U.S. patent application number 15/437887 was filed with the patent office on 2017-08-17 for polynucleotide and polypeptide sequences associated with herbicide tolerance.
The applicant listed for this patent is PIONEER HI-BRED INTERNATIONAL, INC.. Invention is credited to KEVIN A. FENGLER, BAILIN LI.
Application Number | 20170233757 15/437887 |
Document ID | / |
Family ID | 43629267 |
Filed Date | 2017-08-17 |
United States Patent
Application |
20170233757 |
Kind Code |
A1 |
LI; BAILIN ; et al. |
August 17, 2017 |
POLYNUCLEOTIDE AND POLYPEPTIDE SEQUENCES ASSOCIATED WITH HERBICIDE
TOLERANCE
Abstract
This invention relates generally to the detection of genetic
differences among soybeans. More particularly, soybean quantitative
trait loci (QTL) associated with herbicide tolerance, including
tolerance to one or more of an HPPD-inhibitor herbicide, such as
mesotrione and isoxazole herbicides, and/or a PPO inhibitor
herbicide; soybean plants possessing these QTLs; and genetic
markers that are indicative of phenotypes associated with such
herbicide tolerance are provided. Methods and compositions for use
of these markers in genotyping of soybean and selection are also
disclosed, as are methods and compositions for use of herbicides
for weed control. Also disclosed are isolated polynucleotides and
polypeptides relating to such tolerance or sensitivity and methods
of introgressing such tolerance into a plant by breeding or
transgenically, or by a combination thereof. Plant cells, plants,
and seeds produced are also provided.
Inventors: |
LI; BAILIN; (JOHNSTON,
IA) ; FENGLER; KEVIN A.; (CLIVE, IA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PIONEER HI-BRED INTERNATIONAL, INC. |
Johnston |
IA |
US |
|
|
Family ID: |
43629267 |
Appl. No.: |
15/437887 |
Filed: |
February 21, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13013139 |
Jan 25, 2011 |
9611485 |
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15437887 |
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61298528 |
Jan 26, 2010 |
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61298523 |
Jan 26, 2010 |
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61371392 |
Aug 6, 2010 |
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61371454 |
Aug 6, 2010 |
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Current U.S.
Class: |
504/206 |
Current CPC
Class: |
C12N 15/8274 20130101;
C12Q 2600/13 20130101; C12Q 2600/156 20130101; C12Q 2600/172
20130101; A01H 1/04 20130101; C12Q 1/6895 20130101 |
International
Class: |
C12N 15/82 20060101
C12N015/82; C07K 14/415 20060101 C07K014/415; C12Q 1/68 20060101
C12Q001/68 |
Claims
1.-6. (canceled)
7. An isolated polynucleotide comprising a nucleic acid sequence
encoding an ABC transporter polypeptide; wherein the ABC
transporter polypeptide comprises an amino acid sequence at least
80% identical over its full length to SEQ ID NO: 128; wherein the
ABC transporter polypeptide confers tolerance or improved tolerance
to one or more herbicides; and wherein the ABC transporter
polypeptide comprises one or more amino acid substitutions, said
amino acid substitutions selected from the group consisting of
E478X, V520X, L584X, S611X, K961X, L1038X, and G1120X based on the
amino acid positions of SEQ ID NO:128.
8. The isolated polynucleotide of claim 7, wherein the one or more
herbicides are selected from the group consisting of a mesotrione
herbicide, an isoxazole herbicide, and a PPO inhibitor
herbicide.
9. The isolated polynucleotide of claim 7, wherein the ABC
transporter polypeptide comprises an amino acid sequence at least
about 95% identical over its full length to SEQ ID NO:128.
10. (canceled)
11. The isolated polynucleotide of claim 7, wherein the one or more
amino acid substitutions are selected from the group consisting of
E478G, V520A, L584I, S611I, K961R, L1038M, and G1120R based on the
amino acid positions of SEQ ID NO:128.
12. The isolated polynucleotide of claim 7, wherein the one or more
amino acid substitutions comprise all of the amino acid
substitutions E478G, V520A, L584I, S611I, K961R, L1038M, and G1120R
based on the amino acid positions of SEQ ID NO:128.
13. An isolated polynucleotide comprising a nucleic acid sequence
at least about 95% identical to the full length of SEQ ID NO:
124.
14. (canceled)
15. The isolated polynucleotide of claim 7, further comprising an
operably linked promoter functional in a host cell.
16. The isolated polynucleotide of claim 15, wherein the host cell
is a plant cell.
17. The isolated polynucleotide of claim 16, wherein the plant cell
is from corn, rice, sorghum, barley, oats, sugarcane, wheat,
canola, soybean, alfalfa, sunflower, or safflower.
18.-23. (canceled)
24. A plant comprising an isolated polynucleotide, wherein the
isolated polynucleotide is selected from the group consisting of
the isolated polynucleotide of claim 7 and a polynucleotide having
a sequence at least 95% identical to one of SEQ ID NOS:
122-125.
25. A plant comprising an isolated polynucleotide encoding an ABC
transporter polypeptide, wherein said ABC transporter polypeptide
comprises an amino acid sequence at least 80% identical over its
full length to SEQ ID NO: 128 or SEQ ID NO: 129; wherein said
polynucleotide comprises a G at position 1455, a C at position
1581, an A at position 1772, a T at position 1854, a G at position
1954, a C at position 2773, a G at position 2904, a Tat position
3073, an A at position 3134, a G at position 3136, an A at position
3380, and a T at position 3882 based on the nucleotide positions of
the sequence set forth in SEQ ID NO:124; and wherein the plant
shows tolerance to one or more herbicides selected from the group
consisting of a mesotrione herbicide, an isoxazole herbicide, and a
PPO inhibitor herbicide.
26. A cell of the plant of claim 25.
27. A seed of the plant of claim 25.
28.-33. (canceled)
34. A method to confer tolerance or improved tolerance to one or
more herbicides, the method comprising: a. introducing into a plant
cell a polynucleotide of claim 7 operably linked to a promoter
functional in the plant cell to produce a transformed plant cell;
and b. selecting a transformed plant cell having the polynucleotide
stably incorporated into its genome.
35. The method of claim 34, wherein the one or more herbicides are
selected from the group consisting of a mesotrione herbicide, an
isoxazole herbicide, and a PPO inhibitor herbicide.
36. The method of claim 34, wherein the selecting comprises
exposing the transformed plant cell to the one or more herbicides,
wherein the one or more herbicides are at a concentration that
inhibits the growth of an untransformed plant cell, and identifying
the transformed plant cell by its ability to grow in the presence
of the herbicide.
37. (canceled)
38. A method for selectively controlling weeds in a field
containing a crop comprising: (a) planting a field with crop seeds
or plants comprising the isolated polynucleotide of claim 7,
wherein the seeds or plants have tolerance to one or more
herbicides; and (b) applying to the crop and weeds in the field a
sufficient amount of the one or more herbicides to control the
weeds without significantly affecting the crop.
39. The method of claim 38, wherein the one or more herbicides are
selected from the group consisting of a mesotrione herbicide, an
isoxazole herbicide, and a PPO inhibitor herbicide
40. The method of claim 39, wherein the one or more herbicides are
applied as a pre-emergent herbicide.
41. The method of claim 40, further comprising applying to the crop
and weeds in the field a simultaneous or a chronologically
staggered application of the one or more herbicide and an
additional herbicide formulation.
42. The method of claim 41, wherein the additional herbicide
formulation comprises 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.
43. The method of claim 41, wherein the crop seeds or plants
further comprise tolerance to the active ingredient of the
additional herbicide formulation.
44. The method of claim 43, wherein the tolerance to the active
ingredient of the additional herbicide formulation is provided by
insertion of a transgene which confers the tolerance.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a Divisional of U.S. application Ser.
No. 13/013,139, filed Jan. 25, 2011, which claims priority to U.S.
Provisional Application Nos. 61/298,528, filed Jan. 26, 2010;
61/298,523, filed Jan. 26, 2010; 61/371,392, filed Aug. 6, 2010;
and 61/371,454, filed Aug. 6, 2010, each of which is incorporated
by reference in its entirely.
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
[0002] The official copy of the sequence listing is submitted
electronically via EFS-Web as an ASCII formatted sequence listing
with a file named 20170217_3707USDIV_SeqLst.txt created on Feb. 17,
2017 and having a size of 85 kilobytes and is filed concurrently
with the specification. The sequence listing contained in this
ASCII formatted document is part of the specification and is herein
incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0003] This invention relates generally to the detection of genetic
differences among soybeans.
BACKGROUND OF THE INVENTION
[0004] 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.
[0005] 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
[0006] This invention relates generally to polynucleotide and
polypeptide sequences related to tolerance to multiple herbicides,
compositions comprising the same, and methods of their use. In
certain examples, the invention relates to polynucleotide markers
capable of discriminating between soybean that displays tolerance
and soybean that displays susceptibility to one or more herbicides,
such as one or more herbicides selected from the group consisting
of an HPPD-inhibitor herbicide, such as mesotrione and isoxazole
herbicides, and/or a PPO inhibitor herbicide. In certain other
examples, the polynucleotide marker is capable of detecting a
polynucleotide sequence with 80%, 90%, 95%, or better identity to a
polynucleotide sequence provided herein, such as one of SEQ ID NOs:
122-125. In still further examples, the markers contain certain
identified SNPs at given positions, such as a SNP at one or more of
nucleotide position 1455, 1581, 1772, 1854, 1954, 2773, 2904, 3073,
3134, 3136, 3380, and 3882 of the sequence set forth in SEQ ID NO:
124 or SEQ ID NO: 125. Methods and compositions for use of these
markers in genotyping of soybean and selection are also
disclosed.
[0007] In still further examples, isolated polynucleotides encoding
ABC transporter polypeptides are provided. In certain examples, the
ABC transporter polypeptide displays 80%, 90%, 95%, or better
identity to a polypeptide sequence provided herein, such as one of
SEQ ID NOs: 126-129. In other examples, the polynucleotide sequence
possesses certain identified amino acid substitutions at given
amino acid positions, such as a substitution at one or more of
positions E478X, V520X, L584X, S611X, K961X, L1038X, or G1120X, or
positions equivalent thereto, including one or more of the amino
acid substitutions E478G, V520A, L584I, S611I, K961R, L1038M, and
G1120R. In still further examples, the polynucleotide comprises
80%, 90%, 95%, or better identity to a polynucleotide sequence
provided herein, such as one of SEQ ID NOs:122-125; or the
polynucleotide possesses certain identified SNPs at given
positions, such as a SNP at one or more of nucleotide position
1455, 1581, 1772, 1854, 1954, 2773, 2904, 3073, 3134, 3136, 3380,
and 3882 of the sequence set forth in SEQ ID NO: 124 or SEQ ID NO:
125. In certain examples, the one or more SNPs are selected from
the group consisting of a G at position 1455; a C at position 1581;
an A at position 1772; a T at position 1854; a G at position 1954;
a C at position 2773; a G at position 2904; a T at position 3073;
an A at position 3134; a G at position 3136; an A at position 3380;
and a T at position 3882. In yet further examples, the
polynucleotide sequence is operably linked to a promoter functional
in a host cell.
[0008] In additional examples, isolated ABC transporter
polypeptides are provided. In certain examples, the isolated
polypeptide displays 80%, 90%, 95%, or better identity to a
polypeptide sequence provided herein, such as one of SEQ ID NOs:
126-129. In other examples, the polynucleotide sequence possesses
certain identified amino acid substitutions at given amino acid
positions, such as a substitution at one or more of positions
E478X, V520X, L584X, S611X, K961X, L1038X, and/or G1120X or
positions equivalent thereto, including one or more of the amino
acid substitutions E478G, V520A, L584I, S611I, K961R, L1038M, and
G1120R.
[0009] Also described are isolated polynucleotides and isolated
polypeptides relevant to tolerance or sensitivity to one or more
herbicides. 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 a given herbicide with a soybean plant
susceptible to such herbicide 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 nucleotide position 1455, 1581, 1772,
1854, 1954, 2773, 2904, 3073, 3134, 3136, 3380, and 3882 of the
sequence set forth as SEQ ID NOs: 124 or 125, for example as shown
in FIGS. 7A-7F and FIGS. 9A-9I, or a sequence equivalent to SEQ ID
NOs: 124 or 125, for example SEQ ID NOs: 122 or 123, and optionally
selecting, if present, one or more soybean plants of the
segregating population containing the at least one SNP. In some
examples, soybean plant cells, plants, and/or seeds having a
haplotype comprising SNPs at nucleotide positions 1455, 1581, 1772,
1854, 1954, 2773, 2904, 3073, 3134, 3136, 3380, and 3882, or their
equivalent position(s), are selected. In some examples, soybean
plant cells, plants, and/or seeds having a haplotype comprising a G
at position 1455, a C at position 1581, an A at position 1772, a T
at position 1854, a G at position 1954, a C at position 2773, a G
at position 2904, a T at position 3073, an A at position 3134, a G
at position 3136, an A at position 3380, and a T at position 3882,
or their equivalent position(s), are selected. 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.
[0010] Also useful are isolated polynucleotide variants,
polynucleotides encoding polypeptide variants, and polypeptide
variants having sequence identity to the appropriate reference
sequence, such as 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%, 99.75%, or
100% identity.
[0011] Soybean plants, seeds, tissue cultures, variants, and
mutants having herbicide tolerance 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.
[0012] 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.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1A provides marker linkage information for linkage
group L (LG-L) and depicts an integrated genetic map of soybean
markers on LG-L, including the marker type (SSR, ASH, or 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.
[0014] FIG. 1B is a continuation of FIG. 1A and depicts an
integrated genetic map of soybean markers on LG-L.
[0015] FIG. 2 provides a table listing genetic markers that are
linked to the herbicide tolerance markers identified on linkage
group L. These markers are from the soybean public composite map of
Jun. 18, 2008 for linkage group L.
[0016] FIG. 3A provides examples of primer and probe nucleic acid
sequences that are useful for detecting SNP markers associated with
the herbicide tolerance QTL on LG-L.
[0017] FIG. 3B is a continuation of FIG. 3A and provides examples
of primer and probe nucleic acid sequences that are useful for
detecting SNP markers associated with the herbicide tolerance QTL
on LG-L.
[0018] FIG. 3C is a continuation of FIG. 3B and provides examples
of primer and probe nucleic acid sequences that are useful for
detecting SNP markers associated with the herbicide tolerance QTL
on LG-L.
[0019] FIG. 3D is a continuation of FIG. 3C and provides examples
of primer and probe nucleic acid sequences that are useful for
detecting SNP markers associated with the herbicide tolerance QTL
on LG-L.
[0020] FIG. 3E is a continuation of FIG. 3D and provides examples
of primer and probe nucleic acid sequences that are useful for
detecting SNP markers associated with the herbicide tolerance QTL
on LG-L.
[0021] FIG. 4 provides an example of cultivars with vastly
different protoporphyrinogen oxidase (PPO) 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).
[0022] FIG. 5 provides an example of cultivars with vastly
different PPO 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.
[0023] FIG. 6A is an exemplary alignment of Glyma19940 polypeptide
sequences and provides a GAP alignment of tolerant (SEQ ID No: 128)
vs. susceptible (SEQ ID NO: 129) polypeptide sequences.
[0024] FIG. 6B is a continuation of FIG. 6A and provides a GAP
alignment of tolerant (SEQ ID NO: 128) vs. susceptible (SEQ ID NO:
129) polypeptide sequences.
[0025] FIG. 7A is an exemplary alignment of Glyma19940
polynucleotide sequences and provides a GAP alignment of tolerant
(SEQ ID NO: 124) vs. susceptible (SEQ ID NO: 125) polynucleotide
sequences.
[0026] FIG. 7B is a continuation of FIG. 7A and provides a GAP
alignment of tolerant (SEQ ID NO: 124) vs. susceptible (SEQ ID NO:
125) polynucleotide sequences.
[0027] FIG. 7C is a continuation of FIG. 7B and provides a GAP
alignment of tolerant (SEQ ID NO: 124) vs. susceptible (SEQ ID NO:
125) polynucleotide sequences.
[0028] FIG. 7D is a continuation of FIG. 7C and provides a GAP
alignment of tolerant (SEQ ID NO: 124) vs. susceptible (SEQ ID NO:
125) polynucleotide sequences.
[0029] FIG. 7E is a continuation of FIG. 7D and provides a GAP
alignment of tolerant (SEQ ID NO: 124) vs. susceptible (SEQ ID NO:
125) polynucleotide sequences.
[0030] FIG. 7F is a continuation of FIG. 7E and provides a GAP
alignment of tolerant (SEQ ID NO: 124) vs. susceptible (SEQ ID NO:
125) polynucleotide sequences.
[0031] FIG. 8A is an exemplary alignment of Glyma19940 polypeptide
sequences and provides a PILEUP multiple sequence alignment of
tolerant (SEQ ID NOs: 126 and 128) and susceptible (SEQ ID NOs: 127
and 129) polypeptide sequences.
[0032] FIG. 8B is a continuation of FIG. 8A and provides a PILEUP
multiple sequence alignment of tolerant (SEQ ID NOs: 126 and 128)
and susceptible (SEQ ID NOs: 127 and 129) polypeptide
sequences.
[0033] FIG. 8C is a continuation of FIG. 8B and provides a PILEUP
multiple sequence alignment of tolerant (SEQ ID NOs: 126 and 128)
and susceptible (SEQ ID NOs: 127 and 129) polypeptide
sequences.
[0034] FIG. 9A is an exemplary alignment of Glyma19940
polynucleotide sequences and provides a PILEUP multiple sequence
alignment of tolerant (SEQ ID NOs: 122 and 124) and susceptible
(SEQ ID NOs: 123 and 125) polynucleotide sequences.
[0035] FIG. 9B is a continuation of FIG. 9A and provides a PILEUP
multiple sequence alignment of tolerant (SEQ ID NOs: 122 and 124)
and susceptible (SEQ ID NOs: 123 and 125) polynucleotide
sequences.
[0036] FIG. 9C is a continuation of FIG. 9B and provides a PILEUP
multiple sequence alignment of tolerant (SEQ ID NOs: 122 and 124)
and susceptible (SEQ ID NOs: 123 and 125) polynucleotide
sequences.
[0037] FIG. 9D is a continuation of FIG. 9C and provides a PILEUP
multiple sequence alignment of tolerant (SEQ ID NOs: 122 and 124)
and susceptible (SEQ ID NOs: 123 and 125) polynucleotide
sequences.
[0038] FIG. 9E is a continuation of FIG. 9D and provides a PILEUP
multiple sequence alignment of tolerant (SEQ ID NOs: 122 and 124)
and susceptible (SEQ ID NOs: 123 and 125) polynucleotide
sequences.
[0039] FIG. 9F is a continuation of FIG. 9E and provides a PILEUP
multiple sequence alignment of tolerant (SEQ ID NOs: 122 and 124)
and susceptible (SEQ ID NOs: 123 and 125) polynucleotide
sequences.
[0040] FIG. 9G is a continuation of FIG. 9F and provides a PILEUP
multiple sequence alignment of tolerant (SEQ ID NOs: 122 and 124)
and susceptible (SEQ ID NOs: 123 and 125) polynucleotide
sequences.
[0041] FIG. 9H is a continuation of FIG. 9G and provides a PILEUP
multiple sequence alignment of tolerant (SEQ ID NOs: 122 and 124)
and susceptible (SEQ ID NOs: 123 and 125) polynucleotide
sequences.
[0042] FIG. 9I is a continuation of FIG. 9H and provides a PILEUP
multiple sequence alignment of tolerant (SEQ ID NOs: 122 and 124)
and susceptible (SEQ ID NOs: 123 and 125) polynucleotide
sequences.
SUMMARY OF THE SEQUENCES
[0043] SEQ ID NOs: 1-5 comprise nucleotide sequences of regions of
the Soybean genome, each capable of being used as a probe or
primer, either alone or in combination, for the detection of marker
locus P10649C-3 on LG-L. In certain examples, SEQ ID NOs: 1 and 2
are used as primers while SEQ ID NOs: 3-5 are used as probes.
[0044] SEQ ID NOs: 6-9 comprise nucleotide sequences of regions of
the Soybean genome, each capable of being used as a probe or
primer, either alone or in combination, for the detection of marker
locus S00224-1 on LG-L. In certain examples, SEQ ID NOs: 6 and 7
are used as primers while SEQ ID NOs: 8 and 9 are used as
probes.
[0045] SEQ ID NOs: 10-13 comprise nucleotide sequences of regions
of the Soybean genome, each capable of being used as a probe or
primer, either alone or in combination, for the detection of marker
locus P5467-1 on LG-N. In certain examples, SEQ ID NOs: 10 and 11
are used as primers while SEQ ID NOs: 12 and 13 are used as
probes.
[0046] SEQ ID NOs: 14-17 comprise nucleotide sequences of regions
of the Soybean genome, each capable of being used as a probe or
primer, either alone or in combination, for the detection of marker
locus S08101-1-Q1 on LG-L. In certain examples, SEQ ID NOs: 14 and
15 are used as primers while SEQ ID NOs: 16 and 17 are used as
probes.
[0047] SEQ ID NOs: 18-21 comprise nucleotide sequences of regions
of the Soybean genome, each capable of being used as a probe or
primer, either alone or in combination, for the detection of marker
locus S08101-2-Q1 on LG-L. In certain examples, SEQ ID NOs: 18 and
19 are used as primers while SEQ ID NOs: 20 and 21 are used as
probes.
[0048] SEQ ID NOs: 22-25 comprise nucleotide sequences of regions
of the Soybean genome, each capable of being used as a probe or
primer, either alone or in combination, for the detection of marker
locus S08101-3-Q1 on LG-L. In certain examples, SEQ ID NOs: 22 and
23 are used as primers while SEQ ID NOs: 24 and 25 are used as
probes.
[0049] SEQ ID NOs: 26-29 comprise nucleotide sequences of regions
of the Soybean genome, each capable of being used as a probe or
primer, either alone or in combination, for the detection of marker
locus S08101-4-Q1 on LG-L. In certain examples, SEQ ID NOs: 26 and
27 are used as primers while SEQ ID NOs: 28 and 29 are used as
probes.
[0050] SEQ ID NOs: 30-33 comprise nucleotide sequences of regions
of the Soybean genome, each capable of being used as a probe or
primer, either alone or in combination, for the detection of marker
locus S08102-1-Q1 on LG-L. In certain examples, SEQ ID NOs: 30 and
31 are used as primers while SEQ ID NOs: 32 and 33 are used as
probes.
[0051] SEQ ID NOs: 34-37 comprise nucleotide sequences of regions
of the Soybean genome, each capable of being used as a probe or
primer, either alone or in combination, for the detection of marker
locus S08103-1-Q1 on LG-L. In certain examples, SEQ ID NOs: 34 and
35 are used as primers while SEQ ID NOs: 36 and 37 are used as
probes.
[0052] SEQ ID NOs: 38-41 comprise nucleotide sequences of regions
of the Soybean genome, each capable of being used as a probe or
primer, either alone or in combination, for the detection of marker
locus S08104-1-Q1 on LG-L. In certain examples, SEQ ID NOs: 38 and
39 are used as primers while SEQ ID NOs: 40 and 41 are used as
probes.
[0053] SEQ ID NOs: 42-45 comprise nucleotide sequences of regions
of the Soybean genome, each capable of being used as a probe or
primer, either alone or in combination, for the detection of marker
locus S08105-1-Q1 on LG-L. In certain examples, SEQ ID NOs: 42 and
43 are used as primers while SEQ ID NOs: 44 and 45 are used as
probes.
[0054] SEQ ID NOs: 46-49 comprise nucleotide sequences of regions
of the Soybean genome, each capable of being used as a probe or
primer, either alone or in combination, for the detection of marker
locus S08106-1-Q1 on LG-L. In certain examples, SEQ ID NOs: 46 and
47 are used as primers while SEQ ID NOs: 48 and 49 are used as
probes.
[0055] SEQ ID NOs: 50-53 comprise nucleotide sequences of regions
of the Soybean genome, each capable of being used as a probe or
primer, either alone or in combination, for the detection of marker
locus S08107-1-Q1 on LG-L. In certain examples, SEQ ID NOs: 50 and
51 are used as primers while SEQ ID NOs: 52 and 53 are used as
probes.
[0056] SEQ ID NOs: 54-57 comprise nucleotide sequences of regions
of the Soybean genome, each capable of being used as a probe or
primer, either alone or in combination, for the detection of marker
locus S08108-1-Q1 on LG-L. In certain examples, SEQ ID NOs: 54 and
55 are used as primers while SEQ ID NOs: 56 and 57 are used as
probes.
[0057] SEQ ID NOs: 58-61 comprise nucleotide sequences of regions
of the Soybean genome, each capable of being used as a probe or
primer, either alone or in combination, for the detection of marker
locus S08109-1-Q1 on LG-L. In certain examples, SEQ ID NOs: 58 and
59 are used as primers while SEQ ID NOs: 60 and 61 are used as
probes.
[0058] SEQ ID NOs: 62-65 comprise nucleotide sequences of regions
of the Soybean genome, each capable of being used as a probe or
primer, either alone or in combination, for the detection of marker
locus S08110-1-Q1 on LG-L. In certain examples, SEQ ID NOs: 62 and
63 are used as primers while SEQ ID NOs: 64 and 65 are used as
probes.
[0059] SEQ ID NOs: 66-69 comprise nucleotide sequences of regions
of the Soybean genome, each capable of being used as a probe or
primer, either alone or in combination, for the detection of marker
locus S08111-1-Q1 on LG-L. In certain examples, SEQ ID NOs: 66 and
67 are used as primers while SEQ ID NOs: 68 and 69 are used as
probes.
[0060] SEQ ID NOs: 70-73 comprise nucleotide sequences of regions
of the Soybean genome, each capable of being used as a probe or
primer, either alone or in combination, for the detection of marker
locus S08112-1-Q1 on LG-L. In certain examples, SEQ ID NOs: 70 and
71 are used as primers while SEQ ID NOs: 72 and 73 are used as
probes.
[0061] SEQ ID NOs: 74-77 comprise nucleotide sequences of regions
of the Soybean genome, each capable of being used as a probe or
primer, either alone or in combination, for the detection of marker
locus S08115-2-Q1 on LG-L. In certain examples, SEQ ID NOs: 74 and
75 are used as primers while SEQ ID NOs: 76 and 77 are used as
probes.
[0062] SEQ ID NOs: 78-81 comprise nucleotide sequences of regions
of the Soybean genome, each capable of being used as a probe or
primer, either alone or in combination, for the detection of marker
locus S08116-1-Q1 on LG-L. In certain examples, SEQ ID NOs: 78 and
79 are used as primers while SEQ ID NOs: 80 and 81 are used as
probes.
[0063] SEQ ID NOs: 82-85 comprise nucleotide sequences of regions
of the Soybean genome, each capable of being used as a probe or
primer, either alone or in combination, for the detection of marker
locus S08117-1-Q1 on LG-L. In certain examples, SEQ ID NOs: 82 and
83 are used as primers while SEQ ID NOs: 84 and 85 are used as
probes.
[0064] SEQ ID NOs: 86-89 comprise nucleotide sequences of regions
of the Soybean genome, each capable of being used as a probe or
primer, either alone or in combination, for the detection of marker
locus S08118-1-Q1 on LG-L. In certain examples, SEQ ID NOs: 86 and
87 are used as primers while SEQ ID NOs: 88 and 89 are used as
probes.
[0065] SEQ ID NOs: 90-93 comprise nucleotide sequences of regions
of the Soybean genome, each capable of being used as a probe or
primer, either alone or in combination, for the detection of marker
locus S08119-1-Q1 on LG-L. In certain examples, SEQ ID NOs: 90 and
91 are used as primers while SEQ ID NOs: 92 and 93 are used as
probes.
[0066] SEQ ID NOs: 94-97 comprise nucleotide sequences of regions
of the Soybean genome, each capable of being used as a probe or
primer, either alone or in combination, for the detection of marker
locus S04867-1-A on LG-L. In certain examples, SEQ ID NOs: 94 and
95 are used as primers while SEQ ID NOs: 96 and 97 are used as
probes.
[0067] SEQ ID NOs: 98-101 comprise nucleotide sequences of regions
of the Soybean genome, each capable of being used as a probe or
primer, either alone or in combination, for the detection of marker
locus S03859-1-A on LG-L. In certain examples, SEQ ID NOs: 98 and
99 are used as primers while SEQ ID NOs: 100 and 101 are used as
probes.
[0068] SEQ ID NOs: 102-105 comprise nucleotide sequences of regions
of the Soybean genome, each capable of being used as a probe or
primer, either alone or in combination, for the detection of marker
locus S08010-1-Q1 on LG-L. In certain examples, SEQ ID NOs: 102 and
103 are used as primers while SEQ ID NOs: 104 and 105 are used as
probes.
[0069] SEQ ID NOs: 106-109 comprise nucleotide sequences of regions
of the Soybean genome, each capable of being used as a probe or
primer, either alone or in combination, for the detection of marker
locus S08010-2-Q1 on LG-L. In certain examples, SEQ ID NOs: 106 and
107 are used as primers while SEQ ID NOs: 108 and 109 are used as
probes.
[0070] SEQ ID NOs: 110-113 comprise nucleotide sequences of regions
of the Soybean genome, each capable of being used as a probe or
primer, either alone or in combination, for the detection of marker
locus S08114-1-Q1 on LG-L. In certain examples, SEQ ID NOs: 110 and
111 are used as primers while SEQ ID NOs: 112 and 113 are used as
probes.
[0071] SEQ ID NOs: 114-117 comprise nucleotide sequences of regions
of the Soybean genome, each capable of being used as a probe or
primer, either alone or in combination, for the detection of marker
locus S08113-1-Q1 on LG-L. In certain examples, SEQ ID NOs: 114 and
115 are used as primers while SEQ ID NOs: 116 and 117 are used as
probes.
[0072] SEQ ID NOs: 118-121 comprise nucleotide sequences of regions
of the Soybean genome, each capable of being used as a probe or
primer, either alone or in combination, for the detection of marker
locus S08007-1-Q1 on LG-L. In certain examples, SEQ ID NOs: 118 and
119 are used as primers while SEQ ID NOs: 120 and 121 are used as
probes.
[0073] SEQ ID NO: 122 comprises the nucleotide sequence of
approximately 95% of the coding region of Glyma19g1940.1 from a
tolerant soybean line.
[0074] SEQ ID NO: 123 comprises the nucleotide sequence of
approximately 95% of the coding region of Glyma19g1940.1 from a
susceptible soybean line.
[0075] SEQ ID NO: 124 comprises the nucleotide sequence of a full
length cDNA of Glyma19g1940.1 from tolerant soybean line
GEID3495695.
[0076] SEQ ID NO: 125 comprises the nucleotide sequence of a full
length cDNA of Glyma19g1940.1 from susceptible soybean line
GEID1653063.
[0077] SEQ ID NO: 126 comprises the deduced amino acid sequence
from the nucleotide sequence of SEQ ID NO: 122, which comprises the
nucleotide sequence of approximately 95% of the coding region of
Glyma19g1940.1 from a tolerant soybean line.
[0078] SEQ ID NO: 127 comprises the deduced amino acid sequence
from the nucleotide sequence of SEQ ID NO: 123, which comprises the
nucleotide sequence of approximately 95% of the coding region of
Glyma19g1940.1 from a susceptible soybean line.
[0079] SEQ ID NO: 128 comprises the deduced amino acid sequence
from the nucleotide sequence of SEQ ID NO: 124, which comprises the
nucleotide sequence of a full length cDNA of Glyma19g1940.1 from
tolerant soybean line GEID3495695.
[0080] SEQ ID NO: 129 comprises the deduced amino acid sequence
from the nucleotide sequence of SEQ ID NO: 125, which comprises the
nucleotide sequence of a full length cDNA of Glyma19g1940.1 from
susceptible soybean line GEID1653063.
DETAILED DESCRIPTION
[0081] 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. Further, 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.
Definitions
[0082] 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.
[0083] Additionally, as used herein, "comprising" is to be
interpreted as specifying the presence of the stated features,
integers, steps, or components as referred to, but does not
preclude the presence or addition of one or more features,
integers, steps, or components, or groups thereof. Thus, for
example, a kit comprising one pair of oligonucleotide primers may
have two or more pairs of oligonucleotide primers. Additionally,
the term "comprising" is intended to include examples encompassed
by the terms "consisting essentially of" and "consisting of."
Similarly, the term "consisting essentially of" is intended to
include examples encompassed by the term "consisting of."
[0084] Certain definitions used in the specification and claims are
provided below. 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:
[0085] 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 a growing
season. Individual agronomic traits include emergence vigor,
vegetative vigor, stress tolerance, disease resistance or
tolerance, insect resistance or tolerance, herbicide resistance or
tolerance, branching, flowering, seed set, seed size, seed density,
standability, threshability, and the like.
[0086] "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. With regard to a SNP marker, allele refers
to the specific nucleotide base present at that SNP locus in that
individual plant.
[0087] 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).
[0088] An "ancestral line" is a parent line used as a source of
genes, e.g., for the development of elite lines.
[0089] An "ancestral population" is a group of ancestors that have
contributed the bulk of the genetic variation that was used to
develop elite lines.
[0090] "Backcrossing" is a process in which a breeder crosses a
progeny variety back to one of the parental genotypes one or more
times.
[0091] "Breeding" means the genetic manipulation of living
organisms.
[0092] The term "chromosome segment" designates a contiguous linear
span of genomic DNA that resides in planta on a single
chromosome.
[0093] 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).
[0094] "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 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.
[0095] 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.
[0096] 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.
[0097] An "equivalent position" in a polynucleotide and/or
polypeptide sequence is a position that correlates to 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.
[0098] As used herein, the terms "exogenous" or "heterologous," as
applied to polynucleotides or polypeptides, refer 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. For example, exogenous
polynucleotides includes polynucleotides from another organism or
from the same organism which have been modified by linkage to a
distinct non-endogenous polynucleotide and/or inserted to a
distinct non-endogenous locus. 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. The term "introduced," when referring to a heterologous or
exogenous nucleic acids, 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." The term "host cell" means a cell that contains an
exogenous 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.
[0099] An "exotic soybean strain" or an "exotic soybean germplasm"
is a strain or germplasm derived from a soybean not belonging to an
available elite soybean line or strain of germplasm. In the context
of a cross between two soybean plants or strains of germplasm, an
exotic germplasm is not closely related by descent to the elite
germplasm with which it is crossed. Most commonly, the exotic
germplasm is not derived from any known elite line of soybean, but
rather is selected to introduce novel genetic elements (typically
novel alleles) into a breeding program.
[0100] 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.
[0101] "Genotype" refers to the genetic constitution of a cell or
organism.
[0102] "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.
[0103] 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.
[0104] "Haplotype" means a combination of sequence polymorphisms
that are located closely together on the same chromosome and that
can discriminate between different genotypes. The combination
represented by the haplotype tends to be inherited together, and
this combination may represent sequence differences or alleles
within a region. The region may contain one gene, or more than one
gene.
[0105] 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.
[0106] The term "introgression" refers to the transmission of a
desired allele of a genetic locus from one genetic background to
another by sexual crossing, transgenic means, or any other means
known in the art. 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.
[0107] 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.
[0108] A "line" or "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 two 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
(e.g., yield, tolerance, etc.).
[0109] "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).
[0110] The genetic elements or genes located on a single chromosome
segment are physically linked. 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.
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. Closely
linked markers display a cross over frequency with a given marker
of about 10% or less (the given marker is within about 10 cM of a
closely linked marker). Put another way, closely linked loci
co-segregate at least about 90% of the time.
[0111] When referring to the relationship between two genetic
elements, such as a genetic element contributing to resistance and
a proximal marker, "coupling" phase linkage indicates the state
where the "favorable" allele at the resistance 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
resistance) 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).
[0112] "Linkage disequilibrium" refers to a phenomenon wherein
alleles tend to remain together in linkage groups when segregating
from parents to offspring, with a greater frequency than expected
from their individual frequencies.
[0113] "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.
[0114] "Locus" is a defined segment of DNA.
[0115] 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.
[0116] "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.
[0117] "Marker" or "molecular marker" is a term used to denote 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. 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 and/or under specific
conditions 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, New York.), developed a
molecular genetic linkage map that consisted of 25 linkage groups
with about 365 RFLP, 11 RAPD, three classical markers, and four
isozyme loci. See also Shoemaker R. C. (1994) RFLP Map of Soybean.
pp. 299-309 in R. L. Phillips and I. K. Vasil (ed.) DNA-based
markers in plants. Kluwer Academic Press Dordrecht, the
Netherlands.
[0118] "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.
[0119] 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.
[0120] As used herein, the term "plant cell" 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.
[0121] "Plant parts" means any portion or piece of a plant,
including leaves, stems, buds, roots, root tips, anthers, seed,
grain, embryo, pollen, ovules, flowers, cotyledons, hypocotyls,
pods, flowers, shoots, stalks, tissues, tissue cultures, cells and
the like.
[0122] "Polymorphism" means a change or difference between two
related nucleic acids. A "nucleotide polymorphism" refers to a
nucleic acid comprising at least one nucleotide difference when
compared to a related sequence when the two nucleic acids are
aligned for maximal correspondence. A "genetic nucleotide
polymorphism" refers to a nucleic acid comprising at least one
nucleotide difference 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.
[0123] "Polynucleotide," "polynucleotide sequence," "nucleic acid
sequence," "nucleic acid fragment," and "oligonucleotide" are used
interchangeably herein. These terms encompass nucleotide sequences
and the like. A polynucleotide may be a polymer of RNA or DNA that
is single- or double-stranded, that optionally contains synthetic,
non-natural, or altered nucleotide bases. A polynucleotide in the
form of a polymer of DNA may be comprised of one or more strands of
cDNA, genomic DNA, synthetic DNA, or mixtures thereof.
[0124] "Positional cloning" is a cloning procedure in which a
target nucleic acid is identified and isolated by its genomic
proximity to marker nucleic acid. For example, a genomic nucleic
acid clone can include part or all of two more chromosomal regions
that are proximal to one another. If a marker can be used to
identify the genomic nucleic acid clone from a genomic library,
standard methods such as sub-cloning or sequencing can be used to
identify and or isolate subsequences of the clone that are located
near the marker.
[0125] "Primer" refers to an oligonucleotide (synthetic or
occurring naturally), which is capable of acting as a point of
initiation of nucleic acid synthesis or replication along a
complementary strand when placed under conditions in which
synthesis of a complementary strand is catalyzed by a polymerase.
Typically, primers are oligonucleotides from 10 to 30 nucleic acids
in length, but longer or shorter sequences can be employed. Primers
may be provided in double-stranded form, though the single-stranded
form is preferred. A primer can further contain a detectable label,
for example a 5' end label.
[0126] "Probe" refers to an oligonucleotide (synthetic or occurring
naturally) that is complementary (though not necessarily fully
complementary) to a polynucleotide of interest and forms a duplexed
structure by hybridization with at least one strand of the
polynucleotide of interest. Typically, probes are oligonucleotides
from 10 to 50 nucleic acids in length, but longer or shorter
sequences can be employed. A probe can further contain a detectable
label. The terms "label" and "detectable label" refer to a molecule
capable of detection, including, but not limited to, radioactive
isotopes, fluorescers, chemiluminescers, enzymes, enzyme
substrates, enzyme cofactors, enzyme inhibitors, chromophores,
dyes, metal ions, metal sols, semiconductor nanocrystals, ligands
(e.g., biotin, avidin, streptavidin, or haptens), and the like. A
detectable label can also include a combination of a reporter and a
quencher, such as are employed in FRET probes or TaqMan.TM. probes.
The term "reporter" refers to a substance or a portion thereof
which is capable of exhibiting a detectable signal, which signal
can be suppressed by a quencher. The detectable signal of the
reporter is, e.g., fluorescence in the detectable range. The term
"quencher" refers to a substance or portion thereof which is
capable of suppressing, reducing, inhibiting, etc., the detectable
signal produced by the reporter. As used herein, the terms
"quenching" and "fluorescence energy transfer" refer to the process
whereby, when a reporter and a quencher are in close proximity, and
the reporter is excited by an energy source, a substantial portion
of the energy of the excited state nonradiatively transfers to the
quencher where it either dissipates nonradiatively or is emitted at
a different emission wavelength than that of the reporter.
[0127] "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.
[0128] 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.
[0129] "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 segregated 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.
[0130] "RFLP" means restriction fragment length polymorphism. Any
sequence change in DNA, including a single base substitution,
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
[0131] "Self crossing" or "self pollination" or "selfing" a process
through which a breeder crosses progeny with itself; for example, a
second generation hybrid F2 with itself to yield progeny designated
F2:3.
[0132] "SNP" or "single nucleotide polymorphism" means a sequence
variation that occurs when a single nucleotide (A, T, C, or G) in
the genome sequence is altered or variable. "SNP markers" exist
when SNPs are mapped to sites on the soybean genome. Many
techniques for detecting SNPs are known in the art, including
allele specific hybridization, primer extension, direct sequencing,
and real-time PCR, such as the TaqMan.TM. assay.
[0133] "SSR" means short sequence repeats. "SSR markers" 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.
[0134] "Tolerance" and "improved tolerance" are used
interchangeably herein and 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. A "tolerant plant" or "tolerant plant
variety" need not possess absolute or complete tolerance such that
no detrimental effect to the plant or plant variety is observed
when the given herbicide is applied. Instead, a "tolerant plant,"
"tolerant plant variety," or a plant or plant variety with
"improved tolerance" will simply be less affected by the given
herbicide than a comparable susceptible plant or variety.
[0135] "Transgenic plant" refers to a plant that comprises within
its cells an exogenous polynucleotide, e.g., a polynucleotide from
another organism (including a polynucleotide from another soybean
plant). Generally, the exogenous polynucleotide is stably
integrated within a genome such that the polynucleotide is passed
on to successive generations. The exogenous 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 exogenous 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.
[0136] "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.
[0137] The term "vector" is used in reference to polynucleotide or
other molecules that transfer nucleic acid segment(s) into a cell.
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.
[0138] 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.
Mesotrione, Isoxazole, and PPO Inhibitor Herbicides
[0139] Mesotrione and isoxazole are two herbicide classes from
different chemical families. Isoxazole is used as a pre-plant
herbicide while mesotrione is used as either a pre-plant or
post-emergent herbicide. Isoxazole is member of the isoxazole
chemical family. Following either foliar or root uptake, isoxazole
is rapidly converted to a diketonitrile derivative
(2-cyclopropyl-3-(2-mesyl-4-trifluoromethylphenyl)-3-oxopropanenitrile)
by opening of the isoxazole ring. This diketonitrile undergoes
degradation to a benzoic acid derivative (2-mesyl-4-trifluoromethyl
benzoic acid) in treated plants and the extent of this degradation
is correlated to the degree of susceptibility, being most rapid in
tolerant plants and slowest in susceptible plants.
[0140] Mesotrione belongs to the triketone family of herbicides,
which are chemically derived from a natural phytotoxin produced by
the bottlebrush plant Callistemon citrinus. Mesotrione works by
inhibiting HPPD (p-hydroxyphenylpyruvate dioxygenase), an essential
enzyme in the biosynthesis of carotenoids. Carotenoids protect
chlorophyll from excess light energy
[0141] 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 (PPO) 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.
[0142] 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.
[0143] 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.
[0144] The N-phenylphthalimide family is characterized by
phthalimide group wherein the nitrogen is bonded to a benzene ring.
Examples of N-phenylphthalimide protoporphyrinogen oxidase
inhibitors include flumiclorac and flumioxazin.
[0145] 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.
[0146] 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.
[0147] Mesotrione, isoxazole, and PPO inhibitor herbicides are each
useful, for example, as a pre-emergent herbicide.
Molecular Markers and Genetic Linkage
[0148] 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.
[0149] 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. 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.
[0150] Most plant traits of agronomic importance are polygenic,
otherwise known as quantitative traits. A quantitative trait is
controlled by two or more 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),
indicate greater 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.
[0151] The method for determining the presence or absence of a QTL
associated with tolerance or sensitivity to multiple herbicides,
including one or more of a mesotrione, an isoxazole, and a PPO
inhibitor herbicide, 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 is associated with herbicide tolerance or
sensitivity. The term "is associated with" in this context means
that the QTL associated with herbicide tolerance or sensitivity has
been found to be present in soybean plants showing herbicide
tolerance or sensitivity as described herein.
[0152] 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, improved tolerance, or
susceptibility/sensitivity 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 linked and/or closely linked to the given
markers are provided, for example, in FIGS. 1A and 1B. These
include, for example, SATT495, SATT723, Sat_408, A169_1, EV2_1,
Sle3_4s, BLT010_2, BLT007_1, SATT232, S04867-1-A, S08102-1-Q1,
S08103-1-Q1, S08104-1-Q1, S08106-1-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, S08101-3-Q1, S08118-1-Q1, S08114-1-Q1, S08113-1-Q1,
S03859-1-A, Sat 301, SATT446, P10649C-3, SATT232, S08105-1-Q1,
SATT182, 508010-1-Q1, S08010-2-Q1, R176_1, JUBC090, SATT238,
Sat_071, BLT039_1, Bng071_1, SATT388, A264_1, RGA7, RGA7, SATT523,
Sat 134, S00224-1, S01659-1, LbA, i8_2, A450_2, A106_1, Sat_405,
SATT143, B124_2, A459_1, SATT398, SATT694, Sat_195, Sat_388,
SATT652, SATT711, Sat_187, SATT418, SATT278, Sat_397, Sat_191,
Sat_320, O109_1, A204_2, SATT497, G214_17, SATT313, B164_1,
G214_16, SATT613, A023_1, SATT284, AW508247, SATT462, L050_7,
E014_1, A071_5, B046_1, L1, and B162_2.
[0153] 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.
[0154] Many marker alleles can be detected or selected for or
against. Optionally, one, two, three, or more marker allele(s) can
be identified in or introgressed into the plant. Plants or
germplasm frequently are identified that have at least one
favorable allele that positively correlates with tolerance or
improved tolerance. However, it is useful for exclusionary purposes
during breeding to also identify alleles that negatively correlate
with tolerance, to eliminate such plants or germplasm from
subsequent rounds of breeding.
[0155] The identification of favorable marker alleles may be
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 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.
[0156] Numerous markers disclosed herein have been found to be
associated with or to correlate with tolerance, improved tolerance,
or susceptibility/sensitivity to herbicides in soybean, including
one or more of a mesotrione, an isoxazole, and a PPO inhibitor
herbicide. Generally, markers that map closer to the QTL mapped to
linkage group L and associated with herbicide tolerance or
sensitivity 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 associated
with herbicide tolerance or sensitivity includes one or more of
SATT495, P10649C-3, SATT182, S03859-1, S00224-1, SATT388, SATT313,
and SATT613, or other markers above marker SATT613 on LG-L.
Additional useful and/or relevant markers include S03859-1-A,
S08103-1-Q1, S08104-1-Q1, S08106-1-Q1, S08110-1-Q1, S08111-1-Q1,
S08115-2-Q1, S08117-1-Q1, S08119-1-Q1, S08118-1-Q1, S08116-1-Q1,
S08114-1-Q1, S08113-1-Q1, S08112-1-Q1, S08108-1-Q1, S08101-2-Q1,
S08101-3-Q1, S08101-4-Q1, S08105-1-Q1, S08102-1-Q1, S08107-1-Q1,
S08109-1-Q1, and S08101-1-Q1. Any marker assigned to soybean
linkage group L and linked or closely linked to a marker disclosed
herein as associated with herbicide tolerance or sensitivity may be
used. Generally, a linked marker is within 50 cM of the referenced
marker or trait, and a closely linked marker is within 10 cM of the
referenced marker or trait. Updated information regarding markers
assigned to soybean linkage group L may be found on the USDA's
Soybase website. Further, linkage group L is now formally referred
to as chromosome #19.
[0157] Intervals defined by markers flanking the QTL associated
with herbicide tolerance or sensitivity are useful, as well. For
interval determination, 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. Examples of
such intervals include the interval flanked by and including
SATT613 and above on LG-L, the interval flanked by and including
markers SATT495 and SATT613, the interval flanked by and including
SATT313 and above on LG-L, the interval flanked by and including
markers SATT495 and SATT313, the interval flanked by and including
markers SATT495 and SATT388, the interval flanked by and including
markers P10649C-3 and SATT182, the interval flanked by and
including markers S04867-1-A and S03859-1-A, the interval flanked
by and including markers S08110-1-Q1 and S08010-1-Q1, the interval
flanked by and including markers S08117-1-Q1 and S08010-1-Q1, the
interval flanked by and including markers S08110-1-Q1 and
S08105-1-Q1, the interval flanked by and including markers
S08117-1-Q1 and S08105-1-Q1, and the interval flanked by and
including markers S08113-1-Q1 and S08105-1-Q1.
[0158] Initial fine mapping isolated the location of the QTL
associated with herbicide tolerance/sensitivity to a .about.56 kb
interval between marker S08117-1-Q1 and S08105-1-Q1 on linkage
group L. Further fine mapping refined the location of the QTL to a
.about.44 kb interval between marker S08113-1-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 S08105-1-Q1. In some examples, the markers are S08112-1-Q1,
S08108-1-Q1, S08101-2-Q1, S08101-3-Q1, and S08101-4-Q1.
[0159] Methods of introgressing herbicide 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 herbicide
tolerance or sensitivity derived from the QTL mapped to linkage
group L which is associated with herbicide tolerance or sensitivity
and a second soybean germplasm that lacks such tolerance or
sensitivity are provided. The first soybean plant may be crossed
with the second soybean plant to provide progeny soybeans.
Phenotypic and/or marker screening is performed on the progeny
plants to determine the presence of herbicide tolerance or
sensitivity derived from the QTL mapped to linkage group L. Progeny
that test positive for the presence of herbicide tolerance or
sensitivity derived from the QTL mapped to linkage group L can be
selected.
[0160] 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 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, temperature gradient electrophoresis, allelic PCR,
ligase chain reaction direct sequencing, mini sequencing, nucleic
acid hybridization, or micro-array-type detection.
[0161] Amplification primers for amplifying marker loci and
suitable marker probes to detect marker loci or to genotype SNP
alleles are provided, for example, in FIGS. 3A-3E and the related
sequence listing (SEQ ID NOs: 1-121). 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.
[0162] 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 and/or instructions that correlate
label detection to the presence of a particular marker locus or
allele.
[0163] Kits are also provided. For example, a kit can include
appropriate primers or probes for detecting tolerance associated
marker loci and instructions for using the primers or probes for
detecting the marker loci and correlating the loci with predicted
herbicide 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.
[0164] Isolated nucleic acid fragments comprising a nucleic acid
sequence coding for herbicide tolerance or sensitivity are
provided. The nucleic acid fragment comprises at least a portion of
a nucleic acid belonging to linkage group L. The nucleic acid
fragment is capable of hybridizing under stringent conditions to a
nucleic acid of a soybean cultivar possessing a QTL associated with
herbicide tolerance that is located on major linkage group L.
[0165] 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.
[0166] Seed of a soybean produced by crossing a soybean variety
having an herbicide tolerance QTL located on major linkage group L
in its genome with another soybean variety, and progeny thereof,
are provided.
Detection Methods
[0167] Any suitable detection method known in the art can be used
to detect the markers, QTL, or traits discussed herein. 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. In other examples,
amplification based techniques are employed. PCR, RT-PCR, and LCR
are in particularly broad use as amplification and
amplification-detection methods for amplifying nucleic acids of
interest, thus facilitating detection of markers. 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.
[0168] Typically, molecular markers are detected by any established
method available, including, without limitation, allele specific
hybridization (ASH), real-time PCR assays 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 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).
[0169] 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.
[0170] In general, the majority of genetic markers rely on one or
more properties 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.
[0171] 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
enzymes that produce 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.
[0172] In some examples, molecular markers are detected using a
suitable PCR-based detection method. This includes methods 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), as well as methods where a labeled allele-specific probe
is used for detection (e.g., a TaqMan.RTM. assay). In these types
of methods, PCR primers and, optionally, probes 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..
[0173] In some examples, primers are 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.
[0174] 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.
[0175] Nucleic acid probes to the marker loci can also 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.).
[0176] 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).
[0177] 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.
Real Time Amplification/Detection Methods:
[0178] 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."
[0179] PCR detection and quantification using dual-labeled
fluorogenic oligonucleotide probes can be done, using, for example,
TaqMan.RTM. probes. These probes are composed of short (e.g., 10-40
bases) 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 via 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.RTM. 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.RTM. 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).
[0180] 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. Oligonucleotides, including
modified oligonucleotides and PNAs, can also be ordered from a
variety of commercial sources known to persons of skill in the
art.
Additional Details Regarding Amplified Variable Sequences, SSR,
AFLP ASH, SNPs, and Isozyme Markers
[0181] 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.
[0182] 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.
[0183] Amplified fragment length polymorphisms (AFLP), which are
amplified before or after cleavage by a restriction endonuclease,
can also be used as genetic markers (Vos et al. (1995) Nucl Acids
Res 23:4407). 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).
[0184] 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.
[0185] 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.
[0186] 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.
[0187] 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.
[0188] 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, RFLP analysis, or
real-time PCR analysis are also appropriate.
[0189] 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.
Marker Assisted Selection and Breeding of Plants
[0190] The identification of markers associated with a particular
phenotypic trait can allow for selection of plants possessing that
trait, for example, via marker assisted selection (MAS). 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.
[0191] After a desired phenotype (e.g., herbicide tolerance or
sensitivity) 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.
[0192] 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.
[0193] 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, relevant markers include:
SATT495, P10649C-3, SATT182, S03859-1, S00224-1, SATT388, SATT313,
and SATT613 (or other markers above SATT613). Additional relevant
markers on linkage group L include S03859-1-A, S08103-1-Q1,
S08104-1-Q1, S08106-1-Q1, S08110-1-Q1, S08111-1-Q1, S08115-2-Q1,
S08117-1-Q1, S08119-1-Q1, S08118-1-Q1, S08116-1-Q1, S08114-1-Q1,
S08113-1-Q1, S08112-1-Q1, S08108-1-Q1, S08101-2-Q1, S08101-3-Q1,
S08101-4-Q1, S08105-1-Q1, S08102-1-Q1, S08107-1-Q1, S08109-1-Q1,
and S08101-1-Q1. Markers for other traits, transgenes, and/or loci
can be assayed simultaneously or sequentially in a single sample or
population of samples.
[0194] 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 herbicide
tolerance.
[0195] The determination of the presence and/or absence of a
particular genetic marker or allele, e.g., SATT495, P10649C-3,
SATT182, S03859-1, S00224-1, SATT388, SATT313, SATT613 (including
markers above SATT613), S03859-1-A, S08103-1-Q1, S08104-1-Q1,
S08106-1-Q1, S08110-1-Q1, S08111-1-Q1, S08115-2-Q1, S08117-1-Q1,
S08119-1-Q1, S08118-1-Q1, S08116-1-Q1, S08114-1-Q1, S08113-1-Q1,
S08112-1-Q1, S08108-1-Q1, S08101-2-Q1, S08101-3-Q1, S08101-4-Q1,
S08105-1-Q1, S08102-1-Q1, S08107-1-Q1, S08109-1-Q1, or S08101-1-Q1,
in the genome of a plant exhibiting a preferred phenotypic trait
can be 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
[0196] 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.
[0197] 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, for example, introgression, traditional breeding, or
transformation, or a combination thereof, to yield a soybean plant
with superior agronomic performance. The number of alleles
associated with herbicide 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.
[0198] Methods of making a progeny soybean plant, and these progeny
soybean plants having herbicide tolerance or susceptibility, 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 herbicide 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.
[0199] 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).
Positional Cloning
[0200] The molecular marker loci and alleles associated with
herbicide tolerance or susceptibility, e.g., SATT495, P10649C-3,
SATT182, S03859-1, S00224-1, SATT388, SATT313, SATT613 (including
markers above SATT613), S03859-1-A, S08103-1-Q1, S08104-1-Q1,
S08106-1-Q1, S08110-1-Q1, S08111-1-Q1, S08115-2-Q1, S08117-1-Q1,
S08119-1-Q1, S08118-1-Q1, S08116-1-Q1, S08114-1-Q1, S08113-1-Q1,
S08112-1-Q1, S08108-1-Q1, S08101-2-Q1, S08101-3-Q1, S08101-4-Q1,
S08105-1-Q1, S08102-1-Q1, S08107-1-Q1, S08109-1-Q1, and
S08101-1-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.
[0201] 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.
[0202] 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.
[0203] 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.
[0204] 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. 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.
[0205] Variant proteins include proteins 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 can be
biologically active, that is they continue to possess the desired
biological activity of the native protein, for example a variant
recombinase can implement a recombination event between appropriate
recombination sites. Such variants may result from, for example,
genetic polymorphism or from human manipulation. 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.
[0206] 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 polypeptide each encodes. Many programs and
algorithms for the comparison and analysis of sequences are
available.
[0207] 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).
[0208] 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.
[0209] 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).
[0210] Equivalent positions between two or more polynucleotides,
and/or polypeptides can be identified using any searching, sequence
assembly, and/or alignment tool including, but not limited to,
BLAST, GAP, PILEUP, FrameAlign, Sequencher, or similar tools. In
some examples, GAP alignment can be used to identify equivalent
positions, using the following parameters: 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; 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). In some examples, PILEUP can be used to
identify equivalent positions, using the following parameters for a
nucleotide sequence: a gap weight of 5 and a gap length weight of
1, and the pileupdna.cmp scoring matrix; for an amino acid sequence
using a gap weight of 8 and a gap length weight of 2, and the
BLOSUM62 scoring matrix (Henikoff & Henikoff (1989) Proc Natl
Acad Sci USA 89:10915).
[0211] Proteins may be altered in various ways including amino acid
substitutions, deletions, truncations, and insertions. Methods for
such manipulations are generally known. Methods for mutagenesis and
nucleotide sequence alterations are described, for example, in
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
[0212] In some embodiments, the activity and/or level of a
polypeptide provided herein within a cell, such as a plant cell, 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 in the art for providing a polypeptide to a
plant, including, but not limited to, direct introduction of the
polypeptide into the plant or introducing into the plant
(transiently or stably) a polynucleotide construct encoding a
polypeptide having the desired activity.
[0213] The present invention also relates to host cells and
organisms which are transformed with nucleic acids corresponding to
the tolerance, improved tolerance, or susceptibility/sensitivity
markers, traits, or QTLs identified herein. For example, such
nucleic acids include chromosome intervals (e.g., genomic
fragments), ORFs, and/or cDNAs that encode an herbicide tolerance
or improved tolerance trait. Additionally, production of
polypeptides that provide tolerance or improved tolerance by
recombinant techniques are provided.
[0214] 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.
[0215] 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. 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 that provides for effective introduction of a
nucleic acid into a cell or protoplast can be employed.
[0216] 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, 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 New York) and Plant
Molecular Biology (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 the Plant Culture Catalogue and supplement
(e.g., 1997 or later), also from Sigma-Aldrich, Inc (St Louis, Mo.)
("Sigma-PCCS").
[0217] 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. 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). 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 are well known in the art, e.g., The ATCC Catalogue of
Bacteria and Bacteriophage (1992) Gherna et al. (eds), published by
the ATCC.
Polynucleotide Constructs:
[0218] 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 an
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.
[0219] The cassette may additionally contain at least one
additional gene to be co-transformed 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).
[0220] An expression cassette comprising an herbicide tolerance
polynucleotide will include, in the 5'-3' direction of
transcription, a transcriptional and translational initiation
region (i.e., a promoter), an 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.
[0221] 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.
[0222] 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.
[0223] 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.
[0224] 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.
[0225] 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. 14:247-257) 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.
[0226] 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.
[0227] 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(20):9586-9590.
[0228] 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. 20:207-218 (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(3):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(7): 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.
[0229] 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); mi1ps (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.
[0230] 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.
[0231] 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.
[0232] 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 Gowri (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.
[0233] 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 published
application US2007/0061917. 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. Generally, variants of a particular
polynucleotides 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 polynucleotides 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.
[0234] 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.
[0235] 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) (Kong et al. (1988) Arch
Virol 143:1791-1799), 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.
[0236] 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").
[0237] 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. Comm. 196: 1414-1421; and Shah et al.
(1986) Science 233: 478-481.
[0238] 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. Comm. 196:
1414-1421; and Shah et al. (1986) Science 233: 478-481.
[0239] 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.
[0240] 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.
Introducing Nucleic Acids into Plants:
[0241] Methods for the production of transgenic plants comprising
the cloned nucleic acids, e.g., isolated ORFs and cDNAs encoding
herbicide tolerance genes, are provided. Techniques for
transforming plant cells with nucleic acids are widely available
and can be readily adapted. In addition to the Berger, Ausubel, and
Sambrook references, 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.
[0242] 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.
[0243] 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.
[0244] Such methods for introducing polynucleotide or polypeptides
into plants include stable transformation methods, transient
transformation methods, virus-mediated methods, and breeding.
"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.
[0245] 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.
[0246] 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. Nos. 5,886,244; and,
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.
[0247] 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.
[0248] 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.
[0249] 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.
[0250] 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.
[0251] 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.
[0252] 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.
[0253] 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.
Generation/Regeneration of Transgenic Plants:
[0254] 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 New York); 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.
[0255] In addition, the regeneration of plants containing
polynucleotides 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.
[0256] 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 herbicide 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. raga, 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 occidentals), 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).
[0257] 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.
[0258] 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.
[0259] 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 chlorsulforon, 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).
[0260] 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.
[0261] 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.
[0262] 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, such as 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).
[0263] 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.
[0264] 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.
[0265] 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.
[0266] 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.
[0267] 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.
[0268] 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 for Identifying Herbicide Tolerant or Susceptible Soybean
Plants
[0269] 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. In some examples, the tolerance is observed in the
context of herbicide carryover from the previous crop season.
[0270] The screening and selection may also be performed by
exposing plants containing said progeny germplasm to a desired
herbicide, for example, a mesotrione, isoxazole, or PPO inhibitor
herbicide, in an assay and selecting those plants showing herbicide
tolerance or sensitivity as containing soybean germplasm into which
germplasm having tolerance or sensitivity to the given herbicide(s)
derived from the QTL mapped to linkage group L 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.
[0271] 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 the Examples.
Automated Detection/Correlation Systems
[0272] In some examples, the methods include an automated system
for detecting markers and or correlating the markers with a desired
phenotype (e.g., tolerance or susceptibility). 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 herbicide tolerance, improved tolerance, or
sensitivity, such as to one or more of a mesotrione herbicide, an
isoxazole herbicide, or a PPO inhibitor herbicide. These probes or
primers are configured to detect the marker alleles, such as the
marker alleles noted in the tables and examples herein, e.g., using
any available allele detection format, such as solid or liquid
phase array based detection, microfluidic-based sample detection,
etc.
[0273] In some examples, markers involving linkage group L are
used. In some examples a marker linked or closely linked to the
marker locus of SATT495, P10649C-3, SATT182, S03859-1, S00224-1,
SATT388, SATT313, SATT613 (or another marker above SATT613),
S03859-1-A, S08103-1-Q1, S08104-1-Q1, S08106-1-Q1, S08110-1-Q1,
S08111-1-Q1, S08115-2-Q1, S08117-1-Q1, S08119-1-Q1, S08118-1-Q1,
S08116-1-Q1, S08114-1-Q1, S08113-1-Q1, S08112-1-Q1, S08108-1-Q1,
S08101-2-Q1, S08101-3-Q1, S08101-4-Q1, S08105-1-Q1, S08102-1-Q1,
S08107-1-Q1, S08109-1-Q1, and S08101-1-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,
S03859-1, S00224-1, SATT388, SATT313, SATT613 (or another marker
above SATT613), S03859-1-A, S08103-1-Q1, S08104-1-Q1, S08106-1-Q1,
S08110-1-Q1, S08111-1-Q1, S08115-2-Q1, S08117-1-Q1, S08119-1-Q1,
S08118-1-Q1, S08116-1-Q1, S08114-1-Q1, S08113-1-Q1, S08112-1-Q1,
S08108-1-Q1, S08101-2-Q1, S08101-3-Q1, S08101-4-Q1, S08105-1-Q1,
S08102-1-Q1, S08107-1-Q1, S08109-1-Q1, and S08101-1-Q1, and the
probe set is configured to detect the locus. Similarly, alleles of
SATT495, P10649C-3, SATT182, S03859-1, S00224-1, SATT388, SATT313,
SATT613 (or another marker above SATT613), S03859-1-A, S08103-1-Q1,
S08104-1-Q1, S08106-1-Q1, S08110-1-Q1, S08111-1-Q1, S08115-2-Q1,
S08117-1-Q1, S08119-1-Q1, S08118-1-Q1, S08116-1-Q1, S08114-1-Q1,
S08113-1-Q1, S08112-1-Q1, S08108-1-Q1, S08101-2-Q1, S08101-3-Q1,
S08101-4-Q1, S08105-1-Q1, S08102-1-Q1, S08107-1-Q1, S08109-1-Q1,
and S08101-1-Q1 can be detected.
[0274] 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.
[0275] 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.
[0276] The system typically includes components for storing or
transmitting computer readable data representing or designating the
alleles detected, 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 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.
[0277] 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.
[0278] 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 of the invention 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.
[0279] 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.
[0280] 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.
[0281] 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.
[0282] 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.).
[0283] 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).
[0284] 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.
Stacking of Traits and Additional Traits of Interest
[0285] In some embodiments, the polynucleotide conferring the
herbicide 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.
[0286] In some embodiments, an herbicide tolerance polynucleotide
described herein 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.
[0287] 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.
[0288] 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).
[0289] 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.
[0290] 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.
[0291] 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).
[0292] 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.
[0293] 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.
[0294] 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.
[0295] 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.
[0296] 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.
[0297] 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.
Methods of Controlling Weeds
[0298] 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.
[0299] The herbicide tolerant plants display a modified tolerance
to herbicides, such as mesotrione, isoxazole, or PPO inhibitor
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 herbicide 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.
[0300] The methods comprise planting the area of cultivation with
herbicide tolerant crop seeds or plants, and applying to any crop,
crop part, weed or area of cultivation thereof an effective amount
of an herbicide of interest. It is recognized that the herbicide
can be applied before or after the crop is planted in the area of
cultivation. Such herbicide applications can include an application
of a mesotrione chemistry, an isoxazole chemistry, or a PPO
inhibitor chemistry, or any combination thereof.
[0301] In certain 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. In certain 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, 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 an herbicide tolerance allele, an herbicide
tolerance polynucleotide, and/or a polynucleotide encoding an ABC
transporter protein that confers tolerance to herbicide
formulations, including one or more of mesotrione, isoxazole, and
PPO inhibitor herbicides. 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.
[0302] In some examples, the method of controlling weeds comprises
planting the area with herbicide 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 an 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.
[0303] In another embodiment, the method of controlling weeds
comprises planting the area with herbicide 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 an 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 herbicide tolerant crop,
crop part, seed, or the area of cultivation thereof.
[0304] 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 in Table 1.
TABLE-US-00001 TABLE 1 Abbreviated HRAC classification table. 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 B Inhibition of Phenylpyrazoline "DEN"
pinoxaden 2 acetolactate Sulfonylurea amidosulfuron synthase ALS
azimsulfuron (acetohydroxyacid bensulfuron-methyl synthase AHAS)
chlorimuron-ethyl chlorsulfuron cinosulfuron cyclosulfamuron
ethametsulfuron- methyl ethoxysulfuron flazasulfuron
flupyrsulfuron- methyl-Na foramsulfuron halosulfuron- methyl
imazosulfuron iodosulfuron mesosulfuron metsulfuron-methyl
nicosulfuron oxasulfuron 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 isoxazole
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
[0305] 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, an
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, an inhibitor
of HPPD (hydroxyphenyl pyruvate dioxygenase), a sulfonylurea, a
glyphosate, or a synthetic auxin. In other examples, a transgenic
plant is tolerant to more than one of mesotrione, isoxazole, and
PPO inhibitor herbicides. 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
[0306] 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.
[0307] 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 an 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.
[0308] 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.)).
[0309] Also included are plant cells, plants, and/or seeds produced
by any of the foregoing methods.
[0310] 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 Isoxafutole Tolerant and Sensitive
Soybean Lines--Herbicide Screening Bioassay and Intergroup
Association Marker Based Diagnostic
[0311] Two soybean mapping populations were used to confirm
significant QTLs related to tolerance and susceptibility to
mesotrione and/or isoxazole herbicides (HPPD-inhibitors), to
identify any potential QTLs associated with the tolerance or
susceptibility to these herbicides, and to identify any varietal
variation due to differences between the two herbicide chemistries
used in the study. The mesotrione herbicide used in this study was
Callisto.RTM. (referred to as Herbicide B); the isoxazole herbicide
used in this study was Balance Pro.RTM. (referred to as Herbicide
A).
Part 1:
[0312] Studies were conducted using herbicides A and B and were
performed at two locations, Princeton, I L and Johnston, Iowa
Herbicide screening protocols developed in the summer of 2008
determined the optimum herbicide rate, application timing and the
best time to evaluate soybean injury following application.
[0313] Herbicide A and B were applied as a pre-plant incorporated
herbicide. The application rate for Herbicide A was based on soil
organic matter and was applied at half the recommended labeled
rate. Herbicide B was applied at half the pre-plant incorporated
label rate. Both herbicides were applied using an ATV sprayer
outfitted with a 10-foot boom, GPS and a Raven control system. The
herbicides were applied at a rate of 30 gallons of water per acre
and a spray pressure of 35-40 psi. An agitation system was used to
maintain herbicide suspension in the water spray solution. Since
both herbicides A and B were used in the same field, the sprayer
was cleaned out between applications and a 10-foot buffer strip was
used to help separate the two herbicides in the field to ensure no
spray overlap.
[0314] The herbicides were incorporated into the soil to a depth of
1-2 inches using a field cultivator with rolling baskets 2-5 days
following application (Table 2). Incorporation was performed in two
directions to ensure even distribution of the herbicide in the
soil.
[0315] The soybeans were planted into the soil to a depth of 1-1.5
inches using an Almaco 4-row index planter set on 30-inch row
spacing. All plots were planted as single row plots with 25 seeds
for 4.5 feet of planted row with a three-foot alleyway. The planted
population was approximately 90,000 seeds per acre. Both Princeton
and Johnston locations were planted on June 4. The herbicide
application, planting, and rating dates for both Princeton and
Johnston locations are presented in Table 2.
TABLE-US-00002 TABLE 2 Herbicide Application, tillage, planting and
rating dates Untreated Untreated Herbicide Planting 1st Crop 2nd
Crop Location Application Tilled In Date Rating Stage Rating Stage
Princeton, IL May 29, 2009 Jun. 4, 2009 Jun. 4, 2009 Jun. 29, 2009
V3 Jul. 8, 2009 V6 Johnston, IA Jun. 2, 2009 Jun. 4, 2009 Jun. 4,
2009 Jun. 30, 2009 V3 Jul. 7, 2009 V5
[0316] Soybean varietal herbicide reactions were evaluated using
visual scores for plant growth reduction (STNT) and crop injury
rating (HERSC) using descriptions defined below. Two ratings were
conducted at both locations; the initial rating (V3) was based off
of the clearest distinction of symptoms across the experiments. A
second rating (V5 or V6) was conducted to ensure accuracy and note
any varietal variation over time. An untreated check was used as a
guide for the expected plant growth and development over time.
Plant Growth Reduction Rating (STNT)
[0317] 1-9 herbicide reaction scale for plant growth reduction:
[0318] 9=no plant growth reduction from the herbicide [0319]
8=<5% plant growth reduction [0320] 7=>5% and <20% plant
growth reduction [0321] 6=>20% and <35% plant growth
reduction [0322] 5=>35% and <50% plant growth reduction
[0323] 4=>50 and <65% plant growth reduction [0324] 3=>65
and <80% plant growth reduction [0325] 2=>80 and <95%
plant growth reduction [0326] 1=>95% plant growth reduction
Crop Injury Rating (HERSC)
[0327] 1-9 herbicide reaction scale for crop injury (both chlorotic
and necrotic tissue): [0328] 9=no crop injury [0329] 8=<5% crop
injury [0330] 7=>5% and <20% crop injury [0331] 6=>20% and
<35% crop injury [0332] 5=>35% and <50% crop injury [0333]
4=>50 and <65% crop injury [0334] 3=>65 and <80% crop
injury [0335] 2=>80 and <95% crop injury [0336] 1=>95%
crop injury
[0337] Two mapping populations were used that contained known
susceptible and tolerant parents that were fixed and carried
different alleles for two QTLs identified on linkage group L. The
mapping populations were screened using herbicide A and the
herbicide screening protocol described above. Two populations (Pop
A and Pop B) of 90 randomly selected F3:F5 lines were used in the
study. Four replications of the populations were placed in a row by
column design to help adjust means due to field variation. The
parents of each population were replicated 3 times per replication
for a total of 12 times per location. An analysis of variance
(ANOVA) was conducted to identify significant differences between
the varieties within the populations.
[0338] A variety trial was conducted using 144 varieties with
herbicides A and B and the herbicide screening protocol described
above. Four replications of the varieties were placed in a row by
column design to help adjust means due to field variation for both
herbicides. The 144 lines included 52 susceptible and 61 tolerant
lines identified previously as well as lines identified as moderate
but containing the susceptible or tolerant allele for the QTLs.
[0339] An ANOVA was run on the STNT and HERSC data to determine any
significant differences between soybean varieties. The herbicide
response and the varieties were classified into tolerant and
susceptible groups to be analyzed using available SSR and SNP
markers for identification of other potential QTLs associated with
the trait. The tolerant and susceptible classes were analyzed to
observe marker trait associations by comparing the allelic
frequencies of tolerant and susceptible varieties. This analysis
used all available genome wide data produced for the markers and
the varieties to run the analysis. Significant markers were then
identified and potential QTL regions were recognized for candidates
causing tolerant reactions. This data was used to help identify
additional polymorphic markers within the mapping populations.
Table 3 indicates the results of the various varieties tested.
TABLE-US-00003 TABLE 3 Mapping population analysis Grouping HERSC
score Adjusted mean SUS 1 2.2915 SUS 1 2.3105 SUS 1 2.333 SUS 1
2.3955 SUS 1 2.4045 SUS 1 2.4155 SUS 2 2.4825 SEG 2 2.547 SUS 2
2.577 SUS 2 2.888 SUS 2 2.9185 SUS 3 3.083 SEG 3 3.091 SUS 3 3.1775
SEG 3 3.207 SUS 3 3.252 SUS 3 3.3115 SEG 4 3.427 SEG? 4 3.5305 SEG
4 3.544 SUS 4 3.5845 SEG 4 3.589 SUS 4 3.6135 SUS 4 3.6575 SEG 4
3.662 SEG 5 3.729 SEG 5 3.73 SUS 5 3.7435 SEG 5 3.843 TOL 5 3.89
SEG 5 3.9255 SEG 5 3.9925 SEG 5 4.0415 SEG 5 4.0755 SEG 5 4.1025
TOL 5 4.1315 TOL 5 4.171 SEG 6 4.273 SEG 6 4.276 SEG 6 4.325 SEG? 4
4.331 SEG 4 4.375 TOL 6 4.4225 SEG 6 4.436 SEG 6 4.456 SEG 6 4.4955
TOL 6 4.5525 TOL 6 4.5905 TOL 6 4.6135 SEG 6 4.627 TOL 6 4.652 SEG
6 4.652 SEG 6 4.7305 TOL 6 4.7785 TOL 6 4.7805 TOL 6 4.814 SEG? 6
4.815 TOL 6 4.827 TOL 6 4.883 SEG 6 4.955 SEG 7 5.005 SEG 7 5.018
TOL 7 5.0185 SEG 7 5.048 TOL 7 5.053 TOL 7 5.0635 TOL 7 5.0895 TOL
7 5.151 TOL 6 5.239 SEG 7 5.2525 TOL 7 5.258 SEG 7 5.263 TOL 5
5.312 TOL 7 5.357 TOL 7 5.358 SEG? 8 5.4165 TOL 8 5.4475 SEG? 8
5.512 TOL 8 5.5645 TOL 8 5.5975 TOL 8 5.6185 TOL 8 5.707 TOL 8
5.713 TOL 9 5.8935 TOL 9 5.904 TOL 9 6.022 TOL 7 6.0235 SEG? 9
6.1865 TOL 9 6.591 TOL 9 6.842 SUS 1 2.049 TOL 9 5.9145 SUS 2 2.733
SUS 2 2.748 SUS 2 2.801 SUS 2 2.883 SUS 2 2.938 SUS 2 2.9515 SUS 2
2.9955 SEG? 2 3.0555 SUS 1 2.4295 SUS 1 2.5185 SUS 1 2.6205 SUS 1
2.6395 SUS 3 3.1315 SEG? 3 3.1925 SUS 3 3.193 SEG 3 3.216 SEG 3
3.267 SUS 3 3.2875 SUS 3 3.2885 SUS 3 3.3375 SUS 4 3.3435 SUS 4
3.3555 SUS 4 3.361 SUS 4 3.428 SUS 4 3.449 SUS 4 3.539 SUS 4 3.5505
SUS 4 3.6335 SUS 5 3.681 SUS 5 3.793 SUS 5 3.8215 SUS 4 3.8395 SUS
5 4.01 SEG? 5 4.0635 SUS 5 4.072 SEG 5 4.1245 SUS 5 4.1535 TOL 5
4.239 SEG 6 4.3385 SEG 6 4.489 TOL 6 4.5315 SEG 6 4.5915 SEG 6
4.721 TOL 6 4.7345 TOL 6 4.8215 TOL 6 4.8495 TOL 6 4.8625 TOL 6
4.867 TOL 6 4.944 TOL 4 4.973 TOL 7 5.0845 TOL 7 5.0885 TOL 5
5.0955 TOL 7 5.1245 TOL 7 5.1505 TOL 7 5.1695 TOL 4 5.1885 TOL 7
5.221 TOL 7 5.228 SEG 7 5.2285 SEG 7 5.2805 TOL 7 5.3305 SEG 7
5.3345 TOL 8 5.381 TOL 8 5.3835 TOL 8 5.414 TOL 8 5.5165 TOL 8
5.535 TOL 8 5.55 TOL 8 5.5925 TOL 8 5.6125 TOL 8 5.617 TOL 8 5.6275
TOL 8 5.6435 TOL 8 5.67 TOL 8 5.7255 TOL 8 5.7645 TOL 8 5.8155 TOL
8 5.8245 TOL 8 5.8835 TOL 9 5.885 TOL 9 5.8885 TOL 9 5.9165 TOL 9
5.964 TOL 9 6.0095 TOL 9 6.2345 TOL 9 6.2655 TOL 9 6.286 TOL 7
6.3875 TOL 9 6.599 SUS 3 3.069 TOL 7 5.359
[0340] The experimental means for Herbicide A across both locations
for HERSC2 was 4.695 with a standard deviation of 1.94. The
coefficient of variation across the locations was 26.7.
Example 2: Determination of QTL and Marker Associations/Intergroup
Analysis
[0341] There was significant (P<0.001) difference across
varieties for Herbicide A. The LSD was 1.316 across all varieties.
Predicted means by location were calculated using a linear model
for the locations. The varieties were looked at individually by the
adjusted means by location, the LSD value, the average score by
location, and the 2008 data for each variety. This gave an overall
view of each variety and allowed for a simple classification across
all varieties. Any variety that showed a high rate of variability
across the data was automatically placed in the segregating
group.
[0342] The results of the ANOVA for both Herbicides A and B are
reported in Table 4. The model used for the analysis was the
incomplete block design and the affect of the model is described
through the relative efficiency and the Czekanowski Coefficient
(Czek Coeff). The relative efficiency is comparing the error terms
of the more complex block (incomplete block, ICB) to the less
complex model (randomized complete block, RCB). The relative
efficiency for the variety trial using herbicide A was 111% and
herbicide B was 123%. The Czek Coeff which reports the top 10 and
20 percent of the entries that were the same for both the RCB and
ICB designs was 73 and 83% for the top 10% of entries and 86 and
90% for the top 20% of the entries for the variety trials using
herbicides A and B, respectfully.
TABLE-US-00004 TABLE 4 Analysis of variance for the variety trial
Herbicide A Herbicide B HERSC HERSC Experiment Mean 4.695 4.585
CV(%) 26.7 26.6 Model IB IB Rel Eff 111 123 Czek Coeff. 10 0.73
0.87 Czek Coeff. 20 0.86 0.9 # Environments 2 2 Total Blocks 8 8
p.val(Entry) 0 0 % V 85.7 82.9 % VL 0.8 2.9 % VE 13.5 14.1 SED
between 2 entry means 0.658 0.637 2*SED between 2 entry means (LSD)
1.316 1.274
[0343] Using this method of classification the varieties were
grouped according to their reactions to herbicides A and B. For
herbicide A there were 32 tolerant, 67 moderate, 37 susceptible and
8 segregating lines. For herbicide B there were 29 tolerant, 75
moderate, 32 susceptible and 8 segregating lines.
[0344] 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.
[0345] 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.
[0346] 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 or
sensitivity to mesotrione and/or isoxazole herbicides. 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 or sensitivity to mesotrione and/or
isoxazole herbicides. 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.
[0347] 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.
[0348] For the herbicide A variety trial, the analysis identified
275 markers (P<0.05), of these 275 markers identified, 243 of
the markers had been previously mapped to a particular genomic
position. This allowed for further analysis to identify potential
genomic regions for genes and to eliminate marker regions that are
likely not associated with the native tolerance to the herbicide.
There were a total of 6 regions identified where multiple markers
were pointing to a particular genomic region (Table 5). The regions
identified included regions on B2, D2, E, G, L and 7 unmapped (UM)
markers. Of the original 275 markers identified, 112 markers were
used to identify the 6 genomic regions.
TABLE-US-00005 TABLE 5 Potential genomic regions for Herbicide A
Linkage group cM (position) Markers Comment B2 92.2-111.86 31 D2
88.11-92.58 6 E 82.16-100.51 37 G 78.6-85.82 22 L 10.1-14.31 4
Highest significance L 41.09-46.35 5 UM 7 Highly significant
<.008
[0349] A similar analysis was conducted for the tolerant and
susceptible classes to Herbicide B where 274 markers were
identified (P<0.05). Of the 274 markers identified 239 markers
had been previously mapped to a particular genomic position.
Similar regions to Herbicide A were identified that included B2, E,
G, L, and 10 UM markers (Table 6). A potential region on N was
added and the region on D2 was taken off for Herbicide B tolerance.
Of the original 274 markers identified 116 markers were used to
help identify the 6 regions.
TABLE-US-00006 TABLE 6 Potential genomic regions for Herbicide B
Linkage group cM (position) Markers Comment B2 92.2-111.86 20 E
82.16-100.51 41 G 77.87-85.82 17 L 10.1-14.31 4 Highest
significance L 41.09-42.17 3 N 37.11-53.27 21 UM 10 Highly
significant <.008
[0350] Additional observations were made through looking at the
tolerant, moderate, susceptible and segregating classes to each
herbicide as assigned through the variety trial. Of the 144
varieties in the herbicide trial, 21 were tolerant to both
herbicides, 54 displayed moderate tolerance to both herbicides, and
28 displayed susceptible reactions to both herbicides. There were a
total of 31 varieties that were classified one class higher or
lower to herbicide A or B. For example 11 varieties were tolerant
to herbicide A and moderate to herbicide B, where they were lowered
1 class from herbicide A classification to herbicide B
classification. There were zero lines that were tolerant to one
herbicide and susceptible to the other. This is summarized in Table
7.
TABLE-US-00007 TABLE 7 Variety reactions to both herbicides
Herbicide B (mesotrione) Class Tol Moderate Sus Seg Herbicide A Tol
21 11 0 0 (isoxazole) Moderate 8 54 3 2 Sus 0 9 28 0 Seg 0 1 1
6
[0351] The significant markers were observed for both variety
classes to each of the chemistries. Of the 275 and 274 markers that
were significant for the Herbicide A and Herbicide B reactions, 144
markers were significant for both variety reactions. As observed in
the variety trial analysis, the regions of potential QTLs were
observed for both classes on B2, E, G, and L; with the most
significant markers on L from 10.1-14.31 cM (Tables 5 and 6).
[0352] Table 8 shows the allele distribution for marker S03859,
which is closely linked to this region, among the 144 lines
analyzed; 32 tolerant lines, 37 non-tolerant (susceptible) lines,
67 moderate, and 8 segregating lines analyzed. Marker calls for the
S03859 locus were available for 111 of the 144 lines.
TABLE-US-00008 TABLE 8 Allele distribution S03859 allele (LG-L)
Phenotype Adjusted mean 1,1 Susceptible 1.6085 1,1 Susceptible
1.7085 1,1 Susceptible 1.8675 Susceptible 1.9175 1,1 Susceptible
2.006 1,1 Susceptible 2.212 Susceptible 2.272 Susceptible 2.283 1,1
Susceptible 2.2845 1,1 Susceptible 2.327 1,1 Susceptible 2.4255 1,1
Susceptible 2.643 Susceptible 2.6815 1,1 Susceptible 2.781 1,1
Susceptible 2.8325 1,1 Susceptible 2.838 1,1 Susceptible 2.881 1,1
Susceptible 2.883 1,1 Susceptible 2.9005 1,1 Susceptible 2.927 1,1
Susceptible 2.992 1,1 Susceptible 2.994 Susceptible 3.056 1,1
Susceptible 3.1475 3,3 Susceptible 3.215 1,1 Susceptible 3.2835 1,3
Susceptible 3.3085 Susceptible 3.3385 1,1 Susceptible 3.3875 1,1
Susceptible 3.3885 1,1 Susceptible 3.4085 1,1 Segregating 3.5095
1,1 Susceptible 3.6345 1,1 Susceptible 3.711 1,1 Susceptible 3.7595
1,3 Moderate 3.851 Moderate 3.919 Susceptible 3.9385 1,1 Moderate
3.967 3,3 Moderate 4.067 Moderate 4.107 3,3 Susceptible 4.1605 1,1
Susceptible 4.1645 1,3 Moderate 4.17 3,3 Moderate 4.179 3,3
Moderate 4.3415 3,3 Moderate 4.35 1,1 Moderate 4.357 3,3 Moderate
4.4295 3,3 Moderate 4.4495 Moderate 4.4765 3,3 Moderate 4.524 1,1
Moderate 4.555 Moderate 4.6375 3,3 Moderate 4.6615 3,3 Moderate
4.676 3,3 Moderate 4.678 Segregating 4.723 Moderate 4.7575 3,3
Segregating 4.7645 3,3 Moderate 4.7675 Moderate 4.771 Moderate
4.7875 3,3 Moderate 4.797 1,3 Moderate 4.846 Tolerant 4.85 Moderate
4.891 1,1 Moderate 4.9085 3,3 Moderate 4.9095 3,3 Moderate 4.9395
3,3 Tolerant 4.943 1,1 Moderate 4.961 1,1 Moderate 5.015 3,3
Moderate 5.0195 3,3 Moderate 5.051 3,3 Moderate 5.1305 Tolerant
5.1475 3,3 Moderate 5.1495 Moderate 5.2105 3,3 Tolerant 5.2125 3,3
Moderate 5.214 3,3 Tolerant 5.215 3,3 Moderate 5.2565 3,3 Moderate
5.2795 3,3 Moderate 5.2945 3,3 Moderate 5.3125 3,3 Moderate 5.3145
3,3 Moderate 5.3345 3,3 Moderate 5.335 3,3 Tolerant 5.3365 Tolerant
5.377 3,3 Moderate 5.39 3,3 Segregating 5.3905 3,3 Segregating
5.4675 3,3 Moderate 5.4835 3,3 Segregating 5.4925 3,3 Moderate
5.4975 3,3 Tolerant 5.518 3,3 Moderate 5.524 Segregating 5.541
Tolerant 5.574 1,1 Moderate 5.5895 3,3 Moderate 5.6025 Moderate
5.616 3,3 Tolerant 5.6685 3,3 Tolerant 5.6925 3,3 Moderate 5.7005
3,3 Moderate 5.7095 Tolerant 5.7165 3,3 Moderate 5.7315 Moderate
5.741 Moderate 5.75 3,3 Tolerant 5.7545 3,3 Moderate 5.7625 3,3
Moderate 5.7705 3,3 Moderate 5.7715 3,3 Moderate 5.7835 3,3
Segregating 5.7875 3,3 Moderate 5.798 3,3 Moderate 5.851 3,3
Moderate 5.852 3,3 Tolerant 5.857 3,3 Tolerant 5.87 3,3 Moderate
5.8705 Tolerant 5.914 Tolerant 5.948 3,3 Tolerant 5.9525 3,3
Tolerant 5.9675 Tolerant 6.0245 3,3 Moderate 6.026 3,3 Moderate
6.0275 3,3 Tolerant 6.034 3,3 Tolerant 6.042 3,3 Tolerant 6.054
Tolerant 6.092 Tolerant 6.1295 3,3 Moderate 6.2395 3,3 Tolerant
6.2475 3,3 Tolerant 6.2695 Tolerant 6.275 3,3 Tolerant 6.387 3,3
Tolerant 6.431 3,3 Tolerant 6.6535 Tolerant 6.674
[0353] The non-random distribution of alleles between the tolerant
and non-tolerant plant groups at the marker loci in Table 8 is good
evidence that a QTL influencing tolerance or sensitivity to
mesotrione and/or isoxazole herbicides is linked to these marker
loci.
[0354] QTLs related to tolerance and susceptibility to mesotrione
and/or isoxazole herbicides were found to essentially co-localize
with QTLs related to tolerance to PPO inhibitor herbicides to
linkage group L, as shown in the Examples below. Thus, PPO
tolerance could be used to fine map the QTL and to identify
putative candidate genes as shown below.
Example 3: Identification of Sulfentrazone Tolerant and Sensitive
Soybean Lines--Herbicide Screening Bioassay and Intergroup
Association Marker Based Diagnostic
[0355] 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
[0356] 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:
[0357] 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 55960
[0358] 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:
[0359] 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:
[0360] A) Mix a stock solution of 0.926 g Authority.RTM. 75DF (FMC
Corp.), thoroughly dissolved in 1000 ml of water. B) Mix 10 ml of
STOCK SOLUTION in 1000 ml of water to create final solution. C)
Pour 100 ml of FINAL SOLUTION over each pot.
Recording Data:
[0361] 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: [0362] 9=Equivalent or better when compared to the
tolerant check [0363] 7=Very little damage or response noted.
[0364] 5=Intermediate response or damage [0365] 3=Major damage,
including stunting and foliar necrosis [0366] 1=Severe damage,
including severe stunting and necrosis; equivalent or worse when
compared to the non-tolerant check
[0367] 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
[0368] 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.
[0369] 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.
[0370] 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.
[0371] 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:
[0372] Table 9 lists 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 were highly significant, as shown in the table below.
TABLE-US-00009 TABLE 9 Intergroup analysis results for Lg-L markers
Linkage G- Locus Test Group 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
[0373] Table 10 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-00010 TABLE 10 Allele distribution P10649C-3 allele
Phenotype LG-L TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL
1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1
TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1
TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1
TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1
TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1
TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1
TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1
TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL TOL 1 TOL 1 TOL
2 TOL 1 TOL 1 TOL 1 NON 3 NON 3 NON 1 NON 1_2 NON 3 NON 3 NON 1 NON
3 NON 2 NON 3 NON 2 NON 2 NON 2 NON 1 NON 2_3 NON 3 NON 3 NON 2_3
NON 3 NON 3 NON 1 NON 2 NON 3 NON 3 NON 3 NON 2 NON 3 NON 1_3 NON 3
NON 2 NON 1 NON 3
[0374] The non-random distribution of alleles between the tolerant
and non-tolerant plant groups at the marker loci in Table 10 is
good evidence that a QTL influencing tolerance to
protoporphyrinogen oxidase inhibitors is linked to these marker
loci.
Example 4: Predication and Confirmation of Marker Based Selection
for Response to PPO Chemistries in a Set of Diverse Public Soybean
Lines
[0375] Marker haplotype data for a set of 17 diverse public soybean
lines was determined for two QTL identified in Example 3 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 Hulting 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 11. 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 GM and is 100% predictive of non-tolerance to
sulfentrazone when injury is set at 40% or higher GM. 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 GM.
TABLE-US-00011 TABLE 11 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) Linkage Group Growth 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) 24/26 = (allele 3) 23/26 = tolerant 92% 88%
correct (allele 3) 8/8 = (allele 2) = 8/8 = non-tolerant 100% 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)
Example 5: Predication and Confirmation of Marker Based Selection
for Response to PPO Chemistries in a Set of Soybean Commercial
Lines
[0376] 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 3.
In addition, the same scale was used for scoring such that: [0377]
9=Equivalent or better when compared to the tolerant check [0378]
7=Very little damage or response noted. [0379] 5=Intermediate
response or damage [0380] 3=Major damage, including stunting and
foliar necrosis [0381] 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 12. 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-00012 [0381] TABLE 12 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 6: 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
[0382] 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 13. 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-00013 TABLE 13 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 (alleles
1 or 2) 14/14 = (allele 3) 14/14 = tolerant 100% 100% correct
(allele 3) 8/8 = (allele 2) = 8/8 = non-tolerant 100% 100%
Example 7: Pictures of Soybean Variety Response (Tolerant and
Non-Tolerant Check Varieties) to Sulfentrazone Injury in the Field
and in the Greenhouse/Growth Chamber Bioassay
[0383] Known non-tolerant (i.e., Pioneer variety 9692, Asgrow
variety A4715) and tolerant (i.e., Pioneer variety 9584, Syngenta
variety 55960) 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. 4 and 5 show these differences in phenotype. FIG. 4 shows a
field sample, with a non-tolerant variety on the left (stunted,
necrotic) and tolerant variety on the right (normal growth). FIG. 5
shows a greenhouse sample, with non-tolerant (left side) and
tolerant (right side) variety checks, treated in the foreground,
untreated in the background.
Example 8: Fine Mapping of the LG-L Herbicide Tolerance QTL
[0384] The herbicide tolerance QTL on LG-L was initially mapped in
two different soybean mapping populations:
GEID1653063.times.GEID3495695 (F4-derived F6) and
GEID4520632.times.GEID7589905 (F3-derived F5). From these
populations, 184 and 180 lines respectively were genotyped and
scored for PPO herbicide tolerance as described above. This data
was used to map the herbicide tolerance QTL to chromosome GM19 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.
[0385] 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 herbicide
tolerance QTL as 504867-1-A (GM19: 841543-841958) and S03859-1-A
(GM19: 1634882-1635399) (Table 17). The
GEID4520632.times.GEID7589905 population had 42 recombinants that
delimit the QTL to the same interval (Table 18).
[0386] Because S03859-1-A was determined to be closely linked to
the herbicide tolerance 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 (open source
software available from SourceForge.net) 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 14-15). The PCR was then cleaned up using the
ExoSAP-IT.RTM. protocol (USB-Cleveland, Ohio, USA) (Table 15)
before being sequenced by Sanger sequencing.
[0387] 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 86 select recombinants combined from the
two mapping populations to facilitate fine-mapping and to further
delimit the herbicide tolerance QTL interval (Table 19).
[0388] Urea Extraction Protocol [0389] 1. Grind 2 g fresh tissue or
0.5 g lyophilized tissue and add it to 6 mL Urea Extraction Buffer
and mix well. [0390] 2. Add RNase A (100 mg/mL) and incubate @
37.degree. C. for 20 min. [0391] a. 3 uL--Leaf [0392] b. 12
uL--Seed [0393] 3. Add 4-5 mL Phenol:Chloroform:Isoamyl 25:24:1.
Mix well. (Sigma P3803) [0394] 4. Put on rocker inside hood. [0395]
a. Fresh--15 min [0396] b. Lyophilized--30 min [0397] 5. Centrifuge
@ 8000 rpm at 10.degree. C. for 10 min. [0398] 6. Transfer
supernatant to clean tube. [0399] 7. Add 700 uL of 3M NaOAC (pH
5.0) and 5 mL cold isopropanol. Mix well. [0400] 8. Hook DNA and
wash in 70% EtOH. [0401] 9. Repeat 70% wash. [0402] 10. Transfer
pellet to 1.5 mL tube and allow to dry. [0403] 11. Dissolve pellet
in 1 mL 10 mM Tris. [0404] 7 M Urea Extraction Buffer [0405] Water
350 mL [0406] Urea 336 g [0407] 5M NaCl 50 mL (14.61 g) [0408] 1M
Tris 40 mL (pH 8.0) [0409] 0.5M EDTA 32 mL (pH 8.0) [0410] 20%
Sarcosine Sol. 40 mL (8 g) [0411] Adjust volume to 800 mL with
ddH2O
TABLE-US-00014 [0411] TABLE 14 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 (F + R)
4.0 -- -- -- ddH2O 9.9 29,462 38,016 54,173 Total 20.0 41,664
53,760 76,608
TABLE-US-00015 TABLE 15 PCR Setup for SNP Discovery Dipper Setup
PCR conditions Temp Time #Cycles initial denature 94 C. .sup. 3 min
1X denature 94 C. 45 sec 35X anneal 65 C. 60 sec extension 72 C. 75
sec final extension 72 C. .sup. 5 min 1X end
TABLE-US-00016 TABLE 16 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-00017 TABLE 17 Initial recombinants identified from
GEID1653063 .times. GEID3495695 mapping population that delimited
herbicide tolerance QTL to interval between S04867-1-A and
S03859-1-A S04867- S03859- SAMPLE 1-A 1-A Call Average Comment
Genetic Pos 7.81 10.00 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-00018 TABLE 18 Initial recombinants identified from
GEID4520632 .times. GEID7589905 mapping population that delimited
herbicide tolerance QTL to interval between S04867-1-A and
S03859-1-A S04867- S03859- SAMPLE 1-A 1-A Call Ave Comment Genetic
Pos 7.81 10.00 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
[0412] In an initial analysis of the GEID1653063.times.GEID3495695
mapping population, four key recombinants were identified which
served to further fine-map the herbicide tolerance QTL interval
(Table 20). 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 herbicide tolerance QTL to an .about.70 kb
interval. Initial analysis of the GEID4520632.times.GEID7589905
mapping population identified eight key recombinants (Table 13). 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
herbicide tolerance QTL to a .about.526 kb interval. However, when
the data from these two mapping populations are combined into a
single set, the herbicide tolerance QTL interval was delimited to a
.about.56 kb interval between S08117-1-Q1 and S08105-1-Q1 (Table 19
and Table 20).
[0413] To facilitate higher resolution mapping of the herbicide
tolerance QTL interval, lines from the initial set of 86
recombinants were re-scored for herbicide tolerance to confirm
their phenotype. Moreover, new markers were developed and used to
genotype these recombinants. Consequently, this further analysis
resulted in the identification of a key recombinant (SP21669417)
from the GEID4520632.times.GEID7589905 mapping population which set
the left border of the herbicide tolerance QTL interval at
S08113-1-Q1 (Table 13). In summary, key recombinants from the two
mapping populations, scored in two fine-mapping experiments, define
the herbicide tolerance QTL to a .about.44 kb interval between
S08113-1-Q1 and S08105-1-Q1 (Table 19 and Table 20).
TABLE-US-00019 TABLE 19 Summary of SNP markers used for initial QTL
mapping and fine-mapping of herbicide tolerance QTL. Combined data
from the two populations delimits the QTL to a ~44 kb interval
between S08113-1-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 A S08107-1-Q1
PPO_Gm19_1541k3-1 Glymal9g01900.1 1542880 1543693 A S08109-1-Q1
PPO_Gm19_1541k4-1 Glymal9g01900.1 1543868 1544588 A S08110-1-Q1
PPO_Gm19_1548k1-1 Glymal9g01910.1 1548367 1548822 A L border Pop A
S08111-1-Q1 PPO_Gm19_1548k2-1 Glymal9g01910.1 1548902 1549558 A
S08115-2-Q1 PPO_Gm19_1563k1-1 X 1563958 1564512 Both S08117-1-Q1
PPO_Gm19_1563k2-1 X 1564563 1564960 Both L border Pop B 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 Pop A S03859-1-A sbacm.pk005.c3.f X 1634882 1635399
Both S08010-1-Q1 PPO_Gm19_2089k4-1 Glyma19g02370.1 2091644 2092359
Both R border Pop B S08010-2-Q2 PPO_Gm19_2089k4-1 Glyma19g02370.1
2091644 2092359 Both * Population A = GEID1653063/GEID3495695;
Population B = GEID4520632/GEID7589905
Tables 20A-20H. Fine-Mapping of the 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-00020 TABLE 20A Marker S04867-1-A S08102-1-Q1 S08103-1-Q1
S08104-1-Q1 Pos Sample Amplicon 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 SJ22186045 -- -- H H
SJ22186913 -- -- H H SJ22186891 -- -- H H SJ22186879 -- -- H H
SJ22186841 -- -- H H SJ22186057 -- -- H H SJ22186065 -- -- H H
SJ22186951 -- -- H H SJ22186840 -- -- H H SJ22186070 -- -- A A
TABLE-US-00021 TABLE 20B Marker S08106-1-Q1 S08107-1-Q1 S08109-1-Q1
S08110-1-Q1 Pos Sample Amplicon 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 SJ22186045
H -- -- H SJ22186913 H -- -- B SJ22186891 H -- -- H SJ22186879 H --
-- H SJ22186841 H -- -- H SJ22186057 H -- -- H SJ22186065 H -- -- H
SJ22186951 H -- -- H SJ22186840 H -- -- A SJ22186070 A -- -- A
TABLE-US-00022 TABLE 20C Marker S08111-1-Q1 S08115-2-Q1 S08117-1-Q1
S08119-1-Q1 Pos Sample Amplicon 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
SJ22186045 H B B B SJ22186913 B B B B SJ22186891 H H H H SJ22186879
H H H H SJ22186841 H H H H SJ22186057 H H H H SJ22186065 H H H H
SJ22186951 H H H H SJ22186840 A A A A SJ22186070 A A A A
TABLE-US-00023 TABLE 20D Marker S08118-1-Q1 S08116-1-Q1 S08114-1-Q1
S08113-1-Q1 Pos Sample Amplicon PPO_Gm19_1566k4-1 PPO_Gm19_1566k5-1
PPO_Gm19_1571k3-1 PPO_Gm19_1571k3-1 SJ22185925 B B -- -- SJ22186974
-- B -- -- SJ22185946 B B -- -- SJ22186019 -- B B B SJ22186923 B B
B B SJ22185604 H H -- -- SJ22186029 H H -- -- SJ22186052 -- H -- --
SJ22185534 A A -- -- SJ22185552 A A -- -- SJ22186842 A A -- --
SJ22186924 A A -- -- SJ22186873 A A -- -- SJ22186894 A A A A
SJ22185957 A A A A SJ22185941 A A A A SJ22186872 A A -- --
SJ22185984 A A A A SJ22186045 B B B B SJ22186913 B B B B SJ22186891
H H H H SJ22186879 H H H H SJ22186841 H H -- -- SJ22186057 H H H H
SJ22186065 H H H H SJ22186951 H H H H SJ22186840 A A A A SJ22186070
A A A A
TABLE-US-00024 TABLE 20E Marker S08101-1-Q1 S08112-1-Q1 S08108-1-Q1
S08101-2-Q1 Pos Sample Amplicon PPO_Gm19_1586k1-1 PPO_Gm19_1586k1-1
PPO_Gm19_1586k2-1 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 SJ22186045 -- B B B
SJ22186913 -- B B B SJ22186891 -- H H H SJ22186879 -- H H H
SJ22186841 -- H H H SJ22186057 -- H H H SJ22186065 -- H H H
SJ22186951 -- H H H SJ22186840 -- A A A SJ22186070 -- A A A
TABLE-US-00025 TABLE 20F Marker S08101-1-Q1 S08101-2-Q1 S08101-3-Q1
S08101-4-Q1 Pos Sample Amplicon PPO_Gm19_1586k4-1 PPO_Gm19_1586k4-1
PPO_Gm19_1586k4-1 PPO_Gm19_1586k4-1 SJ22185925 B -- B B SJ22186974
B -- B B SJ22185946 B -- B B SJ22186019 B B B B SJ22186923 B B B B
SJ22185604 H -- H H SJ22186029 H -- H H SJ22186052 H -- H H
SJ22185534 A -- A A SJ22185552 A -- A A SJ22186842 A -- A A
SJ22186924 A -- A A SJ22186873 A -- A A SJ22186894 A A A A
SJ22185957 A A A A SJ22185941 A A A A SJ22186872 A -- A A
SJ22185984 A A A A SJ22186045 -- B B B SJ22186913 -- B B B
SJ22186891 -- H H H SJ22186879 -- H H H SJ22186841 -- H H H
SJ22186057 -- H H H SJ22186065 -- H H H SJ22186951 -- H H H
SJ22186840 -- A A A SJ22186070 -- A A A
TABLE-US-00026 TABLE 20G Marker S08105-1-Q1 S08007-1-Q1 S03859-1-A
S08010-1-Q1 Pos Sample Amplicon PPO_Gm19_1618k2-1 PPO_Gm19_2089k3-1
PPO_Gm19_1635140 PPO_Gm19_2089k4-1 SJ22185925 B -- B A SJ22186974 B
-- B A SJ22185946 B -- B A SJ22186019 H H H H SJ22186923 B B B B
SJ22185604 B -- B B SJ22186029 H -- H B SJ22186052 H -- H H
SJ22185534 A -- A B SJ22185552 A -- A B SJ22186842 A -- A B
SJ22186924 A -- A B SJ22186873 A -- A B SJ22186894 B B B B
SJ22185957 A B A B SJ22185941 H H H H SJ22186872 A -- A B
SJ22185984 A A A A SJ22186045 B B -- B SJ22186913 B B -- B
SJ22186891 H A -- A SJ22186879 H B -- B SJ22186841 H A -- A
SJ22186057 H A -- A SJ22186065 H H -- A SJ22186951 H H -- A
SJ22186840 A A -- A SJ22186070 A H -- H
TABLE-US-00027 TABLE 20H 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
SJ22186045 B TOL SJ22186913 B TOL SJ22186891 A SEG SJ22186879 B SEG
SJ22186841 A SEG SJ22186057 A SEG SJ22186065 A SEG SJ22186951 A SEG
SJ22186840 A SUS SJ22186070 H SUS
Tables 20I-20L. Fine-Mapping of the Herbicide Tolerance QTL
Interval with Recombinants from the GEID4520632.times.GEID7589905
Population
TABLE-US-00028 TABLE 20I Marker S08102-1- S08103-1- S08104-1-
S08115-2- S08115-1- S08117-1- Sample Comment Phenotype S04867-1-A
Q1 Q1 Q1 Q1 Q1 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
SP21669417 L Border SEG -- -- A A -- A A SP21669560 SEG -- -- B B
-- H H SP21669331 SEG -- -- H H -- H H
TABLE-US-00029 TABLE 20J Marker S08119-1- S08118-1- S08116-1-
S08114-1- S08113-1- S08101-1- S08112-1- Sample Comment Phenotype Q1
Q1 Q1 Q1 Q1 Q1 Q1 SP21669249 R Border TOL 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 B/H SP21669670 R
Border TOL B B B -- -- B B SP21669503 L Border SEG H H H -- -- H H
SP21669458 R Border SUS A A A -- -- A A SP21669760 R Border SUS A A
A -- -- A A SP21669417 L Border SEG A A A A A -- H SP21669560 SEG
-- H H H H -- H SP21669331 SEG H H H H H -- H
TABLE-US-00030 TABLE 20K Marker S08108-1- S08101-1- S08101-2-
S08101-3- S08101-4- S08105-1- S03859-1-A Sample Comment Phenotype
Q1 Q1 Q1 Q1 Q1 Q1 SP21669249 R Border TOL B 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 B
SP21669616 R Border TOL B B B B B B B SP21669670 R Border TOL B B B
B B B B SP21669503 L Border SEG H H H H H H H SP21669458 R Border
SUS A A A A A A A SP21669760 R Border SUS A A A A A A A SP21669417
L Border SEG H -- H H H H -- SP21669560 SEG H -- H H H H --
SP21669331 SEG H -- H H H H --
TABLE-US-00031 TABLE 20L Marker S08007- S08010- S08010- Sample
Comment Phenotype 1-Q1 1-Q1 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 SP21669417 L Border SEG H H H
SP21669560 SEG H -- H SP21669331 SEG A A A
Example 9: SNP Haplotype Association Analysis
[0414] Association analysis of SNP haplotypes across the herbicide
tolerance QTL region provides an independent method of validating
the herbicide tolerance 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 21).
TABLE-US-00032 TABLE 21 SNP haplotype association analysis of the
herbicide tolerance QTL interval. Perfect association between
haplotype and phenotype between amplicons 1563k1 and 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 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
[0415] Although it is difficult to definitively define the
co-segregating region, it can conservatively be estimated to reside
between amplicons PPO_Gm19_1563k1 and PPO_Gm19_1618k2-1. Within the
borders defined by these loci, there are 38 SNP differences that
are shared between all of the susceptible lines as compared to all
the tolerant lines. This interval overlaps with the .about.44 kb
QTL interval identified by fine-mapping.
Example 10: Identification of Herbicide Tolerance Candidate
Genes
[0416] The .about.44 kb QTL interval defined by fine-mapping with
key recombinants includes only two annotated gene loci based on the
Phytozome database (see, e.g., phytozome.net): Glyma19g01920.1 and
Glyma19g01940.1. The former is functionally annotated as a Histone
deacetylase complex (SIN 3 component) while the latter is annotated
as a multidrug/pheromone exporter (ABC superfamily). Given the
nature of the herbicide tolerance trait and the role of plant ABC
transporters in detoxification and cellular export processes,
Glyma19g01940.1, the ABC transporter gene, may be considered the
more likely candidate gene. Moreover, all but .about.1.2 kb of
genomic sequence at the 5' end of and promoter of the histone
deacetylase are excluded from the QTL interval.
[0417] Based on a gene prediction model for the soybean reference
genome (Williams82), Glyma19g01940.1 appeared to be a 3672 base
pair coding region which encoded a 1224 amino acid polypeptide
(FIGS. 6A-9I, SEQ ID NOs:114-117). To examine sequence differences
between the two susceptible lines (GEID1653063 and GEID7589905) and
the two tolerant lines (GEID3495695 and GEID4520632) used for
mapping, the coding region from Glyma19g01940.1 was targeted for
re-sequencing in these four lines. With 95% of the coding region
re-sequenced, 10 SNPs, which result in 6 amino acid substitutions,
were observed between susceptible and tolerant lines (SEQ ID NOs:
114-117).
[0418] In an effort to obtain the actual coding sequence from the
tolerant and susceptible lines from one of the mapping populations,
full-length cDNAs for Glyma19g01940.1 were isolated and sequenced
for GEID3495695 (Tol) and GEID1653063 (Sus). For each of these
lines, sequence analysis of the cDNAs from Glyma19g01940.1
identified a 3696 base pair coding region which encodes a 1231
amino acid polypeptide (SEQ ID NOs: 118-121), which differs
slightly from the putative sequence obtained from the initial gene
prediction model. These sequence differences did not any indicate a
frameshift in the coding region of the initial sequence as compared
to the full insert sequence obtained. A sequence comparison between
the two sequenced cDNAs identified 11 SNPs between the susceptible
and tolerant lines, which led to seven differences in the 1231
amino acids that comprise the ABC transporter protein
(Glyma19g1940.1, see FIG. 6A-91). None of these SNPs or amino acid
substitutions result in a truncated protein. These amino acid
differences for Glyma19g01940.1 between tolerant and susceptible
lines are summarized in Table 22 for SEQ ID NOs: 119 and 121, and
equivalent positions in SEQ ID NOs: 115 and 117. These nucleotide
differences for Glyma19g01940.1 between tolerant and susceptible
lines are summarized in Table 23 for SEQ ID NOs: 118 and 120, and
equivalent positions in SEQ ID NOs: 114 and 116.
TABLE-US-00033 TABLE 22 Amino acid differences for Glyma19g01940.1
between tolerant and susceptible lines AA Position AA Position SEQ
ID NOs: SEQ ID NOs: 128 or 129 126 or 127 Tolerant Susceptible 478
-- Glycine (G) Glutamine (E) 520 520 Alanine (A) Valine (V) 584 584
Isoleucine (I) Leucine (L) 611 611 Isoleucine (I) Serine (S) 961
953 Arginine (R) Lysine (K) 1038 1030 Methionine (M) Leucine (L)
1120 1112 Arginine (R) Glycine (G)
TABLE-US-00034 TABLE 23 Nucleotide differences for Glyma19g01940.1
between tolerant and susceptible lines Nucleotide Nucleotide
Position Position SEQ ID NOs: SEQ ID NOs: 124 or 125 112 or 113
Tolerant Susceptible 1455 -- G A 1581 1559 C T 1772 1750 A C 1854
1832 T G 1954 1932 G A 2773 2727 C T 2904 2858 G A 3073 3027 T C
3134 3088 A C 3136 3090 G A 3380 3334 A G 3882 n/a T C
Example 11: QTL Analysis
[0419] The F2 population derived from GEID1653063.times.GEID6461257
consisting of 251 progeny and segregating for the herbicide
tolerance trait was used for mapping. The trait has been previously
mapped to LG-L. The population was screened with a total of 15
polymorphic markers from LG-L (Ch 19). Five of these 19 markers
showed severe segregation distortion and were excluded in the
mapping analysis. A significant QTL for herbicide tolerance was
detected on the LG-L (LRS=364) which was closely linked with the
PPO production marker S08101-2-Q1 and flanked by markers S04867-1-A
(7.81 cM) and S03859-1-A (10.00 cM). The QTL explained around 76%
of phenotypic variation.
Material and Methods
[0420] Population: An F2 mapping population GEID1653063/GEID6461257
consisting of 251 F2 progeny was used. DNA extraction of the tissue
was prepared using a citrate extraction protocol and quantified
using the GW DNA quantification protocol. Phenotype: The herbicide
tolerance phenotypes were for each line were evaluated using
chi-square analysis to establish a goodness to fit to the expected
1:2:1 genetic segregation ratio. The goodness of fit test indicated
that the phenotypic data for the 251 progeny follows the expected
1:2:1 genetic ratio (p-value=0.769). Genotype: PolyM was used to
identify polymorphic markers between the two parents. A total of 15
polymorphic markers from LG-L were assayed. Allele nomenclature
used were maternal alleles were assigned "A" and paternal alleles
"B", and heterozygous "H". The 10 of 15 markers were linked
together on LG-L with 5 markers showing severe segregation
distortion in the population. The 5 markers showing severe
segregation distortion were excluded for mapping analysis. Linkage
Analysis: Map Manager QTX.b20 (Manly et al. (2001) Mammalian Genome
12:930-932) was used to construct the linkage map and perform the
QTL analysis. A 1000 permutation test was used to establish the
threshold for statistical significance (LOD ratio statistic--LRS)
to declare a putative QTL. Map Manager parameters were set to:
1) Linkage Evaluation: Intercross
2) Search Criteria: P=1e-5
3) Map Function: Kosambi
4) Cross Type: Line Cross
QTL Analysis
[0421] Permutation Test: The thresholds at, 0.01 and 0.05 level
based on a 1000 permutation test for herbicide tolerance trait are
7.0 and 17.3, respectively. The marker regression analysis showed
that the QTL associated with herbicide tolerance could locate on
the LG-L. Interval mapping showed a highly significant region on LG
L (LRS=346). The QTL was closely linked with marker S08101-2-Q1 and
flanked by markers 504867-1-A (7.81 cM) and S03859-1-A (10.00 cM).
This region was estimated to explain .about.76% of the phenotypic
variation.
Example 12: Characterization of the Glycine max ABC Transporter 1
(GM ABCT1) Resistant and Susceptible Alleles in Transgenic Soy and
Transgenic Tobacco
[0422] Expression of the soybean ABC Transporter 1 Tolerant Allele
(GM-ABCT1 TOL) can be used to engineer plants with multiple
herbicide tolerance. This can be done by controlling expression of
the gene with a plant gene promoter, preferably a constitutive
promoter, such as the Arabidopsis polyubiquitin 10 promoter
(AT-UBQ10 PRO) or the corn polyubiquitin 1 promoter (UBI1ZM PRO),
and joining the promoter to the GM-ABCT1 TOL coding sequence with a
plant derived 5' untranslated leader. Examples of such a leader
include the Arabidopsis polyubiquitin 10 5' untranslated leader or
the corn polyubiquitin 1 5' untranslated leader. 5' untranslated
leaders may contain introns; inclusion of an intron would be the
preferred configuration, particularly for monocot species. The gene
expression cassette would be completed by addition of a 3'
untranslated region and poly-adenylation signal.
[0423] The several genetic elements could be joined by ligation,
Gateway Cloning.TM. (Invitrogen, www.invitrogen.com), or by
In-Fusion.TM. (Clontech, www.clonetech.com). Alternatively, part or
all of the expression cassette DNA sequence could be synthesized.
The expression cassette can then be introduced to plant cells by
agrobacterium mediated transformation or by direct DNA transfer, eg
Biolistic.TM. (DuPont, www.dupont.com) bombardment.
Soybean Experiments:
[0424] Soybean was transformed essentially as described in Li et
al. (2007) Plant Mol Biol 65:329-341. Soybean (cv. Jack)
embryogenic cultures were transformed by particle bombardment with
plant expression vectors using a strong constitutive promoter to
control the GM ABCT1 SUS (susceptible allele, SEQ ID NO:125) or GM
ABCT1 TOL (resistant allele, SEQ ID NO: 124) genes. Stable
transgenic events were tested for presence of the transgene by PCR,
but no confirmation of expression was performed. Three clonal
plants were produced from each transgenic event.
[0425] These T0 plants were grown to the V2 to V8 growth stage and
then sprayed with saflufenacil at a rate of 3 g/ha. All treatments
were applied in a spray volume of 280 L/ha. The spray mixture
contained acetone, water, glycerin and surfactant. Treated plants
were compared to untreated plants of similar genetic background and
evaluated for herbicide injury at seven days after treatment.
Visual injury was on a scale of 0 to 100% injury (0=no effect to
100=dead plant). Plants are also visually evaluated for agronomic
effects such as interveinal and veinal chlorosis, reddening, leaf
malformation, necrosis, apical withering/death/regrowth, and whole
plant height.
[0426] T0 plants carrying the susceptible allele showed a mean
visual injury score of 59% while plants carrying the resistant
allele showed a mean visual injury of 72%. Wild type soy plants
showed a mean visual injury of 84%.
[0427] There are numerous reasons why this result may have been
obtained, including: [0428] 1. T0 plants are highly variable coming
out of tissue culture, so the plants being compared are not uniform
in age, size, or vigor. [0429] 2. The phenotype observed for these
native soy gene alleles was only for pre-emergence treatment with
saflufenacil; resistance to foliar application of this herbicide
was not tested. Thus, these foliar spray experiments were performed
without evidence the gene would provide protection for this
herbicide under such conditions. Pre-emergence experiments were not
performed because seed for these transgenic lines had not yet been
produced. [0430] 3. The soybean genotype used in this experiment,
Jack, carries the resistant allele of GM ABCT1, so the resistance
gene was endogenously present in this experiment. Thus, while it
was possible that adding an additional copy of the gene and
expressing it with a constitutive promoter would have produced a
quantitative change in herbicide tolerance, it was equally possible
that the addition would not produce any measurable difference as
compared to the two copies of the resistant allele already present.
This soybean line was used because a reproducible transformation
procedure for this soybean genotype was known to the inventors.
[0431] 4. To date only 15 T0 plants carrying the resistant allele,
representing 11 events, have been tested. This low number of
replicates could have caused skewed the results of this study.
[0432] 5. As no confirmation of expression was performed, it is
possible that no transcription and/or translation is taking place.
[0433] 6. The putative events were not subjected to quality control
for single and/or intact insertion of the transgene construct.
Therefore, some events may contain deletions, duplications, and/or
rearrangements of all or part of the transgene construct
elements.
Tobacco Experiments:
[0434] Tobacco (cv. Petite Havana) was transformed by particle
bombardment with plant expression vectors using a strong
constitutive promoter to control the GM ABCT1 SUS (susceptible
allele, SEQ ID NO: 125) or GM ABCT1 RES (resistant allele, SEQ ID
NO: 124) genes using the following protocol: [0435] 1) Weigh 1 mg
0.6 micron gold powder (for 8 shots). Add 1 ml 100% ethanol
Sonicate 2 min. Centrifuge at 13,000 rpm.times.5 sec. Remove
Supernatant. Repeat 3 times. [0436] 2) Add 1 ml sterile
double-deionized water. Sonicate 2 min. Centrifuge at 13,000
rpm.times.5 sec. Remove Supernatant. Repeat 2 times. [0437] 3) Add
321 .mu.l sterile water. Vortex and sonicate 2 min. [0438] 4) Add 4
.mu.l of DNA (1 .mu.g/.mu.l concentration) and vortex briefly.
[0439] 5) Add 100 .mu.l of 5M CaCl.sub.2 and 80 .mu.l of 0.1M
Spermidine (14.4 .mu.l of Spermidine+985.6 .mu.l water prepared
freshly). Mix with finger and vortex 2 sec. [0440] 6) Incubate on
ice for 1 min. Spin down at 13,000 rpm for 2 sec and remove
supernatant. [0441] 7) Add 500 .mu.l of 100% ethanol. Pipette up
and down a few times to wash. (It is important to break all big
aggregates at this stage). Vortex 2 sec. Spin down at 13,000 rpm
for 2 sec and remove supernatant. [0442] 8) Re-suspend in 85 .mu.l
of 100% ethanol. Keep on ice. Vortex solution before spreading 10
.mu.l of DNA prep on flying disk. (Sterilize in 100% ethanol for
5-10 min and air dry, also sterilize and air dry 1100 psi rupture
disk). [0443] 9) Place tobacco leaf (cv. Petit Havana) abaxial side
up in the center of a petri plate containing MS01 medium (MS+B5
vitamins (Phytotechnology M519), Sucrose 30 g/1, agar 8 g/1, BA 2
mg/L, NAA 0.05 mg/L). [0444] 10) Bombard with 28 mm Hg vacuum,
position plate at 9 cm (2nd position from bottom), 1100 psi rupture
disk. After bombardment, invert leaves so bombarded side and
petioles are in contact with medium. [0445] 11) Maintain leaves
under 16 hrs/day dim light (<5 .mu.Einstein) for 2-3 days.
[0446] 12) Cut leaves into small pieces (0.5 cm.times.0.5 cm) in
MSB-Co (MS+Vit, Glucose 10 g/L, BA 50 mg/L, pH5.2), then blot dry
on sterile filter paper and place with bombarded side in contact
with the MS01 medium containing hygromycin 25 mg/L under 16 hrs/day
light (80-120 .mu.Einstein). When shoots appear, cut and place
directly in selection media. [0447] 13) Until shoots appear, change
selection medium every 2 weeks. [0448] 14) When entire plantlets
are formed (1-2 cm high), leaves are cut into approx. 1-2 mm
segments and re-cultured on MS01 medium containing hygromycin 25
mg/L and 8 g/L agar. Subculture to fresh medium every 3 weeks. When
new shoots appear, isolate single shoots and transfer onto 1/2 MS
media (M519 2.22 g/L, sucrose 15 g/L, agar 6 g/L).
[0449] Stable transgenic events were tested for presence of the
transgene by PCR, and transcription of the genes was confirmed by
qRT-PCR. Three clonal plants were produced from each transgenic
event; two plants from each event were used for the spray
experiment.
[0450] Plants were grown until 3 to 5 expanded true leaves were
present and then sprayed with saflufenacil at a rate of 12.5 g/ha.
All treatments were applied in a spray volume of 140 L/ha. Ammonium
sulfate was included at 1% w/v. Treated plants were compared to
untreated plants of similar genetic background and evaluated for
herbicide injury at seven and seventeen days after treatment.
Visual injury was on a scale of 0 to 100% injury (0=no effect to
100=dead plant).
[0451] At seven days after treatment T0 plants carrying the
susceptible allele showed a mean visual injury score of 77% while
plants carrying the resistant allele showed a mean visual injury of
63%. Transgenic control plants carrying an unrelated transgene
showed a mean visual injury of 63%.
[0452] At seventeen days after treatment many plants had shown
partial recovery from herbicide damage. T0 plants carrying the
susceptible allele now showed a mean visual injury score of 61%
while plants carrying the resistant allele showed a mean visual
injury of 46%. Transgenic control plants carrying an unrelated
transgene showed a mean visual injury of 53%.
[0453] Again, there are several reasons why this result may have
been obtained, including: [0454] 1. T0 plants are highly variable
coming out of tissue culture, so the plants being compared are not
uniform in age, size, or vigor. [0455] 2. The phenotype observed
for these native soy gene alleles was only for pre-emergence
treatment with saflufenacil; resistance to foliar application of
this herbicide was not tested. Thus, these foliar spray experiments
were performed without evidence the gene would provide protection
for this herbicide under such conditions. Pre-emergence experiments
were not performed because seed for these transgenic lines had not
yet been produced. [0456] 3. Expression of the GM ABCT1 peptide was
not tested, so it is possible that no peptide translation is taking
place.
[0457] Unsprayed clones of the transgenic tobacco plants described
above were grown in a greenhouse until flowering and were allowed
to self pollinate. Seeds were harvested and used for further
experiments. Transgene insertion copy number was not determined for
these plants, but for single copy plants the seeds would be
expected to segregate 1homozygous:2hemizygous:1null for the
transgene.
[0458] T1 seeds from multiple transgenic events carrying the
resistant and susceptible alleles, and wild type seeds, were
germinated on 1.5% Gelrite (no nutrients or minerals) plates and
kept in a growth chamber at 26.degree. C. under a 16 hr light
regime. Plate media contained saflufenacil at concentrations down
to 0.45 parts per billion (ppb). At concentrations above 0.91 ppb
no germination was observed; at 0.91 ppb and 0.45 ppb seeds
germinated but were bleached and failed to grow. No visible
difference was observed between transgenic seed with either GM
ABCT1 allele or wild type seed. All seed germinated and grew
normally on Gelrite plates lacking saflufenacil.
[0459] T1 seeds were sown in four inch pots containing soil and
allowed to germinate for five days. At this point, with cotyledons
visible above the soil, pots were drenched with 100 mls
saflufenacil in water at 0.45 ppb. The solution was applied to
soil, avoiding direct contact with plant cotyledons. Three days
after treatment all seedlings were severely bleached and appeared
dead. No visible differences were observed between any transgenic
lines and wild type seed.
[0460] 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.
[0461] 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
129130DNAArtificial SequencePrimer/Probe Sequence 1gagggctatg
ttttcttctc cagatgtgag 30226DNAArtificial SequencePrimer/Probe
Sequence 2aaggtcggct tggtggttaa aggcag 26313DNAArtificial
SequencePrimer/Probe Sequence 3tcatctgtga taa 13413DNAArtificial
SequencePrimer/Probe Sequence 4tcatgtgtga taa 13513DNAArtificial
SequencePrimer/Probe Sequence 5tcatctctga taa 13622DNAArtificial
SequencePrimer/Probe Sequence 6ctggacctac ccgggatgaa aa
22722DNAArtificial SequencePrimer/Probe Sequence 7tcttcctctc
ccttcctctc gc 22813DNAArtificial SequencePrimer/Probe Sequence
8cgcgactctc ctc 13913DNAArtificial SequencePrimer/Probe Sequence
9cgcgagtctc ctc 131027DNAArtificial SequencePrimer/Probe Sequence
10tcccaggtta gattttctga acgaaga 271124DNAArtificial
SequencePrimer/Probe Sequence 11catcagcaca aaagggcatc ctca
241216DNAArtificial SequencePrimer/Probe Sequence 12cactccttaa
ggtaat 161316DNAArtificial SequencePrimer/Probe Sequence
13cactccttaa gataat 161420DNAArtificial SequencePrimer/Probe
Sequence 14gttatcgtca ccaccaccaa 201521DNAArtificial
SequencePrimer/Probe Sequence 15cacaacacga gtagccgtag g
211616DNAArtificial SequencePrimer/Probe Sequence 16aacggatcat
cacaac 161715DNAArtificial SequencePrimer/Probe Sequence
17aacggctcat cacaa 151820DNAArtificial SequencePrimer/Probe
Sequence 18cgacaatggc ctttacacct 201920DNAArtificial
SequencePrimer/Probe Sequence 19tcgatatgga cgaaggagga
202016DNAArtificial SequencePrimer/Probe Sequence 20acaccatttt
tcatcc 162116DNAArtificial SequencePrimer/Probe Sequence
21acaccctttt tcatcc 162222DNAArtificial SequencePrimer/Probe
Sequence 22gcaatcacat ttgcattcct ta 222321DNAArtificial
SequencePrimer/Probe Sequence 23tctgaacgag ttgtgcaaga a
212416DNAArtificial SequencePrimer/Probe Sequence 24actgctgctt
tgtcta 162516DNAArtificial SequencePrimer/Probe Sequence
25ctactgctac tttgtc 162620DNAArtificial SequencePrimer/Probe
Sequence 26acctcgtatt ggtggtggtg 202720DNAArtificial
SequencePrimer/Probe Sequence 27gaatgttcag tgcgagcaac
202815DNAArtificial SequencePrimer/Probe Sequence 28acttccctcg
tttcg 152914DNAArtificial SequencePrimer/Probe Sequence
29cttccctcat ttcg 143022DNAArtificial SequencePrimer/Probe Sequence
30caaaaggaaa gaagaaccgt gt 223121DNAArtificial SequencePrimer/Probe
Sequence 31tccaacctat gtgttggtgt g 213217DNAArtificial
SequencePrimer/Probe Sequence 32atgattgaag caggaaa
173318DNAArtificial SequencePrimer/Probe Sequence 33tcatgattga
agcagcaa 183427DNAArtificial SequencePrimer/Probe Sequence
34ggagacttga cttaaagaga aagaaaa 273526DNAArtificial
SequencePrimer/Probe Sequence 35cggaaagaaa aacaatagat tgaatg
263619DNAArtificial SequencePrimer/Probe Sequence 36cttgttctag
actgatcat 193716DNAArtificial SequencePrimer/Probe Sequence
37ctagactgat aattca 163826DNAArtificial SequencePrimer/Probe
Sequence 38tcattcaaga ctacatgaaa gacaaa 263920DNAArtificial
SequencePrimer/Probe Sequence 39caagggagag caatccttga
204016DNAArtificial SequencePrimer/Probe Sequence 40atagtctccc
aaacac 164117DNAArtificial SequencePrimer/Probe Sequence
41atagtctctc aaacacc 174222DNAArtificial SequencePrimer/Probe
Sequence 42gaaactttcc attttgccct tc 224318DNAArtificial
SequencePrimer/Probe Sequence 43agaacgcagg ggagaagc
184416DNAArtificial SequencePrimer/Probe Sequence 44cttcttccac
tcttac 164517DNAArtificial SequencePrimer/Probe Sequence
45ccttcttaca ctcttac 174628DNAArtificial SequencePrimer/Probe
Sequence 46tgatatgaca ctctactaag atgtgttg 284720DNAArtificial
SequencePrimer/Probe Sequence 47tgattcatcc gcaaacttga
204817DNAArtificial SequencePrimer/Probe Sequence 48cactctccta
tattgtc 174916DNAArtificial SequencePrimer/Probe Sequence
49ctctcctaca ttgtca 165022DNAArtificial SequencePrimer/Probe
Sequence 50agatccttgt tccaaattcc aa 225120DNAArtificial
SequencePrimer/Probe Sequence 51ccttggctta atgggtgtgt
205216DNAArtificial SequencePrimer/Probe Sequence 52ccaacacaat
ctaact 165314DNAArtificial SequencePrimer/Probe Sequence
53ccaacacaat cgaa 145420DNAArtificial SequencePrimer/Probe Sequence
54atggaggcaa gcttgtgttt 205520DNAArtificial SequencePrimer/Probe
Sequence 55catgctacca gcatctgcaa 205617DNAArtificial
SequencePrimer/Probe Sequence 56cttcataaac gccaaag
175716DNAArtificial SequencePrimer/Probe Sequence 57cataaatgcc
aaagca 165820DNAArtificial SequencePrimer/Probe Sequence
58aatgagcaag ggagaggaca 205920DNAArtificial SequencePrimer/Probe
Sequence 59tcgccgctgc tatttaattt 206018DNAArtificial
SequencePrimer/Probe Sequence 60aagcactact ttcaattg
186114DNAArtificial SequencePrimer/Probe Sequence 61aagcaccact ttca
146220DNAArtificial SequencePrimer/Probe Sequence 62agatgccttg
ctcagtggac 206322DNAArtificial SequencePrimer/Probe Sequence
63atgatgaatg tgttgagcca at 226414DNAArtificial SequencePrimer/Probe
Sequence 64ccccatcacc atac 146514DNAArtificial SequencePrimer/Probe
Sequence 65accccaccac cata 146622DNAArtificial SequencePrimer/Probe
Sequence 66agaaaccttc caaagctctt gg 226720DNAArtificial
SequencePrimer/Probe Sequence 67tagggaggca cttgacaacc
206815DNAArtificial SequencePrimer/Probe Sequence 68caacatccga
gtcca 156915DNAArtificial SequencePrimer/Probe Sequence
69caacatcaga gtcca 157020DNAArtificial SequencePrimer/Probe
Sequence 70ttttgacccc cagagagttg 207120DNAArtificial
SequencePrimer/Probe Sequence 71ttgcaagcct aaaggatggt
207219DNAArtificial SequencePrimer/Probe Sequence 72ctatctctac
acgatgtgt 197316DNAArtificial SequencePrimer/Probe Sequence
73ctatctccac acgatg 167420DNAArtificial SequencePrimer/Probe
Sequence 74tcccacttga tcttgcagaa 207520DNAArtificial
SequencePrimer/Probe Sequence 75tacggtgggt ggattattcg
207615DNAArtificial SequencePrimer/Probe Sequence 76cctccaatgg
catac 157717DNAArtificial SequencePrimer/Probe Sequence
77cctccaatag catacat 177822DNAArtificial SequencePrimer/Probe
Sequence 78agaaaagcag cagaaagagg ac 227922DNAArtificial
SequencePrimer/Probe Sequence 79cttcatgaat cccaacatca ga
228017DNAArtificial SequencePrimer/Probe Sequence 80ctctaattcc
acatctg 178118DNAArtificial SequencePrimer/Probe Sequence
81cctctaattt cacatctg 188222DNAArtificial SequencePrimer/Probe
Sequence 82tcaaaccatt ttgtttccca gt 228321DNAArtificial
SequencePrimer/Probe Sequence 83tgctagcctt tgatacccaa c
218416DNAArtificial SequencePrimer/Probe Sequence 84ttgcattgta
ttctct 168515DNAArtificial SequencePrimer/Probe Sequence
85ttgcattgta ttttc 158621DNAArtificial SequencePrimer/Probe
Sequence 86gtctcaggca gtgaatctgc t 218720DNAArtificial
SequencePrimer/Probe Sequence 87cagccttacc atcaacatcg
208813DNAArtificial SequencePrimer/Probe Sequence 88ttccgtgaag atc
138915DNAArtificial SequencePrimer/Probe Sequence 89atgcttccgc
gaaga 159026DNAArtificial SequencePrimer/Probe Sequence
90ggtagcagtt actttgtgat gtaagc 269122DNAArtificial
SequencePrimer/Probe Sequence 91catgcaataa aatccaaaac ca
229217DNAArtificial SequencePrimer/Probe Sequence 92tactgatcac
aggttat 179317DNAArtificial SequencePrimer/Probe Sequence
93tactgaccac aggttat 179420DNAArtificial SequencePrimer/Probe
Sequence 94ttgctttgga aaggactcca 209520DNAArtificial
SequencePrimer/Probe Sequence 95cctcatcaac tcctgctgct
209614DNAArtificial SequencePrimer/Probe Sequence 96ctcggtgctg tttt
149714DNAArtificial SequencePrimer/Probe Sequence 97ctcggtgctg tctt
149823DNAArtificial SequencePrimer/Probe Sequence 98gaaaccaatt
ttgatgtgaa gga 239920DNAArtificial SequencePrimer/Probe Sequence
99aagtgagagg ggtgcaaaga 2010014DNAArtificial SequencePrimer/Probe
Sequence 100cagccctatc tcac 1410114DNAArtificial
SequencePrimer/Probe Sequence 101agccctgtct cact
1410221DNAArtificial SequencePrimer/Probe Sequence 102gcaaatgaga
aggctgaagc t 2110319DNAArtificial SequencePrimer/Probe Sequence
103gctgtccctc agtccatcc 1910415DNAArtificial SequencePrimer/Probe
Sequence 104cggtatcgct cgtca 1510515DNAArtificial
SequencePrimer/Probe Sequence 105tatcgctcgc caacg
1510623DNAArtificial SequencePrimer/Probe Sequence 106atccacttgc
aagataggac act 2310726DNAArtificial SequencePrimer/Probe Sequence
107gtgtaagtac tgatgtgcag ttttga 2610819DNAArtificial
SequencePrimer/Probe Sequence 108cttgacatta agactatcc
1910919DNAArtificial SequencePrimer/Probe Sequence 109agactaatcc
ttaaacaag 1911027DNAArtificial SequencePrimer/Probe Sequence
110tcaacaggtt atgaatatac aggtcaa 2711122DNAArtificial
SequencePrimer/Probe Sequence 111catcaccaat tgtttggagt tc
2211219DNAArtificial SequencePrimer/Probe Sequence 112ctattactct
ccgttattt 1911318DNAArtificial SequencePrimer/Probe Sequence
113ctattactcc ccgttatt 1811418DNAArtificial SequencePrimer/Probe
Sequence 114ttgttgaatg ggggcact 1811520DNAArtificial
SequencePrimer/Probe Sequence 115ctcgagcaaa tctcgatggt
2011617DNAArtificial SequencePrimer/Probe Sequence 116ttgaatgctt
actctct 1711719DNAArtificial SequencePrimer/Probe Sequence
117ttgaatgttt actctcttt 1911820DNAArtificial SequencePrimer/Probe
Sequence 118ctgtggagga ggagcttgag 2011922DNAArtificial
SequencePrimer/Probe Sequence 119acaagtcaca accgtcaatg at
2212019DNAArtificial SequencePrimer/Probe Sequence 120agtctttgtt
ttctctttt 1912119DNAArtificial SequencePrimer/Probe Sequence
121agtctttgtt ttctctctt 191223672DNAGlycine max 122atgcatgctg
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 36721233672DNAGlycine max
123atgcatgctg 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 36721243920DNAGlycine max 124tggctctctt
cggtctattt tcatgcatgc tgatggctta gactggttcc tcatgatttt 60tggtctcttt
ggggccattg gtgatggcat aggcacccct ttggtgttgt ttatcaccag
120caaaattatg aacaatattg gtggtttttc tagcaacata ggcagcactt
tcatccacag 180catcaatgag aatgccgtgg ttttgttata tttggctggt
gggtctttca ttgcttgttt 240cctagagggt tattgttgga caagaacagg
agaaaggcaa gctgcaagaa tgagagttag 300gtaccttaaa gcagttctca
ggcaagaagt agcatacttt gatttgcatg tcacaagcac 360atcggaggtc
atcaccagcg tctctaatga tagcctcgta attcaagatt gtcttagtga
420aaaggtccca aactttttga tgaatgcgtc catgtttgtt gggagctaca
tagtggcttt 480tgcattattg tggagattgg ccattgtggg gttccctttt
gtggccctac ttgtgatccc 540cggtttcatg tatgggagga cattaatggg
gttggctagc aagataagag aagagtacaa 600taaagcaggc acaatagcag
aacaagcaat atcctccatc agaaccgttt attcttttgt 660gggggaaagc
aagactattg atgctttctc tgaagcccta caagggtctg ttgagttggg
720actgagacaa ggcttagcaa aaggtttagc tattggaagc aatggtgttg
tctttgctat 780atgggcattc atgtcctatt atggtagcag attggtcatg
taccatggag ctaaaggtgg 840gactgtattt gcagttggag cagccatagc
tcttggagga ttggcactag gtgctggttt 900gtcgaacgtg aagtacttct
cagaagcaag taccgcagga gaacgcataa tggaagtgat 960aaaaagggtt
ccaaagattg attctgatag catggctgag gagattctgg agaacgtttc
1020aggggaagtt gaattcaacc atgtggactt tgtgtaccca tcaaggccag
acagtgttat 1080tctgaatgat ttctgcctaa agattccagc agggaaaaca
gtggctttgg ttggagggag 1140tggctctgga aaatccactg tgatatcact
tttgcagagg ttttatgacc caattgaggg 1200agagatattt cttgatggtg
tggccattca caagttgcaa ctcaagtggt tgaggtctca 1260aatgggtttg
gtcagccaag agcctgcact gtttgcaact agcattaaag agaatatact
1320ttttggaaga gaagatgcca ctcaagaaga ggttgtggag gcagcaaaag
cttccaatgc 1380tcataatttc atttcacagt tgccacaagg atatgatact
caggttgggg agagaggagt 1440tcaaatgtca ggtggacaaa agcaaagaat
tgcaatagca cgagcaataa taaaaaagcc 1500acggattctt ctattagatg
aagcaacaag tgcactagat tctgaatctg aacgagttgt 1560gcaagaagca
ttagacaaag cagcagtagg gcgcacaaca atcatcattg cacatagatt
1620atccaccata aggaatgcaa atgtgattgc tgttgtgcaa agtgggaaaa
tcatggagat 1680gggatcacac catgaactaa tccaaaacga caatggcctt
tacacctcac tagttcgtct 1740ccaacaagca aaaaatgaaa aagaagacac
catttttcat cctactcctc cttcgtccat 1800atcgaacaaa gacaatcaca
acacgagtag ccgtaggctc tctgttgtga tgatccgttc 1860tagctccacc
aactcgatac ctcgtattgg tggtggtgac gataacaata ttgttgaaga
1920agtagtggaa gataacaagc caccacttcc ctcgtttcga aggttgctcg
cactgaacat 1980tcccgagtgg aagcaagcat gtttagggtg tttgaatgcg
gtgttgtttg gtgcaattca 2040gcctgtgtat gcatttgcaa tggggtcagt
gatatctgtt tacttcctcc cagaccataa 2100tgagataaag aagaaaacta
tgatctattc actttgtttc ctagggttgg ctgtgttctc 2160cttagtggtt
aatatcctcc agcattacaa ctttgcttac ataggagagt acttgactaa
2220aaggatcaga gaaagaatgt tttccaagat actcactttt gaagttggat
ggtttgatca 2280agatgaaaat tccacaggtg ctgtttgttc tagacttgcc
aaagaagcca atgtggtaag 2340gtctttagtg ggagatagaa tggctctagt
ggtacaaacc atttcagcag tggtaatagc 2400ttttacaatg ggcctaatca
ttgcatggag gttggccatt gttatgatag cagttcaacc 2460cattatcata
gcatgtttct acacaaggcg tgtccttctc aagagcatgt ctagtaaggc
2520catcaaggcc caagatgaaa gtagcaagat agctgttgaa gctgtttcca
acctcagaac 2580aatcacagca ttttcttccc aagataggat ccttaaaatg
ctcgaaaagg cccaagaagg 2640cccgagccgt gaaagcattc gacaatcatg
gtttgcgggc attgggcttg catgttccca 2700aagccttaca ttttgcactt
gggctttgga cttttggtat ggaggcaagc ttgtgtttca 2760gggcttcata
aacgccaaag cattgtttga gaccttcatg attttagtga gcacaggtag
2820ggttattgca gatgctggta gcatgaccaa tgacctagct aaaggggctg
atgctgtggg 2880ctcagttttt gcaatcttag ataggtacac aaaaattgag
ccagatgatg acatagatgg 2940gtacaagcct gaaaagctaa cagggaaaat
agagcttcat gatgtccatt ttgcataccc 3000agctaggccc aatgtgatga
tcttccaagg cttctcaatc aaaattgatg caggcagatc 3060aacagcattg
gttgggcaaa gtggctctgg aaaatcaaca atcataggct taattgagag
3120attctatgac cctatgaaag ggattgtgac cattgatggt agagacataa
aatcatacca 3180ccttaggtca ctaaggaagc atattgctct tgtaagccaa
gagccaacat tgtttggtgg 3240gaccataagg gaaaatattg catatggggc
atctaataat aataacaagg ttgatgaaac 3300tgagatcata gaagcagcta
gggcagctaa tgctcatgat ttcattgcaa gcctaaagga 3360tggttatgac
acatcgtgta gagatagagg agtgcaactc tctgggggtc aaaagcaaag
3420aattgcaata gctagagcca tattgaagaa tccagaagtg ttgttgttgg
atgaagccac 3480aagtgcccta gatagccaat cagaaaaatt ggtgcaagat
gctctagaaa gggtgatggt 3540ggggagaact agtgtggtgg tggctcacag
gttaagcaca atacaaaatt gtgacctaat 3600tgctgtgtta gataagggaa
aagtggtgga gaaagggacc cactcatctt tgttggctca 3660tggaccaggt
ggagcttatt actctttgat aagtttacaa agaagaccag caaattaaac
3720atgaatgtta gttttacgca tgaaatctca gctagctaat caaaacaaac
aaaatgtcac 3780atttattggt gattagtatt aaacctcttt tgtggtaact
tgtgaaagta aattaagaaa 3840aatgaaagaa aagtaaatta agcaaagata
gaagggaaga attatataac agttgtagtc 3900tctcccaact tcctaaattc
39201253920DNAGlycine max 125tggctctctt cggtctattt tcatgcatgc
tgatggctta gactggttcc tcatgatttt 60tggtctcttt ggggccattg gtgatggcat
aggcacccct ttggtgttgt ttatcaccag 120caaaattatg aacaatattg
gtggtttttc tagcaacata ggcagcactt tcatccacag 180catcaatgag
aatgccgtgg ttttgttata tttggctggt gggtctttca ttgcttgttt
240cctagagggt tattgttgga caagaacagg agaaaggcaa gctgcaagaa
tgagagttag 300gtaccttaaa gcagttctca ggcaagaagt agcatacttt
gatttgcatg tcacaagcac 360atcggaggtc atcaccagcg tctctaatga
tagcctcgta attcaagatt gtcttagtga 420aaaggtccca aactttttga
tgaatgcgtc catgtttgtt gggagctaca tagtggcttt 480tgcattattg
tggagattgg ccattgtggg gttccctttt gtggccctac ttgtgatccc
540cggtttcatg tatgggagga cattaatggg gttggctagc aagataagag
aagagtacaa 600taaagcaggc acaatagcag aacaagcaat atcctccatc
agaaccgttt attcttttgt 660gggggaaagc aagactattg atgctttctc
tgaagcccta caagggtctg ttgagttggg 720actgagacaa ggcttagcaa
aaggtttagc tattggaagc aatggtgttg tctttgctat 780atgggcattc
atgtcctatt atggtagcag attggtcatg taccatggag ctaaaggtgg
840gactgtattt gcagttggag cagccatagc tcttggagga ttggcactag
gtgctggttt 900gtcgaacgtg aagtacttct cagaagcaag taccgcagga
gaacgcataa tggaagtgat 960aaaaagggtt ccaaagattg attctgatag
catggctgag gagattctgg agaacgtttc 1020aggggaagtt gaattcaacc
atgtggactt tgtgtaccca tcaaggccag acagtgttat 1080tctgaatgat
ttctgcctaa agattccagc agggaaaaca gtggctttgg ttggagggag
1140tggctctgga aaatccactg tgatatcact tttgcagagg ttttatgacc
caattgaggg 1200agagatattt cttgatggtg tggccattca caagttgcaa
ctcaagtggt tgaggtctca 1260aatgggtttg gtcagccaag agcctgcact
gtttgcaact agcattaaag agaatatact 1320ttttggaaga gaagatgcca
ctcaagaaga ggttgtggag gcagcaaaag cttccaatgc 1380tcataatttc
atttcacagt tgccacaagg atatgatact caggttgggg agagaggagt
1440tcaaatgtca ggtgaacaaa agcaaagaat tgcaatagca cgagcaataa
taaaaaagcc 1500acggattctt ctattagatg aagcaacaag tgcactagat
tctgaatctg aacgagttgt 1560gcaagaagca ttagacaaag tagcagtagg
gcgcacaaca atcatcattg cacatagatt 1620atccaccata aggaatgcaa
atgtgattgc tgttgtgcaa agtgggaaaa tcatggagat 1680gggatcacac
catgaactaa tccaaaacga caatggcctt tacacctcac tagttcgtct
1740ccaacaagca aaaaatgaaa aagaagacac cctttttcat cctactcctc
cttcgtccat 1800atcgaacaaa gacaatcaca acacgagtag ccgtaggctc
tctgttgtga tgagccgttc 1860tagctccacc aactcgatac ctcgtattgg
tggtggtgac gataacaata ttgttgaaga 1920agtagtggaa gataacaagc
caccacttcc ctcatttcga aggttgctcg cactgaacat 1980tcccgagtgg
aagcaagcat gtttagggtg tttgaatgcg gtgttgtttg gtgcaattca
2040gcctgtgtat gcatttgcaa tggggtcagt gatatctgtt tacttcctcc
cagaccataa 2100tgagataaag aagaaaacta tgatctattc actttgtttc
ctagggttgg ctgtgttctc 2160cttagtggtt aatatcctcc agcattacaa
ctttgcttac ataggagagt acttgactaa 2220aaggatcaga gaaagaatgt
tttccaagat actcactttt gaagttggat ggtttgatca 2280agatgaaaat
tccacaggtg ctgtttgttc tagacttgcc aaagaagcca atgtggtaag
2340gtctttagtg ggagatagaa tggctctagt ggtacaaacc atttcagcag
tggtaatagc 2400ttttacaatg ggcctaatca ttgcatggag gttggccatt
gttatgatag cagttcaacc 2460cattatcata gcatgtttct acacaaggcg
tgtccttctc aagagcatgt ctagtaaggc 2520catcaaggcc caagatgaaa
gtagcaagat agctgttgaa gctgtttcca acctcagaac 2580aatcacagca
ttttcttccc aagataggat ccttaaaatg ctcgaaaagg cccaagaagg
2640cccgagccgt gaaagcattc gacaatcatg gtttgcgggc attgggcttg
catgttccca 2700aagccttaca ttttgcactt gggctttgga cttttggtat
ggaggcaagc ttgtgtttca 2760gggcttcata aatgccaaag cattgtttga
gaccttcatg attttagtga gcacaggtag 2820ggttattgca gatgctggta
gcatgaccaa tgacctagct aaaggggctg atgctgtggg 2880ctcagttttt
gcaatcttag ataagtacac aaaaattgag ccagatgatg acatagatgg
2940gtacaagcct gaaaagctaa cagggaaaat agagcttcat gatgtccatt
ttgcataccc 3000agctaggccc aatgtgatga tcttccaagg cttctcaatc
aaaattgatg caggcagatc 3060aacagcattg gtcgggcaaa gtggctctgg
aaaatcaaca atcataggct taattgagag 3120attctatgac cctctaaaag
ggattgtgac cattgatggt agagacataa aatcatacca 3180ccttaggtca
ctaaggaagc atattgctct tgtaagccaa gagccaacat tgtttggtgg
3240gaccataagg gaaaatattg catatggggc atctaataat aataacaagg
ttgatgaaac 3300tgagatcata gaagcagcta gggcagctaa tgctcatgat
ttcattgcaa gcctaaagga 3360tggttatgac acatcgtgtg gagatagagg
agtgcaactc tctgggggtc aaaagcaaag 3420aattgcaata gctagagcca
tattgaagaa tccagaagtg ttgttgttgg atgaagccac 3480aagtgcccta
gatagccaat cagaaaaatt ggtgcaagat gctctagaaa gggtgatggt
3540ggggagaact agtgtggtgg tggctcacag gttaagcaca atacaaaatt
gtgacctaat 3600tgctgtgtta gataagggaa aagtggtgga gaaagggacc
cactcatctt tgttggctca 3660tggaccaggt ggagcttatt actctttgat
aagtttacaa agaagaccag caaattaaac 3720atgaatgtta gttttacgca
tgaaatctca gctagctaat caaaacaaac aaaatgtcac 3780atttattggt
gattagtatt aaacctcttt tgtggtaact tgtgaaagta aattaagaaa
3840aatgaaagaa aagtaaatta agcaaagata gaagggaaga actatataac
agttgtagtc 3900tctcccaact tcctaaattc 39201261223PRTGlycine max
126Met 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 1271223PRTGlycine max 127Met 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
1281231PRTGlycine max 128Met 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 Val Arg Ser Leu Val Gly Asp Arg
Met Ala Leu Val Val 770 775 780 Gln Thr Ile Ser Ala Val Val Ile Ala
Phe Thr Met Gly Leu Ile Ile 785 790 795 800 Ala Trp Arg Leu Ala Ile
Val Met Ile Ala Val Gln Pro Ile Ile Ile 805 810 815 Ala Cys Phe Tyr
Thr Arg Arg Val Leu Leu Lys Ser Met Ser Ser Lys 820 825 830 Ala Ile
Lys Ala Gln Asp Glu Ser Ser Lys Ile Ala Val Glu Ala Val 835 840 845
Ser Asn Leu Arg Thr Ile Thr Ala Phe Ser Ser Gln Asp Arg Ile Leu 850
855 860 Lys Met Leu Glu Lys Ala Gln Glu Gly Pro Ser Arg Glu Ser Ile
Arg 865 870 875 880 Gln Ser Trp Phe Ala Gly Ile Gly Leu Ala Cys Ser
Gln Ser Leu Thr 885 890 895 Phe Cys Thr Trp Ala Leu Asp Phe Trp Tyr
Gly Gly Lys Leu Val Phe 900 905 910 Gln Gly Phe Ile Asn Ala Lys Ala
Leu Phe Glu Thr Phe Met Ile Leu 915 920 925 Val Ser Thr Gly Arg Val
Ile Ala Asp Ala Gly Ser Met Thr Asn Asp 930 935 940 Leu Ala Lys Gly
Ala Asp Ala Val Gly Ser Val Phe Ala Ile Leu Asp 945 950 955 960 Arg
Tyr Thr Lys Ile Glu Pro Asp Asp Asp Ile Asp Gly Tyr Lys Pro 965 970
975 Glu Lys Leu Thr Gly Lys Ile Glu Leu His Asp Val His Phe Ala Tyr
980 985 990 Pro Ala Arg Pro Asn Val Met Ile Phe Gln Gly Phe Ser Ile
Lys Ile 995 1000 1005 Asp Ala Gly Arg Ser Thr Ala Leu Val Gly Gln
Ser Gly Ser Gly 1010 1015 1020 Lys Ser Thr Ile Ile Gly Leu Ile Glu
Arg Phe Tyr Asp Pro Met 1025 1030 1035 Lys Gly Ile Val Thr Ile Asp
Gly Arg Asp Ile Lys Ser Tyr His 1040 1045 1050 Leu Arg Ser Leu Arg
Lys His Ile Ala Leu Val Ser Gln Glu Pro 1055 1060 1065 Thr Leu Phe
Gly Gly Thr Ile Arg Glu Asn Ile Ala Tyr Gly Ala 1070 1075 1080 Ser
Asn Asn Asn Asn Lys Val Asp Glu Thr Glu Ile Ile Glu Ala 1085 1090
1095 Ala Arg Ala Ala Asn Ala His Asp Phe Ile Ala Ser Leu Lys Asp
1100 1105 1110 Gly Tyr Asp Thr Ser Cys Arg Asp Arg Gly Val Gln Leu
Ser Gly 1115 1120 1125 Gly Gln Lys Gln Arg Ile Ala Ile Ala Arg Ala
Ile Leu Lys Asn 1130 1135 1140 Pro Glu Val Leu Leu Leu Asp Glu Ala
Thr Ser Ala Leu Asp Ser 1145 1150 1155 Gln Ser Glu Lys Leu Val Gln
Asp Ala Leu Glu Arg Val Met Val 1160 1165 1170 Gly Arg Thr Ser Val
Val Val Ala His Arg Leu Ser Thr Ile Gln 1175 1180 1185 Asn Cys Asp
Leu Ile Ala Val Leu Asp Lys Gly Lys Val Val Glu 1190 1195 1200 Lys
Gly Thr His Ser Ser Leu Leu Ala His Gly Pro Gly Gly Ala 1205 1210
1215 Tyr Tyr Ser Leu Ile Ser Leu Gln Arg Arg Pro Ala Asn 1220 1225
1230 1291231PRTGlycine max 129Met 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 Glu
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 Val Arg Ser
Leu Val Gly Asp Arg Met Ala Leu Val Val 770 775 780 Gln Thr Ile Ser
Ala Val Val Ile Ala Phe Thr Met Gly Leu Ile Ile 785 790 795 800 Ala
Trp Arg Leu Ala Ile Val Met Ile Ala Val Gln Pro Ile Ile Ile 805 810
815 Ala Cys Phe Tyr Thr Arg Arg Val Leu Leu Lys Ser Met Ser Ser Lys
820 825 830 Ala Ile Lys Ala Gln Asp Glu Ser Ser Lys Ile Ala Val Glu
Ala Val 835 840 845 Ser Asn Leu Arg Thr Ile Thr Ala Phe Ser Ser Gln
Asp Arg Ile Leu 850 855 860 Lys Met Leu Glu Lys Ala Gln Glu Gly Pro
Ser Arg Glu Ser Ile Arg 865 870 875 880 Gln Ser Trp Phe Ala Gly Ile
Gly Leu Ala Cys Ser Gln Ser Leu Thr 885 890 895 Phe Cys Thr Trp Ala
Leu Asp Phe Trp Tyr Gly Gly Lys Leu Val Phe 900 905 910 Gln Gly Phe
Ile Asn Ala Lys Ala Leu Phe Glu Thr Phe Met Ile Leu 915 920 925 Val
Ser Thr Gly Arg Val Ile Ala Asp Ala Gly Ser Met Thr Asn Asp 930 935
940 Leu Ala Lys Gly Ala Asp Ala Val Gly Ser Val Phe Ala Ile Leu Asp
945 950 955 960 Lys Tyr Thr Lys Ile Glu Pro Asp Asp Asp Ile Asp Gly
Tyr Lys Pro 965 970 975 Glu Lys Leu Thr Gly Lys Ile Glu Leu His Asp
Val His Phe Ala Tyr 980 985 990 Pro Ala Arg Pro Asn Val Met Ile Phe
Gln Gly Phe Ser Ile Lys Ile 995 1000 1005 Asp Ala Gly Arg Ser Thr
Ala Leu Val Gly Gln Ser Gly Ser Gly 1010 1015 1020 Lys Ser Thr Ile
Ile Gly Leu Ile Glu Arg Phe Tyr Asp Pro Leu 1025 1030 1035 Lys Gly
Ile Val Thr Ile Asp Gly Arg Asp Ile Lys Ser Tyr His 1040 1045 1050
Leu Arg Ser Leu Arg Lys His Ile Ala Leu Val Ser Gln Glu Pro 1055
1060 1065 Thr Leu Phe Gly Gly Thr Ile Arg Glu Asn Ile Ala Tyr Gly
Ala 1070 1075 1080 Ser Asn Asn Asn Asn Lys Val Asp Glu Thr Glu Ile
Ile Glu Ala 1085 1090 1095 Ala Arg Ala Ala Asn Ala His Asp Phe Ile
Ala Ser Leu Lys Asp 1100 1105 1110 Gly Tyr Asp Thr Ser Cys Gly Asp
Arg Gly Val Gln Leu Ser Gly 1115 1120 1125 Gly Gln Lys Gln Arg Ile
Ala Ile Ala Arg Ala Ile Leu Lys Asn 1130 1135 1140 Pro Glu Val Leu
Leu Leu Asp Glu Ala Thr Ser Ala Leu Asp Ser 1145 1150 1155 Gln Ser
Glu Lys Leu Val Gln
Asp Ala Leu Glu Arg Val Met Val 1160 1165 1170 Gly Arg Thr Ser Val
Val Val Ala His Arg Leu Ser Thr Ile Gln 1175 1180 1185 Asn Cys Asp
Leu Ile Ala Val Leu Asp Lys Gly Lys Val Val Glu 1190 1195 1200 Lys
Gly Thr His Ser Ser Leu Leu Ala His Gly Pro Gly Gly Ala 1205 1210
1215 Tyr Tyr Ser Leu Ile Ser Leu Gln Arg Arg Pro Ala Asn 1220 1225
1230
* * * * *
References