U.S. patent application number 14/623171 was filed with the patent office on 2015-08-06 for molecular markers linked to ppo inhibitor tolerance in soybeans.
This patent application is currently assigned to PIONEER HI-BRED INTERNATIONAL, INC.. The applicant listed for this patent is PIONEER HI-BRED INTERNATIONAL, INC.. Invention is credited to Julian CHAKY, Kevin A. FENGLER, Jennifer A. KLAIBER, Donald KYLE, Balin L I, Mark D. VOGT.
Application Number | 20150218658 14/623171 |
Document ID | / |
Family ID | 42338031 |
Filed Date | 2015-08-06 |
United States Patent
Application |
20150218658 |
Kind Code |
A1 |
CHAKY; Julian ; et
al. |
August 6, 2015 |
MOLECULAR MARKERS LINKED TO PPO INHIBITOR TOLERANCE IN SOYBEANS
Abstract
This invention relates generally to the detection of genetic
differences among soybeans. More particularly, the invention
relates to soybean quantitative trait loci (QTL) for tolerance to
protoporphyrinogen oxidase inhibitors, to soybean plants possessing
these QTLs, which map to a novel chromosomal region, and to genetic
markers that are indicative of phenotypes associated with
protoporphyrinogen oxidase inhibitor tolerance. Methods and
compositions for use of these markers in genotyping of soybean and
selection are also disclosed.
Inventors: |
CHAKY; Julian; (Urbandale,
IA) ; FENGLER; Kevin A.; (Wilmington, DE) ;
KLAIBER; Jennifer A.; (Urbandale, IA) ; KYLE;
Donald; (Princeton, IL) ; L I; Balin;
(Hockessin, DE) ; VOGT; Mark D.; (Ankeny,
IA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PIONEER HI-BRED INTERNATIONAL, INC. |
Johnston |
IA |
US |
|
|
Assignee: |
PIONEER HI-BRED INTERNATIONAL,
INC.
Johnston
IA
|
Family ID: |
42338031 |
Appl. No.: |
14/623171 |
Filed: |
February 16, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14266161 |
Apr 30, 2014 |
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14623171 |
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12694255 |
Jan 26, 2010 |
8748695 |
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14266161 |
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12506498 |
Jul 21, 2009 |
8697941 |
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12694255 |
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61083038 |
Jul 23, 2008 |
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Current U.S.
Class: |
504/273 ;
435/6.11; 506/16; 506/2; 506/9; 800/266; 800/300 |
Current CPC
Class: |
A01N 47/38 20130101;
C12Q 1/6895 20130101; C12Q 2600/172 20130101; C12N 15/8274
20130101; C12Q 2600/13 20130101; C12Q 2600/156 20130101; A01H 1/04
20130101; A01H 1/02 20130101; A01H 5/10 20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; A01H 1/04 20060101 A01H001/04; A01H 1/02 20060101
A01H001/02; A01H 5/10 20060101 A01H005/10; C12N 15/82 20060101
C12N015/82; A01N 47/38 20060101 A01N047/38 |
Claims
1. A method of selecting a soybean plant or germplasm with
tolerance or improved tolerance to herbicides that inhibit
protoporphyrinogen oxidase function, the method comprising: a)
detecting in a soybean plant or germplasm at least one allele of a
marker locus that is associated with the tolerance or improved
tolerance to herbicides that inhibit protoporphyrinogen oxidase
function, wherein the one or more marker locus is a marker locus
localizing within a chromosome interval flanked by and including
S01659-1-A and S03859-1-A on linkage group L; and b) selecting the
soybean plant or germplasm comprising the at least one allele of
one or more marker locus, thereby selecting a soybean plant with
tolerance or improved tolerance to herbicides that inhibit
protoporphyrinogen oxidase function.
2. The method of claim 1, wherein the marker locus is selected from
the group consisting of S08102-1-Q1, S08103-1-Q1. S08104-1-Q1,
S08106-1-Q1, S08107-1-Q1, S08107-1-Q1, S08109-1-Q1, S08110-1-Q1,
S08111-1-Q1, S08115-2-Q1, S08117-1-Q1, S08119-1-Q1, S08116-1-Q1,
S08112-1-Q1, S08108-1-Q1, S08101-4-Q1, S08101-1-Q1, S08101-2-Q1,
and S08101-3-Q1.
3. The method of claim 1, wherein said quantitative trait locus is
localized to a chromosomal interval defined by and including
S08119-1-Q1 and S08101-3-Q1 on linkage group L.
4. The method of claim 1, wherein said quantitative trait locus is
localized to a chromosomal interval defined by and including
markers S08116-1-Q1 and S08101-2-Q1 on linkage group L.
5. The method of claim 1, wherein said quantitative trait locus is
localized to a chromosomal interval defined by and including
markers S08101-1-Q1 and S08101-1-Q1 on linkage group L.
6. The method of claim 1, wherein said quantitative trait locus is
localized to a chromosomal interval defined by and including
markers S08112-1-Q1 and S08108-1-Q1 on linkage group L.
7. The method of claim 1, wherein the herbicide is selected from
the group consisting of diphenylethers, N-phenylpthalamides,
oxadiazole and triazolinones.
8. The method of claim 7, wherein the herbicide is selected from
the group consisting of sulfentrazone, carfentrazone-ethyl,
aciflourfen, lactofen, fomesafen, flumioxazin, flumiclorac-pentyl
and oxyfluorfen.
9. The method of claim 1, wherein the selection occurs as part of
further breeding to improve a soybean variety's tolerance to one or
more herbicides that inhibit protoporphyrinogen oxidase
function.
10. The method of claim 9, wherein the further breeding is selected
from the group consisting of additional crosses with other lines,
hybrids, backcrossing, self-crossing, and combinations thereof.
11. A soybean plant selected by the method of claim 1.
12. A kit for selecting at least one soybean plant by marker
assisted selection of a quantitative trait locus associated with
tolerance to herbicides that inhibit protoporphyrinogen oxidase
function comprising: a. primers for detecting at least one
tolerance-associated marker locus, wherein the tolerance-associated
locus is selected from the group consisting of S08102-1-Q1,
S08103-1-Q1. S08104-1-Q1, S08106-1-Q1, S08107-1-Q1, S08107-1-Q1,
S08109-1-Q1, S08110-1-Q1, S08111-1-Q1, S08115-2-Q1, S08117-1-Q1,
S08119-1-Q1, S08116-1-Q1, S08112-1-Q1, S08108-1-Q1, S08101-4-Q1,
S08101-1-Q1, S08101-2-Q1, and S08101-3-Q1; and b. instructions in
using the primers or probes for detecting the marker loci and
correlating the loci with predicted protoporphyrinogen oxidase
inhibitor tolerance.
13. A method for selectively controlling weeds in a field
containing a soybean crop comprising: (a) planting a field with
crop seeds or plants which are tolerant to herbicides that inhibit
protoporphyrinogen oxidase function as a result of comprising a
tolerance allele in a marker localizing within a chromosomal
interval flanked by and including S01659-1-A and S03859-1-A on
linkage group L; and (b) applying a sufficient amount of a
herbicide that inhibits protoporphyrinogen oxidase function to
control the weeds without significantly affecting the crop.
14. The method of claim 13, wherein the marker is selected from the
group consisting of S08102-1-Q1, S08103-1-Q1. S08104-1-Q1,
S08106-1-Q1, S08107-1-Q1, S08107-1-Q1, S08109-1-Q1, S08110-1-Q1,
S08111-1-Q1, S08115-2-Q1, S08117-1-Q1, S08119-1-Q1, S08116-1-Q1,
S08112-1-Q1, S08108-1-Q1, S08101-4-Q1, S08101-1-Q1, S08101-2-Q1,
and S08101-3-Q1.
15. The method of claim 13, wherein said marker is localized to a
chromosomal interval defined by and including S08119-1-Q1 and
S08101-3-Q1 on linkage group L.
16. The method of claim 13, wherein said marker is localized to a
chromosomal interval defined by and including markers S08116-1-Q1
and S08101-2-Q1 on linkage group L.
17. The method of claim 13, wherein said marker is localized to a
chromosomal interval defined by and including markers S08101-1-Q1
and S08101-1-Q1 on linkage group L.
18. The method of claim 13, wherein said marker is localized to a
chromosomal interval defined by and including markers S08112-1-Q1
and S08108-1-Q1 on linkage group L.
19. The method of claim 13, wherein the herbicide is applied as a
pre-emergent herbicide.
20. The method of claim 13, wherein the herbicide is applied as a
post-emergent herbicide.
21. The method of claim 13, further comprising applying to the crop
and weeds in the field a simultaneous or chronologically staggered
application of a herbicide that inhibits protoporphyrinogen oxidase
function and optionally an additional herbicide formulation.
22. The method of claim 21, wherein the additional herbicide
formulation is applied and the herbicide formulation contains an
active ingredient selected from the group consisting of a
hydroxyphenylpyruvatedioxygenase inhibitor, a glyphosate, a
sulfonylurea, a sulfonamide, an imidazolinone, a bialaphos, a
phosphinothricin, an azafenidin, a butafenacil, a sulfosate, a
glufosinate, a dicamba, and a protox inhibitor.
23. The method of claim 22, wherein said additional herbicide
formulation is applied simultaneously or sequentially.
24. The method of claim 22, wherein said crop seeds or plants
further comprise tolerance to the active ingredient of the
additional herbicide formulation.
25. The method of claim 24, wherein tolerance to the active
ingredient of the additional herbicide formulation is provided by
insertion of a transgene which confers the tolerance.
26. A method for selectively screening soybean plants for herbicide
tolerance comprising: (a) planting soybean seeds or plants
comprising a marker localizing within a chromosomal interval
flanked by and including S01659-1-A and S03859-1-A on linkage group
L; and (b) treating the plants by applying a sufficient amount of a
herbicide that inhibits protoporphyrinogen oxidase function to
differentiate between susceptible and tolerant plants; (c) scoring
the treated plants for tolerance to the herbicide.
27. The method of claim 26, wherein the marker is selected from the
group consisting of S08102-1-Q1, S08103-1-Q1. S08104-1-Q1,
S08106-1-Q1, S08107-1-Q1, S08107-1-Q1, S08109-1-Q1, S08110-1-Q1,
S08111-1-Q1, S08115-2-Q1, S08117-1-Q1, S08119-1-Q1, S08116-1-Q1,
S08112-1-Q1, S08108-1-Q1, S08101-4-Q1, S08101-1-Q1, S08101-2-Q1,
and S08101-3-Q1.
28. The method of claim 26, wherein said marker is localized to a
chromosomal interval defined by and including S08119-1-Q1 and
S08101-3-Q1 on linkage group L.
29. The method of claim 26, wherein said marker is localized to a
chromosomal interval defined by and including markers S08116-1-Q1
and S08101-2-Q1 on linkage group L.
30. The method of claim 26, wherein said marker is localized to a
chromosomal interval defined by and including markers S08101-1-Q1
and S08101-1-Q1 on linkage group L.
31. The method of claim 26, wherein said marker is localized to a
chromosomal interval defined by and including markers S08112-1-Q1
and S08108-1-Q1 on linkage group L.
32. A plant selected by the method of claim 1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/266,161, filed Apr. 30, 2014, now pending,
which is a divisional of U.S. patent application Ser. No.
12/694,255, filed Jan. 26, 2010, now U.S. Pat. No. 8,748,695, which
is a continuation-in-part of U.S. patent application Ser. No.
12/506,498, filed Jul. 21, 2009, now U.S. Pat. No. 8,697,941, which
claims the benefit under 35 U.S.C. .sctn.119(e) of U.S. Provisional
Application Ser. No. 60/083,038 filed Jul. 23, 2008, all herein
incorporated by reference in their entireties.
FIELD OF THE INVENTION
[0002] This invention relates generally to the detection of genetic
differences among soybeans.
BACKGROUND OF THE INVENTION
[0003] Soybeans (Glycine max L. Merr.) are a major cash crop and
investment commodity in North America and elsewhere. Soybean oil is
one of the most widely used edible oils, and soybeans are used
worldwide both in animal feed and in human food production.
Additionally, soybean utilization is expanding to industrial,
manufacturing, and pharmaceutical applications. Weed management in
soybean fields is important to maximizing yields. A recent
development in soybean technology has been the development of
herbicide-tolerant soybean varieties. Glyphosate tolerant soybeans
were commercially introduced in 1996 and accounted for more than
85% percent of U.S. soybean acreage in 2007.
[0004] Some weeds are starting to show increased tolerance to
glyphosate. This increased tolerance decreases the effectiveness of
glyphosate application and results in lower yields for farmers. As
a result there is a need in the art for soybean varieties that are
tolerant to other herbicide chemistry.
SUMMARY OF THE INVENTION
[0005] This invention relates generally to the detection of genetic
differences among soybeans. More particularly, the invention
relates to soybean quantitative trait loci (QTL) for tolerance to
protoporphyrinogen oxidase (PPOase) inhibitors, to soybean plants
possessing these QTLs, which map to a novel chromosomal region, and
to genetic markers that are indicative of phenotypes associated
with protoporphyrinogen oxidase inhibitor tolerance. Methods and
compositions for use of these markers in genotyping of soybean and
selection are also disclosed.
[0006] A novel method is provided for determining the presence or
absence in soybean germplasm of a QTL associated with tolerance to
protoporphyrinogen oxidase inhibitors. The tolerance trait has been
found to be closely linked to a number of molecular markers that
map to linkage groups L and N. Soybean plants, seeds, tissue
cultures, variants and mutants having tolerance to
protoporphyrinogen oxidase inhibitors produced by the foregoing
methods are also provided in this invention.
[0007] The QTL associated with tolerance to protoporphyrinogen
oxidase inhibitors maps to soybean linkage group L and/or N. These
QTL may be mapped by one or more molecular markers. For linkage
group L, the markers include SATT495, P10649C-3, SATT182, SATT388,
SATT313, SATT613, S08102-1-Q1, S08103-1-Q1. S08104-1-Q1,
S08106-1-Q1, S08107-1-Q1, S08107-1-Q1, S08109-1-Q1, S08110-1-Q1,
S08111-1-Q1, S08115-2-Q1, S08117-1-Q1, S08119-1-Q1, S08116-1-Q1,
S08112-1-Q1, S08108-1-Q1, S08101-4-Q1, S08101-1-Q1, S08101-2-Q1,
and S08101-3-Q1, or markers closely linked thereto. Other markers
of linkage group L may also be used to identify the presence or
absence of the gene, including other markers above marker SATT613.
For linkage group N, the markers include Sat.sub.--379, SCT 195,
SATT631, S60167-TB, SATT675, SATT624, SATT080, SATT387, or markers
closely linked thereto. Other markers of linkage group N may also
be used to identify the presence or absence of the gene, including
other markers above marker SATT387.
[0008] The information disclosed herein regarding the QTL for
tolerance to protoporphyrinogen oxidase inhibitors which maps to
soybean linkage group L and/or N is used to aid in the selection of
breeding plants, lines and populations containing tolerance to
protoporphyrinogen oxidase inhibitors for use in introgression of
this trait into elite soybean germplasm, or germplasm of proven
genetic superiority suitable for variety release.
[0009] Also provided is a method for introgressing a soybean QTL
associated with tolerance to protoporphyrinogen oxidase inhibitors
into non-tolerant soybean germplasm or less tolerant soybean
germplasm. According to the method, nucleic acid markers mapping
the QTL are used to select soybean plants containing the QTL.
Plants so selected have a high probability of expressing the trait
tolerance to protoporphyrinogen oxidase inhibitors. Plants so
selected can be used in a soybean breeding program. Through the
process of introgression, the QTL associated with tolerance to
protoporphyrinogen oxidase inhibitors is introduced from plants
identified using marker-assisted selection to other plants.
According to the method, agronomically desirable plants and seeds
can be produced containing the QTL associated with tolerance to
protoporphyrinogen oxidase inhibitors from germplasm containing the
QTL. Sources of tolerance to protoporphyrinogen oxidase inhibitors
are disclosed below.
[0010] Also provided herein is a method for producing a soybean
plant adapted for conferring tolerance to protoporphyrinogen
oxidase inhibitors. First, donor soybean plants for a parental line
containing the tolerance QTL are selected. According to the method,
selection can be accomplished via nucleic acid marker-associated
selection as explained herein. Selected plant material may
represent, among others, an inbred line, a hybrid, a heterogeneous
population of soybean plants, or simply an individual plant.
According to techniques well known in the art of plant breeding,
this donor parental line is crossed with a second parental line.
Typically, the second parental line is a high yielding line. This
cross produces a segregating plant population composed of
genetically heterogeneous plants. Plants of the segregating plant
population are screened for the tolerance QTL and are subjected to
further breeding. This further breeding may include, among other
techniques, additional crosses with other lines, hybrids,
backcrossing, or self-crossing. The result is a line of soybean
plants that is tolerant to protoporphyrinogen oxidase inhibitors
and also has other desirable traits from one or more other soybean
lines.
[0011] Also provided is a method for introgressing a soybean QTL
associated with tolerance or sensitivity to protoporphyrinogen
oxidase inhibitors into non-tolerant soybean germplasm or less
tolerant soybean germplasm. According to the method, nucleic acid
markers mapping the QTL are used to select soybean plants
containing the QTL. Plants so selected have a high probability of
expressing the trait tolerance or sensitivity to protoporphyrinogen
oxidase inhibitors. Plants so selected can be used in a soybean
breeding program. Through the process of introgression, the QTL
associated with tolerance or sensitivity to protoporphyrinogen
oxidase inhibitors is introduced from plants identified using
marker-assisted selection to other plants. According to the method,
agronomically desirable plants and seeds can be produced containing
the QTL associated with tolerance or sensitivity to
protoporphyrinogen oxidase inhibitors from germplasm containing the
QTL. Sources of tolerance or sensitivity to protoporphyrinogen
oxidase inhibitors are disclosed below.
[0012] Also provided herein is a method for producing a soybean
plant adapted for conferring tolerance or sensitivity to
protoporphyrinogen oxidase inhibitors. First, donor soybean plants
for a parental line containing the tolerance QTL are selected.
According to the method, selection can be accomplished via nucleic
acid marker-associated selection as explained herein. Selected
plant material may represent, among others, an inbred line, a
hybrid, a heterogeneous population of soybean plants, or simply an
individual plant. According to techniques well known in the art of
plant breeding, this donor parental line is crossed with a second
parental line. Typically, the second parental line is a high
yielding line. This cross produces a segregating plant population
composed of genetically heterogeneous plants. Plants of the
segregating plant population are screened for the tolerance QTL and
are subjected to further breeding. This further breeding may
include, among other techniques, additional crosses with other
lines, hybrids, backcrossing, or self-crossing. The result is a
line of soybean plants that is tolerant to mesotrione and/or
isoxaflutole herbicides, and also has other desirable traits, such
as yield, from one or more other soybean lines.
[0013] Also described are isolated polynucleotides and isolated
polypeptides relevant to tolerance or sensitivity to
protoporphyrinogen oxidase inhibitors. Additional traits may also
be added to plants having such tolerance or sensitivity, such as
additional herbicide tolerance traits, insect tolerance traits, or
other transgenic traits. Also described are methods of
introgressing a tolerance or susceptibility allele into a plant,
such as by crossing a soybean plant tolerant to an isoflutole
herbicide with a soybean plant susceptible to a isoflutole
herbicide in order to form a segregating population, screening the
segregating population with one or more nucleic acid markers to
determine if plants from the segregating population contains at
least one SNP selected from the group consisting of an SNP at
position #1433, #1559, #1750, #1832, #1932, #2727, #2858, #3027,
#3088, #3090, and #3334 of the sequence set forth as SEQ ID NO: 114
as shown in FIG. 3, or a sequence equivalent to SEQ ID NO: 114, and
optionally selecting, if present, one or more soybean plants of the
segregating population containing the at least one SNP.
Alternatively, such tolerance may be transgenically provided by
introducing into a plant cell a polynucleotide as disclosed herein
operably linked to a promoter functional in the plant cell to
produce a transformed plant cell, and optionally selecting a
transformed plant cell having the polynucleotide stably
incorporated into its genome.
[0014] Compositions include isolated polynucleotides encoding ABC
transporter polypeptides that confer tolerance to such herbicides,
and isolated ABC transporter polypeptides. Compositions include
those polynucleotides encoding polypeptides with amino acid
substitutions at position V520X, L584X, S611X, K953X, L1030X,
and/or G1112X or positions equivalent thereto, as well as
polypeptides with amino acid substitutions at position V520X,
L584X, S611X, K953X, L1030X, and/or G1112X or positions equivalent
thereto. Also useful are isolated polynucleotide variants,
polynucleotides encoding polypeptide variants, and polypeptide
variants having sequence identity to the appropriate reference
sequence, such an ABC transporter polypeptide of at least 60%, 65%,
70%, 75, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.75%.
[0015] Soybean plants, seeds, tissue cultures, variants and mutants
having tolerance or sensitivity to PPO inhibitor herbicides
produced by the foregoing methods are also provided. Also provided
herein are methods for controlling weeds in a crop by applying to
the crop and any weeds affecting such crop an effective amount of
such herbicide(s), either pre-emergent or post-emergent, such that
the weeds are substantially controlled without substantially
negatively impacting the crop.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] The invention can be more fully understood from the
following detailed description and the accompanying drawings and
Sequence Listing, which form a part of this application.
[0017] FIG. 1A provides an integrated genetic map of soybean
markers on linkage group L, including the marker type (SSR or
ASH/SNP). The genetic map positions of the markers are indicated in
centiMorgans (cM), typically with position zero being the first
(most distal) marker on the chromosome. The map includes relative
positions for some markers for which higher resolution genetic
mapping data was not available; no position in cM is provided.
[0018] FIG. 1B provides a table listing genetic markers that are
linked to the protoporphyrinogen oxidase (PPOase) inhibitor
tolerance markers identified on linkage group L. These markers are
from the soybean public composite map of Jun. 18, 2008 for linkage
group L.
[0019] FIG. 2A provides an integrated genetic map of soybean
markers on linkage group N, including the marker type (SSR or
ASH/SNP). The genetic map positions of the markers are indicated in
centiMorgans (cM), typically with position zero being the first
(most distal) marker on the chromosome.
[0020] FIG. 2B provides a table listing genetic markers that are
linked to the protoporphyrinogen oxidase (PPOase) inhibitor
tolerance markers identified by the present invention on linkage
group N. These markers are from the soybean public composite map of
Jun. 18, 2008 for linkage group N.
[0021] FIG. 3 provides a table listing SSR markers, including those
markers that demonstrated linkage disequilibrium with the
protoporphyrinogen oxidase (PPOase) inhibitor tolerance phenotype.
The table provides the sequences of the left and right PCR primers
used in the SSR marker locus genotyping analysis. Also shown is the
pigtail sequence used on the 5' end of the right primer.
[0022] FIG. 4 provides a table listing the SNP markers that
demonstrated linkage disequilibrium with the protoporphyrinogen
oxidase (PPOase) inhibitor tolerance phenotype. The table provides
the sequences of the PCR primers used to generate a SNP-containing
amplicon, and the allele-specific probes that were used to identify
the SNP allele in an allele-specific hybridization assay (ASH
assay).
[0023] FIG. 5 provides an example of cultivars with vastly
different protoporphyrinogen oxidase (PPOase) inhibitor tolerance
phenotypes. Shown are field samples, with a non-tolerant variety on
the left (white circle: stunted, necrotic) and tolerant variety on
the right (normal growth)
[0024] FIG. 6 provides an example of cultivars with vastly
different protoporphyrinogen oxidase (PPOase) inhibitor tolerance
phenotypes. Shown are greenhouse samples, with a non-tolerant
variety with non-tolerant (arrow, left side) and tolerant (right
side) variety checks, showing treated plants in the foreground, and
untreated plants in the background.
DETAILED DESCRIPTION
[0025] It is to be understood that this invention is not limited to
particular embodiments or examples, which can, of course, vary. It
is also to be understood that the terminology used herein is for
the purpose of describing particular embodiments only, and is not
intended to be limiting. As used in this specification and the
appended claims, terms in the singular and the singular forms "a,"
"an" and "the," for example, include plural referents unless the
content clearly dictates otherwise. Thus, for example, reference to
"plant," "the plant" or "a plant" also includes a plurality of
plants; also, depending on the context, use of the term "plant" can
also include genetically similar or identical progeny of that
plant; use of the term "a nucleic acid" optionally includes, as a
practical matter, many copies of that nucleic acid molecule;
similarly, the term "probe" optionally (and typically) encompasses
many similar or identical probe molecules.
[0026] Certain definitions used in the specification are provided
below. Also in the examples which follow, a number of terms are
used. Terms not specifically defined herein should be given their
ordinary meaning to those in the art. In order to provide a clear
and consistent understanding of the specification and claims,
including the scope to be given such terms, the following
definitions are provided:
[0027] AGRONOMICS, AGRONOMIC TRAITS, and AGRONOMIC PERFORMANCE
refer to the traits and underlying genetic elements of a given
plant variety that contribute to yield over the course of growing
season. Individual agronomic traits include emergence vigor,
vegetative vigor, stress tolerance, disease resistance or
tolerance, herbicide resistance or tolerance, branching, flowering,
seed set, seed size, seed density, standability, threshability and
the like.
[0028] ALLELE means any of one or more alternative forms of a
genetic sequence. In a diploid cell or organism, the two alleles of
a given sequence typically occupy corresponding loci on a pair of
homologous chromosomes.
[0029] The term AMPLIFYING in the context of nucleic acid
amplification is any process whereby additional copies of a
selected nucleic acid (or a transcribed form thereof) are produced.
Typical amplification methods include various polymerase based
replication methods, including the polymerase chain reaction (PCR),
ligase mediated methods such as the ligase chain reaction (LCR) and
RNA polymerase based amplification (e.g., by transcription)
methods. An "amplicon" is an amplified nucleic acid, e.g., a
nucleic acid that is produced by amplifying a template nucleic acid
by any available amplification method (e.g., PCR, LCR,
transcription, or the like).
[0030] An ANCESTRAL LINE is a parent line used as a source of
genes.
[0031] An ANCESTRAL POPULATION is a group of ancestors that have
contributed the bulk of the genetic variation that was used to
develop elite lines.
[0032] BACKCROSSING is a process in which a breeder crosses a
progeny variety back to one of the parental genotypes one or more
times.
[0033] BREEDING means the genetic manipulation of living
organisms.
[0034] The term CHROMOSOME SEGMENT designates a contiguous linear
span of genomic DNA that resides in planta on a single
chromosome.
[0035] CULTIVAR and VARIETY are used synonymously and mean a group
of plants within a species (e.g., Glycine max) that share certain
genetic traits that separate them from the typical form and from
other possible varieties within that species. Soybean cultivars are
inbred lines produced after several generations of
self-pollinations. Individuals within a soybean cultivar are
homogeneous, nearly genetically identical, with most loci in the
homozygous state.
[0036] An ELITE LINE is an agronomically superior line that has
resulted from many cycles of breeding and selection for superior
agronomic performance. Numerous elite lines are available and known
to those of skill in the art of soybean breeding.
[0037] An ELITE POPULATION is an assortment of elite individuals or
lines that can be used to represent the state of the art in terms
of agronomically superior genotypes of a given crop species, such
as soybean.
[0038] A GENETIC MAP is a description of genetic linkage
relationships among loci on one or more chromosomes or linkage
groups within a given species, generally depicted in a diagrammatic
or tabular form.
[0039] GENOTYPE refers to the genetic constitution of a cell or
organism.
[0040] GERMPLASM means the genetic material that comprises the
physical foundation of the hereditary qualities of an organism. As
used herein, germplasm includes seeds and living tissue from which
new plants may be grown; or, another plant part, such as leaf,
stem, pollen, or cells, that may be cultured into a whole plant.
Germplasm resources provide sources of genetic traits used by plant
breeders to improve commercial cultivars.
[0041] An individual is HOMOZYGOUS if the individual has only one
type of allele at a given locus (e.g., a diploid individual has a
copy of the same allele at a locus for each of two homologous
chromosomes). An individual is "HETEROZYGOUS" if more than one
allele type is present at a given locus (e.g., a diploid individual
with one copy each of two different alleles). The term
"HOMOGENEITY" indicates that members of a group have the same
genotype at one or more specific loci. In contrast, the term
"HETEROGENEITY" is used to indicate that individuals within the
group differ in genotype at one or more specific loci.
[0042] INTROGRESSION means the entry or introduction of a gene,
QTL, or trait locus from the genome of one plant into the genome of
another plant.
[0043] A LINE or a STRAIN is a group of individuals of identical
parentage that are generally inbred to some degree and that are
generally homozygous and homogeneous at most loci (isogenic or near
isogenic). A "SUBLINE" refers to an inbred subset of descendents
that are genetically distinct from other similarly inbred subsets
descended from the same progenitor. Traditionally, a subline has
been derived by inbreeding the seed from an individual soybean
plant selected at the F3 to F5 generation until the residual
segregating loci are "fixed" or homozygous across most or all loci.
Commercial soybean varieties (or lines) are typically produced by
aggregating ("bulking") the self-pollinated progeny of a single F3
to F5 plant from a controlled cross between 2 genetically different
parents. While the variety typically appears uniform, the
self-pollinating variety derived from the selected plant eventually
(e.g., F8) becomes a mixture of homozygous plants that can vary in
genotype at any locus that was heterozygous in the originally
selected F3 to F5 plant. Marker-based sublines that differ from
each other based on qualitative polymorphism at the DNA level at
one or more specific marker loci are derived by genotyping a sample
of seed derived from individual self-pollinated progeny derived
from a selected F3-F5 plant. The seed sample can be genotyped
directly as seed, or as plant tissue grown from such a seed sample.
Optionally, seed sharing a common genotype at the specified locus
(or loci) are bulked providing a subline that is genetically
homogenous at identified loci important for a trait of interest
(yield, tolerance, etc.).
[0044] LINKAGE refers to a phenomenon wherein alleles on the same
chromosome tend to segregate together more often than expected by
chance if their transmission was independent. Genetic recombination
occurs with an assumed random frequency over the entire genome.
Genetic maps are constructed by measuring the frequency of
recombination between pairs of traits or markers. The closer the
traits or markers lie to each other on the chromosome, the lower
the frequency of recombination, and the greater the degree of
linkage. Traits or markers are considered herein to be linked if
they generally co-segregate. A 1/100 probability of recombination
per generation is defined as a map distance of 1.0 centiMorgan (1.0
cM). For example, in soybean, 1 cM correlates, on average, to about
400,000 base pairs (400 Kb).
[0045] The genetic elements or genes located on a single chromosome
segment are physically linked. In the context of the present
invention the genetic elements located within a chromosome segment
are also genetically linked, typically within a genetic
recombination distance of less than or equal to 50 centimorgans
(cM), e.g., about 49, 40, 30, 20, 10, 5, 4, 3, 2, 1, 0.75, 0.5, or
0.25 cM or less. That is, two genetic elements within a single
chromosome segment undergo recombination during meiosis with each
other at a frequency of less than or equal to about 50%, e.g.,
about 49%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, or
0.25% or less.
[0046] LINKAGE GROUP refers to traits or markers that generally
co-segregate. A linkage group generally corresponds to a
chromosomal region containing genetic material that encodes the
traits or markers.
[0047] LOCUS is a defined segment of DNA.
[0048] A MAP LOCATION is an assigned location on a genetic map
relative to linked genetic markers where a specified marker can be
found within a given species. Markers are frequently described as
being "above" or "below" other markers on the same linkage group; a
marker is "above" another marker if it appears earlier on the
linkage group, whereas a marker is "below" another marker if it
appears later on the linkage group.
[0049] MAPPING is the process of defining the linkage relationships
of loci through the use of genetic markers, populations segregating
for the markers, and standard genetic principles of recombination
frequency.
[0050] MOLECULAR MARKER is a nucleic acid or amino acid sequence
that is sufficiently unique to characterize a specific locus on the
genome. Examples include Restriction Fragment Length Polymorphisms
(RFLPs), Single Sequence Repeats (SSRs), Target Region
Amplification Polymorphisms (TRAPs), Isozyme Electrophoresis,
Randomly Amplified Polymorphic DNAs (RAPDs), Arbitrarily Primed
Polymerase Chain Reaction (AP-PCR), DNA Amplification
Fingerprinting (DAF), Sequence Characterized Amplified Regions
(SCARs), Amplified Fragment Length Polymorphisms (AFLPs), and
Single Nucleotide Polymorphisms (SNPs). Additionally, other types
of molecular markers are known to the art, and phenotypic traits
may also be used as markers in the methods. All markers are used to
define a specific locus on the soybean genome. Large numbers of
these markers have been mapped. Each marker is therefore an
indicator of a specific segment of DNA, having a unique nucleotide
sequence. The map positions provide a measure of the relative
positions of particular markers with respect to one another. When a
trait is stated to be linked to a given marker it will be
understood that the actual DNA segment whose sequence affects the
trait generally co-segregates with the marker. More precise and
definite localization of a trait can be obtained if markers are
identified on both sides of the trait. By measuring the appearance
of the marker(s) in progeny of crosses, the existence of the trait
can be detected by relatively simple molecular tests without
actually evaluating the appearance of the trait itself, which can
be difficult and time-consuming because the actual evaluation of
the trait requires growing plants to a stage where the trait can be
expressed. Molecular markers have been widely used to determine
genetic composition in soybeans. Shoemaker and Olsen, ((1993)
Molecular Linkage Map of Soybean (Glycine max L. Merr.). p.
6.131-6.138. In S. J. O'Brien (ed.) Genetic Maps: Locus Maps of
Complex Genomes. Cold Spring Harbor Laboratory Press. Cold Spring
Harbor, N.Y.), developed a molecular genetic linkage map that
consisted of 25 linkage groups with about 365 RFLP, 11 RAPD (random
amplified polymorphic DNA), three classical markers, and four
isozyme loci. See also Shoemaker R. C. 1994 RFLP Map of Soybean. P.
299-309 In R. L. Phillips and I. K. Vasil (ed.) DNA-based markers
in plants. Kluwer Academic Press Dordrecht, the Netherlands.
[0051] MARKER ASSISTED SELECTION refers to the process of selecting
a desired trait or desired traits in a plant or plants by detecting
one or more molecular markers from the plant, where the molecular
marker is linked to the desired trait.
[0052] The term PHYSICALLY LINKED is used to indicate that two
loci, e.g., two marker loci, are physically present on the same
chromosome. Advantageously, the two loci are located in close
proximity such that recombination between homologous chromosome
pairs does not occur between the two loci during meiosis with high
frequency, e.g., such that linked loci co-segregate at least about
90% of the time, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
99.5%, 99.75%, or more of the time.
[0053] The term PLANT includes reference to an immature or mature
whole plant, including a plant from which seed or grain or anthers
have been removed. Seed or embryo that will produce the plant is
also considered to be the plant.
[0054] PLANT PARTS include leaves, stems, buds, roots, root tips,
anthers, seed, grain, embryo, pollen, ovules, flowers, cotyledons,
hypocotyls, pods, flowers, shoots and stalks, tissues, cells and
the like.
[0055] POLYMORPHISM means a change or difference between two
related nucleic acids. A "NUCLEOTIDE POLYMORPHISM" refers to a
nucleotide that is different in one sequence when compared to a
related sequence when the two nucleic acids are aligned for maximal
correspondence. A "GENETIC NUCLEOTIDE POLYMORPHISM" refers to a
nucleotide that is different in one sequence when compared to a
related sequence when the two nucleic acids are aligned for maximal
correspondence, where the two nucleic acids are genetically
related, i.e., homologous, for example, where the nucleic acids are
isolated from different strains of a soybean plant, or from
different alleles of a single strain, or the like.
[0056] PROBE means a polynucleotide designed to be sufficiently
complementary to a sequence in a denatured nucleic acid to be
probed and to be bound under selected stringency conditions.
[0057] RAPD marker means random amplified polymorphic DNA marker.
Chance pairs of sites complementary to single octa- or
decanucleotides may exist in the correct orientation and close
enough to one another for PCR amplification. With some randomly
chosen decanucleotides no sequences are amplified. With others, the
same length products are generated from DNAs of different
individuals. With still others, patterns of bands are not the same
for every individual in a population. The variable bands are
commonly called random amplified polymorphic DNA (RAPD) bands.
[0058] RECOMBINATION FREQUENCY is the frequency of a crossing over
event (recombination) between two genetic loci. Recombination
frequency can be observed by following the segregation of markers
and/or traits during meiosis. A marker locus is "associated with"
another marker locus or some other locus (for example, a tolerance
locus), when the relevant loci are part of the same linkage group
and are in linkage disequilibrium. This occurs when the marker
locus and a linked locus are found together in progeny plants more
frequently than if the two loci segregate randomly. Similarly, a
marker locus can also be associated with a trait, e.g., a marker
locus can be "associated with tolerance or improved tolerance" when
the marker locus is in linkage disequilibrium with the trait.
[0059] RFLP means restriction fragment length polymorphism.
Molecular markers that occur because any sequence change in DNA,
including a single base change, insertion, deletion or inversion,
can result in loss or gain of a restriction endonuclease
recognition site. The size and number of fragments generated by one
such enzyme is therefore altered. A probe that hybridizes
specifically to DNA in the region of such an alteration can be used
to rapidly and specifically identify a region of DNA that displays
allelic variation between two plant varieties. Isozyme
Electrophoresis and RFLPs have been widely used to determine
genetic composition
[0060] SELF CROSSING or SELF-POLLINATION or SELFING is a process
through which a breeder crosses hybrid progeny with itself; for
example, a second generation hybrid F2 with itself to yield progeny
designated F2:3.
[0061] SNP means single nucleotide polymorphism. SNPs are genetic
markers in which DNA sequence variations that occur when a single
nucleotide (A, T, C, or G) in the genome sequence is altered are
mapped to sites on the soybean genome. Many techniques for
detecting SNPs are known in the art, including allele specific
hybridization, primer extension, and direct sequencing.
[0062] SSR means short sequence repeats. SSRs are genetic markers
based on polymorphisms in repeated nucleotide sequences, such as
microsatellites. A marker system based on SSRs can be highly
informative in linkage analysis relative to other marker systems in
that multiple alleles may be present. The PCR detection is done by
use of two oligonucleotide primers flanking the polymorphic segment
of repetitive DNA. Repeated cycles of heat denaturation of the DNA
followed by annealing of the primers to their complementary
sequences at low temperatures, and extension of the annealed
primers with DNA polymerase, comprise the major part of the
methodology.
[0063] TOLERANT and TOLERANCE refer to plants in which higher doses
of a herbicide are required to produce effects similar to those
seen in non-tolerant plants. Tolerant plants typically exhibit
fewer necrotic, lytic, chlorotic, or other lesions when subjected
to the herbicide at concentrations and rates typically employed by
the agricultural community.
[0064] TRANSGENIC PLANT refers to a plant that comprises within its
cells a heterologous polynucleotide. Generally, the heterologous
polynucleotide is stably integrated within the genome such that the
polynucleotide is passed on to successive generations. The
heterologous polynucleotide may be integrated into the genome alone
or as part of a recombinant expression cassette. TRANSGENIC is used
herein to refer to any cell, cell line, callus, tissue, plant part
or plant, the genotype of which has been altered by the presence of
heterologous nucleic acid including those transgenic organisms or
cells initially so altered, as well as those created by crosses or
asexual propagation from the initial transgenic organism or cell.
The term "transgenic" as used herein does not encompass the
alteration of the genome (chromosomal or extra-chromosomal) by
conventional plant breeding methods (e.g., crosses) or by naturally
occurring events such as random cross-fertilization,
non-recombinant viral infection, non-recombinant bacterial
transformation, non-recombinant transposition, or spontaneous
mutation.
[0065] TRAP marker means target region amplification polymorphism
marker. The TRAP technique employs one fixed primer of known
sequence in combination with a random primer to amplify genomic
fragments. The differences in fragments between alleles can be
detected by gel electrophoresis.
[0066] The term VECTOR is used in reference to polynucleotide or
other molecules that transfer nucleic acid segment(s) into a cell.
The term "vehicle" is sometimes used interchangeably with "vector."
A vector optionally comprises parts which mediate vector
maintenance and enable its intended use (e.g., sequences necessary
for replication, genes imparting drug or antibiotic resistance, a
multiple cloning site, operably linked promoter/enhancer elements
which enable the expression of a cloned gene, etc.). Vectors are
often derived from plasmids, bacteriophages, or plant or animal
viruses. A "cloning vector" or "shuttle vector" or "subcloning
vector" contains operably linked parts that facilitate subcloning
steps (e.g., a multiple cloning site containing multiple
restriction endonuclease sites).
[0067] The term YIELD refers to the productivity per unit area of a
particular plant product of commercial value. For example, yield of
soybean is commonly measured in bushels of seed per acre or metric
tons of seed per hectare per season. Yield is affected by both
genetic and environmental factors. Yield is the final culmination
of all agronomic traits.
[0068] An equivalent position in a polynucleotide and/or
polypeptide sequence is a position that correlates a position in
the reference sequence when the sequences are aligned for a maximum
correspondence. In some examples the sequences are aligned across
their whole length using a global alignment program. In other
examples, a portion of the sequence or sequences may be aligned
using a local alignment program or a global alignment program, for
example a sequence may comprise exons and introns, conserved motifs
or domains, or functional motifs or domains which may be aligned to
the reference sequence(s) to identify equivalent positions.
Equivalent positions in polynucleotides encoding a polypeptide can
be determined using the encoded amino acid, and/or using a
FrameAlign program to align the polynucleotide and polypeptide for
maximal correspondence.
[0069] The term "homologous" refers to nucleic acid sequences that
are derived from a common ancestral gene through natural or
artificial processes (e.g., are members of the same gene family),
and thus, typically share sequence similarity. Typically,
homologous nucleic acids have sufficient sequence identity that one
of the sequences or a subsequence thereof or its complement is able
to selectively hybridize to the other under selective (e.g.,
stringent) hybridization conditions. The term "selectively
hybridizes" includes reference to hybridization, under stringent
hybridization conditions, of a nucleic acid sequence to a specified
nucleic acid target sequence to a detectably greater degree (e.g.,
at least 2-fold over background) than its hybridization to
non-target nucleic acid sequences and to the substantial exclusion
of non-target nucleic acids. Selectively hybridizing nucleic acid
sequences typically have about at least 70% sequence identity, at
least 80% sequence identity, or about 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, 99.5%, 99.75%, or 100% sequence identity with
each other. A nucleic acid that exhibits at least some degree of
homology to a reference nucleic acid can be unique or identical to
the reference nucleic acid or its complementary sequence.
[0070] The term "isolated" refers to material, such as
polynucleotides or polypeptides, which are identified and separated
from at least one contaminant with which it is ordinarily
associated in its natural or original source. Furthermore, an
isolated polynucleotide or polypeptide is typically present in a
form or setting that is different from the form or setting that is
normally found in nature. In some examples, the isolated molecule
is substantially free from components that normally accompany or
interact with it in its naturally occurring environment. In some
embodiments, the isolated material optionally comprises material
not found with the material in its natural environment, e.g., in a
cell.
[0071] As used herein, the terms "exogenous" or "heterologous" as
applied to polynucleotides or polypeptides refers to molecules that
have been artificially supplied to a biological system (e.g., a
plant cell, a plant gene, a particular plant species or a plant
chromosome under study) and are not native to that particular
biological system. The terms indicate that the relevant material
originated from a source other than the naturally occurring source,
or refers to molecules having a non-natural configuration, genetic
location or arrangement of parts. A heterologous polynucleotide
includes polynucleotides from another organism or the same organism
which have been modified by linkage to a distinct non-endogenous
polynucleotide and/or inserted to a distinct non-endogenous locus.
The terms "exogenous" and "heterologous" are sometimes used
interchangeably with "recombinant."
[0072] In contrast, for example, a "native" or "endogenous" gene is
a gene that does not contain nucleic acid elements encoded by
sources other than the chromosome or other genetic element on which
it is normally found in nature. An endogenous gene, transcript or
polypeptide is encoded by its natural chromosomal locus, and not
artificially supplied to the cell.
[0073] The term "recombinant" indicates that the material (e.g., a
recombinant nucleic acid, gene, polynucleotide or polypeptide) has
been altered by human intervention. Generally, the arrangement of
parts of a recombinant molecule is not a native configuration, or
the primary sequence of the recombinant polynucleotide or
polypeptide has in some way been manipulated. The alteration to
yield the recombinant material can be performed on the material
within or removed from its natural environment or state. For
example, a naturally occurring nucleic acid becomes a recombinant
nucleic acid if it is altered, or if it is transcribed from DNA
which has been altered, by means of human intervention performed
within the cell from which it originates. A gene sequence open
reading frame is recombinant if that nucleotide sequence has been
removed from it natural text and cloned into any type of artificial
nucleic acid vector. Protocols and reagents to produce recombinant
molecules, especially recombinant nucleic acids, are common and
routine in the art (see, e.g., Maniatis et al. (eds.), Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press,
NY, [1982]; Sambrook et al. (eds.), Molecular Cloning: A Laboratory
Manual, Second Edition, Volumes 1-3, Cold Spring Harbor Laboratory
Press, NY, [1989]; and Ausubel et al. (eds.), Current Protocols in
Molecular Biology, Vol. 1-4, John Wiley & Sons, Inc., New York
[1994]). The term recombinant can also refer to an organism that
harbors a recombinant material, e.g., a plant that comprises a
recombinant nucleic acid is considered a recombinant plant. In some
embodiments, a recombinant organism is a transgenic organism.
[0074] The term "introduced" when referring to a heterologous or
exogenous nucleic acid refers to the incorporation of a nucleic
acid into a eukaryotic or prokaryotic cell using any type of
suitable vector, e.g., naked linear DNA, plasmid, plastid or
virion), converted into an autonomous replicon, or transiently
expressed (e.g., transfected mRNA). The term includes such nucleic
acid introduction means as "transfection," "transformation" and
"transduction."
[0075] The term "host cell" means a cell that contains a
heterologous nucleic acid, such as a vector, and supports the
replication and/or expression of the nucleic acid. Host cells may
be prokaryotic cells such as E. coli, or eukaryotic cells such as
yeast, insect, amphibian or mammalian cells. In some examples, host
cells are plant cells, including but not limited to dicot and
monocot cells.
[0076] The term "transgenic plant" refers to a plant that comprises
within its cells a heterologous polynucleotide. Generally, the
heterologous polynucleotide is stably integrated within the genome
such that the polynucleotide is passed on to successive
generations. The heterologous polynucleotide may be integrated into
the genome alone or as part of a recombinant expression cassette.
"Transgenic" is used herein to refer to any cell, cell line,
callus, tissue, plant part or plant, the genotype of which has been
altered by the presence of heterologous nucleic acid including
those transgenic organisms or cells initially so altered, as well
as those created by crosses or asexual propagation from the initial
transgenic organism or cell. The term "transgenic" as used herein
does not encompass the alteration of the genome (chromosomal or
extra-chromosomal) by conventional plant breeding methods (e.g.,
crosses) or by naturally occurring events such as random
cross-fertilization, non-recombinant viral infection,
non-recombinant bacterial transformation, non-recombinant
transposition, or spontaneous mutation.
[0077] Plant cell, as used herein includes, without limitation,
cells within or derived from, for example and without limitation,
plant seeds, plant tissue suspension cultures, plant tissue, plant
tissue explants, plant embryos, meristematic tissue, callus tissue,
leaves, roots, shoots, gametophytes, sporophytes, pollen and
microspores.
[0078] The term "crossed" or "cross" means the fusion of gametes
via pollination to produce progeny (e.g., cells, seeds or plants).
The term encompasses both sexual crosses (the pollination of one
plant by another) and selfing (self-pollination, e.g., when the
pollen and ovule are from the same plant).
[0079] The term "introgression" refers to the transmission of a
desired allele of a genetic locus from one genetic background to
another. For example, introgression of a desired allele at a
specified locus can be transmitted to at least one progeny plant
via a sexual cross between two parent plants, at least one of the
parent plants having the desired allele within its genome.
Alternatively, for example, transmission of an allele can occur by
recombination between two donor genomes, e.g., in a fused
protoplast, where at least one of the donor protoplasts has the
desired allele in its genome. The desired allele can be, e.g., a
transgene or a gene allele that imparts resistance to a plant
pathogen.
Protoporphyrinogen Oxidase Inhibitors
[0080] Porphyrins are biologically important organic structures
that are found in plants attached to chlorophyll and cytochrome
pigments. An intermediate in the chlorophyll and cytochrome
synthesis pathway is protoporphyrinogen IX which is converted to
protoporphyrin IX by protoporphyrinogen oxidase. Inhibition of
protoporphyrinogen oxidase prevents this conversion and results in
a buildup of protoporphyrinogen IX in the cytoplasm of the plant.
The protoporphyrinogen then undergoes non-enzymatic auto-oxidation
and becomes protoporphyrin IX. When cytoplasmic protoporphyrin IX
is exposed to sunlight, free radicals are formed which results in
lipid peroxidation reactions that result in plant death.
Protoporphyrinogen oxidase inhibitor chemical families include
diphenyl ether, triazolinone, N-phenylphthalimide, pyrimidindione
and oxadiazole families. There are other families of chemistries
that also belong to this group.
[0081] The diphenyl ether family is characterized by two benzene
rings linked with an ether bridge and a nitro group bonded to the 4
position. Examples of diphenyl ether protoporphyrinogen oxidase
inhibitors include acifluorfen, fomesafen, oxyfluorfen and
lactofen. The diphenyl ethers are typically considered to be
contact herbicides.
[0082] The triazolinone family is characterized by a 5-member ring
containing three nitrogen atoms (two of which are adjacent) and two
carbon atoms, one of the carbon atoms has a double bond with an
oxygen atom and one of the nitrogen atoms is bonded to a benzene
ring. Examples of triazolinone protoprophyrinogen oxidase
inhibitors include sulfentrazone, carfentrasone, and
azafeniden.
[0083] The N-phenylphthalimide family is characterized by
pthalimide group wherein the nitrogen is bonded to a benzene ring.
Examples of N-phenylphthalimide protoporphyrinogen oxidase
inhibitors include flumiclorac and flumioxazin.
[0084] The oxadiazole family is characterized by a five member ring
consisting of two adjacent nitrogen atoms, two carbon atoms, and an
oxygen or sulfur atom. Examples of oxadiazole protoporphyrinogen
oxidase inhibitors include oxadiazon and fluthiacet.
[0085] The various families of protoporphyrinogen oxidase
inhibitors provide a wide variety in application options.
Sulfentrazone, for example, has a relatively long half-life
(approximately 280 days), is known to have residual soil activity
and is frequently used as a pre-emergence herbicide. Carfentrazone
has a considerably shorter half-life (approximately 4 days) has no
residual soil activity, and is used as a contact/post-emergence
herbicide. The pyrimidindiones family of PPO herbicides is a rather
small class that includes benzfendizone, butagenacil and
saflufenacil. This diversity in chemical characteristics combined
with protoporphyrinogen oxidase inhibitor tolerance provides
farmers with a wide variety of weed management options.
Molecular Markers and Genetic Linkage
[0086] Molecular markers have been used to selectively improve
soybean crops through the use of marker assisted selection. Any
detectable polymorphic trait can be used as a marker so long as it
is inherited differentially and exhibits linkage disequilibrium
with a phenotypic trait of interest. A number of soybean markers
have been mapped and linkage groups created, as described in
Cregan, P. B. et al., "An Integrated Genetic Linkage Map of the
Soybean Genome" (1999) Crop Science 39:1464-90, and more recently
in Choi et al., "A Soybean Transcript Map: Gene Distribution,
Haplotype and Single-Nucleotide Polymorphism Analysis" (2007)
Genetics 176:685-96. Many soybean markers are publicly available at
the USDA affiliated soybase website.
[0087] Most plant traits of agronomic importance are polygenic,
otherwise known as quantitative, traits. A quantitative trait is
controlled by several genes located at various locations, or loci,
in the plant's genome. The multiple genes have a cumulative effect
which contributes to the continuous range of phenotypes observed in
many plant traits. These genes are referred to as quantitative
trait loci (QTL). Recombination frequency measures the extent to
which a molecular marker is linked with a QTL. Lower recombination
frequencies, typically measured in centiMorgans (cM), indicates
greater the linkage between the QTL and the molecular marker. The
extent to which two features are linked is often referred to as the
genetic distance. The genetic distance is also typically related to
the physical distance between the marker and the QTL, however,
certain biological phenomenon (including recombinational "hot
spots") can affect the relationship between physical distance and
genetic distance. Generally, the usefulness of a molecular marker
is determined by the genetic and physical distance between the
marker and the selectable trait of interest.
[0088] The method for determining the presence or absence of a QTL
associated with tolerance to protoporphyrinogen oxidase inhibitors
in soybean germplasm, comprises analyzing genomic DNA from a
soybean germplasm for the presence of at least one molecular
marker, wherein at least one molecular marker is linked to the QTL,
and wherein the QTL maps to soybean major linkage group L and N and
is associated with tolerance to protoporphyrinogen oxidase
inhibitors. The term "is associated with" in this context means
that the QTL associated with tolerance to protoporphyrinogen
oxidase inhibitors has been found, using marker-assisted analysis,
to be present in soybean plants showing tolerance to
protoporphyrinogen oxidase inhibitors in live bioassays as
described herein.
[0089] Generally, markers that map closer to the QTL mapped to
linkage group L and N and associated with tolerance to
protoporphyrinogen oxidase inhibitors are superior to markers that
map farther from the QTL. In some examples a marker used to
determine the presence or absence of a QTL mapping to soybean
linkage group L and/or N and associated with tolerance to
protoporphyrinogen oxidase inhibitors maps to soybean linkage group
L are SATT495, P10649C-3, SATT182, SATT388, SATT313, SATT613 (or
other markers above marker SATT613), S08102-1-Q1, S08103-1-Q1.
S08104-1-Q1, S08106-1-Q1, S08107-1-Q1, S08107-1-Q1, S08109-1-Q1,
S08110-1-Q1, S08111-1-Q1, S08115-2-Q1, S08117-1-Q1, S08119-1-Q1,
S08116-1-Q1, S08112-1-Q1, S08108-1-Q1, S08101-4-Q1, S08101-1-Q1,
S08101-2-Q1, and S08101-3-Q1, and those mapped to linkage group N
are Sat.sub.--379, SCT.sub.--195, SATT631, S60167-TB, SATT675,
SATT624, SATT080, and SATT387 (or other markers above SATT387). Any
marker assigned to soybean linkage group L and/or N and linked to a
marker disclosed herein as associated with tolerance to
protoporphyrinogen oxidase inhibitors may be used with the
invention. Generally, a linked marker is within 50 cM of the
referenced marker. Updated information regarding markers assigned
to soybean linkage group L and N may be found on the USDA's Soybase
website. Further, linkage group L is now formally referred to as
chromosome #19 and linkage group N is now formally referred to as
chromosome #3.
[0090] Markers flanking the QTL associated with tolerance to
protoporphyrinogen oxidase inhibitors are used in the
marker-assisted selection processes provided. The genomic DNA of
soybean germplasm is typically tested for the presence of at least
two of the foregoing molecular markers, one marker on each side of
the QTL. In some examples a QTL on linkage group L is used. Useful
markers on linkage group L include SATT495, P10649C-3, SATT182,
SATT388, SATT313, and SATT613, including markers above SATT613.
Markers that map close to SATT495, P10649C-3, SATT182, SATT388,
SATT313, and SATT613 can also be used. In some examples a QTL on
linkage group N is used. Useful markers on linkage group N include
Sat.sub.--379, SCT.sub.--195, SATT631, S60167-TB, SATT675, SATT624,
SATT080, and SATT387, including markers above SATT387. Markers that
map close to Sat.sub.--379, SCT.sub.--195, SATT631, S60167-TB,
SATT675, SATT624, SATT080, and SATT387 can also be used.
[0091] Fine mapping further isolated the location of the QTL to a
56 kb interval between marker S08117-1-Q1 and S08105-1-Q1 on
linkage group L. Accordingly, markers that map within the interval
defined by and including these markers are particularly useful for
selecting for this QTL. These markers include S08117-1-Q1,
S08119-1-Q1, S08118-1-Q1, S08116-1-Q1, S08101-1-Q1, S08112-1-Q1,
S08108-1-Q1, S08101-1-Q1, S08101-2-Q1, S08101-3-Q1, S08101-4-Q1,
and S08105-4-Q1.
[0092] Methods of introgressing protoporphyrinogen oxidase
inhibitor tolerance into non-tolerant or less-tolerant soybean
germplasm are provided. Any method for introgressing QTLs into
soybean plants can be used. In some examples, a first soybean
germplasm that contains tolerance to protoporphyrinogen oxidase
inhibitors derived from the QTL mapped to linkage group L and/or N
which is associated with tolerance to protoporphyrinogen oxidase
inhibitors and a second soybean germplasm that lacks tolerance to
protoporphyrinogen oxidase inhibitors derived from the QTL mapped
to linkage group L and/or N are provided. The first soybean plant
may be crossed with the second soybean plant to provide progeny
soybeans. Phenotypic and/or marker screening is then performed on
the progeny plants to determine the presence of tolerance to
protoporphyrinogen oxidase inhibitors derived from the QTL mapped
to linkage group L and/or N. Progeny that test positive for the
presence of tolerance to protoporphyrinogen oxidase inhibitors
derived from the QTL mapped to linkage group L and/or N can be
selected.
[0093] In some examples, the screening and selection are performed
by using marker-assisted selection using any marker or combination
of markers on major linkage group L and/or N provided. Any method
of identifying the presence or absence of these markers may be
used, including for example single-strand conformation polymorphism
(SSCP) analysis, base excision sequence scanning (BESS), RFLP
analysis, heteroduplex analysis, denaturing gradient gel
electrophoresis, and temperature gradient electrophoresis, allelic
PCR, ligase chain reaction direct sequencing, mini sequencing,
nucleic acid hybridization, or micro-array-type detection.
[0094] Systems, including automated systems for selecting plants
that comprise a marker of interest and/or for correlating presence
of the marker with tolerance are also provided. These systems can
include probes relevant to marker locus detection, detectors for
detecting labels on the probes, appropriate fluid handling elements
and temperature controllers that mix probes and templates and/or
amplify templates, and systems instructions that correlate label
detection to the presence of a particular marker locus or
allele.
[0095] Kits are also provided. For example, a kit can include
appropriate primers or probes for detecting tolerance associated
marker loci and instructions in using the primers or probes for
detecting the marker loci and correlating the loci with predicted
protoporphyrinogen oxidase inhibitor tolerance. The kits can
further include packaging materials for packaging the probes,
primers or instructions, controls such as control amplification
reactions that include probes, primers or template nucleic acids
for amplifications, molecular size markers, or the like.
[0096] Isolated nucleic acid fragments comprising a nucleic acid
sequence coding for soybean tolerance to protoporphyrinogen oxidase
inhibitors, are provided. The nucleic acid fragment comprises at
least a portion of nucleic acid belonging to linkage group L and/or
N. The nucleic acid fragment is capable of hybridizing under
stringent conditions to nucleic acid of a soybean cultivar tolerant
to protoporphyrinogen oxidase inhibitors containing a QTL
associated with protoporphyrinogen oxidase inhibitor tolerance that
is located on major linkage group L and/or N.
[0097] Vectors comprising such nucleic acid fragments, expression
products of such vectors expressed in a host compatible therewith,
antibodies to the expression product (both polyclonal and
monoclonal), and antisense nucleic acid to the nucleic acid
fragment are also provided.
[0098] Seed of a soybean produced by crossing a soybean variety
having protoporphyrinogen oxidase inhibitor tolerance QTL located
on major linkage group L and/or N in its genome with another
soybean variety, and progeny thereof, are provided.
Tolerance Markers and Favorable Alleles
[0099] In traditional linkage analysis, no direct knowledge of the
physical relationship of genes on a chromosome is required.
Mendel's first law is that factors of pairs of characteristics are
segregated, meaning that alleles of a diploid trait separate into
two gametes and then into different offspring. Classical linkage
analysis can be thought of as a statistical description of the
relative frequencies of cosegregation of different traits. Linkage
analysis, as described previously, is the well-characterized
descriptive framework of how traits are grouped together based upon
the frequency with which they segregate together. Because
chromosomal distance is approximately proportional to the frequency
of crossing over events between traits, there is an approximate
physical distance that correlates with recombination frequency.
[0100] Marker loci are traits, and can be assessed according to
standard linkage analysis by tracking the marker loci during
segregation. Thus, one cM is equal to a 1% chance that a marker
locus will be separated from another locus (which can be any other
trait, e.g., another marker locus, or another trait locus that
encodes a QTL), due to crossing over in a single generation. The
markers herein, e.g., for linkage group L: SATT495, P10649C-3,
SATT182, SATT388, SATT313, SATT613 (and other markers above
SATT613), S08102-1-Q1, S08103-1-Q1. S08104-1-Q1, S08106-1-Q1,
S08107-1-Q1, S08107-1-Q1, S08109-1-Q1, S08110-1-Q1, S08111-1-Q1,
S08115-2-Q1, S08117-1-Q1, S08119-1-Q1, S08116-1-Q1, S08112-1-Q1,
S08108-1-Q1, S08101-4-Q1, S08101-1-Q1, S08101-2-Q1, and
S08101-3-Q1, and for linkage group N: Sat.sub.--379, SCT.sub.--195,
SATT631, S60167-TB, SATT675, SATT624, SATT080, and SATT387 (and
other markers above SATT387), have been found to correlate with
tolerance or improved tolerance to protoporphyrinogen oxidase
inhibitors in soybean. This means that the markers are sufficiently
proximal to a tolerance trait that they can be used as a predictor
for the tolerance trait itself, using, for example, marker assisted
selection (MAS). Soybean plants or germplasm can be selected for
markers or marker alleles that positively correlate with tolerance,
without actually raising soybean and measuring for tolerance or
improved tolerance (or, contrawise, soybean plants can be selected
against if they possess markers that negatively correlate with
tolerance or improved tolerance. MAS is a powerful shortcut to
selecting for desired phenotypes and for introgressing desired
traits into cultivars of soybean (e.g., introgressing desired
traits into elite lines). MAS is easily adapted to high throughput
molecular analysis methods that can quickly screen large numbers of
plant or germplasm genetic material for the markers of interest and
is much more cost effective than raising and observing plants for
visible traits.
[0101] Any marker that is linked to a trait of interest (e.g., in
the present case, a tolerance or improved tolerance trait) can be
used as a marker for that trait. Thus, in addition to the markers
described herein, markers linked to the markers itemized herein can
also be used to predict the tolerance or improved tolerance trait.
Such linked markers are particularly useful when they are
sufficiently proximal to a given marker so that they display a low
recombination frequency with the given marker. Markers closely
linked to the markers on linkage group L and/or linkage group N are
also provided. Closely linked markers display a cross over
frequency with a given marker of about 10% or less (the given
marker is within 10 cM of the given marker). Put another way,
closely linked loci co-segregate at least 90% of the time.
[0102] Marker loci are especially useful when they are closely
linked to target loci (e.g., QTL for tolerance, or, alternatively,
simply other marker loci, such as those identified herein, that are
linked to such QTL) for which they are being used as markers. A
marker more closely linked to a target locus is a better indicator
for the target locus (due to the reduced cross-over frequency
between the target locus and the marker). Thus, in one example,
closely linked loci such as a marker locus and a second locus
(e.g., a given marker or a QTL) display an inter-locus cross-over
frequency of about 10% or less, about 9% or less, about 8% or less,
about 7% or less, about 6% or less, about 5% or less, about 4% or
less, about 3% or less, or about 2% or less. In some examples, the
relevant loci (e.g., a marker locus and a target locus such as a
QTL) display a recombination a frequency of about 1% or less, e.g.,
about 0.75% or less, about 0.5% or less, or about 0.25% or less.
Thus, the loci are about 10 cM, 9 cM, 8 cM, 7 cM, 6 cM, 5 cM, 4 cM,
3 cM, 2 cM, 1 cM, 0.75 cM, 0.5 cM or 0.25 cM or less apart. Put
another way, two loci that are localized to the same chromosome,
and at such a distance that recombination between the two loci
occurs at a frequency of no more than 10% (e.g., about 9%, 8%, 7%,
6%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.25%, or less) are said to be
proximal to each other.
[0103] When referring to the relationship between two genetic
elements, such as a genetic element contributing to tolerance and a
proximal marker, "coupling" phase linkage indicates the state where
the "favorable" allele at the tolerance locus is physically
associated on the same chromosome strand as the "favorable" allele
of the respective linked marker locus. In coupling phase, both
favorable alleles are inherited together by progeny that inherit
that chromosome strand. In "repulsion" phase linkage, the
"favorable" allele at the locus of interest (e.g., a QTL for
tolerance) is physically linked with an "unfavorable" allele at the
proximal marker locus, and the two "favorable" alleles are not
inherited together (i.e., the two loci are "out of phase" with each
other).
[0104] Optionally, one, two, three or more favorable allele(s) are
identified in, or introgressed into the plant. Many marker alleles
can be selected for or against during MAS. Plants or germplasm are
identified that have at least one such favorable allele that
positively correlates with tolerance or improved tolerance.
However, it is useful for exclusionary purposes during breeding to
identify alleles that negatively correlate with tolerance, to
eliminate such plants or germplasm from subsequent rounds of
breeding.
[0105] The identification of favorable marker alleles is
germplasm-specific. The determination of which marker alleles
correlate with tolerance (or non-tolerance) is determined for the
particular germplasm under study. One of skill recognizes that
methods for identifying the favorable alleles are routine and well
known, and furthermore, that the identification and use of such
favorable alleles is well within the scope of the invention.
[0106] Amplification primers for amplifying marker loci and
suitable marker probes to detect marker loci or to genotype SNP
alleles are provided. Optionally, other sequences to either side of
the given primers can be used in place of the given primers, so
long as the primers can amplify a region that includes the allele
to be detected. Further, it will be appreciated that the precise
probe to be used for detection can vary, e.g., any probe that can
identify the region of a marker amplicon to be detected can be
substituted for those examples provided herein. The configuration
of the amplification primers and detection probes can, of course,
vary. Thus, the invention is not limited to the primers and probes
specifically recited herein.
[0107] In some examples the presence of marker loci is directly
detected in unamplified genomic DNA by performing a Southern blot
on a sample of genomic DNA using probes to the marker loci.
Procedures for performing Southern blotting, amplification (PCR,
LCR, or the like) and many other nucleic acid detection methods are
well established and are taught, e.g., in Sambrook et al.,
Molecular Cloning-A Laboratory Manual (3d ed.), Vol. 1-3, Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 2000
("Sambrook"); Current Protocols in Molecular Biology, F. M. Ausubel
et al., eds., Current Protocols, a joint venture between Greene
Publishing Associates, Inc. and John Wiley & Sons, Inc.,
(supplemented through 2002) ("Ausubel")) and PCR Protocols A Guide
to Methods and Applications (Innis et al. eds) Academic Press Inc.
San Diego, Calif. (1990) (Innis). Additional details regarding
detection of nucleic acids in plants can also be found, e.g., in
Plant Molecular Biology (1993) Croy (ed.) BIOS Scientific
Publishers, Inc.
[0108] Separate detection probes can also be omitted in
amplification/detection methods, e.g., by performing a real time
amplification reaction that detects product formation by
modification of the relevant amplification primer upon
incorporation into a product, incorporation of labeled nucleotides
into an amplicon, or by monitoring changes in molecular rotation
properties of amplicons as compared to unamplified precursors
(e.g., by fluorescence polarization).
[0109] Typically, molecular markers are detected by any established
method available, including, without limitation, allele specific
hybridization (ASH) or other methods for detecting single
nucleotide polymorphisms (SNP), amplified fragment length
polymorphism (AFLP) detection, amplified variable sequence
detection, randomly amplified polymorphic DNA (RAPD) detection,
restriction fragment length polymorphism (RFLP) detection,
self-sustained sequence replication detection, simple sequence
repeat (SSR) detection, single-strand conformation polymorphisms
(SSCP) detection, isozyme markers detection, or the like. While the
exemplary markers provided in the tables herein are either SSR or
SNP (ASH) markers, any of the aforementioned marker types can be
employed to identify chromosome segments encompassing genetic
element that contribute to superior agronomic performance (e.g.,
tolerance or improved tolerance).
[0110] In another example, the presence or absence of a molecular
marker is determined by nucleotide sequencing of the polymorphic
marker region. This method is readily adapted to high throughput
analysis as are the other methods noted above, e.g., using
available high throughput sequencing methods such as sequencing by
hybridization.
[0111] In general, the majority of genetic markers rely on one or
more property of nucleic acids for their detection. For example,
some techniques for detecting genetic markers utilize hybridization
of a probe nucleic acid to nucleic acids corresponding to the
genetic marker (e.g., amplified nucleic acids produced using
genomic soybean DNA as a template). Hybridization formats,
including but not limited to solution phase, solid phase, mixed
phase, or in situ hybridization assays are useful for allele
detection. An extensive guide to the hybridization of nucleic acids
is found in Tijssen (1993) Laboratory Techniques in Biochemistry
and Molecular Biology--Hybridization with Nucleic Acid Probes
Elsevier, New York, as well as in Sambrook, and Ausubel.
[0112] For example, markers that comprise restriction fragment
length polymorphisms (RFLP) are detected, e.g., by hybridizing a
probe which is typically a sub-fragment (or a synthetic
oligonucleotide corresponding to a sub-fragment) of the nucleic
acid to be detected to restriction digested genomic DNA. The
restriction enzyme is selected to provide restriction fragments of
at least two alternative (or polymorphic) lengths in different
individuals or populations. Determining one or more restriction
enzyme that produces informative fragments for each cross is a
simple procedure. After separation by length in an appropriate
matrix (e.g., agarose, polyacrylamide, etc.) and transfer to a
membrane (e.g., nitrocellulose, nylon, etc.), the labeled probe is
hybridized under conditions which result in equilibrium binding of
the probe to the target followed by removal of excess probe by
washing.
[0113] Nucleic acid probes to the marker loci can be cloned and/or
synthesized. Any suitable label can be used with a probe.
Detectable labels suitable for use with nucleic acid probes
include, for example, any composition detectable by spectroscopic,
radioisotopic, photochemical, biochemical, immunochemical,
electrical, optical or chemical means. Useful labels include biotin
for staining with labeled streptavidin conjugate, magnetic beads,
fluorescent dyes, radiolabels, enzymes, and colorimetric labels.
Other labels include ligands, which bind to antibodies labeled with
fluorophores, chemiluminescent agents, and enzymes. A probe can
also constitute radiolabelled PCR primers that are used to generate
a radiolabelled amplicon. Methods and reagents for labeling nucleic
acids and corresponding detection strategies can be found, e.g., in
Haugland (1996) Handbook of Fluorescent Probes and Research
Chemicals Sixth Edition by Molecular Probes, Inc. (Eugene Oreg.);
or Haugland (2001) Handbook of Fluorescent Probes and Research
Chemicals Eighth Edition by Molecular Probes, Inc. (Eugene
Oreg.).
Amplification-Based Detection Methods
[0114] PCR, RT-PCR and LCR are in particularly broad use as
amplification and amplification-detection methods for amplifying
nucleic acids of interest (e.g., those comprising marker loci),
facilitating detection of the markers. Details regarding the use of
these and other amplification methods can be found in any of a
variety of standard texts, including, e.g., Sambrook, Ausubel,
Berger and Croy, supra. Many available biology texts also have
extended discussions regarding PCR and related amplification
methods. Any RNA can be converted into a double stranded DNA
suitable for restriction digestion, PCR expansion and sequencing
using reverse transcriptase and a polymerase ("Reverse
Transcription-PCR, or "RT-PCR"). See also Ausubel and Sambrook,
supra.
Real Time Amplification/Detection Methods
[0115] In one aspect, real time PCR or LCR is performed on the
amplification mixtures described herein, e.g., using molecular
beacons or TaqMan.TM. probes. A molecular beacon (MB) is an
oligonucleotide or peptide nucleic acid (PNA) which, under
appropriate hybridization conditions, self-hybridizes to form a
stem and loop structure. The MB has a label and a quencher at the
termini of the oligonucleotide or PNA; thus, under conditions that
permit intra-molecular hybridization, the label is typically
quenched (or at least altered in its fluorescence) by the quencher.
Under conditions where the MB does not display intra-molecular
hybridization (e.g., when bound to a target nucleic acid, e.g., to
a region of an amplicon during amplification), the MB label is
unquenched and signal is detected. Standard methods of making and
using MBs are known and MBs and reagents are commercially
available. See also, e.g., Leone et al. (1995) "Molecular beacon
probes combined with amplification by NASBA enable homogenous
real-time detection of RNA." Nucleic Acids Res. 26:2150-2155; Tyagi
and Kramer (1996) "Molecular beacons: probes that fluoresce upon
hybridization" Nature Biotechnology 14:303-308; Blok and Kramer
(1997) "Amplifiable hybridization probes containing a molecular
switch" Mol Cell Probes 11:187-194; Hsuih et al. (1997) "Novel,
ligation-dependent PCR assay for detection of hepatitis C in serum"
J Clin Microbiol 34:501-507; Kostrikis et al. (1998) "Molecular
beacons: spectral genotyping of human alleles" Science
279:1228-1229; Sokol et al. (1998) "Real time detection of DNA:RNA
hybridization in living cells" Proc. Natl. Acad. Sci. U.S.A.
95:11538-11543; Tyagi et al. (1998) "Multicolor molecular beacons
for allele discrimination" Nature Biotechnology 16:49-53; Bonnet et
al. (1999) "Thermodynamic basis of the chemical specificity of
structured DNA probes" Proc. Natl. Acad. Sci. U.S.A. 96:6171-6176;
Fang et al. (1999) "Designing a novel molecular beacon for
surface-immobilized DNA hybridization studies" J. Am. Chem. Soc.
121:2921-2922; Marras et al. (1999) "Multiplex detection of
single-nucleotide variation using molecular beacons" Genet. Anal.
Biomol. Eng. 14:151-156; and Vet et al. (1999) "Multiplex detection
of four pathogenic retroviruses using molecular beacons" Proc.
Natl. Acad. Sci. U.S.A. 96:6394-6399. See also, e.g., U.S. Pat. No.
5,925,517 (Jul. 20, 1999) to Tyagi et al. entitled "Detectably
labeled dual conformation oligonucleotide probes, assays and kits;"
U.S. Pat. No. 6,150,097 to Tyagi et al. (Nov. 21, 2000) entitled
"Nucleic acid detection probes having non-FRET fluorescence
quenching and kits and assays including such probes" and U.S. Pat.
No. 6,037,130 to Tyagi et al. (Mar. 14, 2000), entitled
"Wavelength-shifting probes and primers and their use in assays and
kits."
[0116] PCR detection and quantification using dual-labeled
fluorogenic oligonucleotide probes can be done, using for example
TaqMan.TM. probes. These probes are composed of short (e.g., 20-25
base) oligodeoxynucleotides that are labeled with two different
fluorescent dyes. On the 5' terminus of each probe is a reporter
dye, and on the 3' terminus of each probe a quenching dye is found.
The oligonucleotide probe sequence is complementary to an internal
target sequence present in a PCR amplicon. When the probe is
intact, energy transfer occurs between the two fluorophores and
emission from the reporter is quenched by the quencher by FRET.
During the extension phase of PCR, the probe is cleaved by 5'
nuclease activity of the polymerase used in the reaction, thereby
releasing the reporter from the oligonucleotide-quencher and
producing an increase in reporter emission intensity. Accordingly,
TaqMan.TM. probes are oligonucleotides that have a label and a
quencher, where the label is released during amplification by the
exonuclease action of the polymerase used in amplification. This
provides a real time measure of amplification during synthesis. A
variety of TaqMan.TM. reagents are commercially available, e.g.,
from Applied Biosystems (Division Headquarters in Foster City,
Calif.) as well as from a variety of specialty vendors such as
Biosearch Technologies (e.g., black hole quencher probes).
Additional Details Regarding Amplified Variable Sequences, SSR,
AFLP ASH, SNPs and Isozyme Markers
[0117] Amplified variable sequences refer to amplified sequences of
the plant genome, which exhibit high nucleic acid residue
variability between members of the same species. All organisms have
variable genomic sequences and each organism (with the exception of
a clone) has a different set of variable sequences. Once
identified, the presence of specific variable sequence can be used
to predict phenotypic traits. Typically, DNA from the plant serves
as a template for amplification with primers that flank a variable
sequence of DNA. The variable sequence is amplified and then
sequenced.
[0118] Alternatively, self-sustained sequence replication can be
used to identify genetic markers. Self-sustained sequence
replication refers to a method of nucleic acid amplification using
target nucleic acid sequences which are replicated exponentially in
vitro under substantially isothermal conditions by using three
enzymatic activities involved in retroviral replication: (1)
reverse transcriptase, (2) Rnase H, and (3) a DNA-dependent RNA
polymerase (Guatelli et al. (1990) Proc Natl Acad Sci USA 87:1874).
By mimicking the retroviral strategy of RNA replication by means of
cDNA intermediates, this reaction accumulates cDNA and RNA copies
of the original target.
[0119] Amplified fragment length polymophisms (AFLP) can also be
used as genetic markers (Vos et al. (1995) Nucl Acids Res 23:4407).
The phrase "amplified fragment length polymorphism" refers to
selected restriction fragments, which are amplified before or after
cleavage by a restriction endonuclease. The amplification step
allows easier detection of specific restriction fragments. AFLP
allows the detection large numbers of polymorphic markers and has
been used for genetic mapping of plants (Becker et al. (1995) Mol
Gen Genet 249:65; and Meksem et al. (1995) Mol Gen Genet
249:74).
[0120] Allele-specific hybridization (ASH) can be used to identify
the genetic markers. ASH technology is based on the stable
annealing of a short, single-stranded, oligonucleotide probe to a
completely complementary single-strand target nucleic acid.
Detection is via an isotopic or non-isotopic label attached to the
probe.
[0121] For each polymorphism, two or more different ASH probes are
designed to have identical DNA sequences except at the polymorphic
nucleotides. Each probe will have exact homology with one allele
sequence so that the range of probes can distinguish all the known
alternative allele sequences. Each probe is hybridized to the
target DNA. With appropriate probe design and hybridization
conditions, a single-base mismatch between the probe and target DNA
will prevent hybridization. In this manner, only one of the
alternative probes will hybridize to a target sample that is
homozygous or homogenous for an allele. Samples that are
heterozygous or heterogeneous for two alleles will hybridize to
both of two alternative probes.
[0122] ASH markers are used as dominant markers where the presence
or absence of only one allele is determined from hybridization or
lack of hybridization by only one probe. The alternative allele may
be inferred from the lack of hybridization. ASH probe and target
molecules are optionally RNA or DNA; the target molecules are any
length of nucleotides beyond the sequence that is complementary to
the probe; the probe is designed to hybridize with either strand of
a DNA target; the probe ranges in size to conform to variously
stringent hybridization conditions, etc.
[0123] PCR allows the target sequence for ASH to be amplified from
low concentrations of nucleic acid in relatively small volumes.
Otherwise, the target sequence from genomic DNA is digested with a
restriction endonuclease and size separated by gel electrophoresis.
Hybridizations typically occur with the target sequence bound to
the surface of a membrane or, as described in U.S. Pat. No.
5,468,613, the ASH probe sequence may be bound to a membrane. In
one example, ASH data are typically obtained by amplifying nucleic
acid fragments (amplicons) from genomic DNA using PCR, transferring
the amplicon target DNA to a membrane in a dot-blot format,
hybridizing a labeled oligonucleotide probe to the amplicon target,
and observing the hybridization dots by autoradiography.
[0124] Single nucleotide polymorphisms (SNP) are markers that
consist of a shared sequence differentiated on the basis of a
single nucleotide. Typically, this distinction is detected by
differential migration patterns of an amplicon comprising the SNP
on, e.g., an acrylamide gel. However, alternative modes of
detection, such as hybridization, e.g., ASH, or RFLP analysis are
also appropriate.
[0125] Isozyme markers can be employed as genetic markers, e.g., to
track markers other than the tolerance markers herein, or to track
isozyme markers linked to the markers herein. Isozymes are multiple
forms of enzymes that differ from one another in their amino acid
sequence, and therefore their nucleic acid sequences. Some isozymes
are multimeric enzymes containing slightly different subunits.
Other isozymes are either multimeric or monomeric but have been
cleaved from the proenzyme at different sites in the amino acid
sequence. Isozymes can be characterized and analyzed at the protein
level, or alternatively, isozymes, which differ at the nucleic acid
level, can be determined. In such cases any of the nucleic acid
based methods described herein can be used to analyze isozyme
markers.
Probe/Primer Synthesis Methods
[0126] In general, synthetic methods for making oligonucleotides,
including probes, primers, molecular beacons, PNAs, LNAs (locked
nucleic acids), etc., are well known. For example, oligonucleotides
can be synthesized chemically according to the solid phase
phosphoramidite triester method described by Beaucage and Caruthers
(1981) Tetrahedron Letts 22:1859-1862, e.g., using a commercially
available automated synthesizer, e.g., as described in
Needham-VanDevanter et al. (1984) Nucleic Acids Res. 12:6159-6168.
Oligonucleotides, including modified oligonucleotides can also be
ordered from a variety of commercial sources known to persons of
skill. There are many commercial providers of oligo synthesis
services, and thus this is a broadly accessible technology. Any
nucleic acid can be custom ordered from any of a variety of
commercial sources, such as The Midland Certified Reagent Company
(mcrc@oligos.com), The Great American Gene Company (genco.com),
ExpressGen Inc. (expressgen.com), Operon Technologies Inc.
(Alameda, Calif.) and many others. Similarly, PNAs can be custom
ordered from any of a variety of sources, such as PeptidoGenic
(pkim@ccnet.com), HTI Bio-products, inc. (htibio.com), BMA
Biomedicals Ltd (U.K.), Bio. Synthesis, Inc., and many others.
In Silico Marker Detection
[0127] In alternative embodiments, in silico methods can be used to
detect the marker loci of interest. For example, the sequence of a
nucleic acid comprising the marker locus of interest can be stored
in a computer. The desired marker locus sequence or its homolog can
be identified using an appropriate nucleic acid search algorithm as
provided by, for example, in such readily available programs as
BLAST, or even simple word processors.
Amplification Primers for Marker Detection
[0128] In some examples, molecular markers are detected using a
suitable PCR-based detection method, where the size or sequence of
the PCR amplicon is indicative of the absence or presence of the
marker (e.g., a particular marker allele). In these types of
methods, PCR primers are hybridized to the conserved regions
flanking the polymorphic marker region. Suitable primers can be
designed using any suitable method. It is not intended that the
invention be limited to any particular primer or primer pair. For
example, primers can be designed using any suitable software
program, such as LASERGENE.RTM..
[0129] In some examples, the primers are radiolabelled, or labeled
by any suitable means (e.g., using a non-radioactive fluorescent
tag), to allow for rapid visualization of the different size
amplicons following an amplification reaction without any
additional labeling step or visualization step. In some examples,
the primers are not labeled, and the amplicons are visualized
following their size resolution, e.g., following agarose gel
electrophoresis. In some examples, ethidium bromide staining of the
PCR amplicons following size resolution allows visualization of the
different size amplicons.
[0130] The primers used to amplify the marker loci and alleles
herein are not limited to amplifying the entire region of the
relevant locus. In some examples, marker amplification produces an
amplicon at least 20 nucleotides in length, or alternatively, at
least 50 nucleotides in length, or alternatively, at least 100
nucleotides in length, or alternatively, at least 200 nucleotides
in length, or up to and including the full length of the
amplicon.
Marker Assisted Selection and Breeding of Plants
[0131] A primary motivation for development of molecular markers in
crop species is the potential for increased efficiency in plant
breeding through marker assisted selection (MAS). Genetic markers
are used to identify plants that contain a desired genotype at one
or more loci, and that are expected to transfer the desired
genotype, along with a desired phenotype to their progeny. Genetic
markers can be used to identify plants that contain a desired
genotype at one locus, or at several unlinked or linked loci (e.g.,
a haplotype), and that would be expected to transfer the desired
genotype, along with a desired phenotype to their progeny. Means to
identify plants, particularly soybean plants, that are tolerant, or
that exhibit improved tolerance to protoporphyrinogen oxidase
inhibitors are provided, for example by identifying plants having a
specified marker loci e.g., for linkage group L: SATT495,
P10649C-3, SATT182, SATT388, SATT313, SATT613 (and other markers
above SATT613), S08102-1-Q1, S08103-1-Q1. S08104-1-Q1, S08106-1-Q1,
S08107-1-Q1, S08107-1-Q1, S08109-1-Q1, S08110-1-Q1, S08111-1-Q1,
S08115-2-Q1, S08117-1-Q1, S08119-1-Q1, S08116-1-Q1, S08112-1-Q1,
S08108-1-Q1, S08101-4-Q1, S08101-1-Q1, S08101-2-Q1, and
S08101-3-Q1, and/or for linkage group N: Sat.sub.--379,
SCT.sub.--195, SATT631, S60167-TB, SATT675, SATT624, SATT080, and
SATT387 (and other markers above SATT387). Similarly, by
identifying plants lacking the desired marker locus, non-tolerant
or less tolerant plants can be identified, and, e.g., eliminated
from subsequent crosses. Similarly, these marker loci can be
introgressed into any desired genomic background, germplasm, plant,
line, variety, etc., as part of an overall MAS breeding program
designed to enhance soybean yield.
[0132] In general, the application of MAS uses the identification
of a population of tolerant plants and genetic mapping of the
tolerance trait. Polymorphic loci in the vicinity of the mapped
tolerance trait are chosen as potential tolerance markers.
Typically, a marker locus closest to the tolerance locus is a
preferred marker. Linkage analysis is then used to determine which
polymorphic marker allele sequence demonstrates a statistical
likelihood of co-segregation with the tolerant phenotype (thus, a
"tolerance marker allele"). Following identification of a marker
allele for co-segregation with the tolerance allele, it is possible
to use this marker for rapid, accurate screening of plant lines for
the tolerance allele without the need to grow the plants through
their life cycle and await phenotypic evaluations, and furthermore,
permits genetic selection for the particular tolerance allele even
when the molecular identity of the actual tolerance QTL is
anonymous. Tissue samples can be taken, for example, from the first
leaf of the plant and screened with the appropriate molecular
marker, and within days it is determined which progeny will
advance. Linked markers also remove the impact of environmental
factors that can often influence phenotypic expression.
[0133] After a desired phenotype (e.g., tolerance to
protoporphyrinogen oxidase inhibitors) and a polymorphic
chromosomal marker locus are determined to cosegregate, the
polymorphic marker locus can be used to select for marker alleles
that segregate with the desired tolerance phenotype. This general
process is typically called marker-assisted selection (MAS). In
brief, a nucleic acid corresponding to the marker nucleic acid is
detected in a biological sample from a plant to be selected. This
detection can take the form of hybridization of a probe nucleic
acid to a marker allele or amplicon thereof, e.g., using
allele-specific hybridization, Southern analysis, northern
analysis, in situ hybridization, hybridization of primers followed
by PCR amplification of a region of the marker, or the like. After
the presence (or absence) of a particular marker in the biological
sample is verified, the plant is selected, e.g., used to make
progeny plants by selective breeding.
[0134] Soybean plant breeders desire combinations of tolerance loci
with genes for high yield and other desirable traits to develop
improved soybean varieties. Screening large numbers of samples by
non-molecular methods (e.g., trait evaluation in soybean plants)
can be expensive, time consuming, and unreliable. Use of the
polymorphic markers described herein genetically linked to
tolerance loci provide effective methods for selecting tolerant
varieties in breeding programs. For example, one advantage of
marker-assisted selection over field evaluations for tolerance is
that MAS can be done at any time of year, regardless of the growing
season. Moreover, environmental effects are largely irrelevant to
marker-assisted selection.
[0135] When a population is segregating for multiple loci affecting
one or multiple traits, e.g., multiple loci involved in tolerance,
or multiple loci each involved in tolerance or tolerance to
different herbicides, the efficiency of MAS compared to phenotypic
screening becomes even greater, because all of the loci can be
evaluated in the lab together from a single sample of DNA. In the
present instance, for linkage group L: SATT495, P10649C-3, SATT182,
SATT388, SATT313, SATT613 (or other markers above SATT613),
S08102-1-Q1, S08103-1-Q1. S08104-1-Q1, S08106-1-Q1, S08107-1-Q1,
S08107-1-Q1, S08109-1-Q1, S08110-1-Q1, S08111-1-Q1, S08115-2-Q1,
S08117-1-Q1, S08119-1-Q1, S08116-1-Q1, S08112-1-Q1, S08108-1-Q1,
S08101-4-Q1, S08101-1-Q1, S08101-2-Q1, and S08101-3-Q1; and for
linkage group N: Sat.sub.--379, SCT.sub.--195, SATT631, S60167-TB,
SATT675, SATT624, SATT080, and SATT387 (or other markers above
SATT387) markers, and markers for other traits, transgenes, and/or
loci can be assayed simultaneously or sequentially in a single
sample or population of samples.
[0136] Another use of MAS in plant breeding is to assist the
recovery of the recurrent parent genotype by backcross breeding.
Backcross breeding is the process of crossing a progeny back to one
of its parents or parent lines. Backcrossing is usually done for
the purpose of introgressing one or a few loci from a donor parent
(e.g., a parent comprising desirable tolerance marker loci) into an
otherwise desirable genetic background from the recurrent parent
(e.g., an otherwise high yielding soybean line). The more cycles of
backcrossing that are done, the greater the genetic contribution of
the recurrent parent to the resulting introgressed variety. This is
often necessary, because tolerant plants may be otherwise
undesirable, e.g., due to low yield, low fecundity, or the like. In
contrast, strains which are the result of intensive breeding
programs may have excellent yield, fecundity or the like, merely
being deficient in one desired trait such as tolerance to
protoporphyrinogen oxidase inhibitors.
[0137] The presence and/or absence of a particular genetic marker
or allele, e.g., for linkage group L: SATT495, P10649C-3, SATT182,
SATT388, SATT313, SATT613 (including markers above SATT613),
S08102-1-Q1, S08103-1-Q1. S08104-1-Q1, S08106-1-Q1, S08107-1-Q1,
S08107-1-Q1, S08109-1-Q1, S08110-1-Q1, S08111-1-Q1, S08115-2-Q1,
S08117-1-Q1, S08119-1-Q1, S08116-1-Q1, S08112-1-Q1, S08108-1-Q1,
S08101-4-Q1, S08101-1-Q1, S08101-2-Q1, and S08101-3-Q1, and for
linkage group N: Sat.sub.--379, SCT 195, SATT631, S60167-TB,
SATT675, SATT624, SATT080, and SATT387 (including markers above
SATT387) in the genome of a plant exhibiting a preferred phenotypic
trait is made by any method noted herein. If the nucleic acids from
the plant are positive for a desired genetic marker, the plant can
be self fertilized to create a true breeding line with the same
genotype, or it can be crossed with a plant with the same marker or
with other desired characteristics to create a sexually crossed
hybrid generation.
Introgression of Favorable Alleles--Efficient Crossing of Tolerance
Markers into Other Lines
[0138] One application of MAS is to use the tolerance or improved
tolerance markers to increase the efficiency of an introgression or
backcrossing effort aimed at introducing a tolerance QTL into a
desired (typically high yielding) background. In marker assisted
backcrossing of specific markers (and associated QTL) from a donor
source, e.g., to an elite genetic background, one selects among
progeny or backcross progeny for the donor trait.
[0139] Thus, the markers and methods can be utilized to guide
marker assisted selection or breeding of soybean varieties with the
desired complement (set) of allelic forms of chromosome segments
associated with herbicide tolerance as well as markers associated
with superior agronomic performance (tolerance, along with any
other available markers for yield, disease tolerance, etc.). Any of
the disclosed marker alleles can be introduced into a soybean line
via introgression, by traditional breeding (or introduced via
transformation, or both) to yield a soybean plant with superior
agronomic performance. The number of alleles associated with
tolerance that can be introduced or be present in a soybean plant
ranges from 1 to the number of alleles disclosed herein, each
integer of which is incorporated herein as if explicitly
recited.
[0140] Methods of making a progeny soybean plant and these progeny
soybean plants having tolerance to PPO inhibitors are provided.
These methods comprise crossing a first parent soybean plant with a
second soybean plant and growing the female soybean plant under
plant growth conditions to yield soybean plant progeny. Such
soybean plant progeny can be assayed for alleles associated with
tolerance and, thereby, the desired progeny selected. Such progeny
plants or seed can be sold commercially for soybean production,
used for food, processed to obtain a desired constituent of the
soybean, or further utilized in subsequent rounds of breeding. At
least one of the first or second soybean plants is a soybean plant
comprising at least one of the allelic forms of the markers
provided, such that the progeny are capable of inheriting the
allele.
[0141] Inheritance of the desired tolerance allele can be traced,
such as from progenitor or descendant lines in the subject soybean
plants pedigree such that the number of generations separating the
soybean plants being subject to the methods will generally be from
1 to 20, commonly 1 to 5, and typically 1, 2, or 3 generations of
separation, and quite often a direct descendant or parent of the
soybean plant will be subject to the method (i.e., 1 generation of
separation).
Methods for Identifying Protoporphyrinogen Oxidase Inhibitor
Tolerant Soybean Plants
[0142] Experienced plant breeders can recognize tolerant soybean
plants in the field, and can select the tolerant individuals or
populations for breeding purposes or for propagation. In this
context, the plant breeder recognizes tolerant, and non-tolerant
soybean plants.
[0143] The screening and selection may also be performed by
exposing plants containing said progeny germplasm to
protoporphyrinogen oxidase inhibitors in an assay and selecting
those plants showing tolerance to protoporphyrinogen oxidase
inhibitors as containing soybean germplasm into which germplasm
having tolerance to protoporphyrinogen oxidase inhibitors derived
from the QTL mapped to linkage group L and/or N has been
introgressed. The live assay may be any such assay known to the
art, e.g., Taylor-Lovell et al. (2001) Weed Tech 15:95-102.
[0144] However, plant tolerance is a phenotypic spectrum consisting
of extremes of high tolerance to non-tolerance with a continuum of
intermediate tolerance phenotypes. Evaluation of these intermediate
phenotypes using reproducible assays are of value to scientists who
seek to identify genetic loci that impart tolerance, conduct marker
assisted selection for tolerant population, and for introgression
techniques to breed a tolerance trait into an elite soybean line,
for example. Describing the continuum of tolerance can be done
using any known scoring system or derivative thereof, including the
scoring systems described in Examples 1-4.
Automated Detection/Correlation Systems
[0145] In some examples, the methods include an automated system
for detecting markers and or correlating the markers with a desired
phenotype (e.g., tolerance). Thus, a typical system can include a
set of marker probes or primers configured to detect at least one
favorable allele of one or more marker locus associated with
tolerance or improved tolerance to protoporphyrinogen oxidase
inhibitors. These probes or primers are configured to detect the
marker alleles noted in the tables and examples herein, e.g., using
any available allele detection format, e.g., solid or liquid phase
array based detection, microfluidic-based sample detection,
etc.
[0146] In some examples markers involving linkage group L are used.
In some examples a marker closely linked to the marker locus of
SATT495, P10649C-3, SATT182, SATT388, SATT313, SATT613,
S08102-1-Q1, S08103-1-Q1. S08104-1-Q1, S08106-1-Q1, S08107-1-Q1,
S08107-1-Q1, S08109-1-Q1, S08110-1-Q1, S08111-1-Q1, S08115-2-Q1,
S08117-1-Q1, S08119-1-Q1, S08116-1-Q1, S08112-1-Q1, S08108-1-Q1,
S08101-4-Q1, S08101-1-Q1, S08101-2-Q1, and S08101-3-Q1 is used, and
the probe set is configured to detect the closely linked marker(s).
In some examples, the marker locus is SATT495, P10649C-3, SATT182,
SATT388, SATT313, SATT613 (or another marker above SATT613),
S08102-1-Q1, S08103-1-Q1. S08104-1-Q1, S08106-1-Q1, S08107-1-Q1,
S08107-1-Q1, S08109-1-Q1, S08110-1-Q1, S08111-1-Q1, S08115-2-Q1,
S08117-1-Q1, S08119-1-Q1, S08116-1-Q1, S08112-1-Q1, S08108-1-Q1,
S08101-4-Q1, S08101-1-Q1, S08101-2-Q1, and S08101-3-Q1 and the
probe set is configured to detect the locus. Similarly, alleles of
SATT495, P10649C-3, SATT182, SATT388, SATT313, and SATT613 can be
detected.
[0147] In some examples markers involving linkage group N are used.
In some examples a marker closely linked to the marker locus of
Sat.sub.--379, SCT.sub.--195, SATT631, S60167-TB, SATT675, SATT624,
SATT080, and SATT387 (or another marker above SATT387) is used, and
the probe set is configured to detect the closely linked marker(s).
In some examples the marker locus is Sat.sub.--379, SCT.sub.--195,
SATT631, S60167-TB, SATT675, SATT624, SATT080, and SATT387 and the
probe set is configured to detect the locus. Similarly, alleles of
Sat.sub.--379, SCT.sub.--195, SATT631, S60167-TB, SATT675, SATT624,
SATT080, and SATT387 can be detected.
[0148] The typical system includes a detector that is configured to
detect one or more signal outputs from the set of marker probes or
primers, or amplicon thereof, thereby identifying the presence or
absence of the allele. A wide variety of signal detection apparatus
are available, including photo multiplier tubes,
spectrophotometers, CCD arrays, arrays and array scanners, scanning
detectors, phototubes and photodiodes, microscope stations,
galvo-scans, microfluidic nucleic acid amplification detection
appliances and the like. The precise configuration of the detector
will depend, in part, on the type of label used to detect the
marker allele, as well as the instrumentation that is most
conveniently obtained for the user. Detectors that detect
fluorescence, phosphorescence, radioactivity, pH, charge,
absorbance, luminescence, temperature, magnetism or the like can be
used. Typical detector examples include light (e.g., fluorescence)
detectors or radioactivity detectors. For example, detection of a
light emission (e.g., a fluorescence emission) or other probe label
is indicative of the presence or absence of a marker allele.
Fluorescent detection is generally used for detection of amplified
nucleic acids (however, upstream and/or downstream operations can
also be performed on amplicons, which can involve other detection
methods). In general, the detector detects one or more label (e.g.,
light) emission from a probe label, which is indicative of the
presence or absence of a marker allele. The detector(s) optionally
monitors one or a plurality of signals from an amplification
reaction. For example, the detector can monitor optical signals
which correspond to "real time" amplification assay results.
[0149] System instructions that correlate the presence or absence
of the favorable allele with the predicted tolerance are also
provided. For example, the instructions can include at least one
look-up table that includes a correlation between the presence or
absence of the favorable alleles and the predicted tolerance or
improved tolerance. The precise form of the instructions can vary
depending on the components of the system, e.g., they can be
present as system software in one or more integrated unit of the
system (e.g., a microprocessor, computer or computer readable
medium), or can be present in one or more units (e.g., computers or
computer readable media) operably coupled to the detector. As
noted, in one typical example, the system instructions include at
least one look-up table that includes a correlation between the
presence or absence of the favorable alleles and predicted
tolerance or improved tolerance. The instructions also typically
include instructions providing a user interface with the system,
e.g., to permit a user to view results of a sample analysis and to
input parameters into the system.
[0150] The system typically includes components for storing or
transmitting computer readable data representing or designating the
alleles detected by the methods, e.g., in an automated system. The
computer readable media can include cache, main, and storage memory
and/or other electronic data storage components (hard drives,
floppy drives, storage drives, etc.) for storage of computer code.
Data representing alleles detected by the methods can also be
electronically, optically, magnetically o transmitted in a computer
data signal embodied in a transmission medium over a network such
as an intranet or internet or combinations thereof. The system can
also or alternatively transmit data via wireless, IR, or other
available transmission alternatives.
[0151] During operation, the system typically comprises a sample
that is to be analyzed, such as a plant tissue, or material
isolated from the tissue such as genomic DNA, amplified genomic
DNA, cDNA, amplified cDNA, RNA, amplified RNA, or the like.
[0152] The phrase "allele detection/correlation system" refers to a
system in which data entering a computer corresponds to physical
objects or processes external to the computer, e.g., a marker
allele, and a process that, within a computer, causes a physical
transformation of the input signals to different output signals. In
other words, the input data, e.g., amplification of a particular
marker allele is transformed to output data, e.g., the
identification of the allelic form of a chromosome segment. The
process within the computer is a set of instructions, or "program,"
by which positive amplification or hybridization signals are
recognized by the integrated system and attributed to individual
samples as a genotype. Additional programs correlate the identity
of individual samples with phenotypic values or marker alleles,
e.g., statistical methods. In addition there are numerous e.g.,
C/C++ programs for computing, Delphi and/or Java programs for GUI
interfaces, and productivity tools (e.g., Microsoft Excel and/or
SigmaPlot) for charting or creating look up tables of relevant
allele-trait correlations. Other useful software tools in the
context of the integrated systems include statistical packages such
as SAS, Genstat, Matlab, Mathematica, and S-Plus and genetic
modeling packages such as QU-GENE. Furthermore, additional
programming languages such as visual basic are also suitably
employed in the integrated systems.
[0153] For example, tolerance marker allele values assigned to a
population of progeny descending from crosses between elite lines
are recorded in a computer readable medium, thereby establishing a
database corresponding tolerance alleles with unique identifiers
for members of the population of progeny. Any file or folder,
whether custom-made or commercially available (e.g., from Oracle or
Sybase) suitable for recording data in a computer readable medium
is acceptable as a database. Data regarding genotype for one or
more molecular markers, e.g., ASH, SSR, RFLP, RAPD, AFLP, SNP,
isozyme markers or other markers as described herein, are similarly
recorded in a computer accessible database. Optionally, marker data
is obtained using an integrated system that automates one or more
aspects of the assay (or assays) used to determine marker(s)
genotype. In such a system, input data corresponding to genotypes
for molecular markers are relayed from a detector, e.g., an array,
a scanner, a CCD, or other detection device directly to files in a
computer readable medium accessible to the central processing unit.
A set of system instructions (typically embodied in one or more
programs) encoding the correlations between tolerance and the
alleles of the invention is then executed by the computational
device to identify correlations between marker alleles and
predicted trait phenotypes.
[0154] Typically, the system also includes a user input device,
such as a keyboard, a mouse, a touchscreen, or the like, for, e.g.,
selecting files, retrieving data, reviewing tables of maker
information, etc., and an output device (e.g., a monitor, a
printer, etc.) for viewing or recovering the product of the
statistical analysis.
[0155] Integrated systems comprising a computer or computer
readable medium comprising set of files and/or a database with at
least one data set that corresponds to the marker alleles herein
are provided. The systems optionally also includes a user interface
allowing a user to selectively view one or more of these databases.
In addition, standard text manipulation software such as word
processing software (e.g., Microsoft Word.TM. or Corel
Wordperfect.TM.) and database or spreadsheet software (e.g.,
spreadsheet software such as Microsoft Excel.TM., Corel Quattro
Pro.TM., or database programs such as Microsoft Access.TM. or
Paradox.TM.) can be used in conjunction with a user interface
(e.g., a GUI in a standard operating system such as a Windows,
Macintosh, Unix or Linux system) to manipulate strings of
characters corresponding to the alleles or other features of the
database.
[0156] The systems optionally include components for sample
manipulation, e.g., incorporating robotic devices. For example, a
robotic liquid control armature for transferring solutions (e.g.,
plant cell extracts) from a source to a destination, e.g., from a
microtiter plate to an array substrate, is optionally operably
linked to the digital computer (or to an additional computer in the
integrated system). An input device for entering data to the
digital computer to control high throughput liquid transfer by the
robotic liquid control armature and, optionally, to control
transfer by the armature to the solid support is commonly a feature
of the integrated system. Many such automated robotic fluid
handling systems are commercially available. For example, a variety
of automated systems are available from Caliper Technologies
(Hopkinton, Mass.), which utilize various Zymate systems, which
typically include, e.g., robotics and fluid handling modules.
Similarly, the common ORCA.RTM. robot, which is used in a variety
of laboratory systems, e.g., for microtiter tray manipulation, is
also commercially available, e.g., from Beckman Coulter, Inc.
(Fullerton, Calif.). As an alternative to conventional robotics,
microfluidic systems for performing fluid handling and detection
are now widely available, e.g., from Caliper Technologies Corp.
(Hopkinton, Mass.) and Agilent technologies (Palo Alto,
Calif.).
[0157] Systems for molecular marker analysis can include a digital
computer with one or more of high-throughput liquid control
software, image analysis software for analyzing data from marker
labels, data interpretation software, a robotic liquid control
armature for transferring solutions from a source to a destination
operably linked to the digital computer, an input device (e.g., a
computer keyboard) for entering data to the digital computer to
control high throughput liquid transfer by the robotic liquid
control armature and, optionally, an image scanner for digitizing
label signals from labeled probes hybridized, e.g., to markers on a
solid support operably linked to the digital computer. The image
scanner interfaces with the image analysis software to provide a
measurement of, e.g., nucleic acid probe label intensity upon
hybridization to an arrayed sample nucleic acid population (e.g.,
comprising one or more markers), where the probe label intensity
measurement is interpreted by the data interpretation software to
show whether, and to what degree, the labeled probe hybridizes to a
marker nucleic acid (e.g., an amplified marker allele). The data so
derived is then correlated with sample identity, to determine the
identity of a plant with a particular genotype(s) for particular
markers or alleles, e.g., to facilitate marker assisted selection
of soybean plants with favorable allelic forms of chromosome
segments involved in agronomic performance (e.g., tolerance or
improved tolerance).
[0158] Optical images, e.g., hybridization patterns viewed (and,
optionally, recorded) by a camera or other recording device (e.g.,
a photodiode and data storage device) are optionally further
processed in any of the embodiments herein, e.g., by digitizing the
image and/or storing and analyzing the image on a computer. A
variety of commercially available peripheral equipment and software
is available for digitizing, storing and analyzing a digitized
video or digitized optical image.
Positional Cloning
[0159] The molecular marker loci and alleles associated with
tolerance to PPO inhibitors, e.g., SATT495, P10649C-3, SATT182,
S03859-1, S00224-1, SATT388, SATT313, and SATT613 (including
markers above SATT613), S08102-1-Q1, S08103-1-Q1. S08104-1-Q1,
S08106-1-Q1, S08107-1-Q1, S08107-1-Q1, S08109-1-Q1, S08110-1-Q1,
S08111-1-Q1, S08115-2-Q1, S08117-1-Q1, S08119-1-Q1, S08116-1-Q1,
S08112-1-Q1, S08108-1-Q1, S08101-4-Q1, S08101-1-Q1, S08101-2-Q1, or
S08101-3-Q1 can be used, as indicated previously, to identify a
tolerance QTL, which can be cloned by well-established procedures,
e.g., as described in detail in Ausubel, Berger and Sambrook,
herein.
[0160] These tolerance clones are first identified by their genetic
linkage to markers provided herein. Isolation of a nucleic acid of
interest is achieved by any number of methods as discussed in
detail in such references as Ausubel, Berger and Sambrook, herein,
and Clark, ed. (1997) Plant Molecular Biology: A Laboratory Manual
Springer-Verlag, Berlin.
[0161] For example, "positional gene cloning" uses the proximity of
a tolerance marker to physically define an isolated chromosomal
fragment containing a tolerance QTL gene. The isolated chromosomal
fragment can be produced by such well known methods as digesting
chromosomal DNA with one or more restriction enzymes, or by
amplifying a chromosomal region in a polymerase chain reaction
(PCR), or any suitable alternative amplification reaction. The
digested or amplified fragment is typically ligated into a vector
suitable for replication, and, e.g., expression, of the inserted
fragment. Markers that are adjacent to an open reading frame (ORF)
associated with a phenotypic trait can hybridize to a DNA clone
(e.g., a clone from a genomic DNA library), thereby identifying a
clone on which an ORF (or a fragment of an ORF) is located. If the
marker is more distant, a fragment containing the open reading
frame is identified by successive rounds of screening and isolation
of clones which together comprise a contiguous sequence of DNA, a
process termed "chromosome walking", resulting in a "contig" or
"contig map." Protocols sufficient to guide one of skill through
the isolation of clones associated with linked markers are found
in, e.g. Berger, Sambrook and Ausubel, all herein.
[0162] Variant sequences have a high degree of sequence similarity.
For polynucleotides, conservative variants include those sequences
that, because of the degeneracy of the genetic code, encode the
amino acid sequence of one of the native recombinase polypeptides.
Variants such as these can be identified with the use of well-known
molecular biology techniques, as, for example, with polymerase
chain reaction (PCR) and hybridization techniques. Variant
polynucleotides also include synthetically derived nucleotide
sequences, such as those generated, for example, by using
site-directed mutagenesis but which still encode a recombinase
protein. Generally, variants of a particular polynucleotide will
have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more
sequence identity to that particular polynucleotide as determined
by known sequence alignment programs and parameters.
[0163] Variants of a particular polynucleotide (the reference
nucleotide sequence) can also be evaluated by comparison of the
percent sequence identity between the polypeptide encoded by a
variant polynucleotide and the polypeptide encoded by the reference
polynucleotide. Thus, for example, isolated polynucleotides that
encode a polypeptide with a given percent sequence identity to the
recombinase are known. Percent sequence identity between any two
polypeptides can be calculated using sequence alignment programs
and parameters described. Where any given pair of polynucleotides
is evaluated by comparison of the percent sequence identity shared
by the two polypeptides they encode, the percent sequence identity
between the two encoded polypeptides is at least about 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99% or more sequence identity.
[0164] A variant protein is intended a protein derived from the
native protein by deletion, addition, and/or substitution of one or
more amino acids to the N-terminal, internal region(s), and/or
C-terminal end of the native protein. Variant proteins are
biologically active, that is they continue to possess the desired
biological activity of the native protein, for example a variant
recombinase will implement a recombination event between
appropriate recombination sites. Such variants may result from, for
example, genetic polymorphism or from human manipulation.
Biologically active variants of a native recombinase protein will
have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more
sequence identity to the amino acid sequence for the native protein
as determined by known sequence alignment programs and parameters.
A biologically active variant of a protein may differ from that
protein by as few as 1-15 amino acid residues, as few as 1-10, such
as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid
residue.
[0165] Sequence relationships can be analyzed and described using
computer-implemented algorithms. The sequence relationship between
two or more polynucleotides, or two or more polypeptides can be
determined by generating the best alignment of the sequences, and
scoring the matches and the gaps in the alignment, which yields the
percent sequence identity, and the percent sequence similarity.
Polynucleotide relationships can also be described based on a
comparison of the polypeptides each encodes. Many programs and
algorithms for the comparison and analysis of sequences are
available.
[0166] Unless otherwise stated, sequence identity/similarity values
provided herein refer to the value obtained using GAP Version 10
(GCG, Accelrys, San Diego, Calif.) using the following parameters:
% identity and % similarity for a nucleotide sequence using a gap
creation penalty weight of 50 and a gap length extension penalty
weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and %
similarity for an amino acid sequence using a GAP creation penalty
weight of 8 and a gap length extension penalty of 2, and the
BLOSUM62 scoring matrix (Henikoff & Henikoff (1989) Proc Natl
Acad Sci USA 89:10915).
[0167] GAP uses the algorithm of Needleman & Wunsch (1970) J
Mol Biol 48:443-453, to find an alignment of two complete sequences
that maximizes the number of matches and minimizes the number of
gaps. GAP considers all possible alignments and gap positions and
creates the alignment with the largest number of matched bases and
the fewest gaps. It allows for the provision of a gap creation
penalty and a gap extension penalty in units of matched bases. GAP
must make a profit of gap creation penalty number of matches for
each gap it inserts. If a gap extension penalty greater than zero
is chosen, GAP must, in addition, make a profit for each gap
inserted of the length of the gap times the gap extension penalty.
GAP presents one member of the family of best alignments.
[0168] Sequence identity, or identity, is a measure of the residues
in the two sequences that are the same when aligned for maximum
correspondence. Sequences, particularly polypeptides, that differ
by conservative substitutions are said to have sequence similarity
or similarity. Means for making this adjustment are known, and
typically involve scoring a conservative substitution as a partial
rather than a full mismatch. For example, where an identical amino
acid is given a score of 1 and a non-conservative substitution is
given a score of zero, a conservative substitution is given a score
between zero and 1. The scoring of conservative substitutions is
calculated using the selected scoring matrix (BLOSUM62 by default
for GAP).
[0169] Proteins may be altered in various ways including amino acid
substitutions, deletions, truncations, and insertions. Methods for
such manipulations are generally known. For example, amino acid
sequence variants of the recombinase proteins can be prepared by
mutations in the DNA. Methods for mutagenesis and nucleotide
sequence alterations include for example, Kunkel (1985) Proc Natl
Acad Sci USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol
154:367-382; U.S. Pat. No. 4,873,192; Walker & Gaastra, eds.
(1983) Techniques in Molecular Biology (MacMillan Publishing
Company, New York) and the references cited therein. Guidance as to
appropriate amino acid substitutions that do not affect biological
activity of the protein of interest may be found in the model of
Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl
Biomed Res Found, Washington, D.C.). Conservative substitutions,
such as exchanging one amino acid with another having similar
properties, may be preferable.
Generation of Transgenic Cells and Plants
[0170] The present invention also relates to host cells and
organisms which are transformed with nucleic acids corresponding to
tolerance QTL identified herein. For example, such nucleic acids
include chromosome intervals (e.g., genomic fragments), ORFs and/or
cDNAs that encode a tolerance or improved tolerance trait.
Additionally, production of polypeptides that provide tolerance or
improved tolerance by recombinant techniques are provided.
[0171] General texts which describe molecular biological techniques
for the cloning and manipulation of nucleic acids and production of
encoded polypeptides include Berger and Kimmel, Guide to Molecular
Cloning Techniques, Methods in Enzymology volume 152 Academic
Press, Inc., San Diego, Calif. (Berger); Sambrook et al., Molecular
Cloning--A Laboratory Manual (3rd Ed.), Vol. 1-3, Cold Spring
Harbor Laboratory, Cold Spring Harbor, N.Y., 2001 ("Sambrook") and
Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.,
Current Protocols, a joint venture between Greene Publishing
Associates, Inc. and John Wiley & Sons, Inc., (supplemented
through 2004 or later) ("Ausubel")). These texts describe
mutagenesis, the use of vectors, promoters and many other relevant
topics related to, e.g., the generation of clones that comprise
nucleic acids of interest, e.g., marker loci, marker probes, QTL
that segregate with marker loci, etc.
[0172] Host cells are genetically engineered (e.g., transduced,
transfected, transformed, etc.) with the vectors (e.g., vectors,
such as expression vectors which comprise an ORF derived from or
related to a tolerance QTL) which can be, for example, a cloning
vector, a shuttle vector or an expression vector. Such vectors are,
for example, in the form of a plasmid, a phagemid, an
agrobacterium, a virus, a naked polynucleotide (linear or
circular), or a conjugated polynucleotide. Vectors can be
introduced into bacteria, especially for the purpose of propagation
and expansion. The vectors are also introduced into plant tissues,
cultured plant cells or plant protoplasts by a variety of standard
methods known in the art, including but not limited to
electroporation (From et al. (1985) Proc. Natl. Acad. Sci. USA 82;
5824), infection by viral vectors such as cauliflower mosaic virus
(CaMV) (Hohn et al. (1982) Molecular Biology of Plant Tumors
(Academic Press, New York, pp. 549-560; Howell U.S. Pat. No.
4,407,956), high velocity ballistic penetration by small particles
with the nucleic acid either within the matrix of small beads or
particles, or on the surface (Klein et al. (1987) Nature 327; 70),
use of pollen as vector (WO 85/01856), or use of Agrobacterium
tumefaciens or A. rhizogenes carrying a T-DNA plasmid in which DNA
fragments are cloned. The T-DNA plasmid is transmitted to plant
cells upon infection by Agrobacterium tumefaciens, and a portion is
stably integrated into the plant genome (Horsch et al. (1984)
Science 233; 496; Fraley et al. (1983) Proc. Natl. Acad. Sci. USA
80; 4803). Additional details regarding nucleic acid introduction
methods are found in Sambrook, Berger and Ausubel, infra. The
method of introducing a nucleic acid into a host cell is not
critical, and therefore should not be limited to any particular
method for introducing exogenous genetic material into a host cell.
Thus, any suitable method, e.g., including but not limited to the
methods provided herein, which provides for effective introduction
of a nucleic acid into a cell or protoplast can be employed.
[0173] The engineered host cells can be cultured in conventional
nutrient media modified as appropriate for such activities as, for
example, activating promoters or selecting transformants. These
cells can optionally be cultured into transgenic plants. In
addition to Sambrook, Berger and Ausubel, all infra. Plant
regeneration from cultured protoplasts is described in Evans et al.
(1983) "Protoplast Isolation and Culture," Handbook of Plant Cell
Cultures 1, 124-176 (MacMillan Publishing Co., New York; Davey
(1983) "Recent Developments in the Culture and Regeneration of
Plant Protoplasts," Protoplasts, pp. 12-29, (Birkhauser, Basel);
Dale (1983) "Protoplast Culture and Plant Regeneration of Cereals
and Other Recalcitrant Crops," Protoplasts pp. 31-41, (Birkhauser,
Basel); Binding (1985) "Regeneration of Plants," Plant Protoplasts,
pp. 21-73, (CRC Press, Boca Raton, Fla.). Additional details
regarding plant cell culture and regeneration include Payne et al.
(1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley
& Sons, Inc. New York, N.Y.; Gamborg and Phillips (eds) (1995)
Plant Cell, Tissue and Organ Culture; Fundamental Methods Springer
Lab Manual, Springer-Verlag (Berlin Heidelberg N.Y.) and Plant
Molecular Biolgy (1993) R. R. D. Croy, Ed. Bios Scientific
Publishers, Oxford, U.K. ISBN 0 12 198370 6. Cell culture media in
general are also set forth in Atlas and Parks (eds) The Handbook of
Microbiological Media (1993) CRC Press, Boca Raton, Fla. Additional
information for cell culture is found in available commercial
literature such as the Life Science Research Cell Culture Catalogue
(1998) from Sigma-Aldrich, Inc (St Louis, Mo.) ("Sigma-LSRCCC")
and, e.g., the Plant Culture Catalogue and supplement (e.g., 1997
or later) also from Sigma-Aldrich, Inc (St Louis, Mo.)
("Sigma-PCCS").
[0174] The production of transgenic organisms is provided, which
may be bacteria, yeast, fungi, animals or plants, transduced with
the nucleic acids (e.g., nucleic acids comprising the marker loci
and/or QTL noted herein). A thorough discussion of techniques
relevant to bacteria, unicellular eukaryotes and cell culture is
found in references enumerated herein and are briefly outlined as
follows. Several well-known methods of introducing target nucleic
acids into bacterial cells are available, any of which may be used.
These include: fusion of the recipient cells with bacterial
protoplasts containing the DNA, treatment of the cells with
liposomes containing the DNA, electroporation, microinjection, cell
fusions, projectile bombardment (biolistics), carbon fiber
delivery, and infection with viral vectors (discussed further,
below), etc. Bacterial cells can be used to amplify the number of
plasmids containing DNA constructs. The bacteria are grown to log
phase and the plasmids within the bacteria can be isolated by a
variety of methods known in the art (see, for instance, Sambrook).
In addition, a plethora of kits are commercially available for the
purification of plasmids from bacteria. For their proper use,
follow the manufacturer's instructions (see, for example,
EasyPrep.TM., FlexiPrep.TM., both from Pharmacia Biotech;
StrataClean.TM., from Stratagene; and, QIAprep.TM. from Qiagen).
The isolated and purified plasmids are then further manipulated to
produce other plasmids, used to transfect plant cells or
incorporated into Agrobacterium tumefaciens related vectors to
infect plants. Typical vectors contain transcription and
translation terminators, transcription and translation initiation
sequences, and promoters useful for regulation of the expression of
the particular target nucleic acid. The vectors optionally comprise
generic expression cassettes containing at least one independent
terminator sequence, sequences permitting replication of the
cassette in eukaryotes, or prokaryotes, or both, (e.g., shuttle
vectors) and selection markers for both prokaryotic and eukaryotic
systems. Vectors are suitable for replication and integration in
prokaryotes, eukaryotes, or both. See, Giliman & Smith (1979)
Gene 8:81; Roberts et al. (1987) Nature 328:731; Schneider et al.
(1995) Protein Expr. Purif. 6435:10; Ausubel, Sambrook, Berger (all
infra). A catalogue of bacteria and bacteriophages useful for
cloning is provided, e.g., by the ATCC, e.g., The ATCC Catalogue of
Bacteria and Bacteriophage (1992) Gherna et al. (eds) published by
the ATCC. Additional basic procedures for sequencing, cloning and
other aspects of molecular biology and underlying theoretical
considerations are also found in Watson et al. (1992) Recombinant
DNA, Second Edition, Scientific American Books, N.Y. In addition,
essentially any nucleic acid (and virtually any labeled nucleic
acid, whether standard or non-standard) can be custom or standard
ordered from any of a variety of commercial sources, such as the
Midland Certified Reagent Company (Midland, Tex.), The Great
American Gene Company (Ramona, Calif.), ExpressGen Inc. (Chicago,
Ill.), Operon Technologies Inc. (Alameda, Calif.) and many
others.
[0175] Introducing Nucleic Acids into Plants
[0176] Embodiments include the production of transgenic plants
comprising the cloned nucleic acids, e.g., isolated ORFs and cDNAs
encoding tolerance genes. Techniques for transforming plant cells
with nucleic acids are widely available and can be readily adapted.
In addition to Berger, Ausubel and Sambrook, all infra, useful
general references for plant cell cloning, culture and regeneration
include Jones (ed) (1995) Plant Gene Transfer and Expression
Protocols--Methods in Molecular Biology, Volume 49 Humana Press
Towata N.J.; Payne et al. (1992) Plant Cell and Tissue Culture in
Liquid Systems John Wiley & Sons, Inc. New York, N.Y. (Payne);
and Gamborg and Phillips (eds) (1995) Plant Cell, Tissue and Organ
Culture; Fundamental Methods Springer Lab Manual, Springer-Verlag
(Berlin Heidelberg N.Y.) (Gamborg). A variety of cell culture media
are described in Atlas and Parks (eds) The Handbook of
Microbiological Media (1993) CRC Press, Boca Raton, Fla. (Atlas).
Additional information for plant cell culture is found in available
commercial literature such as the Life Science Research Cell
Culture Catalogue (1998) from Sigma-Aldrich, Inc (St Louis, Mo.)
(Sigma-LSRCCC) and, e.g., the Plant Culture Catalogue and
supplement (1997) also from Sigma-Aldrich, Inc (St Louis, Mo.)
(Sigma-PCCS). Additional details regarding plant cell culture are
found in Croy, (ed.) (1993) Plant Molecular Biology, Bios
Scientific Publishers, Oxford, U.K.
[0177] The nucleic acid constructs, e.g., DNA molecules, plasmids,
cosmids, artificial chromosomes, DNA and RNA polynucleotides, are
introduced into plant cells, either in culture or in the organs of
a plant by a variety of conventional techniques. Where the sequence
is expressed, the sequence is optionally combined with
transcriptional and translational initiation regulatory sequences
which direct the transcription or translation of the sequence from
the exogenous DNA in the intended tissues of the transformed
plant.
[0178] Isolated nucleic acid acids can be introduced into plants
according to any of a variety of techniques known in the art.
Techniques for transforming a wide variety of higher plant species
are also well known and described in widely available technical,
scientific, and patent literature. See, e.g., Weising et al. (1988)
Ann. Rev. Genet. 22:421-477.
[0179] The DNA constructs, for example DNA fragments, plasmids,
phagemids, cosmids, phage, naked or variously conjugated-DNA
polynucleotides, (e.g., polylysine-conjugated DNA,
peptide-conjugated DNA, liposome-conjugated DNA, etc.), or
artificial chromosomes, can be introduced directly into the genomic
DNA of the plant cell using techniques such as electroporation and
microinjection of plant cell protoplasts, or the DNA constructs can
be introduced directly to plant cells using ballistic methods, such
as DNA particle bombardment.
[0180] Microinjection techniques for injecting plant, e.g., cells,
embryos, callus and protoplasts, are known in the art and well
described in the scientific and patent literature. For example, a
number of methods are described in Jones (ed) (1995) Plant Gene
Transfer and Expression Protocols--Methods in Molecular Biology,
Volume 49 Humana Press, Towata, N.J., as well as in the other
references noted herein and available in the literature.
[0181] For example, the introduction of DNA constructs using
polyethylene glycol precipitation is described in Paszkowski et
al., EMBO J. 3:2717 (1984). Electroporation techniques are
described in Fromm et al., Proc. Natl. Acad. Sci. USA 82:5824
(1985). Ballistic transformation techniques are described in Klein
et al., Nature 327:70-73 (1987). Additional details are found in
Jones (1995) and Gamborg and Phillips (1995), supra, and in U.S.
Pat. No. 5,990,387.
[0182] Alternatively, Agrobacterium-mediated transformation is
employed to generate transgenic plants. Agrobacterium-mediated
transformation techniques, including disarming and use of binary
vectors, are also well described in the scientific literature. See,
e.g., Horsch, et al. (1984) Science 233:496; and Fraley et al.
(1984) Proc. Natl. Acad. Sci. USA 80:4803 and recently reviewed in
Hansen and Chilton (1998) Current Topics in Microbiology 240:22 and
Das (1998) Subcellular Biochemistry 29: Plant Microbe Interactions,
pp 343-363.
[0183] DNA constructs are optionally combined with suitable T-DNA
flanking regions and introduced into a conventional Agrobacterium
tumefaciens host vector. The virulence functions of the
Agrobacterium tumefaciens host will direct the insertion of the
construct and adjacent marker into the plant cell DNA when the cell
is infected by the bacteria. See, U.S. Pat. No. 5,591,616. Although
Agrobacterium is useful primarily in dicots, certain monocots can
be transformed by Agrobacterium. For instance, Agrobacterium
transformation of maize is described in U.S. Pat. No.
5,550,318.
[0184] Other methods of transfection or transformation include (1)
Agrobacterium rhizogenes-mediated transformation (see, e.g.,
Lichtenstein and Fuller (1987) In: Genetic Engineering, vol. 6, P W
J Rigby, Ed., London, Academic Press; and Lichtenstein; C. P., and
Draper (1985) In: DNA Cloning, Vol. II, D. M. Glover, Ed., Oxford,
IRI Press; WO 88/02405, published Apr. 7, 1988, describes the use
of A. rhizogenes strain A4 and its Ri plasmid along with A.
tumefaciens vectors pARC8 or pARC16 (2) liposome-mediated DNA
uptake (see, e.g., Freeman et al. (1984) Plant Cell Physiol.
25:1353), (3) the vortexing method (see, e.g., Kindle (1990) Proc.
Natl. Acad. Sci., (USA) 87:1228.
[0185] DNA can also be introduced into plants by direct DNA
transfer into pollen as described by Zhou et al. (1983) Methods in
Enzymology, 101:433; D. Hess (1987) Intern Rev. Cytol. 107:367; Luo
et al. (1988) Plant Mol. Biol. Reporter 6:165. Expression of
polypeptide coding genes can be obtained by injection of the DNA
into reproductive organs of a plant as described by Pena et al.
(1987) Nature 325:274. DNA can also be injected directly into the
cells of immature embryos and the desiccated embryos rehydrated as
described by Neuhaus et al. (1987) Theor. Appl. Genet. 75:30; and
Benbrook et al. (1986) in Proceedings Bio Expo Butterworth,
Stoneham, Mass., pp. 27-54. A variety of plant viruses that can be
employed as vectors are known in the art and include cauliflower
mosaic virus (CaMV), geminivirus, brome mosaic virus, and tobacco
mosaic virus.
Generation/Regeneration of Transgenic Plants
[0186] Transformed plant cells which are derived by any of the
above transformation techniques can be cultured to regenerate a
whole plant that possesses the transformed genotype and thus the
desired phenotype. Such regeneration techniques rely on
manipulation of certain phytohormones in a tissue culture growth
medium, typically relying on a biocide and/or herbicide marker
which has been introduced together with the desired nucleotide
sequences. Plant regeneration from cultured protoplasts is
described in Payne et al. (1992) Plant Cell and Tissue Culture in
Liquid Systems John Wiley & Sons, Inc. New York, N.Y.; Gamborg
and Phillips (eds) (1995) Plant Cell, Tissue and Organ Culture;
Fundamental Methods Springer Lab Manual, Springer-Verlag (Berlin
Heidelberg N.Y.); Evans et al. (1983) Protoplasts Isolation and
Culture, Handbook of Plant Cell Culture pp. 124-176, Macmillian
Publishing Company, New York; and Binding (1985) Regeneration of
Plants, Plant Protoplasts pp. 21-73, CRC Press, Boca Raton.
Regeneration can also be obtained from plant callus, explants,
somatic embryos (Dandekar et al. (1989) J. Tissue Cult. Meth.
12:145; McGranahan, et al. (1990) Plant Cell Rep. 8:512) organs, or
parts thereof. Such regeneration techniques are described generally
in Klee et al. (1987)., Ann. Rev. of Plant Phys. 38:467-486.
Additional details are found in Payne (1992) and Jones (1995), both
supra, and Weissbach and Weissbach, eds. (1988) Methods for Plant
Molecular Biology Academic Press, Inc., San Diego, Calif. This
regeneration and growth process includes the steps of selection of
transformant cells and shoots, rooting the transformant shoots and
growth of the plantlets in soil. These methods are adapted to
produce transgenic plants bearing QTLs and other genes isolated
according to the methods.
[0187] In addition, the regeneration of plants containing the
polynucleotides and introduced by Agrobacterium into cells of leaf
explants can be achieved as described by Horsch et al. (1985)
Science 227:1229-1231. In this procedure, transformants are grown
in the presence of a selection agent and in a medium that induces
the regeneration of shoots in the plant species being transformed
as described by Fraley et al. (1983) Proc. Natl. Acad. Sci.
(U.S.A.) 80:4803. This procedure typically produces shoots within
two to four weeks and these transformant shoots are then
transferred to an appropriate root-inducing medium containing the
selective agent and an antibiotic to prevent bacterial growth.
Transgenic plants may be fertile or sterile.
[0188] It is not intended that plant transformation and expression
of polypeptides that provide herbicide tolerance be limited to
soybean species. Indeed, it is contemplated that the polypeptides
that provide tolerance in soybean can also provide a similar
phenotype when transformed and expressed in other plants. Examples
of plant genuses and species of interest include, but are not
limited to, monocots and dicots such as corn (Zea mays), Brassica
sp. (e.g., B. napus, B. rapa, B. juncea), particularly those
Brassica species useful as sources of seed oil, alfalfa (Medicago
sativa), rice (Oryza sativa), rye (Secale cereale), sorghum
(Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet
(Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail
millet (Setaria italica), finger millet (Eleusine coracana)),
sunflower (Helianthus annuus), safflower (Carthamus tinctorius),
wheat (Triticum aestivum), soybean (Glycine max), tobacco
(Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis
hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet
potato (Ipomoea batatus), cassava (Manihot esculenta), coffee
(Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas
comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea
(Camellia sinensis), banana (Musa spp.), avocado (Persea
americana), fig (Ficus casica), guava (Psidium guajava), mango
(Mangifera indica), olive (Olea europaea), papaya (Carica papaya),
cashew (Anacardium occidentale), macadamia (Macadamia
integrifolia), almond (Prunus amygdalus), sugar beets (Beta
vulgaris), sugarcane (Saccharum spp.), oats (Avena), barley
(Hordeum), palm, legumes including beans and peas such as guar,
locust bean, fenugreek, garden beans, cowpea, mungbean, lima bean,
fava bean, lentils, chickpea, and castor, Arabidopsis, vegetables,
ornamentals, grasses, conifers, crop and grain plants that provide
seeds of interest, oil-seed plants, and other leguminous plants.
Vegetables include tomatoes (Lycopersicon esculentum), lettuce
(e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lima
beans (Phaseolus limensis), peas (Lathyrus spp.), and members of
the genus Cucumis such as cucumber (C. sativus), cantaloupe (C.
cantalupensis), and musk melon (C. melo). Ornamentals include
azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea),
hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa
spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida),
carnation (Dianthus caryophyllus), poinsettia (Euphorbia
pulcherrima), and chrysanthemum. Conifers include, for example,
pines such as loblolly pine (Pinus taeda), slash pine (Pinus
elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus
contorta), and Monterey pine (Pinus radiata); Douglas-fir
(Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitka
spruce (Picea glauca); redwood (Sequoia sempervirens); true firs
such as silver fir (Abies amabilis) and balsam fir (Abies
balsamea); and cedars such as Western red cedar (Thuja plicata) and
Alaska yellow-cedar (Chamaecyparis nootkatensis).
[0189] Promoters of bacterial origin that operate in plants include
the octopine synthase promoter, the nopaline synthase promoter and
other promoters derived from native Ti plasmids. See,
Herrara-Estrella et al. (1983), Nature, 303:209. Viral promoters
include the 35S and 19S RNA promoters of cauliflower mosaic virus.
See, Odell et al. (1985) Nature, 313:810. Other plant promoters
include Kunitz trypsin inhibitor promoter (KTI), SCP1, SUP, UCD3,
the ribulose-1,3-bisphosphate carboxylase small subunit promoter
and the phaseolin promoter. The promoter sequence from the E8 gene
and other genes may also be used. The isolation and sequence of the
E8 promoter is described in detail in Deikman and Fischer (1988)
EMBO J. 7:3315. Many other promoters are in current use and can be
coupled to an exogenous DNA sequence to direct expression of the
nucleic acid.
[0190] If expression of a polypeptide from a cDNA is desired, a
polyadenylation region at the 3'-end of the coding region is
typically included. The polyadenylation region can be derived from
the natural gene, from a variety of other plant genes, or from,
e.g., T-DNA.
[0191] The vector comprising the sequences (e.g., promoters or
coding regions) from genes encoding expression products and
transgenes will typically include a nucleic acid subsequence, a
marker gene which confers a selectable, or alternatively, a
screenable, phenotype on plant cells. For example, the marker can
encode biocide tolerance, particularly antibiotic tolerance, such
as tolerance to kanamycin, G418, bleomycin, hygromycin, or
herbicide tolerance, such as tolerance to chlorosluforon, or
phosphinothricin (the active ingredient in the herbicides bialaphos
or Basta). See, e.g., Padgette et al. (1996) In:
Herbicide-Resistant Crops (Duke, ed.), pp 53-84, CRC Lewis
Publishers, Boca Raton ("Padgette, 1996"). For example, crop
selectivity to specific herbicides can be conferred by engineering
genes into crops that encode appropriate herbicide metabolizing
enzymes from other organisms, such as microbes. See Vasil (1996)
In: Herbicide-Resistant Crops (Duke, ed.), pp 85-91, CRC Lewis
Publishers, Boca Raton) ("Vasil", 1996).
[0192] One of skill will recognize that after the recombinant
expression cassette is stably incorporated in transgenic plants and
confirmed to be operable, it can be introduced into other plants by
sexual crossing. Any of a number of standard breeding techniques
can be used, depending upon the species to be crossed. In
vegetatively propagated crops, mature transgenic plants can be
propagated by the taking of cuttings or by tissue culture
techniques to produce multiple identical plants. Selection of
desirable transgenics is made and new varieties are obtained and
propagated vegetatively for commercial use. In seed propagated
crops, mature transgenic plants can be self crossed to produce a
homozygous inbred plant. The inbred plant produces seed containing
the newly introduced heterologous nucleic acid. These seeds can be
grown to produce plants that would produce the selected phenotype.
Parts obtained from the regenerated plant, such as flowers, seeds,
leaves, branches, fruit, and the like are included, provided that
these parts comprise cells comprising the isolated nucleic acid.
Progeny and variants, and mutants of the regenerated plants are
also included, provided that these parts comprise the introduced
nucleic acid sequences.
[0193] Transgenic or introgressed plants expressing a
polynucleotide can be screened for transmission of the nucleic acid
by, for example, standard nucleic acid detection methods or by
immunoblot protocols. Expression at the RNA level can be determined
to identify and quantitate expression-positive plants. Standard
techniques for RNA analysis can be employed and include RT-PCR
amplification assays using oligonucleotide primers designed to
amplify only heterologous or introgressed RNA templates and
solution hybridization assays using marker or linked QTL specific
probes. Plants can also be analyzed for protein expression, e.g.,
by Western immunoblot analysis using antibodies that recognize the
encoded polypeptides. In addition, in situ hybridization and
immunocytochemistry according to standard protocols can be done
using heterologous nucleic acid specific polynucleotide probes and
antibodies, respectively, to localize sites of expression within
transgenic tissue. Generally, a number of transgenic lines are
usually screened for the incorporated nucleic acid to identify and
select plants with the most appropriate expression profiles.
[0194] In one example, a transgenic plant that is homozygous for
the added heterologous nucleic acid; e.g., a transgenic plant that
contains two added nucleic acid sequence copies, e.g., a gene at
the same locus on each chromosome of a homologous chromosome pair
is provided. A homozygous transgenic plant can be obtained by
sexually mating (self-fertilizing) a heterozygous transgenic plant
that contains a single added heterologous nucleic acid, germinating
some of the seed produced and analyzing the resulting plants
produced for altered expression of a polynucleotide relative to a
control plant (e.g., a native, non-transgenic plant). Back-crossing
to a parental plant and out-crossing with a non-transgenic plant
can be used to introgress the heterologous nucleic acid into a
selected background (e.g., an elite or exotic soybean line).
Stacking of Traits and Additional Traits of Interest
[0195] In some embodiments, the polynucleotide conferring the
tolerance in the plants are engineered into a molecular stack with
at least one additional polynucleotide. The additional
polynucleotide may confer any additional trait of interest, such as
tolerance to an additional herbicide, insects, disease, or any
other desirable trait. A trait, as used herein, refers to the
phenotype derived from a particular sequence or groups of
sequences. For example, herbicide-tolerance polynucleotides may be
stacked with any other polynucleotides encoding polypeptides having
pesticidal and/or insecticidal activity, such as Bacillus
thuringiensis toxic proteins (described in U.S. Pat. Nos.
5,366,892; 5,747,450; 5,737,514; 5,723,756; 5,593,881; Geiser et
al. (1986) Gene 48: 109; Lee et al. (2003) Appl. Environ.
Microbiol. 69: 4648-4657 (Vip3A); Galitzky et al. (2001) Acta
Crystallogr. D. Biol. Crystallogr. 57:1101-1109 (Cry3Bb1); and
Herman et al. (2004) J. Agric. Food Chem. 52: 2726-2734 (Cry1F)),
lectins (Van Damme et al. (1994) Plant Mol. Biol. 24: 825, pentin
(described in U.S. Pat. No. 5,981,722), and the like. The
combinations generated can also include multiple copies of any one
of the polynucleotides of interest.
[0196] In some embodiments, herbicide-tolerance polynucleotide may
be stacked with other herbicide-tolerance traits to create a
transgenic plant with further improved properties. Other
herbicide-tolerance polynucleotides that could be used in such
embodiments include those conferring tolerance to the same
herbicide by other modes of action, or a different herbicide. Other
traits that could be combined with herbicide-tolerance
polynucleotides include those derived from polynucleotides that
confer on the plant the capacity to produce a higher level of
5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), for example,
as more fully described in U.S. Pat. Nos. 6,248,876 B1; 5,627,061;
5,804,425; 5,633,435; 5,145,783; 4,971,908; 5,312,910; 5,188,642;
4,940,835; 5,866,775; 6,225,114 B1; 6,130,366; 5,310,667;
4,535,060; 4,769,061; 5,633,448; 5,510,471; U.S. Pat. No. Re.
36,449; U.S. Pat. Nos. RE 37,287 E; and 5,491,288; and WO 97/04103;
WO 00/66746; WO 01/66704; and WO 00/66747. Other traits that could
be combined with herbicide-tolerance polynucleotides include those
conferring tolerance to sulfonylurea and/or imidazolinone, for
example, as described more fully in U.S. Pat. Nos. 5,605,011;
5,013,659; 5,141,870; 5,767,361; 5,731,180; 5,304,732; 4,761,373;
5,331,107; 5,928,937; and 5,378,824; and international publication
WO 96/33270.
[0197] In some embodiments, herbicide-tolerance polynucleotides of
the plants may be stacked with, for example,
hydroxyphenylpyruvatedioxygenases which are enzymes that catalyze
the reaction in which para-hydroxyphenylpyruvate (HPP) is
transformed into homogentisate. Molecules which inhibit this enzyme
and which bind to the enzyme in order to inhibit transformation of
the HPP into homogentisate are useful as herbicides. Traits
conferring tolerance to such herbicides in plants are described in
U.S. Pat. Nos. 6,245,968 B1; 6,268,549; and 6,069,115; and WO
99/23886. Other examples of suitable herbicide-tolerance traits
that could be stacked with herbicide-tolerance polynucleotides
include aryloxyalkanoate dioxygenase polynucleotides (which
reportedly confer tolerance to 2,4-D and other phenoxy auxin
herbicides as well as to aryloxyphenoxypropionate herbicides as
described, for example, in WO2005/107437) and dicamba-tolerance
polynucleotides as described, for example, in Herman et al. (2005)
J. Biol. Chem. 280: 24759-24767.
[0198] Other examples of herbicide-tolerance traits that could be
combined with herbicide-tolerance polynucleotides include those
conferred by polynucleotides encoding an exogenous phosphinothricin
acetyltransferase, as described in U.S. Pat. Nos. 5,969,213;
5,489,520; 5,550,318; 5,874,265; 5,919,675; 5,561,236; 5,648,477;
5,646,024; 6,177,616; and 5,879,903. Plants containing an exogenous
phosphinothricin acetyltransferase can exhibit improved tolerance
to glufosinate herbicides, which inhibit the enzyme glutamine
synthase. Other examples of herbicide-tolerance traits that could
be combined with the herbicide-tolerance polynucleotides include
those conferred by polynucleotides conferring altered
protoporphyrinogen oxidase (protox) activity, as described in U.S.
Pat. Nos. 6,288,306 B1; 6,282,837 B1; and 5,767,373; and WO
01/12825. Plants containing such polynucleotides can exhibit
improved tolerance to any of a variety of herbicides which target
the protox enzyme (also referred to as "protox inhibitors").
[0199] Other examples of herbicide-tolerance traits that could be
combined with herbicide-tolerance polynucleotides include those
conferring tolerance to at least one herbicide in a plant such as,
for example, a maize plant or horseweed. Herbicide-tolerant weeds
are known in the art, as are plants that vary in their tolerance to
particular herbicides. See, e.g., Green and Williams (2004)
"Correlation of Corn (Zea mays) Inbred Response to Nicosulfuron and
Mesotrione," poster presented at the WSSA Annual Meeting in Kansas
City, Mo., Feb. 9-12, 2004; Green (1998) Weed Technology 12:
474-477; Green and Ulrich (1993) Weed Science 41: 508-516. The
trait(s) responsible for these tolerances can be combined by
breeding or via other methods with herbicide-tolerance
polynucleotides to provide a plant as well as methods of use
thereof.
[0200] In this manner, plants that are more tolerant to multiple
herbicides are disclosed. Accordingly, methods for growing a crop
(i.e., for selectively controlling weeds in an area of cultivation)
that comprise treating an area of interest (e.g., a field or area
of cultivation) with at least one herbicide to which the plant is
tolerant are likewise disclosed. In some embodiments, methods
further comprise treatment with additional herbicides to which the
plant is tolerant. In such embodiments, generally the methods
permit selective control of weeds without significantly damaging
the crop. As used herein, an "area of cultivation" comprises any
region in which one desires to grow a plant. Such areas of
cultivations include, but are not limited to, a field in which a
plant is cultivated (such as a crop field, a sod field, a tree
field, a managed forest, a field for culturing fruits and
vegetables, etc), a greenhouse, a growth chamber, etc.
[0201] Herbicide-tolerant traits can also be combined with at least
one other trait to produce plants that further comprise a variety
of desired trait combinations including, but not limited to, traits
desirable for animal feed such as high oil content (e.g., U.S. Pat.
No. 6,232,529); increased digestibility (e.g., modified storage
proteins (U.S. application Ser. No. 10/053,410, filed Nov. 7,
2001); and thioredoxins (U.S. application Ser. No. 10/005,429,
filed Dec. 3, 2001)); the disclosures of which are herein
incorporated by reference. Desired trait combinations also include
LLNC (low linolenic acid content; see, e.g., Dyer et al. (2002)
Appl. Microbiol. Biotechnol. 59: 224-230) and OLCH (high oleic acid
content; see, e.g., Fernandez-Moya et al. (2005) J. Agric. Food
Chem. 53: 5326-5330).
[0202] Herbicide-tolerant traits of interest can also be combined
with other desirable traits such as, for example, fumonisim
detoxification genes (U.S. Pat. No. 5,792,931), avirulence and
disease resistance genes (Jones et al. (1994) Science 266: 789;
Martin et al. (1993) Science 262: 1432; Mindrinos et al. (1994)
Cell 78: 1089), and traits desirable for processing or process
products such as modified oils (e.g., fatty acid desaturase genes
(U.S. Pat. No. 5,952,544; WO 94/11516)); modified starches (e.g.,
ADPG pyrophosphorylases (AGPase), starch synthases (SS), starch
branching enzymes (SBE), and starch debranching enzymes (SDBE));
and polymers or bioplastics (e.g., U.S. Pat. No. 5,602,321;
beta-ketothiolase, polyhydroxybutyrate synthase, and
acetoacetyl-CoA reductase (Schubert et al. (1988) J. Bacteriol.
170:5837-5847) facilitate expression of polyhydroxyalkanoates
(PHAs)); the disclosures of which are herein incorporated by
reference. One could also combine herbicide-tolerant
polynucleotides with polynucleotides providing agronomic traits
such as male sterility (e.g., see U.S. Pat. No. 5,583,210), stalk
strength, flowering time, or transformation technology traits such
as cell cycle regulation or gene targeting (e.g., WO 99/61619, WO
00/17364, and WO 99/25821); the disclosures of which are herein
incorporated by reference.
[0203] In another embodiment, the herbicide-tolerant traits of
interest can also be combined with the Rcg1 sequence or
biologically active variant or fragment thereof. The Rcg1 sequence
is an anthracnose stalk rot resistance gene in corn. See, e.g.,
U.S. patent application Ser. Nos. 11/397,153, 11/397,275, and
11/397,247, each of which is herein incorporated by reference.
[0204] These stacked combinations can be created by any method
including, but not limited to, breeding plants by any conventional
or TopCross methodology, or genetic transformation. If the
sequences are stacked by genetically transforming the plants, the
polynucleotide sequences of interest can be combined at any time
and in any order. For example, a transgenic plant comprising one or
more desired traits can be used as the target to introduce further
traits by subsequent transformation. The traits can be introduced
simultaneously in a co-transformation protocol with the
polynucleotides of interest provided by any combination of
transformation cassettes. For example, if two sequences will be
introduced, the two sequences can be contained in separate
transformation cassettes (trans) or contained on the same
transformation cassette (cis). Expression of the sequences can be
driven by the same promoter or by different promoters. In certain
cases, it may be desirable to introduce a transformation cassette
that will suppress the expression of the polynucleotide of
interest. This may be combined with any combination of other
suppression cassettes or overexpression cassettes to generate the
desired combination of traits in the plant. It is further
recognized that polynucleotide sequences can be stacked at a
desired genomic location using a site-specific recombination
system. See, e.g., WO99/25821, WO99/25854, WO99/25840, WO99/25855,
and WO99/25853, all of which are herein incorporated by
reference.
[0205] Insect resistance genes may encode resistance to pests that
have great yield drag such as rootworm, cutworm, European Corn
Borer, and the like. Such genes include, for example, Bacillus
thuringiensis toxic protein genes (U.S. Pat. Nos. 5,366,892;
5,747,450; 5,736,514; 5,723,756; 5,593,881; and Geiser et al.
(1986) Gene 48: 109); and the like.
[0206] Genes encoding disease resistance traits include
detoxification genes, such as against fumonosin (U.S. Pat. No.
5,792,931); avirulence (avr) and disease resistance (R) genes
(Jones et al. (1994) Science 266: 789; Martin et al. (1993) Science
262: 1432; and Mindrinos et al. (1994) Cell 78: 1089); and the
like.
[0207] Sterility genes can also be encoded in an expression
cassette and provide an alternative to physical detasseling.
Examples of genes used in such ways include male tissue-preferred
genes and genes with male sterility phenotypes such as QM,
described in U.S. Pat. No. 5,583,210. Other genes include kinases
and those encoding compounds toxic to either male or female
gametophytic development.
Polynucleotide Constructs
[0208] In specific embodiments, one or more of the
herbicide-tolerant polynucleotides employed in the methods and
compositions can be provided in an expression cassette for
expression in the plant or other organism of interest. The cassette
will include 5' and 3' regulatory sequences operably linked to a
herbicide-tolerance polynucleotide. "Operably linked" is intended
to mean a functional linkage between two or more elements. For
example, an operable linkage between a polynucleotide of interest
and a regulatory sequence (e.g., a promoter) is functional link
that allows for expression of the polynucleotide of interest.
Operably linked elements may be contiguous or non-contiguous. When
used to refer to the joining of two protein coding regions, by
"operably linked" is intended that the coding regions are in the
same reading frame. When used to refer to the effect of an
enhancer, "operably linked" indicates that the enhancer increases
the expression of a particular polynucleotide or polynucleotides of
interest. Where the polynucleotide or polynucleotides of interest
encode a polypeptide, the encoded polypeptide is produced at a
higher level.
[0209] The cassette may additionally contain at least one
additional gene to be cotransformed into the organism.
Alternatively, the additional gene(s) can be provided on multiple
expression cassettes. Such an expression cassette is provided with
a plurality of restriction sites and/or recombination sites for
insertion of the herbicide-tolerance polynucleotide to be under the
transcriptional regulation of the regulatory regions. The
expression cassette may additionally contain other genes, including
other selectable marker genes. Where a cassette contains more than
one polynucleotide, the polynucleotides in the cassette may be
transcribed in the same direction or in different directions (also
called "divergent" transcription).
[0210] An expression cassette comprising a herbicide-tolerance
polynucleotide will include in the 5'-3' direction of transcription
a transcriptional and translational initiation region (i.e., a
promoter), a herbicide-tolerance polynucleotide, and a
transcriptional and translational termination region (i.e.,
termination region) functional in plants or the other organism of
interest. Accordingly, plants having such expression cassettes are
also provided. The regulatory regions (i.e., promoters,
transcriptional regulatory regions, and translational termination
regions) and/or the herbicide-tolerance polynucleotide may be
native (i.e., analogous) to the host cell or to each other.
Alternatively, the regulatory regions and/or the
herbicide-tolerance polynucleotide may be heterologous to the host
cell or to each other.
[0211] While it may be optimal to express polynucleotides using
heterologous promoters, native promoter sequences may be used. Such
constructs can change expression levels and/or expression patterns
of the encoded polypeptide in the plant or plant cell. Expression
levels and/or expression patterns of the encoded polypeptide may
also be changed as a result of an additional regulatory element
that is part of the construct, such as, for example, an enhancer.
Thus, the phenotype of the plant or cell can be altered even though
a native promoter is used.
[0212] The termination region may be native with the
transcriptional initiation region, may be native with the operably
linked herbicide-tolerance polynucleotide of interest, may be
native with the plant host, or may be derived from another source
(i.e., foreign or heterologous) to the promoter, the
herbicide-tolerance polynucleotide of interest, the plant host, or
any combination thereof. Convenient termination regions are
available from the Ti-plasmid of A. tumefaciens, such as the
octopine synthase and nopaline synthase termination regions, or can
be obtained from plant genes such as the Solanum tuberosum
proteinase inhibitor II gene. See Guerineau et al. (1991) Mol. Gen.
Genet. 262: 141-144; Proudfoot (1991) Cell 64: 671-674; Sanfacon et
al. (1991) Genes Dev. 5: 141-149; Mogen et al. (1990) Plant Cell 2:
1261-1272; Munroe et al. (1990) Gene 91: 151-158; Ballas et al.
(1989) Nucleic Acids Res. 17: 7891-7903; and Joshi et al. (1987)
Nucleic Acids Res. 15: 9627-9639.
[0213] A number of promoters can be used, including the native
promoter of the polynucleotide sequence of interest. The promoters
can be selected based on the desired outcome. The polynucleotides
of interest can be combined with constitutive, tissue-preferred, or
other promoters for expression in plants.
[0214] Such constitutive promoters include, for example, the core
promoter of the Rsyn7 promoter and other constitutive promoters
disclosed in WO 99/43838 and U.S. Pat. No. 6,072,050; the core CaMV
35S promoter (Odell et al. (1985) Nature 313: 810-812); rice actin
(McElroy et al. (1990) Plant Cell 2: 163-171); the maize actin
promoter; the ubiquitin promoter (see, e.g., Christensen et al.
(1989) Plant Mol. Biol. 12: 619-632; Christensen et al. (1992)
Plant Mol. Biol. 18: 675-689; Callis et al. (1995) Genetics 139:
921-39); pEMU (Last et al. (1991) Theor. Appl. Genet. 81: 581-588);
MAS (Velten et al. (1984) EMBO J. 3: 2723-2730); ALS promoter (U.S.
Pat. No. 5,659,026), and the like. Other constitutive promoters
include, for example, those described in U.S. Pat. Nos. 5,608,149;
5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463;
5,608,142; and 6,177,611. Some promoters show improved expression
when they are used in conjunction with a native 5' untranslated
region and/or other elements such as, for example, an intron. For
example, the maize ubiquitin promoter is often placed upstream of a
polynucleotide of interest along with at least a portion of the 5'
untranslated region of the ubiquitin gene, including the first
intron of the maize ubiquitin gene.
[0215] Chemical-regulated promoters can be used to modulate the
expression of a gene in a plant through the application of an
exogenous chemical regulator. Depending upon the objective, the
promoter may be a chemical-inducible promoter for which application
of the chemical induces gene expression or the promoter may be a
chemical-repressible promoter for which application of the chemical
represses gene expression. Chemical-inducible promoters are known
in the art and include, but are not limited to, the maize In2-2
promoter, which is activated by benzenesulfonamide herbicide
safeners, the maize GST promoter, which is activated by hydrophobic
electrophilic compounds that are used as pre-emergent herbicides,
and the tobacco PR-1a promoter, which is activated by salicylic
acid. Other chemical-regulated promoters of interest include
steroid-responsive promoters (see, e.g., the
glucocorticoid-inducible promoter in Schena et al. (1991) Proc.
Natl. Acad. Sci. USA 88: 10421-10425 and McNellis et al. (1998)
Plant J. 14(2): 247-257) and tetracycline-inducible and
tetracycline-repressible promoters (see, e.g., Gatz et al. (1991)
Mol. Gen. Genet. 227: 229-237, and U.S. Pat. Nos. 5,814,618 and
5,789,156), herein incorporated by reference).
[0216] Tissue-preferred promoters can be utilized to target
enhanced herbicide-tolerance polypeptide expression within a
particular plant tissue. Tissue-preferred promoters include
Yamamoto et al. (1997) Plant J. 12: 255-265; Kawamata et al. (1997)
Plant Cell Physiol. 38: 792-803; Hansen et al. (1997) Mol. Gen
Genet. 254: 337-343; Russell et al. (1997) Transgenic Res. 6:
157-168; Rinehart et al. (1996) Plant Physiol. 112:1331-1341; Van
Camp et al. (1996) Plant Physiol. 112: 525-535; Canevascini et al.
(1996) Plant Physiol. 112: 513-524; Yamamoto et al. (1994) Plant
Cell Physiol. 35: 773-778; Lam (1994) Results Probl. Cell Differ.
20: 181-196; Orozco et al. (1993) Plant Mol Biol. 23: 1129-1138;
Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA 90: 9586-9590; and
Guevara-Garcia et al. (1993) Plant J. 4: 495-505. Such promoters
can be modified, if necessary, for weak expression.
[0217] Leaf-preferred promoters are known in the art. See, e.g.,
Yamamoto et al. (1997) Plant J. 12: 255-265; Kwon et al. (1994)
Plant Physiol. 105: 357-67; Yamamoto et al. (1994) Plant Cell
Physiol. 35: 773-778; Gotor et al. (1993) Plant J. 3: 509-18;
Orozco et al. (1993) Plant Mol. Biol. 23: 1129-1138; and Matsuoka
et al. (1993) Proc. Natl. Acad. Sci. USA 90: 9586-9590.
[0218] Root-preferred promoters are known and can be selected from
the many available from the literature or isolated de novo from
various compatible species. See, e.g., Hire et al. (1992) Plant
Mol. Biol. 20(2): 207-218 (soybean root-specific glutamine
synthetase gene); Keller and Baumgartner (1991) Plant Cell 3:
1051-1061 (root-specific control element in the GRP 1.8 gene of
French bean); Sanger et al. (1990) Plant Mol. Biol. 14: 433-443
(root-specific promoter of the mannopine synthase (MAS) gene of
Agrobacterium tumefaciens); and Miao et al. (1991) Plant Cell 3:
11-22 (full-length cDNA clone encoding cytosolic glutamine
synthetase (GS), which is expressed in roots and root nodules of
soybean). See also Bogusz et al. (1990) Plant Cell 2: 633-641,
where two root-specific promoters are described. Leach and Aoyagi
(1991) describe their analysis of the promoters of the highly
expressed rolC and rolD root-inducing genes of Agrobacterium
rhizogenes (see Plant Science (Limerick) 79: 69-76). They concluded
that enhancer and tissue-preferred DNA determinants are dissociated
in those promoters. Teeri et al. (1989) used gene fusion to lacZ to
show that the Agrobacterium T-DNA gene encoding octopine synthase
is especially active in the epidermis of the root tip and that the
TR2' gene is root specific in the intact plant and stimulated by
wounding in leaf tissue, an especially desirable combination of
characteristics for use with an insecticidal or larvicidal gene
(see EMBO J. 8: 343-350). The TR1' gene, fused to nptII (neomycin
phosphotransferase II) showed similar characteristics. Additional
root-preferred promoters include the VfENOD-GRP3 gene promoter
(Kuster et al. (1995) Plant Mol. Biol. 29: 759-772); and rolB
promoter (Capana et al. (1994) Plant Mol. Biol. 25: 681-691. See
also U.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252;
5,401,836; 5,110,732; and 5,023,179.
[0219] Seed-preferred promoters include both seed-specific
promoters (those promoters active during seed development such as
promoters of seed storage proteins) as well as "seed-germinating"
promoters (those promoters active during seed germination). See
Thompson et al. (1989) BioEssays 10: 108, herein incorporated by
reference. Such seed-preferred promoters include, but are not
limited to, Cim1 (cytokinin-induced message); cZ19B1 (maize 19 kDa
zein); milps (myo-inositol-1-phosphate synthase) (see WO 00/11177
and U.S. Pat. No. 6,225,529; herein incorporated by reference).
Gamma-zein is an endosperm-specific promoter. Globulin 1 (Glb-1) is
a representative embryo-specific promoter. For dicots,
seed-specific promoters include, but are not limited to, bean
.beta.-phaseolin, napin, .beta.-conglycinin, soybean lectin,
cruciferin, and the like. For monocots, seed-specific promoters
include, but are not limited to, maize 15 kDa zein, 22 kDa zein, 27
kDa zein, gamma-zein, waxy, shrunken 1, shrunken 2, Globulin 1,
etc. See also WO 00/12733, where seed-preferred promoters from end1
and end2 genes are disclosed; herein incorporated by reference.
[0220] Additional promoters of interest include the SCP1 promoter
(U.S. Pat. No. 6,072,050), the HB2 promoter (U.S. Pat. No.
6,177,611) and the SAMS promoter (US20030226166 and SEQ ID NO: 87
and biologically active variants and fragments thereof); each of
which is herein incorporated by reference. In addition, as
discussed elsewhere herein, various enhancers can be used with
these promoters including, for example, the ubiquitin intron (i.e,
the maize ubiquitin intron 1 (see, e.g., NCBI sequence S94464), the
omega enhancer or the omega prime enhancer (Gallie et al. (1989)
Molecular Biology of RNA ed. Cech (Liss, N.Y.) 237-256 and Gallie
et al. Gene (1987) 60:217-25), or the 35S enhancer; each of which
is incorporated by reference.
[0221] The expression cassette can also comprise a selectable
marker gene for the selection of transformed cells. Selectable
marker genes are utilized for the selection of transformed cells or
tissues. Marker genes include genes encoding antibiotic resistance,
such as those encoding neomycin phosphotransferase II (NEO) and
hygromycin phosphotransferase (HPT), as well as genes conferring
resistance to herbicidal compounds, such as glufosinate ammonium,
bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D).
Additional selectable markers include phenotypic markers such as
.beta.-galactosidase and fluorescent proteins such as green
fluorescent protein (GFP) (Su et al. (2004) Biotechnol Bioeng 85:
610-9 and Fetter et al. (2004) Plant Cell 16: 215-28), cyan
florescent protein (CYP) (Bolte et al. (2004) J. Cell Science 117:
943-54 and Kato et al. (2002) Plant Physiol 129: 913-42), and
yellow fluorescent protein (PhiYFP from Evrogen, see, Bolte et al.
(2004) J. Cell Science 117: 943-54). For additional selectable
markers, see generally Yarranton (1992) Curr. Opin. Biotech. 3:
506-511; Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA
89: 6314-6318; Yao et al. (1992) Cell 71: 63-72; Reznikoff (1992)
Mol. Microbiol. 6: 2419-2422; Barkley et al. (1980) in The Operon,
pp. 177-220; Hu et al. (1987) Cell 48: 555-566; Brown et al. (1987)
Cell 49: 603-612; Figge et al. (1988) Cell 52: 713-722; Deuschle et
al. (1989) Proc. Natl. Acad. Aci. USA 86: 5400-5404; Fuerst et al.
(1989) Proc. Natl. Acad. Sci. USA 86: 2549-2553; Deuschle et al.
(1990) Science 248: 480-483; Gossen (1993) Ph.D. Thesis, University
of Heidelberg; Reines et al. (1993) Proc. Natl. Acad. Sci. USA 90:
1917-1921; Labow et al. (1990) Mol. Cell. Biol. 10: 3343-3356;
Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA 89: 3952-3956;
Baim et al. (1991) Proc. Natl. Acad. Sci. USA 88: 5072-5076;
Wyborski et al. (1991) Nucleic Acids Res. 19: 4647-4653;
Hillenand-Wissman (1989) Topics Mol. Struc. Biol. 10: 143-162;
Degenkolb et al. (1991) Antimicrob. Agents Chemother. 35:
1591-1595; Kleinschnidt et al. (1988) Biochemistry 27: 1094-1104;
Bonin (1993) Ph.D. Thesis, University of Heidelberg; Gossen et al.
(1992) Proc. Natl. Acad. Sci. USA 89: 5547-5551; Oliva et al.
(1992) Antimicrob. Agents Chemother. 36: 913-919; Hlavka et al.
(1985) Handbook of Experimental Pharmacology, Vol. 78
(Springer-Verlag, Berlin); Gill et al. (1988) Nature 334: 721-724.
Such disclosures are herein incorporated by reference. The above
list of selectable marker genes is not meant to be limiting. Any
selectable marker gene can be used, including the GAT gene and/or
HRA gene.
[0222] Methods are known in the art of increasing the expression
level of a polypeptide in a plant or plant cell, for example, by
inserting into the polypeptide coding sequence one or two G/C-rich
codons (such as GCG or GCT) immediately adjacent to and downstream
of the initiating methionine ATG codon. Where appropriate, the
polynucleotides may be modified for increased expression in the
transformed plant. That is, the polynucleotides can be synthesized
substituting in the polypeptide coding sequence one or more codons
which are less frequently utilized in plants for codons encoding
the same amino acid(s) which are more frequently utilized in
plants, and introducing the modified coding sequence into a plant
or plant cell and expressing the modified coding sequence. See,
e.g., Campbell and Gown (1990) Plant Physiol. 92: 1-11 for a
discussion of host-preferred codon usage. Methods are available in
the art for synthesizing plant-preferred genes. See, e.g., U.S.
Pat. Nos. 5,380,831, and 5,436,391, and Murray et al. (1989)
Nucleic Acids Res. 17: 477-498, herein incorporated by reference.
Embodiments comprising such modifications are also a feature
disclosed.
[0223] Additional sequence modifications are known to enhance gene
expression in a cellular host. These include elimination of
sequences encoding spurious polyadenylation signals, exon-intron
splice site signals, transposon-like repeats, and other such
well-characterized sequences that may be deleterious to gene
expression. The G-C content of the sequence may be adjusted to
levels average for a given cellular host, as calculated by
reference to known genes expressed in the host cell. When possible,
the sequence is modified to avoid predicted hairpin secondary mRNA
structures. "Enhancers" such as the CaMV 35S enhancer may also be
used (see, e.g., Benfey et al. (1990) EMBO J. 9: 1685-96), or other
enhancers may be used. For example, the sequence set forth in SEQ
ID NO: 1, 72, 79, 84, 85, 88, or 89 or a biologically active
variant or fragment thereof can be used. See also U.S. Utility
application Ser. No. 11/508,045, entitled "Methods and Compositions
for the Expression of a Polynucleotide of Interest." As used
herein, an enhancer, when operably linked to an appropriate
promoter, will modulate the level of transcription of an operably
linked polynucleotide of interest. Biologically active fragments
and variants of the enhancer domain may retain the biological
activity of modulating (increase or decrease) the level of
transcription when operably linked to an appropriate promoter.
[0224] Generally, variants of a particular polynucleotide will have
at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence
identity to another polynucleotide as determined by sequence
alignment programs and parameters. Variants of a particular
polynucleotides also include those encoding a polypeptide having at
least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence
identity to a reference polypeptide as determined by sequence
alignment programs and parameters. Polypeptide variants include
those encoded by variant polynucleotides, and those having at least
about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to
a reference polypeptide as determined by sequence alignment
programs and parameters.
[0225] The expression cassette may additionally contain 5' leader
sequences. Such leader sequences can act to enhance translation.
Translation leaders are known in the art and include: picornavirus
leaders, for example, EMCV leader (Encephalomyocarditis 5'
noncoding region) (Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci.
USA 86: 6126-6130); potyvirus leaders, for example, TEV leader
(Tobacco Etch Virus) (Gallie et al. (1995) Gene 165(2): 233-238),
MDMV leader (Maize Dwarf Mosaic Virus) (Virology 154: 9-20), and
human immunoglobulin heavy-chain binding protein (BiP) (Macejak et
al. (1991) Nature 353: 90-94); untranslated leader from the coat
protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling et al.
(1987) Nature 325: 622-625); tobacco mosaic virus leader (TMV)
(Gallie et al. (1989) in Molecular Biology of RNA, ed. Cech (Liss,
N.Y.), pp. 237-256); and maize chlorotic mottle virus leader (MCMV)
(Lommel et al. (1991) Virology 81: 382-385). See also, Della-Cioppa
et al. (1987) Plant Physiol. 84: 965-968.
[0226] In preparing the expression cassette, the various
polynucleotide fragments may be manipulated, so as to provide for
sequences to be in the proper orientation and, as appropriate, in
the proper reading frame. Toward this end, adapters or linkers may
be employed to join the fragments or other manipulations may be
involved to provide for convenient restriction sites, removal of
superfluous material such as the removal of restriction sites, or
the like. For this purpose, in vitro mutagenesis, primer repair,
restriction, annealing, resubstitutions, e.g., transitions and
transversions, may be involved. Standard recombinant DNA and
molecular cloning techniques used herein are well known in the art
and are described more fully, for example, in Sambrook et al.
(1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor
Laboratory Press, Cold Spring Harbor) (also known as
"Maniatis").
[0227] In some embodiments, the polynucleotide of interest is
targeted to the chloroplast for expression. In this manner, where
the polynucleotide of interest is not directly inserted into the
chloroplast, the expression cassette will additionally contain a
nucleic acid encoding a transit peptide to direct the gene product
of interest to the chloroplasts. Such transit peptides are known in
the art. See, e.g., Von Heijne et al. (1991) Plant Mol. Biol. Rep.
9: 104-126; Clark et al. (1989) J. Biol. Chem. 264: 17544-17550;
Della-Cioppa et al. (1987) Plant Physiol. 84: 965-968; Romer et al.
(1993) Biochem. Biophys. Res. Commun 196: 1414-1421; and Shah et
al. (1986) Science 233: 478-481.
[0228] Chloroplast targeting sequences are known in the art and
include the chloroplast small subunit of ribulose-1,5-bisphosphate
carboxylase (Rubisco) (de Castro Silva Filho et al. (1996) Plant
Mol. Biol. 30: 769-780; Schnell et al. (1991) J. Biol. Chem.
266(5): 3335-3342); 5-(enolpyruvyl)shikimate-3-phosphate synthase
(EPSPS) (Archer et al. (1990) J. Bioenerg Biomemb. 22(6): 789-810);
tryptophan synthase (Zhao et al. (1995) J. Biol. Chem. 270(11):
6081-6087); plastocyanin (Lawrence et al. (1997) J. Biol. Chem.
272(33): 20357-20363); chorismate synthase (Schmidt et al. (1993)
J. Biol. Chem. 268(36): 27447-27457); and the light harvesting
chlorophyll a/b binding protein (LHBP) (Lamppa et al. (1988) J.
Biol. Chem. 263: 14996-14999). See also Von Heijne et al. (1991)
Plant Mol. Biol. Rep. 9: 104-126; Clark et al. (1989) J. Biol.
Chem. 264: 17544-17550; Della-Cioppa et al. (1987) Plant Physiol.
84: 965-968; Romer et al. (1993) Biochem. Biophys. Res. Commun 196:
1414-1421; and Shah et al. (1986) Science 233: 478-481.
[0229] Methods for transformation of chloroplasts are known in the
art. See, e.g., Svab et al. (1990) Proc. Natl. Acad. Sci. USA 87:
8526-8530; Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:
913-917; Svab and Maliga (1993) EMBO J. 12: 601-606. The method
relies on particle gun delivery of DNA containing a selectable
marker and targeting of the DNA to the plastid genome through
homologous recombination. Additionally, plastid transformation can
be accomplished by transactivation of a silent plastid-borne
transgene by tissue-preferred expression of a nuclear-encoded and
plastid-directed RNA polymerase. Such a system has been reported in
McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91: 7301-7305.
[0230] The polynucleotides of interest to be targeted to the
chloroplast may be optimized for expression in the chloroplast to
account for differences in codon usage between the plant nucleus
and this organelle. In this manner, the polynucleotide of interest
may be synthesized using chloroplast-preferred codons. See, e.g.,
U.S. Pat. No. 5,380,831, herein incorporated by reference.
[0231] Methods of Introducing
[0232] Compositions include plants generated by introducing a
polypeptide or polynucleotide into a plant. "Introducing" is
intended to mean presenting to the plant the polynucleotide or
polypeptide in such a manner that the sequence gains access to the
interior of a cell of the plant. The methods do not depend on a
particular method for introducing a sequence into a plant, only
that the polynucleotide or polypeptides gains access to the
interior of at least one cell of the plant. Methods for introducing
polynucleotide or polypeptides into plants are known in the art
including, but not limited to, stable transformation methods,
transient transformation methods, virus-mediated methods, and
breeding.
[0233] "Stable transformation" is intended to mean that the
nucleotide construct introduced into a plant integrates into the
genome of the plant and is capable of being inherited by the
progeny thereof "Transient transformation" is intended to mean that
a polynucleotide is introduced into the plant and does not
integrate into the genome of the plant or a polypeptide is
introduced into a plant.
[0234] Transformation protocols as well as protocols for
introducing polypeptides or polynucleotide sequences into plants
may vary depending on the type of plant or plant cell (i.e.,
monocot or dicot) targeted for transformation. Suitable methods of
introducing polypeptides and polynucleotides into plant cells
include microinjection (Crossway et al. (1986) Biotechniques 4:
320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad.
Sci. USA 83: 5602-5606, Agrobacterium-mediated transformation (U.S.
Pat. No. 5,563,055 and U.S. Pat. No. 5,981,840), direct gene
transfer (Paszkowski et al. (1984) EMBO J. 3: 2717-2722), and
ballistic particle acceleration (see, for example, U.S. Pat. No.
4,945,050; U.S. Pat. No. 5,879,918; U.S. Pat. No. 5,886,244; and,
U.S. Pat. No. 5,932,782; Tomes et al. (1995) in Plant Cell, Tissue,
and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips
(Springer-Verlag, Berlin); McCabe et al. (1988) Biotechnology 6:
923-926); and Lec1 transformation (WO 00/28058). Also see
Weissinger et al. (1988) Ann. Rev. Genet. 22: 421-477; Sanford et
al. (1987) Particulate Science and Technology 5: 27-37 (onion);
Christou et al. (1988) Plant Physiol. 87: 671-674 (soybean); McCabe
et al. (1988) Bio/Technology 6: 923-926 (soybean); Finer and
McMullen (1991) In Vitro Cell Dev. Biol. 27P: 175-182 (soybean);
Singh et al. (1998) Theor. Appl. Genet. 96:319-324 (soybean); Datta
et al. (1990) Biotechnology 8: 736-740 (rice); Klein et al. (1988)
Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein et al.
(1988) Biotechnology 6:559-563 (maize); U.S. Pat. Nos. 5,240,855;
5,322,783; and, 5,324,646; Klein et al. (1988) Plant Physiol. 91:
440-444 (maize); Fromm et al. (1990) Biotechnology 8: 833-839
(maize); protocols published electronically by "IP.com" under the
permanent publication identifiers IPCOM000033402D, IPCOM000033402D,
and IPCOM000033402D and available at the "IP.com" website (cotton);
Hooykaas-Van Slogteren et al. (1984) Nature (London) 311: 763-764;
U.S. Pat. No. 5,736,369 (cereals); Bytebier et al. (1987) Proc.
Natl. Acad. Sci. USA 84: 5345-5349 (Liliaceae); De Wet et al.
(1985) in The Experimental Manipulation of Ovule Tissues, ed.
Chapman et al. (Longman, N.Y.), pp. 197-209 (pollen); Kaeppler et
al. (1990) Plant Cell Reports 9: 415-418 and Kaeppler et al. (1992)
Theor. Appl. Genet. 84: 560-566 (whisker-mediated transformation);
D'Halluin et al. (1992) Plant Cell 4: 1495-1505 (electroporation);
Li et al. (1993) Plant Cell Reports 12: 250-255 and Christou and
Ford (1995) Annals of Botany 75: 407-413 (rice); Osjoda et al.
(1996) Nature Biotechnology 14: 745-750 (maize via Agrobacterium
tumefaciens); all of which are herein incorporated by
reference.
[0235] In specific embodiments, herbicide-tolerance or other
desirable sequences can be provided to a plant using a variety of
transient transformation methods. Such transient transformation
methods include, but are not limited to, the introduction of the
polypeptide or variants and fragments thereof directly into the
plant or the introduction of a transcript into the plant. Such
methods include, for example, microinjection or particle
bombardment. See, e.g., Crossway et al. (1986) Mol Gen. Genet. 202:
179-185; Nomura et al. (1986) Plant Sci. 44: 53-58; Hepler et al.
(1994) Proc. Natl. Acad. Sci. 91: 2176-2180 and Hush et al. (1994)
The Journal of Cell Science 107: 775-784, all of which are herein
incorporated by reference. Alternatively, a herbicide-tolerance
polynucleotide can be transiently transformed into the plant using
techniques known in the art. Such techniques include viral vector
system and the precipitation of the polynucleotide in a manner that
precludes subsequent release of the DNA. Thus, the transcription
from the particle-bound DNA can occur, but the frequency with which
it is released to become integrated into the genome is greatly
reduced. Such methods include the use particles coated with
polyethylimine (PEI; Sigma #P3143).
[0236] In other embodiments, polynucleotides may be introduced into
plants by contacting plants with a virus or viral nucleic acids.
Generally, such methods involve incorporating a nucleotide
construct within a viral DNA or RNA molecule. It is recognized that
a polypeptide of interest may be initially synthesized as part of a
viral polyprotein, which later may be processed by proteolysis in
vivo or in vitro to produce the desired recombinant protein.
Further, it is recognized that useful promoters may include
promoters utilized for transcription by viral RNA polymerases.
Methods for introducing polynucleotides into plants and expressing
a polypeptide encoded thereby, involving viral DNA or RNA
molecules, are known in the art. See, e.g., U.S. Pat. Nos.
5,889,191, 5,889,190, 5,866,785, 5,589,367, 5,316,931, and Porta et
al. (1996) Molecular Biotechnology 5: 209-221; herein incorporated
by reference.
[0237] Methods are known in the art for the targeted insertion of a
polynucleotide at a specific location in the plant genome. In one
embodiment, the insertion of the polynucleotide at a desired
genomic location is achieved using a site-specific recombination
system. See, e.g., WO99/25821, WO99/25854, WO99/25840, WO99/25855,
and WO99/25853, all of which are herein incorporated by reference.
Briefly, a polynucleotide can be contained in transfer cassette
flanked by two non-recombinogenic recombination sites. The transfer
cassette is introduced into a plant having stably incorporated into
its genome a target site which is flanked by two non-recombinogenic
recombination sites that correspond to the sites of the transfer
cassette. An appropriate recombinase is provided and the transfer
cassette is integrated at the target site. The polynucleotide of
interest is thereby integrated at a specific chromosomal position
in the plant genome.
[0238] The cells that have been transformed may be grown into
plants in accordance with conventional ways. See, e.g., McCormick
et al. (1986) Plant Cell Reports 5: 81-84. These plants may then be
grown, and either pollinated with the same transformed strain or
different strains, and the resulting progeny having constitutive
expression of the desired phenotypic characteristic identified. Two
or more generations may be grown to ensure that expression of the
desired phenotypic characteristic is stably maintained and
inherited and then seeds harvested to ensure expression of the
desired phenotypic characteristic has been achieved. In this
manner, transformed seed (also referred to as "transgenic seed")
having a polynucleotide conferring tolerance to a PPO inhibitor
stably incorporated into their genome are provided.
[0239] In specific embodiments, a polypeptide or the polynucleotide
of interest is introduced into the plant cell. Subsequently, a
plant cell having the introduced sequence is selected using methods
known to those of skill in the art such as, but not limited to,
Southern blot analysis, DNA sequencing, PCR analysis, or phenotypic
analysis. A plant or plant part altered or modified by the
foregoing embodiments is grown under plant forming conditions for a
time sufficient to modulate the concentration and/or activity of
polypeptides in the plant. Plant forming conditions are well known
in the art and discussed briefly elsewhere herein.
[0240] It is also recognized that the level and/or activity of a
polypeptide of interest may be modulated by employing a
polynucleotide that is not capable of directing, in a transformed
plant, the expression of a protein or an RNA. For example, the
polynucleotides may be used to design polynucleotide constructs
that can be employed in methods for altering or mutating a genomic
nucleotide sequence in an organism. Such polynucleotide constructs
include, but are not limited to, RNA:DNA vectors, RNA:DNA
mutational vectors, RNA:DNA repair vectors, mixed-duplex
oligonucleotides, self-complementary RNA:DNA oligonucleotides, and
recombinogenic oligonucleobases. Such nucleotide constructs and
methods of use are known in the art. See, U.S. Pat. Nos. 5,565,350;
5,731,181; 5,756,325; 5,760,012; 5,795,972; and 5,871,984; all of
which are herein incorporated by reference. See also, WO 98/49350,
WO 99/07865, WO 99/25821, and Beetham et al. (1999) Proc. Natl.
Acad. Sci. USA 96: 8774-8778; herein incorporated by reference.
[0241] It is therefore recognized that methods disclosed do not
depend on the incorporation of the entire polynucleotide into the
genome, only that the plant or cell thereof is altered as a result
of the introduction of the polynucleotide into a cell. In one
embodiment, the genome may be altered following the introduction of
the polynucleotide into a cell. For example, the polynucleotide, or
any part thereof, may incorporate into the genome of the plant.
Alterations to the genome include, but are not limited to,
additions, deletions, and substitutions of nucleotides into the
genome. While the methods disclosed do not depend on additions,
deletions, and substitutions of any particular number of
nucleotides, it is recognized that such additions, deletions, or
substitutions comprises at least one nucleotide.
[0242] Plants may be produced by any suitable method, including
breeding. Plant breeding can be used to introduce desired
characteristics (e.g., a stably incorporated transgene or a genetic
variant or genetic alteration of interest) into a particular plant
line of interest, and can be performed in any of several different
ways. Pedigree breeding starts with the crossing of two genotypes,
such as an elite line of interest and one other elite inbred line
having one or more desirable characteristics (i.e., having stably
incorporated a polynucleotide of interest, having a modulated
activity and/or level of the polypeptide of interest, etc.) which
complements the elite plant line of interest. If the two original
parents do not provide all the desired characteristics, other
sources can be included in the breeding population. In the pedigree
method, superior plants are selfed and selected in successive
filial generations. After a sufficient amount of inbreeding,
successive filial generations will serve to increase seed of the
developed inbred. In specific embodiments, the inbred line
comprises homozygous alleles at about 95% or more of its loci.
Various techniques known in the art can be used to facilitate and
accelerate the breeding (e.g., backcrossing) process, including,
for example, the use of a greenhouse or growth chamber with
accelerated day/night cycles, the analysis of molecular markers to
identify desirable progeny, and the like.
[0243] In addition to being used to create a backcross conversion,
backcrossing can also be used in combination with pedigree breeding
to modify an elite line of interest and a hybrid that is made using
the modified elite line. As discussed previously, backcrossing can
be used to transfer one or more specifically desirable traits from
one line, the donor parent, to an inbred called the recurrent
parent, which has overall good agronomic characteristics yet lacks
that desirable trait or traits. However, the same procedure can be
used to move the progeny toward the genotype of the recurrent
parent but at the same time retain many components of the
non-recurrent parent by stopping the backcrossing at an early stage
and proceeding with selfing and selection. For example, an F1, such
as a commercial hybrid, is created. This commercial hybrid may be
backcrossed to one of its parent lines to create a BC1 or BC2.
Progeny are selfed and selected so that the newly developed inbred
has many of the attributes of the recurrent parent and yet several
of the desired attributes of the non-recurrent parent. This
approach leverages the value and strengths of the recurrent parent
for use in new hybrids and breeding.
[0244] Therefore, a method of making a backcross conversion of an
inbred line of interest comprising the steps of crossing a plant
from the inbred line of interest with a donor plant comprising at
least one mutant gene or transgene conferring a desired trait
(e.g., herbicide tolerance), selecting an F1 progeny plant
comprising the mutant gene or transgene conferring the desired
trait, and backcrossing the selected F1 progeny plant to a plant of
the inbred line of interest is provided. This method may further
comprise the step of obtaining a molecular marker profile of the
inbred line of interest and using the molecular marker profile to
select for a progeny plant with the desired trait and the molecular
marker profile of the inbred line of interest. In the same manner,
this method may be used to produce an F1 hybrid seed by adding a
final step of crossing the desired trait conversion of the inbred
line of interest with a different plant to make F1 hybrid seed
comprising a mutant gene or transgene conferring the desired
trait.
[0245] Recurrent selection is a method used in a plant breeding
program to improve a population of plants. The method entails
individual plants cross pollinating with each other to form
progeny. The progeny are grown and the superior progeny selected by
any number of selection methods, which include individual plant,
half-sib progeny, full-sib progeny, selfed progeny and topcrossing.
The selected progeny are cross-pollinated with each other to form
progeny for another population. This population is planted and
again superior plants are selected to cross pollinate with each
other. Recurrent selection is a cyclical process and therefore can
be repeated as many times as desired. The objective of recurrent
selection is to improve the traits of a population. The improved
population can then be used as a source of breeding material to
obtain inbred lines to be used in hybrids or used as parents for a
synthetic cultivar. A synthetic cultivar is the resultant progeny
formed by the intercrossing of several selected inbreds.
[0246] Mass selection is a useful technique when used in
conjunction with molecular marker enhanced selection. In mass
selection seeds from individuals are selected based on phenotype
and/or genotype. These selected seeds are then bulked and used to
grow the next generation. Bulk selection requires growing a
population of plants in a bulk plot, allowing the plants to
self-pollinate, harvesting the seed in bulk and then using a sample
of the seed harvested in bulk to plant the next generation. Instead
of self pollination, directed pollination could be used as part of
the breeding program.
[0247] Mutation breeding is one of many methods that could be used
to introduce new traits into an elite line. Mutations that occur
spontaneously or are artificially induced can be useful sources of
variability for a plant breeder. The goal of artificial mutagenesis
is to increase the rate of mutation for a desired characteristic.
Mutation rates can be increased by many different means including
temperature, long-term seed storage, tissue culture conditions,
radiation such as X-rays, Gamma rays (e.g., cobalt 60 or cesium
137), neutrons, (product of nuclear fission of uranium 235 in an
atomic reactor), Beta radiation (emitted from radioisotopes such as
phosphorus 32 or carbon 14), or ultraviolet radiation (typically
from 2500 to 2900 nm), or chemical mutagens (such as base analogues
(5-bromo-uracil), related compounds (8-ethoxy caffeine),
antibiotics (streptonigrin), alkylating agents (sulfur mustards,
nitrogen mustards, epoxides, ethylenamines, sulfates, sulfonates,
sulfones, lactones), azide, hydroxylamine, nitrous acid, or
acridines. Once a desired trait is observed through mutagenesis the
trait may then be incorporated into existing germplasm by
traditional breeding techniques, such as backcrossing. Details of
mutation breeding can be found in "Principals of Cultivar
Development" Fehr, 1993 Macmillan Publishing Company the disclosure
of which is incorporated herein by reference. In addition,
mutations created in other lines may be used to produce a backcross
conversion of elite lines that comprises such mutations.
Methods of Modulating Expression
[0248] In some embodiments, the activity and/or level of the
polypeptide is modulated (i.e., increased or decreased). An
increase in the level and/or activity of the polypeptide can be
achieved by providing the polypeptide to the plant. As discussed
elsewhere herein, many methods are known the art for providing a
polypeptide to a plant including, but not limited to, direct
introduction of the polypeptide into the plant, introducing into
the plant (transiently or stably) a polynucleotide construct
encoding a polypeptide having the desired activity.
Methods of Controlling Weeds
[0249] Methods are provided for controlling weeds in an area of
cultivation, preventing the development or the appearance of
herbicide resistant weeds in an area of cultivation, producing a
crop, and increasing crop safety. The term "controlling," and
derivations thereof, for example, as in "controlling weeds" refers
to one or more of inhibiting the growth, germination, reproduction,
and/or proliferation of and/or killing, removing, destroying, or
otherwise diminishing the occurrence and/or activity of a weed.
[0250] The PPO inhibitor plants display tolerance to herbicides and
therefore allow for the application of one or more herbicides at
rates that would significantly damage control plants and further
allow for the application of combinations of herbicides at lower
concentrations than normally applied which still continue to
selectively control weeds. In addition, the PPO inhibitor-tolerant
plants can be used in combination with herbicide blends technology
and thereby make the application of chemical pesticides more
convenient, economical, and effective for the producer.
[0251] The methods comprise planting the area of cultivation with
PPO inhibitor-tolerant crop seeds or plants, and applying to any
crop, crop part, weed or area of cultivation thereof an effective
amount of a PPO inhibitor containing herbicide of interest. It is
recognized that the herbicide can be applied before and/or after
the crop is planted in the area of cultivation. Such herbicide
applications can include an application of any PPO inhibitor
chemistry, or any combination thereof. In some examples, a diphenyl
ether, triazolinone, N-phenylphthalimide, pyrimidindione and/or
oxadiazole containing herbicide formulation is applied. In some
examples the herbicide formulation comprises acifluorfen,
fomesafen, oxyfluorfen, lactofen, sulfentrazone, carfentrasone,
azafeniden flumiclorac, flumioxazin, oxadiazon, fluthiacet,
benzfendizone, butagenacil and/or saflufenacil.
[0252] In other examples, the combination of herbicides comprises a
glyphosate, a glufosinate, a dicamba, a bialaphos, a
phosphinothricin, a protox inhibitor, a sulfonylurea, an
imidazolinone, a chlorsulfuron, an imazapyr, a chlorimuron-ethyl, a
quizalofop, an HPPD, a PPO inhibitor, and/or a fomesafen, or
combinations thereof, wherein said effective amount is tolerated by
the crop and controls weeds. Any effective amount of these
herbicides can be applied, wherein the effective amount is any
amount that differentiates between plant cells, plants, and/or seed
comprising a PPO inhibitor tolerance allele, a PPO inhibitor
polynucleotide, and/or a polynucleotide encoding an ABC transporter
protein that confers tolerance to herbicide formulations comprising
a PPO inhibitor. In some examples the herbicides are applied
simultaneously, in some examples the herbicides are applied
sequentially, in some examples the herbicides are applied as
pre-emergent treatments, in some examples the herbicides are
applied as post-emergent treatments, in some examples the
herbicides are applied as a combination of pre- and post-emergent
treatments.
[0253] In some examples, the method of controlling weeds comprises
planting the area with PPO inhibitor tolerant crop seeds or plants
and applying to the crop, crop part, seed of said crop or the area
under cultivation, an effective amount of a herbicide, wherein said
effective amount comprises an amount that is not tolerated by a
control crop when applied to the control crop, crop part, seed or
the area of cultivation, wherein the control crop does not express
a polynucleotide that encodes an herbicide-tolerance polypeptide.
In specific embodiments, combinations of herbicides may be used,
such as when an additional tolerance trait is incorporated into the
plant.
[0254] In another embodiment, the method of controlling weeds
comprises planting the area with a PPO inhibitor-tolerant crop
seeds or plant and applying to the crop, crop part, seed of said
crop or the area under cultivation, an effective amount of a
herbicide, wherein said effective amount comprises a level that is
above the recommended label use rate for the crop, wherein said
effective amount is tolerated when applied to the PPO
inhibitor-tolerant crop, crop part, seed, or the area of
cultivation thereof.
[0255] Any herbicide can be applied to the tolerant crop, crop
part, or the area of cultivation containing said crop plant.
Classifications of herbicides (i.e., the grouping of herbicides
into classes and subclasses) is well-known in the art and includes
classifications by HRAC (Herbicide Resistance Action Committee) and
WSSA (the Weed Science Society of America) (see also, Retzinger and
Mallory-Smith (1997) Weed Technology 11: 384-393). An abbreviated
version of the HRAC classification (with notes regarding the
corresponding WSSA group) is set forth below:
TABLE-US-00001 HRAC WSSA Group Mode of Action Chemical Family
Active Ingredient Group A Inhibition of acetyl Aryloxyphenoxy-
clodinafop- 1 CoA carboxylase propionate "FOPs" propargyl (ACCase)
cyhalofop-butyl diclofop-methyl fenoxaprop-P-ethyl
fluazifop-P-butyl haloxyfop-R- methyl propaquizafop
quizalofop-P-ethyl Cyclohexanedione alloxydim "DIMs" butroxydim
clethodim cycloxydim profoxydim sethoxydim tepraloxydin tralkoxydim
Phenylpyrazoline "DEN" pinoxaden B Inhibition of Sulfonylurea
amidosulfuron 2 acetolactate azimsulfuron synthase ALS
bensulfuron-methyl (acetohydroxyacid chlorimuron-ethyl synthase
AHAS) chlorsulfuron cinosulfuron cyclosulfamuron ethametsulfuron-
methyl ethoxysulfuron flazasulfuron flupyrsulfuron- methyl-Na
foramsulfuron halosulfuron- methyl imazosulfuron iodosulfuron
mesosulfuron metsulfuron-methyl nicosulforon oxasulforon
primisulfuron- methyl prosulfuron pyrazosulfuron- ethyl rimsulfuron
sulfometuron- methyl sulfosulfuron thifensulfuron- methyl
triasulfuron tribenuron-methyl trifloxysulfuron triflusulfuron-
methyl tritosulfuron Imidazolinone imazapic imazamethabenz- methyl
imazamox imazapyr imazaquin imazethapyr Triazolopyrimidine
cloransulam-methyl diclosulam florasulam flumetsulam metosulam
penoxsulam Pyrimidinyl(thio)benzoate bispyribac-Na pyribenzoxim
pyriftalid pyrithiobac-Na pyriminobac- methyl
Sulfonylaminocarbonyl- flucarbazone-Na triazolinone
propoxycarbazone- Na C1 Inhibition of Triazine ametryne 5
photosynthesis at atrazine photosystem II cyanazine desmetryne
dimethametryne prometon prometryne propazine simazine simetryne
terbumeton terbuthylazine terbutryne trietazine Triazinone
hexazinone metamitron metribuzin Triazolinone amicarbazone Uracil
bromacil lenacil terbacil Pyridazinone pyrazon = chloridazon
Phenyl-carbamate desmedipham phenmedipham C2 Inhibition of Urea
chlorobromuron 7 photosynthesis at chlorotoluron photosystem II
chloroxuron dimefuron diuron ethidimuron fenuron fluometuron (see
F3) isoproturon isouron linuron methabenzthiazuron metobromuron
metoxuron monolinuron neburon siduron tebuthiuron Amide propanil
pentanochlor C3 Inhibition of Nitrile bromofenoxim 6 photosynthesis
at bromoxynil photosystem II ioxynil Benzothiadiazinone bentazon
Phenyl-pyridazine pyridate pyridafol D Photosystem-I- Bipyridylium
diquat 22 electron diversion paraquat E Inhibition of Diphenylether
acifluorfen-Na 14 protoporphyrinogen bifenox oxidase (PPO)
chlomethoxyfen fluoroglycofen- ethyl fomesafen halosafen lactofen
oxyfluorfen Phenylpyrazole fluazolate pyraflufen-ethyl
N-phenylphthalimide cinidon-ethyl flumioxazin flumiclorac-pentyl
Thiadiazole fluthiacet-methyl thidiazimin Oxadiazole oxadiazon
oxadiargyl Triazolinone azafenidin carfentrazone-ethyl
sulfentrazone Oxazolidinedione pentoxazone Pyrimidindione
benzfendizone butafenacil Other pyraclonil profluazol
flufenpyr-ethyl F1 Bleaching: Pyridazinone norflurazon 12
Inhibition of carotenoid biosynthesis at the phytoene desaturase
step (PDS) Pyridinecarboxamide diflufenican picolinafen Other
beflubutamid fluridone flurochloridone flurtamone F2 Bleaching:
Triketone mesotrione 27 Inhibition of 4- sulcotrione hydroxyphenyl-
pyruvate- dioxygenase (4- HPPD) Isoxazole isoxachlortole
isoxaflutole Pyrazole benzofenap pyrazolynate pyrazoxyfen Other
benzobicyclon F3 Bleaching: Triazole amitrole 11 Inhibition of (in
vivo inhibition carotenoid of lycopene cyclase biosynthesis
(unknown target) Isoxazolidinone clomazone 13 Urea fluometuron (see
C2) Diphenylether aclonifen G Inhibition of EPSP Glycine glyphosate
9 synthase sulfosate H Inhibition of Phosphinic acid glufosinate-
10 glutamine ammonium synthetase bialaphos = bilanaphos I
Inhibition of DHP Carbamate asulam 18 (dihydropteroate) synthase K1
Microtubule Dinitroaniline benefin = 3 assembly inhibition
benfluralin butralin dinitramine ethalfluralin oryzalin
pendimethalin trifluralin Phosphoroamidate amiprophos-methyl
butamiphos Pyridine dithiopyr thiazopyr Benzamide propyzamide =
pronamide tebutam Benzoic acid DCPA = chlorthal- dimethyl K2
Inhibition of Carbamate chlorpropham 23 mitosis/ propham
microtubule carbetamide organisation K3 Inhibition of
Chloroacetamide acetochlor 15 VLCFAs alachlor (Inhibition of cell
butachlor division) dimethachlor dimethanamid metazachlor
metolachlor pethoxamid pretilachlor propachlor propisochlor
thenylchlor Acetamide diphenamid napropamide naproanilide
Oxyacetamide flufenacet mefenacet Tetrazolinone fentrazamide Other
anilofos
cafenstrole piperophos L Inhibition of cell Nitrile dichlobenil 20
wall (cellulose) chlorthiamid synthesis Benzamide isoxaben 21
Triazolocarboxamide flupoxam Quinoline carboxylic acid quinclorac
(for 26 monocots) (also group O) M Uncoupling Dinitrophenol DNOC 24
(Membrane dinoseb disruption) dinoterb N Inhibition of lipid
Thiocarbamate butylate 8 synthesis - not cycloate ACCase inhibition
dimepiperate EPTC esprocarb molinate orbencarb pebulate
prosulfocarb thiobencarb = benthiocarb tiocarbazil triallate
vernolate Phosphorodithioate bensulide Benzofuran benfuresate
ethofumesate Chloro-Carbonic-acid TCA 26 dalapon flupropanate O
Action like indole Phenoxy-carboxylic-acid clomeprop 4 acetic acid
2,4-D (synthetic auxins) 2,4-DB dichlorprop = 2,4- DP MCPA MCPB
mecoprop = MCPP = CMPP Benzoic acid chloramben dicamba TBA Pyridine
carboxylic acid clopyralid fluroxypyr picloram triclopyr Quinoline
carboxylic acid quinclorac (also group L) quinmerac Other
benazolin-ethyl P Inhibition of auxin Phthalamate naptalam 19
transport Semicarbazone diflufenzopyr-Na Z Unknown (actual
Arylaminopropionic acid Flamprop-M- 25 mode of action
methyl/-isopropyl unknown, but likely that they differ in mode of
action between themselves and from other groups) Pyrazolium
difenzoquat 26 Organoarsenical DSMA 17 MSMA Other bromobutide 27
(chloro)-flurenol cinmethylin cumyluron dazomet dymron = daimuron
methyl-dimuron = methyl-dymron etobenzanid fosamine indanofan metam
oxaziclomefone oleic acid pelargonic acid pyributicarb
[0256] Herbicides can be classified by their mode of action and/or
site of action and can also be classified by the time at which they
are applied (e.g., pre-emergent or post-emergent), by the method of
application (e.g., foliar application or soil application), or by
how they are taken up by or affect the plant. Mode of action
generally refers to the metabolic or physiological process within
the plant that the herbicide inhibits or otherwise impairs, whereas
site of action generally refers to the physical location or
biochemical site within the plant where the herbicide acts or
directly interacts. Herbicides can be classified in various ways,
including by mode of action and/or site of action. Often, a
herbicide-tolerance gene that confers tolerance to a particular
herbicide or other chemical on a plant expressing it will also
confer tolerance to other herbicides or chemicals in the same class
or subclass, for example, a class or subclass set forth in the
table above. Thus, in some examples, a transgenic plant is tolerant
to more than one herbicide or chemical in the same class or
subclass, such as, for example, an inhibitor of PPO, a
sulfonylurea, or a synthetic auxin. In some examples the plant is
transgenic for one or more of the herbicide tolerance traits,
non-transgenic for one of more of the tolerance traits, or any
combination thereof.
[0257] Typically, the plants provided can tolerate treatment with
different types of herbicides (i.e., herbicides having different
modes of action and/or different sites of action) as well as with
higher amounts of herbicides than previously known plants, thereby
permitting improved weed management strategies that are recommended
in order to reduce the incidence and prevalence of
herbicide-tolerant weeds. Specific herbicide combinations can be
employed to effectively control weeds.
[0258] A transgenic crop plant which can be selected for use in
crop production based on the prevalence of herbicide-tolerant weed
species in the area where the transgenic crop is to be grown is
provided. Methods are known in the art for assessing the herbicide
tolerance of various weed species. Weed management techniques are
also known in the art, such as for example, crop rotation using a
crop that is tolerant to a herbicide to which the local weed
species are not tolerant. A number of entities monitor and publicly
report the incidence and characteristics of herbicide-tolerant
weeds, including the Herbicide Resistance Action Committee (HRAC),
the Weed Science Society of America, and various state agencies
(see, e.g., herbicide tolerance scores for various broadleaf weeds
from the 2004 Illinois Agricultural Pest Management Handbook), and
one of skill in the art would be able to use this information to
determine which crop and herbicide combinations should be used in a
particular location.
[0259] These entities also publish advice and guidelines for
preventing the development and/or appearance of and controlling the
spread of herbicide tolerant weeds (see, e.g., Owen and Hartzler
(2004), 2005 Herbicide Manual for Agricultural Professionals, Pub.
WC 92 Revised (Iowa State University Extension, Iowa State
University of Science and Technology, Ames, Iowa); Weed Control for
Corn, Soybeans, and Sorghum, Chapter 2 of "2004 Illinois
Agricultural Pest Management Handbook" (University of Illinois
Extension, University of Illinois at Urbana-Champaign, Ill.); Weed
Control Guide for Field Crops, MSU Extension Bulletin E434
(Michigan State University, East Lansing, Mich.)).
[0260] Also included are plant cells, plants, and/or seeds produced
by any of the foregoing methods.
[0261] The present invention is illustrated by the following
examples. The foregoing and following description of the present
invention and the various embodiments are not intended to be
limiting of the invention but rather are illustrative thereof.
Hence, it will be understood that the invention is not limited to
the specific details of these examples.
EXAMPLES
Example 1
Identification of Sulfentrazone Tolerant and Sensitive Soybean
Lines--Herbicide Screening Bioassay and Intergroup Association
Marker Based Diagnostic
[0262] Sulfentrazone is a PPO inhibitor and is the active
ingredient in Authority.RTM. herbicide. Authority.RTM. 75DF (FMC
Corp., Philadelphia, Pa., USA) is a 75% active ingredient
formulation of sulfentrazone containing no other active
ingredients.
Part 1: Herbicide Bioassay
[0263] One hundred sixteen (116) elite soybean lines were screened
for sulfentrazone tolerance using the following protocol. Seed of
soybean varieties with adequate seed quality, having greater than
85% warm germination were used.
Design and Replication:
[0264] After planting, entries were set up in a randomized complete
block design, blocked by replication. Three replications per
experiment were used. One or more of well established check variety
were included in the experiment, such as available public sector
check lines.
Non-tolerant check: Pioneer 9692, Asgrow A4715 Tolerant check:
Pioneer 9584, Syngenta S5960 Growing conditions were as follows
(greenhouse/growth chamber): 16 hr photoperiod @ 85.degree. F.
(w/75.degree. nighttime set back). Lighting is critical to the
success of the screening as stated below.
Method of Screening:
[0265] Four inch plastic pots were filled with a high quality
universal potting soil. Entries were planted 1 inch deep at the
rate of 5 seeds/pot. A bar-coded plastic stake was used to identify
each entry. After planting the pots were allowed to sit in
greenhouse overnight to acclimate to soil and improve germination.
The following day a sulfentrazone herbicide solution was slowly
poured over each pot and allowed to evenly soak through entire soil
profile. This ensured that each seed was exposed to an equal amount
of sulfentrazone. Pots were placed on aluminum trays and placed in
a greenhouse or growth chamber under high intensity light
conditions with photosynthetic photon flux density (PPFD) of at
least 500 .mu.mol/m/s. Proper lighting conditions were necessary
for this screening due to the nature of the PPO inhibitor used.
Pots were lightly watered so that herbicide was not leached from
the soil profile. After soybean emergence the pots were watered by
keeping aluminum trays filled with 3/4'' of water under each
pot.
Herbicide Solution:
[0266] A) Mix a stock solution of 0.926 g Authority.RTM. 75DF (FMC
Corp.), thoroughly dissolved in 1000 ml of water. [0267] B) Mix 10
ml of STOCK SOLUTION in 1000 ml of water to create final solution.
[0268] C) Pour 100 ml of FINAL SOLUTION over each pot.
Recording Data:
[0269] 10-14 days after treatment, plants were ready to be scored.
All scores were based on a comparison to the checks and evaluated
as follows: [0270] 9=Equivalent or better when compared to the
tolerant check [0271] 7=Very little damage or response noted.
[0272] 5=Intermediate response or damage [0273] 3=Major damage,
including stunting and foliar necrosis [0274] 1=Severe damage,
including severe stunting and necrosis; equivalent or worse when
compared to the non-tolerant check
[0275] Of the 116 soybean lines screened, 102 showed at least some
tolerance to sulfentrazone based herbicides and 11 showed high
sensitivity. A reference relevant to this protocol would be: Dayan
et al. (1997) `Soybean (Glycine max) cultivar differences in
response to sulfentrazone` Weed Science 45:634-641.
Part 2: Intergroup Analysis
[0276] An "Intergroup Allele Frequency Distribution" analysis was
conducted using GeneFlow.TM. version 7.0 software. An intergroup
allele frequency distribution analysis provides a method for
finding non-random distributions of alleles between two phenotypic
groups.
[0277] During processing, a contingency table of allele frequencies
was constructed and from this a G-statistic and probability were
calculated. The G statistic was adjusted by using the William's
correction factor. The probability value was adjusted to take into
account the fact that multiple tests are being done (thus, there is
some expected rate of false positives). The adjusted probability is
proportional to the probability that the observed allele
distribution differences between the two classes would occur by
chance alone. The lower that probability value, the greater the
likelihood that the tolerance phenotype and the marker will
co-segregate. A more complete discussion of the derivation of the
probability values can be found in the GeneFlow.TM. version 7.0
software documentation. See also Sokal and Rolf (1981), Biometry:
The Principles and Practices of Statistics in Biological Research,
2nd ed., San Francisco, W. H. Freeman and Co.
[0278] The underlying logic is that markers with significantly
different allele distributions between the tolerant and
non-tolerant groups (i.e., non-random distributions) might be
associated with the trait and can be used to separate them for
purposes of marker assisted selection of soybean lines with
previously uncharacterized tolerance or non-tolerance to
protoporphyrinogen oxidase inhibitors. The present analysis
examined one marker locus at a time and determined if the allele
distribution within the tolerant group is significantly different
from the allele distribution within the non-tolerant group. A
statistically different allele distribution is an indication that
the marker is linked to a locus that is associated with tolerance
or non-tolerance to protoporphyrinogen oxidase inhibitors. In this
analysis, unadjusted probabilities less than one are considered
significant (the marker and the phenotype show linkage
disequilibrium), and adjusted probabilities less than approximately
0.05 are considered highly significant. Allele classes represented
by less than 5 observations across both groups were not included in
the statistical analysis. In this analysis, 1043 marker loci had
enough observations for analysis.
[0279] This analysis compares the plants' phenotypic score with the
genotypes at the various loci. This type of intergroup analysis
neither generates nor requires any map data. Subsequently, map data
(for example, a composite soybean genetic map) is relevant in that
multiple significant markers that are also genetically linked can
be considered as collaborating evidence that a given chromosomal
region is associated with the trait of interest.
Results
[0280] Table 1 below provides a table listing the soybean markers
that demonstrated linkage disequilibrium with the tolerance to
protoporphyrinogen oxidase inhibitor phenotype. There were 1043
markers used in this analysis. Also indicated in that table are the
chromosomes on which the markers are located and their approximate
map position relative to other known markers, given in cM, with
position zero being the first (most distal) marker known at the
beginning of the chromosome. These map positions are not absolute,
and represent an estimate of map position. The statistical
probabilities that the marker allele and tolerance phenotype are
segregating independently are reflected in the Adjusted Probability
values. Out of 584 loci studied in 38 sensitive and 160 tolerant
soybean lines, QTLs on Lg L and on Lg N were highly significant, as
shown in the table below.
TABLE-US-00002 TABLE 1 Intergroup analysis results for LgL and LgN
markers G- Locus Test Chrom# Position value df Prob (G) Adj Prob
S00224-1 GW L 12.03 89.87 -1 0 0 P10649C- ASH L 3.6 86.01 -1 0 0 3
SATT523 SSR L 32.4 24.02 -1 0.000001 0.000592 S60167- SSR N 26
62.35 -1 0 0 TB P5467A-1 ASH N 25 16.25 -1 0.000056 0.032192
P5467A-2 ASH N 25 16.2 -1 0.000057 0.032731
Table 2 below shows the allele distribution between 101 tolerant
lines and 32/33 non-tolerant lines analyzed. Lines exhibiting
tolerance are indicated in the first column as "TOL," and lines
exhibiting non-tolerance are indicated in the first column as
"NON." Marker calls for the P10649C-3 locus and the S60167-TB locus
were available for 132 and 63 of the lines respectively.
TABLE-US-00003 TABLE 2 Allele distribution P10649C-3 allele
S60167-TB allele Phenotype LG-L LG-N TOL 1 1 TOL 1 1 TOL 1 TOL 1
TOL 1 TOL 1 2 TOL 1 TOL 1 TOL 1 1 TOL 1 TOL 1 1 TOL 1 TOL 1 1 TOL 1
1 TOL 1 1 TOL 1 1 TOL 1 TOL 1 1 TOL 1 TOL 1 TOL 1 1 TOL 1 1 TOL 1
TOL 1 1 TOL 1 1 TOL 1 1 TOL 1 1 TOL 1 TOL 1 1 TOL 1 1 TOL 1 TOL 1
TOL 1 1 TOL 1 1 TOL 1 TOL 1 1 TOL 1 TOL 1 TOL 1 TOL 1 1 TOL 1 1 TOL
1 1 TOL 1 1 TOL 1 TOL 1 1 TOL 1 1 TOL 1 TOL 1 1 TOL 1 TOL 1 TOL 1 1
TOL 1 1 TOL 1 TOL 1 1 TOL 1 1 TOL 1 1 TOL 1 1 TOL 1 TOL 1 1 TOL 1 1
TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 1 TOL 1 TOL 1 1 TOL 1 1
TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1
1 TOL 1 1 TOL 1 1 TOL 1 TOL 1 TOL 1 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1
TOL 1 TOL 1 TOL 1 TOL TOL 1 TOL 1 TOL 2 2 TOL 1 TOL 1 TOL 1 NON 3 2
NON 3 NON 1 1 NON 1_2 2 NON 3 2 NON 3 NON 1 1_2 NON 3 2 NON 2 1_2
NON 3 2 NON 2 NON 2 2 NON 2 2 NON 1 1 NON 2_3 NON 3 2 NON 3 2 NON
2_3 NON 3 NON 3 NON 1 1 NON 2 NON 3 NON 3 2 NON 3 NON 2 2 NON 3 2
NON 1_3 2 NON 3 NON 2 NON 1 NON 3
[0281] The non-random distribution of alleles between the tolerant
and non-tolerant plant groups at the marker loci in Table 2 is good
evidence that a QTL influencing tolerance to protoporphyrinogen
oxidase inhibitors is linked to these marker loci.
Example 2
Predication and Confirmation of Marker Based Selection for Response
to PPO Chemistries in a Set of Diverse Public Soybean Lines
[0282] Marker haplotype data for a set of 17 diverse public soybean
lines was determined for two QTL identified in Example 1 for
Linkage Group L molecular markers P10649C-3 (approximate position
3.6) and S00224-1 (approximate position 12.0). The response of
these lines to sulfentrazone herbicide was published by Hulling et
al. (Soybean (Glycine max (L.) Merr.) cultivar tolerance to
sulfentrazone. 2001 Science Direct, Vol. 20(8): 679-683). The
phenotypic response was reported as a growth reduction index: plant
height and visual injury as expressed as a percentage of check plot
of each cultivar. Data for the marker haplotype on Linkage Group L
and the herbicide bioassay results are presented in Table 3. Use of
the molecular diagnostic P10649C-3 (linked QTL on Linkage Group L,
approximate position 3.6) for this set of phentoyped soybean lines
is 92% predictive of tolerance to sulfentrazone when injury is set
at 39% or less GRI and is 100% predictive of non-tolerance to
sulfentrazone when injury is set at 40% or higher GRI. Use of the
S00224-1 marker (approximate position 12.0) for this set of soybean
lines is 88% predictive of tolerance to sulfentrazone when injury
is set at 39% or less GRI and is 100% predictive of non-tolerance
to sulfentrazone when injury is set at 40% or more GRI.
TABLE-US-00004 TABLE 3 Marker haplotype at/near QTL on Linkage
Group L for PPO herbicide (sulfentrazone) response and phenotypic
measure of crop response, expressed in terms of Growth Reduction
Index, for soybean cultivars (italicized items indicate deviations
from expected) Growth Linkage Group L QTLs Reduction Position 3.6
Position 12.0 Cultivar Index* P10649C-3 S00224-1 PI88788 2 1, 1 3,
3 Richland 4 1, 1 3, 3 Lincoln 5 1, 1 3, 3 PI180501 8 1, 1 3, 3
Illini 8 1, 1 3, 3 S100 8 1, 1 3, 3 Mukden 8 1, 1 3, 3 Arksoy 10 1,
1 3, 3 Capital 10 1, 1 3, 3 Haberlandt 10 3, 3 2, 2 Ralsoy 13 1, 1
2, 3 Dunfield 16 1, 1 3, 3 Peking 22 1, 1 3, 3 Roanoke 40 3, 3 2, 2
Ogden 42 3, 3 2, 2 Hutcheson 46 3, 3 2, 2 Ransom 52 3, 3 2, 2
allele call load percent accuracy correct (alleles 1) (allele 3)
tolerant 24/26 = 92% 23/26 = 88% correct (allele 3) (allele 2) =
non-tolerant 8/8 = 100% 8/8 = 100% *growth reduction index (plant
height and visual injury as expressed as a percentage of check plot
of each cultivar); Pre-emergence sulfentrazone application of 0.28
kg ai/ha, from Hulting, et al. (supra)
[0283] Haplotype data for a set of 15 diverse public soybean lines
was determined for two QTL identified in Example 1 for Linkage
Group N molecular marker 560167 (approximate position 26.0). The
response of these 15 lines to sulfentrazone herbicide was
determined and published upon by Hulling et al. (Soybean (Glycine
max (L.) Merr.) cultivar tolerance to sulfentrazone. 2001 Science
Direct, Vol. 20(8): 679-683). The phenotypic response was reported
as a growth reduction index: plant height and visual injury as
expressed as a percentage of check plot of each cultivar. Data for
the marker haplotype on Linkage Group N and the herbicide bioassay
results are presented in Table 4. The cultivar Ralsoy is
heterozygous for the S60167 marker. Use of the S60167 marker for
this set of phentoyped soybean lines is 88% predictive of tolerance
to sulfentrazone when injury is set at 39% or less GRI and is 100%
predictive of tolerance to sulfentrazone when injury is set at 40%
or higher GRI.
TABLE-US-00005 TABLE 4 Marker haplotype at/near QTL on Linkage
Group N for PPO herbicide (sulfentrazone) response and phenotypic
measure of crop response, expressed in terms of Growth Reduction
Index, for soybean cultivars (italicized items indicate deviations
from expected) Growth Linkage Group N QTL Reduction Position 26
Cultivar Index* S60167-TB PI88788 2 1, 1 Richland 4 1, 1 Lincoln 5
1, 1 Illini 8 1, 1 S100 8 1, 1 Mukden 8 1, 1 Arksoy 10 1, 1
Haberlandt 10 1, 1 Ralsoy 13 1, 2 Dunfield 16 1, 1 CNS 20 2, 2
Peking 22 1, 1 Roanoke 40 2, 2 Ogden 42 2, 2 Hutcheson 46 2, 2
allele call load percent accuracy correct (allele 1) tolerant 21/24
= 88% correct (allele 2) non-tolerant 6/6 = 100%
Example 3
Predication and Confirmation of Marker Based Selection for Response
to PPO Chemistries in a Set of Soybean Commercial Lines
[0284] Haplotype data for a set of 7 commercial soybean lines was
determined for two QTL identified in the previous example for
Linkage Group L molecular markers P10649C-3 (position 3.6) and
S00224-1 (position 12.0). The response of these lines to
sulfentrazone herbicide was determined by method used in Example 1.
In addition, the same scale was used for scoring such that: [0285]
9=Equivalent or better when compared to the tolerant check [0286]
7=Very little damage or response noted. [0287] 5=Intermediate
response or damage [0288] 3=Major damage, including stunting and
foliar necrosis [0289] 1=Severe damage, including severe stunting
and necrosis; equivalent or worse when compared to the non-tolerant
check Data for the marker haplotype on Linkage Group L and the
herbicide bioassay results are presented in Table 5. Use of
either/both of these markers for this set of phentoyped soybean
lines is 100% predictive of both tolerance (score of a 7 or 9) and
non-tolerance (score of a 1 for the non-tolerant check).
TABLE-US-00006 [0289] TABLE 5 Prediction and confirmation of marker
based selection at QTL for linkage group L for response to PPO
chemistry (sulfentrazone) in a set of commercial soybean varieties.
sulfentrazone Position 3.6 Position 12.0 Variety injury score
P10649C-3 S00224-1 93B41 9 1, 1 3, 3 93B82 9 1, 1 3, 3 9281 9 1, 1
3, 3 9584 9 1, 1 3, 3 92B52 7 1, 1 3, 3 92B61 7 1, 1 3, 3 9692 1 3,
3 2, 2
Example 4
Predication and Confirmation of Marker Based Selection for Response
to PPO Chemistries (Sulfentrazone) in Ten Lines from a Set of
Soybean Lines Phenotyped at the University of Illinois
[0290] A comparison for the marker predictiveness of PPO response
was conducted. The herbicide bioassay experiment used is described
in Phytoxic Response and Yield of Soybean (Glycine max) Varieties
Treated with Sulfentrazone or Flumioxazin (Taylor-Lovell et al.,
2001 Weed Technology 15:96-102). Phenotypic data was taken from
Table 2 of the publication for those varieties for which in-house
marker data was available. Phenotypic score and haplotype data for
a set of 10 soybean lines (1 public and 9 commercial) in the
chromosomal regions around the QTL for Linkage group L is presented
in Table 6. The phenotypic score was determined as percent injury
which is defined as visible injury ratings including stunting,
chlorosis, and bronzing symptomology (0=no injury; 100=complete
death) with 448 g ai/ha field application. Ratings were taken 12
days after treatment. Use of marker P10649C (linked QTL on Linkage
Group L, approximate position 3.6, allele call 1) for this set of
phentoyped soybean lines is 100% predictive of tolerance (allele
call 1) to sulfentrazone when injury is 21% or less and is 100%
predictive of non-tolerance (allele call 2 or 3) to sulfentrazone
when injury is 43% or greater. The predictiveness of marker
S00224-1 is also 100% accurate for tolerance (allele 3) and
non-tolerance (allele 2) for this set of material.
TABLE-US-00007 TABLE 6 Marker haplotype at/near QTL on Linkage
Group L for PPO herbicide (sulfentrazone) response and phenotypic
measure of crop injury sulfentrazone Position 3.6 Position 12.0
Variety injury score P10649C-3 S00224-1 P9584 5 1, 1 3, 3 P9671 5
1, 1 3, 3 P9151 8 1, 1 3, 3 P9306 15 1, 1 3, 3 Elgin 18 1, 1 3, 3
P9282 19 1, 1 3, 3 P9352 21 1, 1 3, 3 P9362 43 2, 2 2, 2 91B01 58
3, 3 2, 2 P9552 61 3, 3 2, 2 LSD (0.05) 8 allele call load percent
accuracy correct tolerant (alleles 1 or 2) (allele 3) 14/14 = 100%
14/14 = 100% correct non-tolerant (allele 3) (allele 2) = 8/8 =
100% 8/8 = 100%
Example 5
Pictures of Soybean Variety Response (Tolerant and Non-Tolerant
Check Varieties) to Sulfentrazone Injury in the Field and in the
Greenhouse/Growth Chamber Bioassay
[0291] Known non-tolerant (i.e., Pioneer variety 9692, Asgrow
variety A4715) and tolerant (i.e., Pioneer variety 9584, Syngenta
variety S5960) germplasm can exhibit severe differences in
symptomology when field conditions are conducive to damage and when
lab conditions for bioassays are optimized for selection purposes.
FIGS. 5 and 6 show these differences in phenotype. FIG. 5 shows a
field sample, with a non-tolerant variety on the left (stunted,
necrotic) and tolerant variety on the right (normal growth). FIG. 6
shows a greenhouse sample, with non-tolerant (left side) and
tolerant (right side) variety checks, treated in the foreground,
untreated in the background.
Example 6
Fine Mapping of PPO Herbicide Tolerance QTL
[0292] The PPO herbicide tolerance QTL was mapped in two mapping
populations, GEID1653063.times.GEID3495695 and
GEID4520632.times.GEID7589905 PPO tolerance mapped to chromosome
GM19 (LG-L) near the closely linked marker S03859-1-A, which
explains 80% of the phenotypic variation. From these two
populations, lines with recombination breakpoints near S03859-1-A
were identified to define the borders of the QTL and to facilitate
fine-mapping.
[0293] Subsequent analysis of the recombinants indicated that the
closely linked marker S03859-1-A was actually the left flanking
marker. The GEID1653063.times.GEID3495695 population had 37
recombinants that set the flanking markers for the PPO QTL as
504867-1-A (GM19: 841543-841958) and S03859-1-A (GM19:
1634882-1635399) (Table 10). The GEID4520632.times.GEID7589905
population had 42 recombinants that delimit the QTL to the same
interval (Table 11).
[0294] Because S03859-1-A was determined to be closely linked to
the PPO QTL, annotated loci in the vicinity of this marker were
targeted for SNP discovery and marker development. Primers were
designed from target loci using Primer3 and checked for uniqueness
using bioinformatics software. A panel composed of 20 PPO tolerant
and 8 PPO susceptible lines, including the four mapping parents
from the mapping population, was re-sequenced at the target loci to
identify informative SNPs. DNA was extracted using the urea
extraction protocol below and PCR amplified using standard lab
protocols (see Tables 7-8). The PCR was then cleaned up using the
ExoSAP-IT.RTM. protocol (USB-Cleveland, Ohio, USA) (Table 9) before
being sequenced by Sanger sequencing.
[0295] In total, 104 loci were re-sequenced and 235 informative
SNPs were identified. From these SNPs, 22 Taqman.RTM. probe markers
were designed to distinguish between tolerant versus susceptible
alleles in the mapping populations. Taqman.RTM. assays were
designed generally following ABI suggested parameters. These
markers were then run on the recombinants from the two mapping
populations to facilitate fine-mapping and to further delimit the
PPO QTL interval.
[0296] Urea Extraction Protocol [0297] 1. Grind 2 g fresh tissue or
0.5 g lyophilized tissue and add it to 6 mL Urea Extraction Buffer
and mix well. [0298] 2. Add RNase A (100 mg/mL) and incubate @
37.degree. C. for 20 min. [0299] a. 3 uL--Leaf [0300] b. 12
uL--Seed [0301] 3. Add 4-5 mL Phenol:Chloroform:Isoamyl 25:24:1.
Mix well. (Sigma P3803) [0302] 4. Put on rocker inside hood. [0303]
a. Fresh--15 min [0304] b. Lyophilized--30 min [0305] 5. Centrifuge
@ 8000 rpm at 10.degree. C. for 10 min. [0306] 6. Transfer
supernatant to clean tube. [0307] 7. Add 700 uL of 3M NaOAC (pH
5.0) and 5 mL cold isopropanol. Mix well. [0308] 8. Hook DNA and
wash in 70% EtOH. [0309] 9. Repeat 70% wash. [0310] 10. Transfer
pellet to 1.5 mL tube and allow to dry. [0311] 11. Dissolve pellet
in 1 mL 10 mM Tris.
[0312] 7 M Urea Extraction Buffer [0313] Water 350 mL [0314] Urea
336g [0315] 5M NaCl 50 mL (14.61g) [0316] 1M Tris 40 mL (pH 8.0)
[0317] 0.5M EDTA 32 mL (pH 8.0) [0318] 20% Sarcosine Sol. 40 mL
(8g) [0319] Adjust volume to 800 mL with ddH2O
TABLE-US-00008 [0319] TABLE 7 PCR Reaction Mix for SNP Discovery.
1X (uL) 24 plate(ul) 36 plate(uL) 48 plate(uL) gDNA (~50-100 ng)
2.0 -- -- -- 10x PCR Buffer 2.0 5,952 7,680 10,944 1 mM dNTP 2.0
5,952 7,680 10,944 Taq 0.1 297.6 384 547.2 0.5 uM Primer 4.0 -- --
-- (F + R) ddH2O 9.9 29,462 38,016 54,173 Total 20.0 41,664 53,760
76,608
TABLE-US-00009 TABLE 8 PCR Setup for SNP Discovery. Dipper Setup
PCR conditions Temp Time #Cycles initial denature 94 C. 3 min 1X
denature 94 C. 45 sec anneal 65 C. 60 sec 35X extension 72 C. 75
sec 1X final extension 72 C. 5 min end
TABLE-US-00010 TABLE 9 Exo/SAP Protocol for PCR clean up. PCR clean
up Exo/SAP Mix (pre-sequencing) add 3.6 ul of mastermix to 7.mu.l
final PCR product 24 plate(.mu.l) 36 plate(.mu.l) 48 plate(.mu.l)
ddH2O 4,285.4 5,944.3 7,326.7 SAP 4,285.4 5,944.3 7,326.7 Exo
2,142.7 2,972.2 3,663.4 total 10,714 14,861 18,317
TABLE-US-00011 TABLE 10 Initial recombinants identified from
GEID1653063 .times. GEID3495695 mapping population that delimited
PPO herbicide tolerance QTL to interval between S01659-1-A and
S03859-1-A. SAMPLE S04867-1-A S03859-1-A Genetic Pos 12.55 16.08
Call Average Comment GEID1653063 A A SUS 1 Control GEID3495695 B B
TOL 9 Control SJ22185567 A B TOL 9 L border SJ22185980 A B TOL 9 L
border SJ22186045 A B TOL 9 L border SJ22186929 A B TOL 9 L border
SJ22186019 B H TOL 9 R border SJ22185608 H B TOL 9 L border
SJ22186913 H B TOL 9 L border SJ22185928 H B TOL 9 L border
SJ22186923 H B TOL 8.333333 L border SJ22185569 A H SEG 5 L border
SJ22186052 A H SEG 6.333333 L border SJ22186882 A H SEG 5 L border
SJ22186919 B H SEG 5.666667 L border SJ22186968 B H SEG 6.333333 L
border SJ22186824 B H SEG 6.333333 L border SJ22185604 H B SEG
6.333333 R border SJ22185573 H A SEG? 3.666667 R border SJ22185983
A B SUS 1 R border SJ22186894 A B SUS 2.333333 R border SJ22185562
A H SUS 1.666667 R border SJ22185941 A H SUS 1 R border SJ22185534
B A SUS 3 L border SJ22185545 B A SUS 1.666667 L border SJ22185559
B A SUS 2.333333 L border SJ22186023 B A SUS 3 L border SJ22186057
B A SUS 1 L border SJ22186065 B A SUS 1 L border SJ22186837 B A SUS
3 L border SJ22185957 B A SUS 1 L border SJ22186846 B A SUS
1.666667 L border SJ22186840 H A SUS 1 L border SJ22186950 H A SUS
1 L border SJ22186872 H A SUS 2.333333 L border SJ22186836 H A SUS
1.666667 L border SJ22186074 H A SUS 1 L border SJ22186906 H A SUS
1 L border SJ22185984 H A SUS 1 L border
TABLE-US-00012 TABLE 11 Initial recombinants identified from
GEID4520632 .times. GEID7589905 mapping population that delimited
PPO herbicide tolerance QTL to interval between S04867-1-A and
S03859-1-A SAMPLE S04867-1-A S03859-1-A Genetic Pos 12.55 16.08
Call Ave Comment GEID7589905 A A SUS 1 Control GEID4520632 B B TOL
9 Control SP21669231 A B TOL 9 L border SP21669401 A B TOL 9 L
border SP21669240 A B TOL 9 L border SP21669613 A B TOL 9 L border
SP21669249 H B TOL 9 L border SP21669645 H B TOL 9 L border
SP21669670 H B TOL 9 L border SP21669563 H B TOL 9 L border
SP21669592 H B TOL 9 L border SP21669260 B A SUS 1 L border
SP21669265 B A SUS 1 L border SP21669778 B A SUS 1.666667 L border
SP21669590 B A SUS 1 L border SP21669751 A H SUS 1 R border
SP21669380 H A SUS 2.666667 L border SP21669679 H A SUS 1 L border
SP21669708 H A SUS 1 L border SP21669755 H A SUS 1 L border
SP21669214 H A SUS 1 L border SP21669573 H A SUS 1.666667 L border
SP21669612 H A SUS 2.333333 L border SP21669336 H A SUS 3.666667 L
border SP21669201 B H SEG 5 L border SP21669503 B H SEG 5 L border
SP21669664 B H SEG 5 L border SP21669540 B H SEG 5 L border
SP21669752 B H SEG 5.666667 L border SP21669230 B H SEG 5.666667 L
border SP21669331 A H SEG 6.333333 L border SP21669371 A H SEG 5 L
border SP21669542 A H SEG 6.333333 L border SP21669584 A H SEG 5 L
border SP21669694 A H SEG 5.666667 L border SP21669763 A H SEG 5 L
border SP21669533 A H SEG 5 L border SP21669417 A H SEG 6.333333 L
border SP21669647 A H SEG? 7.666667 L border SP21669651 A H SEG?
7.666667 L border SP21669541 H B SEG? 7.666667 R border SP21669749
H A SEG 5 R border SP21669356 H A SEG 5 R border SP21669674 H A
SEG? 3.666667 R border
[0320] From the GEID1653063.times.GEID3495695 mapping population,
four key recombinants were identified which served to further
fine-map the PPO QTL interval (Table 13). A recombination
breakpoint at S08110-1-Q1 in line SJ22186052 set the left border,
while breakpoints at S08105-1-Q1 in SJ22186019, SJ22186894, and
SJ22185941 set the right border. These recombinants delimit the PPO
QTL to .about.70 kb interval. From the
GEID4520632.times.GEID7589905 mapping population, eight key
recombinants were identified (Table 14). A recombination breakpoint
in line SP21669503 at S08117-1-Q1 set the left border, while
breakpoints in SP21669249, SP21669332, SP21669615, SP21669616,
SP21669670, SP21669458, and SP21669760 set the right border at
S08010-1-Q1. These recombinants delimit the PPO QTL to a .about.526
kb interval. However, when the data from these two mapping
populations are combined into a single set, the PPO QTL interval is
delimited to a .about.56 kb interval between S08117-1-Q1 and
S08105-1-Q1 (Table 12).
TABLE-US-00013 TABLE 12 Summary of SNP markers used for initial QTL
mapping and fine-mapping of PPO herbicide tolerance QTL. Combined
data from the two populations delimits the QTL to a ~56 kb interval
between S08117-1-Q1 and S8105-1-Q1. First Base Last Base Marker
Amplicon Loci coordinate coordinate Population Fine-mapping Comment
S04867-1-A Glyma19g01220.1 841543 841958 Both S08102-1-Q1
PPO_Gm19_1487k3-1 Glyma19g01860.1 1489113 1489545 Both S08103-1-Q1
PPO_Gm19_1491k1-1 X 1491603 1492136 Both S08104-1-Q1
PPO_Gm19_1491k2-1 Glyma19g01870.1 1492364 1492948 Both S08106-1-Q1
PPO_Gm19_1499k2-1 Glyma19g01880.1 1500732 1501392 GEID1653063/
GEID3495695 S08107-1-Q1 PPO_Gm19_1541k3-1 Glyma19g01900.1 1542880
1543693 GEID1653063/ GEID3495695 S08109-1-Q1 PPO_Gm19_1541k4-1
Glyma19g01900.1 1543868 1544588 GEID1653063/ GEID3495695
S08110-1-Q1 PPO_Gm19_1548k1-1 Glyma19g01910.1 1548367 1548822
GEID1653063/ L border GEID3495695 GEID1653063/ GEID3495695
S08111-1-Q1 PPO_Gm19_1548k2-1 Glyma19g01910.1 1548902 1549558
GEID1653063/ GEID3495695 S08115-2-Q1 PPO_Gm19_1563k1-1 X 1563958
1564512 Both 508117-1-Q1 PPO_Gm19_1563k2-1 X 1564563 1564960 Both L
border GEID4520632/ GEID7589905 S08119-1-Q1 PPO_Gm19_1566k2-1
Glyma19g01920.1 1567791 1568282 Both histone deacetylase
S08118-1-Q1 PPO_Gm19_1566k4-1 Glyma19g01920.1 1569273 1569748 Both
histone deacetylase S08116-1-Q1 PPO_Gm19_1566k5-1 Glyma19g01920.1
1570198 1570729 Both histone deacetylase S08101-1-Q1
PPO_Gm19_1586k1-1 Glyma19g01940.1 1587051 1587687 Both multidrug/
pheromone exporter, ABC superfamily S08112-1-Q1 PPO_Gm19_1586k1-1
Glyma19g01940.1 1587051 1587687 Both multidrug/ pheromone exporter,
ABC superfamily S08108-1-Q1 PPO_Gm19_1586k2-1 Glyma19g01940.1
1587805 1588500 Both multidrug/ pheromone exporter, ABC superfamily
S08101-1-Q1 PPO_Gm19_1586k4-1 Glyma19g01940.1 1589409 1590062 Both
multidrug/ pheromone exporter, ABC superfamily S08101-2-Q1
PPO_Gm19_1586k4-1 Glyma19g01940.1 1589409 1590062 Both multidrug/
pheromone exporter, ABC superfamily S08101-3-Q1 PPO_Gm19_1586k4-1
Glyma19g01940.1 1589409 1590062 Both multidrug/ pheromone exporter,
ABC superfamily S08101-4-Q1 PPO_Gm19_1586k4-1 Glyma19g01940.1
1589409 1590062 Both multidrug/ pheromone exporter, ABC superfamily
S08105-1-Q1 PPO_Gm19_1618k2-1 X 1619657 1620279 Both R border
GEID1653063/ GEID3495695 S03859-1-A sbacm.pk005.c3.f X 1634882
1635399 Both S08010-1-Q1 PPO_Gm19_2089k4-1 G1yma19g02370.1 2091644
2092359 Both R border GEID4520632/ GEID7589905 S08010-2-Q2
PPO_Gm19_2089k4-1 G1yma19g02370.1 2091644 2092359 Both
[0321] Tables 13A-13G: Fine-mapping of the PPO herbicide tolerance
QTL interval with recombinants from the
GEID1653063.times.GEID3495695 population. Key recombinants delimit
the QTL to the .about.70 kb interval between S08110-1-Q1 and
S08105-1-Q1.
TABLE-US-00014 TABLE 13A Marker Amplicon/Pos S04867-1-A S08102-1-Q1
S08103-1-Q1 S08104-1-Q1 Sample Gm19:841750 PPO_Gm19_1487k3-1
PPO_Gm19_1491k1-1 PPO_Gm19_1491k2-1 SJ22185925 B B B B SJ22186974 B
B B B SJ22185946 B B B B SJ22186019 B B B B SJ22186923 H H H H
SJ22185604 H H H H SJ22186029 H H H H SJ22186052 A A A A SJ22185534
B A A A SJ22185552 A A A A SJ22186842 A A -- A SJ22186924 A A A A
SJ22186873 A A A A SJ22186894 A A A A SJ22185957 B A A A SJ22185941
A A A A SJ22186872 H A A A SJ22185984 H H H H
TABLE-US-00015 TABLE 13B Marker Amplicon/POS S08106-1-Q1
S08107-1-Q1 S08109-1-Q1 S08110-1-Q1 Sample PPO_Gm19_1499k2-1
PPO_Gm19_1541k3-1 PPO_Gm19_1541k4-1 PPO_Gm19_1548k1-1 SJ22185925 B2
B B B SJ22186974 B1 B B B SJ22185946 B2 B B B SJ22186019 B2 B B B
SJ22186923 H B -- B SJ22185604 H H H H SJ22186029 H H H H
SJ22186052 A A A A SJ22185534 A A A A SJ22185552 A A A A SJ22186842
A A A A SJ22186924 A A A A SJ22186873 A A A A SJ22186894 H A A A
SJ22185957 A A A A SJ22185941 A A A A SJ22186872 A A A A SJ22185984
H A A A
TABLE-US-00016 TABLE 13C Marker Amplicon/Pos S08111-1-Q1
S08115-2-Q1 S08117-1-Q1 S08119-1-Q1 Sample PPO_Gm19_1548k2-1
PPO_Gm19_1563k1-1 PPO_Gm19_1563k2-1 PPO_Gm19_1566k2-1 SJ22185925 B
B B B SJ22186974 B B/H B B SJ22185946 B B B B SJ22186019 B B B B
SJ22186923 B B B B SJ22185604 H H H H SJ22186029 H H H H SJ22186052
-- H H H SJ22185534 A A A A SJ22185552 A A A A SJ22186842 A A A A
SJ22186924 A A A A SJ22186873 A A A A SJ22186894 A A A A SJ22185957
A A A -- SJ22185941 A A A A SJ22186872 A A A -- SJ22185984 A A A
A
TABLE-US-00017 TABLE 13D Marker Amplicon/Pos S08118-1-Q1
S08116-1-Q1 S08101-1-Q1 S08112-1-Q1 Sample PPO_Gm19_1566k4-1
PPO_Gm19_1566k5-1 PPO_Gm19_1586k1-1 PPO_Gm19_1586k1-1 SJ22185925 B
B B B SJ22186974 -- B B B SJ22185946 B B B B SJ22186019 -- B B B
SJ22186923 B B B B SJ22185604 H H H H SJ22186029 H H H H SJ22186052
-- H H H SJ22185534 A A A A SJ22185552 A A A A SJ22186842 A A A A
SJ22186924 A A A A SJ22186873 A A A A SJ22186894 A A A A SJ22185957
A A A A SJ22185941 A A A A SJ22186872 A A A A SJ22185984 A A A
A
TABLE-US-00018 TABLE 13E Marker Amplicon/Pos S08108-1-Q1
S08101-1-Q1 S08101-2-Q1 S08101-3-Q1 Sample PPO_Gm19_1586k2-1
PPO_Gm19_1586k4-1 PPO_Gm19_1586k4-1 PPO_Gm19_1586k4-1 SJ22185925 B
B B B SJ22186974 B B B B SJ22185946 B B B B SJ22186019 B B B B
SJ22186923 B B B B SJ22185604 H H H H SJ22186029 H H H H SJ22186052
H H H H SJ22185534 A A A A SJ22185552 A A A A SJ22186842 A A A A
SJ22186924 A A A A SJ22186873 A A A A SJ22186894 A A A A SJ22185957
A A A A SJ22185941 A A A A SJ22186872 A A A A SJ22185984 A A A
A
TABLE-US-00019 TABLE 13F Marker Amplicon/Pos S08101-4-Q1
S08105-1-Q1 S03859-1-A S08010-1-Q1 Sample PPO_Gm19_1586k4-1
PPO_Gm19_1618k2-1 PPO_Gm19_1635140 PPO_Gm19_ 2089k4-1 SJ22185925 B
B B A SJ22186974 B B B A SJ22185946 B B B A SJ22186019 B H H H
SJ22186923 B B B B SJ22185604 H B B B SJ22186029 H H H B SJ22186052
H H H H SJ22185534 A A A B SJ22185552 A A A B SJ22186842 A A A B
SJ22186924 A A A B SJ22186873 A A A B SJ22186894 A B B B SJ22185957
A A A B SJ22185941 A H H H SJ22186872 A A A B SJ22185984 A A A
A
TABLE-US-00020 TABLE 13G Marker S08010-2-Q2 Amplicon/Pos
PPO_Gm19_2089k4-1 Comment Phenotype SJ22185925 A TOL SJ22186974 A
TOL SJ22185946 H TOL SJ22186019 H R Border TOL SJ22186923 B TOL
SJ22185604 B SEG SJ22186029 B SEG SJ22186052 H L Border SEG
SJ22185534 B SUS SJ22185552 B SUS SJ22186842 B SUS SJ22186924 B SUS
SJ22186873 B SUS SJ22186894 B R Border SUS SJ22185957 B SUS
SJ22185941 H R Border SUS SJ22186872 B SUS SJ22185984 A SUS
Tables 14A-14C: Fine-mapping of the PPO herbicide tolerance QTL
interval with recombinants from the GEID4520632.times.GEID7589905
population.
TABLE-US-00021 TABLE 14A Marker Sample Comment Phenotype S04867-1-A
S08102-1-Q1 S08103-1-Q1 S08104-1-Q1 S08115-2-Q1 S08117-1-Q1
SP21669249 R Border TOL H B B B B B SP21669332 R Border TOL B B --
B B B SP21669615 R Border TOL B -- B B B B SP21669616 R Border TOL
B B -- B B/H B SP21669670 R Border TOL H B B B -- B SP21669503 L
Border SEG B B B B B B SP21669458 R Border SUS A A A A A A
SP21669760 R Border SUS A A A A A A
TABLE-US-00022 TABLE 14B Marker Sample Comment Phenotype
S08119-1-Q1 S08118-1-Q1 S08116-1-Q1 S08101-1-Q1 S08112-1-Q1
S08108-1-Q1 SP21669249 R Border TOL B B B B B B SP21669332 R Border
TOL B B B B B B SP21669615 R Border TOL B B B B B B SP21669616 R
Border TOL B B B B B/H B SP21669670 R Border TOL B B B B B B
SP21669503 L Border SEG H H H H H H SP21669458 R Border SUS A A A A
A A SP21669760 R Border SUS A A A A A A
TABLE-US-00023 TABLE 14C Marker Sample Comment Phenotype
S08101-1-Q1 S08101-2-Q1 S08101-3-Q1 S08101-4-Q1 S08105-1-Q1
S03859-1-A SP21669249 R Border TOL B B B B B B SP21669332 R Border
TOL B B B B B B SP21669615 R Border TOL B B B B B B SP21669616 R
Border TOL B B B B B B SP21669670 R Border TOL B B B B B B
SP21669503 L Border SEG H H H H H H SP21669458 R Border SUS A A A A
A A SP21669760 R Border SUS A A A A A A
TABLE-US-00024 TABLE 14D Marker Sample Comment Phenotype
S08010-1-Q1 S08010-2-Q2 SP21669249 R Border TOL H H SP21669332 R
Border TOL H H SP21669615 R Border TOL B B SP21669616 R Border TOL
H H SP21669670 R Border TOL B B SP21669503 L Border SEG H H
SP21669458 R Border SUS H H SP21669760 R Border SUS H H
Example 7
SNP Haplotype Association Analysis
[0322] Association analysis of SNP haplotypes across the PPO QTL
region provides an independent method of validating the PPO
interval. From the panel of susceptible and tolerant lines used to
identify SNPs for Taqman.RTM. probe development, 235 SNPs from 49
amplicons were identified in the vicinity of the closely linked
marker S03859-1-A. The resulting SNP haplotype data was analyzed to
identify an interval in which all of the haplotypes from the
susceptible and tolerant lines co-segregated with each other (Table
15).
TABLE-US-00025 TABLE 15 SNP haplotype association analysis of the
PPO herbicide tolerance QTL interval. Perfect association between
haplotype and phenotype between amplicons Gm19_1563k1 and
Gm19_1618k2 defines the QTL interval. GEID Amplicon 1563k1 1563k1
1563k1 1563k1 1618k2 1618k2 627002 TOL (PPO) G G A C * C 3911338
TOL (PPO) G G A C * C 1564727 TOL (PPO) G G A C * C 4230314 TOL
(PPO) G G A C * C 4135359 TOL (PPO) G G A C * C 4611588 TOL (PPO) G
G A C * C 1590166 TOL (PPO) G G A C * C 3395451 TOL (PPO) G G A C *
C 2322432 TOL (PPO) G G A C * C 4520632 TOL (PPO) G G A C * C
632343 TOL (PPO) G G A C * C 1770139 TOL (PPO) G G A C * C 3587853
TOL (PPO) G G A C * C 4553991 TOL (PPO) G G A C * C 5183219 TOL
(PPO) G G A C * C 2636517 TOL (PPO) G G A C * C 3495695 TOL (PPO) G
G A C * C 1737165 SUS (PPO) A * G T A A 1653063 SUS (PPO) A A A
4501774 SUS (PPO) A * G T A A 7589905 SUS (PPO) A * G T N A 4832982
SUS (PPO) A * G T N A 2839548 SUS (PPO) A * G T 3958440 SUS (PPO) A
* G T A A 6116656 SUS (PPO) A * G T A A
[0323] Although it is difficult to definitively define the
co-segregating region, it can conservatively be estimated to reside
between amplicons PPO_Gm19.sub.--1563 k1 and PPO_Gm19.sub.--1618
k2. Within the borders defined by these loci, there are 38 SNP
differences that are shared between all of the susceptible lines
compared to all the tolerant lines. This interval is approximately
the same-56 kb interval identified by fine-mapping.
[0324] It will be apparent to those of skill in the art that it is
not intended that the invention be limited by such illustrative
embodiments or mechanisms, and that modifications can be made
without departing from the scope or spirit of the invention, as
defined by the appended claims. It is intended that all such
obvious modifications and variations be included within the scope
of the present invention as defined in the appended claims. The
claims are meant to cover the claimed components and steps in any
sequence which is effective to meet the objectives there intended,
unless the context specifically indicates to the contrary.
[0325] All publications referred to herein are incorporated by
reference herein for the purpose cited to the same extent as if
each was specifically and individually indicated to be incorporated
by reference herein.
Sequence CWU 1
1
117122DNAGlycine max 1ttattgaggt gggcaaggtg tg 22223DNAGlycine max
2catgaacgtc tggtggttga aca 23327DNAGlycine max 3gcgatttctt
ccttgaagaa ttttctg 27427DNAGlycine max 4gcgctttttc ggctgttatt
tttaact 27530DNAGlycine max 5gagggctatg ttttcttctc cagatgtgag
30626DNAGlycine max 6aaggtcggct tggtggttaa aggcag 26713DNAGlycine
max 7tcatctgtga taa 13813DNAGlycine max 8tcatgtgtga taa
13913DNAGlycine max 9tcatctctga taa 131022DNAGlycine max
10ctggacctac ccgggatgaa aa 221122DNAGlycine max 11tcttcctctc
ccttcctctc gc 221213DNAGlycine max 12cgcgactctc ctc
131313DNAGlycine max 13cgcgagtctc ctc 131427DNAGlycine max
14tcccaggtta gattttctga acgaaga 271524DNAGlycine max 15catcagcaca
aaagggcatc ctca 241616DNAGlycine max 16cactccttaa ggtaat
161716DNAGlycine max 17cactccttaa gataat 161820DNAGlycine max
18gttatcgtca ccaccaccaa 201921DNAGlycine max 19cacaacacga
gtagccgtag g 212016DNAGlycine max 20aacggatcat cacaac
162115DNAGlycine max 21aacggctcat cacaa 152220DNAGlycine max
22cgacaatggc ctttacacct 202320DNAGlycine max 23tcgatatgga
cgaaggagga 202416DNAGlycine max 24acaccatttt tcatcc
162516DNAGlycine max 25acaccctttt tcatcc 162622DNAGlycine max
26gcaatcacat ttgcattcct ta 222721DNAGlycine max 27tctgaacgag
ttgtgcaaga a 212816DNAGlycine max 28actgctgctt tgtcta
162916DNAGlycine max 29ctactgctac tttgtc 163020DNAGlycine max
30acctcgtatt ggtggtggtg 203120DNAGlycine max 31gaatgttcag
tgcgagcaac 203215DNAGlycine max 32acttccctcg tttcg 153314DNAGlycine
max 33cttccctcat ttcg 143422DNAGlycine max 34caaaaggaaa gaagaaccgt
gt 223521DNAGlycine max 35tccaacctat gtgttggtgt g 213617DNAGlycine
max 36atgattgaag caggaaa 173718DNAGlycine max 37tcatgattga agcagcaa
183827DNAGlycine max 38ggagacttga cttaaagaga aagaaaa
273926DNAGlycine max 39cggaaagaaa aacaatagat tgaatg
264019DNAGlycine max 40cttgttctag actgatcat 194116DNAGlycine max
41ctagactgat aattca 164226DNAGlycine max 42tcattcaaga ctacatgaaa
gacaaa 264320DNAGlycine max 43caagggagag caatccttga
204416DNAGlycine max 44atagtctccc aaacac 164517DNAGlycine max
45atagtctctc aaacacc 174622DNAGlycine max 46gaaactttcc attttgccct
tc 224718DNAGlycine max 47agaacgcagg ggagaagc 184816DNAGlycine max
48cttcttccac tcttac 164917DNAGlycine max 49ccttcttaca ctcttac
175028DNAGlycine max 50tgatatgaca ctctactaag atgtgttg
285120DNAGlycine max 51tgattcatcc gcaaacttga 205217DNAGlycine max
52cactctccta tattgtc 175316DNAGlycine max 53ctctcctaca ttgtca
165422DNAGlycine max 54agatccttgt tccaaattcc aa 225520DNAGlycine
max 55ccttggctta atgggtgtgt 205616DNAGlycine max 56ccaacacaat
ctaact 165714DNAGlycine max 57ccaacacaat cgaa 145820DNAGlycine max
58atggaggcaa gcttgtgttt 205920DNAGlycine max 59catgctacca
gcatctgcaa 206017DNAGlycine max 60cttcataaac gccaaag
176116DNAGlycine max 61cataaatgcc aaagca 166220DNAGlycine max
62aatgagcaag ggagaggaca 206320DNAGlycine max 63tcgccgctgc
tatttaattt 206418DNAGlycine max 64aagcactact ttcaattg
186514DNAGlycine max 65aagcaccact ttca 146620DNAGlycine max
66agatgccttg ctcagtggac 206722DNAGlycine max 67atgatgaatg
tgttgagcca at 226814DNAGlycine max 68ccccatcacc atac
146914DNAGlycine max 69accccaccac cata 147022DNAGlycine max
70agaaaccttc caaagctctt gg 227120DNAGlycine max 71tagggaggca
cttgacaacc 207215DNAGlycine max 72caacatccga gtcca 157315DNAGlycine
max 73caacatcaga gtcca 157420DNAGlycine max 74ttttgacccc cagagagttg
207520DNAGlycine max 75ttgcaagcct aaaggatggt 207619DNAGlycine max
76ctatctctac acgatgtgt 197716DNAGlycine max 77ctatctccac acgatg
167820DNAGlycine max 78tcccacttga tcttgcagaa 207920DNAGlycine max
79tacggtgggt ggattattcg 208015DNAGlycine max 80cctccaatgg catac
158117DNAGlycine max 81cctccaatag catacat 178222DNAGlycine max
82agaaaagcag cagaaagagg ac 228322DNAGlycine max 83cttcatgaat
cccaacatca ga 228417DNAGlycine max 84ctctaattcc acatctg
178518DNAGlycine max 85cctctaattt cacatctg 188622DNAGlycine max
86tcaaaccatt ttgtttccca gt 228721DNAGlycine max 87tgctagcctt
tgatacccaa c 218816DNAGlycine max 88ttgcattgta ttctct
168915DNAGlycine max 89ttgcattgta ttttc 159021DNAGlycine max
90gtctcaggca gtgaatctgc t 219120DNAGlycine max 91cagccttacc
atcaacatcg 209213DNAGlycine max 92ttccgtgaag atc 139315DNAGlycine
max 93atgcttccgc gaaga 159426DNAGlycine max 94ggtagcagtt actttgtgat
gtaagc 269522DNAGlycine max 95catgcaataa aatccaaaac ca
229617DNAGlycine max 96tactgatcac aggttat 179717DNAGlycine max
97tactgaccac aggttat 179820DNAGlycine max 98ttgctttgga aaggactcca
209920DNAGlycine max 99cctcatcaac tcctgctgct 2010014DNAGlycine max
100ctcggtgctg tttt 1410114DNAGlycine max 101ctcggtgctg tctt
1410223DNAGlycine max 102gaaaccaatt ttgatgtgaa gga
2310320DNAGlycine max 103aagtgagagg ggtgcaaaga 2010414DNAGlycine
max 104cagccctatc tcac 1410514DNAGlycine max 105agccctgtct cact
1410621DNAGlycine max 106gcaaatgaga aggctgaagc t 2110719DNAGlycine
max 107gctgtccctc agtccatcc 1910815DNAGlycine max 108cggtatcgct
cgtca 1510915DNAGlycine max 109tatcgctcgc caacg 1511023DNAGlycine
max 110atccacttgc aagataggac act 2311126DNAGlycine max
111gtgtaagtac tgatgtgcag ttttga 2611219DNAGlycine max 112cttgacatta
agactatcc 1911319DNAGlycine max 113agactaatcc ttaaacaag
191143672DNAGlycine max 114atgcatgctg atggcttaga ctggttcctc
atgatttttg gtctctttgg ggccattggt 60gatggcatag gcaccccttt ggtgttgttt
atcaccagca aaattatgaa caatattggt 120ggtttttcta gcaacatagg
cagcactttc atccacagca tcaatgagaa tgccgtggtt 180ttgttatatt
tggctggtgg gtctttcatt gcttgtttcc tagagggtta ttgttggaca
240agaacaggag aaaggcaagc tgcaagaatg agagttaggt accttaaagc
agttctcagg 300caagaagtag catactttga tttgcatgtc acaagcacat
cggaggtcat caccagcgtc 360tctaatgata gcctcgtaat tcaagattgt
cttagtgaaa aggtcccaaa ctttttgatg 420aatgcgtcca tgtttgttgg
gagctacata gtggcttttg cattattgtg gagattggcc 480attgtggggt
tcccttttgt ggccctactt gtgatccccg gtttcatgta tgggaggaca
540ttaatggggt tggctagcaa gataagagaa gagtacaata aagcaggcac
aatagcagaa 600caagcaatat cctccatcag aaccgtttat tcttttgtgg
gggaaagcaa gactattgat 660gctttctctg aagccctaca agggtctgtt
gagttgggac tgagacaagg cttagcaaaa 720ggtttagcta ttggaagcaa
tggtgttgtc tttgctatat gggcattcat gtcctattat 780ggtagcagat
tggtcatgta ccatggagct aaaggtggga ctgtatttgc agttggagca
840gccatagctc ttggaggatt ggcactaggt gctggtttgt cgaacgtgaa
gtacttctca 900gaagcaagta ccgcaggaga acgcataatg gaagtgataa
aaagggttcc aaagattgat 960tctgatagca tggctgagga gattctggag
aacgtttcag gggaagttga attcaaccat 1020gtggactttg tgtacccatc
aaggccagac agtgttattc tgaatgattt ctgcctaaag 1080attccagcag
ggaaaacagt ggctttggtt ggagggagtg gctctggaaa atccactgtg
1140atatcacttt tgcagaggtt ttatgaccca attgagggag agatatttct
tgatggtgtg 1200gccattcaca agttgcaact caagtggttg aggtctcaaa
tgggtttggt cagccaagag 1260cctgcactgt ttgcaactag cattaaagag
aatatacttt ttggaagaga agatgccact 1320caagaagagg ttgtggaggc
agcaaaagct tccaatgctc ataatttcat ttcacagttg 1380ccacaaggat
atgatactca ggttggggag agaggagttc aaatgtcagg tggacaaaag
1440caaagaattg caatagcacg agcaataata aaaaagccac ggattcttct
attagatgaa 1500gcaacaagtg cactagattc tgaatctgaa cgagttgtgc
aagaagcatt agacaaagca 1560gcagtagggc gcacaacaat catcattgca
catagattat ccaccataag gaatgcaaat 1620gtgattgctg ttgtgcaaag
tgggaaaatc atggagatgg gatcacacca tgaactaatc 1680caaaacgaca
atggccttta cacctcacta gttcgtctcc aacaagcaaa aaatgaaaaa
1740gaagacacca tttttcatcc tactcctcct tcgtccatat cgaacaaaga
caatcacaac 1800acgagtagcc gtaggctctc tgttgtgatg atccgttcta
gctccaccaa ctcgatacct 1860cgtattggtg gtggtgacga taacaatatt
gttgaagaag tagtggaaga taacaagcca 1920ccacttccct cgtttcgaag
gttgctcgca ctgaacattc ccgagtggaa gcaagcatgt 1980ttagggtgtt
tgaatgcggt gttgtttggt gcaattcagc ctgtgtatgc atttgcaatg
2040gggtcagtga tatctgttta cttcctccca gaccataatg agataaagaa
gaaaactatg 2100atctattcac tttgtttcct agggttggct gtgttctcct
tagtggttaa tatcctccag 2160cattacaact ttgcttacat aggagagtac
ttgactaaaa ggatcagaga aagaatgttt 2220tccaagatac tcacttttga
agttggatgg tttgatcaag atgaaaattc cacaggtgct 2280gtttgttcta
gacttgccaa agaagccaat gtgaatggtc tagtggtaca aaccatttca
2340gcagtggtaa tagcttttac aatgggccta atcattgcat ggaggttggc
cattgttatg 2400atagcagttc aacccattat catagcatgt ttctacacaa
ggcgtgtcct tctcaagagc 2460atgtctagta aggccatcaa ggcccaagat
gaaagtagca agatagctgt tgaagctgtt 2520tccaacctca gaacaatcac
agcattttct tcccaagata ggatccttaa aatgctcgaa 2580aaggcccaag
aaggcccgag ccgtgaaagc attcgacaat catggtttgc gggcattggg
2640cttgcatgtt cccaaagcct tacattttgc acttgggctt tggacttttg
gtatggaggc 2700aagcttgtgt ttcagggctt cataaacgcc aaagcattgt
ttgagacctt catgatttta 2760gtgagcacag gtagggttat tgcagatgct
ggtagcatga ccaatgacct agctaaaggg 2820gctgatgctg tgggctcagt
ttttgcaatc ttagataggt acacaaaaat tgagccagat 2880gatgacatag
atgggtacaa gcctgaaaag ctaacaggga aaatagagct tcatgatgtc
2940cattttgcat acccagctag gcccaatgtg atgatcttcc aaggcttctc
aatcaaaatt 3000gatgcaggca gatcaacagc attggttggg caaagtggct
ctggaaaatc aacaatcata 3060ggcttaattg agagattcta tgaccctatg
aaagggattg tgaccattga tggtagagac 3120ataaaatcat accaccttag
gtcactaagg aagcatattg ctcttgtaag ccaagagcca 3180acattgtttg
gtgggaccat aagggaaaat attgcatatg gggcatctaa taataataac
3240aaggttgatg aaactgagat catagaagca gctagggcag ctaatgctca
tgatttcatt 3300gcaagcctaa aggatggtta tgacacatcg tgtagagata
gaggagtgca actctctggg 3360ggtcaaaagc aaagaattgc aatagctaga
gccatattga agaatccaga agtgttgttg 3420ttggatgaag ccacaagtgc
cctagatagc caatcagaaa aattggtgca agatgctcta 3480gaaagggtga
tggtggggag aactagtgtg gtggtggctc acaggttaag cacaatacaa
3540aattgtgacc taattgctgt gttagataag ggaaaagtgg tggagaaagg
gacccactca 3600tctttgttgg ctcatggacc aggtggagct tattactctt
tgataagttt acaaagaaga 3660ccagcaaatt aa 36721151223PRTGlycine max
115Met His Ala Asp Gly Leu Asp Trp Phe Leu Met Ile Phe Gly Leu Phe
1 5 10 15 Gly Ala Ile Gly Asp Gly Ile Gly Thr Pro Leu Val Leu Phe
Ile Thr 20 25 30 Ser Lys Ile Met Asn Asn Ile Gly Gly Phe Ser Ser
Asn Ile Gly Ser 35 40 45 Thr Phe Ile His Ser Ile Asn Glu Asn Ala
Val Val Leu Leu Tyr Leu 50 55 60 Ala Gly Gly Ser Phe Ile Ala Cys
Phe Leu Glu Gly Tyr Cys Trp Thr 65 70 75 80 Arg Thr Gly Glu Arg Gln
Ala Ala Arg Met Arg Val Arg Tyr Leu Lys 85 90 95 Ala Val Leu Arg
Gln Glu Val Ala Tyr Phe Asp Leu His Val Thr Ser 100 105 110 Thr Ser
Glu Val Ile Thr Ser Val Ser Asn Asp Ser Leu Val Ile Gln 115 120 125
Asp Cys Leu Ser Glu Lys Val Pro Asn Phe Leu Met Asn Ala Ser Met 130
135 140 Phe Val Gly Ser Tyr Ile Val Ala Phe Ala Leu Leu Trp Arg Leu
Ala 145 150 155 160 Ile Val Gly Phe Pro Phe Val Ala Leu Leu Val Ile
Pro Gly Phe Met 165 170 175 Tyr Gly Arg Thr Leu Met Gly Leu Ala Ser
Lys Ile Arg Glu Glu Tyr 180 185 190 Asn Lys Ala Gly Thr Ile Ala Glu
Gln Ala Ile Ser Ser Ile Arg Thr 195 200 205 Val Tyr Ser Phe Val Gly
Glu Ser Lys Thr Ile Asp Ala Phe Ser Glu 210 215 220 Ala Leu Gln Gly
Ser Val Glu Leu Gly Leu Arg Gln Gly Leu Ala Lys 225 230 235 240 Gly
Leu Ala Ile Gly Ser Asn Gly Val Val Phe Ala Ile Trp Ala Phe 245 250
255 Met Ser Tyr Tyr Gly Ser Arg Leu Val Met Tyr His Gly Ala Lys Gly
260 265 270 Gly Thr Val Phe Ala Val Gly Ala Ala Ile Ala Leu Gly Gly
Leu Ala 275 280 285 Leu Gly Ala Gly Leu Ser Asn Val Lys Tyr Phe Ser
Glu Ala Ser Thr 290 295 300 Ala Gly Glu Arg Ile Met Glu Val Ile Lys
Arg Val Pro Lys Ile Asp 305 310 315 320 Ser Asp Ser Met Ala Glu Glu
Ile Leu Glu Asn Val Ser Gly Glu Val 325 330 335 Glu Phe Asn His Val
Asp Phe Val Tyr Pro Ser Arg Pro Asp Ser Val 340 345
350 Ile Leu Asn Asp Phe Cys Leu Lys Ile Pro Ala Gly Lys Thr Val Ala
355 360 365 Leu Val Gly Gly Ser Gly Ser Gly Lys Ser Thr Val Ile Ser
Leu Leu 370 375 380 Gln Arg Phe Tyr Asp Pro Ile Glu Gly Glu Ile Phe
Leu Asp Gly Val 385 390 395 400 Ala Ile His Lys Leu Gln Leu Lys Trp
Leu Arg Ser Gln Met Gly Leu 405 410 415 Val Ser Gln Glu Pro Ala Leu
Phe Ala Thr Ser Ile Lys Glu Asn Ile 420 425 430 Leu Phe Gly Arg Glu
Asp Ala Thr Gln Glu Glu Val Val Glu Ala Ala 435 440 445 Lys Ala Ser
Asn Ala His Asn Phe Ile Ser Gln Leu Pro Gln Gly Tyr 450 455 460 Asp
Thr Gln Val Gly Glu Arg Gly Val Gln Met Ser Gly Gly Gln Lys 465 470
475 480 Gln Arg Ile Ala Ile Ala Arg Ala Ile Ile Lys Lys Pro Arg Ile
Leu 485 490 495 Leu Leu Asp Glu Ala Thr Ser Ala Leu Asp Ser Glu Ser
Glu Arg Val 500 505 510 Val Gln Glu Ala Leu Asp Lys Ala Ala Val Gly
Arg Thr Thr Ile Ile 515 520 525 Ile Ala His Arg Leu Ser Thr Ile Arg
Asn Ala Asn Val Ile Ala Val 530 535 540 Val Gln Ser Gly Lys Ile Met
Glu Met Gly Ser His His Glu Leu Ile 545 550 555 560 Gln Asn Asp Asn
Gly Leu Tyr Thr Ser Leu Val Arg Leu Gln Gln Ala 565 570 575 Lys Asn
Glu Lys Glu Asp Thr Ile Phe His Pro Thr Pro Pro Ser Ser 580 585 590
Ile Ser Asn Lys Asp Asn His Asn Thr Ser Ser Arg Arg Leu Ser Val 595
600 605 Val Met Ile Arg Ser Ser Ser Thr Asn Ser Ile Pro Arg Ile Gly
Gly 610 615 620 Gly Asp Asp Asn Asn Ile Val Glu Glu Val Val Glu Asp
Asn Lys Pro 625 630 635 640 Pro Leu Pro Ser Phe Arg Arg Leu Leu Ala
Leu Asn Ile Pro Glu Trp 645 650 655 Lys Gln Ala Cys Leu Gly Cys Leu
Asn Ala Val Leu Phe Gly Ala Ile 660 665 670 Gln Pro Val Tyr Ala Phe
Ala Met Gly Ser Val Ile Ser Val Tyr Phe 675 680 685 Leu Pro Asp His
Asn Glu Ile Lys Lys Lys Thr Met Ile Tyr Ser Leu 690 695 700 Cys Phe
Leu Gly Leu Ala Val Phe Ser Leu Val Val Asn Ile Leu Gln 705 710 715
720 His Tyr Asn Phe Ala Tyr Ile Gly Glu Tyr Leu Thr Lys Arg Ile Arg
725 730 735 Glu Arg Met Phe Ser Lys Ile Leu Thr Phe Glu Val Gly Trp
Phe Asp 740 745 750 Gln Asp Glu Asn Ser Thr Gly Ala Val Cys Ser Arg
Leu Ala Lys Glu 755 760 765 Ala Asn Val Asn Gly Leu Val Val Gln Thr
Ile Ser Ala Val Val Ile 770 775 780 Ala Phe Thr Met Gly Leu Ile Ile
Ala Trp Arg Leu Ala Ile Val Met 785 790 795 800 Ile Ala Val Gln Pro
Ile Ile Ile Ala Cys Phe Tyr Thr Arg Arg Val 805 810 815 Leu Leu Lys
Ser Met Ser Ser Lys Ala Ile Lys Ala Gln Asp Glu Ser 820 825 830 Ser
Lys Ile Ala Val Glu Ala Val Ser Asn Leu Arg Thr Ile Thr Ala 835 840
845 Phe Ser Ser Gln Asp Arg Ile Leu Lys Met Leu Glu Lys Ala Gln Glu
850 855 860 Gly Pro Ser Arg Glu Ser Ile Arg Gln Ser Trp Phe Ala Gly
Ile Gly 865 870 875 880 Leu Ala Cys Ser Gln Ser Leu Thr Phe Cys Thr
Trp Ala Leu Asp Phe 885 890 895 Trp Tyr Gly Gly Lys Leu Val Phe Gln
Gly Phe Ile Asn Ala Lys Ala 900 905 910 Leu Phe Glu Thr Phe Met Ile
Leu Val Ser Thr Gly Arg Val Ile Ala 915 920 925 Asp Ala Gly Ser Met
Thr Asn Asp Leu Ala Lys Gly Ala Asp Ala Val 930 935 940 Gly Ser Val
Phe Ala Ile Leu Asp Arg Tyr Thr Lys Ile Glu Pro Asp 945 950 955 960
Asp Asp Ile Asp Gly Tyr Lys Pro Glu Lys Leu Thr Gly Lys Ile Glu 965
970 975 Leu His Asp Val His Phe Ala Tyr Pro Ala Arg Pro Asn Val Met
Ile 980 985 990 Phe Gln Gly Phe Ser Ile Lys Ile Asp Ala Gly Arg Ser
Thr Ala Leu 995 1000 1005 Val Gly Gln Ser Gly Ser Gly Lys Ser Thr
Ile Ile Gly Leu Ile 1010 1015 1020 Glu Arg Phe Tyr Asp Pro Met Lys
Gly Ile Val Thr Ile Asp Gly 1025 1030 1035 Arg Asp Ile Lys Ser Tyr
His Leu Arg Ser Leu Arg Lys His Ile 1040 1045 1050 Ala Leu Val Ser
Gln Glu Pro Thr Leu Phe Gly Gly Thr Ile Arg 1055 1060 1065 Glu Asn
Ile Ala Tyr Gly Ala Ser Asn Asn Asn Asn Lys Val Asp 1070 1075 1080
Glu Thr Glu Ile Ile Glu Ala Ala Arg Ala Ala Asn Ala His Asp 1085
1090 1095 Phe Ile Ala Ser Leu Lys Asp Gly Tyr Asp Thr Ser Cys Arg
Asp 1100 1105 1110 Arg Gly Val Gln Leu Ser Gly Gly Gln Lys Gln Arg
Ile Ala Ile 1115 1120 1125 Ala Arg Ala Ile Leu Lys Asn Pro Glu Val
Leu Leu Leu Asp Glu 1130 1135 1140 Ala Thr Ser Ala Leu Asp Ser Gln
Ser Glu Lys Leu Val Gln Asp 1145 1150 1155 Ala Leu Glu Arg Val Met
Val Gly Arg Thr Ser Val Val Val Ala 1160 1165 1170 His Arg Leu Ser
Thr Ile Gln Asn Cys Asp Leu Ile Ala Val Leu 1175 1180 1185 Asp Lys
Gly Lys Val Val Glu Lys Gly Thr His Ser Ser Leu Leu 1190 1195 1200
Ala His Gly Pro Gly Gly Ala Tyr Tyr Ser Leu Ile Ser Leu Gln 1205
1210 1215 Arg Arg Pro Ala Asn 1220 1163672DNAGlycine max
116atgcatgctg atggcttaga ctggttcctc atgatttttg gtctctttgg
ggccattggt 60gatggcatag gcaccccttt ggtgttgttt atcaccagca aaattatgaa
caatattggt 120ggtttttcta gcaacatagg cagcactttc atccacagca
tcaatgagaa tgccgtggtt 180ttgttatatt tggctggtgg gtctttcatt
gcttgtttcc tagagggtta ttgttggaca 240agaacaggag aaaggcaagc
tgcaagaatg agagttaggt accttaaagc agttctcagg 300caagaagtag
catactttga tttgcatgtc acaagcacat cggaggtcat caccagcgtc
360tctaatgata gcctcgtaat tcaagattgt cttagtgaaa aggtcccaaa
ctttttgatg 420aatgcgtcca tgtttgttgg gagctacata gtggcttttg
cattattgtg gagattggcc 480attgtggggt tcccttttgt ggccctactt
gtgatccccg gtttcatgta tgggaggaca 540ttaatggggt tggctagcaa
gataagagaa gagtacaata aagcaggcac aatagcagaa 600caagcaatat
cctccatcag aaccgtttat tcttttgtgg gggaaagcaa gactattgat
660gctttctctg aagccctaca agggtctgtt gagttgggac tgagacaagg
cttagcaaaa 720ggtttagcta ttggaagcaa tggtgttgtc tttgctatat
gggcattcat gtcctattat 780ggtagcagat tggtcatgta ccatggagct
aaaggtggga ctgtatttgc agttggagca 840gccatagctc ttggaggatt
ggcactaggt gctggtttgt cgaacgtgaa gtacttctca 900gaagcaagta
ccgcaggaga acgcataatg gaagtgataa aaagggttcc aaagattgat
960tctgatagca tggctgagga gattctggag aacgtttcag gggaagttga
attcaaccat 1020gtggactttg tgtacccatc aaggccagac agtgttattc
tgaatgattt ctgcctaaag 1080attccagcag ggaaaacagt ggctttggtt
ggagggagtg gctctggaaa atccactgtg 1140atatcacttt tgcagaggtt
ttatgaccca attgagggag agatatttct tgatggtgtg 1200gccattcaca
agttgcaact caagtggttg aggtctcaaa tgggtttggt cagccaagag
1260cctgcactgt ttgcaactag cattaaagag aatatacttt ttggaagaga
agatgccact 1320caagaagagg ttgtggaggc agcaaaagct tccaatgctc
ataatttcat ttcacagttg 1380ccacaaggat atgatactca ggttggggag
agaggagttc aaatgtcagg tggacaaaag 1440caaagaattg caatagcacg
agcaataata aaaaagccac ggattcttct attagatgaa 1500gcaacaagtg
cactagattc tgaatctgaa cgagttgtgc aagaagcatt agacaaagta
1560gcagtagggc gcacaacaat catcattgca catagattat ccaccataag
gaatgcaaat 1620gtgattgctg ttgtgcaaag tgggaaaatc atggagatgg
gatcacacca tgaactaatc 1680caaaacgaca atggccttta cacctcacta
gttcgtctcc aacaagcaaa aaatgaaaaa 1740gaagacaccc tttttcatcc
tactcctcct tcgtccatat cgaacaaaga caatcacaac 1800acgagtagcc
gtaggctctc tgttgtgatg agccgttcta gctccaccaa ctcgatacct
1860cgtattggtg gtggtgacga taacaatatt gttgaagaag tagtggaaga
taacaagcca 1920ccacttccct catttcgaag gttgctcgca ctgaacattc
ccgagtggaa gcaagcatgt 1980ttagggtgtt tgaatgcggt gttgtttggt
gcaattcagc ctgtgtatgc atttgcaatg 2040gggtcagtga tatctgttta
cttcctccca gaccataatg agataaagaa gaaaactatg 2100atctattcac
tttgtttcct agggttggct gtgttctcct tagtggttaa tatcctccag
2160cattacaact ttgcttacat aggagagtac ttgactaaaa ggatcagaga
aagaatgttt 2220tccaagatac tcacttttga agttggatgg tttgatcaag
atgaaaattc cacaggtgct 2280gtttgttcta gacttgccaa agaagccaat
gtgaatggtc tagtggtaca aaccatttca 2340gcagtggtaa tagcttttac
aatgggccta atcattgcat ggaggttggc cattgttatg 2400atagcagttc
aacccattat catagcatgt ttctacacaa ggcgtgtcct tctcaagagc
2460atgtctagta aggccatcaa ggcccaagat gaaagtagca agatagctgt
tgaagctgtt 2520tccaacctca gaacaatcac agcattttct tcccaagata
ggatccttaa aatgctcgaa 2580aaggcccaag aaggcccgag ccgtgaaagc
attcgacaat catggtttgc gggcattggg 2640cttgcatgtt cccaaagcct
tacattttgc acttgggctt tggacttttg gtatggaggc 2700aagcttgtgt
ttcagggctt cataaatgcc aaagcattgt ttgagacctt catgatttta
2760gtgagcacag gtagggttat tgcagatgct ggtagcatga ccaatgacct
agctaaaggg 2820gctgatgctg tgggctcagt ttttgcaatc ttagataagt
acacaaaaat tgagccagat 2880gatgacatag atgggtacaa gcctgaaaag
ctaacaggga aaatagagct tcatgatgtc 2940cattttgcat acccagctag
gcccaatgtg atgatcttcc aaggcttctc aatcaaaatt 3000gatgcaggca
gatcaacagc attggtcggg caaagtggct ctggaaaatc aacaatcata
3060ggcttaattg agagattcta tgaccctcta aaagggattg tgaccattga
tggtagagac 3120ataaaatcat accaccttag gtcactaagg aagcatattg
ctcttgtaag ccaagagcca 3180acattgtttg gtgggaccat aagggaaaat
attgcatatg gggcatctaa taataataac 3240aaggttgatg aaactgagat
catagaagca gctagggcag ctaatgctca tgatttcatt 3300gcaagcctaa
aggatggtta tgacacatcg tgtggagata gaggagtgca actctctggg
3360ggtcaaaagc aaagaattgc aatagctaga gccatattga agaatccaga
agtgttgttg 3420ttggatgaag ccacaagtgc cctagatagc caatcagaaa
aattggtgca agatgctcta 3480gaaagggtga tggtggggag aactagtgtg
gtggtggctc acaggttaag cacaatacaa 3540aattgtgacc taattgctgt
gttagataag ggaaaagtgg tggagaaagg gacccactca 3600tctttgttgg
ctcatggacc aggtggagct tattactctt tgataagttt acaaagaaga
3660ccagcaaatt aa 36721171223PRTGlycine max 117Met His Ala Asp Gly
Leu Asp Trp Phe Leu Met Ile Phe Gly Leu Phe 1 5 10 15 Gly Ala Ile
Gly Asp Gly Ile Gly Thr Pro Leu Val Leu Phe Ile Thr 20 25 30 Ser
Lys Ile Met Asn Asn Ile Gly Gly Phe Ser Ser Asn Ile Gly Ser 35 40
45 Thr Phe Ile His Ser Ile Asn Glu Asn Ala Val Val Leu Leu Tyr Leu
50 55 60 Ala Gly Gly Ser Phe Ile Ala Cys Phe Leu Glu Gly Tyr Cys
Trp Thr 65 70 75 80 Arg Thr Gly Glu Arg Gln Ala Ala Arg Met Arg Val
Arg Tyr Leu Lys 85 90 95 Ala Val Leu Arg Gln Glu Val Ala Tyr Phe
Asp Leu His Val Thr Ser 100 105 110 Thr Ser Glu Val Ile Thr Ser Val
Ser Asn Asp Ser Leu Val Ile Gln 115 120 125 Asp Cys Leu Ser Glu Lys
Val Pro Asn Phe Leu Met Asn Ala Ser Met 130 135 140 Phe Val Gly Ser
Tyr Ile Val Ala Phe Ala Leu Leu Trp Arg Leu Ala 145 150 155 160 Ile
Val Gly Phe Pro Phe Val Ala Leu Leu Val Ile Pro Gly Phe Met 165 170
175 Tyr Gly Arg Thr Leu Met Gly Leu Ala Ser Lys Ile Arg Glu Glu Tyr
180 185 190 Asn Lys Ala Gly Thr Ile Ala Glu Gln Ala Ile Ser Ser Ile
Arg Thr 195 200 205 Val Tyr Ser Phe Val Gly Glu Ser Lys Thr Ile Asp
Ala Phe Ser Glu 210 215 220 Ala Leu Gln Gly Ser Val Glu Leu Gly Leu
Arg Gln Gly Leu Ala Lys 225 230 235 240 Gly Leu Ala Ile Gly Ser Asn
Gly Val Val Phe Ala Ile Trp Ala Phe 245 250 255 Met Ser Tyr Tyr Gly
Ser Arg Leu Val Met Tyr His Gly Ala Lys Gly 260 265 270 Gly Thr Val
Phe Ala Val Gly Ala Ala Ile Ala Leu Gly Gly Leu Ala 275 280 285 Leu
Gly Ala Gly Leu Ser Asn Val Lys Tyr Phe Ser Glu Ala Ser Thr 290 295
300 Ala Gly Glu Arg Ile Met Glu Val Ile Lys Arg Val Pro Lys Ile Asp
305 310 315 320 Ser Asp Ser Met Ala Glu Glu Ile Leu Glu Asn Val Ser
Gly Glu Val 325 330 335 Glu Phe Asn His Val Asp Phe Val Tyr Pro Ser
Arg Pro Asp Ser Val 340 345 350 Ile Leu Asn Asp Phe Cys Leu Lys Ile
Pro Ala Gly Lys Thr Val Ala 355 360 365 Leu Val Gly Gly Ser Gly Ser
Gly Lys Ser Thr Val Ile Ser Leu Leu 370 375 380 Gln Arg Phe Tyr Asp
Pro Ile Glu Gly Glu Ile Phe Leu Asp Gly Val 385 390 395 400 Ala Ile
His Lys Leu Gln Leu Lys Trp Leu Arg Ser Gln Met Gly Leu 405 410 415
Val Ser Gln Glu Pro Ala Leu Phe Ala Thr Ser Ile Lys Glu Asn Ile 420
425 430 Leu Phe Gly Arg Glu Asp Ala Thr Gln Glu Glu Val Val Glu Ala
Ala 435 440 445 Lys Ala Ser Asn Ala His Asn Phe Ile Ser Gln Leu Pro
Gln Gly Tyr 450 455 460 Asp Thr Gln Val Gly Glu Arg Gly Val Gln Met
Ser Gly Gly Gln Lys 465 470 475 480 Gln Arg Ile Ala Ile Ala Arg Ala
Ile Ile Lys Lys Pro Arg Ile Leu 485 490 495 Leu Leu Asp Glu Ala Thr
Ser Ala Leu Asp Ser Glu Ser Glu Arg Val 500 505 510 Val Gln Glu Ala
Leu Asp Lys Val Ala Val Gly Arg Thr Thr Ile Ile 515 520 525 Ile Ala
His Arg Leu Ser Thr Ile Arg Asn Ala Asn Val Ile Ala Val 530 535 540
Val Gln Ser Gly Lys Ile Met Glu Met Gly Ser His His Glu Leu Ile 545
550 555 560 Gln Asn Asp Asn Gly Leu Tyr Thr Ser Leu Val Arg Leu Gln
Gln Ala 565 570 575 Lys Asn Glu Lys Glu Asp Thr Leu Phe His Pro Thr
Pro Pro Ser Ser 580 585 590 Ile Ser Asn Lys Asp Asn His Asn Thr Ser
Ser Arg Arg Leu Ser Val 595 600 605 Val Met Ser Arg Ser Ser Ser Thr
Asn Ser Ile Pro Arg Ile Gly Gly 610 615 620 Gly Asp Asp Asn Asn Ile
Val Glu Glu Val Val Glu Asp Asn Lys Pro 625 630 635 640 Pro Leu Pro
Ser Phe Arg Arg Leu Leu Ala Leu Asn Ile Pro Glu Trp 645 650 655 Lys
Gln Ala Cys Leu Gly Cys Leu Asn Ala Val Leu Phe Gly Ala Ile 660 665
670 Gln Pro Val Tyr Ala Phe Ala Met Gly Ser Val Ile Ser Val Tyr Phe
675 680 685 Leu Pro Asp His Asn Glu Ile Lys Lys Lys Thr Met Ile Tyr
Ser Leu 690 695 700 Cys Phe Leu Gly Leu Ala Val Phe Ser Leu Val Val
Asn Ile Leu Gln 705 710 715 720 His Tyr Asn Phe Ala Tyr Ile Gly Glu
Tyr Leu Thr Lys Arg Ile Arg 725 730 735 Glu Arg Met Phe Ser Lys Ile
Leu Thr Phe Glu Val Gly Trp Phe Asp 740 745 750 Gln Asp Glu Asn Ser
Thr Gly Ala Val Cys Ser Arg Leu Ala Lys Glu 755 760 765 Ala Asn Val
Asn Gly Leu Val Val Gln Thr Ile Ser Ala Val Val Ile 770 775 780 Ala
Phe Thr Met Gly Leu Ile Ile Ala Trp Arg Leu Ala Ile Val Met 785 790
795 800 Ile Ala Val Gln Pro Ile Ile Ile Ala Cys Phe Tyr Thr Arg Arg
Val 805 810 815 Leu Leu Lys Ser Met Ser Ser Lys Ala Ile Lys Ala Gln
Asp Glu Ser 820 825 830 Ser Lys Ile Ala Val Glu Ala Val Ser Asn Leu
Arg Thr Ile Thr Ala 835 840
845 Phe Ser Ser Gln Asp Arg Ile Leu Lys Met Leu Glu Lys Ala Gln Glu
850 855 860 Gly Pro Ser Arg Glu Ser Ile Arg Gln Ser Trp Phe Ala Gly
Ile Gly 865 870 875 880 Leu Ala Cys Ser Gln Ser Leu Thr Phe Cys Thr
Trp Ala Leu Asp Phe 885 890 895 Trp Tyr Gly Gly Lys Leu Val Phe Gln
Gly Phe Ile Asn Ala Lys Ala 900 905 910 Leu Phe Glu Thr Phe Met Ile
Leu Val Ser Thr Gly Arg Val Ile Ala 915 920 925 Asp Ala Gly Ser Met
Thr Asn Asp Leu Ala Lys Gly Ala Asp Ala Val 930 935 940 Gly Ser Val
Phe Ala Ile Leu Asp Lys Tyr Thr Lys Ile Glu Pro Asp 945 950 955 960
Asp Asp Ile Asp Gly Tyr Lys Pro Glu Lys Leu Thr Gly Lys Ile Glu 965
970 975 Leu His Asp Val His Phe Ala Tyr Pro Ala Arg Pro Asn Val Met
Ile 980 985 990 Phe Gln Gly Phe Ser Ile Lys Ile Asp Ala Gly Arg Ser
Thr Ala Leu 995 1000 1005 Val Gly Gln Ser Gly Ser Gly Lys Ser Thr
Ile Ile Gly Leu Ile 1010 1015 1020 Glu Arg Phe Tyr Asp Pro Leu Lys
Gly Ile Val Thr Ile Asp Gly 1025 1030 1035 Arg Asp Ile Lys Ser Tyr
His Leu Arg Ser Leu Arg Lys His Ile 1040 1045 1050 Ala Leu Val Ser
Gln Glu Pro Thr Leu Phe Gly Gly Thr Ile Arg 1055 1060 1065 Glu Asn
Ile Ala Tyr Gly Ala Ser Asn Asn Asn Asn Lys Val Asp 1070 1075 1080
Glu Thr Glu Ile Ile Glu Ala Ala Arg Ala Ala Asn Ala His Asp 1085
1090 1095 Phe Ile Ala Ser Leu Lys Asp Gly Tyr Asp Thr Ser Cys Gly
Asp 1100 1105 1110 Arg Gly Val Gln Leu Ser Gly Gly Gln Lys Gln Arg
Ile Ala Ile 1115 1120 1125 Ala Arg Ala Ile Leu Lys Asn Pro Glu Val
Leu Leu Leu Asp Glu 1130 1135 1140 Ala Thr Ser Ala Leu Asp Ser Gln
Ser Glu Lys Leu Val Gln Asp 1145 1150 1155 Ala Leu Glu Arg Val Met
Val Gly Arg Thr Ser Val Val Val Ala 1160 1165 1170 His Arg Leu Ser
Thr Ile Gln Asn Cys Asp Leu Ile Ala Val Leu 1175 1180 1185 Asp Lys
Gly Lys Val Val Glu Lys Gly Thr His Ser Ser Leu Leu 1190 1195 1200
Ala His Gly Pro Gly Gly Ala Tyr Tyr Ser Leu Ile Ser Leu Gln 1205
1210 1215 Arg Arg Pro Ala Asn 1220
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