U.S. patent application number 16/059570 was filed with the patent office on 2019-02-07 for soybean resistant to cyst nematodes.
This patent application is currently assigned to Board of Trustees of Southern Illinois University. The applicant listed for this patent is Board of Trustees of Southern Illinois University, The Curators of the University of Missouri. Invention is credited to Pramod Kaitheri Kandoth, Shiming Liu, Khalid Meksem, Melissa G. Mitchum.
Application Number | 20190037795 16/059570 |
Document ID | / |
Family ID | 49673904 |
Filed Date | 2019-02-07 |
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United States Patent
Application |
20190037795 |
Kind Code |
A1 |
Meksem; Khalid ; et
al. |
February 7, 2019 |
SOYBEAN RESISTANT TO CYST NEMATODES
Abstract
A transgenic soybean resistant to soybean cyst nematode (SCN),
or parts thereof, including an artificial DNA construct encoding a
serine hydroxymethyltransferase protein (e.g., GmSHMT). Also
provided are GmSHMT alleles containing mutations R130P and Y358N
along with research and breeding methods and compositions including
such
Inventors: |
Meksem; Khalid; (Carbondale,
IL) ; Liu; Shiming; (Carbondale, IL) ;
Kandoth; Pramod Kaitheri; (Columbia, MO) ; Mitchum;
Melissa G.; (Columbia, MO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Board of Trustees of Southern Illinois University
The Curators of the University of Missouri |
Carbondale
Columbia |
IL
MO |
US
US |
|
|
Assignee: |
Board of Trustees of Southern
Illinois University
Carbondale
IL
The Curators of the University of Missouri
Columbia
MO
|
Family ID: |
49673904 |
Appl. No.: |
16/059570 |
Filed: |
August 9, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14404559 |
Nov 28, 2014 |
10070614 |
|
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PCT/US2013/043392 |
May 30, 2013 |
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16059570 |
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61653227 |
May 30, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
Y02A 40/146 20180101;
C12N 15/8218 20130101; C12N 15/8285 20130101; Y02A 40/164 20180101;
A01H 1/04 20130101; A01H 5/10 20130101; C12N 15/8227 20130101; A01H
6/542 20180501 |
International
Class: |
A01H 6/54 20060101
A01H006/54; C12N 15/82 20060101 C12N015/82; A01H 1/04 20060101
A01H001/04; A01H 5/10 20060101 A01H005/10 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under grant
number 0820642 awarded by National Science Foundation Plant Genome
Research Program and DBI-0845196 awarded by National Science
Foundation. The government has certain rights in the invention.
Claims
1. An agronomically elite soybean plant with soybean cyst nematode
(SCN) resistance, comprising a GmSHMT allele comprising mutations
P130R and N358Y operably linked to a promoter functional in said
soybean plant.
2. The soybean plant of claim 1, wherein said GmSHMT allele
comprises an amino acid sequence having 95% identity to the amino
acid sequence of SEQ ID NO:2, wherein amino acid position 130 is an
arginine residue and amino acid position 358 is a tyrosine
residue.
3. The soybean plant of claim 2, wherein said GmSHMT allele
comprises the amino acid sequence of SEQ ID NO:2, wherein amino
acid position 130 is an arginine residue and amino acid position
358 is a tyrosine residue.
4. The soybean plant of claim 2, wherein said GmSHMT allele is
encoded by a polynucleotide having 95% identity to the nucleotide
sequence of SEQ ID NO:1, wherein the codon corresponding to amino
acid position 130 in SEQ ID NO:2 codes for an arginine residue and
the codon corresponding to amino acid position 358 in SEQ ID NO:2
codes for a tyrosine residue.
5. The soybean plant of claim 4, wherein said GmSHMT allele is
encoded by a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:1, wherein the codon corresponding to amino acid position
130 in SEQ ID NO:2 codes for an arginine residue and the codon
corresponding to amino acid position 358 in SEQ ID NO:2 codes for a
tyrosine residue.
6. The soybean plant of claim 1, wherein said promoter is a
heterologous promoter.
7. The soybean plant of claim 1, wherein said promoter is an
inducible promoter.
8. The soybean plant of claim 1, wherein said promoter is a
tissue-specific promoter.
9. The soybean plant of claim 1, wherein said promoter is a
nematode-inducible promoter.
10. A plant part of the soybean plant of claim 1.
11. The plant part of claim 10, wherein the plant part is a cell,
pollen, a leaf, a flower, a stem, a seed, or an ovule.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/404,559 filed Nov. 28, 2014, which is the
Section 371 National Stage of PCT/US2013/043392 filed May 30, 2013,
which claims the benefit of U.S. Provisional Application Ser. No.
61/653,227 filed 30 May 2012; which are incorporated herein by
reference in their entireties.
MATERIAL INCORPORATED-BY-REFERENCE
[0003] The Sequence Listing, which is a part of the present
disclosure, includes a computer readable form comprising nucleotide
and/or amino acid sequences of the present invention. The subject
matter of the Sequence Listing is incorporated herein by reference
in its entirety.
FIELD OF THE INVENTION
[0004] The present disclosure generally relates to methods of
conferring resistance to nematodes in soybeans.
BACKGROUND OF THE INVENTION
[0005] Soybean cyst nematode causes more than one billion dollars
in annual yield losses to US soybean producers and is a continuing
problem in soybean producing regions throughout the world. Virulent
populations of Heterodera glycines, the nematode responsible for
this yield loss, have been identified on most known resistance
sources. But the soybean genes which confer resistance to soybean
cyst nematode have not been previously cloned.
[0006] Cyst nematodes belonging to genera Heterodera and Globodera
include some of the most economically important classes of
plant-parasitic nematodes. Different species of these nematodes
feed selectively on certain host plants as obligate sedentary
endoparasites by inducing specialized feedings sites (syncytia)
within host roots. The soybean cyst nematode (SCN) Heterodera
glycines Ichinohe has a host range limited to plants in the family
Leguminosae and some weeds. It is consistently the most
economically important pathogen on soybean (Glycine max (L.) Merr.)
(Koenning and Wrather, 2010). Infective second-stage juveniles (J2)
hatch from eggs in the soil and penetrate plant roots utilizing a
hollow mouth spear (stylet) to mechanically perforate the plant
cell wall and to secrete cell wall digesting enzymes that
facilitate intracellular migration through the root cortex to a
pericycle or endodermal cell near the vasculature. Once a cell has
been selected for feeding, the nematode delivers a suite of
effector proteins into the cell leading to its transformation into
a unique, highly metabolically active feeding cell. The now
sedentary juvenile feeds from this cell as it progresses through a
25-30 day life cycle divided into four juvenile (J1-J4) and the
sexually dimorphic adult life stages. After fertilization by a
male, the adult female retains hundreds of eggs in her uterus and
following her death forms a protective cyst that allows the eggs to
survive for years in the soil in the absence of a host. The host
plant suffers from loss of nutrients and reduced transport through
the vasculature, manifested as less vigorous growth and reduced
yield.
[0007] Its amphimictic lifestyle and unique survival strategy
complicate current measures to control SCN. The use of nematicides
is restricted and not cost-effective for soybean producers. Current
methods to control SCN include a combination of nonhost crop
rotation and planting of resistant cultivars. Soybean breeders have
been successful in developing SCN resistant cultivars. The first
Rhg (for resistance to Heterodera glycines) genes were identified
in the early 1960's (Caldwell et al., 1960; Matson and Williams,
1965) and since then numerous papers on the identification and
localization of QTL (quantitative trait loci) underlying resistance
to SCN from a variety of different germplasm sources have been
published. QTL on chromosome 18 (rhg1) and chromosome 8 (Rhg4) have
been consistently mapped in a variety of germplasm sources
(Concibido et al., 2004). In some sources, such as P188788, rhg1 is
sufficient for full resistance and displays incomplete dominance
(Concibido et al., 2004). In other cases, such as the soybean
cultivar (cv.) Forrest, full resistance to SCN requires both rhg1
and Rhg4, with Rhg4 exhibiting dominant gene action (Meksem et al.,
2001).
[0008] Plants carrying Rhg genes display an incompatible
interaction between host and parasite. The roots of plants carrying
Rhg genes are penetrated by infective J2s, but feeding cells
ultimately degenerate and most of the nematodes die before reaching
adult stages (Endo, 1965; Riggs et al., 1973). Genetic variability
in H. glycines is prevalent and nematodes that survive on resistant
cultivars carry the undefined ror (reproduction on a resistant
host) alleles (Dong and Opperman, 1997) leading to population
shifts in the field as a consequence of soybean resistance
monoculture (Niblack et al., 2008). Understanding of resistance to
SCN is limited because the genes underlying identified SCN
resistance QTL have not been cloned (Melito et al., 2010; Liu et
al., 2010).
SUMMARY OF THE INVENTION
[0009] Among the various aspects of the present disclosure is the
provision of a soybean plant having resistance to soybean cyst
nematode (SCN).
[0010] One aspect of the present disclosure provides a transgenic
soybean resistant to soybean cyst nematode (SCN). The SCN resistant
soybean includes a serine hydroxymethyltransferase gene (GmSHMT)
conferring the resistant phenotype. In some embodiments, the GmSHMT
gene is exogenous to the soybean and thereby provides SCN
resistance. In some embodiments, the GmSHMT gene is endogenous to
the soybean and transformation increases expression of GmSHMT,
thereby increasing SCN resistance.
[0011] In some embodiments, the soybean plant is transformed with
an artificial DNA construct comprising, as operably associated
components in the 5' to 3' direction of transcription: a promoter
that functions in soybean; a polynucleotide comprising a sequence
selected from the group consisting of (a) SEQ ID NO: 1 or a
sequence 95% identical thereto having serine
hydroxymethyltransferase activity; (b) a nucleotide sequence
encoding a polypeptide of SEQ ID NO: 2 or a sequence 95% identical
thereto having serine hydroxymethyltransferase activity; (c) a
nucleotide sequence that hybridizes under stringent conditions to a
nucleic acid sequence of SEQ ID NO: 1, wherein the polynucleotide
encodes a polypeptide having serine hydroxymethyltransferase
activity, wherein said stringent conditions comprise incubation at
65.degree. C. in a solution comprising 6.times.SSC (0.9 M sodium
chloride and 0.09 M sodium citrate); and (d) a polynucleotide
complementary to the polynucleotide sequence of (a), (b), or (c);
and a transcriptional termination sequence; wherein the transgenic
soybean exhibits increased SCN resistance.
[0012] In some embodiments, the soybean plant is an agronomically
elite soybean variety with soybean cyst nematode (SCN) resistance,
comprising an GmSHMT allele containing mutations R130P and Y358N.
In some embodiments, the soybean plant is prepared by (a) crossing
first and second soybean plants, wherein the first and second
plants collectively comprise a GmSHMT allele containing mutations
R130P and Y358N resulting in an SCN resistant phenotype, and
wherein the first and second plants collectively comprise germplasm
capable of conferring agronomically elite characteristics to a
progeny plant of said plants; and (b) assaying progeny soybean
plants resulting from the crossing for agronomically elite
characteristics and for SCN resistance; and (c) selecting at least
a first progeny plant comprising said SCN resistant phenotype and
agronomically elite characteristics to obtain the desired
plant.
[0013] Another aspect of the present disclosure is a plant part of
any SCN resistant soybean plant described herein.
[0014] Another aspect of the present disclosure is an artificial
DNA construct including a promoter that functions in soybean; a
polynucleotide encoding a polypeptide having serine
hydroxymethyltransferase activity; and a transcriptional
termination sequence.
[0015] Another aspect of the present disclosure is a method of
increasing soybean cyst nematode (SCN) of a soybean including
transforming a plant an artificial DNA construct of the present
disclosure.
[0016] Other objects and features will be in part apparent and in
part pointed out hereinafter.
DESCRIPTION OF THE DRAWINGS
[0017] Those of skill in the art will understand that the drawings,
described below, are for illustrative purposes only. The drawings
are not intended to limit the scope of the present teachings in any
way.
[0018] FIGS. 1A-1D are a series of drawings and a sequence listing
illustrating the positional cloning of the Rhg4 gene. FIG. 1A shows
a high density genetic map of the Rhg4 locus. The black horizontal
line represents approximately 300 Kbp of the Rhg4 chromosomal
interval. The arrows under the black horizontal line designate the
position of each DNA marker and its name. Numbers above the black
horizontal line denote the position of each marker relative to the
LRR-RLK marker (an LRR-RLK gene at the Rhg4 locus described
previously in Liu et al. 2011), which was assigned the position
`0`. Solid arrows designate polymorphic alleles between Essex (E)
or Williams 82 (W) and Forrest (F), dashed arrows represent the DNA
markers with Forrest (F) alleles found in the double recombinants
E.times.F74 and F.times.W5093. FIG. 1B shows that a BAC clone
1001310 was identified by screening three soybean BAC libraries
(Hindlll, EcoRl and BamHI BAC libraries; Meksem et al., 2000) using
a GmSHMT probe. The BAC clone 1001310 encompasses the markers
LRR-RLK, SHMT and partial sequence of GmSHMT (the first two exons
and part of the 2.sup.nd intron). FIG. 1C shows a gene model for
the GmSHMT genomic DNA sequence. The gene is 2189 bp from start
codon to stop codon and contains three exons (boxes) and two
introns (solid black lines). The numbers above the solid black
lines indicate the nucleotide position relative to the first
nucleotide of the start codon. Comparison of the GmSHMT gene
sequences between Forrest and Essex identified three SNPs (G389C,
T1165A and G1473C) and two InDels (at position -1384C and -1385T).
FIG. 1D shows comparison of the predicted GmSHMT protein sequence
between Forrest and Essex (SEQ ID NO: 2) with the amino acids
differences (R130P and Y358N) highlighted.
[0019] FIGS. 2A-2C are a drawing, sequence listing, and bar graph
demonstrating the functional validation of GmSHMT by mutational
analysis. FIG. 2A shows an EMS-mutagenized population of soybean
cv. Forrest was used to screen for mutations in GmSHMT by TILLING.
The gene was divided into 3 TILLING intervals (1, 2 and 3) for
screening. Two missense mutants, F6266 (E61K) and F6756 (M125I),
were identified. FIG. 2B shows an amino acid alignment of GmSHMT
sequences including the resistant wild type allele from Forrest
(SEQ ID NOs: 99-101), the resistant allele from the donor parent
P1548402 (Peking) (SEQ ID NOs: 102-104), the F6266 (SEQ ID NO: 105)
and F6756 (SEQ ID NO: 106) Forrest mutant alleles, and the
susceptible alleles from cultivars Essex (SEQ ID NO: 2) and
Williams 82 (SEQ ID NOs: 107-109). The numbers above the alignment
correspond to the amino acid position within the Forrest GmSHMT
protein sequence. The arrows denote the position of two amino acids
that differ between SCN resistant and susceptible lines (R130P and
Y358N) and the position of the mutations in the two new Forrest
alleles (E61K and M125I) induced by EMS. FIG. 2C shows a SCN
phenotype of the GmSHMT TILLING mutants. Compared to the SCN
resistant cv. Forrest (wild type) and the SCN susceptible cv.
Essex, both the E61K and M125I mutations produced a shift from
resistant to moderately susceptible (FI.gtoreq.20% in homozygote
mutant lines) and from resistant (FI.ltoreq.10%) to moderately
resistant (FI between 10-20% in heterozygote mutant lines).
[0020] FIG. 3 is a table depicting haplotypes identified at GmSHMT
in 28 plant introductions (PIs). A total of 81 soybean lines (plant
introductions, landraces and elite cultivars) were scored for their
SCN phenotype and SNP-genotyped at the rhg1 and Rhg4 loci (for a
list see e.g., TABLE 1). The coding region of GmSHMT for 28 lines
shown here was sequenced. Lines were classified resistant (R) to
SCN if the FI.ltoreq.10% and susceptible (S) if the FI.gtoreq.10%.
Polymorphic sites were positioned relative to the first nucleotide
of the start codon in Forrest. Boxes indicate the G to C and T to A
transitions resulting in the amino acid substitutions R130P and
Y358N, respectively linked to SCN phenotype.
[0021] FIGs. A-4G are a series of graphs and images demonstrating
the functional validation of GmSHMT by VIGS, RNAi and
complementation. FIG. 4A shows SCN reproduction in soybean roots
silenced for GmSHMT using virus-induced gene silencing. Nematode
reproduction was measured on SCN resistant RIL E.times.F67
inoculated with either BPMV (Bean pod mottle virus) or BPMV
containing a fragment of the SHMT gene sequence (BPMV-SHMT) and SCN
susceptible RIL E.times.F63 inoculated with BPMV only. Diamonds
represent the number of cysts on a single root system. At least
twelve plants per treatment were used. Four independent experiments
were performed showing similar results. Representative data from
one experiment are presented. Different letters denote a
significant difference at P<0.0001. FIG. 4B shows qPCR analysis
of GmSHMT transcript levels in control and GmSHMT-silenced roots.
The value for GmSHMT-silenced roots represents the mean.+-.SE of
five samples, normalized relative to soybean ubiquitin and
calibrated to the expression in the BPMV control sample. FIG. 4C
shows SCN reproduction in soybean roots silenced for GmSHMT using
RNA interference. Nematode reproduction was measured on transgenic
E.times.F67 hairy root lines transformed with a GmSHMT RNAi
construct under control of a nematode-inducible promoter, pZF
(Kandoth et al., 2011; pZF-SHMTi). Transgenic E.times.F67 and
E.times.F63 hairy root lines transformed with the vector containing
a portion of the GUS gene (GUSi) were used as resistant and
susceptible controls, respectively. At least twelve independent
transgenic hairy root lines were generated per genotype treatment.
Diamonds represent the number of cysts on a single hairy root line.
Three independent experiments were performed showing similar
results. Data from one experiment are presented. Different letters
denote a significant difference at P<0.01. FIG. 4D, FIG. 4E, and
FIG. 4F show GmSHMT promoter-GUS analysis in SCN resistant
E.times.F67 (FIG. 4D and FIG. 4E) and SCN susceptible E.times.F63
(FIG. 4F) showing expression in syncytial feeding cells at 3 days
post-inoculation with SCN (FIG. 4G) SCN reproduction on SCN
susceptible E.times.F63 hairy root lines transformed with the a
full length GmSHMT gene fragment under control of the native
promoter. Transgenic E.times.F67 and E.times.F63 hairy root lines
transformed with the vector containing only promoter sequence were
used as resistant and susceptible controls, respectively. At least
fourteen independent transgenic hairy root lines were generated per
genotype treatment. Diamonds represent the number of cysts on a
single hairy root. Four independent experiments were performed
showing similar results. Data from one experiment are presented.
Different letters denote a significant difference at P<0.001.
N=nematode; Syn=syncytium. In graphs, the bars indicate the mean
values.
[0022] FIGS. 5A-5B are a series of views of the computer-modeled
structure of GmSHMT. FIG. 5A shows a homology model of the Essex
GmSHMT homodimer showing that the corresponding Forrest mutations,
P130R and N358Y are located on the surface of the dimer, whereas
the position of F6266 (E61K) mutation is buried in the dimeric
interaction interface and the F6756 (M1251) mutation is located in
the core of each monomeric subunit. FIG. 5B shows that three of the
four mutations overlap with GmSHMT ligand binding sites. The ligand
binding sites are mapped on each of the two monomers forming the
dimer. When two ligand binding sites overlap only the highlighted
surface for one of the sites is shown. Both Forrest mutations,
P130R and N358Y, overlap with the THF/MTHF/FTHF binding site and
are in close proximity to the PLS and PLG binding sites. In
addition, Forrest P130R is in a close proximity to one of the two
glycine binding sites. The F6266 (E61K) mutation overlaps with both
the THF/MTHF/FTHF and PLS binding sites and is in a close proximity
to PLG binding site.
[0023] FIG. 6 depicts the sequence analysis of the GmSUB gene
mapped to the Rhg4 locus. FIG. 6 shows a comparison of GmSUB cDNA
and predicted protein sequences between the SCN resistant soybean
cv. Forrest (F) (SEQ ID NOs: 110-113) and the SCN susceptible cvs.
Essex (E) (SEQ ID NOs: 114-117) and Williams 82 (W) (SEQ ID NOs:
118-121). The nucleotide differences and the corresponding amino
acid differences are boxed. An alignment of GmSUB promoter
sequences from soybean cvs. Forrest, Essex, and Williams identified
no nucleotide differences in 1,766 bp of sequence 5' of the start
codon.
[0024] FIG. 7A shows an alignment of GmSHMT promoter sequences from
soybean cvs. Forrest (SEQ ID NO: 122), Essex (SEQ ID NO: 4), and
Williams 82 (SEQ ID NO: 123). Nucleotide differences are
highlighted. FIG. 7B is an image of Essex pSHMT-GUS.
DETAILED DESCRIPTION OF THE INVENTION
[0025] The present disclosure is based, at least in part, on the
discovery that a serine hydroxymethyltransferase (GmSHMT) gene
mapped to the Rhg4 locus confers resistance to the soybean cyst
nematode, Heterodera glycines. While the Rhg4 locus was previously
identified as a major quantitative trait locus contributing to
resistance of this pathogen, the present disclosure is the first
report of map-based cloning of the gene at the Rhg4 locus
responsible for conferring a resistance phenotype.
[0026] The GmSHMT gene encodes a serine hydroxymethyltransferase,
an enzyme responsible for interconversion of serine and glycine and
essential for one-carbon folate metabolism. As reported herein,
alleles of Rhg4 conferring resistance or susceptibility differ by
two genetic polymorphisms predicted to compromise folate binding
affinity. Also as reported herein, two independent point mutations
identified by TILLING, gene knockdown by VIGS and RNAi, and
transgenic complementation of the susceptible line confirmed that
the GmSHMT gene confers resistance to soybean cyst nematode.
[0027] According to the approach described herein, a soybean cell
or plant can be transformed so as to provide for SCN resistance. In
some embodiments, a soybean host cell or plant can be transformed
with a nucleic acid molecule encoding a polypeptide having serine
hydroxymethyltransferase activity. A nucleic acid encoding a
polypeptide having serine hydroxymethyltransferase activity can
have a substantial effect on the resistance of a plant to SCN.
Definitions
[0028] The following definitions and methods are provided to better
define the present invention and to guide those of ordinary skill
in the art in the practice of the present invention. Unless
otherwise noted, terms are to be understood according to
conventional usage by those of ordinary skill in the relevant
art.
[0029] The terms "heterologous DNA sequence", "exogenous DNA
segment" or "heterologous nucleic acid," as used herein, each refer
to a sequence that originates from a source foreign to the
particular host cell or, if from the same source, is modified from
its original form. Thus, a heterologous gene in a host cell
includes a gene that is endogenous to the particular host cell but
has been modified through, for example, the use of DNA shuffling.
The terms also include non-naturally occurring multiple copies of a
naturally occurring DNA sequence. Thus, the terms refer to a DNA
segment that is foreign or heterologous to the cell, or homologous
to the cell but in a position within the host cell nucleic acid in
which the element is not ordinarily found. Exogenous DNA segments
are expressed to yield exogenous polypeptides. A "homologous" DNA
sequence is a DNA sequence that is naturally associated with a host
cell into which it is introduced.
[0030] Expression vector, expression construct, plasmid, or
recombinant DNA construct is generally understood to refer to a
nucleic acid that has been generated via human intervention,
including by recombinant means or direct chemical synthesis, with a
series of specified nucleic acid elements that permit transcription
or translation of a particular nucleic acid in, for example, a host
cell. The expression vector can be part of a plasmid, virus, or
nucleic acid fragment. Typically, the expression vector can include
a nucleic acid to be transcribed operably linked to a promoter.
[0031] A "promoter" is generally understood as a nucleic acid
control sequence that directs transcription of a nucleic acid. An
inducible promoter is generally understood as a promoter that
mediates transcription of an operably linked gene in response to a
particular stimulus. A promoter can include necessary nucleic acid
sequences near the start site of transcription, such as, in the
case of a polymerase II type promoter, a TATA element. A promoter
can optionally include distal enhancer or repressor elements, which
can be located as much as several thousand base pairs from the
start site of transcription.
[0032] A "transcribable nucleic acid molecule" as used herein
refers to any nucleic acid molecule capable of being transcribed
into a RNA molecule. Methods are known for introducing constructs
into a cell in such a manner that the transcribable nucleic acid
molecule is transcribed into a functional mRNA molecule that is
translated and therefore expressed as a protein product. Constructs
may also be constructed to be capable of expressing antisense RNA
molecules, in order to inhibit translation of a specific RNA
molecule of interest. For the practice of the present disclosure,
conventional compositions and methods for preparing and using
constructs and host cells are well known to one skilled in the art
(see e.g., Sambrook and Russel (2006) Condensed Protocols from
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short
Protocols in Molecular Biology, 5th ed., Current Protocols,
ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning:
A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press,
ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in
Enzymology 167, 747-754).
[0033] The "transcription start site" or "initiation site" is the
position surrounding the first nucleotide that is part of the
transcribed sequence, which is also defined as position+1. With
respect to this site all other sequences of the gene and its
controlling regions can be numbered. Downstream sequences (i.e.,
further protein encoding sequences in the 3' direction) can be
denominated positive, while upstream sequences (mostly of the
controlling regions in the 5' direction) are denominated
negative.
[0034] "Operably-linked" or "functionally linked" refers preferably
to the association of nucleic acid sequences on a single nucleic
acid fragment so that the function of one is affected by the other.
For example, a regulatory DNA sequence is said to be "operably
linked to" or "associated with" a DNA sequence that codes for an
RNA or a polypeptide if the two sequences are situated such that
the regulatory DNA sequence affects expression of the coding DNA
sequence (i.e., that the coding sequence or functional RNA is under
the transcriptional control of the promoter). Coding sequences can
be operably-linked to regulatory sequences in sense or antisense
orientation. The two nucleic acid molecules may be part of a single
contiguous nucleic acid molecule and may be adjacent. For example,
a promoter is operably linked to a gene of interest if the promoter
regulates or mediates transcription of the gene of interest in a
cell.
[0035] A "construct" is generally understood as any recombinant
nucleic acid molecule such as a plasmid, cosmid, virus,
autonomously replicating nucleic acid molecule, phage, or linear or
circular single-stranded or double-stranded DNA or RNA nucleic acid
molecule, derived from any source, capable of genomic integration
or autonomous replication, comprising a nucleic acid molecule where
one or more nucleic acid molecule has been operably linked.
[0036] A constructs of the present disclosure can contain a
promoter operably linked to a transcribable nucleic acid molecule
operably linked to a 3' transcription termination nucleic acid
molecule. In addition, constructs can include but are not limited
to additional regulatory nucleic acid molecules from, e.g., the
3'-untranslated region (3' UTR). Constructs can include but are not
limited to the 5' untranslated regions (5' UTR) of an mRNA nucleic
acid molecule which can play an important role in translation
initiation and can also be a genetic component in an expression
construct. These additional upstream and downstream regulatory
nucleic acid molecules may be derived from a source that is native
or heterologous with respect to the other elements present on the
promoter construct.
[0037] The term "transformation" refers to the transfer of a
nucleic acid fragment into the genome of a host cell, resulting in
genetically stable inheritance. Host cells containing the
transformed nucleic acid fragments are referred to as "transgenic"
cells, and organisms comprising transgenic cells are referred to as
"transgenic organisms".
[0038] "Transformed," "transgenic," and "recombinant" refer to a
host cell or organism such as a plant into which a heterologous
nucleic acid molecule has been introduced. The nucleic acid
molecule can be stably integrated into the genome as generally
known in the art and disclosed (Sambrook 1989; Innis 1995; Gelfand
1995; Innis & Gelfand 1999). Known methods of PCR include, but
are not limited to, methods using paired primers, nested primers,
single specific primers, degenerate primers, gene-specific primers,
vector-specific primers, partially mismatched primers, and the
like. The term "untransformed" refers to normal cells that have not
been through the transformation process.
[0039] "Wild-type" refers to a virus or organism found in nature
without any known mutation.
[0040] Transformed Organism
[0041] Provided herein is a soybean plant genetically engineered to
be resistant to soybean cyst nematode (e.g., Heterodera glycines).
The host genetically engineered to resist SCN can be any soybean
plant or cell.
[0042] Assays to assess SCN resistance are well known in the art
(see Examples). Except as otherwise noted herein, therefore, SCN
resistance of a plant can be carried out in accordance with such
assays.
[0043] One aspect of the current invention is therefore directed to
the aforementioned plants and parts thereof and methods for using
these plants and plant parts. The term "plant" can include plant
cells, plant protoplasts, plant cells of tissue culture from which
a plant can be regenerated, plant calli, plant clumps and plant
cells that are intact in plants or parts of plants such as pollen,
flowers, seeds, leaves, stems, and the like. Each of these terms
can apply to a soybean "plant". Plant parts (e.g., soybean parts)
include, but are not limited to, pollen, an ovule and a cell. The
invention further provides tissue cultures of regenerable cells of
these plants, which cultures regenerate soybean plants capable of
expressing all the physiological and morphological characteristics
of the starting variety. Such regenerable cells may include
embryos, meristematic cells, pollen, leaves, roots, root tips or
flowers, or protoplasts or callus derived therefrom. Also provided
by the invention are soybean plants regenerated from such a tissue
culture, wherein the plants are capable of expressing all the
physiological and morphological characteristics of the starting
plant variety from which the regenerable cells were obtained.
[0044] Such SCN resistant plants can have a commercially
significant yield, for example, a yield of at least 90% to at least
110% (e.g., at least 95%, 100%, 105%) of a soybean check line.
Plants are provided comprising the GmSHMT alleles and SCN
resistance and a grain yield of at least about 90%, at least about
94%, at least about 98%, at least about 100%, at least about 105%
or at least about 110% of these lines.
[0045] As reported herein, transformation of a SCN susceptible
soybean (RIL E.times.F63) with a GmSHMT gene construct (2.3-kb of
sequence upstream of the start through 0.57-kb downstream of the
stop codon) so as to express GmSHMT within syncytia provided SCN
resistance in hairy roots according to the nematode infection
assays with H. glycines.
[0046] Further experiments showed that silencing of the GmSHMT gene
in the SCN-resistant RIL E.times.F67 resulted in a 74% reduced
GmSHMT expression in the roots of plants and a 29% increase in
susceptibility to SCN. Addition of a complementary targeted RNAi
gene silencing approach increased nematode reproduction on hairy
roots of the SCN resistant RIL E.times.F67.
[0047] In various embodiments, a gene encoding a polypeptide having
serine hydroxymethyltransferase activity is engineered in a host
plant (e.g., a soybean plant) so as to result in an SCN resistant
phenotype. In some embodiments, the gene encoding a polypeptide
having serine hydroxymethyltransferase activity is expressed in the
host plant. In some embodiments, the gene encoding a polypeptide
having serine hydroxymethyltransferase activity is overexpressed in
the host plant.
[0048] A gene encoding a polypeptide having serine
hydroxymethyltransferase activity can be endogenous or exogenous to
the host plant. Transformation of a plant to express polypeptide
having serine hydroxymethyltransferase activity can convey SCN
resistance to a host lacking such phenotype. Transformation of a
plant to express polypeptide having serine hydroxymethyltransferase
activity can increase SCN resistance to a host already possessing
such phenotype.
[0049] A transformed plant or plant cell can be analyzed for the
presence of a gene of interest and the expression level or profile
conferred by the construct of the present disclosure. Those of
skill in the art are aware of the numerous methods available for
the analysis of transformed hosts. For example, methods for host
analysis include, but are not limited to Southern blots or northern
blots, PCR-based approaches, biochemical analyses, phenotypic
screening methods, and immunodiagnostic assays.
[0050] In some embodiments, a host plant transformed to express a
polypeptide having serine hydroxymethyltransferase activity can
exhibit at least about 10% decrease in susceptibility to SCN. For
example, a host plant transformed to express a polypeptide having
serine hydroxymethyltransferase activity can exhibit at least about
20%, at least about 30%, at least about 40%, at least about 50%, at
least about 60%, at least about 70%, at least about 80%, at least
about 90%, or at least about 100% decrease in susceptibility to SCN
as compared to a non-transformed control. As another example, a
host plant transformed to express a polypeptide having serine
hydroxymethyltransferase activity can exhibit at least about 100%,
at least about 200%, at least about 300%, at least about 400%, at
least about 500%, at least about 600%, at least about 700%, at
least about 800%, at least about 900%, or at least about 1000%
decrease in susceptibility to SCN as compared to a non-transformed
control.
[0051] A gene of particular interest for engineering a soybean
plant to exhibit SCN resistance is GmSHMT (SEQ ID NO: 1). As
described herein, GmSHMT has been mapped to the Rhg4 locus and
confers resistance to the soybean cyst nematode, Heterodera
glycines.
[0052] In some embodiments, a transformed host soybean plant
comprises an GmSHMT polynucleotide of SEQ ID NO: 1, or a functional
fragment thereof. In some embodiments, a transformed host soybean
plant comprises an GmSHMT polynucleotide of SEQ ID NO: 3, or a
functional fragment thereof. In some embodiments, a soybean plant
is transformed with a nucleotide sequence encoding GmSHMT
polypeptide of SEQ ID NO: 2, or a functional fragment thereof.
[0053] In further embodiments, a transformed host soybean plant
comprises a nucleotide sequence having at least about 80% sequence
identity to a GmSHMT polynucleotide of SEQ ID NO: 1 or SEQ ID NO:
3, or a functional fragment thereof, or a nucleotide sequence
encoding a polypeptide having serine hydroxymethyltransferase
activity and at least about 80% sequence identity to the GmSHMT
polypeptide SEQ ID NO: 2, or a functional fragment thereof. As an
example, a transformed host soybean plant can comprise a nucleotide
sequence having at least about 85%, at least about 90%, at least
about 95%, or at least about 99% sequence identity to a GmSHMT
polynucleotide of SEQ ID NO: 1 or SEQ ID NO: 3, or a functional
fragment thereof, wherein the transformed soybean exhibits SCN
resistance or serine hydroxymethyltransferase activity. As an
example, a transformed soybean can comprise a nucleotide sequence
encoding a polypeptide having at least about 85%, at least about
90%, at least about 95%, or at least about 99% sequence identity to
a GmSHMT polypeptide of SEQ ID NO: 2, or a functional fragment
thereof, wherein the transformed soybean exhibits SCN resistance or
serine hydroxymethyltransferase activity.
[0054] As another example, a transformed soybean can comprise a
nucleotide sequence that hybridizes under stringent conditions to a
GmSHMT polynucleotide of SEQ ID NO: 1 or SEQ ID NO: 3 over the
entire length of SEQ ID NO: 1 or SEQ ID NO: 3, respectively, or a
functional fragment thereof, and which encodes a polypeptide having
serine hydroxymethyltransferase activity.
[0055] As a further example, a transformed soybean can comprise the
complement to any of the above sequences.
[0056] Variant Sequences
[0057] As describe above, a plant can be transformed with a variant
of the GmSHMT polynucleotide SEQ ID NO: 1 or SEQ ID NO: 3 or with a
polynucleotide encoding a variant of the GmSHMT polypeptide SEQ ID
NO: 2. The species of SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 2
are representative of the genus of variant nucleic acid and
polypeptides, respectively, because all variants must possess the
specified catalytic activity (e.g., serine hydroxymethyltransferase
activity) and must have the percent identity required above to the
reference sequence.
[0058] Furthermore, the present disclosure provides guidance as to
regions of the sequences important to activity.
[0059] As reported herein, genomic DNA sequences of GmSHMT from
Forrest and Essex showed 5 nucleotides differences (3 SNPs and 2
Ins/Dels) between the resistant and susceptible alleles. Two of the
nucleotide differences found between the Forrest and Essex GmSHMT
cDNAs resulted in an amino acid change in the predicted protein
sequences (R130P and Y358N). The amino acid sequence at these two
positions in Williams 82 was consistent with that of Essex.
[0060] Also reported herein are two mutations in the GmSHMT gene on
chromosome 8 that lead to missense mutations at E61K and M125I.
SIFT predictions were performed on both mutations. Based on SIFT
predictions (i.e., whether an amino acid substitution affects
protein function based on sequence homology and the physical
properties of amino acids), the M125I mutation was predicted to be
deleterious to the protein and both mutants were more susceptible
to SCN in nematode infection assays. Additionally, the F6756
(M1251) mutation was correlated with the SCN resistance phenotype
of individual plants.
[0061] Based on SCN female index score and SNP-based GmSHMT
haplotype, 13 polymorphisms were identified in the coding regions
and 6 in the non coding DNA sequences. Two coding polymorphisms,
R130P and Y358N substitutions, were shown to produce amino acid
changes responsible for the "Peking type resistance" SCN phenotype.
Eight different GmSHMT haplotypes were identified.
[0062] Homology modeling of the structure of GmSHMT provides
guidance as to how variant genotypes affect structural and
functional properties. Mapping of ligand binding sites of SHMT
homologs onto the surface of a GmSHMT model identified five
putative binding sites, including two glycine (GS.sub.A and
GS.sub.2), one PLP-serine (PLS), one PLP-glycine (PLG), and one
THF/MTHF/5-formylTHF (FTHF) binding site, with the latter three
binding sites physically co-localized in the binding pocket formed
by the SHMT dimer molecule. Both Forrest mutations and TILLING
mutation F6266 E61K were shown to be in close proximity to the
tentative ligand binding sites.
[0063] Forrest mutations P130R and N358Y were co-localized with the
THF/MTHF/FTHF binding site and in close proximity to PLS, PLG and
one of the two glycine binding sites. The position of the F6266
E61K mutation overlapped with the PLS and THF/MTHF/FTHF binding
sites. Thus, the three mutations may directly affect the reversible
interconversion of L-serine and THF to glycine and MTHF. TILLING
mutation F6756 M125I was shown to be in an interior beta sheet,
suggesting structural instability of the GmSHMT region affected by
the TILLING mutation.
[0064] Variant polynucleic acid molecules and corresponding encoded
polypeptides discussed herein can contain one or more of the above
described mutations.
[0065] Thus is provided guidance as to regions of the sequences
important to activity.
[0066] Promoters
[0067] One or more of the nucleotide sequences discussed above
(e.g., GmSHMT or a variant thereof) can be operably linked to a
promoter that can function in a plant, such as soybean. Promoter
selection can allow expression of a desired gene product under a
variety of conditions.
[0068] Promoters can be selected for optimal function in a soybean
host cell into which the vector construct will be inserted.
Promoters can also be selected on the basis of their regulatory
features. Examples of such features include enhancement of
transcriptional activity and inducibility.
[0069] Numerous promoters functional in a soybean plant will be
known to one of skill in the art (see e.g., Weise et al. Applied
Microbiology and Biotechnology 70(3), 337-345; Saidi et al. 2005
Plant Molecular Biology 59(5), 697-711; Horstmann et al. 2004 BMC
Biotechnology 4; Holtorf et al. 2002 Plant Cell Reports 21(4),
341-346; Zeidler et al. 1996 Plant Molecular Biology 30(1),
199-205). Except as otherwise noted herein, therefore, the
processes and compositions of the present disclosure can be carried
out in accordance with such known promoters. Examples of promoters
than can be used in accord with methods and compositions described
herein include, but are not limited to, factor EF1.alpha. gene
promoter (US App Pub No. 2008/0313776); rice tungro bacilliform
virus (RTBV) gene promoter (US App Pub No. 2008/0282431); cestrum
yellow leaf curling virus (CmYLCV) promoter (Stavolone et al. Plant
Molecular Biology 53(5), 663-673); tCUP cryptic promoter system
(Malik et al. 2002 Theoretical and Applied Genetics 105(4),
505-514); T6P-3 promoter (JP2002238564); S-adenosyl-L-methionine
synthetase promoter (WO/2000/037662); Raspberry E4 gene promoter
(U.S. Pat. No. 6,054,635); cauliflower mosaic virus 35S promoter
(Benfey et al. 1990 Science 250(4983), 959-966); figwort mosaic
virus promoter (U.S. Pat. No. 5,378,619); conditional heat-shock
promoter (Saidi et al. 2005 Plant Molecular Biology 59(5),
697-711); promoter subfragments of the sugar beet V-type H+-ATPase
subunit c isoform (Holtorf et al. 2002 Plant Cell Reports 21(4),
341-346); beta-tubulin promoter (Jost et al. 2005 Current Genetics
47(2), 111-120); and bacterial quorum-sensing components (You et
al. 2006 Plant Physiology 140 (4), 1205-1212).
[0070] The promoter can be an inducible promoter. For example, the
promoter can be induced according to temperature, pH, a hormone, a
metabolite (e.g., lactose, mannitol, an amino acid), light (e.g.,
wavelength specific), osmotic potential (e.g., salt induced), a
heavy metal, or an antibiotic. In some embodiments, the promoter
comprises a nematode-inducible promoter (e.g., Glyma15g04570.1, see
Example 8). Numerous standard inducible promoters will be known to
one of skill in the art.
[0071] The promoter can be a tissue-specific promoter. For example,
a transcribable nucleic acid molecule described herein can be
operably linked to a pollen-, flower-, seed-, leaf-, or
stem-specific promoter. As another example, a transcribable nucleic
acid molecule described herein can be operably linked to a
seed-specific promoter. For example, the promoter can be a
root-specific promoter. Numerous standard tissue-specific promoters
will be known to one of skill in the art.
[0072] The term "chimeric" is understood to refer to the product of
the fusion of portions of two or more different polynucleotide
molecules. "Chimeric promoter" is understood to refer to a promoter
produced through the manipulation of known promoters or other
polynucleotide molecules. Such chimeric promoters can combine
enhancer domains that can confer or modulate gene expression from
one or more promoters or regulatory elements, for example, by
fusing a heterologous enhancer domain from a first promoter to a
second promoter with its own partial or complete regulatory
elements. Thus, the design, construction, and use of chimeric
promoters according to the methods disclosed herein for modulating
the expression of operably linked polynucleotide sequences are
encompassed by the present invention.
[0073] Novel chimeric promoters can be designed or engineered by a
number of methods. For example, a chimeric promoter may be produced
by fusing an enhancer domain from a first promoter to a second
promoter. The resultant chimeric promoter may have novel expression
properties relative to the first or second promoters. Novel
chimeric promoters can be constructed such that the enhancer domain
from a first promoter is fused at the 5' end, at the 3' end, or at
any position internal to the second promoter.
[0074] The promoter can be any promoter endogenously associated
with SHMT, or a variant thereof. In some embodiments, the promoter
can comprises SEQ ID NO: 4, which is the native Essex Gm08-A2 SHMT
promoter. In other embodiments, the promoter can comprise a variant
of SEQ ID NO: 4 having at least about 80% identity thereto (e.g.,
at least about 85%, at least about 90%, at least about 95%, at
least about 99%) and retaining promoter function.
[0075] Inclusion of a termination region control sequence is
optional, and if employed, then the choice is be primarily one of
convenience, as the termination region is relatively
interchangeable. The termination region may be native to the
transcriptional initiation region (the promoter), may be native to
the nucleic acid sequence of interest, or may be obtainable from
another source.
[0076] A promoter of the present disclosure can be incorporated
into a construct using marker genes as described and tested for an
indication of gene expression in a stable host system. As used
herein the term "marker gene" refers to any transcribable nucleic
acid molecule whose expression can be screened for or scored in
some way.
[0077] Constructs
[0078] Any of the transcribable polynucleotide molecule sequences
described above can be provided in a construct. Constructs of the
present invention generally include a promoter functional in the
host plant, such as soybean, operably linked to a transcribable
polynucleotide molecule encoding a polypeptide with serine
hydroxymethyltransferase activity (e.g., GmSHMT), such as provided
in SEQ ID NO: 1, and variants thereof as discussed above.
[0079] Exemplary promoters are discussed above. One or more
additional promoters may also be provided in the recombinant
construct. These promoters can be operably linked to any of the
transcribable polynucleotide molecule sequences described
above.
[0080] The term "construct" is understood to refer to any
recombinant polynucleotide molecule such as a plasmid, cosmid,
virus, autonomously replicating polynucleotide molecule, phage, or
linear or circular single-stranded or double-stranded DNA or RNA
polynucleotide molecule, derived from any source, capable of
genomic integration or autonomous replication, comprising a
polynucleotide molecule where one or more polynucleotide molecule
has been linked in a functionally operative manner, i.e. operably
linked. The term "vector" or "vector construct" is understood to
refer to any recombinant polynucleotide construct that may be used
for the purpose of transformation, i.e., the introduction of
heterologous DNA into a host plant, such as a soybean.
[0081] In addition, constructs may include, but are not limited to,
additional polynucleotide molecules from an untranslated region of
the gene of interest. These additional polynucleotide molecules can
be derived from a source that is native or heterologous with
respect to the other elements present in the construct.
[0082] Host cells developed according to the approaches described
herein can be evaluated by a number of means known in the art (see
e.g., Studier (2005) Protein Expr Purif. 41(1), 207-234; Gellissen,
ed. (2005) Production of Recombinant Proteins: Novel Microbial and
Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363;
Baneyx (2004) Protein Expression Technologies, Taylor &
Francis, ISBN-10: 0954523253).
[0083] Molecular Engineering
[0084] Design, generation, and testing of the variant nucleotides,
and their encoded polypeptides, having the above required percent
identities and retaining a required activity of the expressed
protein is within the skill of the art. For example, directed
evolution and rapid isolation of mutants can be according to
methods described in references including, but not limited to, Link
et al. (2007) Nature Reviews 5(9), 680-688; Sanger et al. (1991)
Gene 97(1), 119-123; Ghadessy et al. (2001) Proc Natl Acad Sci USA
98(8) 4552-4557. Thus, one skilled in the art could generate a
large number of nucleotide and/or polypeptide variants having, for
example, at least 95-99% identity to the gmSHMT sequence described
herein and screen such for desired phenotypes according to methods
routine in the art.
[0085] Nucleotide and/or amino acid sequence identity percent (%)
is understood as the percentage of nucleotide or amino acid
residues that are identical with nucleotide or amino acid residues
in a candidate sequence in comparison to a reference sequence when
the two sequences are aligned. To determine percent identity,
sequences are aligned and if necessary, gaps are introduced to
achieve the maximum percent sequence identity. Sequence alignment
procedures to determine percent identity are well known to those of
skill in the art. Often publicly available computer software such
as BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR) software is used to
align sequences. Those skilled in the art can determine appropriate
parameters for measuring alignment, including any algorithms needed
to achieve maximal alignment over the full-length of the sequences
being compared. When sequences are aligned, the percent sequence
identity of a given sequence A to, with, or against a given
sequence B (which can alternatively be phrased as a given sequence
A that has or comprises a certain percent sequence identity to,
with, or against a given sequence B) can be calculated as: percent
sequence identity=X/Y100, where X is the number of residues scored
as identical matches by the sequence alignment program's or
algorithm's alignment of A and B and Y is the total number of
residues in B. If the length of sequence A is not equal to the
length of sequence B, the percent sequence identity of A to B will
not equal the percent sequence identity of B to A.
[0086] Generally, conservative substitutions can be made at any
position so long as the required activity is retained. So-called
conservative exchanges can be carried out in which the amino acid
which is replaced has a similar property as the original amino
acid, for example the exchange of Glu by Asp, Gln by Asn, Val by
Ile, Leu by Ile, and Ser by Thr. Deletion is the replacement of an
amino acid by a direct bond. Positions for deletions include the
termini of a polypeptide and linkages between individual protein
domains. Insertions are introductions of amino acids into the
polypeptide chain, a direct bond formally being replaced by one or
more amino acids. Amino acid sequence can be modulated with the
help of art-known computer simulation programs that can produce a
polypeptide with, for example, improved activity or altered
regulation. On the basis of this artificially generated polypeptide
sequences, a corresponding nucleic acid molecule coding for such a
modulated polypeptide can be synthesized in-vitro using the
specific codon-usage of the desired host cell.
[0087] "Highly stringent hybridization conditions" are defined as
hybridization at 65.degree. C. in a 6.times.SSC buffer (i.e., 0.9 M
sodium chloride and 0.09 M sodium citrate). Given these conditions,
a determination can be made as to whether a given set of sequences
will hybridize by calculating the melting temperature (T.sub.m) of
a DNA duplex between the two sequences. If a particular duplex has
a melting temperature lower than 65.degree. C. in the salt
conditions of a 6.times.SSC, then the two sequences will not
hybridize. On the other hand, if the melting temperature is above
65.degree. C. in the same salt conditions, then the sequences will
hybridize. In general, the melting temperature for any hybridized
DNA:DNA sequence can be determined using the following formula:
T.sub.m=81.5.degree. C.+16.6(log.sub.10[Na.sup.+])+0.41 (fraction
G/C content)-0.63 (% formamide)-(600/l). Furthermore, the T.sub.m
of a DNA:DNA hybrid is decreased by 1-1.5.degree. C. for every 1%
decrease in nucleotide identity (see e.g., Sambrook and Russel,
2006).
[0088] Host cells can be transformed using a variety of standard
techniques known to the art (see, e.g., Sambrook and Russel (2006)
Condensed Protocols from Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel
et al. (2002) Short Protocols in Molecular Biology, 5th ed.,
Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001)
Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor
Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P.
1988. Methods in Enzymology 167, 747-754). Such techniques include,
but are not limited to, viral infection, calcium phosphate
transfection, liposome-mediated transfection,
microprojectile-mediated delivery, receptor-mediated uptake, cell
fusion, electroporation, and the like. The transfected cells can be
selected and propagated to provide recombinant host cells that
comprise the expression vector stably integrated in the host cell
genome.
[0089] Exemplary nucleic acids which may be introduced to a host
cell include, for example, DNA sequences or genes from another
species, or even genes or sequences which originate with or are
present in the same species, but are incorporated into recipient
cells by genetic engineering methods. The term "exogenous" is also
intended to refer to genes that are not normally present in the
cell being transformed, or perhaps simply not present in the form,
structure, etc., as found in the transforming DNA segment or gene,
or genes which are normally present and that one desires to express
in a manner that differs from the natural expression pattern, e.g.,
to over-express. Thus, the term "exogenous" gene or DNA is intended
to refer to any gene or DNA segment that is introduced into a
recipient cell, regardless of whether a similar gene may already be
present in such a cell. The type of DNA included in the exogenous
DNA can include DNA which is already present in the plant cell, DNA
from another plant, DNA from a different organism, or a DNA
generated externally, such as a DNA sequence containing an
antisense message of a gene, or a DNA sequence encoding a synthetic
or modified version of a gene.
[0090] Host strains developed according to the approaches described
herein can be evaluated by a number of means known in the art (see
e.g., Studier (2005) Protein Expr Purif. 41(1), 207-234; Gellissen,
ed. (2005) Production of Recombinant Proteins: Novel Microbial and
Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363;
Baneyx (2004) Protein Expression Technologies, Taylor &
Francis, ISBN-10: 0954523253).
[0091] Methods of down-regulation or silencing genes are known in
the art. For example, expressed protein activity can be
down-regulated or eliminated using antisense oligonucleotides,
protein aptamers, nucleotide aptamers, and RNA interference (RNAi)
(e.g., small interfering RNAs (siRNA), short hairpin RNA (shRNA),
and micro RNAs (miRNA) (see e.g., Fanning and Symonds (2006) Handb
Exp Pharmacol. 173, 289-303G, describing hammerhead ribozymes and
small hairpin RNA; Helene, C., et al. (1992) Ann. N.Y. Acad. Sci.
660, 27-36; Maher (1992) Bioassays 14(12): 807-15, describing
targeting deoxyribonucleotide sequences; Lee et al. (2006) Curr
Opin Chem Biol. 10, 1-8, describing aptamers; Reynolds et al.
(2004) Nature Biotechnology 22(3), 326-330, describing RNAi;
Pushparaj and Melendez (2006) Clinical and Experimental
Pharmacology and Physiology 33(5-6), 504-510, describing RNAi;
Dillon et al. (2005) Annual Review of Physiology 67, 147-173,
describing RNAi; Dykxhoorn and Lieberman (2005) Annual Review of
Medicine 56, 401-423, describing RNAi). RNAi molecules are
commercially available from a variety of sources (e.g., Ambion, TX;
Sigma Aldrich, MO; Invitrogen). Several siRNA molecule design
programs using a variety of algorithms are known to the art (see
e.g., Cenix algorithm, Ambion; BLOCK-IT.TM. RNAi Designer,
Invitrogen; siRNA Whitehead Institute Design Tools, Bioinoformatics
& Research Computing). Traits influential in defining optimal
siRNA sequences include G/C content at the termini of the siRNAs,
Tm of specific internal domains of the siRNA, siRNA length,
position of the target sequence within the CDS (coding region), and
nucleotide content of the 3' overhangs.
[0092] Breeding
[0093] The present disclosure provides genetic markers and methods
for the introduction of GmSHMT alleles into agronomically elite
soybean plants. The invention therefore allows the creation of
plants that combine these GmSHMT alleles that confer SCN resistance
with a commercially significant yield and an agronomically elite
genetic background. Using the methods of the invention, loci
conferring the SCN phenotype may be introduced into a desired
soybean genetic background, for example, in the production of new
varieties with commercially significant yield and SCN
resistance.
[0094] Marker assisted introgression involves the transfer of a
chromosome region defined by one or more markers from one germplasm
to a second germplasm. The initial step in that process is the
localization of the trait by gene mapping, which is the process of
determining the position of a gene relative to other genes and
genetic markers through linkage analysis. The basic principle for
linkage mapping is that the closer together two genes are on the
chromosome, the more likely they are to be inherited together.
Briefly, a cross is generally made between two genetically
compatible but divergent parents relative to traits under study.
Genetic markers can then be used to follow the segregation of
traits under study in the progeny from the cross, often a backcross
(BC1), F.sub.2, or recombinant inbred population.
[0095] The term quantitative trait loci, or QTL, is used to
describe regions of a genome showing quantitative or additive
effects upon a phenotype. The Rhg4 loci, containing GmSHMT alleles,
represent exemplary QTL because GmSHMT alleles result in SCN
resistance. Herein identified are genetic markers for
non-transgenic, GmSHMT alleles that enable breeding of soybean
plants comprising the GmSHMT alleles with agronomically superior
plants, and selection of progeny that inherited the mutant GmSHMT
alleles. Thus, the invention allows the use of molecular tools to
combine these QTLs with desired agronomic characteristics.
[0096] Processes for marker assisted breeding are well known in the
art. Except as otherwise noted herein, therefore, the process of
the present disclosure can be carried out in accordance with such
processes.
[0097] Research Tool
[0098] The SHMT gene can be used to find or characterize related
(interactive) genes or identify or further characterize the cascade
for SCN resistance. The discovery of a SHMT as part of the
resistance signaling pathway against SCN provides novel insight
into this complex host-pathogen interaction. Insights reported
herein can be used to discern the relationship between SHMT and
metabolism.
[0099] In some embodiments, the SHMT gene can be used in a
genomics, proteomics, bioinformatics, or statistical modeling
approach to fish or isolate candidate genes or encoded proteins or
other molecules with a direct or indirect function in mediating
disease resistance to SCN in soybeans. In some embodiments, the
SHMT gene can be used in a genomics, proteomics, bioinformatics, or
statistical modeling approach to fish or isolate candidate genes or
encoded proteins or other molecules with a direct or indirect
function in mediating compatible or incompatible responses of
soybeans to SCN (e.g., to a nematode or any intermediate). Thus is
provided various methods to find or characterize related
(interactive) genes involved with SCN resistance.
[0100] With the exception of Hs1.sup.pro-1, a gene for H. schachtii
resistance in sugar beet that encodes a 282-aa transmembrane
protein with imperfect leucine-rich repeats (Cai et al., 1997), all
other genes for nematode resistance identified to date belong to
the canonical NB-LRR (nucleotide binding site, leucine-rich repeat)
class of plant resistance genes (Milligan et al., 1998; van der
Vossin et al., 2000; Ernst et al., 2002; Paal et al., 2004).
Conversely, SHMT is a metabolic enzyme with a key role in
one-carbon folate metabolism. Although the enzyme has multiple
catalytic activities, one of its main roles is to catalyze the
reversible conversion of serine and tetrahydrofolate (THF) to
glycine and 5,10-methyleneTHF (MTHF) to supply one-carbon units for
de novo purine, thymidylate, and methionine synthesis underlying
its importance in DNA synthesis and cellular methylation
reactions.
[0101] SHMT is a ubiquitous enzyme in nature that is structurally
conserved across kingdoms. With the exception of a mung bean SHMT
(Sukanya et al., 1991), all other SHMTs characterized to date
require pyridoxal 5'-phosphate (PLP), the active form of vitamin
B6. The 3-D structures of SHMT from a variety of organisms
including humans, rabbit, mouse, and E. coli have been determined
(Renwick et al., 1998; Scarsdale et al, 2000). Dimeric SHMTs are
mainly found in prokaryotes, while eukaryotic SHMTs form a dimer of
dimers.
[0102] Arabidopsis has seven SHMT family members that encode
predicted mitochondrial, plastid or cytoplasmic localized enzymes
which exhibit organ-specific expression patterns during plant
development (McClung et al., 2000; Moreno et al., 2004). SHMT1 is
highly expressed in leaves, stems and flowers, and is undetectable
in roots, consistent with its role as a photorespiratory
mitochondrial family member (Moreno et al., 2004). In mitochondria,
glycine is decarboxylated by the glycine decarboxylase complex
(GDC) to form MTHF. SHMT1 then transfers a one-carbon unit from
MTHF to glycine for the production of serine which gets recycled
into the chloroplast Calvin cycle. Arabidopsis plants harboring a
null allele in SHMT1 are dwarf and chlorotic and die before
producing progeny. Additionally, an Arabidopsis EMS mutant
exhibiting reduced SHMT1 activity were not able to mount an
efficient defense response to limit invasion by biotrophic and
necrotrophic foliar pathogens, highlighting the importance of the
photorespiratory pathway in plant resistance to pathogens. The role
of plant cytosolic SHMTs and one-carbon flux in the cytoplasm,
however, is not well understood. It is also not known how
alterations in one-carbon flux in one compartment may influence
one-carbon flux in other compartments. The compartmentalization of
nucleotide synthesis in the plastids and mitochondria of plants
suggests that one-carbon flux in the cytoplasm may be biased toward
the re-methylation of homocysteine to methionine and highly active
in cells types actively producing methylated compounds such as
lignins and alkaloids (Christensen and MacKenzie, 2006).
[0103] Within a few days after establishment, feeding cells induced
in plants carrying Rhg genes began to degenerate by what has been
described as a hypersensitive response (HR), a form of localized
programmed cell death (PCD) in plants to ward off invading
pathogens. The mechanism could be attributed to the plant
activating HR-like PCD to kill the feeding cell causing the
nematode to starve and die or the nematode death occurs prior to
activation of HR-like PCD. Localized necrosis at the feeding site
in response to SCN is a common theme among resistant soybean
cultivars, but the timing of necrosis and the degeneration of
syncytia vary depending on the source of resistance (Acedo et al.,
1984). Extensive histological studies have documented the cellular
changes associated with degenerating syncytia in soybean, including
the deposition of secondary cell wall material and formation of
lipid-like globules (Endo, 1965; Riggs et al., 1973; Acedo et al.,
1984). Comparative analyses of syncytia transcriptional profiles in
resistant and susceptible soybean have identified increased
defense-related gene expression associated with apoptotic cell
death and the plant hypersensitive response in syncytia formed in
resistant plants (Kandoth et al., 2011).
[0104] A biochemical analysis of the Essex and Forrest SHMTs can be
used to determine more specifically how the observed amino acid
differences may be altering GmSHMT function to contribute to
resistance against SCN. The evidence herein suggests that altered
folate metabolism is likely a contributing factor in soybean
resistance to SCN, providing new insight into the molecular basis
of this agronomically important pathosystem.
[0105] Active Organism Suppression
[0106] Folate (vitamin B) is critical for nematode health. If
expression of the SHMT or its regulation is impacting the folate
pathway in a plant tissue, then it may impact a nematode feeding on
that plant tissue. It is hypothesized that a nematode may acquire
folate or a precursor or derivative thereof from the soybean plant
itself. It is further hypothesized that the SHMT gene or gene
product may interfere with the nematode's folate pathway.
[0107] In humans, mutations in SHMT have been linked with a wide
range of disease states including adult lymphocytic leukemia
(Skibola et al., 2002), cardiovascular disease (Lim et al, 2005),
and neural tube defects (Heil et al., 2001). This is not surprising
considering the importance of SHMT in supplying one-carbon units
for multiple folate pathways. A reduction in the availability of
one-carbon units as a result of altered SHMT protein expression,
stability, or activity could mimic a folate deficiency. Folate is
essential for the maintenance of DNA integrity and stability and a
cellular deficiency in this important B vitamin can lead to an
imbalance of DNA precursors and altered DNA synthesis and repair.
Consequently, folate deficiency has been shown to mediate a variety
of malignancies (Kim, 2003). Under folate sufficient conditions,
thymidylate synthase utilizes MTHF as a methyl donor to convert
dUMP (deoxyuridylate monophosphate) to the pyrimidine nucleotide
dTMP (deoxythymidylate monophosphate). However, if MTHF is limited
under folate deficient conditions this reaction is blocked and
increased dUMP levels lead to uracil misincorporation into DNA
resulting in strand breaks and chromosomal aberrations. Such
cellular alterations have been shown to induce apoptosis in mammals
(Novakovic et al., 2006). Folate deficiency can also alter normal
DNA methylation. Low levels of MTHF lead to a reduction in
availability of 5-methylTHF required for homocysteine conversion
into methionine. Reductions in methionine deplete the pool of
S-adenosylmethionine which results in hypomethylation of DNA and
other cellular compounds, leading to improper gene expression and
other cellular abnormalities.
[0108] The role of SHMTs and one-carbon folate metabolism in plants
is much less characterized than in yeast and mammals, nonetheless
significant differences among these organisms have been recognized
(reviewed in Christensen and MacKenzie, 2006). In yeast and
mammals, one-carbon folate metabolism is divided between the
mitochondrial and cytoplasmic compartments. In contrast, one-carbon
folate metabolism is divided among the cytoplasmic, plastid, and
mitochondrial compartments in plants. Consistent with this
compartmentalization, cytoplasmic, mitochondrial and plastid
isoforms of one-carbon folate enzymes, including SHMT, have been
identified. These observations suggest that there are likely to be
differences in the metabolic roles of these enzymes, as well as
one-carbon flux among these organisms.
[0109] The GmSHMT identified and demonstrated herein to play a role
in soybean resistance to SCN is predicted to be a cytosolic enzyme.
LCM-microarray analysis of developing syncytia (Ithal et al., 2007)
and the promoter-GUS analyses described herein indicate that GmSHMT
is upregulated in feeding cells formed in SCN-susceptible soybean
cultivars. The feeding cells induced by cyst nematode undergo
dramatic changes in gene expression, organelles proliferate, and
they become metabolically highly active nutrient sinks. Syncytia
contain multiple large and amoeboid nuclei as a consequence of
repeated rounds of DNA synthesis (i.e., endoreduplication) in the
absence of mitosis and it has been demonstrated that blocking DNA
synthesis negatively affects the establishment and development of
syncytia (de Almeida Engler et al., 2011). Transcriptional and
metabolic profiling studies of syncytia support increased rates of
amino acid, DNA, and secondary cell wall biosynthesis (Ithal et
al., 2007; Hofmann et al., 2010). Thus, one might predict a high
demand on folate one-carbon metabolism for syncytium development
and maintenance.
[0110] Computational analysis predicted that the mutations in
Forrest SHMT may negatively impact its activity, which could
ultimately lead to folate deficiency, especially in a cell type
with a higher demand for one-carbon metabolism. The nematode's
nutritional requirements may also be influencing folate metabolism
in developing syncytia. Although the nutritional requirements of
plant-parasitic nematodes are not well defined, it is assumed that
like other animals, they acquire folate from their diet. Folate
deficiency, which has been shown to induce apoptosis in mammalian
cells (Novakovic et al., 2006), may ultimately lead to HR-like PCD
or nematode starvation. The ability, however, to restore resistance
in a genetic background containing a functional Essex GmSHMT by
transformation of a full length genomic clone corresponding to the
Forrest GmSHMT resistance allele, also supports a model wherein a
host-pathogen recognition event must occur for resistance to be
triggered.
[0111] Compositions and methods described herein utilizing
molecular biology protocols can be according to a variety of
standard techniques known to the art (see, e.g., Sambrook and
Russel (2006) Condensed Protocols from Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10:
0879697717; Ausubel et al. (2002) Short Protocols in Molecular
Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook
and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed.,
Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J.
and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754; Studier
(2005) Protein Expr Purif. 41(1), 207-234; Gellissen, ed. (2005)
Production of Recombinant Proteins: Novel Microbial and Eukaryotic
Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004)
Protein Expression Technologies, Taylor & Francis, ISBN-10:
0954523253).
[0112] Definitions and methods described herein are provided to
better define the present disclosure and to guide those of ordinary
skill in the art in the practice of the present disclosure. Unless
otherwise noted, terms are to be understood according to
conventional usage by those of ordinary skill in the relevant
art.
[0113] In some embodiments, numbers expressing quantities of
ingredients, properties such as molecular weight, reaction
conditions, and so forth, used to describe and claim certain
embodiments of the present disclosure are to be understood as being
modified in some instances by the term "about." In some
embodiments, the term "about" is used to indicate that a value
includes the standard deviation of the mean for the device or
method being employed to determine the value. In some embodiments,
the numerical parameters set forth in the written description and
attached claims are approximations that can vary depending upon the
desired properties sought to be obtained by a particular
embodiment. In some embodiments, the numerical parameters should be
construed in light of the number of reported significant digits and
by applying ordinary rounding techniques. Notwithstanding that the
numerical ranges and parameters setting forth the broad scope of
some embodiments of the present disclosure are approximations, the
numerical values set forth in the specific examples are reported as
precisely as practicable. The numerical values presented in some
embodiments of the present disclosure may contain certain errors
necessarily resulting from the standard deviation found in their
respective testing measurements. The recitation of ranges of values
herein is merely intended to serve as a shorthand method of
referring individually to each separate value falling within the
range. Unless otherwise indicated herein, each individual value is
incorporated into the specification as if it were individually
recited herein.
[0114] In some embodiments, the terms "a" and "an" and "the" and
similar references used in the context of describing a particular
embodiment (especially in the context of certain of the following
claims) can be construed to cover both the singular and the plural,
unless specifically noted otherwise. In some embodiments, the term
"or" as used herein, including the claims, is used to mean "and/or"
unless explicitly indicated to refer to alternatives only or the
alternatives are mutually exclusive.
[0115] The terms "comprise," "have" and "include" are open-ended
linking verbs. Any forms or tenses of one or more of these verbs,
such as "comprises," "comprising," "has," "having," "includes" and
"including," are also open-ended. For example, any method that
"comprises," "has" or "includes" one or more steps is not limited
to possessing only those one or more steps and can also cover other
unlisted steps. Similarly, any composition or device that
"comprises," "has" or "includes" one or more features is not
limited to possessing only those one or more features and can cover
other unlisted features.
[0116] All methods described herein can be performed in any
suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g. "such as") provided with respect to
certain embodiments herein is intended merely to better illuminate
the present disclosure and does not pose a limitation on the scope
of the present disclosure otherwise claimed. No language in the
specification should be construed as indicating any non-claimed
element essential to the practice of the present disclosure.
[0117] Groupings of alternative elements or embodiments of the
present disclosure disclosed herein are not to be construed as
limitations. Each group member can be referred to and claimed
individually or in any combination with other members of the group
or other elements found herein. One or more members of a group can
be included in, or deleted from, a group for reasons of convenience
or patentability. When any such inclusion or deletion occurs, the
specification is herein deemed to contain the group as modified
thus fulfilling the written description of all Markush groups used
in the appended claims.
[0118] All publications, patents, patent applications, and other
references cited in this application are incorporated herein by
reference in their entirety for all purposes to the same extent as
if each individual publication, patent, patent application or other
reference was specifically and individually indicated to be
incorporated by reference in its entirety for all purposes.
Citation of a reference herein shall not be construed as an
admission that such is prior art to the present disclosure.
[0119] Having described the present disclosure in detail, it will
be apparent that modifications, variations, and equivalent
embodiments are possible without departing the scope of the present
disclosure defined in the appended claims. Furthermore, it should
be appreciated that all examples in the present disclosure are
provided as non-limiting examples.
EXAMPLES
[0120] The following non-limiting examples are provided to further
illustrate the present disclosure. It should be appreciated by
those of skill in the art that the techniques disclosed in the
examples that follow represent approaches the inventors have found
function well in the practice of the present disclosure, and thus
can be considered to constitute examples of modes for its practice.
However, those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments that are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
present disclosure.
Example 1: Haplotyping of Plant Introductions (PIs) at Rhg4 and
Rhg1 Loci
[0121] A total of 81 soybean lines (plant introductions, landraces
and elite cultivars) were scored for their SCN phenotype and
SNP-genotyped at the rhg1 and Rhg4 loci. Lines were classified
resistant (R) to SCN if the F1 was .ltoreq.10% and susceptible (S)
if the F1 was .gtoreq.10%. Soybean lines were genotyped at the Rhg4
locus using the DNA markers Sat_162, SHMT and SUB1 and at the rhg1
locus using the DNA markers 560, 570 and Satt309 (see e.g., TABLE
1).
TABLE-US-00001 TABLE 1 Haplotyping of plant introductions (PIs) at
Rhg4 and rhg1 loci. SCN Infection Rhg4 Locus rhg1 Locus PI Name
phenotype Sat_162 SUB1 560 570 Satt309 PI 90406 S S S R PI 59645 S
S S R PI 594788 S S S R PI 594629 S S S R PI 603521 S S S S PI
603596 S S S R PI 406342 S S S S PI 594871 S S S R PI 594562 A S S
S S PI 594707 S S S R PI 603428 C S R R S S R PI 587849 S S S S PI
602991 S R R S S S PI 567503 S S S S PI 603698 G S S S R PI 594451
S S S S PI 603516 S S S S PI 587752 S S S R PI 594770 A S R R S S R
PI 603785 S S S S PI 603424 A S S S S PI 594777 S S S R PI 603502 A
S S S S S R PI 603318 S S S S PI 603384 S S S R R S PI 603712 S S S
S PI 588040 S S S R PI 587666 S S S S PI 567359 S R R S S R PI
587799 S S S S PI 603704 A S S S R PI 567368 S S S S S R PI 603420
S R R S S R PI 587993 S S S S S S PI 407801 S S S S PI 603408 S S S
S PI 587700 C S S S S PI 587552 S S S R PI 587823 S S S S PI 567568
B S S S R R S PI 603357 S S S S S R PI 603337 B S S S R PI 79691 S
S S S PI 567293 S S S S PI 567364 S S S R PI 567395 S S S R PI
567631 S S S R PI 86145 S S S R PI 567481 S S S S S R PI 567525 S S
S S PI 567700 S S S S PI 594615 S S S R PI 603147 S S S R PI 603336
S S S R PI 603372 S R R R R S PI 603419 B S S S R PI 603479 S S S S
PI 603587 A R R R R R S PI 603656 S R R S S S PI 603675 S S S R PI
603756 S S S R PI 603784 S S S R PI 97094 S S S R R R PI 228056 S S
S R PI 594773 S R R S S R PI 588000 S S S S PI 588047 S S S R PI
594554 S S S S PI 594597 S S S S PI 416937 S S S R RESSEQ R R R R R
R Essex S S S S S S Forrest R R R R R R Williams 82 S S S S S S PI
548402 R R R R R R (Peking) PI 88788 R R R R R S PI 90763 R R R R R
R PI 437654 R R R R R R PI 209332 R S S R R S PI 89772 R R R R R R
PI 548316 R R R R R S (Cloud)
Example 2: Primers
[0122] References are listed below for primers described herein. I.
Primers for the initial screening of Rhg4 recombinants; II. Primers
for high resolution genetic mapping of Rhg4 recombinants and
haplotyping of plant introductions (PIs); III. Primers for TILLING,
SHMT cloning, and sequencing and IV. Primers for promoter cloning,
complementation, RNAi, VIGS and qPCR
TABLE-US-00002 TABLE 2 Primers. I. Primers for screening of Rhg4
recombinants Annealing Name Forward Primer Reverse Primer Temp
Comments Satt632.sup.[4] GGGCTATGAAGGGAATGGAAAGGA [SEQ ID NO.: 5]
CCCATATTGAAGATTTGAAGTAAT [SEQ ID NO.: 6] 50 For Rhg4
Sat_162.sup.[4] GCGTGGTTTTTCGCTGGATATA [SEQ ID NO.: 7]
GCGCATTTCGTAACATATTTTTCAC [SEQ ID NO.: 8] 50 GMES6186.sup.[3]
AGCGGGAATTGAAGGTTTTT [SEQ ID NO.: 9] GGAATCTCATCTGAAAATAATGGA [SEQ
ID NO.: 10] 50 Sat_210.sup.[4] GCGCCAGCAACAAAGTTCCTGACAAA [SEQ ID
NO.: 11] GCGCATGCAAATGAAATAATAA [SEQ ID NO.: 12] 50 For rhg1
Satt309.sup.[4] GCGCCTTCAAATTGGCGTCTT [SEQ ID NO.: 13]
GCGCCTTAAATAAAACCCGAAACT [SEQ ID NO.: 14] 50 SIUC-SAT143
TGTTACTTAGTAATTATGAAG [SEQ ID NO.: 15] AATAATGATTTGTTGATCGAT [SEQ
ID NO.: 16] 50 II. Primers for high resolution genetic mapping of
Rhg4 recombinants and haplotyping of soybean lines Annealing Name
Forward Primer Reverse Primer Temp Comments 16683.sup.[2]
GCGAAGCCCATACTCCGAACCTGCCA [SEQ ID GCCTCCAAAAACTCAACCCCATCAA [SEQ
ID 58 For Rhg4 NO.: 17] NO.: 18] Satt632.sup.[4]
GGGCTATGAAGGGAATGGAAAGGA [SEQ ID NO.: 19] CCCATATTGAAGATTTGAAGTAAT
[SEQ ID NO.: 20] 50 25005.sup.[2] GATGCCTTACGCCTGTCACTAAC [SEQ ID
NO.: 21] GCAGAACAGTAGAACAAGTCCAGT [SEQ ID NO.: 22] 58 10931.sup.[2]
GCCCACCAGTTGTTGTGTAAGAC [SEQ ID NO.: 23] GCGTGCGATGAGAAACTCAGAC
[SEQ ID NO.: 24] 60 Sat_162.sup.[4] GCGTGGTTTTTCGCTGGATATA [SEQ ID
NO.: 25] GCGCATTTCGTAACATATTTTTCAC [SEQ ID NO.: 26] 50
LRR-RLK.sup.[1] GAAGTTGGTGACTGCGGGAAATGC [SEQ ID NO.: 27]
TTCAATGCACCGATCCAACAAGGA [SEQ ID NO.: 28] 65 8B.GA
TACAAGTCAGTAATATAACCT [SEQ ID NO.: 29] CTGAGTAGATAGCAGTGACAT [SEQ
ID NO.: 30] 55 SAT6 ACTGCTTATGGTTGCAGAATC [SEQ ID NO.: 31]
GAGTATGTAAATGACATCTT [SEQ ID NO.: 32] 55 SCN6 TATGACTGCAGAAGTCAAGTC
[SEQ ID NO.: 33] TGACCTTGAAGAGGAGATAGA [SEQ ID NO.: 34] 60 SHMT
ACAACACTCTCTCTTCTCGC [SEQ ID NO.: 35] CAGATTATGAGTTTTGGCCTG [SEQ ID
NO.: 36] 60 8K.GA ATTTCACTTATATAAATATGC [SEQ ID NO.: 37]
TCTCTTTTATATGCTACAATA [SEQ ID NO.: 38] 55 SUB1
GGTACCATCTTCCTTAGAATGG [SEQ ID NO.: 39] TGTGGGAAAGAGACAACAAACC [SEQ
ID NO.: 40] 60 SCN8 TTCGTTGGCTCCCACTGCTC [SEQ ID NO.: 41]
TCTGGTACACGTCAATGGGC [SEQ ID NO.: 42] 60 SCN9 ACGAAGAGATCCTGAAGGAG
[SEQ ID NO.: 43] ATTCCCAAGGGTTGGAAGGC [SEQ ID NO.: 44] 60 CS1
TCAAGCATTGTTTGGAGATGG [SEQ ID NO.: 45] ACAGAAGCATTTGCAGGGCAG [SEQ
ID NO.: 46] 60 SCN13 ACCTTCGTTGGATGCAAGGC [SEQ ID NO.: 47]
CTTGGTCCAAAATTGCGGGTC [SEQ ID NO.: 48] 60 SCN10
CGTGGCAATTTTTCGAAGGTAG [SEQ ID NO.: 49] CAACTCAAAACCACATTGAGGC [SEQ
ID NO.: 50] 60 HSP1 AGCAACACACGCAAACCAAATC [SEQ ID NO.: 51]
TGCAATTCATCCTACGGTGGC [SEQ ID NO.: 52] 60 SCN11
TCAGGACATGTTTGTTGGTGG [SEQ ID NO.: 53] CACACTCAGTTCAGCTTATAG [SEQ
ID NO.: 54] 60 SCN14 ATACGTGGGCCCAACTAAGAC [SEQ ID NO.: 55]
TGTCGTCTTAGGTGAGAGGC [SEQ ID NO.: 56] 60 25103.sup.[2]
TGGCTGTTCCTAGAAGGCTGTG [SEQ ID NO.: 57] TGGAGTTGGATCGGAGGATTAAGG
[SEQ ID NO.: 58] 65 SCN12 AAGGGAGACTGGATAACCATC [SEQ ID NO.: 59]
CCGCTCATTTGGTGAGTCATG [SEQ ID NO.: 60] 60 SCN15
ATGTGCTCGCTGTTGGTGATG [SEQ ID NO.: 61] GCACCATGGAGGTGAAAAAAATA [SEQ
ID NO.: 62] 60 25131.sup.[2] GGACGGTTCGCTGGCTAAGA [SEQ ID NO.: 63]
TCACTGCCTTCCTCTTCTTCTTCA [SEQ ID NO.: 64] 58 25133.sup.[2]
TCCACCGAGCAACTACCATATCTT [SEQ ID NO.: 65] ACGAGCACATAGCCAGGCATTA
[SEQ ID NO.: 66] 58 Satt309.sup.[4] GCGCCTTCAAATTGGCGTCTT [SEQ ID
NO.: 67] GCGCCTTAAATAAAACCCGAAACT [SEQ ID NO.: 68] 50 For rhg1 560
TTACTTTTGGTCAGCATTTTGGC [SEQ ID NO.: 69] TATTGTTGATATATTATATTGTCC
[SEQ ID NO.: 70] 55 570 ACCCTTTTTGCAGTATTTATGC [SEQ ID NO.: 71]
CTAGGTAACTCTTTTAGCCGTGA [SEQ ID NO.: 72] 55 III. Primers for
TILLING, SHMT cloning, and sequencing Annealing Name Forward Primer
Reverse Primer Temp Comments SHMT ACAACACTCTCTCTTCTCGC [SEQ ID NO.:
73] CAGATTATGAGTTTTGGCCTG [SEQ ID NO.: 74] 60 For TILLING 1 and
sequencing SHMT2 CAGGCCAAAACTCATAATCTG [SEQ ID NO.: 75]
CAGATTATGAGTTTTGGCCTG [SEQ ID NO.: 76] 60 For TILLING 2 and
sequencing SHMT3 TAATTTTGGTTGGAGAACAATG [SEQ ID NO.: 77]
CTAATCCTTGTACTTCATTTC [SEQ ID NO.: 78] 60 For TILLING 3 and
sequencing SHMTcDNA ATGGATCCAGTAAGCGTGTGG [SEQ ID NO.: 79]
CTAATCCTTGTACTTCATTTCAG [SEQ ID NO.: 80] 60 For SHMT cDNA cloning
IV. Primers for promoter cloning, complementation, RNAi, VIGS and
qPCR Name Forward Primer* Reverse Primer* Comments Promoter cloning
GmSUB1 promoter GTTAACCTTCAAGTCCCAATCTG [SEQ ID NO.: 81]
AGAAGAATTTGGAGCAGAAAGTG [SEQ ID NO.: 82] SHMT promoter-1
AATTGAGCTCCAATGGCACCAATGCCCA AATTGGTACCGAACGGTGGAAATGAATGAATG [SEQ
ID NO.: 83] [SEQ ID NO.: 84] SHMT promoter-2
AAAAAAGCAGGCTATCAATGGCACCAATGCCCA
AAGAAAGCTGGGTAGAACGGTGGAAATGAATGAATG Gateway [SEQ ID NO.: 85] [SEQ
ID NO.: 86] cloning attB1 and attB2 GGGGACAAGTTTGTACAAAAAAGCAGGCT
GGGGACCACTTTGTACAAGAAAGCTGGGT [SEQ ID Gateway [SEQ ID NO.: 87] NO.:
88] cloning gDNA construct for complementation gSHMT-5'
AATTGGCGCGCCTGCAGGCAATGGCACCAATGCCCA AATTGAGCTCGATTCCGCGGCA
Fragment 1 [SEQ ID NO.: 89] [SEQ ID NO.: 90] (5)cloning gSHMT-3'
AATTGAGCTCATCGCCTCCGAGA AATTGGTACCTGCAGGCCAGATTTTATGGTGCCCAA
Fragment 2 [SEQ ID NO.: 91] [SEQ ID NO.: 92] (3)cloning RNAi
cloning SHMT-Ri AAAAAAGCAGGCTATTACGGCGGCAATGAATACAT
AAGAAAGCTGGGTACTGAAGTCTAGGGCTTTTTCT Gateway [SEQ ID NO.: 93] [SEQ
ID NO.: 94] cloning VIGS cloning SHMT-VIGS
ATGCGGATTCGGCAATGAATACATCGACCAG [SEQ ID
TTGGGTACCTGTCTAGGGCTTTTTCTTCCAAG [SEQ ID NO.: 95] NO.: 96] qPCR
primers qSHMT TGAAAAAGACTTTGAGCAGATTGG [SEQ ID NO.: 97]
TTGCCATGCTCCTTCTGGAT [SEQ ID NO.: 98] *Restriction
sites/recombination sites are underlined References: 1. Liu et al.
Functional and Integrative Genomics, 2011, 11: 539-549 2. Hyten et
al. Genetics, 2007, 175: 1937-1944 3. Hwang et al. DNA Research,
2009, 16: 213-225 4. http://soybase.org
Example 3: Nematode and Plant Material
[0123] The SCN (Heterodera glycines `Ichinohe`) inbred population
PA3 (Hg type 0) used was mass-selected on soybean cv. Williams 82
according to standard procedures (Niblack et al., 1993) at the
University of Missouri. The soybean cultivar Forrest (Hartwig and
Epps, 1973) is resistant to SCN PA3. The soybean cultivars Essex
(Smith and Camper, 1973) and Williams 82 (Bernard and Cremeens,
1988) are susceptible to SCN PA3. Forrest was used to develop an
ethylmethane sulphonate (EMS)-mutagenized M2 population of 2,000
lines for TILLING. The two F2:6 RIL lines, E.times.F67
(rhg1.sub.Frhg1.sub.FRhg4.sub.FRhg4.sub.F) and E.times.F63
(rhg1.sub.Frhg1.sub.FRhg4.sub.ERhg4.sub.E), are resistant and
susceptible to PA3, respectively. These two RIL lines differed at
the majority of markers assigned to the Rhg4 region and appeared to
be nearly opposite recombination events. The collection of plant
introductions used in this study was obtained from Dr. Randal
Nelson, USDA Soybean Germplasm curator, UI, Urbana, Ill.
Example 4: Map-Based Positional Cloning of the Rhg4 Gene
[0124] Three genetic populations segregating for resistance to SCN
PA3 (Hg type 0) were used for mapping. These included an F2:6
recombinant inbred line (RIL) population from a cross between
Forrest and Essex (98 individuals; Meksem et al., 2001), and two
large F2 populations generated from crosses between Forrest and
either Essex (1,755 lines) or Williams 82 (2,060 lines), to enrich
the chromosomal interval carrying the Rhg4 gene with recombinants.
SCN phenotyping was conducted according to Brown et al. (2010).
[0125] Because Forrest resistance to SCN requires both rhg1 and
Rhg4 (Meksem et al., 2001), genotyping was conducted using DNA
markers flanking both loci to detect informative recombinants at
the Rhg4 locus (see e.g., TABLE 2). The SSR markers, Satt632,
Sat_162 and GMES6186 (website soybase.org and Hwang et al., 2009)
were used to identify chromosomal breakpoints at the Rhg4 locus.
PCR amplifications were performed using DNA from individuals for
each of the three genetic populations. Cycling parameters were as
follows: 35 cycles of 94.degree. C. 30 sec, 50.degree. C. 30 sec
and 72.degree. C. 30 sec with 7 min of extension at 72.degree. C.
The PCR products were separated on 3%-4% metaphor agarose gels. The
identified recombinants were subject to a second screening using
SSR markers, Sat_210 and Satt309 (website soybase.org), and
SIUC-SAT143 to identify the rhg1 genotype of each recombinant.
[0126] To enrich the chromosomal regions carrying the Rhg4 locus
with DNA markers, the Genbank published sequences AX196297 and
AX197417 were used to design PCR primers every 5 to 10 Kbp of the
300 Kbp carrying the Rhg4 locus (see e.g., TABLE 2). DNA from
Forrest and Essex were tested with each primer using a modified
EcoTILLING protocol to find and map polymorphic sequences at the
Rhg4 locus (Meksem et al., 2008; Liu et al., 2011). The identified
SNP and InDel DNA markers were integrated into the informative
recombinants to identify chromosomal breakpoints and the interval
that carried the Rhg4 gene.
[0127] The closest DNA markers harboring the Rhg4 locus were used
to screen a Forrest BAC library (Meksem et al., 2001, Liu et al.,
2011). The BAC clone 100610 was identified, integrated with the
developed genetic map and partially sequenced.
Example 5: Isolation of the GmSHMT Genomic and cDNA Sequences
[0128] A 5.103 kb Forrest SHMT genomic DNA fragment (Genbank
Accession No. JQ714083) spanning 2.339 kb of sequence 5' of the ATG
start site, 2.189 kb of sequence from start to stop including 3
exon and 2 introns, and 0.675 kb of sequence 3' of the stop codon
was cloned and sequenced. Because the 1001310 BAC clone contained
only a partial SHMT gene sequence that included the 2.339 kb of
sequence 5' of the ATG start site and 1.315 bp downstream of the
ATG start site (see e.g., FIGS. 1A-1C), an internal Sac I site at
position 108 from the ATG start was used for a PCR-based cloning
approach of the full length genomic sequence. First, a 2.447 kb
fragment including the 2.339 kb of sequence 5' of the ATG start
site and 108 bp of exon 1 was PCR amplified using a forward primer
designed with an Asc I site and a reverse primer spanning the
internal Sac I site. The fragment was digested and cloned into the
CGT35S vector (Wang et al., 2010) using Asc I and Sac I. An Sbf I
site was also introduced into the forward primer internal to Asc I
for subsequent subcloning for complementation analysis (see e.g.,
TABLE 2). The remainder of the SHMT gDNA fragment, including the
unique internal Sac I site, was amplified from Forrest genomic DNA
by PCR with a forward primer spanning the internal Sac I site and a
reverse primer designed with a Kpn I site. The fragment was
digested and cloned into the Sac I and Kpn I sites downstream of
the 5'fragment in the above CGT35S clone. The reverse primer was
designed with a Sbf I site internal to Kpn I for the purpose of
subsequent subcloning for complementation analysis (see e.g., TABLE
2). The fragments were ligated together utilizing the internal Sac
I restriction site to generate the 5.103 kb SHMT genomic DNA
fragment and sequenced. Primers designed to the Forrest genomic DNA
sequence were used to clone the Essex SHMT genomic DNA sequence.
PCR primers designed based on the Forrest and Essex genomic DNA
sequences were used to amplify the corresponding cDNA sequences.
Genomic DNA was isolated from young leaves using the DNeasy Plant
Mini Kit (Qiagen Science, USA). Total RNA was isolated from roots
using the RNeasy Plant Mini Kit (Qiagen) and cDNA was synthesized
using a cDNA synthesis kit (Invitrogen).
Example 6: Mutation Screening of GmSHMT
[0129] An EMS-mutagenized M2 population from SCN resistant cultivar
Forrest containing 1,920 M2 families (Cooper et al., 2008, Meksem
et al., 2008) was used to screen for mutations within the GmSHMT
gene sequence. The gene was divided into 3 intervals (see e.g.,
FIG. 2A) and TILLING was performed as previously described (Meksem
et al., 2008). The GmSHMT gene of each mutant was sequenced to
characterize the identified allele and its subsequent amino acid
changes within the predicted protein sequences.
Example 7: Phenotype and Zygosity Analyses of GmSHMT Tilling
Mutants
[0130] Mutants seeds were planted and scored for their SCN female
index according to Brown et al. (2010). DNA from each plant was
subjected to TILLING analysis without adding the reference wild
type DNA of Forrest to the reaction tube before mismatch analyses
to detect the zygosity level of the identified mutant.
Example 6: Haplotyping of Plant Introductions
[0131] A total of 81 soybean lines (plant introductions, landraces
and elite cultivars) representing 90% of the genetic variability in
soybean were scored for their SCN female index. Lines were
classified resistant (R) to SCN if the FI was .ltoreq.10% and
susceptible (S) if the FI was .gtoreq.10%. Soybean lines were
genotyped at the Rhg4 locus using the DNA markers Sat_162, SHMT and
SUB1 and at the rhg1 locus using the DNA markers 560, 570 and
Satt309. The coding region of GmSHMT for 28 lines was sequenced.
Common SNPs and Indels were identified and used to determine the
different GmSHMT haplotypes.
Example 7: Virus-Induced Gene Silencing (VIGS)
[0132] Bean pod mottle virus (BPMV) VIGS vectors, pBPMV IA-R1M and
pBPMV-IA-D35 were used in this example (Zhang et. al., 2010).
pBPMV-IA-D35 is a derivative of pBPMV-IA-R2 containing Bam HI and
Kpn I restriction sites between the cistrons encoding movement
protein and the large coat protein subunit. Briefly, a 328 base
pair (bp) fragment (spanning bps 210-537) of the GmSHMT cDNA
sequence (Genbank Accession No. JQ714080) was amplified from
soybean (cv. Forrest) root cDNA by RT-PCR. PCR products were
digested with Bam HI and Kpn I and ligated into pBPMV-IA-D35
digested with the same enzymes to generate pBPMV-IA-SHMT. Gold
particles coated with plasmid DNA corresponding to pBPMV-IA-R1 M
and pBPMV-IA-SHMT were co-bombarded into soybean leaf tissue as
described in Zhang et al. (2010). At 3-4 weeks post-inoculation,
BPMV-infected leaves were collected, lyophilized, and stored at
-20.degree. C. for future experiments. Infected soybean leaf
tissues were ground in a mortar and pestle with 0.05 M potassium
phosphate buffer (pH 7.0) and used as virus inoculum for VIGS
assays.
[0133] The SCN-resistant RIL E.times.F67 was inoculated with
pBPMV-IA-SHMT. Control plants were infected with BPMV only. Each
treatment consisted of at least 12 plants. Unifoliate leaves of
9-dy-old plants were rub inoculated with virus using carborundum
according to Zhang et al. (2010). Plants were grown in a growth
chamber set to the following conditions: 20-21.degree. C., 16 h
light/8 h dark, and 100 mE m.sup.-2s.sup.-1 light intensity.
Twenty-one days post virus inoculation, plants were inoculated with
1500 SCN eggs and maintained at 20.degree. C. for 35 days. Cysts
were isolated from the roots systems of individual plants by
decanting and sieving and counted under a stereomicroscope. The
results were plotted and analyzed for statistical significance by
an unpaired t-test using GraphPad PRISM.RTM. software. To estimate
GmSHMT gene silencing in roots, root tissues were harvested at
21-days post-virus inoculation (the time of nematode inoculation)
from two representative plants inoculated with either pBPMV-IA-SHMT
or BPMV only and frozen at -80.degree. C. for RNA isolation and
qPCR analysis.
Example 8: Hairy Root RNA.sub.I Experiments
[0134] A 338 bp fragment (spanning bps 205-542) of the GmSHMT cDNA
sequence was amplified from soybean (cv. Forrest) root cDNA by
RT-PCR, cloned into the pDONR-zeo gateway cloning vector
(Invitrogen), and subsequently moved to a gateway RNAi binary
vector under the control of the nematode-inducible Glyma15g04570.1
promoter (Kandoth et. al., 2011) (pZF-RNAi vector) to generate
pZF-SHMTi. The pZF-RNAi vector was constructed by introducing
gateway cloning sites flanking the FADR intron downstream of pZF
promoter in the pAKK vector (Wang et al., 2010) which has a GFP
selectable marker in planta. Transgenic E.times.F67 hairy roots
transformed with pZF-SHMTi were produced from soybean cotyledons
according to Kandoth et al. (2011). E.times.F63 and E.times.F67
hairy roots transformed with pZF-GUSi (the pZF-RNAi vector
containing a portion of the GUS gene) were used as susceptible and
resistant controls, respectively. GFP-positive hairy roots were
root tip propagated three times on media containing antibiotic to
clear Agrobacterium prior to nematode inoculation as described
previously (Kandoth et al., 2011). Briefly, hairy roots (3-4 cm in
length) were grown in square Petri plates and infected with
approximately 400 sterile infective second-stage nematode juveniles
one centimeter above the root tip. The plates were incubated in the
dark at room temperature for 30 days. After 30 days, cysts were
counted under a stereomicroscope. The experiment was conducted
independently three times with a minimum of 12 independent hairy
root lines per treatment. The results were plotted and analyzed for
statistical significance by an unpaired t-test using GraphPad
PRISM.RTM. software.
Example 9: Promoter-Gus Analysis
[0135] A 2.339 kb fragment corresponding to sequence 5' of the ATG
start site of the Forrest SHMT gene (Genbank Accession No.
JQ714083) and the same region from the Essex SHMT gene (Genbank
Accession No. JQ714084) were amplified by PCR from the 1001310 BAC
clone and Essex gDNA, respectively, and cloned into the pYXT1
vector (Xiao et al., 2005) to generate transcriptional fusions with
the .beta.-glucuronidase (GUS) gene. Soybean hairy roots
transformed with these constructs were generated and infected with
SCN. At two and four days post inoculation, root pieces excised
from the infection zone were stained for GUS activity (Jefferson et
al., 1987). Multiple roots from at least five independent lines
were stained for each construct. Root pieces were fixed with 4% v/v
paraformaldehyde in phosphate-buffered saline overnight at room
temperature, embedded in paraffin, and sectioned longitudinally to
a thickness of 10 .mu.m. The sections were observed using
differential interference contrast microscopy on a Vanox (Olympus)
microscope and photographed with CMOS color digital camera.
Example 10: Genomic Complementation Experiments
[0136] The 5.103 kb Forrest SHMT genomic DNA fragment was subcloned
into the Sbf 1 restriction site of the pAKK binary vector which has
GFP selection for transgenic events. Transgenic hairy roots were
produced and infected with SCN as described for RNAi experiments.
The SCN-susceptible RIL E.times.F63 was used for the
complementation experiment. Control hairy roots were produced by
transforming E.times.F63 and E.times.F67 hairy roots with the pAKK
binary vector carrying only the SHMT promoter sequence. The
experiment was conducted independently five times with a minimum of
15 independent hairy root lines per treatment. The results were
plotted and analyzed for statistical significance by an unpaired
t-test using GraphPad PRISM.RTM. software.
Example 11: RNA Isolation and qPCR Analysis
[0137] Total RNA was isolated from root tissues using the RNeasy
plant miniprep kit (Qiagen) according to the manufacturer's
instructions. Real-time qRT-PCR was conducted as described in
Kandoth et al. (2011).
Example 12: Computational Methods
[0138] A computational approach was performed to structurally and
functionally annotate the identified GmSHMT protein and to estimate
the effect of the mutations on GmSHMT function. Our approach
consisted of two stages. First, a homology model of GmSHMT was
obtained using Essex sequence as a target. Second, functional sites
were mapped onto the surface of GmSHMT using the structural
information of ligand binding by SHMT homologs. The homology
analysis of GmSHMT has determined 43 structurally resolved SMHT
homologs from a diverse set of bacterial and mammalian species; no
structurally resolved plant SHMTs were found. Among the group of
four homologs with the highest sequence similarities, the mouse
SHMT (PDB ID 1 EJI) with the largest coverage of the GmSHMT
sequence (sequence identity 57%, template coverage 100%) was
selected as a template for homology modeling of GmSHMT. Homology
modeling was done using MODELLER-9 (Sali and Blundell, 1993) and
the top-ranked model was selected from the set of candidate models
using the building MODELLER scoring function. To determine the
ligand binding sites for PLP-serine, PLP-glycine, and
THF/MTHF)/FTHF, the obtained model of GmSHMT was structurally
aligned with each of the orthologous SHMTs known to interact with
the small ligands, and the ligand binding site from each homolog
was mapped onto the surface of GmSHMT model through the structural
alignment. The residues constituting the glycine binding sites,
GBS1 and GBS2, in the SHMT homologs were identified in the
literature and then mapped onto the structure of GmSHMT in a
similar way, using the structural alignment of GmSHMT with its
homolog.
Example 13: Positional Cloning of the Rhg4 Gene from SCN Resistant
Soybean Cv. Forrest
[0139] Three genetic populations segregating for resistance to SCN
were used for mapping. These included an F6 recombinant inbred line
(RIL) population from a cross between Forrest and the SCN
susceptible cv. Essex (98 individuals; Meksem et al., 2001), and
two large F2 populations generated from crosses between Forrest and
either Essex (1,755 lines) or the SCN susceptible cv. Williams 82
(2,060 lines), to enrich the chromosomal interval carrying the Rhg4
gene with recombinants.
[0140] Methods were according to Examples 1-12, unless described
otherwise.
[0141] Because Forrest resistance to SCN requires both rhg1 and
Rhg4 (Meksem et al., 2001), genotyping was conducted using DNA
markers flanking both loci to detect informative recombinants at
the Rhg4 locus (see e.g., TABLE 2). From a total of 355 recombinant
lines identified with chromosomal breakpoints at the Rhg4 locus,
two recombinants (E.times.F74 and F.times.W5093) were used to
define the interval carrying the Rhg4 gene. Both lines carried the
resistant allele at the rhg1 locus and were double recombinants for
an 8 kb interval carrying the Rhg4 resistant allele (see e.g.,
FIGS. 1A-1C). Two genes, one coding for a serine
hydroxymethyltransferase (GmSHMT) and the other a subtilisin-like
protease (GmSUB), were identified in the 8 kb interval. A
comparison between the genomic DNA sequences of GmSHMT from Forrest
and Essex showed 5 nucleotides differences (3 SNPs and 2 Ins/Dels)
between the resistant and susceptible alleles; however, only two of
the nucleotide differences found between the Forrest and Essex
GmSHMT cDNAs resulted in an amino acid change in the predicted
protein sequences (R130P and Y358N) (see e.g., FIG. 1D). The amino
acid sequence at these two positions in Williams 82 was consistent
with that of Essex (see e.g., FIG. 1D).
Example 14: GmSHMT Characterization
[0142] Three nucleotide differences were identified in 2,339 bp of
sequence 5' of the predicted start site for GmSHMT between Forrest
and Essex (see e.g., FIG. 7). Based on these findings, GmSHMT was
characterized further for a role in SCN resistance.
[0143] Methods were according to Examples 1-12, unless described
otherwise.
[0144] Primers specific for GmSHMT (see e.g., TABLE 2) were used to
screen a population of 1920 ethyl methane-sulfonate
(EMS)-mutagenized M2 lines from the SCN resistant cultivar Forrest.
Using a TILLING (Targeting Induced Local Lesions In Genomes)
approach, two mutations in the GmSHMT gene were identified on
chromosome 8 that led to missense mutations at E61K and M125I. SIFT
predictions were performed on both mutations. SIFT predicts whether
an amino acid substitution affects protein function based on
sequence homology and the physical properties of amino acids (Kumar
et al., 2009). SIFT predictions with IC<3.25 are considered
confident. Changes with a SIFT score <0.05 are predicted to be
damaging to the protein. Both missense mutations identified had IC
values <3.25 (see e.g., FIGS. 2A-2C), thus the SIFT predictions
can be considered confident. Of the two TILLING mutants, the M1251
mutation (SIFT score=0.02) was predicted to be deleterious to the
protein. Both mutants were more susceptible to SCN in nematode
infection assays (see e.g., FIGS. 2A-2C). Additionally, in the
segregating M3 mutant seed, the F6756 (M1251) mutation was
correlated with the SCN resistance phenotype of individual
plants.
[0145] These data indicated that GmSHMT at the Rhg4 locus plays a
role in resistance to SCN.
Example 15: Identification of GmSHMT Haplotypes
[0146] A link was established between GmSHMT alleles and soybean
resistance to SCN by scoring 81 soybean lines (including plant
introductions, landraces and elite cultivars) representing 90% of
the genetic variability in soybean for their SCN female index and
determining their SNP-based GmSHMT haplotype.
[0147] Methods were according to Examples 1-12, unless described
otherwise.
[0148] The GmSHMT gene was fully sequenced from 28 selected plant
introductions including all known SCN reporter lines. 13
polymorphisms were identified in the coding regions and 6 in the
non coding DNA sequences. Of the thirteen coding polymorphisms, two
produced amino acid changes. The R130P and Y358N substitutions were
responsible for the SCN phenotype except when SCN resistance is
derived from plant introduction P188788. Eight different GmSHMT
haplotypes were identified (see e.g., FIG. 3).
[0149] Soybean lines with haplotypes H1-H3 carried resistant
alleles at GmSHMT and rhg1, and were resistant to SCN. These
included soybean lines P1548402 (Peking), Forrest, P190763,
P1437654, and P189772, all previously reported to exhibit what is
known as "Peking type resistance" which requires both rhg1 and Rhg4
(Meksem et al., 2001). Soybean lines with haplotypes H4-H5 carried
the susceptible allele at GmSHMT, but varied for either the
resistant or susceptible allele at the rhg1 locus, those lines were
susceptible to SCN regardless of the rhg1 allele, and included
soybean cvs. Essex and Williams 82, both well known for their
susceptibility to SCN. H6-H8 carried the susceptible allele at
GmSHMT, but varied for either the resistant or susceptible allele
at the rhg1 locus, those that carried the rhg1 susceptible allele
were susceptible and those that carried the rhg1 resistant allele
were resistant to SCN. These included P188788, P1209332, and
P1548316 (cv. Cloud), all previously reported to exhibit what is
known as "P188788 type resistance", which has been shown to require
rhg1, but not Rhg4 for resistance to SCN (Concibidio et al.,
2004).
[0150] In summary, the GmSHMT haplotyping analysis is in agreement
with the previous SCN-resistant QTL reports (see e.g., review of
Concibido et al., 2004) and confirms the requirement of Rhg4 for
the Peking type of resistance to SCN.
Example 16: Validation of GmSHMT by VIGS, RNA.sub.I and
Complementation
[0151] Knock down studies using VIGS (Virus Induced Gene Silencing)
and targeted RNAi (RNA interference) provided further evidence that
the GmSHMT gene confers resistance to H. glycines.
[0152] Methods were according to Examples 1-12, unless described
otherwise.
[0153] It has been shown Bean pod mottle virus (BPMV, genus
Comovirus) is an effective VIGS vector for soybean (Meyer et al.,
2009; Pandey et al., 2011). Further, BPMV has a bipartite
positive-strand RNA genome consisting of RNA-1 and RNA-2. A
DNA-based system was used to place the cDNAs of BPMV genomic RNA1
and RNA2 under control of the Cauliflower mosaic Virus (CaMV 35S)
promoter (Zhang et al., 2009) to clone 328-bp of GmSHMT into RNA2
and generate infectious tissue by biolistic delivery into soybean
leaf tissues.
[0154] Nematode infection assays were conducted on soybean plants
either inoculated with BPMV-SHMT or BPMV only. Two RIL lines were
used which differed at the majority of markers assigned to the Rhg4
region and appeared to be nearly opposite recombination events. RIL
lines, E.times.F67 (rhg1.sub.Frhg1.sub.FRhg4.sub.FRhg4.sub.F) and
E.times.F63 (rhg1.sub.Frhg1.sub.FRhg4.sub.ERhg4.sub.E), are
SCN-resistant and SCN-susceptible, respectively (Liu et al.,
2011).
[0155] Results showed that silencing of the GmSHMT gene in the
SCN-resistant RIL E.times.F67 resulted in a 29% increase in
susceptibility to SCN compared to E.times.F67 inoculated with BPMV
only (see e.g., FIG. 4A; P<0.0001). At the time of nematode
inoculation of the plants, GmSHMT expression was determined by
qRT-PCR to be reduced by an average of 74% in the roots of plants
inoculated with BPMV-SHMT compared with those inoculated with BPMV
only (see e.g., FIG. 4B).
[0156] Given the partial transcript reduction, transient nature,
and inability to control spatial silencing of GmSHMT in roots with
respect to the nematode using VIGS, a complementary targeted RNAi
gene silencing approach was employed to test GmSHMT for a role in
resistance to SCN. A 338-bp dsRNA corresponding to GmSHMT was
expressed under control of a SCN-inducible zinc finger
transcription factor promoter (Kandoth et al., 2011) in soybean
hairy roots.
[0157] Results showed that nematode reproduction on hairy roots of
the SCN resistant RIL E.times.F67 transformed with pZF-SHMTi was
greater than on E.times.F67 hairy roots transformed with the
pZF-GUSi control (see e.g., FIG. 4C; P<0.01). No statistically
significant difference in nematode reproduction was observed
between E.times.F67 hairy roots transformed with pZF-SHMTi and
E.times.F63 hairy roots transformed with pZF-GUSi (see e.g., FIG.
4C).
[0158] These data further confirmed a role for GmSHMT in conferring
SCN resistance.
[0159] 2.3-kb of putative promoter sequence of GmSHMT from Forrest
and Essex was fused with the .beta.-glucuronidase (GUS) reporter
gene to determine whether the GmSHMT gene is expressed in syncytial
feeding cells induced by SCN and to determine whether the
difference in resistance between the susceptible and resistant
cultivars is related to the expression level of Rhg4, (Jefferson,
1987).
[0160] Results from nematode infection assays of soybean hairy
roots transformed with the pSHMT-GUS constructs confirmed
expression of GmSHMT within syncytia (see e.g., FIGS. 4D-4F). The
same pattern of GUS expression was also observed in
nematode-infected soybean hairy roots of E.times.F63 transformed
with the Essex pSHMT-GUS construct (see e.g., FIG. 7B). No visible
difference was detected between the resistant and susceptible line,
which is in agreement with only three polymorphisms between Forrest
and Essex in the putative 2.3-kb promoter region of GmSHMT (see
e.g., FIG. 7A).
[0161] To confirm that GmSHMT was Rhg4, SCN susceptible RIL
E.times.F63 was transformed with a 5.1-kb genomic fragment that
included the Forrest SHMT gene with 2.3-kb of sequence upstream of
the start and 0.57-kb downstream of the stop codon (gSHMT).
[0162] Results of nematode infection assays with H. glycines
demonstrated restored resistance in the complemented transformed
hairy roots (see e.g., FIG. 4G; P<0.0001), confirming that
GmSHMT is the Rhg4 gene.
Example 16: Modeled Structure of GmSHMT
[0163] Using homology modeling, the structure of GmSHMT was
predicted to examine how the variant genotypes (from the resistant
and susceptible cultivars and TILLING mutants) may be affecting its
structural and functional properties.
[0164] Methods were according to Examples 1-12, unless described
otherwise.
[0165] The available structural information about small ligand
binding by SHMT homologs was analyzed and mapped the ligand binding
sites onto the surface of the GmSHMT model. As a result, five
putative binding sites were determined, including two glycine
(GS.sub.1 and GS.sub.2), one PLP-serine (PLS), one PLP-glycine
(PLG), and one THF/MTHF/5-formylTHF (FTHF) binding site (see e.g.,
FIG. 5B), with the latter three binding sites physically
co-localized in the binding pocket formed by the SHMT dimer
molecule. When mapping the Forrest and TILLING mutations onto the
structural model of GmSHMT, both Forrest mutations and TILLING
mutation F6266 E61K were found to be in close proximity to the
tentative ligand binding sites. Specifically, it was found that the
Forrest mutations P130R and N358Y were co-localized with the
THF/MTHF/FTHF binding site and in close proximity to PLS, PLG and
one of the two glycine binding sites. The position of the F6266
E61K mutation overlapped with the PLS and THF/MTHF/FTHF binding
sites. These findings suggest that the three mutations may directly
affect the reversible interconversion of L-serine and THF to
glycine and MTHF. On the other hand, TILLING mutation F6756 M1251
was found in an interior beta sheet (see e.g., FIGS. 5A-5B),
suggesting that there may be a different mechanism altering the
function of GmSHMT in this mutant, perhaps through the structural
instability of the region affected by the TILLING mutation. This
hypothesis is in agreement with the previous functional genomics
analysis using SIFT software (Kumar et al., 2009), which suggested
that F6756 M1251 is likely to be a deleterious mutation.
GENBANK ACCESSION NUMBERS
[0166] Forrest full-length genomic DNA, JQ714083; Essex full-length
genomic DNA, JQ714084; Forrest cDNA sequence, JQ714080; Essex cDNA
sequence, JQ714079; Forrest TILLING mutant F6266 sequence,
JQ714081; Forrest TILLING mutant F6756 sequence, JQ714082.
TABLE-US-00003 SEQUENCES SEQ ID NO: 1 Essex Gm08-A2 SHMTcDNA
sequence (1416 bases) 1 ATGGATCCAG TAAGCGTGTG GGGTAACACG CCCTTGGCGA
CGGTGGATCC CGAGATCCAT GACCTCATCG AGAAGGAGAA 81 GCGCCGTCAA
TGCCGCGGAA TCGAGCTCAT CGCCTCCGAG AACTTCACCT CCTTCGCCGT CATCGAGGCC
CTCGGCAGCG 161 CTCTCACGAA CAAATACTCC GAGGGCATGC CGGGCAACCG
CTACTACGGC GGCAATGAAT ACATCGACCA GATCGAAAAC 241 CTCTGCCGCT
CACGCGCCCT CCAAGCCTTC CACCTCGACG CCCAATCCTG GGGCGTCAAC GTCCAGCCCT
ACTCCGGCTC 321 CCCGGCCAAC TTCGCCGCCT ACACCGCCGT CCTCAACCCC
CACGACCGCA TCATGGGGCT AGATCTCCCC TCCGGCGGCC 401 ACCTCACCCA
CGGCTACTAC ACCTCCGGCG GAAAGAAGAT CTCCGCCACC TCCATTTACT TCGAGAGTCT
CCCTTACAAG 481 GTAAACTCCA CCACCGGCTA CATCGACTAC GACCGCTTGG
AAGAAAAAGC CCTAGACTTC AGGCCAAAAC TCATAATCTG 561 CGGTGGCAGC
GCGTACCCTC GCGATTGGGA CTACAAACGT TTCAGGGAAG TCGCTGATAA GTGCGGAGCA
TTGCTTCTCT 641 GCGACATGGC GCACACTAGC GGCCTTGTGG CCGCGCAGGA
AGTGAACAGC CCCTTCGAGT ATTGCGACAT TGTGACCACC 721 ACGACTCACA
AGAGCTTGCG GGGCCCACGT GCGGGGATGA TCTTTTACCG GAAGGGCCCC AAGCCGCCGA
AGAAGGGGCA 801 GCCGGAGAAC GCGGTTTATG ATTTCGAGGA CAAGATTAAC
TTCGCGGTGT TCCCTTCGCT GCAGGGTGGG CCCCACAACC 881 ACCAGATCGG
TGCTCTCGCC GTGGCGCTGA AGCAGGCCGC GTCGCCCGGG TTTAAGGCCT ACGCGAAGCA
GGTTAAGGCG 961 AACGCCGTTG CGCTTGGAAA ATACTTGATG GGGAAAGGGT
ACAGCCTTGT CACTGGCGGA ACGGAGAACC ATCTTGTTTT 1041 GTGGGATCTG
AGACCTCTTG GATTGACTGG GAATAAGGTG GAGAAACTCT GTGATCTCTG TAACATTACT
GTTAACAAGA 1121 ACGCTGTTTT TGGTGATAGC AGTGCCTTGG CCCCTGGTGG
AGTGCGAATT GGTGCCCCTG CCATGACTTC TAGGGGTTTG 1201 GTTGAAAAAG
ACTTTGAGCA GATTGGTGAG TTCCTTCACC GTGCTGTGAC TCTCACACTG GAGATCCAGA
AGGAGCATGG 1281 CAAACTTCTC AAGGATTTCA ACAAGGGTCT CGTCAACAAC
AAGGCTATTG AAGATCTCAA AGCTGATGTT GAGAAGTTCT 1361 CTGCCTTGTT
TGACATGCCT GGCTTCCTGG TATCTGAAAT GAAGTACAAG GATTAG SEQ ID NO: 2
Essex Gm08-A2 SHMT protein sequence (471 aa) 1 MDPVSVWGNT
PLATVDPEIH DLIEKEKRRQ CRGIELIASE NFTSFAVIEA LGSALTNKYS EGMPGNRYYG
GNEYIDQIEN 81 LCRSRALQAF HLDAQSWGVN VQPYSGSPAN FAAYTAVLNP
HDRIMGLDLP SGGHLTHGYY TSGGKKISAT SIYFESLPYK 161 VNSTTGYIDY
DRLEEKALDF RPKLIICGGS AYPRDWDYKR FREVADKCGA LLLCDMAHTS GLVAAQEVNS
PFEYCDIVTT 241 TTHKSLRGPR AGMIFYRKGP KPPKKGQPEN AVYDFEDKIN
FAVFPSLQGG PHNHQIGALA VALKQAASPG FKAYAKQVKA 321 NAVALGKYLM
GKGYSLVTGG TENHLVLWDL RPLGLTGNKV EKLCDLCNIT VNKNAVFGDS SALAPGGVRI
GAPAMTSRGL 401 VEKDFEQIGE FLHRAVTLTL EIQKEHGKLL KDFNKGLVNN
KAIEDLKADV EKFSALFDMP GFLVSEMKYK D SEQ ID NO: 3 Essex
GM08-A2-SHMTgDNA (5105 bases) 1 CAATGGCACC AATGCCCAAT GGGAGATTTA
AGTCAAGCCC AACATCAACC TCTGAAATTA TGAATTATGA AATTAAAATG 81
CTTCCTAGTA AGTGAACTAG TTGCATCTCA TTTATATCAT AAATTTCGAA CTACGACTTT
CTTGGCCATG TTAGTAAAGT 161 TTGGGGGATT GTTCAAAATT GGTGGAGTGG
TTCAGCTTAA TCTCCAAATT ATTTGTTCTA AGTTGTTTTG GTAGGCAGGT 241
TTAATTTTTT CCTGATCCTG GGAAAAAAAT TATTGATACC ATATTAACAT CTCTTGACGA
TGCTACGAGA TTTCTCATGA 321 TTATAGAACT GAGTAGGGTG GCTTAAAAGG
TTTTATTTTA AATATAATTT CACCACATTG AATTGGGTAT TAGTAAACTG 401
GTTACTGGTA TGCCTGTAAA GTGGACAATG ATAAATGTTT TTATAGAAGT TGGTATGGAT
TTTAAAATAG CTCATGTATA 481 AAATGTGAAA AAGGAAACGT GAACTAAAAT
GCTAATAATA AAAGATAAAG ACTAAATTAA TTAAAGTTAA AGGATAAAAT 561
GCTTGTTACA TCAAGTCATT TTAAAGGTGC ACTATTAGAG GCTGCACAGT AAAAGTTAAC
ACTGATATAT TTTTAAAGAT 641 GTTCTTAGTT AAATAGCTTT TGACTTGATG
GGGTGAAGAC ACAAGAGGTT GTTGTTGCGA TGTGATTTTG GCTGAATATG 721
CATGCCTGCT GAACATTGAC TTCATTGTTA AATCAAAATT AATCCCATAG ACCTATTGTA
TTATTTAAGG GGATCAATTT 801 CATAAATCAA AATTTATTGG TTGGGGAAAA
AAACAATGTT TAGTAGTTCC CAGTCATATT CAGAAACCTA CAAATTAACT 881
ATCCCCCATG TTAATGAAGC AAGGTGTGGG GGAAGGAAAG AGTCAGCATC AGTGAAGTAG
AGAGGGGGGT TGGTGATTTT 961 GGTGGGAATA AATTGGCTAT ATTGCCCCCA
CCAACCTCGT TGCTACCAAA TACCAACAAC ACTGACTCAC TGAGAATTGG 1041
GAAAGAAACT TAAAACCAAG TCTTGCAGTG ACGTACATGC AGTGTGTGCA TCACACATTC
AGGTTTCCAG TCAAATTGTA 1121 GAACAAATGA ATTTCTTGCT TTAACTTAAG
TTGAAGTTTA AGAAGTGAAG CTGATGCTTG TTTTTGAATG AAAAGCCTTT 1201
GATAGTTTGA TGTAAGCATT TTCCAAATTT AACTCTTCCC ATGCTTGACA GAGCCAATTA
AGCTAACTGG TTTGATAACA 1281 AGTAAACTTC TAAATCTATG AGTATGAGTG
CATGCAGCAC ACCTTTTAAA CACAAGCCAC TGTTTTGTCT TTTTTATCAA 1361
CAGAAAGAGA ATCCTACTAA TAACACTAAT CAAGATCGCT GCTCTTTTCT GTTTATTTTT
CTTAATAAAT TAACTTTTGT 1441 TTTGTACTCC TGTTAAACAA CTGCTCTATT
TGTTTCATGT GTTGCATTAA ATAACATGGT TTTATTCACA TCTACAAGCA 1521
AAATTTCCTA AAAACTGTGA ATGATGTAGA AGCAAGTCAT TTATGTTTTG AAATTCACGC
ATTGGAGTTT CTAACGCCCA 1601 ACCAACCAAA CGGTAATATG AATATCGTGT
TTGGAACAAA TTAGAATTTA GGACATAATT TTTCACATCA GAATAAATGT 1681
TAGGAATTTT TGCTTTTACG TTTTTCGCAT TAAAATAATG TGATTTATCG GTTGTTCCTG
AACAATAACC ATCGATGTAA 1761 TTATAAAATT CTAATTTGTC CTATCCTGGG
GCGTCAACGT CCAGCCAAAT GCGTAACATT TATTCTGATG TAAAAAATTA 1841
TTATTATTAT TATAGATAAT AAAATCTTGT TCCTGAACAA TAACCATCAA TGTAATTATA
AAATTGAATC TTAGACTCAA 1921 AACTAGTTAT TAATCTGGAA CAATGTTTAC
TCAAAACTAG TTATTAATAG TATTTTTAAG TTAATTTGAA ATTTTTTTTT 2001
CGGCGTTAAA CAAATACTAG ATGTTTATAC TACAAATATT GATTATTGAT TATAAATTTA
TAAATGTTAA AAAAAAAAAA 2081 AAGAGAAAAC AAAGAATTGA AGTTGTGGTT
GGTAGTAAAC CAGCACCAGG CGAACAAGTG GACACAATTT ACCTACAAGT 2161
AACTAACCAA CCGGAAGCAC AGGCTACAAC GGTCCTTTCA CACCCGGTCT CAAAGCTTTT
AAAAACGAAC ACATACGCAC 2241 TCACATTTCC ATTCCACCTC AACAAACACA
ACAACACTCT CTCTTCTCGC TCTTGGCTTT TCGCTCTTCA CTCACTCTCA 2321
TTCATTCATT TCCACCGTTC ATGGATCCAG TAAGCGTGTG GGGTAACACG CCCTTGGCGA
CGGTGGATCC CGAGATCCAT 2401 GACCTCATCG AGAAGGAGAA GCGCCGTCAA
TGCCGCGGAA TCGAGCTCAT CGCCTCCGAG AACTTCACCT CCTTCGCCGT 2481
CATCGAGGCC CTCGGCAGCG CTCTCACGAA CAAATACTCC GAGGGCATGC CGGGCAACCG
CTACTACGGC GGCAATGAAT 2561 ACATCGACCA GATCGAAAAC CTCTGCCGCT
CACGCGCCCT CCAAGCCTTC CACCTCGACG CCCAATCCTG GGGCGTCAAC 2641
GTCCAGCCCT ACTCCGGCTC CCCGGCCAAC TTCGCCGCCT ACACCGCCGT CCTCAACCCC
CACGACCGCA TCATGGGGCT 2721 AGATCTCCCC TCCGGCGGCC ACCTCACCCA
CGGCTACTAC ACCTCCGGCG GAAAGAAGAT CTCCGCCACC TCCATTTACT 2801
TCGAGAGTCT CCCTTACAAG GTAAACTCCA CCACCGGCTA CATCGACTAC GACCGCTTGG
AAGAAAAAGC CCTAGACTTC 2881 AGGCCAAAAC TCATAATCTG CGGTGGCAGC
GCGTACCCTC GCGATTGGGA CTACAAACGT TTCAGGGAAG TCGCTGATAA 2961
GTGCGGAGCA TTGCTTCTCT GCGACATGGC GCACACTAGC GGCCTTGTGG CCGCGCAGGA
AGTGAACAGC CCCTTCGAGT 3041 ATTGCGACAT TGTGACCACC ACGACTCACA
AGAGCTTGCG GGGCCCACGT GCGGGGATGA TCTTTTACCG GAAGGGCCCC 3121
AAGCCGCCGA AGAAGGGGCA GCCGGAGAAC GCGGTTTATG ATTTCGAGGA CAAGATTAAC
TTCGCGGTGT TCCCTTCGCT 3201 GCAGGGTGGG CCCCACAACC ACCAGATCGG
TGCTCTCGCC GTGGCGCTGA AGCAGGCCGC GTCGCCCGGG TTTAAGGCCT 3281
ACGCGAAGCA GGTTAAGGCG AACGCCGTTG CGCTTGGAAA ATACTTGATG GGGAAAGGGT
ACAGCCTTGT CACTGGCGGA 3361 ACGGAGAACC ATCTTGTTTT GTGGGATCTG
AGACCTCTTG GATTGACTGG TAATATATAT AGGATTGGAT CTCTACCTTC 3441
TGGTTTTGAT TTGTTACAAA TGTCTATAAA TCTGACTTGT TCGTTGTGTG ATTGTTTTGC
AGGGAATAAG GTGGAGAAAC 3521 TCTGTGATCT CTGTAACATT ACTGTTAACA
AGAACGCTGT TTTTGGTGAT AGCAGTGCCT TGGCCCCTGG TGGAGTGCGA 3601
ATTGGTAACG ATCTTACTTC TCTTTTATAT GCTACAATAC AAATCTTGCT TTACTAACTC
AATTGGAAAC AAGATCTCAT 3681 TTATAAGATT ATAAAAATGA TTTCCTTAGG
CTAGGACTAT ATCCTCTCTC TCTCTCTCTC TTTTTCTTTT TTATCATCGC 3761
AGAACTTAGA TGAATTTTCT TACGTAATTT TAGTACTGTT CTCTTATCAG AGTTCGAAAG
TAAGTTATAA AATTTCTATT 3841 GAAGGCTTGC ATATTTATAT AAGTGAAATT
TTAATTTTGG TTGGAGAACA ATGTCCAAAA CACCAAAGTG ATTGCATCTA 3921
AGTTTTTTGG ATTTTTTAAT GTATTTGTAT TTTGTACAAG GTATCTTAGT AAGTTGTTGT
AGATTAGTAT TGAAAGAGAT 4001 TTCATTGAGG ATGTGTTTTT TAGTGCTTTA
ACAAAGGAGG TATGTTAGTT CGGGCTAAAG CTTGCAGACT GCCTTTGTTA 4081
AAGAATTTCG AGTTGTTGTC GTGCAATATG ATTGGCAAAT CAATTATAAA CTAATCTGTT
ATTTTGTTTT TCTGATACTT 4161 TTCCCTAGAA ATGAATTATT TTGATGTATC
AATTACCAAA ATGGTTTTTT TGTGCCCCCG TTTCTGTATT TTTCTCTGAT 4241
GTGTTAGATA AATGTGAGTG CCCCTGACTG GAGTTTCTGT GAACAGGTGC CCCTGCCATG
ACTTCTAGGG GTTTGGTTGA 4321 AAAAGACTTT GAGCAGATTG GTGAGTTCCT
TCACCGTGCT GTGACTCTCA CACTGGAGAT CCAGAAGGAG CATGGCAAAC 4401
TTCTCAAGGA TTTCAACAAG GGTCTCGTCA ACAACAAGGC TATTGAAGAT CTCAAAGCTG
ATGTTGAGAA GTTCTCTGCC 4481 TTGTTTGACA TGCCTGGCTT CCTGGTATCT
GAAATGAAGT ACAAGGATTA GGTTCAACCA
TACCACTTTC TACTAAATTG 4561 TGTCACTCAA GTTCGACACA AAGTGCAGAA
ATGGAGAAAA AGGAAATATG TGTCTTCCTT TCCTGGGAGT GATAGGGTTT 4641
ATCGCCATGG TGTTTCAATT CAAAAGTTTG AAGTTTCTTT GTCTTTGATT TCATGTTTAA
TTTTGTTAGC CTGATTGATA 4721 TCATATTTTT TTTCTTATTT AACAATTGAA
ATAATACGTG CTGCCTTTCT TTCTTTTTTT TTCCTCGCTA GCTAGTAGTA 4801
TGTTTCATGA TTTCATCTTC TAATATTGCT CAACAGAACA TCTTAATTCT TAACAACCAT
GAGTTTTAGT GGAGTTAAGC 4881 AAAAGAAAAA GTTATTCTAA TAAATCTATC
GTCTTTCTTA TGCCTCAATG TCCTATGCCT CTCCCCCCTA TTTGAAAACC 4961
AAAATGCTCC ATGTCTAATT GTGATAAGCT GACAATACCC GTCTGGCAAA TTATGAAGTC
AACATTTTTT TTTAGCTCAG 5041 CAATAACAAA TAATATTAAT TGCACAAGTG
CTAAAATAAC AATTGTTGGG CACCATAAAA TCTGG SEQ ID NO: 4 Essex Gm08-A2
SHMT promoter 1 CAATGGCACC AATGCCCAAT GGGAGATTTA AGTCAAGCCC
AACATCAACC TCTGAAATTA TGAATTATGA AATTAAAATG 81 CTTCCTAGTA
AGTGAACTAG TTGCATCTCA TTTATATCAT AAATTTCGAA CTACGACTTT CTTGGCCATG
TTAGTAAAGT 161 TTGGGGGATT GTTCAAAATT GGTGGAGTGG TTCAGCTTAA
TCTCCAAATT ATTTGTTCTA AGTTGTTTTG GTAGGCAGGT 241 TTAATTTTTT
CCTGATCCTG GGAAAAAAAT TATTGATACC ATATTAACAT CTCTTGACGA TGCTACGAGA
TTTCTCATGA 321 TTATAGAACT GAGTAGGGTG GCTTAAAAGG TTTTATTTTA
AATATAATTT CACCACATTG AATTGGGTAT TAGTAAACTG 401 GTTACTGGTA
TGCCTGTAAA GTGGACAATG ATAAATGTTT TTATAGAAGT TGGTATGGAT TTTAAAATAG
CTCATGTATA 481 AAATGTGAAA AAGGAAACGT GAACTAAAAT GCTAATAATA
AAAGATAAAG ACTAAATTAA TTAAAGTTAA AGGATAAAAT 561 GCTTGTTACA
TCAAGTCATT TTAAAGGTGC ACTATTAGAG GCTGCACAGT AAAAGTTAAC ACTGATATAT
TTTTAAAGAT 641 GTTCTTAGTT AAATAGCTTT TGACTTGATG GGGTGAAGAC
ACAAGAGGTT GTTGTTGCGA TGTGATTTTG GCTGAATATG 721 CATGCCTGCT
GAACATTGAC TTCATTGTTA AATCAAAATT AATCCCATAG ACCTATTGTA TTATTTAAGG
GGATCAATTT 801 CATAAATCAA AATTTATTGG TTGGGGAAAA AAACAATGTT
TAGTAGTTCC CAGTCATATT CAGAAACCTA CAAATTAACT 881 ATCCCCCATG
TTAATGAAGC AAGGTGTGGG GGAAGGAAAG AGTCAGCATC AGTGAAGTAG AGAGGGGGGT
TGGTGATTTT 961 GGTGGGAATA AATTGGCTAT ATTGCCCCCA CCAACCTCGT
TGCTACCAAA TACCAACAAC ACTGACTCAC TGAGAATTGG 1041 GAAAGAAACT
TAAAACCAAG TCTTGCAGTG ACGTACATGC AGTGTGTGCA TCACACATTC AGGTTTCCAG
TCAAATTGTA 1121 GAACAAATGA ATTTCTTGCT TTAACTTAAG TTGAAGTTTA
AGAAGTGAAG CTGATGCTTG TTTTTGAATG AAAAGCCTTT 1201 GATAGTTTGA
TGTAAGCATT TTCCAAATTT AACTCTTCCC ATGCTTGACA GAGCCAATTA AGCTAACTGG
TTTGATAACA 1281 AGTAAACTTC TAAATCTATG AGTATGAGTG CATGCAGCAC
ACCTTTTAAA CACAAGCCAC TGTTTTGTCT TTTTTATCAA 1361 CAGAAAGAGA
ATCCTACTAA TAACACTAAT CAAGATCGCT GCTCTTTTCT GTTTATTTTT CTTAATAAAT
TAACTTTTGT 1441 TTTGTACTCC TGTTAAACAA CTGCTCTATT TGTTTCATGT
GTTGCATTAA ATAACATGGT TTTATTCACA TCTACAAGCA 1521 AAATTTCCTA
AAAACTGTGA ATGATGTAGA AGCAAGTCAT TTATGTTTTG AAATTCACGC ATTGGAGTTT
CTAACGCCCA 1601 ACCAACCAAA CGGTAATATG AATATCGTGT TTGGAACAAA
TTAGAATTTA GGACATAATT TTTCACATCA GAATAAATGT 1681 TAGGAATTTT
TGCTTTTACG TTTTTCGCAT TAAAATAATG TGATTTATCG GTTGTTCCTG AACAATAACC
ATCGATGTAA 1761 TTATAAAATT CTAATTTGTC CTATCCTGGG GCGTCAACGT
CCAGCCAAAT GCGTAACATT TATTCTGATG TAAAAAATTA 1841 TTATTATTAT
TATAGATAAT AAAATCTTGT TCCTGAACAA TAACCATCAA TGTAATTATA AAATTGAATC
TTAGACTCAA 1921 AACTAGTTAT TAATCTGGAA CAATGTTTAC TCAAAACTAG
TTATTAATAG TATTTTTAAG TTAATTTGAA ATTTTTTTTT 2001 CGGCGTTAAA
CAAATACTAG ATGTTTATAC TACAAATATT GATTATTGAT TATAAATTTA TAAATGTTAA
AAAAAAAAAA 2081 AAGAGAAAAC AAAGAATTGA AGTTGTGGTT GGTAGTAAAC
CAGCACCAGG CGAACAAGTG GACACAATTT ACCTACAAGT 2161 AACTAACCAA
CCGGAAGCAC AGGCTACAAC GGTCCTTTCA CACCCGGTCT CAAAGCTTTT AAAAACGAAC
ACATACGCAC 2241 TCACATTTCC ATTCCACCTC AACAAACACA ACAACACTCT
CTCTTCTCGC TCTTGGCTTT TCGCTCTTCA CTCACTCTCA 2321 TTCATTCATT
TCCACCGTTC
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Sequence CWU 1
1
12311416DNAGlycine max 1atggatccag taagcgtgtg gggtaacacg cccttggcga
cggtggatcc cgagatccat 60gacctcatcg agaaggagaa gcgccgtcaa tgccgcggaa
tcgagctcat cgcctccgag 120aacttcacct ccttcgccgt catcgaggcc
ctcggcagcg ctctcacgaa caaatactcc 180gagggcatgc cgggcaaccg
ctactacggc ggcaatgaat acatcgacca gatcgaaaac 240ctctgccgct
cacgcgccct ccaagccttc cacctcgacg cccaatcctg gggcgtcaac
300gtccagccct actccggctc cccggccaac ttcgccgcct acaccgccgt
cctcaacccc 360cacgaccgca tcatggggct agatctcccc tccggcggcc
acctcaccca cggctactac 420acctccggcg gaaagaagat ctccgccacc
tccatttact tcgagagtct cccttacaag 480gtaaactcca ccaccggcta
catcgactac gaccgcttgg aagaaaaagc cctagacttc 540aggccaaaac
tcataatctg cggtggcagc gcgtaccctc gcgattggga ctacaaacgt
600ttcagggaag tcgctgataa gtgcggagca ttgcttctct gcgacatggc
gcacactagc 660ggccttgtgg ccgcgcagga agtgaacagc cccttcgagt
attgcgacat tgtgaccacc 720acgactcaca agagcttgcg gggcccacgt
gcggggatga tcttttaccg gaagggcccc 780aagccgccga agaaggggca
gccggagaac gcggtttatg atttcgagga caagattaac 840ttcgcggtgt
tcccttcgct gcagggtggg ccccacaacc accagatcgg tgctctcgcc
900gtggcgctga agcaggccgc gtcgcccggg tttaaggcct acgcgaagca
ggttaaggcg 960aacgccgttg cgcttggaaa atacttgatg gggaaagggt
acagccttgt cactggcgga 1020acggagaacc atcttgtttt gtgggatctg
agacctcttg gattgactgg gaataaggtg 1080gagaaactct gtgatctctg
taacattact gttaacaaga acgctgtttt tggtgatagc 1140agtgccttgg
cccctggtgg agtgcgaatt ggtgcccctg ccatgacttc taggggtttg
1200gttgaaaaag actttgagca gattggtgag ttccttcacc gtgctgtgac
tctcacactg 1260gagatccaga aggagcatgg caaacttctc aaggatttca
acaagggtct cgtcaacaac 1320aaggctattg aagatctcaa agctgatgtt
gagaagttct ctgccttgtt tgacatgcct 1380ggcttcctgg tatctgaaat
gaagtacaag gattag 14162471PRTGlycine max 2Met Asp Pro Val Ser Val
Trp Gly Asn Thr Pro Leu Ala Thr Val Asp 1 5 10 15 Pro Glu Ile His
Asp Leu Ile Glu Lys Glu Lys Arg Arg Gln Cys Arg 20 25 30 Gly Ile
Glu Leu Ile Ala Ser Glu Asn Phe Thr Ser Phe Ala Val Ile 35 40 45
Glu Ala Leu Gly Ser Ala Leu Thr Asn Lys Tyr Ser Glu Gly Met Pro 50
55 60 Gly Asn Arg Tyr Tyr Gly Gly Asn Glu Tyr Ile Asp Gln Ile Glu
Asn 65 70 75 80 Leu Cys Arg Ser Arg Ala Leu Gln Ala Phe His Leu Asp
Ala Gln Ser 85 90 95 Trp Gly Val Asn Val Gln Pro Tyr Ser Gly Ser
Pro Ala Asn Phe Ala 100 105 110 Ala Tyr Thr Ala Val Leu Asn Pro His
Asp Arg Ile Met Gly Leu Asp 115 120 125 Leu Pro Ser Gly Gly His Leu
Thr His Gly Tyr Tyr Thr Ser Gly Gly 130 135 140 Lys Lys Ile Ser Ala
Thr Ser Ile Tyr Phe Glu Ser Leu Pro Tyr Lys 145 150 155 160 Val Asn
Ser Thr Thr Gly Tyr Ile Asp Tyr Asp Arg Leu Glu Glu Lys 165 170 175
Ala Leu Asp Phe Arg Pro Lys Leu Ile Ile Cys Gly Gly Ser Ala Tyr 180
185 190 Pro Arg Asp Trp Asp Tyr Lys Arg Phe Arg Glu Val Ala Asp Lys
Cys 195 200 205 Gly Ala Leu Leu Leu Cys Asp Met Ala His Thr Ser Gly
Leu Val Ala 210 215 220 Ala Gln Glu Val Asn Ser Pro Phe Glu Tyr Cys
Asp Ile Val Thr Thr 225 230 235 240 Thr Thr His Lys Ser Leu Arg Gly
Pro Arg Ala Gly Met Ile Phe Tyr 245 250 255 Arg Lys Gly Pro Lys Pro
Pro Lys Lys Gly Gln Pro Glu Asn Ala Val 260 265 270 Tyr Asp Phe Glu
Asp Lys Ile Asn Phe Ala Val Phe Pro Ser Leu Gln 275 280 285 Gly Gly
Pro His Asn His Gln Ile Gly Ala Leu Ala Val Ala Leu Lys 290 295 300
Gln Ala Ala Ser Pro Gly Phe Lys Ala Tyr Ala Lys Gln Val Lys Ala 305
310 315 320 Asn Ala Val Ala Leu Gly Lys Tyr Leu Met Gly Lys Gly Tyr
Ser Leu 325 330 335 Val Thr Gly Gly Thr Glu Asn His Leu Val Leu Trp
Asp Leu Arg Pro 340 345 350 Leu Gly Leu Thr Gly Asn Lys Val Glu Lys
Leu Cys Asp Leu Cys Asn 355 360 365 Ile Thr Val Asn Lys Asn Ala Val
Phe Gly Asp Ser Ser Ala Leu Ala 370 375 380 Pro Gly Gly Val Arg Ile
Gly Ala Pro Ala Met Thr Ser Arg Gly Leu 385 390 395 400 Val Glu Lys
Asp Phe Glu Gln Ile Gly Glu Phe Leu His Arg Ala Val 405 410 415 Thr
Leu Thr Leu Glu Ile Gln Lys Glu His Gly Lys Leu Leu Lys Asp 420 425
430 Phe Asn Lys Gly Leu Val Asn Asn Lys Ala Ile Glu Asp Leu Lys Ala
435 440 445 Asp Val Glu Lys Phe Ser Ala Leu Phe Asp Met Pro Gly Phe
Leu Val 450 455 460 Ser Glu Met Lys Tyr Lys Asp 465 470
35105DNAGlycine max 3caatggcacc aatgcccaat gggagattta agtcaagccc
aacatcaacc tctgaaatta 60tgaattatga aattaaaatg cttcctagta agtgaactag
ttgcatctca tttatatcat 120aaatttcgaa ctacgacttt cttggccatg
ttagtaaagt ttgggggatt gttcaaaatt 180ggtggagtgg ttcagcttaa
tctccaaatt atttgttcta agttgttttg gtaggcaggt 240ttaatttttt
cctgatcctg ggaaaaaaat tattgatacc atattaacat ctcttgacga
300tgctacgaga tttctcatga ttatagaact gagtagggtg gcttaaaagg
ttttatttta 360aatataattt caccacattg aattgggtat tagtaaactg
gttactggta tgcctgtaaa 420gtggacaatg ataaatgttt ttatagaagt
tggtatggat tttaaaatag ctcatgtata 480aaatgtgaaa aaggaaacgt
gaactaaaat gctaataata aaagataaag actaaattaa 540ttaaagttaa
aggataaaat gcttgttaca tcaagtcatt ttaaaggtgc actattagag
600gctgcacagt aaaagttaac actgatatat ttttaaagat gttcttagtt
aaatagcttt 660tgacttgatg gggtgaagac acaagaggtt gttgttgcga
tgtgattttg gctgaatatg 720catgcctgct gaacattgac ttcattgtta
aatcaaaatt aatcccatag acctattgta 780ttatttaagg ggatcaattt
cataaatcaa aatttattgg ttggggaaaa aaacaatgtt 840tagtagttcc
cagtcatatt cagaaaccta caaattaact atcccccatg ttaatgaagc
900aaggtgtggg ggaaggaaag agtcagcatc agtgaagtag agaggggggt
tggtgatttt 960ggtgggaata aattggctat attgccccca ccaacctcgt
tgctaccaaa taccaacaac 1020actgactcac tgagaattgg gaaagaaact
taaaaccaag tcttgcagtg acgtacatgc 1080agtgtgtgca tcacacattc
aggtttccag tcaaattgta gaacaaatga atttcttgct 1140ttaacttaag
ttgaagttta agaagtgaag ctgatgcttg tttttgaatg aaaagccttt
1200gatagtttga tgtaagcatt ttccaaattt aactcttccc atgcttgaca
gagccaatta 1260agctaactgg tttgataaca agtaaacttc taaatctatg
agtatgagtg catgcagcac 1320accttttaaa cacaagccac tgttttgtct
tttttatcaa cagaaagaga atcctactaa 1380taacactaat caagatcgct
gctcttttct gtttattttt cttaataaat taacttttgt 1440tttgtactcc
tgttaaacaa ctgctctatt tgtttcatgt gttgcattaa ataacatggt
1500tttattcaca tctacaagca aaatttccta aaaactgtga atgatgtaga
agcaagtcat 1560ttatgttttg aaattcacgc attggagttt ctaacgccca
accaaccaaa cggtaatatg 1620aatatcgtgt ttggaacaaa ttagaattta
ggacataatt tttcacatca gaataaatgt 1680taggaatttt tgcttttacg
tttttcgcat taaaataatg tgatttatcg gttgttcctg 1740aacaataacc
atcgatgtaa ttataaaatt ctaatttgtc ctatcctggg gcgtcaacgt
1800ccagccaaat gcgtaacatt tattctgatg taaaaaatta ttattattat
tatagataat 1860aaaatcttgt tcctgaacaa taaccatcaa tgtaattata
aaattgaatc ttagactcaa 1920aactagttat taatctggaa caatgtttac
tcaaaactag ttattaatag tatttttaag 1980ttaatttgaa attttttttt
cggcgttaaa caaatactag atgtttatac tacaaatatt 2040gattattgat
tataaattta taaatgttaa aaaaaaaaaa aagagaaaac aaagaattga
2100agttgtggtt ggtagtaaac cagcaccagg cgaacaagtg gacacaattt
acctacaagt 2160aactaaccaa ccggaagcac aggctacaac ggtcctttca
cacccggtct caaagctttt 2220aaaaacgaac acatacgcac tcacatttcc
attccacctc aacaaacaca acaacactct 2280ctcttctcgc tcttggcttt
tcgctcttca ctcactctca ttcattcatt tccaccgttc 2340atggatccag
taagcgtgtg gggtaacacg cccttggcga cggtggatcc cgagatccat
2400gacctcatcg agaaggagaa gcgccgtcaa tgccgcggaa tcgagctcat
cgcctccgag 2460aacttcacct ccttcgccgt catcgaggcc ctcggcagcg
ctctcacgaa caaatactcc 2520gagggcatgc cgggcaaccg ctactacggc
ggcaatgaat acatcgacca gatcgaaaac 2580ctctgccgct cacgcgccct
ccaagccttc cacctcgacg cccaatcctg gggcgtcaac 2640gtccagccct
actccggctc cccggccaac ttcgccgcct acaccgccgt cctcaacccc
2700cacgaccgca tcatggggct agatctcccc tccggcggcc acctcaccca
cggctactac 2760acctccggcg gaaagaagat ctccgccacc tccatttact
tcgagagtct cccttacaag 2820gtaaactcca ccaccggcta catcgactac
gaccgcttgg aagaaaaagc cctagacttc 2880aggccaaaac tcataatctg
cggtggcagc gcgtaccctc gcgattggga ctacaaacgt 2940ttcagggaag
tcgctgataa gtgcggagca ttgcttctct gcgacatggc gcacactagc
3000ggccttgtgg ccgcgcagga agtgaacagc cccttcgagt attgcgacat
tgtgaccacc 3060acgactcaca agagcttgcg gggcccacgt gcggggatga
tcttttaccg gaagggcccc 3120aagccgccga agaaggggca gccggagaac
gcggtttatg atttcgagga caagattaac 3180ttcgcggtgt tcccttcgct
gcagggtggg ccccacaacc accagatcgg tgctctcgcc 3240gtggcgctga
agcaggccgc gtcgcccggg tttaaggcct acgcgaagca ggttaaggcg
3300aacgccgttg cgcttggaaa atacttgatg gggaaagggt acagccttgt
cactggcgga 3360acggagaacc atcttgtttt gtgggatctg agacctcttg
gattgactgg taatatatat 3420aggattggat ctctaccttc tggttttgat
ttgttacaaa tgtctataaa tctgacttgt 3480tcgttgtgtg attgttttgc
agggaataag gtggagaaac tctgtgatct ctgtaacatt 3540actgttaaca
agaacgctgt ttttggtgat agcagtgcct tggcccctgg tggagtgcga
3600attggtaacg atcttacttc tcttttatat gctacaatac aaatcttgct
ttactaactc 3660aattggaaac aagatctcat ttataagatt ataaaaatga
tttccttagg ctaggactat 3720atcctctctc tctctctctc tttttctttt
ttatcatcgc agaacttaga tgaattttct 3780tacgtaattt tagtactgtt
ctcttatcag agttcgaaag taagttataa aatttctatt 3840gaaggcttgc
atatttatat aagtgaaatt ttaattttgg ttggagaaca atgtccaaaa
3900caccaaagtg attgcatcta agttttttgg attttttaat gtatttgtat
tttgtacaag 3960gtatcttagt aagttgttgt agattagtat tgaaagagat
ttcattgagg atgtgttttt 4020tagtgcttta acaaaggagg tatgttagtt
cgggctaaag cttgcagact gcctttgtta 4080aagaatttcg agttgttgtc
gtgcaatatg attggcaaat caattataaa ctaatctgtt 4140attttgtttt
tctgatactt ttccctagaa atgaattatt ttgatgtatc aattaccaaa
4200atggtttttt tgtgcccccg tttctgtatt tttctctgat gtgttagata
aatgtgagtg 4260cccctgactg gagtttctgt gaacaggtgc ccctgccatg
acttctaggg gtttggttga 4320aaaagacttt gagcagattg gtgagttcct
tcaccgtgct gtgactctca cactggagat 4380ccagaaggag catggcaaac
ttctcaagga tttcaacaag ggtctcgtca acaacaaggc 4440tattgaagat
ctcaaagctg atgttgagaa gttctctgcc ttgtttgaca tgcctggctt
4500cctggtatct gaaatgaagt acaaggatta ggttcaacca taccactttc
tactaaattg 4560tgtcactcaa gttcgacaca aagtgcagaa atggagaaaa
aggaaatatg tgtcttcctt 4620tcctgggagt gatagggttt atcgccatgg
tgtttcaatt caaaagtttg aagtttcttt 4680gtctttgatt tcatgtttaa
ttttgttagc ctgattgata tcatattttt tttcttattt 4740aacaattgaa
ataatacgtg ctgcctttct ttcttttttt ttcctcgcta gctagtagta
4800tgtttcatga tttcatcttc taatattgct caacagaaca tcttaattct
taacaaccat 4860gagttttagt ggagttaagc aaaagaaaaa gttattctaa
taaatctatc gtctttctta 4920tgcctcaatg tcctatgcct ctccccccta
tttgaaaacc aaaatgctcc atgtctaatt 4980gtgataagct gacaataccc
gtctggcaaa ttatgaagtc aacatttttt tttagctcag 5040caataacaaa
taatattaat tgcacaagtg ctaaaataac aattgttggg caccataaaa 5100tctgg
510542340DNAGlycine max 4caatggcacc aatgcccaat gggagattta
agtcaagccc aacatcaacc tctgaaatta 60tgaattatga aattaaaatg cttcctagta
agtgaactag ttgcatctca tttatatcat 120aaatttcgaa ctacgacttt
cttggccatg ttagtaaagt ttgggggatt gttcaaaatt 180ggtggagtgg
ttcagcttaa tctccaaatt atttgttcta agttgttttg gtaggcaggt
240ttaatttttt cctgatcctg ggaaaaaaat tattgatacc atattaacat
ctcttgacga 300tgctacgaga tttctcatga ttatagaact gagtagggtg
gcttaaaagg ttttatttta 360aatataattt caccacattg aattgggtat
tagtaaactg gttactggta tgcctgtaaa 420gtggacaatg ataaatgttt
ttatagaagt tggtatggat tttaaaatag ctcatgtata 480aaatgtgaaa
aaggaaacgt gaactaaaat gctaataata aaagataaag actaaattaa
540ttaaagttaa aggataaaat gcttgttaca tcaagtcatt ttaaaggtgc
actattagag 600gctgcacagt aaaagttaac actgatatat ttttaaagat
gttcttagtt aaatagcttt 660tgacttgatg gggtgaagac acaagaggtt
gttgttgcga tgtgattttg gctgaatatg 720catgcctgct gaacattgac
ttcattgtta aatcaaaatt aatcccatag acctattgta 780ttatttaagg
ggatcaattt cataaatcaa aatttattgg ttggggaaaa aaacaatgtt
840tagtagttcc cagtcatatt cagaaaccta caaattaact atcccccatg
ttaatgaagc 900aaggtgtggg ggaaggaaag agtcagcatc agtgaagtag
agaggggggt tggtgatttt 960ggtgggaata aattggctat attgccccca
ccaacctcgt tgctaccaaa taccaacaac 1020actgactcac tgagaattgg
gaaagaaact taaaaccaag tcttgcagtg acgtacatgc 1080agtgtgtgca
tcacacattc aggtttccag tcaaattgta gaacaaatga atttcttgct
1140ttaacttaag ttgaagttta agaagtgaag ctgatgcttg tttttgaatg
aaaagccttt 1200gatagtttga tgtaagcatt ttccaaattt aactcttccc
atgcttgaca gagccaatta 1260agctaactgg tttgataaca agtaaacttc
taaatctatg agtatgagtg catgcagcac 1320accttttaaa cacaagccac
tgttttgtct tttttatcaa cagaaagaga atcctactaa 1380taacactaat
caagatcgct gctcttttct gtttattttt cttaataaat taacttttgt
1440tttgtactcc tgttaaacaa ctgctctatt tgtttcatgt gttgcattaa
ataacatggt 1500tttattcaca tctacaagca aaatttccta aaaactgtga
atgatgtaga agcaagtcat 1560ttatgttttg aaattcacgc attggagttt
ctaacgccca accaaccaaa cggtaatatg 1620aatatcgtgt ttggaacaaa
ttagaattta ggacataatt tttcacatca gaataaatgt 1680taggaatttt
tgcttttacg tttttcgcat taaaataatg tgatttatcg gttgttcctg
1740aacaataacc atcgatgtaa ttataaaatt ctaatttgtc ctatcctggg
gcgtcaacgt 1800ccagccaaat gcgtaacatt tattctgatg taaaaaatta
ttattattat tatagataat 1860aaaatcttgt tcctgaacaa taaccatcaa
tgtaattata aaattgaatc ttagactcaa 1920aactagttat taatctggaa
caatgtttac tcaaaactag ttattaatag tatttttaag 1980ttaatttgaa
attttttttt cggcgttaaa caaatactag atgtttatac tacaaatatt
2040gattattgat tataaattta taaatgttaa aaaaaaaaaa aagagaaaac
aaagaattga 2100agttgtggtt ggtagtaaac cagcaccagg cgaacaagtg
gacacaattt acctacaagt 2160aactaaccaa ccggaagcac aggctacaac
ggtcctttca cacccggtct caaagctttt 2220aaaaacgaac acatacgcac
tcacatttcc attccacctc aacaaacaca acaacactct 2280ctcttctcgc
tcttggcttt tcgctcttca ctcactctca ttcattcatt tccaccgttc
2340524DNAArtificial SequenceDNA Primer 5gggctatgaa gggaatggaa agga
24624DNAArtificial SequenceDNA Primer 6cccatattga agatttgaag taat
24722DNAArtificial SequenceDNA Primer 7gcgtggtttt tcgctggata ta
22825DNAArtificial SequenceDNA Primer 8gcgcatttcg taacatattt ttcac
25920DNAArtificial SequenceDNA Primer 9agcgggaatt gaaggttttt
201024DNAArtificial SequenceDNA Primer 10ggaatctcat ctgaaaataa tgga
241126DNAArtificial SequenceDNA Primer 11gcgccagcaa caaagttcct
gacaaa 261222DNAArtificial SequenceDNA Primer 12gcgcatgcaa
atgaaataat aa 221321DNAArtificial SequenceDNA Primer 13gcgccttcaa
attggcgtct t 211424DNAArtificial SequenceDNA Primer 14gcgccttaaa
taaaacccga aact 241521DNAArtificial SequenceDNA Primer 15tgttacttag
taattatgaa g 211621DNAArtificial SequenceDNA Primer 16aataatgatt
tgttgatcga t 211726DNAArtificial SequenceDNA Primer 17gcgaagccca
tactccgaac ctgcca 261825DNAArtificial SequenceDNA Primer
18gcctccaaaa actcaacccc atcaa 251924DNAArtificial SequenceDNA
Primer 19gggctatgaa gggaatggaa agga 242024DNAArtificial SequenceDNA
Primer 20cccatattga agatttgaag taat 242123DNAArtificial SequenceDNA
Primer 21gatgccttac gcctgtcact aac 232224DNAArtificial SequenceDNA
Primer 22gcagaacagt agaacaagtc cagt 242323DNAArtificial SequenceDNA
Primer 23gcccaccagt tgttgtgtaa gac 232422DNAArtificial SequenceDNA
Primer 24gcgtgcgatg agaaactcag ac 222522DNAArtificial SequenceDNA
Primer 25gcgtggtttt tcgctggata ta 222625DNAArtificial SequenceDNA
Primer 26gcgcatttcg taacatattt ttcac 252724DNAArtificial
SequenceDNA Primer 27gaagttggtg actgcgggaa atgc 242824DNAArtificial
SequenceDNA Primer 28ttcaatgcac cgatccaaca agga 242921DNAArtificial
SequenceDNA Primer 29tacaagtcag taatataacc t 213021DNAArtificial
SequenceDNA Primer 30ctgagtagat agcagtgaca t 213121DNAArtificial
SequenceDNA Primer 31actgcttatg gttgcagaat c 213220DNAArtificial
SequenceDNA Primer 32gagtatgtaa atgacatctt 203321DNAArtificial
SequenceDNA Primer 33tatgactgca gaagtcaagt c 213421DNAArtificial
SequenceDNA Primer 34tgaccttgaa gaggagatag a 213520DNAArtificial
SequenceDNA Primer 35acaacactct ctcttctcgc 203621DNAArtificial
SequenceDNA Primer 36cagattatga gttttggcct g 213721DNAArtificial
SequenceDNA
Primer 37atttcactta tataaatatg c 213821DNAArtificial SequenceDNA
Primer 38tctcttttat atgctacaat a 213922DNAArtificial SequenceDNA
Primer 39ggtaccatct tccttagaat gg 224022DNAArtificial SequenceDNA
Primer 40tgtgggaaag agacaacaaa cc 224120DNAArtificial SequenceDNA
Primer 41ttcgttggct cccactgctc 204220DNAArtificial SequenceDNA
Primer 42tctggtacac gtcaatgggc 204320DNAArtificial SequenceDNA
Primer 43acgaagagat cctgaaggag 204420DNAArtificial SequenceDNA
Primer 44attcccaagg gttggaaggc 204521DNAArtificial SequenceDNA
Primer 45tcaagcattg tttggagatg g 214621DNAArtificial SequenceDNA
Primer 46acagaagcat ttgcagggca g 214720DNAArtificial SequenceDNA
Primer 47accttcgttg gatgcaaggc 204821DNAArtificial SequenceDNA
Primer 48cttggtccaa aattgcgggt c 214922DNAArtificial SequenceDNA
Primer 49cgtggcaatt tttcgaaggt ag 225022DNAArtificial SequenceDNA
Primer 50caactcaaaa ccacattgag gc 225122DNAArtificial SequenceDNA
Primer 51agcaacacac gcaaaccaaa tc 225221DNAArtificial SequenceDNA
Primer 52tgcaattcat cctacggtgg c 215321DNAArtificial SequenceDNA
Primer 53tcaggacatg tttgttggtg g 215421DNAArtificial SequenceDNA
Primer 54cacactcagt tcagcttata g 215521DNAArtificial SequenceDNA
Primer 55atacgtgggc ccaactaaga c 215620DNAArtificial SequenceDNA
Primer 56tgtcgtctta ggtgagaggc 205722DNAArtificial SequenceDNA
Primer 57tggctgttcc tagaaggctg tg 225824DNAArtificial SequenceDNA
Primer 58tggagttgga tcggaggatt aagg 245921DNAArtificial SequenceDNA
Primer 59aagggagact ggataaccat c 216021DNAArtificial SequenceDNA
Primer 60ccgctcattt ggtgagtcat g 216121DNAArtificial SequenceDNA
Primer 61atgtgctcgc tgttggtgat g 216223DNAArtificial SequenceDNA
Primer 62gcaccatgga ggtgaaaaaa ata 236320DNAArtificial SequenceDNA
Primer 63ggacggttcg ctggctaaga 206424DNAArtificial SequenceDNA
Primer 64tcactgcctt cctcttcttc ttca 246524DNAArtificial SequenceDNA
Primer 65tccaccgagc aactaccata tctt 246622DNAArtificial SequenceDNA
Primer 66acgagcacat agccaggcat ta 226721DNAArtificial SequenceDNA
Primer 67gcgccttcaa attggcgtct t 216824DNAArtificial SequenceDNA
Primer 68gcgccttaaa taaaacccga aact 246923DNAArtificial SequenceDNA
Primer 69ttacttttgg tcagcatttt ggc 237024DNAArtificial SequenceDNA
Primer 70tattgttgat atattatatt gtcc 247122DNAArtificial SequenceDNA
Primer 71accctttttg cagtatttat gc 227223DNAArtificial SequenceDNA
Primer 72ctaggtaact cttttagccg tga 237320DNAArtificial SequenceDNA
Primer 73acaacactct ctcttctcgc 207421DNAArtificial SequenceDNA
Primer 74cagattatga gttttggcct g 217521DNAArtificial SequenceDNA
Primer 75caggccaaaa ctcataatct g 217621DNAArtificial SequenceDNA
Primer 76cagattatga gttttggcct g 217722DNAArtificial SequenceDNA
Primer 77taattttggt tggagaacaa tg 227821DNAArtificial SequenceDNA
Primer 78ctaatccttg tacttcattt c 217921DNAArtificial SequenceDNA
Primer 79atggatccag taagcgtgtg g 218023DNAArtificial SequenceDNA
Primer 80ctaatccttg tacttcattt cag 238123DNAArtificial SequenceDNA
Primer 81gttaaccttc aagtcccaat ctg 238223DNAArtificial SequenceDNA
Primer 82agaagaattt ggagcagaaa gtg 238328DNAArtificial SequenceDNA
Primer 83aattgagctc caatggcacc aatgccca 288432DNAArtificial
SequenceDNA Primer 84aattggtacc gaacggtgga aatgaatgaa tg
328533DNAArtificial SequenceDNA Primer 85aaaaaagcag gctatcaatg
gcaccaatgc cca 338636DNAArtificial SequenceDNA Primer 86aagaaagctg
ggtagaacgg tggaaatgaa tgaatg 368729DNAArtificial SequenceDNA Primer
87ggggacaagt ttgtacaaaa aagcaggct 298829DNAArtificial SequenceDNA
Primer 88ggggaccact ttgtacaaga aagctgggt 298936DNAArtificial
SequenceDNA Primer 89aattggcgcg cctgcaggca atggcaccaa tgccca
369022DNAArtificial SequenceDNA Primer 90aattgagctc gattccgcgg ca
229123DNAArtificial SequenceDNA Primer 91aattgagctc atcgcctccg aga
239236DNAArtificial SequenceDNA Primer 92aattggtacc tgcaggccag
attttatggt gcccaa 369335DNAArtificial SequenceDNA Primer
93aaaaaagcag gctattacgg cggcaatgaa tacat 359435DNAArtificial
SequenceDNA Primer 94aagaaagctg ggtactgaag tctagggctt tttct
359531DNAArtificial SequenceDNA Primer 95atgcggattc ggcaatgaat
acatcgacca g 319632DNAArtificial SequenceDNA Primer 96ttgggtacct
gtctagggct ttttcttcca ag 329724DNAArtificial SequenceDNA Primer
97tgaaaaagac tttgagcaga ttgg 249820DNAArtificial SequenceDNA Primer
98ttgccatgct ccttctggat 209940PRTGlycine max 99Cys Arg Gly Ile Glu
Leu Ile Ala Ser Glu Asn Phe Thr Ser Phe Ala 1 5 10 15 Val Ile Glu
Ala Leu Gly Ser Ala Leu Thr Asn Lys Tyr Ser Glu Gly 20 25 30 Met
Pro Gly Asn Arg Tyr Tyr Gly 35 40 10050PRTGlycine max 100His Leu
Asp Ala Gln Ser Trp Gly Val Asn Val Gln Pro Tyr Ser Gly 1 5 10 15
Ser Pro Ala Asn Phe Ala Ala Tyr Thr Ala Val Leu Asn Pro His Asp 20
25 30 Arg Ile Met Gly Leu Asp Leu Arg Ser Gly Gly His Leu Thr His
Gly 35 40 45 Tyr Tyr 50 10120PRTGlycine max 101Arg Pro Leu Gly Leu
Thr Gly Tyr Lys Val Glu Lys Leu Cys Asp Leu 1 5 10 15 Cys Asn Ile
Thr 20 10240PRTGlycine max 102Cys Arg Gly Ile Glu Leu Ile Ala Ser
Glu Asn Phe Thr Ser Phe Ala 1 5 10 15 Val Ile Glu Ala Leu Gly Ser
Ala Leu Thr Asn Lys Tyr Ser Glu Gly 20 25 30 Met Pro Gly Asn Arg
Tyr Tyr Gly 35 40 10350PRTGlycine max 103His Leu Asp Ala Gln Ser
Trp Gly Val Asn Val Gln Pro Tyr Ser Gly 1 5 10 15 Ser Pro Ala Asn
Phe Ala Ala Tyr Thr Ala Val Leu Asn Pro His Asp 20 25 30 Arg Ile
Met Gly Leu Asp Leu Arg Ser Gly Gly His Leu Thr His Gly 35 40 45
Tyr Tyr 50 10420PRTGlycine max 104Arg Pro Leu Gly Leu Thr Gly Tyr
Lys Val Glu Lys Leu Cys Asp Leu 1 5 10 15 Cys Asn Ile Thr 20
105471PRTGlycine max 105Met Asp Pro Val Ser Val Trp Gly Asn Thr Pro
Leu Ala Thr Val Asp 1 5 10 15 Pro Glu Ile His Asp Leu Ile Glu Lys
Glu Lys Arg Arg Gln Cys Arg 20 25 30 Gly Ile Glu Leu Ile Ala Ser
Glu Asn Phe Thr Ser Phe Ala Val Ile 35 40 45 Glu Ala Leu Gly Ser
Ala Leu Thr Asn Lys Tyr Ser Lys Gly Met Pro 50 55 60 Gly Asn Arg
Tyr Tyr Gly Gly Asn Glu Tyr Ile Asp Gln Ile Glu Asn 65 70 75 80 Leu
Cys Arg Ser Arg Ala Leu Gln Ala Phe His Leu Asp Ala Gln Ser 85 90
95 Trp Gly Val Asn Val Gln Pro Tyr Ser Gly Ser Pro Ala Asn Phe Ala
100 105 110 Ala Tyr Thr Ala Val Leu Asn Pro His Asp Arg Ile Met Gly
Leu Asp 115 120 125 Leu Arg Ser Gly Gly His Leu Thr His Gly Tyr Tyr
Thr Ser Gly Gly 130 135 140 Lys Lys Ile Ser Ala Thr Ser Ile Tyr Phe
Glu Ser Leu Pro Tyr Lys 145 150 155 160 Val Asn Ser Thr Thr Gly Tyr
Ile Asp Tyr Asp Arg Leu Glu Glu Lys 165 170 175 Ala Leu Asp Phe Arg
Pro Lys Leu Ile Ile Cys Gly Gly Ser Ala Tyr 180 185 190 Pro Arg Asp
Trp Asp Tyr Lys Arg Phe Arg Glu Val Ala Asp Lys Cys 195 200 205 Gly
Ala Leu Leu Leu Cys Asp Met Ala His Thr Ser Gly Leu Val Ala 210 215
220 Ala Gln Glu Val Asn Ser Pro Phe Glu Tyr Cys Asp Ile Val Thr Thr
225 230 235 240 Thr Thr His Lys Ser Leu Arg Gly Pro Arg Ala Gly Met
Ile Phe Tyr 245 250 255 Arg Lys Gly Pro Lys Pro Pro Lys Lys Gly Gln
Pro Glu Asn Ala Val 260 265 270 Tyr Asp Phe Glu Asp Lys Ile Asn Phe
Ala Val Phe Pro Ser Leu Gln 275 280 285 Gly Gly Pro His Asn His Gln
Ile Gly Ala Leu Ala Val Ala Leu Lys 290 295 300 Gln Ala Ala Ser Pro
Gly Phe Lys Ala Tyr Ala Lys Gln Val Lys Ala 305 310 315 320 Asn Ala
Val Ala Leu Gly Lys Tyr Leu Met Gly Lys Gly Tyr Ser Leu 325 330 335
Val Thr Gly Gly Thr Glu Asn His Leu Val Leu Trp Asp Leu Arg Pro 340
345 350 Leu Gly Leu Thr Gly Tyr Lys Val Glu Lys Leu Cys Asp Leu Cys
Asn 355 360 365 Ile Thr Val Asn Lys Asn Ala Val Phe Gly Asp Ser Ser
Ala Leu Ala 370 375 380 Pro Gly Gly Val Arg Ile Gly Ala Pro Ala Met
Thr Ser Arg Gly Leu 385 390 395 400 Val Glu Lys Asp Phe Glu Gln Ile
Gly Glu Phe Leu His Arg Ala Val 405 410 415 Thr Leu Thr Leu Glu Ile
Gln Lys Glu His Gly Lys Leu Leu Lys Asp 420 425 430 Phe Asn Lys Gly
Leu Val Asn Asn Lys Ala Ile Glu Asp Leu Lys Ala 435 440 445 Asp Val
Glu Lys Phe Ser Ala Leu Phe Asp Met Pro Gly Phe Leu Val 450 455 460
Ser Glu Met Lys Tyr Lys Asp 465 470 106471PRTGlycine max 106Met Asp
Pro Val Ser Val Trp Gly Asn Thr Pro Leu Ala Thr Val Asp 1 5 10 15
Pro Glu Ile His Asp Leu Ile Glu Lys Glu Lys Arg Arg Gln Cys Arg 20
25 30 Gly Ile Glu Leu Ile Ala Ser Glu Asn Phe Thr Ser Phe Ala Val
Ile 35 40 45 Glu Ala Leu Gly Ser Ala Leu Thr Asn Lys Tyr Ser Glu
Gly Met Pro 50 55 60 Gly Asn Arg Tyr Tyr Gly Gly Asn Glu Tyr Ile
Asp Gln Ile Glu Asn 65 70 75 80 Leu Cys Arg Ser Arg Ala Leu Gln Ala
Phe His Leu Asp Ala Gln Ser 85 90 95 Trp Gly Val Asn Val Gln Pro
Tyr Ser Gly Ser Pro Ala Asn Phe Ala 100 105 110 Ala Tyr Thr Ala Val
Leu Asn Pro His Asp Arg Ile Ile Gly Leu Asp 115 120 125 Leu Arg Ser
Gly Gly His Leu Thr His Gly Tyr Tyr Thr Ser Gly Gly 130 135 140 Lys
Lys Ile Ser Ala Thr Ser Ile Tyr Phe Glu Ser Leu Pro Tyr Lys 145 150
155 160 Val Asn Ser Thr Thr Gly Tyr Ile Asp Tyr Asp Arg Leu Glu Glu
Lys 165 170 175 Ala Leu Asp Phe Arg Pro Lys Leu Ile Ile Cys Gly Gly
Ser Ala Tyr 180 185 190 Pro Arg Asp Trp Asp Tyr Lys Arg Phe Arg Glu
Val Ala Asp Lys Cys 195 200 205 Gly Ala Leu Leu Leu Cys Asp Met Ala
His Thr Ser Gly Leu Val Ala 210 215 220 Ala Gln Glu Val Asn Ser Pro
Phe Glu Tyr Cys Asp Ile Val Thr Thr 225 230 235 240 Thr Thr His Lys
Ser Leu Arg Gly Pro Arg Ala Gly Met Ile Phe Tyr 245 250 255 Arg Lys
Gly Pro Lys Pro Pro Lys Lys Gly Gln Pro Glu Asn Ala Val 260 265 270
Tyr Asp Phe Glu Asp Lys Ile Asn Phe Ala Val Phe Pro Ser Leu Gln 275
280 285 Gly Gly Pro His Asn His Gln Ile Gly Ala Leu Ala Val Ala Leu
Lys 290 295 300 Gln Ala Ala Ser Pro Gly Phe Lys Ala Tyr Ala Lys Gln
Val Lys Ala 305 310 315 320 Asn Ala Val Ala Leu Gly Lys Tyr Leu Met
Gly Lys Gly Tyr Ser Leu 325 330 335 Val Thr Gly Gly Thr Glu Asn His
Leu Val Leu Trp Asp Leu Arg Pro 340 345 350 Leu Gly Leu Thr Gly Tyr
Lys Val Glu Lys Leu Cys Asp Leu Cys Asn 355 360 365 Ile Thr Val Asn
Lys Asn Ala Val Phe Gly Asp Ser Ser Ala Leu Ala 370 375 380 Pro Gly
Gly Val Arg Ile Gly Ala Pro Ala Met Thr Ser Arg Gly Leu 385 390 395
400 Val Glu Lys Asp Phe Glu Gln Ile Gly Glu Phe Leu His Arg Ala Val
405 410 415 Thr Leu Thr Leu Glu Ile Gln Lys Glu His Gly Lys Leu Leu
Lys Asp 420 425 430 Phe Asn Lys Gly Leu Val Asn Asn Lys Ala Ile Glu
Asp Leu Lys Ala 435 440 445 Asp Val Glu Lys Phe Ser Ala Leu Phe Asp
Met Pro Gly Phe Leu Val 450 455 460 Ser Glu Met Lys Tyr Lys Asp 465
470 10740PRTGlycine max 107Cys Arg Gly Ile Glu Leu Ile Ala Ser Glu
Asn Phe Thr Ser Phe Ala 1 5 10 15 Val Ile Glu Ala Leu Gly Ser Ala
Leu Thr Asn Lys Tyr Ser Glu Gly 20 25 30 Met Pro Gly Asn Arg Tyr
Tyr Gly 35 40 10850PRTGlycine max 108His Leu Asp Ala Gln Ser Trp
Gly Val Asn Val Gln Pro Tyr Ser Gly 1 5 10 15 Ser Pro Ala Asn Phe
Ala Ala Tyr Thr Ala Val Leu Asn Pro His Asp 20 25 30 Arg Ile Met
Gly Leu Asp Leu Pro Ser Gly Gly His Leu Thr His Gly 35 40 45 Tyr
Tyr 50 10920PRTGlycine max 109Arg Pro Leu Gly Leu Thr Gly Asn Lys
Val Glu Lys Leu Cys Asp Leu 1 5 10 15 Cys Asn Ile Thr 20
11060DNAGlycine max 110cccaaagtgt tgtcagtgtt cgagaaccgt gggagaaagc
tacacaccac tcgatcatgg 6011160DNAGlycine max 111cttggtaacg
atattacctt caagggggaa agcttatcag ctacaaaatt ggcacacaag
6011260DNAGlycine max 112tataaatgcc gcaagaaatt tagtctcctc
aacctgaact atccctcaat cacagtccca 6011360DNAGlycine max
113tacattgctc atgttcagaa cccgtatgga atcaccgttt ctgtgaagcc
aagcatcttg 6011460DNAGlycine max 114cccaaagtgt tgtcagcgtt
cgagaaccgt gggagaaagc tacacaccac tcgatcatgg 6011560DNAGlycine max
115cttggtaacg atattacctt caagggggaa agcttatcag ctacaaaatt
ggcacacaag 6011660DNAGlycine max 116tataaatgcc gcaagaaatt
tagtctcctc aacctgaact atcccctaat cacagtccca 6011760DNAGlycine max
117tacattgctc atgttcaaaa cccgtatgga atcaccgttt ctgtgaagcc
aagcatcttg 6011860DNAGlycine max 118cccaaagtgt tgtcagtgtt
cgagaaccgt gggagaaagc tacacaccac tcgatcatgg 6011960DNAGlycine max
119cttggtaacg atattacctt gaagggggaa agcttatcag ctacaaaatt
ggcacacaag 6012060DNAGlycine max 120tataaatgcc gcaagaaatt
tagtctcctc aacctgaact atccctcaat cacagtccca
6012160DNAGlycine max 121tacattgctc atgttcaaaa cccgtatgga
atcaccgttt ctgtgaagcc aagcatcttg 601222339DNAGlycine max
122caatggcacc aatgcccaat gggagattta agtcaagccc aacatcaacc
tctgaaatta 60tgaattatga aattaaaatg cttcctagta agtgaactag ttgcatctca
tttatatcat 120aaatttcgaa ctacgacttt cttggccatg ttagtaaagt
ttgggggatt gttcaaaatt 180ggtggagtgg ttcagcttaa tctccaaatt
atttgttcta agttgttttg gtaggcaggt 240ttaatttttt cctgatcctg
ggaaaaaaat tattgatacc atattaacat ctcttgacga 300tgctacgaga
tttctcatga ttatagaact gagtagggtg gcttaaaagg ttttatttta
360aatataattt caccacattg aattgggtat tagtaaactg gttactggta
tgcctgtaaa 420gtggacaatg ataaatgttt ttatagaagt tggtatggat
tttaaaatag ctcatgtata 480aaatgtgaaa aaggaaacgt gaactaaaat
gctaataata aaagataaag actaaattaa 540ttaaagttaa aggataaaat
gcttgttaca tcaagtcatt ttaaaggtgc actattagag 600gctgcacagt
aaaagttaac actgatatat ttttaaagat gttcttagtt aaatagcttt
660tgacttgatg gggtgaagac acaagaggtt gttgttgcga tgtgattttg
gctgaatatg 720catgcctgct gaacattgac ttcattgtta aatcaaaatt
aatcccatag acctattgta 780ttatttaagg ggatcaattt cataaatcaa
aatttattgg ttggggaaaa aaacaatgtt 840tagtagttcc cagtcatatt
cagaaaccta caaattaact atcccccatg ttaatgaagc 900aaggtgtggg
ggaaggaaag agtcagcatc agtgaagtag agaggggggt tggtgatttt
960ggtgggaata aattggctat attgccccca ccaacctcgt tgctaccaaa
taccaacaac 1020actgactcac tgagaattgg gaaagaaact taaaaccaag
tcttgcagtg acgtacatgt 1080agtgtgtgca tcacacattc aggtttccag
tcaaattgta gaacaaatga atttcttgct 1140ttaacttaag ttgaagttta
agaagtgaag ctgatgcttg tttttgaatg aaaagccttt 1200gatagtttga
tgtaagcatt ttccaaattt aactcttccc atgcttgaca gagccaatta
1260agctaactgg tttgataaca agtaaacttc taaatctatg agtatgagtg
catgcagcac 1320accttttaaa cacaagccac tgttttgtct tttttatcaa
cagaaagaga atcctactaa 1380taacactaat caagatcgct gctcttttct
gtttattttt cttaataaat taacttttgt 1440tttgtactcc tgttaaacaa
ctgctctatt tgtttcatgt gttgcattaa ataacatggt 1500tttattcaca
tctacaagca aaatttccta aaaactgtga atgatgtaga agcaagtcat
1560ttatgttttg aaattcacgc attggagttt ctaacgccca accaaccaaa
cggtaatatg 1620aatatcgtgt ttggaacaaa ttagaattta ggacataatt
tttcacatca gaataaatgt 1680taggaatttt tgcttttacg tttttcgcat
taaaataatg tgatttatcg gttgttcctg 1740aacaataacc atcgatgtaa
ttataaaatt ctaatttgtc ctatcctggg gcgtcaacgt 1800ccagccaaat
gcgtaacatt tattctgatg taaaaaatta ttattattat tatagataat
1860aaaatcttgt tcctgaacaa taaccatcaa tgtaattata aaattgaatc
ttagactcaa 1920aactagttat taatctggaa caatgtttac tcaaaactag
ttattaatag tatttttaag 1980ttaatttgaa attttttttt cggcgttaaa
caaatactag atgtttatac tacaaatatt 2040gattattgat tataaattta
taaatgttaa aaaaaaaaaa agagaaaaca aagaattgaa 2100gttgtggttg
gtagtaaacc agcaccaggc gaacaagtgg acacaattta cctacaagta
2160actaaccaac cggaagcaca ggctacaacg gtcctttcac acccggtctc
aaagctttta 2220aaaacgaaca catacgcact catatttcca ttccacctca
acaaacacaa caacactctc 2280tcttctcgct cttggctttt cgctcttcac
tcactctcat tcattcattt ccaccgttc 23391232340DNAGlycine max
123caatggcacc aatgcccaat gggagattta agtcaagccc aacatcaacc
tctgaaatta 60tgaattatga aattaaaatg cttcctagta agtgaactag ttgcatctca
tttatatcat 120aaatttcgaa ctacgacttt cttggccatg ttagtaaagt
ttgggggatt gttcaaaatt 180ggtggagtgg ttcagcttaa tctccaaatt
atttgttcta agttgttttg gtaggcaggt 240ttaatttttt cctgatcctg
ggaaaaaaat tattgatacc atattaacat ctcttgacga 300tgctacgaga
tttctcatga ttatagaact gagtagggtg gcttaaaagg ttttatttta
360aatataattt caccacattg aattgggtat tagtaaactg gttactggta
tgcctgtaaa 420gtggacaatg ataaatgttt ttatagaagt tggtatggat
tttaaaatag ctcatgtata 480aaatgtgaaa aaggaaacgt gaactaaaat
gctaataata aaagataaag actaaattaa 540ttaaagttaa aggataaaat
gcttgttaca tcaagtcatt ttaaaggtgc actattagag 600gctgcacagt
aaaagttaac actgatatat ttttaaagat gttcttagtt aaatagcttt
660tgacttgatg gggtgaagac acaagaggtt gttgttgcga tgtgattttg
gctgaatatg 720catgcctgct gaacattgac ttcattgtta aatcaaaatt
aatcccatag acctattgta 780ttatttaagg ggatcaattt cataaatcaa
aatttattgg ttggggaaaa aaacaatgtt 840tagtagttcc cagtcatatt
cagaaaccta caaattaact atcccccatg ttaatgaagc 900aaggtgtggg
ggaaggaaag agtcagcatc agtgaagtag agaggggggt tggtgatttt
960ggtgggaata aattggctat attgccccca ccaacctcgt tgctaccaaa
taccaacaac 1020actgactcac tgagaattgg gaaagaaact taaaaccaag
tcttgcagtg acgtacatgc 1080agtgtgtgca tcacacattc aggtttccag
tcaaattgta gaacaaatga atttcttgct 1140ttaacttaag ttgaagttta
agaagtgaag ctgatgcttg tttttgaatg aaaagccttt 1200gatagtttga
tgtaagcatt ttccaaattt aactcttccc atgcttgaca gagccaatta
1260agctaactgg tttgataaca agtaaacttc taaatctatg agtatgagtg
catgcagcac 1320accttttaaa cacaagccac tgttttgtct tttttatcaa
cagaaagaga atcctactaa 1380taacactaat caagatcgct gctcttttct
gtttattttt cttaataaat taacttttgt 1440tttgtactcc tgttaaacaa
ctgctctatt tgtttcatgt gttgcattaa ataacatggt 1500tttattcaca
tctacaagca aaatttccta aaaactgtga atgatgtaga agcaagtcat
1560ttatgttttg aaattcacgc attggagttt ctaacgccca accaaccaaa
cggtaatatg 1620aatatcgtgt ttggaacaaa ttagaattta ggacataatt
tttcacatca gaataaatgt 1680taggaatttt tgcttttacg tttttcgcat
taaaataatg tgatttatcg gttgttcctg 1740aacaataacc atcgatgtaa
ttataaaatt ctaatttgtc ctatcctggg gcgtcaacgt 1800ccagccaaat
gcgtaacatt tattctgatg taaaaaatta ttattattat tatagataat
1860aaaatcttgt tcctgaacaa taaccatcaa tgtaattata aaattgaatc
ttagactcaa 1920aactagttat taatctggaa caatgtttac tcaaaactag
ttattaatag tatttttaag 1980ttaatttgaa attttttttt cggcgttaaa
caaatactag atgtttatac tacaaatatt 2040gattattgat tataaattta
taaatgttaa aaaaaaaaaa aagagaaaac aaagaattga 2100agttgtggtt
ggtagtaaac cagcaccagg cgaacaagtg gacacaattt acctacaagt
2160aactaaccaa ccggaagcac aggctacaac ggtcctttca cacccggtct
caaagctttt 2220aaaaacgaac acatacgcac tcacatttcc attccacctc
aacaaacaca acaacactct 2280ctcttctcgc tcttggcttt tcgctcttca
ctcactctca ttcattcatt tccaccgttc 2340
* * * * *
References