggaacttg
120 aggctggtga ggatgttgtt gttgacgggg tcggggaggt gatcg 165
<210> SEQ ID NO 108 <211> LENGTH: 166 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 108
catgctaaag attgggtgag ttaggtaggg gctgtcgcgc acaaggctgc taggaatgga
60 gcttgagact tcaggtgcaa tggattcagc tgtgaagccc actggctttc
caccagagaa 120 caccttgaac agctggtcaa catcctccaa ggtggtggtc tcatcg
166 <210> SEQ ID NO 109 <211> LENGTH: 310 <212>
TYPE: DNA <213> ORGANISM: Festuca mairei <400>
SEQUENCE: 109 cgaataagga agcattaaag tcaggctgaa ctccatgtgt
gcaatatatg gtttgcctag 60 tccagcgaaa tcaagttgta gcagatgttg
gcacttatgc ggttgtccta gagaagtaga 120 agaagcttag ataacgagtt
ctccggttag ctacactcct ctcagtcttg actgtgttct 180 tacaagagat
ggctgcagcg cgtcatagtg cccataaccg tcgtagagaa cacggactgg 240
attatccttg gcatactcct gaccatattc tgctatgatc ctggggccat cagacccctt
300 ggtgtacatg 310 <210> SEQ ID NO 110 <211> LENGTH:
206 <212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 110 cgaacaaggc ctgcatctct ctttgcttca
catctgacga ccctgctcca atcactatgt 60 ctatgattat cacctttccc
ccatcctctc ttgaaggaat agctttcttg cagttcttta 120 gtatcttgac
acactcttgg tcgccccagt catgcataac ccacttgagg aagacaacgt 180
ttgccggagg aacgctctca aacatg 206 <210> SEQ ID NO 111
<211> LENGTH: 449 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 111 cgaagaagag tacctgcacg
atgacacgca gagggagacg ctcattctga gcggcgtggg 60 tgcaagcctc
cagggagagt ttctggcagt ccattacacg gcaaagttct tccttctctg 120
actccgggag atggggatgc gccttcagat agatgtcaac agcacgataa agtccatcgt
180 ctattggccg agcataatct ggtatggcag cagccaaaga cttgaacttt
ggcaacttta 240 ggttggcatc tggcgcaact tcagctaggt agccgtcaat
caacttagca accatagtta 300 ccggcattag agatggagaa gctaatagtt
gcccatcgtc gccaaggcca ggggaagttc 360 caccagtttc ttgatccatt
gccaagaagt gtccaagaat cctatggacg caatccacat 420 cgtagagcgt
ttcatcagat tcagacatg 449 <210> SEQ ID NO 112 <211>
LENGTH: 233 <212> TYPE: DNA <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 112 catgcttgtc caggagcagg acaatgttaa
gcgtgtgcag cttgctgaca cttacatgag 60 ccaggcagct ctgggtgatg
ctaaccagga tgccacgaag actggttcct tctacggtta 120 gaacactctt
catacaccca ccatctctag ctgcatagga ggaggtaaag gagcacaaca 180
aagaactttg cctgtgccgg aaggttgtac cgaccgggaa gccaagaact tcg 233
<210> SEQ ID NO 113 <211> LENGTH: 206 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 113
cgaacaaggc ctgcatctct ctttgcttca catctgacga ccctgctcca atcactatgt
60 ctatgattat cacctttccc ccatcctctc ttgaaggaat agctttcttg
cagttcttta 120 gtatcttgac acactcttgg tcgccccagt catgcataac
ccacttgagg aagacaacgt 180 ttgccggagg aacgctctca aacatg 206
<210> SEQ ID NO 114 <211> LENGTH: 251 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 114
catgttgaac agcctttgcc gcgacaagaa caacatggtc ttgctcgcta gcacgaagac
60 tcgggcgatg ttaagcgaat ggttttcgcc atgtgagaac ctagggctgg
ctgctgagca 120 cggctatttc ctcaggctga gaggagatgc agagtgggag
acgtgcgctc ctgcgcctga 180 ctctggctgg aagcagattg tggagcctgt
gatgaaaacc tacacggaga caaccgacgg 240 gtcaacgatc g 251 <210>
SEQ ID NO 115 <211> LENGTH: 280 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 115
catgctatcc gatcagagca gcaactcatt tggctctacc gactttgggt gggatgatga
60 ggccatgaca ccggactaca catccgtctt cgttccaaat gctgccatgc
cagcatatgg 120 cgggcccgct tacctgcaag gcggagcgcc aaagaggatg
aggaacaatt tcggtgtagc 180 tgtgcttcct cagggaaatg atgcgccaca
agatgtctgt gcttttgacc atgagatgaa 240 gtattcactg ccttacgttg
agagtagctc agacggatcg 280 <210> SEQ ID NO 116 <211>
LENGTH: 199 <212> TYPE: DNA <213> ORGANISM: Festuca
mairei
<400> SEQUENCE: 116 catgtactta ccagctagct gttggtccgg
tcgtcgttaa gaagcaatta accacagctt 60 aattgaagtg atcgtgacga
gtaactaaac caaactaggg taggtagacg gacgggtccg 120 ggacgtccgt
ccagcagctc ccggcgttcc agtacgcggc cggcgacgcg tcgtccccga 180
gctcgttcag gactcatcg 199 <210> SEQ ID NO 117 <211>
LENGTH: 76 <212> TYPE: DNA <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 117 catgtgtggt ctctagagga acttgaacag
caggcctccg tacgtggcaa agaacggcgc 60 aaacacaagg gattcg 76
<210> SEQ ID NO 118 <211> LENGTH: 309 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 118
catgcgacag gtagtagtac aaaccaacag actacaagat tagtcaggac aacagctaca
60 gagcgtattc ctactatgta cacatatatg gcaccatcta tacgtagtag
taacttaatg 120 tgtgcaatgc atgtccacat caccagccat atacagggtg
ctgtacctgg ggaggcagca 180 ggcccatatc agcacggtgt tgttcatcgt
agtcacgtgt accaggaatc gcgcctgtag 240 ccaacctgag gacctcacta
gaggtcaggc aacgatgcgc aaatgccgat ccactgtcca 300 cgacattcg 309
<210> SEQ ID NO 119 <211> LENGTH: 273 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 119
catgtggtat ccagggtcca tttatccaca caaatgcaca atcggcaata catacgtaag
60 cacagactgg tcactgggtt cagcgatgaa tactgatcac tgggtttcaa
ggctggggca 120 tttgttcagt gcttgtgttt tggcttcttc acaatcatca
ccgtgcagtg cgcgtgatgg 180 gtgcagtaat cgctcacact tccaagaaca
gcccttttaa ttgctccata gccatggttg 240 cccacaacca acatctccgc
gtgatgccgt tcg 273 <210> SEQ ID NO 120 <211> LENGTH:
231 <212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 120 catgtggtgc tgtctctcca cgaagtgaag
gtgaagctga agccaaagcg cttttgacat 60 ggaagtctac tttgatgttc
tccgacgtca acggctcttc tccgctctcg tcatgatcac 120 cggccaactc
cctctgcaat tcttggtctg gcatcacgtg caacacggct ggccatatcg 180
tggagctcac ggttcccgga gctggtgtcg caggcacgct ggacgccttc g 231
<210> SEQ ID NO 121 <211> LENGTH: 128 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 121
cgcagggcaa actgcaagca gatgcaaagg atcctgagac agcactggcc ccaagcattg
60 tagagatctc tccagcacca agtttgtctc tgagtaactc aacactctgg
tcataggaag 120 cgagcatg 128 <210> SEQ ID NO 122 <211>
LENGTH: 103 <212> TYPE: DNA <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 122 catgttaagg cttatagcaa tgtgaacaag
tatctactta ctggtaggta cagaaacatc 60 atggacatga acgcaggctt
tgggggtttc gctgcagcga tcg 103 <210> SEQ ID NO 123 <211>
LENGTH: 256 <212> TYPE: DNA <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 123 catgctaagc gcactgtttc ataaatataa
tgttgtgcag acgatgataa atacagtaga 60 tgcaaccaga ggcgactggt
aacccagctt cattatccag ggaagtgggc gaacccttgg 120 tcctaaagca
gtcgctcact gcttaggaga gtgccaagga tcaatctgat ctcacaggag 180
atgcagaacc ggataagctc ttgctagggc ttctgctcac tgattttcca ggagaggccg
240 aagaaactgg ggatcg 256 <210> SEQ ID NO 124 <211>
LENGTH: 156 <212> TYPE: DNA <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 124 ccatgatgct gaagggatgg tgaatgttat
atctgagaag gaaactgaca gaatcctcgg 60 cgtacacatt atgtcccctg
gcgcgggaga gatcatccat gaggctgtgc ttgcgcttca 120 gtatggagct
tccagcgagg acattgcccg tacatg 156 <210> SEQ ID NO 125
<211> LENGTH: 120 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 125 cgacagcaac ggacagggtg
tacagcgggg ccaccttata gtcaactacc agttcgtcaa 60 ctgcggcgac
aacgagctgc tgctccagcg cgaagagaaa taagaagcta ccagtacatg 120
<210> SEQ ID NO 126 <211> LENGTH: 306 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 126
cgaatgagaa cgcggcatcc tccgtcgcca agaccatctc cggtcagccc ccactgaaga
60 taccgatcag gagcgacaac gccgggtcct ggctgctcac aacctacctt
gatgacgagc 120 ttagaatctc cagaggagat ggcagcagca tctttgtgct
gttcaaggaa gggagcactc 180 tcttaatata ggcttacgtg tatctcttct
cagagtagaa tttgggcgaa tccaatagat 240 agttgtggct atgtgtttgt
tttgttagcc cgtgcgttta tagttcgttc ttgtgtgttg 300 tgcatg 306
<210> SEQ ID NO 127 <211> LENGTH: 168 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 127
catgcgtaga attcttcgcg aagtaaacac gacatgatac gtacgtaaag catcacgtat
60 acgtagctaa tctcggttga ttctgtcctc gcaacctaca taaactggct
gcaaggacgc 120 ggtactagtt aatttcgcaa aaagtatatc ggccacgtgt acgattcg
168 <210> SEQ ID NO 128 <211> LENGTH: 230 <212>
TYPE: DNA <213> ORGANISM: Festuca mairei <400>
SEQUENCE: 128 cgaactcgtg ccgcagcgtg gcgacccaga acccgacggt
ggcgatggcg agcgacttgc 60 cggggcagct ccgcctgccc gacccgaacg
gcgccagcct gaggtctgag cccgttatgg 120 agaactcggc ggcgccggcg
tgatcccgcg acggtccggc gaggaaccgg tcaggcctga 180 actctgccgg
ctcggtccag acggccgggt cgtgcgttat ggcccacatg 230 <210> SEQ ID
NO 129 <211> LENGTH: 185 <212> TYPE: DNA <213>
ORGANISM: Festuca mairei <400> SEQUENCE: 129 cgaagggaag
agatagacaa atgtatccat aaagccgatg gcttcattca gagtattcaa 60
agaagtgacg gttcatggta cggctcctgg ggtgtttgtt tcacatatgg gacatggtat
120 gcagtgaggg gattagttgc cgctggaagg acattcaaga actgtcctgc
tatcaggaag 180 gcatg 185 <210> SEQ ID NO 130 <211>
LENGTH: 201 <212> TYPE: DNA <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 130 cgataattgc tcttatcgtc tgcataccac
cagcagttat tattgaaggt ccccaactta 60 tgcagtatgg attaaatgac
gcaattgcaa aagtaggtct gacaaagttt gtttcagacc 120 ttttcctggt
cggactgttc taccatctgt ataaccagct tgctacaaac acattggagc 180
gggtggcccc tctgacacat g 201 <210> SEQ ID NO 131 <211>
LENGTH: 390 <212> TYPE: DNA <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 131 catgcgtgaa gatggaggtt ttgaagtgat
taagaaagca atcctgaacc tttcacttcg 60 tcacgacttg cacataagtg
aatatggtga aggaaatgaa cggaggttga cagggttaca 120 tgagacagct
agcatatcag acttttcatg gggtgtagca aaccgtggtt gctctattcg 180
ggtggggcga gacactgagg caaaagggaa aggatacctg gaagaccgtc gtccggcctc
240 aaacatggac ccatacactg tgacggccct actggctgaa accacaattc
tctgggagcc 300 gacccttgaa gcagaggctc ttgctgccaa gaagctggcg
atgaacgtat gaaggactga 360 aaaggatgaa tttctgggga aaataaatcg 390
<210> SEQ ID NO 132 <211> LENGTH: 69 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 132
catgttcggc ggcggcaagt tcaagaagtg gaagtaatct gccagtagct ttccatagct
60 gatggatcg 69 <210> SEQ ID NO 133 <211> LENGTH: 238
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 133 catgcccgcc aacaaccgga acactgactt
ctctaggatc aactccaagc acttcagcca 60 caaaggtgtt agccctcgct
acatcaagag ttgtcactcc aaggagacgt ttggggcagt 120 aagttccagc
cctcttgaaa actttcgccg caatggggac agttgagttc acagggttgc 180
tgatcaaatt cataattgca ttagggcagc tcttggcaac gccctcacag attgatcg 238
<210> SEQ ID NO 134 <211> LENGTH: 119 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 134
catgtacacg caagcccccc taatacaggt cgccttcctt gtgggtgtgg atgatgcagt
60 cagacttggg gtatgagacg caggtcagca cgtagccttc ctcctgctgg ttgtcatcg
119 <210> SEQ ID NO 135 <211> LENGTH: 122 <212>
TYPE: DNA <213> ORGANISM: Festuca mairei <400>
SEQUENCE: 135 catgtaccac actgagcacg agcttctccg ttacctacac
aagttgcaaa ccaaggatct 60 ctcactgtgc cacagtatga ttcctcttgg
ttcttgcacc atgaaactaa atgctactgt 120 cg 122 <210> SEQ ID NO
136 <211> LENGTH: 153 <212> TYPE: DNA <213>
ORGANISM: Festuca mairei <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (11)..(11) <223> OTHER
INFORMATION: n is a, c, g, or t <400> SEQUENCE: 136
cgaaacgggg ngatttcttt ttctttttat ggaggaaaag aacattcaag tgaacaacat
60 cccagcagaa gatggggaga aagagagatg aataagaatt attccgatca
ggggaggaac 120 aaacaagctc cctttcttaa ttatgatgac atg 153 <210>
SEQ ID NO 137 <211> LENGTH: 80 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 137
cgaatcctgg ctgtgcaata ccccggaccg aatctattga cagatcatcc atccttggtt
60 tcttgggaga aggctgcatg 80 <210> SEQ ID NO 138 <211>
LENGTH: 473 <212> TYPE: DNA <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 138 catgcatcct taccagcttc ctcagttgtc
ctgttgctct ccaatgtaac aactgctgat 60 ttatcacctt cctccaaacc
tgcgacccct ttaatctcat tgctatgcca agactcagca 120 ttagcatcac
aagctaactg aggctgagaa ttttcagtct tcattttcac ccgccgacca 180
ttctgttcat gcttatcagc aagcacagga gatgaagatc tactcccagt gacagatggg
240 tcgtcaaatg agccaccact cacacttcta gcaggactac gacttggatt
tgaaaagcga 300 actatcaatc tatggctatt accattatca ggaggtgtat
cacctacttt ctcagacacc 360 attccaggtt gtgatgcttt ttcctggaat
gaggaccgat caagtgaagt agatcttccc 420 acggtagctt ctttctttac
cccagaacca agacgggcac tgtttgccct tcg 473 <210> SEQ ID NO 139
<211> LENGTH: 91 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 139 cgacttcacc gggggcatct
catcctaccc gctcctcgtc gcccaggtga cccacttcaa 60 gtgcggaggc
gtggccctcg gcataggcat g 91 <210> SEQ ID NO 140 <211>
LENGTH: 166 <212> TYPE: DNA <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 140 cgatccggtg aagcaatccc tccgccgctt
cgccgaagaa aggagaaaag ccattggtga 60 agagatagcc cggctgctcg
cagccggctt tatcatggaa gtgctgcccc cagactggtt 120 ggctaaccca
gtcctggtct tgaagaagaa tgacacctgg cacatg 166 <210> SEQ ID NO
141 <211> LENGTH: 259 <212> TYPE: DNA <213>
ORGANISM: Festuca mairei <400> SEQUENCE: 141 cgatcgccgt
ctccgcactg cagcagctac catggcgtcc accgcgctct ccaccgcctc 60
caaccctacc cagctctgca ggtccagagc ttcgccgtgc aagcccatca agggcctggg
120 catcggccgg gagcgcgtcc cgaggaacat cacatgcatg gccggcagca
tctccgccga 180 ccgcgtgccg gacatgagca agagggagac gatgaacctc
ctcctgctcg gcgccatctc 240 gctccccacc ttcggcatg 259 <210> SEQ
ID NO 142 <211> LENGTH: 276 <212> TYPE: DNA <213>
ORGANISM: Festuca mairei <400> SEQUENCE: 142 catgttctct
tttgcaggaa gttaggacag gagcgaagcc gaacgtcttt agcttgataa 60
aaaagatgaa gcaaaggaag acgccgcatc ctgagacggg gtccttgtgg gttaacgagc
120 aatccgggac ccagtgtgcg gcgtatgtct tgaagttcaa gcagaagcac
ggcgagagct 180 ccaacccaga ggccgaggat tttgacgttg aggttgcggt
gcttgcggga gaaggcatga 240 agcatggccg cctatggctt ggtgatgggt gtgtcg
276 <210> SEQ ID NO 143 <211> LENGTH: 399 <212>
TYPE: DNA <213> ORGANISM: Festuca mairei <400>
SEQUENCE: 143 cgaagaggtt attcgtgata aggaggccca gttcagcagc
cccaacctca atgttgttta 60 ccgcatgaat gtgcgggagt accaggcact
aaccccctat gcctccatgc tggaggagaa 120 ctggggcaag gcacctgggc
atctcaattc tgatggcgag aacctccttg tctatgggaa 180 gcagtatgga
aacatcttca tcggagtgca gcccactttt ggttatgaag gtgatcctat 240
gcggctcctg ttctcaaaat ctgccagccc tcaccatgga tttgcagcat actacaccta
300 tgttgagaag atcttcaagg cagatgctgt tctgcatttc ggcacacacg
gatcccttga 360 gttcatgccc gggaaacaag tcgggacgag tgatgcatg 399
<210> SEQ ID NO 144 <211> LENGTH: 204 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 144
catgtataca aaacctggac ctcagaatac aacacatcca gtaataaggt aaaaacaaat
60 taactcttaa caggatggaa aacatcatct atctagctct tggggatgtt
cttgccaacg 120 atcttggcag gtgtgatgcg gagaaggttc ccctgcttcc
caggaatggc tcccttgatc 180 atcacaactt taagatcgtt atcg 204
<210> SEQ ID NO 145 <211> LENGTH: 285 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 145
cgatcggact atcctcacca ccaattgcat gaataaccac cgggatgtcg ctcaggtagc
60 gtatcaccag gggcagagcc gctggggcaa catcagcttt cactacgtca
tcgctcagca 120 aaactgaatc ggcagcagcg ggaccgccag cctctattgc
tcctggttca tctttgtctt 180 cagataacat taactcggat gggttcacca
ccactgcgac ttcctcaatt gctatgctac 240 atttgaactg gcttcccctc
ctttgggcag gcttgttcct gcatg 285 <210> SEQ ID NO 146
<211> LENGTH: 82 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 146 catgccggcg tgcccttgta
atagccacat tcatgagcct gctgtcaccc atgaacccta 60 cagctcctag
agggtttgat cg 82 <210> SEQ ID NO 147 <211> LENGTH: 224
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 147 cgaaatcata cttaacctcc ttcccttgct
tgtttatttc tttcaacccc ttcctgaaaa 60 agatcatggc tccacgtggt
ccacggagtg acttgtgagt agtggtagta acaacatctg 120 catactcaaa
aggagatgga atgacaccag cagcaactag gccactgata tgtgccatgt 180
ctgcgagaag tattgccttc tgcttgttac agatcttccg catg 224 <210>
SEQ ID NO 148 <211> LENGTH: 120 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 148
cgaattccat aatgaaatat gttgtaattg ctcatgtgaa cgaatggaga acaggagacc
60 tccatgggcg gcccagaaat tcagcaatga cgcggacctg cgcttccgcc
tccaagcatg 120 <210> SEQ ID NO 149 <211> LENGTH: 460
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 149 catgtttgaa ggaaatgacg tgtcagatgg
tatgggtttc ggaatgctaa ccgagggtga 60 gagatccctg gttgagcgtg
taaggcaaga gctgaagcac gagcttaaac aggggtacag 120 agaaaagctt
gtggacatta gggaagagat acttcggaag cgaagagccg gaaagctccc 180
aggagacaca gcgtctactc tgaaagcctg gtggcaagct catgcaaaat ggccataccc
240 gactgaggag gacagggccc gcctggtgca ggaaacaggg ctgcaactga
agcagatcaa 300 caattggttc atcaaccaac gcaagcgcaa ctggcacagc
aaccccacct catcctcatc 360 agacaagagc aagagaaaaa gaaacaatgc
aggtgatggc aacgccgagc ggtcttggta 420 ggacatggtt ggagaagaac
acgcgtgtgt aaacagttcg 460 <210> SEQ ID NO 150 <211>
LENGTH: 177 <212> TYPE: DNA <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 150 ccaaccgctc ataccagcat gatgatttgg
aggccttgct gatgatatct ctccccccct 60 tcctgttcat ataggaagga
tttagtgtac ctattgcccg aatagtgtat ttctggtgca 120 cctgccggtt
ccctgggtac tggcttgaat atgtgaatac tgtgcatatg gggcatg 177 <210>
SEQ ID NO 151 <211> LENGTH: 287 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 151
catgcatgca gcctggggcg tacagattga caggcctatg tgtagctcag ccctcatcag
60 gtagcgatac atggtggtaa ttaagtagtg atgcaagcgg ccagatcata
gctcgttgac 120 tgatgatcta gcaggtgcag caggagcagc cacagctggt
gctgcagttg cacttgctgc 180 aggggcagcc gccgttctcc gcctccgcgg
ccatgtccat cccaccggcg ctcgccttgt 240 gggtggcggc ggcgacgagg
aagacgttgc cgttgccggc ggcttcg 287 <210> SEQ ID NO 152
<211> LENGTH: 91 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 152 cgataagaag gacagcgagg
aggccaagca ggcgctagac cagctgaagg agctcggctg 60 ggccaagcga
tggagctcgc agccctacat g 91 <210> SEQ ID NO 153 <211>
LENGTH: 128 <212> TYPE: DNA <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 153 catgccgggg ctggggaagg agcacgtcaa
ggtgtgggcg gagcagaaca gcctggtgat 60 caagggcgag ggcgagaagg
actccgagga ggagggcgtc gccgccccga ggtacagcgg 120 ccgtatcg 128
<210> SEQ ID NO 154 <211> LENGTH: 132 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 154
catgtagccg atctttgttc caagagatgg taaagctttg ctttcataga tgcacctata
60 tgacctcttc ccaggtggta gtcatcccag gcggcgacga ggtgttcagg
ggagaggccc 120 ttggcgcgat cg 132 <210> SEQ ID NO 155
<211> LENGTH: 223 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 155 catgcggcaa acaatcttcc
cactccttca agttcttctt gatcatggat ctcaacagtt 60 gtgacaatgt
tctattcacc acctcagttt gaccatcagt ttggggatga caagtagtgt 120
tgaaaagtag cttcgtcccc agctttctcc aaagcgtctt ccagaagtag ctcatgaact
180 tcacgtcacg atcagaaaca atagtcttcg ggactccatg tcg 223 <210>
SEQ ID NO 156 <211> LENGTH: 100 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 156
cgacttgcgc cacaccacct gcgtgtagtg cccgcacacc ttgccggcgt cgcaggtgtt
60 gctgctgagg tggtagttct tcttctcgtc cacccacatg 100 <210> SEQ
ID NO 157 <211> LENGTH: 70 <212> TYPE: DNA <213>
ORGANISM: Festuca mairei <400> SEQUENCE: 157 catgttagcc
ccgagcagga agcccacggc gagcggcccg atggtgccca cgtggcccct 60
cttggggtcg 70 <210> SEQ ID NO 158 <211> LENGTH: 582
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 158 catgttgttg gccagaactt ggcaccacgg
agccacgccg cagggaacgc ggcgtactcc 60 ctggagtatg gcttagtcca
tgcgtcactc atcaggagtt ggggtgggtg gggagcgccc 120 ttcaggacat
tgttgttcac atctgctacg ccattttcta cctgtgcaat ttcttccctg 180
atggaaataa gggcatcaca gaacctgtct agttcagcct tgctttcact ttcagtgggt
240 tcaatcataa gtgtgcctgg aacaggccat gacatggttg gtccatggaa
cccatagtcc 300 atcaagcgct tcgccacatc ctcaggctct ataccagcag
tcgccttgag ccctcttaaa 360 tcaatgatga attcatgggc aacagttcca
ttgactccac ggaaaagaac tgggtagtgt 420 ttctccagac gctttgccat
gtagtttgca ttcaagatcg caatctttga agcatcagtg 480 agtccctgag
accccatcat ggctatgtat gtataggaaa ttggaagaat caaagcagat 540
ccccatggag cagcagaaat ggaacccagg aggtcggttt tc 582 <210> SEQ
ID NO 159 <211> LENGTH: 130 <212> TYPE: DNA <213>
ORGANISM: Festuca mairei <400> SEQUENCE: 159 catgccgctg
tactgatcta ccggaagctg cagctggttg cagaacaagc tgctgctgga 60
tctcctcctc ttcatcccat tatacttgtt accagtagcg taatcacggt catctaactg
120 cggcacgtcg 130 <210> SEQ ID NO 160 <211> LENGTH:
122 <212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 160 catgtggcat caattttaca tcaacctgcc
gcagctgtcc tgatcatacg actaattagc 60 cggagaaggt ctggatgatg
gtgttgtgcc acgggtcaga caagtgctgg aacaggttct 120 cg 122 <210>
SEQ ID NO 161 <211> LENGTH: 166 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 161
catgcggcag catccatcag caatgaagtt gtcggccaag cacgcgcgcg cacgcccgcg
60 ctactgctag agagctgaca aagctcactt tccggggacg aagttggtgg
cgaaggccca 120 ggcgttgttg ttgacggggt cggcgaggtg gtcggcgagg ttctcg
166 <210> SEQ ID NO 162 <211> LENGTH: 178 <212>
TYPE: DNA <213> ORGANISM: Festuca mairei <400>
SEQUENCE: 162 cgacaaagaa cacggccggc gaccatggca gcagcggttt
tggaactgct atactttgaa 60 gtttgaacag cgccttgacc tcagatgctg
gtggaattag ctatttgcgt gccaaatgta 120 gcgggtaaaa aatagctgtg
gtggttccag gattgtgtat tcggtaccgt gccacatg 178 <210> SEQ ID NO
163 <211> LENGTH: 178 <212> TYPE: DNA <213>
ORGANISM: Festuca mairei <400> SEQUENCE: 163 catgcggtac
ctcccatcca tggggctgga agacacactg aacgggtgcc actgcaagaa 60
cgacagctcc cgcacttgaa caaagatgaa gctgagagca ctgtaccgga ggcttgctgg
120 ctttgaggag actagctcca cagttccgca ggggcggcag gcagcagaaa caatgtcg
178 <210> SEQ ID NO 164 <211> LENGTH: 278
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 164 catgtgtctc tttgtccaaa tcagtttctt
gaagaggtgc tttctcagtg gtcttgcttt 60 cttcacaaag ctgctgtggc
gtcaccgttg gatcaacagg taaggcagtg cagcttccat 120 ctttctctaa
tttctcatct gctgcatcgg tagcgacagg ttcttcatca gtgacagcag 180
cagcagaagc ggatacatct tcagactctt gtttctcagc tggagctgcc tgcttgttct
240 tgccatgcat tgccttgtag gtggcgacgg cgtggtcg 278 <210> SEQ
ID NO 165 <211> LENGTH: 278 <212> TYPE: DNA <213>
ORGANISM: Festuca mairei <400> SEQUENCE: 165 catgtgtctc
tttgtccaaa tcagtttctt gaagaggtgc tttctcagtg gtcttgcttt 60
cttcacaaag ctgctgtggc gtcaccgttg gatcaacagg taaggcagtg cagcttccat
120 ctttctctaa tttctcatct gctgcatcgg tagcgacagg ttcttcatca
gtgacagcag 180 cagcagaagc ggatacatct tcagactctt gtttctcagc
tggagctgcc tgcttgttct 240 tgccatgcat tgccttgtag gtggcgacgg cgtggtcg
278 <210> SEQ ID NO 166 <211> LENGTH: 215 <212>
TYPE: DNA <213> ORGANISM: Festuca mairei <400>
SEQUENCE: 166 cgacgtactt taatatccgt aaaggcctag acggctccct
agacaaggca attaatgctc 60 tttgtgaaga agctgacgct gctgtgcgga
gtggttctca acttctggtc ctttctgatc 120 gttctgaagc acttgaacca
acacggcctg ccatcccaat acttctagcc gttggtgcca 180 tccaccagca
tctgattcaa aatggcctcc gcatg 215 <210> SEQ ID NO 167
<211> LENGTH: 338 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 167 catgtaccaa caacgaggcc
tgcaacgacg ccattgtccc tgccagccca accggcaaag 60 acgaagagtc
ctatcttgcc atagttataa cccatgttga tatcggtaag ccctgtccca 120
taggaattgg cgaatccaag catgggggcg acgacatagg ctataaccac gtagtaccat
180 ttcacctgtc ggaacattat tggcgtggta accactgcaa cagcacttaa
caaggcatac 240 ccggtgtacg ccaaccaaga ggggatatgg cccttccgga
agatctcgtc gcgctgcaga 300 tcctcaagtg agaccgtatt gtccacatct ttcactcg
338 <210> SEQ ID NO 168 <211> LENGTH: 167 <212>
TYPE: DNA <213> ORGANISM: Festuca mairei <400>
SEQUENCE: 168 cgacacgctc actgatatct gatggaggtg aattcagctt
gacaaatgag caggaaaagt 60 gggttgtaga cattatgctc tcagtaacgc
tggtgaaact tgctctagct ttatattgcc 120 gcacattcac caatgaaatt
gtcaaggctt atgcgcagga tcacatg 167 <210> SEQ ID NO 169
<211> LENGTH: 134 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 169 catgcggagg gaaggatatg
aatttatgat tggacctcca aaggtcataa acaagagtgt 60 agatgggaag
ctactggagc cgtatgagat agctgctata gaggtaccag aggaatatat 120
gtgatcagct gtcg 134 <210> SEQ ID NO 170 <211> LENGTH:
121 <212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 170 cgactgcggc gtctccatcg gaacgatctt
tgggatttac atgatcaaga actttgacac 60 cgtgaccctt gaggaagtgc
cgctgcctgg gaaggacatg attgctgctg gatactgcat 120 g 121 <210>
SEQ ID NO 171 <211> LENGTH: 201 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 171
catgcatatg ctgcaaatgt ttcttcccac ctagtgtttc ttttttcctt ttaccccgca
60 attgaaccgt gcaaagctca aggctccgat catatatacg ccttcgtatc
tagcgacaag 120 agtgaatgag cgcggtaagc tgttatggaa tctccttggc
acgtctgatc aatgtacata 180 ctgacactcg catttgtctc g 201 <210>
SEQ ID NO 172 <211> LENGTH: 113 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 172
cgagtcgcag tcgcagacca acgatctgga gtcggacagt ctgcaggtgt acagcttctc
60 cgggctgttc ctcatctgcg gcgtggcgtg cgtgatcacc ctcgccatac atg 113
<210> SEQ ID NO 173 <211> LENGTH: 148 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 173
catgtcgttg gacggcagca ccatcaccac atggagagaa aggagccgtt ggacaatctg
60 tgcaggagct cgccgaggca caggatggcg tagaccagca ggatgaactt
gaagaggaac 120 gggaagtagt gcgagagccc ggcgctcg 148 <210> SEQ
ID NO 174 <211> LENGTH: 398 <212> TYPE: DNA <213>
ORGANISM: Festuca mairei <400> SEQUENCE: 174 catgcaccca
tttgccccta ttgatcaggc tgcaggctat catgaaatgt ttgacaactt 60
gggtgatctg ttgaacacga tcaccggttt tgattccttc tctctgcaac caaatgctgg
120 tgcttcagga gagtatgctg gactgatggt tattcgggcc caccacaggg
caagaggaga 180 ccatcaccga aatgtctgca tcattcctgt ctcggcacac
ggtacaaatc ctgcaagtgc 240 tgctatgtgt ggaatgaaga ttattactgt
cggaactgac tccaaaggta acattaacat 300 tgcggagttg aagaaagctg
ctgaagcaaa caaggacaac ctgtctgctc tgatggttac 360 ctatccttca
acccatggag tctatgaaga aagcatcg 398 <210> SEQ ID NO 175
<211> LENGTH: 318 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 175 cgacattatg cacaggcaga
ggaagctgaa gaggatgatg aaattgagcg gctctttagt 60 agtaagaaag
agaagaagaa tgatcggcca cgagcagata ttggtcttat cgttgagcag 120
ttcattgccg agtttgaagt agcgtctgaa gaagatgcaa acctaaatag gcaatccaaa
180 ccggccatta acaaacttat gaagcttcca ctgctcatag aggttctctc
aaagaagaat 240 ctccagcagg aattccttga tcatggaatt ctcactcttc
tgaaaaactg gcttgaacct 300 ttacctgatt gaagcatg 318 <210> SEQ
ID NO 176 <211> LENGTH: 86 <212> TYPE: DNA <213>
ORGANISM: Festuca mairei <400> SEQUENCE: 176 catgtacgtg
cttcgttgca tcttctgaac agcctcggtg acctccttac gtacgccaag 60
ccatcgcact gagctgagct cagtcg 86 <210> SEQ ID NO 177
<211> LENGTH: 116 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 177 catgcatatc tcgcccggcc
ggtggtcgga cggatggcta actcatttaa catccaggag 60 cgaagttggt
ggcgaaagcc catgcgttgt tgttgactgg gtcagcaacg cagtcg 116 <210>
SEQ ID NO 178 <211> LENGTH: 194 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 178
cgaccataaa cgccccgtga tcggcggcaa ggagcacccc taccctcgcc gatgccgcac
60 cggtcgccct aaaaccatca ttgactcaga gacggagaag aggagctcac
cagtgtatgt 120 gccacgtgac gagcagttct cggacattaa agggcagaca
ttcagcgcga cgacactgcg 180 gtctggattg catg 194 <210> SEQ ID NO
179 <211> LENGTH: 290 <212> TYPE: DNA <213>
ORGANISM: Festuca mairei <400> SEQUENCE: 179 cgacgaccta
aaggctcacg cagaatcaaa tgtgactgat aagatgatgt caaatgcaaa 60
gttcatctac ccacacaaca ccccgacaac aaaggaggca tactgttaca gaacgatctt
120 tgagaggttc ttcccccaga actcggcgat cctgacagtg ccaggtggac
caagcgtcgc 180 atgcagcacg gcgaaggcgg tagagtggga tgctcagtgg
tcggggaacc tggatccctc 240
agggagagca gcgcttggag tccatctctc agcctatgaa caagagcatg 290
<210> SEQ ID NO 180 <211> LENGTH: 170 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 180
catgcacgta cggggttcgt aacactactc tagcttaatt aatctagacg ttgacaaagg
60 gcgtgccggt gatgaactcg gtgacggcga gcgcgacgag tccgagcatg
gcgaagcggc 120 cgttccagag ctcggcgtcg gcgctccaga cgccgctgga
cttgctctcg 170 <210> SEQ ID NO 181 <211> LENGTH: 204
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 181 catgttggtg cttacaaata tggcagctgg
ggacgaatta agcaaggaag ctgtaatgga 60 tgttattgtt cctcacagat
cagatcgcat caagccatct tttgtggtca actttctgca 120 gagcaaggac
gaacaattga gagttgcatc tttgtggtgc attcttaact tagcttaccc 180
aaaaagtgat gcttcatcta ctcg 204 <210> SEQ ID NO 182
<211> LENGTH: 77 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 182 catgcttggg aataggtgtg
tcatcttcaa tcacctcgca ttcgtagtca tcaggaacgc 60 cccagggatc ccactcg
77 <210> SEQ ID NO 183 <211> LENGTH: 114 <212>
TYPE: DNA <213> ORGANISM: Festuca mairei <400>
SEQUENCE: 183 catgctgtcg ttcaggactc atcgtgactg tagactgcgt
tcccatgctt tctcctccaa 60 agtgagttgc acatccttca tctcacactg
gactgatgcc atttccccgt gtcg 114 <210> SEQ ID NO 184
<211> LENGTH: 431 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 184 catgttcctt ctacgttgat
atgtacggtg catacacaaa caattatacg tggaactaaa 60 agtaaaaccc
ggaaagcgga gttgtcatca aaaactaaac caagagactc cacatggatt 120
cctagctcgc ggcttatgcc ttgccggact cctcacagcc tggtggcttg aaggcgatga
180 agctgacgca ctgcacctga cggatgttgt caaaaccgat gatgcggaca
taggcgtcag 240 ggtactcctt cttgacctcc tccacctcct tgaggacctg
ggtggcgtct gtgcagccga 300 acatgggcag cttccacatt gtccagtacc
tgccgtcgta gtatccggga gtgctgccgt 360 gctcacggaa gatgaagcca
accttgctga actccaggca gggaacccat ttggagcgga 420 tcaagaagtc g 431
<210> SEQ ID NO 185 <211> LENGTH: 84 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 185
catgcagtgg aatgtcttct gataaacgta ggggagaaca tgactgggga aggacttcgg
60 tggaagctat tttcttgcca gtcg 84 <210> SEQ ID NO 186
<211> LENGTH: 398 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 186 catgcttgcg cacatagttc
atctctactg gctcgtccag ctcagcaacc atctttactg 60 cccggtccag
cagattcatc tggttgatga aaagacctct gaacacgccc gagcagttga 120
cgacgacatc aaccctagga cgtccaagct cctcaatgct gacaggctcc acacggttga
180 cacggccaag gccatcagta accggctcca caccaagcat ccaaaacacc
tgggccaggg 240 actcgccgta ggtcttgatg ttgtcagtac cccacaagac
aagagcaatt gtctcaggat 300 acttgccacc attgtcagcc ttttgccgct
ccagcagacg ttccacaaca accttggcac 360 tcttcgtggc cgctgcggtc
gggattgact gcgggtcg 398 <210> SEQ ID NO 187 <211>
LENGTH: 231 <212> TYPE: DNA <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 187 cgacgtccag cgctctcttc tccttcttca
ccttgggctg cgtgatcaca gtggcatcac 60 ccggcccaga gaccgccttg
gcgccgacgt ctgtgcctgg agccgccgtg gaggccatca 120 tcgcccggcg
cgccttccgc tgcctgatgc cacaagcgtt gcaaagtgac ttggggccac 180
atggaccact cctccacaag ggggttttgg tggtgttgca gtcggagcat g 231
<210> SEQ ID NO 188 <211> LENGTH: 178 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 188
catgttcaaa ctagattgat acgacaccag gtccacatga tcactgcggt tccgaagagc
60 tgaagatcct acagtgcctt gaggatctcc tcagcaccag agattgtgaa
ctggccgccc 120 tcctcaatgt ttgcaacagt gaccttgagg tcatcagcaa
ggagagcaaa ccgtctcg 178 <210> SEQ ID NO 189 <211>
LENGTH: 232 <212> TYPE: DNA <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 189 cgagagtact ttgggcttca attttgggtc
attcaataga gaatctagtt caccttcatt 60 cagtacagga cttgatagag
taacctggtc agcattttca gggccaacct ccaagatgtt 120 gcctcgttta
ccaatattaa cttcaagaga catgactaaa ccttctcgga gtggatcaat 180
tgcagggttt gtaacctgtg caaatcgctg cttgaaataa tcaaagagca tg 232
<210> SEQ ID NO 190 <211> LENGTH: 356 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 190
catgccactt gcccttgtag gcagggttct gcttcgttgg cttcttccat tcaccacatc
60 caggtgcctc ttcgcacttg gggttgtcaa tcttcggtgc ctcccattca
ccatcctcct 120 catcatccca gtctttaggc ttagcagcct caggatcgtc
aatttcatca ggctcatcat 180 ccagccatcc ttctggcttg gtggcctcct
catccacaat ctctattggg gcatcctcat 240 cccagtcgtc aggcttagta
gcatctggat cagggatctt agctctctcg tcccagtcct 300 ctggcttctt
gtcgtcaggg tcaggaatcg tctttgatgg aataagtgct gactcg 356 <210>
SEQ ID NO 191 <211> LENGTH: 122 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 191
catgccttca tatctgcaac aactggtcag tagggcaatc acatcagaag ataaattcgt
60 tgatgatgtt ggtccctcca ggcaaacaat aagcaagggt gtcccatcag
acaacaatgt 120 cg 122 <210> SEQ ID NO 192 <211> LENGTH:
390 <212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 192 cgactccagc agcaacccga gaagatcgtc
gctcgttgct tcgccagctt ttaaggcgtt 60 ctctcttttg gtgatgatcc
tcttaggatc ctcccaatct ccgcagcaat ctgcttcatc 120 cttctgttgg
ctttggttgg caagaacagg taaccaggga tatgtatctt gttcatggcc 180
tgcatgacga gcaggatctg ctccccctga agctggaata tcctcacgcc ctcaaggtag
240 ctgctgccga atgcggcgcg ggagatgaca tcccctgtca ggttctgcat
atcaggccag 300 acatctacct cgcatggcag gtcaccggtg actaaacctt
cccatctgtg taccagctcc 360 gtgcaacatt cggcgaaagc cggcaacatg 390
<210> SEQ ID NO 193 <211> LENGTH: 400 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 193
catgctgtca aagtggcagc tgatggctga acaagctagg cagaagcgag caccagtaca
60 gaggccagca catggatctg gaaaaggttc ggcagaacag aatgaagctt
caaagaggag 120 ccattttgca gccttcggaa ctggaggcac gaaaaggcaa
ggcaagggtt cattcgctac 180 gcgtcactcg catgggccac aacgaactgt
ttccgtgaag gatgtaatct gcgtcctgga 240 gagggagcct cagatgacga
aatcacggct aatttatcgg ctgtacgagc gattgcctgg 300 agatttcacc
acagattagg ctgaattatg tagtgtaact tatagcgtgt aactgtttgt 360
tgatgcacag cccgtcgctc agactgacgt gttccagtcg 400 <210> SEQ ID
NO 194 <211> LENGTH: 192 <212> TYPE: DNA <213>
ORGANISM: Festuca mairei <400> SEQUENCE: 194 cgacccatgc
cgtgagcggg tcgcggaagt ccttgagccc gatcccgggt ggcgggaaga 60
ggatggggtt ggcggggtgc ttgatccagc tgcggaggag cgggtcggat gggtcggcgg
120 gcacggccag gcactggacc tgcgcgaagg tgtcagtgtt ccccgtgtcg
agcaggatca 180
cgcgcccgtc ag 192 <210> SEQ ID NO 195 <211> LENGTH: 209
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 195 catgctattg gagaacggtc ccaacgaggc
ccaggcaaag aaggcccgcc aggtcctgca 60 ggcctgcggc gataggaaag
acggctacca gctgaactac gacttcagga acccgttcgt 120 tgtgtgcggg
gcgacctttg tcccgatcta ccgcgggcag aaggacgtct cctgccccta 180
ctgcacttcc cggttcgtgc cctccgtcg 209 <210> SEQ ID NO 196
<211> LENGTH: 172 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 196 cgagatagac gagcagacct
tcctgaccaa cagggagagg gcggtggact acctcaactc 60 cctggacaag
gtgttcgtga acgaccagtt cctcaactgg gacccggaga accgcatcaa 120
ggtgcgcatc atctccgcca gggcctacca ctcgctcttc atgcacaaca tg 172
<210> SEQ ID NO 197 <211> LENGTH: 109 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 197
cgagagaatc acggagcaag ctggtgtagt gctcactctt gacccaaaac caatccaggg
60 tgactggaat ggagctggct gccacacaaa ttacagcaca aagagcatg 109
<210> SEQ ID NO 198 <211> LENGTH: 235 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 198
cgagtcagcg ccactactgt gccattacag ttggagagga tgctgtcgtt tcagcatata
60 ggttatcaga agacagaggc aggtcattag ttggagcaat tttgtcaagg
ggtgtagctg 120 caacattttc aacaatatca tctttgtcca aaattctatg
gcggagtgaa ccatcaccaa 180 ctaagaagcc acggccaaaa cctcaatcct
ttgcaaaaac ttcacctctg acatg 235 <210> SEQ ID NO 199
<211> LENGTH: 398 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 199 cgaggttcgc gttttggagt
tggagaaacc tggtggacgt tgggagacaa ggtccactat 60 tccaatgcaa
ccatttgaaa atgctctgac tgtgcgcatt gttacattac ataacacaac 120
caccaaggaa aatgaaaccc tgatggccat cgggactgct tatgtccaag gagaggatgt
180 agctgctaga ggacgggtgc ttctgttctc tttcacgaaa agtgaaaatt
ctcaaaatct 240 ggtgacagaa gtctactcaa aagagagtaa aggtgctgta
tcagctgttg catcgcttca 300 aggtcatctt gtgatagctt ctggcccaaa
aatcacattg aacaaatggt ccggttctga 360 attgacagct gttgcattct
atgatgcccc tttgcatg 398 <210> SEQ ID NO 200 <211>
LENGTH: 114 <212> TYPE: DNA <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 200 catgcctcat ctataggcaa gaaggcagta
gcctttgact ggatgtccat aaatgcgcca 60 tttgaagtag tcatgaatac
tgttccttta atgagtgagc cgatttcagt gtcg 114 <210> SEQ ID NO 201
<211> LENGTH: 90 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 201 catgttcacg cctagctgga
tctgaaccct tactgtgcct ggaagcagga ggtctacttc 60 cccgaatcca
gcagcagatg atcgcagtcg 90 <210> SEQ ID NO 202 <211>
LENGTH: 129 <212> TYPE: DNA <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 202 catgcttcca tctgcaagtg aggattccac
aacactgcat ctgaccaatt tgtatttgag 60 atgacaattt tgtcacccaa
tccattgtcc agggtaagct cactcggtgc gccaaggtaa 120 acgcagtcg 129
<210> SEQ ID NO 203 <211> LENGTH: 108 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 203
catgccatgt gaccatcaca caggatgcgg atgctgatca gatgcttgac aaggtcattg
60 ggtacatcaa ggcagagtac aacatcagtc atgtgaccat tcaggtcg 108
<210> SEQ ID NO 204 <211> LENGTH: 119 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 204
cgagacactg gctacagtgc cctcaagctg ctcaaggcgc tactagcact ctctccatag
60 gtagatataa gatagctcgc cggccaatgg atcagtagct gtagttcttg acgaacatg
119 <210> SEQ ID NO 205 <211> LENGTH: 245 <212>
TYPE: DNA <213> ORGANISM: Festuca mairei <400>
SEQUENCE: 205 cgagaagtcc gggcacaggt tcctgtgggt gctgcgtgcg
cctcctgcct tcgctgcggc 60 cgccgctgaa ccggatgcgg cgctttctct
cctcccagag gggttcttgg cacggaccgc 120 agacaggggc ctcgtggtga
ccgcgtcctg ggtgccgcag gtggacgtgc ggcgtcacgc 180 ctccactggt
gccttcgtaa cgcactgcgg atggaactca acactggagg caggcgaccg 240 gcatg
245 <210> SEQ ID NO 206 <211> LENGTH: 345 <212>
TYPE: DNA <213> ORGANISM: Festuca mairei <400>
SEQUENCE: 206 cgagtcacga tgagtcctga acgagtgttt tcatcttcat
aatggatatg ttgcatggca 60 tttttcaaag ctttttccct caatttgctt
cccggccaat gcgacagaac tggtacaaca 120 aatttgttaa gagcagccca
tatgatatct tgtcccagtg gatgtggaca gtacagatcc 180 tccttagcac
attggttccg agccttgtcc caatcaacct catcataggg tactttatag 240
agctccttcc gtaagttcaa tacaactggt gtaattgggc caacaaacct ctttccatat
300 acgtaagaca tgggaaagta taccattcgg caatgggacc acatg 345
<210> SEQ ID NO 207 <211> LENGTH: 323 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 207
cgagttacaa ttatatgagt actactgacc agacctgtgt atactggaaa gaactgggtc
60 gctaagaggc tgctgagaaa gaattaccta caagccgtgg attacatggt
gaagattgct 120 tttatggaga gagaaaaaaa ggagaaaaat cagagatatg
tgtatgttat atgtactctc 180 agcaggggaa caacaaaaac gcagcctccc
tgtggatcct cctattctct accagtatga 240 tcttgtccag cttcgccttg
caccactgca gctgctcgct ggtgatcctc ggcatccgca 300 gcacccgcgc
caccagccgc atg 323 <210> SEQ ID NO 208 <211> LENGTH:
357 <212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 208 catgcacccg gagtggcaag agcaggcaag
aaaggaagtg ttgcaccact tcggaagaat 60 cacaccagac tttgagaact
agagtcggct gaagatagta acgatggttc tatatgaggt 120 tcttaggttg
tacccgccag caatctttgt taccagaaga acatacaaga caatggagct 180
tggtggcatc acatatccgg caggagtgaa ccttatgttg cccattctct ttatccacca
240 tgaccccaat atatggggaa aagatgcaag cgagttcaat ccacagaggt
ttgctgatgg 300 catctcaagt gccgtgaagc atccggctgc gttcttccca
tttggagggg gtcctcg 357 <210> SEQ ID NO 209 <211>
LENGTH: 245 <212> TYPE: DNA <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 209 cgactgtcca tgcgattgaa atcggtttgt
aacctgacca tggattttct gaattccact 60 tcttcaatcc atgatccatc
aaagtatgtc cggtgcaaat gccctgggtc tcaactctca 120 caacaccctt
cttaaatgtg aagtactcag gatgaactaa ggccgtaaag ctcacaggat 180
catggaggaa aatcccatgg aagccgtcag acttggtatg ccaatctctg tagaacttgc
240 acatg 245 <210> SEQ ID NO 210 <211> LENGTH: 232
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 210 catgccataa aggctcatat atcctactac
tctacaactt gagctgccta tacaaacgta 60
ttacatctgt ggtctagtct ggactacgta gatcttccca tccttcacta ctccctcaac
120 gtcctgtggt gacccataga gctcctcaat ggcatggcca gcccgtgcaa
tgcttgagag 180 aattgagctt cggaatccag agtctgtgat gagagcgtcg
gtcgtgtagt cg 232 <210> SEQ ID NO 211 <211> LENGTH: 258
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 211 catgcctgac atggcgaacc catccaaatt
gcaaagagga tttgggaagt ttctgacaac 60 ctttcctgca aatatcttgg
tagccacaat gctcaggggg cccaatattg tttgattctt 120 caaagcacct
gctaacagtt catgagactc caaggaacgc tcagcttgct tgatcatctc 180
atctaaacat gctttctcct ccaaagtgag ttgcacatcc ttcatctcac actggactga
240 tgccattttc ccgcgtcg 258 <210> SEQ ID NO 212 <211>
LENGTH: 161 <212> TYPE: DNA <213> ORGANISM: Festuca
mairei <400> SEQUENCE: 212 catgtgatcc aggcgccacc cttatgtcca
ccttgtaacg aggaggaagg ctccgcatga 60 gcttcacacg taagcaaagg
ccaataatag tcgccatact gcaatgctcc actgttgggg 120 tgaaagtaac
ccgcacatga ctaagatcat cgctgatctc g 161 <210> SEQ ID NO 213
<211> LENGTH: 223 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 213 cgagatctgc agtctattgt
gttgtatgat caaaatggta agtttgtggg ggttcgtcgg 60 ccaagctcaa
aactccccat tgaaatcaat ggtaatgaaa tactaattga agacgctatt 120
ggcagtactg gtctggatct taagaccgat ccaggaattc ctgtcgtgta tgctggattt
180 ggcgcgctca tgttgacgac ctgcattagc tatctttcgc atg 223 <210>
SEQ ID NO 214 <211> LENGTH: 309 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 214
catgtgctct ggatgtacca gtgtcgctgg cgaagggaat gcaggccaag cttatcggtg
60 atttcagcag cgttcatggc attaggaaga tcttgtttgt ttgcaaacac
cagaagcaca 120 gcatcacgca actcatcctc attgagcatt cggtgaagct
catctctggc ctcaacaaca 180 cgctctctgt cgttgctgtc caccacaaaa
ataaggccct gggtgttctg gaagtagtgc 240 ctccacaggg gcctgatctt
gtcctgaccc ccgacatccc aaactgtgaa actaatgttc 300 ttgtactcg 309
<210> SEQ ID NO 215 <211> LENGTH: 185 <212> TYPE:
DNA <213> ORGANISM: Festuca mairei <400> SEQUENCE: 215
catgtactta ccagctaggt gttggttcgg tcgtcgttaa gaagcattta accacagctt
60 aattgaagtg atcgtgatga gaaagtaagc caaactaggg taggtagacg
gatggatccg 120 ggacgtccgt ccagcagctc ccggcgttcc agtacgcggc
cggcgacgcg tcgtcgccga 180 gctcg 185 <210> SEQ ID NO 216
<211> LENGTH: 254 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 216 ccagggtttg cacccaatgt
acgacgaacg tgtgagagac ctccttgatt tcagcatcta 60 cttagacatc
agcaatgagg ttaagtttgc atggaaaatt cagagagaca tggcagagcg 120
tgggcacagc cttgaaagca tcaaggctag cattgaagcc aggaaaccaa attttgatgc
180 atttattcgt agtgcctttt tgccatctga aaacaataat tgtttgccat
aaacccaact 240 taacatgggg catg 254 <210> SEQ ID NO 217
<211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic <400> SEQUENCE: 217 gtagactgcg
ttcccatg 18 <210> SEQ ID NO 218 <211> LENGTH: 17
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
<400> SEQUENCE: 218 ggaacgcagt ctacgag 17 <210> SEQ ID
NO 219 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic <400> SEQUENCE: 219 gtagactgcg
ttcccatg 18 <210> SEQ ID NO 220 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
<400> SEQUENCE: 220 gtagactgcg ttcccatgta 20 <210> SEQ
ID NO 221 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic <400> SEQUENCE: 221 gtagactgcg
ttcccatgtt 20 <210> SEQ ID NO 222 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
<400> SEQUENCE: 222 gtagactgcg ttcccatgtc 20 <210> SEQ
ID NO 223 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic <400> SEQUENCE: 223 gtagactgcg
ttcccatgtg 20 <210> SEQ ID NO 224 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
<400> SEQUENCE: 224 gtagactgcg ttcccatgca 20 <210> SEQ
ID NO 225 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic <400> SEQUENCE: 225 gtagactgcg
ttcccatgct 20 <210> SEQ ID NO 226 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
<400> SEQUENCE: 226 gtagactgcg ttcccatgcc 20 <210> SEQ
ID NO 227 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic <400> SEQUENCE: 227 gtagactgcg
ttcccatgcg 20 <210> SEQ ID NO 228 <211> LENGTH: 16
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
<400> SEQUENCE: 228 aagtcctgag tagcac 16
<210> SEQ ID NO 229 <211> LENGTH: 15 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 229
cgttcaggac tcatc 15 <210> SEQ ID NO 230 <211> LENGTH:
18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
<400> SEQUENCE: 230 cacgatgagt cctgaacg 18 <210> SEQ ID
NO 231 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic <400> SEQUENCE: 231 cacgatgagt
cctgaacgaa a 21 <210> SEQ ID NO 232 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
<400> SEQUENCE: 232 cacgatgagt cctgaacgaa t 21 <210>
SEQ ID NO 233 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 233
cacgatgagt cctgaacgaa c 21 <210> SEQ ID NO 234 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic <400> SEQUENCE: 234 cacgatgagt cctgaacgaa g 21
<210> SEQ ID NO 235 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 235
cacgatgagt cctgaacgat a 21 <210> SEQ ID NO 236 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic <400> SEQUENCE: 236 cacgatgagt cctgaacgat t 21
<210> SEQ ID NO 237 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 237
cacgatgagt cctgaacgat c 21 <210> SEQ ID NO 238 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic <400> SEQUENCE: 238 cacgatgagt cctgaacgat g 21
<210> SEQ ID NO 239 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 239
cacgatgagt cctgaacgac a 21 <210> SEQ ID NO 240 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic <400> SEQUENCE: 240 cacgatgagt cctgaacgac t 21
<210> SEQ ID NO 241 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 241
cacgatgagt cctgaacgac c 21 <210> SEQ ID NO 242 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic <400> SEQUENCE: 242 cacgatgagt cctgaacgac g 21
<210> SEQ ID NO 243 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 243
cacgatgagt cctgaacgag a 21 <210> SEQ ID NO 244 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic <400> SEQUENCE: 244 cacgatgagt cctgaacgag t 21
<210> SEQ ID NO 245 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 245
cacgatgagt cctgaacgag c 21 <210> SEQ ID NO 246 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic <400> SEQUENCE: 246 cacgatgagt cctgaacgag g 21
<210> SEQ ID NO 247 <211> LENGTH: 17 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 247
ctcgtagact gcgtacc 17 <210> SEQ ID NO 248 <211> LENGTH:
18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
<400> SEQUENCE: 248 aattggtacg cagtctac 18 <210> SEQ ID
NO 249 <211> LENGTH: 16 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic <400> SEQUENCE: 249
cactgcgtac caattc 16 <210> SEQ ID NO 250 <211> LENGTH:
18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
<400> SEQUENCE: 250 cactgcgtac caattcaa 18 <210> SEQ ID
NO 251 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic <400> SEQUENCE: 251 cactgcgtac
caattcat 18 <210> SEQ ID NO 252 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
<400> SEQUENCE: 252 cactgcgtac caattcac 18 <210> SEQ ID
NO 253 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic <400> SEQUENCE: 253 cactgcgtac
caattcag 18 <210> SEQ ID NO 254 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
<400> SEQUENCE: 254 cactgcgtac caattcta 18 <210> SEQ ID
NO 255 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic <400> SEQUENCE: 255 cactgcgtac
caattctt 18 <210> SEQ ID NO 256 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
<400> SEQUENCE: 256 cactgcgtac caattctc 18 <210> SEQ ID
NO 257 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic <400> SEQUENCE: 257 cactgcgtac
caattctg 18 <210> SEQ ID NO 258 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
<400> SEQUENCE: 258 cactgcgtac caattcca 18 <210> SEQ ID
NO 259 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic <400> SEQUENCE: 259 cactgcgtac
caattcct 18 <210> SEQ ID NO 260 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
<400> SEQUENCE: 260 cactgcgtac caattccc 18 <210> SEQ ID
NO 261 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic <400> SEQUENCE: 261 cactgcgtac
caattccg 18 <210> SEQ ID NO 262 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
<400> SEQUENCE: 262 cactgcgtac caattcga 18 <210> SEQ ID
NO 263 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic <400> SEQUENCE: 263 cactgcgtac
caattcgt 18 <210> SEQ ID NO 264 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
<400> SEQUENCE: 264 cactgcgtac caattcgc 18 <210> SEQ ID
NO 265 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic <400> SEQUENCE: 265 cactgcgtac
caattcgg 18 <210> SEQ ID NO 266 <211> LENGTH: 10
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 266 aatcgggctg 10 <210> SEQ ID NO 267
<211> LENGTH: 10 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 267 aggggtcttg 10 <210>
SEQ ID NO 268 <211> LENGTH: 10 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 268
gaaacgggtg 10 <210> SEQ ID NO 269 <211> LENGTH: 10
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 269 gtgacgtagg 10 <210> SEQ ID NO 270
<211> LENGTH: 10 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 270 gttgcgatcc 10 <210>
SEQ ID NO 271 <211> LENGTH: 10 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 271
ccttgacgca 10 <210> SEQ ID NO 272 <211> LENGTH: 10
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 272 ttcgagccag 10 <210> SEQ ID NO 273
<211> LENGTH: 10 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 273 gtgaggcgtc 10 <210>
SEQ ID NO 274 <211> LENGTH: 10 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 274
ccgcatctac 10 <210> SEQ ID NO 275 <211> LENGTH: 10
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 275 gatgaccgcc 10 <210> SEQ ID NO 276
<211> LENGTH: 10 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 276 gaacggactc 10 <210>
SEQ ID NO 277 <211> LENGTH: 10 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 277
gtcccgacga 10 <210> SEQ ID NO 278 <211> LENGTH: 10
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 278 tggaccggtg 10 <210> SEQ ID NO 279
<211> LENGTH: 10 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 279 ctcaccgtcc 10 <210>
SEQ ID NO 280 <211> LENGTH: 10 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 280
tgtctgggtg 10 <210> SEQ ID NO 281 <211> LENGTH: 10
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 281 aaagctgcgg 10 <210> SEQ ID NO 282
<211> LENGTH: 10 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 282 aagcctcgtc 10 <210>
SEQ ID NO 283 <211> LENGTH: 10 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 283
gacggatcag 10 <210> SEQ ID NO 284 <211> LENGTH: 10
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 284 cacactccag 10 <210> SEQ ID NO 285
<211> LENGTH: 10 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 285 gttgccagcc 10 <210>
SEQ ID NO 286 <211> LENGTH: 10 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 286
acttcgccac 10 <210> SEQ ID NO 287 <211> LENGTH: 10
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 287 cttcacccga 10 <210> SEQ ID NO 288
<211> LENGTH: 10 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 288 gtggcatctc 10 <210>
SEQ ID NO 289 <211> LENGTH: 10 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 289
catcgccgca 10 <210> SEQ ID NO 290 <211> LENGTH: 10
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 290 acagcctgct 10 <210> SEQ ID NO 291
<211> LENGTH: 10 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 291 ggctgcgaca 10 <210>
SEQ ID NO 292 <211> LENGTH: 10 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 292
aaggctcacc 10 <210> SEQ ID NO 293 <211> LENGTH: 10
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 293 agagccgtca 10 <210> SEQ ID NO 294
<211> LENGTH: 10 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 294 agcagcgcac 10 <210>
SEQ ID NO 295 <211> LENGTH: 10 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 295
caaacgtggg 10 <210> SEQ ID NO 296 <211> LENGTH: 10
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 296
gggtctcggt 10 <210> SEQ ID NO 297 <211> LENGTH: 10
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 297 ggtcgatctg 10 <210> SEQ ID NO 298
<211> LENGTH: 10 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 298 agtcgccctt 10 <210>
SEQ ID NO 299 <211> LENGTH: 10 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 299
gggccaatgt 10 <210> SEQ ID NO 300 <211> LENGTH: 10
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 300 gacgtggtga 10 <210> SEQ ID NO 301
<211> LENGTH: 10 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 301 gtggagtcag 10 <210>
SEQ ID NO 302 <211> LENGTH: 10 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 302
tgagggtccc 10 <210> SEQ ID NO 303 <211> LENGTH: 10
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 303 agccgtggaa 10 <210> SEQ ID NO 304
<211> LENGTH: 10 <212> TYPE: DNA <213> ORGANISM:
Festuca mairei <400> SEQUENCE: 304 ctgggcacga 10 <210>
SEQ ID NO 305 <211> LENGTH: 10 <212> TYPE: DNA
<213> ORGANISM: Festuca mairei <400> SEQUENCE: 305
acgccagagg 10 <210> SEQ ID NO 306 <211> LENGTH: 10
<212> TYPE: DNA <213> ORGANISM: Festuca mairei
<400> SEQUENCE: 306 acgggagcaa 10
* * * * *
References