U.S. patent application number 13/042123 was filed with the patent office on 2012-03-08 for isolated polynucleotides and polypeptides relating to loci underlying resistance to soybean cyst nematode and soybean sudden death syndrome and methods employing same.
Invention is credited to David A. Lightfoot, Khalid Meksem.
Application Number | 20120060240 13/042123 |
Document ID | / |
Family ID | 22654034 |
Filed Date | 2012-03-08 |
United States Patent
Application |
20120060240 |
Kind Code |
A1 |
Lightfoot; David A. ; et
al. |
March 8, 2012 |
ISOLATED POLYNUCLEOTIDES AND POLYPEPTIDES RELATING TO LOCI
UNDERLYING RESISTANCE TO SOYBEAN CYST NEMATODE AND SOYBEAN SUDDEN
DEATH SYNDROME AND METHODS EMPLOYING SAME
Abstract
Soybean cyst nematode and soybean sudden death syndrome
resistance genes, soybean cyst nematode and soybean sudden death
syndrome resistant plant lines, and methods of breeding and
engineering same.
Inventors: |
Lightfoot; David A.;
(Carbondale, IL) ; Meksem; Khalid; (Carbondale,
IL) |
Family ID: |
22654034 |
Appl. No.: |
13/042123 |
Filed: |
March 7, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11704728 |
Feb 9, 2007 |
7902337 |
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13042123 |
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09772134 |
Jan 29, 2001 |
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11704728 |
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60178811 |
Jan 28, 2000 |
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Current U.S.
Class: |
800/298 ;
435/320.1; 435/419; 536/23.6 |
Current CPC
Class: |
C12N 15/8285 20130101;
C07K 14/415 20130101; Y02A 40/164 20180101 |
Class at
Publication: |
800/298 ;
536/23.6; 435/320.1; 435/419 |
International
Class: |
A01H 5/10 20060101
A01H005/10; A01H 5/00 20060101 A01H005/00; C12N 5/10 20060101
C12N005/10; C07H 21/04 20060101 C07H021/04; C12N 15/63 20060101
C12N015/63 |
Claims
1-10. (canceled)
11. An isolated and purified nucleic acid molecule encoding a
biologically active SCN/SDS resistance polypeptide and further
comprising an isolated soybean rhg1 and SDS resistance gene, said
gene capable of conveying Heterodera glycines-infestation
resistance, Fusarium solani-infection resistance, or both
Heterodera glycines-infestation resistance and Fusarium
solani-infection resistance to a non-resistant soybean germplasm,
said gene located within a quantitative trait locus mapping to
linkage group G and mapped by genetic markers of SEQ ID NOs:1-6,
said gene located along said quantitative trait locus between said
markers, wherein said SCN/SDS resistance polypeptide has at least
95% sequence identity to SEQ ID NO 14.
12. The nucleic acid molecule of claim 11, wherein the encoded
polypeptide comprises a soybean SCN/SDS resistance polypeptide.
13. (canceled)
14. The nucleic acid molecule of claim 11, further defined as
comprising: (a) the nucleotide sequence of SEQ ID NO:13 or (b) a
nucleotide sequence that has at least 95% sequence identity to SEQ
ID NO:13.
15-16. (canceled)
17. The nucleic acid molecule of claim 11, further defined as a DNA
segment.
18. The nucleic acid molecule of claim 11, further defined as
positioned under the control of a promoter.
19. The nucleic acid molecule of claim 18, wherein said DNA segment
and promoter are operationally inserted into a recombinant
vector.
20. A recombinant host cell comprising the nucleic acid molecule of
claim 11.
21. A transgenic plant having incorporated into its genome a
nucleic acid molecule of claim 11, the nucleic acid molecule being
present in said genome in a copy number effective to confer
expression in the plant of an SCN/SDS resistance polypeptide.
22. Plant seeds, parts, or progeny of a plant as claimed in claim
20.
23. An isolated and purified nucleic acid molecule encoding a
biologically active SCN/SDS resistance polypeptide and further
comprising an isolated soybean Rhg4 gene, said gene capable of
conveying Heterodera glycines-infestation resistance to a
non-resistant soybean germplasm, said gene located within a
quantitative trait locus mapping to linkage group A2 and mapped by
the AFLP markers of SEQ ID NOs:7-12, said gene located along said
quantitative trait locus between said markers.
24. The isolated nucleic acid molecule of claim 23, further
comprising: (a) the nucleotide sequence of any one of SEQ ID
NOs:16-19; or (b) a nucleotide sequence substantially similar to
any one of SEQ ID NOs:16-19.
25. A transgenic plant comprising the isolated soybean Rhg4 gene of
claim 23.
26. Seeds, parts or progeny of a plant as claimed in claim 25.
27-80. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of the U.S. Utility
application Ser. No. 09/772,134, filed Jan. 29, 2001, herein
incorporated by reference in its entirety which claims priority to
the U.S. Provisional Application Ser. No. 60/178,811, filed Jan.
28, 2000, herein incorporated by reference in its entirety.
TECHNICAL FIELD
[0002] The present invention relates to plant breeding and plant
genetics. More particularly, the invention relates to soybean cyst
nematode and soybean sudden death syndrome resistance genes,
soybean cyst nematode and soybean sudden death syndrome resistant
soybean lines, and methods of breeding and engineering the
same.
TABLE-US-00001 Table of Abbreviations NAFLP amplified fragment
length polymorphism BAC bacterial artificial chromosome bp base
pair Cf tomato genes for resistance to Cladosporium fulvus FAM
6-carboxyfluorescein FI female index of parasitism indel a
nucleotide insertion or deletion MMAS molecular marker-assisted
selection QTL quantitative trait loci RAPD random amplified
polymorphic DNA RFLP restriction fragment length polymorphism rhg1
and Rhg4 genetic loci conferring resistance to Heterodera glycines
RIL recombinant inbred line SCN soybean cyst nematode SDS sudden
death syndrome SSR microsatellite TAMRA 6-carboxy-N,N,N'5N'
tetrachlorofluorescein TET 6-carboxy-4,7,2',7',
tetrachlorofluorescein
BACKGROUND OF THE INVENTION
[0003] Soybeans are a major cash crop and investment commodity in
North America and elsewhere. Soybean oil is one of the most widely
used edible oils, and soybeans are used worldwide both in animal
feed and in human food production.
[0004] The soybean cyst nematode (SON), Heterodera glycines, is a
widespread pest of soybeans in the American continent. Reported
first in Japan more than 75 years ago, since the first reports in
North Carolina in 1954, SCN continues its spread toward almost all
soybean-cultivated soils. Known as a small plant-parasitic
roundworm that attacks the roots of soybeans, it reproduces very
quickly, survives in the soil for many years in the absence of a
soybean crop, and can cause substantial soybean crop yield
losses.
[0005] Resistant soybean varieties are an effective tool available
for SCN management. There are multiple sources for soybean cyst
nematode resistance genes in commercial soybean varieties (PI88788,
Peking and PI209332), and several have been used to develop
cultivars (Myers & Anand (1991), Euphytica 55:197-201;
Rao-Arrelli et al. (1988) Crop Sci 28:650-652). All the described
loci involved in the resistance to SCN are reported to be
quantitative. (Concibido et al. (1997) Crop Sci 37:258-264;
Concibido (1996) Theor Appl Genet. 93:234-241; Webb et al. (1995)
Theor Appl Genet. 91:574-581; Rao-Arrelli et al. (1992) Crop Sci
32:862-864; Matthews et al. (1991) Soybean Genetics Newsletter,
Rao-Arrelli et al., 1988). They differ by their chromosomal
position (LG A2, G, B, I, F, J and E) and race of the pathogen
against which they confer the resistance (e.g. Race 1, 3, 5 or 14).
SCN resistance is simply inherited, but field resistance is
oligogenic due to the existence of variation among SCN populations
that are described as "races" (Riggs and Schmidt (1988) J Nematol
20:392-395).
[0006] One gene, rhg1, provides the major portion of resistance to
SCN race 3 across many genotypes derived from Peking (Chang et al.
(1997) Crop Sci 372:965-971; Mathews et al. (1998) Theor Appl
Genet. 97:1047-1052; Mahalingam et al. (1995) Breed Sci
45:435-445); PI437654 (Prabhu et al. (1999) Crop Sci 39:982-987;
Webb et al., 1995), >PI88788= (Bell-Johnson et al. (1998)
Soybean Genet Newslett 25:115-118; Concibido et al., 1997; Cregan
et al. (1999a) Crop Sci 39:1464-1490; Cregan et al. (1999b) Theor
Appl Genet. 99:811-818; Cregan et al. (1999c) Theor Appl Genet.
99:918-928), >PI209332= (Concibido et al., 1996), or
>PI90763= (Concibido et al., 1997). A second gene for SCN
resistance, Rhg4, provides an equal portion of resistance to SCN
race 3 across genotypes derived from Peking (Chang et al., 1997;
Mathews et al., 1998; Mahalingam et al., 1995); and PI437654
(Prabhu et al., 1999; Webb et al., 1995) but not PI88788, PI209332
or PI90763 (Concibido et al., 1996; Concibido et al., 1997).
Cytological studies suggest PI437654 and Peking derived resistances
share mechanisms (pronounced necrosis and cell wall appositions)
not seen in PI88788 in response to race 3 (Mahalingham et al.
(1996) Genome 39:986-998). These differences in mechanism may
derive from distinct alleles at Rhg4, rhg1 and/or other defense
associated loci.
[0007] DNA molecular markers linked to SCN/SDS resistance loci can
be used to develop effective plant breeding strategies. In general,
molecular markers are abundant, often co-dominant, and suitable for
rapid screening at the seedling stage. Genetic linkage maps of
soybean based on RFLP, RAPD, AFLP, and microsatellite markers have
been described. See Brown et al. (1987) Principles and Practice of
Nematode Control in Crops, pp 179-232, Academic Press, Orlando
Fla.; Concibido et al., 1996; Concibido et al., 1997; Mahalingham
et al., 1995; Meksem et al. (1999) Theor Appl Genet. 99:1131-1142;
Meksem et al. (2000) Theor Appl Genet. 101: 747-755; Webb et al.,
1995; Weiseman et al. (1992) Theor Appl Genet. 85:136-138; Lark et
al. (1993) Theor Appl Genet. 86:901-906; Shoemaker and Specht
(1995) Crop Sci 35:436-446; Chang et al., 1997; Keim et al. (1997)
Crop Sci 37:537-543).
[0008] All such markers have a limit of resistance trait
predictability based principally on proximity of the marker to the
resistance locus. In some cases, the interpretative value of
genetic linkage experiments can be augmented through the
simultaneous or serial detection of more than one genetic marker,
although this also incurs additional time and resources. Thus,
there is a need for a reliable cost-effective method for detecting
SCN or SDS resistance using genetic markers. Optimally, a genetic
marker comprises a resistance gene.
[0009] Therefore, it is of particular importance, both to the
soybean breeders and to farmers, to identify, genetic loci for
resistance to SCN and SDS. Having knowledge of the loci for
resistance to SCN and SDS, those of ordinary skill in the art can
breed or engineer SCN and SDS resistant soybeans. Soybean
resistance can be further provided to a non-resistant cultivar in
combination with other genotypic and phenotypic characteristics
required for commercial soybean lines.
SUMMARY OF THE INVENTION
[0010] The present invention discloses an isolated and purified
genetic marker associated with SCN/SDS resistance in soybeans, said
marker mapping to linkage group G in the soybean genome.
Preferably, the marker has a sequence identical to any one of SEQ
ID NOs:1, 3, and 5. Representative corresponding markers associated
with SCN/SDS susceptibility are set forth as SEQ ID NOs:2, 4, and
6.
[0011] Also disclosed is an isolated and purified genetic marker
associated with SCN/SDS resistance in soybeans, said marker mapping
to linkage group A2 in the soybean genome. Preferably, the marker
has a sequence identical to any one of SEQ ID NOs:7, 9, and 11.
Representative corresponding markers associated with SCN/SDS
susceptibility are set forth as SEQ ID NOs:8, 10, and 12.
[0012] The present invention further provides a plant, or parts
thereof, which evidences an SCN/SDS resistance response comprising
a genome, homozygous with respect to genetic alleles which are
native to a first parent and normative to a second parent of the
plant, wherein said second parent evidences significantly less
resistant response to SCN/SDS than said first parent and said
improved plant comprises alleles from said first parent that
evidences resistance to SCN/SDS in hybrid combination in at least
one locus selected from: a locus mapping to linkage group G and
mapped by one or more of the markers set forth as SEQ ID NOs:1, 3,
and 5, a locus mapping to linkage group A2 and mapped by one or
more of the markers set forth as SEQ ID NOs:7, 9, and 11; or
combinations thereof, said resistance not significantly less than
that of the first parent in the same hybrid combination, and yield
characteristics which are not significantly different than those of
the second parent in the same hybrid combination.
[0013] In another embodiment, a plant of the present invention, or
parts thereof, comprises the progeny of a cross between first and
second inbred lines, alleles conferring SCN/SDS resistance being
present in the homozygous state in the genome of one or the other
or both of said first and second inbred lines such that the genome
of said first and second inbreds together donate to the hybrid a
complement of alleles necessary to confer the SCN/SDS resistance.
Further disclosed are hybrid plants derived therefrom.
[0014] Also disclosed herein are isolated and purified biologically
active SCN/SDS resistance polypeptide and an isolated and purified
nucleic acid molecule encoding the same are disclosed. Preferably,
the polypeptide comprises a soybean SCN/SDS resistance polypeptide.
Chimeric genes comprising the isolated and purified nucleic acid
molecules encoding a SCN/SDS resistance polypeptide are also
provided.
[0015] In one embodiment, the nucleic acid molecule encoding a
SCN/SDS resistance gene comprises an isolated soybean rhg1 gene
that confers SCN/SDS resistance to a non-resistant host organism.
The gene is capable of conveying Heterodera glycines-infestation
resistance, Fusarium solani-infection resistance, or both
Heterodera glycines-infestation resistance or Fusarium
solani-infection resistance to a non-resistant plant germplasm, the
gene located within a quantitative trait locus mapping to linkage
group G and mapped by genetic markers of SEQ ID NOs:1, 3, and 5,
said gene located along said quantitative trait locus between said
markers. Preferably, the polypeptide comprises (a) a polypeptide
encoded by a nucleic acid sequence set forth as SEQ ID NO:13; (b) a
polypeptide encoded by a nucleic acid having homology to a DNA
sequence set forth as SEQ ID NO:13; (c) a polypeptide encoded by a
nucleic acid capable of hybridizing under stringent conditions to a
nucleic acid comprising a sequence or the complement of a sequence
set forth as SEQ ID NO:13; (d) a polypeptide which is a
biologically functional equivalent of a peptide set forth as SEQ ID
NO:14; or (e) a polypeptide comprising a fragment of a polypeptide
of (a), (b), (c) or (d).
[0016] In another embodiment, the nucleic acid molecule encoding a
SCN resistance polypeptide comprises an isolated soybean Rhg4 gene
that is capable of conveying Heterodera glycines-infestation
resistance to a non-resistant plant germplasm, said gene located
within a quantitative trait locus mapping to linkage group A2 and
mapped by the AFLP markers of SEQ ID NOs:7, 9, and 11, said gene
located along said quantitative trait locus between said markers.
Preferably, the nucleic acid molecule comprises any one of SEQ ID
NOs:16-19.
[0017] The present invention further provides an isolated SCN/SDS
resistance gene promoter region, or functional portion thereof,
comprising an about 90 kb fragment of soybean genomic clone 73P6
between BamHI restriction sites and 21d9 between HinDIII
restriction site. The genomic clone is available from the Forrest
BAC library described in Meksem et al (2000) Theor Appl Genet. 101
5/6:747-755, available through Southern Illinois
University-Carbondale (Carbondale, Ill.), Texas A&M University
BAC center (College Station, Tex.), and Research Genetics
(Huntsville, Ala.). Preferably, the isolated promoter region
comprises the nucleotide sequence of SEQ ID NO:15 or a sequence
substantially similar to SEQ ID NO:15. The SCN/SDS resistance gene
promoter region can be operably linked to heterologous
sequence.
[0018] A recombinant host cell comprising an isolated and purified
nucleic acid molecule of the present invention is also disclosed,
as is a transgenic plant having incorporated into its genome an
isolated and purified nucleic acid molecule. In one embodiment, the
nucleic acid molecule comprises encodes a SCN/SDS resistance
polypeptide and is present in said genome in a copy number
effective to confer expression in the plant of the SCN/SDS
resistance polypeptide. Seeds, parts or progeny of the transgenic
plant are also disclosed.
[0019] Further provided is a method for detecting a nucleic acid
molecule that encodes an SCN/SDS resistance polypeptide in a
biological sample comprising nucleic acid material is also
disclosed. The method comprises: (a) hybridizing an isolated and
purified nucleic acid molecule of the present invention under
stringent hybridization conditions to the nucleic acid material of
the biological sample, thereby forming a hybridization duplex; and
(b) detecting the hybridization duplex. Preferably, the isolated
and purified nucleic acid molecule comprises any of SEQ ID NOs: 13
and 16-19.
[0020] An assay kit for detecting the presence, in biological
samples, of an SCN/SDS resistance polypeptide is also disclosed. In
one embodiment, the kit comprises a first container that contains a
nucleic acid probe identical or complementary to a segment of at
least ten contiguous nucleotide bases of a nucleic acid molecule of
the present invention, preferably a nucleotide sequence of any one
of SEQ ID NOs:13 and 16-19. In another embodiment, the kit
comprises a nucleic acid probe or primer identical to any one of
SEQ ID NOs:1, 3, 5, 7, 9, and 11, or portion thereof.
[0021] A method for identifying soybean sudden death syndrome (SDS)
resistance or soybean cyst nematode (SCN) resistance in a soybean
plant using a SDS resistance gene, a SCN resistance gene, or DNA
segments having homology to a SDS resistance gene or to an SCN
resistance gene is also disclosed. In one embodiment, the method
comprises: (a) probing nucleic acids obtained from the soybean
plant with a probe derived from said SDS resistance gene or from
said SCN resistance gene or from said DNA segment having homology
to said SDS resistance gene or to said SCN resistance gene; and
observing hybridization of said probe to said nucleic acids, the
presence of said hybridization indicating SDS or SCN resistance in
said soybean plant. In another embodiment, the method comprises (a)
detecting a molecular marker linked to a quantitative trait locus
associated with SCN/SDS resistance, wherein the molecular marker is
the sequence set forth as any one of SEQ ID NOs:1, 3, 5, 7, 9, and
11; and (b) determining the presence of SCN/SDS resistance as
detection of the molecular marker and determining the absence of
SCN/SDS resistance as failure to detect the molecular marker of
(b).
[0022] A method of reliably and predictably introgressing SCN/SDS
resistance genes into non-resistant soybean germplasm is also
disclosed. The method comprises: using one or more nucleic acid
markers for marker assisted selection among soybean lines to be
used in a soybean breeding program, wherein the nucleic acid
markers map to linkage groups G or A2 and wherein the nucleic acid
markers are selected from among any of SEQ ID NOs: 1, 3, 5, 7, 9,
and 11; and introgressing said resistance gene into said
non-resistant soybean germplasm.
[0023] A soybean plant, or parts thereof, which evidences a SCN/SDS
resistance response is also disclosed. The plant comprises a
genome, homozygous with respect to genetic alleles which are native
to a first parent and non-native to a second parent of the soybean
plant, wherein said second parent evidences significantly less
resistant response to SCN/SDS than said first parent, and said
improved plant comprises alleles from said first parent that
evidences resistance to SCN/SDS in hybrid combination of at least
one locus selected from: a locus mapping to linkage group G and
mapped by one or more of the markers set forth as SEQ ID NOs:1, 3,
and 5, a locus mapping to linkage group A2 and mapped by one or
more of the markers set forth in SEQ ID NOs:7, 9, and 11; or
combinations thereof, said resistance not significantly less than
that of the first parent in the same hybrid combination, and yield
characteristics which are not significantly different than those of
the second parent in the same hybrid combination.
[0024] The soybean plant, or parts thereof, can further comprise
the progeny of a cross between first and second inbred lines,
alleles conferring SCN/SDS resistance being present in a homozygous
state in the genome of one or the other or both of said first and
second inbred lines such that the genome of said first and second
inbreds together donate to the hybrid a complement of alleles
necessary to confer the SCN/SDS resistance. Thus, an SCN/SDS
resistant hybrid, or parts thereof, formed with the soybean plant
is also disclosed, as is a soybean plant, or parts thereof, formed
by selfing the SCN/SDS resistant hybrid.
[0025] A method of positional cloning of a nucleic acid is also
disclosed. The method comprises: (a) identifying a first nucleic
acid genetically linked to a SCN/SDS resistance locus, wherein the
first nucleic acid maps between two markers selected from SEQ ID
NOs:1-12; and (b) cloning the first nucleic acid. Optionally, the
first nucleic acid can comprise the rhg1 locus or the Rhg4
locus.
[0026] A method for producing an antibody that specifically
recognizes a SCN/SDS resistance polypeptide is also disclosed. The
method comprises (a) recombinantly or synthetically producing a
SCN/SDS resistance polypeptide, or portion thereof; (b) formulating
the polypeptide of (a) whereby it is an effective immunogen; (c)
administering to an animal the formulation of (b) to generate an
immune response in the animal comprising production of antibodies,
wherein antibodies are present in the blood serum of the animal;
and (d) collecting the blood serum from the animal of (c)
comprising antibodies that specifically recognize a SCN/SDS
resistance polypeptide. Also provided is an antibody produced by
the disclosed method.
[0027] Methods for identifying a candidate compound as a modulator
of SCN/SDS resistance activity is also disclosed. Such methods
include but are not limited to cell-based assays of SCN/SDS
resistance gene expression, assays of specific binding to SCN/SDS
regulatory elements, and assays of specific binding to SCN/SDS
polypeptides. Optionally, the screening methods are adapted to a
high-throughput format.
[0028] In one embodiment, the method comprises: (a) exposing a cell
sample with a candidate compound to be tested, the cell sample
containing at least one cell containing a DNA construct comprising
a modulatable transcriptional regulatory sequence of an SCN/SDS
resistance-encoding nucleic acid and a reporter gene which is
capable of producing a detectable signal; (b) evaluating an amount
of signal produced in relation to a control sample; and (c)
identifying a candidate compound as a modulator of SCN/SDS
resistance activity based on the amount of signal produced in
relation to a control sample.
[0029] The present invention also provides a method for identifying
a substance that regulates SCN/SDS resistance gene expression using
a chimeric gene that includes an isolated SCN/SDS resistance gene
promoter region operably linked to a reporter gene. According to
this method, a gene expression system is established that includes
the chimeric gene and components required for gene transcription
and translation so that reporter gene expression is assayable. To
select a substance that regulates SCN/SDS resistance gene
expression, the method further provides the steps of using the gene
expression system to determine a baseline level of reporter gene
expression in the absence of a candidate regulator; providing a
plurality of candidate regulators to the gene expression system;
and assaying a level of reporter gene expression in the presence of
a candidate regulator. A candidate regulator is selected whose
presence results in an altered level of reporter gene expression
when compared to the baseline level. Preferably, the isolated
SCN/SDS resistance gene promoter region used in this method
comprises the sequence of SEQ ID NO:15, or functional portion
thereof.
[0030] In another embodiment, the method comprises using an SCN/SDS
regulatory sequence to identify a candidate substance that
specifically binds to the regulatory sequence. According to the
method, a SCN/SDS regulatory gene sequence is exposed to a
candidate substance under conditions suitable for binding to a
nucleic acid sequence, and a candidate regulator is selected that
specifically binds to the SCN/SDS resistance gene promoter region.
Preferably, the isolated SCN/SDS resistance gene promoter region
used in this method comprises the sequence of SEQ ID NO:15, or
functional portion thereof.
[0031] In another embodiment, a cell-free assay system is used and
comprises: (a) exposing a SCN/SDS polypeptide of the present
invention to a candidate compound; (b) assaying binding of the
candidate compound to the SCN/SDS polypeptide; and (c) identifying
a candidate compound as a putative modulator of SCN/SDS resistance
activity based on specific binding of the candidate compound to the
SCN/SDS polypeptide. Preferably, the SCN/SDS polypeptide comprises
some or all of the amino acids of SEQ ID NO:14.
[0032] A method of modulating SCN/SDS resistance in a plant is also
disclosed. The method comprises administering to the plant an
effective amount of a substance that modulates expression of an
SCN/SDS resistance activity-encoding nucleic acid molecule in the
plant to thereby modulate SCN/SDS resistance in the plant.
Preferably, the substance that modulates expression of an SCN/SDS
resistance activity is discovered by a disclosed method of the
present invention.
[0033] A method for providing a resistance characteristic to a
plant is also disclosed. The method comprises introducing to said
plant a construct comprising a nucleic acid sequence encoding an
SCN/SDS resistance gene product operatively linked to a promoter,
wherein production of the SCN/SDS resistance gene product in the
plant provides a resistance characteristic to the plant. The
construct can further comprises a vector selected from the group
consisting of a plasmid vector or a viral vector. The SCN/SDS
resistance gene product comprises a protein having an amino acid
sequence of SEQ ID NO:14. The nucleic acid sequence comprises the
nucleotide sequence of SEQ ID NO:13 or a nucleic acid that is
substantially similar to SEQ ID NO:13, and which encodes an SCN/SDS
resistance polypeptide.
[0034] The resistance characteristic is preferably nematode
resistance, fungal resistance or combinations thereof. More
preferably, the nematode resistance is H. glycines resistance, even
more preferably race 3 H. glycines resistance.
[0035] In an alternative embodiment the construct further comprises
another nucleic acid molecule encoding a polypeptide that provides
an additional desired characteristic to the plant. Optionally, the
method further comprises monitoring an insertion point for the
construct in the plant genome; and providing for insertion of the
construct into the plant genome at a location not associated with
the resistance characteristic, the desired characteristic, or both
the resistance and the desired characteristic. Preferably, the
plant is a soybean plant.
[0036] The present invention also provides methods for providing a
resistance characteristic to a plant is also disclosed, wherein a
combination of genetic and non-genetic techniques is employed. The
method comprises introducing to said plant a construct comprising a
nucleic acid sequence encoding an SCN/SDS resistance gene product
operatively linked to a promoter and provision of a substance that
modulates SCS/SDS resistance gene activity, wherein production of
the SCN/SDS resistance gene product in the plant, in combination
with provision of the SCN/SDS resistance gene modulator, provides a
resistance characteristic to the plant.
[0037] Accordingly, it is an object of the present invention to
provide novel isolated polynucleotides and polypeptides relating to
loci underlying resistance to soybean cyst nematode and soybean
sudden death syndrome and methods employing same. The object is
achieved in whole or in part by the present invention.
[0038] An object of the invention having been stated hereinabove,
other objects and advantages will become evident as the description
proceeds, when taken in connection with the accompanying Drawings
and Examples as best described hereinbelow.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] FIG. 1 depicts new AFLP genetic markers for SCN/SDS
resistance.
[0040] FIG. 1A presents genomic sequences of the both alleles
(resistant Forrest and susceptible Essex) of the converted AFLP
markers E.sub.ATGM.sub.CGA87 (SEQ ID NOs:1-2);
E.sub.CTAM.sub.AGG113 (SEQ ID NOs:3-4); E.sub.CGGM.sub.AGA116 (SEQ
ID NOs:5-6); E.sub.CCGM.sub.AAC405 (SEQ ID NOs:7-8),
E.sub.CCCM.sub.ATG161 (SEQ ID NOs:9-10), E.sub.CCAM.sub.AGC114 (SEQ
ID NOs:11-12. The italicized and underlined sequences represent the
forward and reverse sequence specific primers used. The bold
capital sequences represent the original AFLP restriction site. The
bold letters indicate the difference in sequences between the two
alleles.
[0041] FIG. 1B presents genomic sequences of the two alleles
(resistant and susceptible) of the converted E.sub.ATGM.sub.CGA87
markers. The italic sequences represent the resistance specific
TaqMan.TM. probes TMA5-RE and the susceptible allele specific probe
TMA5-S. The standard font underlined sequence represent the
TaqMan.TM. forward and reverse primers assay, the underlined italic
sequence is the ATG4BACF primer used for sequence extension of the
E.sub.ATGM.sub.CGA87 marker, the BAC derived extended sequences are
in small font capitals.
[0042] FIG. 2 depicts AFLPs for selecting SCN/SDS resistance.
[0043] FIG. 2A shows PCR amplification products using
E.sub.ATGM.sub.CGA87 sequence specific primers TMA5 forward and
reverse: Lane 1-40 represent 40 RIL DNA, 41 and 42 are the two
parents. F: Forrest; E: Essex; 1: resistant allele; 2: susceptible
allele; H: heterozygote lines. The PCR products were separated by
electrophoresis on a 4% (w/v) Metaphor gel.
[0044] FIG. 2B shows a partial AFLP autoradiograph profile of the
E.sub.CGGM.sub.AGA116 marker. The six selective nucleotides step
was replaced by MseI primer M.sub.AGAGACT and EcoRI primer E. Lane
7: Essex; Lane 8: Forrest; Lane 1 to 6 and 9 to 20 represent RIL
DNA; 1: resistant allele; 2: susceptible allele
[0045] FIG. 2C shows PCR amplification products using
E.sub.CTAM.sub.AGG113 sequence specific primers CTA forward and
reverse: Lane 1-40 represent 40 RIL DNA, 41 and 42 are the two
parent. F: Forrest; E: Essex; 1: resistant allele; 2: susceptible
allele; H: heterozygote lines. The PCR products were separated by
electrophoresis on a 4% (w/v) Metaphor gel.
[0046] FIG. 2D shows PCR amplification products using
E.sub.CCGM.sub.AAC405 sequence specific primers A2D8 forward and
reverse: Lane 1-40 represent 40 RIL DNA, 41 and 42 are the two
parents F: Forrest; E: Essex; 1: resistant allele; 2: susceptible
allele; H: heterozygote lines. The PCR products were separated by
electrophoresis on a 4% (w/v) Metaphor gel.
[0047] FIG. 3 depicts a genetic and physical map showing the
location of an Rhg4 gene relative to DNA markers. The location of
the aspartokinase serine dehydrogenase (AK-HSDH) and the A2D8
marker are indicated as determined by restriction mapping of BAC
DNA. The A2D8 sequences for Essex and Forrest alleles are deposited
in GenBank as Accession Nos. AF286701 and AF286700, respectively.
The l locus (I) position was estimated by relation to
BARC-SAT.sub.--162 (Cregan et al., 1999c). Genetic mapping shows
Rhg4 and A2D8 are both within the interval shown by the horizontal
line and within a large insert clone, 100B10, that contains a 140
kbp insert (Zobrist et al. (2000) Soybean Genet Newslett
27:10-15).
[0048] FIG. 4 depicts the gene structure of the rhg1 gene and
clones derived from Forrest genomic DNA.
[0049] FIG. 5 depicts detection of the A2D8 marker polymorphism
using the TaqMan.TM. assay and manual selection of genotypes.
Eighty-six individuals from an F5 derived population of recombinant
inbred lines from the cross of Essex.times.Forrest that segregate
for resistance to SON are shown.
[0050] FIG. 5A is an image of fluorescent signals viewed under the
"dye component" field of the sequence detection software and the
A2D8 genotypes were manually selected based on the ratio of FAM and
TET signals. Allele 1 homozygous, Forrest type; FAM<<TET.
Allele 2 homozygous, Essex type; TET<<FAM. Alleles 1 and 2
heterogeneous, Essex and Forrest type; TET less than 2 fold greater
or lesser than FAM. Two selections were used, in the first
(TaqMan.TM. assay1) group of genotypes FAM 6-8 and TET 8-9 were
considered susceptible. In the second (TaqMan.TM. assay 2) group,
they were considered heterogeneous.
[0051] FIG. 5B is a spreadsheet that contains scores (allele
designations) for the samples as they were arranged in the 96 well
plate. There was no DNA in wells E12, F12 and G12 (negative
controls). There was Essex DNA in wells A1, C12 and D12. There was
Forrest DNA in wells B2, A12 and B12. The RIL DNA was in well A3 to
H11 in order by row from RIL1-RIL86 except samples E1 (RIL3) and E6
(RIL 43) that did not amplify. The RILs resistant to SCN had an
index of parasitism FI <10% of the susceptible check resistant
lines.
[0052] FIG. 6 depicts detection of the A2D8 marker polymorphism by
PCR amplification and gel electrophoresis of soybean genotypes.
Seventy-eight individuals from an F5 derived population of
recombinant inbred lines from the cross of Essex.times.Forrest that
segregate for resistance to SCN are shown.
[0053] FIG. 6A is an image of fluorescent signals viewed under the
"dye component" field of the sequence detection software and the
A2D8 genotypes were manually selected based on the ratio of FAM and
TET signals. Lane 1, 42 Essex; Lane 2 and 41 Forrest; Lanes 3-40
RILS 1-38.
[0054] FIG. 6B is a picture of an ehtidium-stained gel, showing
resolution of gel electrophoresis markers. Lane 42 Essex; Lane 41
Forrest; Lanes 1-40 RILS 39-78. Asterisks indicate disagreements
with the TaqMan.TM. assay 1.
[0055] FIG. 7A-B presents the rhg1 gene sequence (SEQ ID
NO:13).
[0056] FIG. 7C presents the rhg1 polypeptide (SEQ ID NO:14).
[0057] FIG. 7D shows sequences producing significant alignments
using BLAST analysis.
[0058] FIG. 7E-F is an alignment between rhg1 protein (SEQ ID
NO:14) and Arabidopsis thaliana hypothetical protein T18N14.120
(Gen Bank Accession T46070).
DETAILED DESCRIPTION OF THE INVENTION
[0059] Disclosed herein is the identification of AFLP markers that
are genetically linked to the SCN/SDS resistance loci of Forrest.
Further disclosed are purified and isolated SCN or SDS resistance
genes, proximal sequences to SCN/SDS resistance genes, and SCN/SDS
resistance-related genes.
[0060] The isolated and purified polynucleotide sequences disclosed
herein can thus be used in a variety of applications pertaining to
breeding and engineering soybeans having SCN and SDS resistance.
For example, the isolated polynucleotides disclosed herein can be
used in position-based or homology-based cloning of additional
SCN/SDS resistance genes, including regulatory elements; in gene
structure determination; in studies of genome organization and gene
expression; in gene complementation experiments; in the isolation
of additional DNA markers for gene manipulation and molecular
marker assisted breeding; and in plant transformation and the
production of transgenic plants.
[0061] The present invention also pertains to a soybean plant and
methods of producing the same, which is resistant to soybean cyst
nematodes (SCN). In one embodiment, the method comprises stable
transformation of a plant with an rhg1 gene, disclosed herein. In
another embodiment, the method comprises introgression in soybean
of a trait enabling the plant to resist soybean cyst nematode (SCN)
infestation. Additionally, the present invention relates to method
of precise and accurate introgression of the genetic material
conferring SCN resistance from one or more parent plants into the
progeny.
[0062] The present invention also pertains to a soybean plant and
methods of producing the same, which is resistant to soybean sudden
death syndrome (SDS). In one embodiment, the method comprises
stable transformation of a plant with an rhg1 gene, disclosed
herein. In another embodiment, the method comprises introgression
of the genetic material conferring SDS resistance from one or more
parent plants into the progeny with precision and accuracy.
[0063] The invention differs from present technology in several
regards. In one aspect, the present invention provides the first
disclosure of the rhg1 gene sequence, thereby enabling transgenic
approaches for providing SCN/SDS resistance. Further, the present
invention provides a non-electorphoretic selection assay using
nucleotide sequences of SCN/SDS resistance gene alleles. The
disclosed nucleotide sequences of SCN/SDS resistance genes and
associated genetic markers provide means for easily selecting
resistant cultivars, for assembling many resistance genes in a
single cultivar, for combining resistance genes in novel
combinations, for identifying genes that confer resistance in new
cultivars, and for predicting resistance in cultivars. The
invention is used to improve selection for SDS and SCN resistance
in soybean in breeding programs.
I. Traits
[0064] The term "phenotype" or "trait" each refer to any observable
property of an organism, produced by the interaction of the
genotype of the organism and the environment. A phenotype can
encompass variable expressivity and penetrance of the phenotype.
Exemplary phenotypes include but are not limited to a visible
phenotype, a physiological phenotype, a susceptibility phenotype, a
cellular phenotype, a molecular phenotype, and combinations
thereof. Preferably, the phenotype is related to SCN/SDS
resistance. The term "susceptibility phenotype" refers to an
increased capacity or risk for displaying a phenotype, i.e. a
susceptibility to SCN/SDS infection.
[0065] The term "complex trait" as used herein refers to a trait
that is not inherited as predicted by classical Mendelian genetics.
A complex trait results from the interaction of multiple genes,
each gene contributing to the phenotype. Complex traits can be
continuous or show threshold penetrance. In the field, SCN/SDS
resistance is inherited as a complex trait.
[0066] The term "quantitative trait" is a complex trait that can be
assessed quantitatively. Quantitation entails measurement of a
trait across a continuous distribution of values. SCN/SDS
resistance is a quantitative trait.
[0067] The term "SCN/SDS resistance" or "SCN/SDS resistance trait"
as used herein refers to a cellular or organismal capacity for
resistance to nematode or fungal infection, or both. Preferably,
the nematode resistance is Heterodera glycines (the organism that
causes SCN in soybeans) resistance, even more preferably, race 3
Heterodera glycines resistance. The fungal resistance is preferably
Fusarium solani (the organism that causes SDS in
soybeans)-infection resistance. SCN resistance can be assayed in
the field or in the greenhouse by methods known in the art,
including but not limited to determination of an SCN index of
parasitism as disclosed in Example 2, Meksem et al. (1999), and
U.S. Pat. No. 6,096,944. SDS resistance can be scored by
determination of disease incidence, disease severity, and disease
index values as disclosed in Hnetkovsky et al. (1996) Crop Sci
36(2):393-400, Njiti et al. (1996) Crop Sci 36:1165-1170; and
Matthews et al. (1991).
[0068] The term "SCN/SDS resistance" is used herein for convenience
to describe traits, transgenic plants, polynucleotides, and
polypeptides of the present invention. Therefore, the resistance
characteristic conveyed by the polynucleotides and polypeptides of
the present invention refers to any resistance characteristic as
set forth herein and as would be apparent to one of ordinary skill
in the art after reviewing the disclosure of the present
invention.
[0069] The term "molecular phenotype" refers to a detectable
feature of molecules in a cell or organism. Exemplary molecular
phenotypes include but are not limited to a presence of a genetic
marker nucleotide sequence, a presence of a SCN/SDS resistance gene
sequence, a level of gene expression, a splice selection, a level
of protein, a protein type, a protein modification, a level of
lipid, a lipid type, a lipid modification, a level of carbohydrate,
a carbohydrate type, a carbohydrate modification, and combinations
thereof. Methods for observing, detecting, and quantitating
molecular phenotypes are well known to one skilled in the art. See
Sambrook et al., eds. (1989) Molecular Cloning, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, New York, N.Y.; by Silhavy et
al. (1984) Experiments with Gene Fusions, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, New York, N.Y.; by Ausubel et
al. (1992) Current Protocols in Molecular Biology, John Wylie and
Sons, Inc. New York, N.Y.; Landgren et. al. (1988) Science
242:229-237; Bodanszky, et al. (1976) Peptide Synthesis, John Wiley
and Sons, Second Edition, New York, N.Y.; Harlow and Lane (1988)
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, New York; Ochman et al. (1990) in PCR
protocols: a Guide to Methods and Applications, Innis et al.
(eds.), pp. 219-227, Academic Press, San Diego, Calif.; Koduri and
Poola (2001) Steroids 66(1):17-23; Regan et al. (2000) Anal Biochem
286(2):265-276; U.S. Pat. Nos. 6,096,555; 5,958,624; and
5,629,158.
II. Genetic Mapping
[0070] For genetic mapping, a representative population was
generated as in Example 1. To detect genomic regions associated
with resistance to SCN and resistance to SDS, the RILs were
classified as Essex type or Forrest type for each marker. In some
cases, SCN susceptibility and resistance was quantitatively
determined according to a SCN female index (F1) of parasitism
(Meksem, 1999) as described in Example 2. Markers were compared
with SCN or SDS response scores by the F-test in analysis of
variance (ANOVA) done with SAS (SAS Institute Inc., Cary, N.C.,
1988). The probability of association of each marker with each
trait was determined and a significant association was declared if
P.ltoreq.0.05 (unless noted otherwise in the text) since the
detection of false associations is reduced in isogenic lines
(Landers & Botstein (1989) Genetics 121:185-199; Paterson et
al. (1990) Genetics 124:735-742).
[0071] Selected pairs of markers were analyzed by the two-way ANOVA
using the general linear model (PROC GLM) procedure to detect
non-additive interactions between the unlinked QTL (Chang et al.
(1996) Crop Sci 36:965-971) or Epistat (Chase et al. (1997) Theor
Appl Genet. 94:724-730). Non-additive interactions between markers
which were significantly associated with SCN/SDS response were
excluded when P.gtoreq.0.05. Selected groups of markers were
analyzed by multi-way ANOVA to estimate joint heritabilities for
traits associated with multiple QTL. Joint heritability was
determined from the R.sup.2 term for the joint model in multi-way
ANOVA.
[0072] Mapmaker-EXP 3.0 (Lander et al. 1987) was used to calculate
map distances (cM, Haldane units) between linked markers and to
construct a linkage map including traits as genes. The RIL
(recombinant inbred line) and F.sub.3 self genetic models were
used. The log.sub.10 of the odds ratio (LOD) for grouping markers
was set minimally at 2.0, and maximum distance was set at 30 cM.
Conflicts were resolved in favor of the highest LOD score after
checking the raw data for errors. Marker order within groups was
determined by comparing the likelihood of many map orders. A
maximum likelihood map was computed with error detection. Trait
data were used for QTL analysis (Webb et al. 1995; Chang et al.
1997). The data were subjected to ANOVA (SAS Institute Inc., Cary,
N.C.) with mean separation by LSD (Gomez and Gomez (1984). Graphs
were constructed by Quattro Pro version 5.0 (Novell Inc., Orem,
Utah).
III. Nucleotide Sequences of SCN/SDS Resistance Genes and
Associated Genetic Markers
[0073] The nucleic acid molecules provided by the present invention
include the isolated nucleic acid molecules of SEQ ID NOs: 1-13 and
15-114, sequences substantially similar to sequences of SEQ ID
NOs:1-13 and 15-114, conservative variants thereof,
plant-expressible variants thereof, subsequences and elongated
sequences thereof, complementary DNA molecules, and corresponding
RNA molecules. The present invention also encompasses genes, cDNAs,
promoters, chimeric genes, and vectors comprising disclosed SCN/SDS
resistance gene and SCN/SDS resistance gene marker nucleic acid
sequences.
[0074] III.A. General Considerations
[0075] The term "nucleic acid molecule" refers to
deoxyribonucleotides or ribonucleotides and polymers thereof in
either single- or double-stranded form. Unless specifically
limited, the term encompasses nucleic acids containing known
analogues of natural nucleotides that have similar properties as
the reference natural nucleic acid. Unless otherwise indicated, a
particular nucleotide sequence also implicitly encompasses
conservatively modified variants thereof (e.g. degenerate codon
substitutions), complementary sequences, subsequences, elongated
sequences, as well as the sequence explicitly indicated. The terms
"nucleic acid molecule" or "nucleotide sequence" can also be used
in place of "gene", "cDNA", or "mRNA". Nucleic acids can be derived
from any source, including any organism.
[0076] The term "isolated", as used in the context of a nucleic
acid molecule, indicates that the nucleic acid molecule exists
apart from its native environment and is not a product of nature.
An isolated DNA molecule can exist in a purified form or can exist
in a non-native environment such as a transgenic host cell.
[0077] The term "purified", when applied to a nucleic acid, denotes
that the nucleic acid is essentially free of other cellular
components with which it is associated in the natural state.
Preferably, a purified nucleic acid molecule is a homogeneous dry
or aqueous solution. The term "purified" denotes that a nucleic
acid or protein gives rise to essentially one band in an
electrophoretic gel. Particularly, it means that the nucleic acid
is at least about 50% pure, more preferably at least about 85%
pure, and most preferably at least about 99% pure.
[0078] The term "substantially identical", in the context of two
nucleotide or amino acid sequences, can also be defined as two or
more sequences or subsequences that have at least 60%, preferably
80%, more preferably 90-95%, and most preferably at least 99%
nucleotide or amino acid sequence identity, when compared and
aligned for maximum correspondence, as measured using one of the
following sequence comparison algorithms (described herein below
under the heading Nucleotide and Amino Acid Sequence Comparisons)
or by visual inspection. Preferably, the substantial identity
exists in nucleotide sequences of at least 50 residues, more
preferably in nucleotide sequence of at least about 100 residues,
more preferably in nucleotide sequences of at least about 150
residues, and most preferably in nucleotide sequences comprising
complete coding sequences.
[0079] In one aspect, polymorphic sequences can be substantially
identical sequences. The term "polymorphic" refers to the
occurrence of two or more genetically determined alternative
sequences or alleles in a population. An allelic difference can be
as small as one base pair.
[0080] Another indication that two nucleotide sequences are
substantially identical is that the two molecules specifically or
substantially hybridize to each other under stringent conditions.
In the context of nucleic acid hybridization, two nucleic acid
sequences being compared can be designated a "probe" and a
"target". A "probe" is a reference nucleic acid molecule, and a
"target" is a test, nucleic acid molecule, often found within a
heterogenous population of nucleic acid molecules. "Target
sequence" is synonymous with "test sequence".
[0081] A preferred nucleotide sequence employed for hybridization
studies or assays includes probe sequences that are complementary
to or mimic at least an about 14 to 40 nucleotide sequence of a
nucleic acid molecule of the present invention. Preferably, a probe
comprises 14 to 20 nucleotides, or even longer where desired, such
as 30, 40, 50, 60, 100, 200, 300, or 500 nucleotides or up to the
full length of any of SEQ ID NOs:1-13, 15-114. Such fragments can
be readily prepared by, for example, directly synthesizing the
fragment by chemical synthesis, by application of nucleic acid
amplification technology, or by introducing selected sequences into
recombinant vectors for recombinant production. The phrase
"hybridizing specifically to" refers to the binding, duplexing, or
hybridizing of a molecule only to a particular nucleotide sequence
under stringent conditions when that sequence is present in a
complex nucleic acid mixture (e.g., total cellular DNA or RNA). The
phrase "binds substantially to" refers to complementary
hybridization between a probe nucleic acid molecule and a target
nucleic acid molecule and embraces minor mismatches that can be
accommodated by reducing the stringency of the hybridization media
to achieve the desired hybridization. Probe sequences can also
hybridize specifically to duplex DNA under certain conditions to
form triplex or other higher order DNA complexes. The preparation
of such probes and suitable hybridization conditions are well known
in the art.
[0082] "Stringent hybridization conditions" and "stringent
hybridization wash conditions" in the context of nucleic acid
hybridization experiments such as Southern and Northern blot
analysis are both sequence- and environment-dependent. Longer
sequences hybridize specifically at higher temperatures. An
extensive guide to the hybridization of nucleic acids is found in
Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular
Biology-Hybridization with Nucleic Acid Probes part I chapter 2,
Elsevier, New York, N.Y. Generally, highly stringent hybridization
and wash conditions are selected to be about 5.degree. C. lower
than the thermal melting point (T.sub.m) for the specific sequence
at a defined ionic strength and pH. Typically, under "stringent
conditions" a probe will hybridize specifically to its target
subsequence, but to no other sequences.
[0083] The T.sub.m is the temperature (under defined ionic strength
and pH) at which 50% of the target sequence hybridizes to a
perfectly matched probe. Very stringent conditions are selected to
be equal to the T.sub.m for a particular probe. An example of
stringent hybridization conditions for Southern or Northern Blot
analysis of complementary nucleic acids having more than about 100
complementary residues is overnight hybridization in 50% formamide
with 1 mg of heparin at 42.degree. C. An example of highly
stringent wash conditions is 15 minutes in 0.15 M NaCl at
65.degree. C. An example of stringent wash conditions is 15 minutes
in 0.2.times.SSC buffer at 65.degree. C. (See Sambrook et al.,
1989) for a description of SSC buffer). Often, a high stringency
wash is preceded by a low stringency wash to remove background
probe signal. An example of medium stringency wash conditions for a
duplex of more than about 100 nucleotides, is 15 minutes in
1.times.SSC at 45.degree. C. An example of low stringency wash for
a duplex of more than about 100 nucleotides, is 15 minutes in
4-6.times.SSC at 40.degree. C. For short probes (e.g., about 10 to
50 nucleotides), stringent conditions typically involve salt
concentrations of less than about 1.0 M Na ion, typically about
0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0-8.3,
and the temperature is typically at least about 30.degree. C.
Stringent conditions can also be achieved with the addition of
destabilizing agents such as formamide. In general, a signal to
noise ratio of 2-fold (or higher) than that observed for an
unrelated probe in the particular hybridization assay indicates
detection of a specific hybridization.
[0084] The following are examples of hybridization and wash
conditions that can be used to clone homologous nucleotide
sequences that are substantially identical to reference nucleotide
sequences of the present invention: a probe nucleotide sequence
preferably hybridizes to a target nucleotide sequence in 7% sodium
dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C.
followed by washing in 2.times.SSC, 0.1% SDS at 50.degree. C.; more
preferably, a probe and target sequence hybridize in 7% sodium
dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C.
followed by washing in 1.times.SSC, 0.1% SDS at 50.degree. C.; more
preferably, a probe and target sequence hybridize in 7% sodium
dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C.
followed by washing in 0.5.times.SSC, 0.1% SDS at 50.degree. C.;
more preferably, a probe and target sequence hybridize in 7% sodium
dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C.
followed by washing in 0.1.times.SSC, 0.1% SDS at 50.degree. C.;
more preferably, a probe and target sequence hybridize in 7% sodium
dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C.
followed by washing in 0.1.times.SSC, 0.1% SDS at 65.degree. C.
[0085] A further indication that two nucleic acid sequences are
substantially identical is that proteins encoded by the nucleic
acids are substantially identical, share an overall
three-dimensional structure, are biologically functional
equivalents; or are immunologically cross-reactive. These terms are
defined further under the heading SCN/SDS Resistance Polypeptides
herein below. Nucleic acid molecules that do not hybridize to each
other under stringent conditions are still substantially identical
if the corresponding proteins are substantially identical. This can
occur, for example, when two nucleotide sequences are significantly
degenerate as permitted by the genetic code.
[0086] The term "conservatively substituted variants" refers to
nucleic acid sequences having degenerate codon substitutions
wherein the third position of one or more selected (or all) codons
is substituted with mixed-base and/or deoxyinosine residues (Batzer
et al. (1991) Nucleic Acid Res. 19:5081; Ohtsuka et al. (1985) J
Biol Chem 260:2605-2608; Rossolini et al. (1994) Mol Cell Probes
8:91-98).
[0087] The term "plant-expressible variant" means a substantially
similar sequence that has been modified to comprise a coding
sequence (nucleotide sequence) can be efficiently expressed by
plant cells, tissue and whole plants. The art understands that a
plant-expressible coding sequence has a GC composition consistent
with good gene expression in plant cells, a sufficiently low CpG
content so that expression of that coding sequence is not
restricted by plant cells, and codon usage which is consistent with
that of plant genes. Where it is desired that the properties of the
plant-expressible SCN/SDS resistance gene are identical to those of
the naturally occurring SCN/SDS resistance gene, the
plant-expressible homolog will have an identical coding sequence or
a substantially identical coding sequence.
[0088] The term "subsequence" refers to a sequence of nucleic acids
that comprises a part of a longer nucleic acid sequence. An
exemplary subsequence is a probe, described herein above, or a
primer. The term "primer" as used herein refers to a contiguous
sequence comprising about 8 or more deoxyribonucleotides or
ribonucleotides, preferably 10-20 nucleotides, and more preferably
20-30 nucleotides of a selected nucleic acid molecule. The primers
of the present invention encompass oligonucleotides of sufficient
length and appropriate sequence so as to provide initiation of
polymerization on a nucleic acid molecule of the present
invention.
[0089] The term "elongated sequence" refers to an addition of
nucleotides (or other analogous molecules) incorporated into the
nucleic acid. For example, a polymerase (e.g., a DNA polymerase),
e.g., a polymerase that adds sequences at the 3' terminus of the
nucleic acid molecule can be employed to prepare an elongated
sequence. In addition, the nucleotide sequence can be combined with
other DNA sequences, such as promoters, promoter regions,
enhancers, polyadenylation signals, intronic sequences, additional
restriction enzyme sites, multiple cloning sites, and other coding
segments.
[0090] The term "complementary sequence", as used herein, indicates
two nucleotide sequences that comprise anti-parallel nucleotide
sequences capable of pairing with one another upon formation of
hydrogen bonds between base pairs. As used herein, the term
"complementary sequences" means nucleotide sequences which are
substantially complementary, as can be assessed by the same
nucleotide comparison set forth above, or is defined as being
capable of hybridizing to the nucleic acid segment in question
under relatively stringent conditions such as those described
herein. A particular example of a complementary nucleic acid
segment is an antisense oligonucleotide.
[0091] The present invention further includes vectors comprising
the disclosed SCN/SDS resistance gene sequences, including
plasmids, cosmids, and viral vectors. The term "vector", as used
herein refers to a DNA molecule having sequences that enable its
replication in a compatible host cell. A vector also includes
nucleotide sequences to permit ligation of nucleotide sequences
within the vector, wherein such nucleotide sequences are also
replicated in a compatible host cell. A vector can also mediate
recombinant production of an SCN/SDS resistance gene polypeptide,
as described further herein below.
[0092] Nucleic acids of the present invention can be cloned,
synthesized, recombinantly altered, mutagenized, or combinations
thereof. Standard recombinant DNA and molecular cloning techniques
used to isolate nucleic acids are well known in the art. Exemplary,
non-limiting methods are described by Sambrook et al., eds., 1989;
by Silhavy et al., 1984; by Ausubel et al., 1992; and by Glover,
ed. (1985) DNA Cloning: A Practical Approach, MRL Press, Ltd.,
Oxford, United Kingdom. Site-specific mutagenesis to create base
pair changes, deletions, or small insertions are also well known in
the art as exemplified by publications, see e.g., Adelman et al.,
(1983) DNA 2:183; Sambrook et al. (1989).
[0093] Nucleotide sequences of the present invention can detected,
subcloned, sequenced, and further evaluated by any measure well
known in the art using any method usually applied to the detection
of a specific DNA sequence including but not limited to dideoxy
sequencing, PCR, oligomer restriction (Saiki et al., Bio/Technology
3:1008-1012 (1985), allele-specific oligonucleotide (ASO) probe
analysis (Conner et al. (1983) Proc Natl Acad Sci USA 80:278), and
oligonucleotide ligation assays (OLAs) (Landgren et. al. (1988)
Science 241:1007). Molecular techniques for DNA analysis have been
reviewed (Landgren et. al. (1988) Science 242:229-237).
TABLE-US-00002 Table of Functionally Equivalent Codons Amino Acids
Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys C UGC UGU
Aspartic Acid Asp D GAC GAU Glumatic acid Glu E GAA GAG
Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine
His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG
Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUG
Asparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine
Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S
ACG AGU UCA UCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val
V GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine Tyr Y UAC UAU
[0094] III.B. Genetic Markers
[0095] The term "genetic marker", as used herein generally refers
to a genetic locus, a phenotype conferred by locus, or a nucleotide
sequence residing at a locus, wherein the locus is genetically
linked to a trait of interest. The term "genetically linked" as
used herein refers to two or more loci that are predictably
inherited together during random crossing or intercrossing.
Quantitative linkage analysis is further described in the section
Genetic Mapping herein above. Preferably, genetically linked loci
are less than about 10 cM apart, more preferably less than about 5
cM apart, and even more preferably less than about 1cM apart.
Optimally, the genetic marker and the gene conferring a trait of
interest comprise the same or overlapping nucleotide sequence.
[0096] An embodiment of the present invention comprises genetic
markers associated with SCN resistance and SDS resistance that are
isolatable from soybeans, and which are free from total genomic
DNA. Disclosed herein are sequences of AFLP markers mapped in
soybean to the chromosomal segments carrying rhg1 and SDS loci on
molecular linkage group G and the Rhg4 locus on molecular linkage
group A2. Representative markers for SCN/SDS resistance are set
forth as SEQ ID NOs:1, 3, 5, 7, 9, and 11. Respresentative
corresponding markers for SCN/SDS susceptibility are set forth as
SEQ ID NOs:2, 4, 6, 8, 10, and 12.
[0097] AFLP bands were obtained as described in Example 3. From
each AFLP band, 4-30 clones were sequenced (mean 15.6) depending on
the sequence complexity of the originating band. The sequence
analysis showed that each AFLP band can be composed of a number of
different DNA sequences from fragments of identical size. A mean of
6 sequences per band with a range of 1-15 sequences per band was
detected. From a single AFLP band only one sequence corresponded
with the original AFLP marker. The other sequences were bands that
shared not only the same size within 1-2 bp but also the same
selective bases at the EcoRI and MseI sites (100%). Further, some
of the cloned sequences from within a band shared between 6 to 15
bp in common to each side (EcoRI and MseI) of the original AFLP
polymorphism (about 30% of bands).
[0098] To identify polymorphisms within the AFLP, the AFLP sequence
was used to design primers to screen the Forrest BamHI BAC library
by PCR. For example, E.sub.ATGM.sub.CGA87 was a dominant AFLP band
in coupling phase with the rhg1 locus, and screening with a
E.sub.ATGM.sub.CGA87 AFLP band primer yielded a single clone. Two
internal primers were designed from the E.sub.ATGM.sub.CGA87
resistant allele and DNA from the corresponding BAC was used as
template to extend the sequence from the AFLP marker both up and
down stream by sequencing. The sequence showed a single 5 bp indel
underlay the polymorphic band and no SNPs were present. As used
herein, an "indel" refers to a nucleotide insertion or a deletion
(FIG. 1B). No additional polymorphisms were detected in about 1,250
bp of flanking sequence.
[0099] Sequence comparison of both, resistant and the susceptible
alleles of the co-dominant AFLP marker E.sub.CTAM.sub.AGG113 found
polymorphisms including both indels and SNPs. There were 4 SNPs
within 113 bp and 1 indel (21 bp) (FIG. 1A). Primer sets were
designed around the indel site and used to map the genetic
position. The genetic position of the identified indel mapped to
the region of the original AFLP.
[0100] Sequence comparison of resistant and the susceptible alleles
of the dominant AFLP marker E.sub.CCCM.sub.ATG161 found SNP
polymorphism. There were 2 SNPs within 116 bp (FIG. 1A). Primer
sets were designed around the SNP site and used to map the genetic
position. The genetic position of the identified indel mapped to
the region of the original AFLP.
[0101] Sequence comparison of both resistant and susceptible
alleles of the dominant AFLP marker E.sub.CCAM.sub.AGC114 found SNP
polymorphism adjacent to the EcoRI site. There was 1 SNP within 114
bp (FIG. 1A).
[0102] Sequence comparison of resistant and susceptible alleles of
the co-dominant AFLP marker E.sub.CCGM.sub.AAC405 found
polymorphisms including both indels and SNPs. There were 2 indels
(12 bp and 4 bp) and 4 SNPs within 405 bp (FIG. 1A). The 4 bp indel
was two AG repeats in an [AG].sub.5 complex micro-satellite
sequence. Primer sets were designed around both indel sites and
used to map the genetic position. In both cases, the genetic
position of the identified indel mapped to the region of the
original AFLP.
[0103] For the AFLP marker E.sub.CGGM.sub.AGA116, the polymorphisms
were found adjacent to both the EcoRI and MseI restriction sites
(FIG. 1A). The six selective nucleotide step was replaced by
M.sub.AGAGACT and E.sub.C. Using this primer set the detection of
the polymorphism on sequencing gels as well as the mapping of this
sequence to the same location as the original AFLP was successful
(FIG. 2B). There was 1 indel (2 bp) and 1 SNPs within 116 bp (FIG.
1A). The 2 bp indel was the [A].sub.2 extension of an [A].sub.8
repeat. Primer sets were designed around the indel and SNP sites
and used to map their genetic positions. In both cases, the genetic
position of the identified polymorphism was identical to the region
of the original AFLP.
[0104] Comparison of both alleles of the AFLP marker
E.sub.CCGM.sub.AAC405 provided four SNPs, two indels and one SSR.
The insertion of [AG].sub.2 in the [AG].sub.8 repeat of the
resistance allele created a microsatellite polymorphism that was
designated SIUC-SAG405 bp the present co-inventors. The difference
of 4 bp between the two alleles at position 224 bp to 228 bp was
enough to discriminate between the resistant and susceptible allele
after electrophoresis through a 4% (v/w) Metaphor7 agarose gel. The
12 bp indel at 42 bp to 54 bp was used to design a sequence
specific PCR marker (FIG. 2D), and to develop a TaqMan.TM. assay
for the Rhg4 locus. SNPs were found within the
E.sub.CCGM.sub.AAC405. The transversions of T at position 327 in
the resistant allele to C at position 337 in the susceptible
allele; and A at position 358 bp in the resistance allele to C at
position 366 bp in the susceptible allele can also be used for
high-throughput screening SNPs based assay.
[0105] An indel of 21 bp was responsible for the polymorphism at
the E.sub.CTAM.sub.AGG113 AFLP locus between Essex and Forrest. PCR
based markers were designed to flank the 21 bp indel and shown to
be polymorphic, the new marker was named CTA (FIG. 2C).
[0106] In the E.sub.ATGM.sub.CGA87 marker the insertion of CTTAT to
form a tandem repeat in the Forrest allele at position 20 bp to 25
bp created a 5 bp polymorphism that was suitable for marker
development. PCR primers were designed to develop a sequence
specific PCR assay (FIG. 2A), the new marker was named ATG4. The
same indel was used to develop a TaqMan.TM. probe named TMA5 to
discriminate between the two alleles.
[0107] The genetic markers of the present invention can be used to
reliably select SCN/SDS resistance, as described herein.
III.C. SCN/SDS Resistance Genes
[0108] The term "gene" refers broadly to any segment of DNA
associated with a biological function. A gene encompasses sequences
including but not limited to a coding sequence, a promoter region,
a cis-regulatory sequence, a non-expressed DNA segment, a
non-expressed DNA segment that contributes to gene expression, a
DNA segment designed to have desired parameters, or combinations
thereof. A gene can be obtained by a variety of methods, including
cloning from a biological sample, synthesis based on known or
predicted sequence information, and recombinant derivation of an
existing sequence.
[0109] The term "gene" thus includes an isolated soybean rhg1 and
SDS resistance gene as disclosed herein (FIG. 3). The gene is
capable of conveying Heterodera glycines-infestation resistance or
Fusarium solani-infection resistance to a non-resistant soybean
germplasm, the gene located within a quantitative trait locus
mapping to linkage group G and mapped by genetic markers of SEQ ID
NOs:1-6, said gene located along said quantitative trait locus
between said markers. Positional cloning methods were used to
isolate genomic sequences in the chromosomal regions of Forrest
that confers SCN/SDS resistance, as further described in Example 4.
Specifically, rhg1 sequences were derived from BAC clones 21D9 and
73P6 of the Forrest BamHI or HindIII BAC libraries (Meksem et al.,
2000). Preferably, the gene comprises the nucleotide sequence set
forth as SEQ ID:13 (FIG. 7A-B). BLASTP analysis of the conceptual
translation of the rhg1 gene (FIG. 7C), set forth as SEQ ID:14
shows high homology to the T46070 GenBank entry described as
hypothetical protein T18N14.120 from Arabidopsis thaliana (FIG.
7E-F), high homology to the rice Xa21 disease resistance gene
encoding a leucine-rich repeat protein, and high homology to the
tomato CF-2 gene for resistance to Cladosporium fulvus (FIG.
7D).
[0110] The rhg1 sequences disclosed herein can also be used to
isolate rhg1 cDNAs according to methods well-known in the art. A
representative rhg1 partial cDNA is set forth as SEQ ID NO:122.
This segment of the rhg1 gene shows homology to the leucine-rich
regions of the Arabidopsis hypothetical protein T18N14.120 (Gen
Bank T46070) and tomato CF-2 resistance genes.
[0111] For example, the term "gene" also includes an isolated
soybean Rhg4 gene. The gene is capable of conveying Heterodera
glycines-infestation resistance to a non-resistant soybean
germplasm, said gene located within a quantitative trait locus
mapping to linkage group A2 and mapped by the AFLP markers of SEQ
ID NOs:6-12, said gene located, along said quantitative trait locus
between said markers. Preferably, the gene comprises a nucleotide
sequence set forth as any one of SEQ ID NOs:16-19.
[0112] Genes underlying quantitative traits, or genes with related
function, such as disease resistance, are often organized in
clusters within the genome (e.g., Staskawicz (1995) Science
268:661-667). In the case of SCN/SDS resistance, previous studies
by the co-inventors of the present invention have suggested that
the resistance trait in Forrest may be caused by four genes in a
cluster with two pairs in close linkage or by a two-gene cluster
with each gene displaying pleitropy (Meksem et al., 1999). Thus,
genomic DNA isolated and disclosed herein comprise multiple
resistance gene sequences. Additional sequences derived from the
SCN/SDS resistance locus are set forth as SEQ ID NOs:20-66. BLASTX
analysis of these sequences reveals further homology to known
proteins in other organisms, supporting that they comprise new
partial gene sequences (Table 1). Of particular interest, BLASTX
analysis of the sequences set forth as SEQ ID NOs:67-114 reveals
that several of the disclosed sequences have high homology to the
T46070 GenBank entry described as hypothetical protein T18N14.120
from Arabidopsis thaliana, high homology to the tomato CF-2 disease
resistance genes encoding leucine-rich repeat proteins, and to the
tomato CF-9 gene for resistance to Cladosporium fulvus (Table
1).
[0113] The present invention also pertains to resistance genes
related to rhg1 and Rhg4. Partial cDNAs of additional putative
SCN/SDS resistance genes, set forth as SEQ ID NOs:67-114, were
identified based on hybridization to rhg1 and Rhg4 sequences, as
further described in Example 5. BLASTX analysis of these sequences
reveals further homology to known proteins in other organisms,
supporting that they comprise new partial gene sequences (Table 2).
Of particular interest, BLASTX analysis of the sequences set forth
as SEQ ID NOs:67-114 reveals that several of the disclosed
sequences have high homology to the T46070 GenBank entry described
as hypothetical protein T18N14.120 from Arabidopsis thaliana, high
homology to the tomato CF-2 disease resistance genes encoding
leucine-rich repeat proteins, and to the tomato CF-9 gene for
resistance to Cladosporium fulvus (Table 2). Based on their
hybridization to rhg1 and Rhg4 sequences, genes comprising any of
SEQ ID NOs:67-114 may also confer resistance to race 3 Heterodera
glycines. It will be apparent to one having ordinary skill in the
art that the disclosed sequences, or portion thereof, can be used
to identify, confirm and/or screen for SDS, SCN and/or other
resistance or for loci that confer SDS, SCN and/or other
resistance.
TABLE-US-00003 TABLE 1 best BLAST hit Score SEQ ID NO. inventor's
reference (ACCESSION) (bits) E value Identities Positives 20
III-00_F2-3RCF1900-2450 T47727 230 9e-60 114/170 (67%) 134/170
(78%) 21 III-01_21d9A1, 1A1 no significant similarity 22
III-01_21d9A2, 11F11Rlaccase AC007063 97 1e-19 62/166 (37%) 92/166
(55%) 23 III-01_21d9A2, 4A4Mic no significant similarity 24
III-01_CMG, smalF1-1F T46070 67 4e-13 49/147 (33%) 62/147 (41%) 25
III-02_21d9A2, 12A12FNaH + hypoth T00576 67 2e-10 57/188 (30%)
87/188 (45%) 26 III-02_F3-1RCF2000-2500 T46070 170 7e-42 79/105
(75%) 93/105 (88%) 27 III-03_21d9A1, 1E1Flaccase AC007020 61 1e-08
37/65 (56%) 43/65 (65%) 28 III-03_21d9A2, 12A12RNaH + hypothet
AC007063 116 2e-25 61/165 (36%) 95/165 (56%) 29 III-03_21d9A2,
4B4ESTM no significant similarity 30 III-03_21d9A2, 8F8CF1a T47727
187 53-48 95/142 (66%) 106/142 (73%) 31 III-03_21d9A2, 8F8CFHomol
T47727 177 5e-45 90/132 (68%) 100/132 (75%) 32 III-03_CMG,
smalF1-3FCF300-1100 T46070 107 4e-27 67/189 (35%) 89/189 (46%) 33
III-03_F3-2R1800-Cterm T47727 201 1e-64 97/129 (75%) 113/129 (87%)
34 III-04_21d9A1, 1E1R no significant similarity 35 III-04_21d9A2,
1B1 no significant similarity 36 III-04_21d9A2, 6D6mic no
significant similarity 37 III-05_21d9A1, 1C1GmxLaccase AB010692 153
2e-36 80/124 (64%) 90/124 (72%) 38 III-05_21d9A2, 4C4CFHomol T46070
125 6e-28 65/106 (61%) 72/106 (67%) 39 III-06_21d9A2,
11A11laccasegene AC007020 67 3e-12 30/49 (61%) 35/49 (71%) 40
III-07_21d9A1, 2A2F no significant similarity 41 III-08_21d9A1,
2A2R no significant similarity 42 III-08_21d9A2, 6F6 no significant
similarity 43 III-09_21d9A1, 1E1 no significant similarity 44
III-09_21d9A1, 2D2FNaH + hypothe AC007063 84 93-17 44/127 (34%)
74/127 (57%) 45 III-09_21d9A2, 4E4Laccase AC007020 90 1e-32 43/53
(81%) 46/53 (86%) 46 III-09_21d9A2, 9A9 no significant similarity
47 III-10_21d9A2, 11C11 T47325 53 3e-06 45/132 (34%) 65/132 (49%)
48 III-10_21d9A2, 11C11hypothetical T47325 53 3e-06 45/132 (34%)
65/132 (49%) 49 III-11_21d9A1, 1F1SatAT no significant similarity
50 III-11_21d9A2, 4A4F no significant similarity 51 III-11_21d9A2,
4F4SatTA no significant similarity 52 III-12_21d9A2,
1F1NaHexchangine AC007063 126 3e-28 72/181 (39%) 108/181(58%) 53
III-12_21d9A2, 4A4RSatTAGA no significant similarity 54
III-13_21d9A1, 1G1NaHexchanHypothe T00576 50 2e-05 31/83 (37%)
44/83 (52%) 55 III-13_21d9A1, 8D8CF500-1000 T46070 84 4e-24 48/127
(37%) 66/127 (51%) 56 III-13_21d9A2, 4B4FSatGAAAA no significant
similarity 57 III-14_21d9A2, 11E11GmxEST no significant similarity
58 III-14_21d9A2, 1G1 no significant similarity 59 III-15_21d9A1,
8E8 no significant similarity 60 III-15_21d9A2, 4C4FCF1600-1000
T46070 158 6e-38 99/215 (46%) 113/215 (52%) 61 III-15_21d9A2,
9D9NaHlonexch AC007063 64 1e-09 38/118 (32%) 59/118 (49%) 62
III-16_21d9A1, 11D11laccase CAA74104 82 4e-17 35/49 (71%) 43/49
(87%) 63 III-16_21d9A2, 11F11MicSatTA no significant similarity 64
III-16_21d9A2, 4C4R300-1000 T46070 110 3e-32 67/178 (37%) 86/178
(47%) 65 III-17_21d9A1, 2A2SatGA no significant similarity 66
III-17_21d9A1, 2A2SatTAA no significant similarity 73
II-01F2-4RCf1900-2400 T46070 187 6e-47 99/183 (54%) 123/183
(67%)
TABLE-US-00004 TABLE 2 SEQ best BLAST hit Score ID NO. inventor's
reference (ACCESSION) (bits) E value Identities Positives 67 3A Cf2
homologues to the +2ORF clone ID: 07d9 T47727 189 4e-47 103/215
(47%) 127/215 (58%) 68 3B Cf2 homologues to the -2ORF clone ID:
05d7 T46070 148 8e-35 76/157 (48%) 98/157 (62%) 69 3C Cf2
homologues to the +3 ORF clone ID: 17P9 T47727 200 2e-50 100/136
(73%) 113/136 (82%) 70 3D Cf2 homologues to the -3ORF clone ID:
06d8 T46070 163 2e-39 86/179 (48%) 110/179 (61%) 71
II-00_F2-3RCF1900-2450 T47727 230 9e-60 114/170 (67%) 134/170 (78%)
72 II-01CMGsmalF1-1F300-1000 T46070 76 4e-13 49/147 (33%) 62/147
(41%) 73 II-01F2-4RCf1900-2400 T46070 187 6e-47 99/183 (54%)
123/183 (67%) 74 II-02F3-1RCF2000-2500 T46070 170 7e-42 79/105
(75%) 93/105 (88%) 75 II-03.21dA2, 8F8CF1-500 T47727 187 5e-48
95/142 (66%) 106/142 (73%) 76 II-03CMG, smalF1-3FCF300-1100 T46070
107 4e-27 67/189 (35%) 89/189 (46%) 77 II-03F3-2R1800-Cterm T47727
201 1e-64 97/129 (75%) 113/129 (87%) 78 II-04.21dA1, 1E1R no
significant similarity 79 II-05.21dA2, 4C4CFhomol T46070 125 6e-28
65/106 (61%) 72/106 (67%) 80 II-12CFLNO1F-CFNOIF T46070 135 2e-33
74/165 (44%) 97/165 (57%) 81 II-12CFLNO1F-CFLNOIR T46070 273 2e-72
133/183 (72%) 156/183 (84%) 82 II-12CFLNO1F-CFLNNIF T46070 184
73-46 91/128 (71%) 100/128 (78%) 83 II-12CFLNO1F-CFLNN2F T46070 109
3e-24 69/189 (36%) 89/189 (46%) 84 II-13.21dA1, 8D8CF500-1000
T46070 84 4e-24 48/127 (37%) 66/127 (51%) 85 II-15.21dA2,
4C4FCF1600-1000 T46070 158 6e-38 99/215 (46%) 113/215 (52%) 86
II-29.21dA2, 8F8FCF500upstream T47727 102 2e-39 56/105 (53%) 67/105
(63%) 87 II-30.21d9A2, 12E12ESTMedicago T47731 238 6e-62 119/163
(73%) 132/163 (80%) 88 II-30.21d9A2, 8F8RCFpromoter no significant
similarity 89 II-30.E2, TetRP1downstreamtoRhg1 S05434 35 1.0 30/109
(27%) 49/109 (44%) 90 II-32.E3, TetRP1CF1115-1249 no significant
similarity 91 II-Cf homol-01CMGsmalF1-2F T46070 76 4e-13 49/147
(33%) 62/147 (41%) 92 II-Cf homol-CMGsmalF1-2F T46070 125 8e-32
74/188 (39%) 95/188 (50%) 93 II-Cf homol-03CMGsmalF1-3 T46070 105
1e-26 66/188 (35%) 88/188 (46%) 94 II-Cf homol-06CMGsmalF2-2F
T46070 123 2e-27 80/224 (35%) 105/224 (46%) 95 II-Cf
homol-07CMGsmalF2-3F T46070 123 2e-27 80/224 (35%) 105/224 (46%) 96
II-Cf homol-08CMGsmalF2-4F03 T46070 118 6e-29 71/183 (38%) 90/183
(48%) 97 II-Cf homol-10CMGsmalF3-2F T46070 184 7e-46 91/128 (71%)
100/128 (78%) 98 II-Cf homol-09CMGsmalF3-1F T46070 184 6e-46 91/128
(71%) 100/128 (78%) 99 II-Cf homol-smalF3-3F T46070 265 2e-70
128/174 (73%) 151/174 (86%) 100 II-Cf homol-12CMGsmalF3-4F T46070
184 7e-46 89/107 (83%) 97/107 (90%) 101 II-Cf homol-13CMGsmalF1-1R
T46070 279 3e-74 136/191 (71%) 159/191 (83%) 102 II-Cf
homol-14CMGsmalF1-2R T46070 261 3e-69 127/176 (72%) 148/176 (83%)
103 II-Cf homol-15CMGsmalF1-3R T47727 246 1e-64 120/162 (74%)
140/162 (86%) 104 II-Cf homol-16CMGsmalF1-4R T46070 263 1e-70
128/176 (72%) 149/176 (83%) 105 II-Cf homol-17CMGsmalF2-1R T46070
268 5e-71 131/183 (71%) 155/183 (84%) 106 II-Cf
homol-18CMGsmalF2-2R T46070 244 4e-65 118/159 (74%) 137/159 (85%)
107 II-Cf homol-05F3-4R T46070 187 6e-47 90/136 (66%) 111/136 (81%)
108 II-Cf homol-00F2-3R T46070 224 3e-58 108/148 (72%) 127/148
(84%) 109 II-Cf homol-01F2-4R T46070 187 6e-47 99/183 (54%) 123/183
(67%) 110 II-Cf homol-02F3-1R T46070 170 7e-42 79/105 (75%) 93/105
(88%) 111 II-Cf homol-03F3-2R T47727 202 9e-65 97/133 (72%) 11/133
(84%) 114 II-Cf homol-04F3-3R T46070 128 1e-30 65/108 (60%) 72/108
(66%) 114 II-Cf homol-05CMGsmalF2-F T46070 184 6e-46 91/128 (71%)
100/128 (78%) 114 II-downstream to Rhg1 no significant
similarity
[0114] III.D. SCN/SDS Resistance Gene Promoters
[0115] The term "promoter region" defines a nucleotide sequence
within a gene that is positioned 5' to a coding sequence of a same
gene and functions to direct transcription of the coding sequence.
The promoter region includes a transcriptional start site and at
least one cis-regulatory element. The present invention encompasses
nucleic acid sequences that comprise a promoter region of an
SCN/SDS resistance gene, or functional portion thereof.
[0116] The terms "cis-acting regulatory sequence" or
"cis-regulatory motif" or "response element", as used herein, each
refer to a nucleotide sequence that enables responsiveness to a
regulatory transcription factor. Responsiveness can encompass a
decrease or an increase in transcriptional output and is mediated
by binding of the transcription factor to the DNA molecule
comprising the response element.
[0117] The term "transcription factor" generally refers to a
protein that modulates gene expression by interaction with the
cis-regulatory element and cellular components for transcription,
including RNA Polymerase, Transcription Associated Factors (TAFs),
chromatin-remodeling proteins, and any other relevant protein that
impacts gene transcription.
[0118] The term "gene expression" generally refers to the cellular
processes by which a biologically active polypeptide is produced
from a DNA sequence.
[0119] A "functional portion" of a promoter gene fragment is a
nucleotide sequence within a promoter region that is required for
normal gene transcription. To determine nucleotide sequences that
are functional, the expression of a reporter gene is assayed when
variably placed under the direction of a promoter region
fragment.
[0120] Promoter region fragments can be conveniently made by
enzymatic digestion of a larger fragment using restriction
endonucleases or DNAse I. Preferably, a functional promoter region
fragment comprises about 5,000 nucleotides, more preferably 2,000
nucleotides, more preferably about 1,000 nucleotides, more
preferably a functional promoter region fragment comprises about
500 nucleotides, even more preferably a functional promoter region
fragment comprises about 100 nucleotides, and even more preferably
a functional promoter region fragment comprises about 20
nucleotides.
[0121] Within a candidate promoter region or response element, the
presence of regulatory proteins bound to a nucleic acid sequence
can be detected using a variety of methods well known to those
skilled in the art (Ausubel et al., 1992). Briefly, in vivo
footprinting assays demonstrate protection of DNA sequences from
chemical and enzymatic modification within living or permeabilized
cells. Similarly, in vitro footprinting assays show protection of
DNA sequences from chemical or enzymatic modification using protein
extracts. Nitrocellulose filter-binding assays and gel
electrophoresis mobility shift assays (EMSAs) track the presence of
radiolabeled regulatory DNA elements based on provision of
candidate transcription factors.
[0122] The terms "reporter gene" or "marker gene" or "selectable
marker" each refer to a heterologous gene encoding a product that
is readily observed and/or quantitated. A reporter gene is
heterologous in that it originates from a source foreign to an
intended host cell or, if from the same source, is modified from
its original form. Non-limiting examples of detectable reporter
genes that can be operably linked to a transcriptional regulatory
region can be found in brown and PCT International Publication No.
WO 97/47763. Preferred reporter genes for transcriptional analyses
include the lacZ gene (See, e.g., Rose & Botstein (1983) Meth
Enzymol 101:167-180), Green Fluorescent Protein (GFP) (Cubitt et
al. (1995) Trends Biochem Sci 20:448-455), luciferase, or
chloramphenicol acetyl transferase (CAT). Preferred reporter genes
for stable transformation include but are not limited to antibiotic
resistance genes. Any suitable reporter and detection method can be
used, and it will be appreciated by one of skill in the art that no
particular choice is essential to or a limitation of the present
invention.
[0123] An amount of reporter gene can be assayed by any method for
qualitatively or preferably, quantitatively determining presence or
activity of the reporter gene product. The amount of reporter gene
expression directed by each test promoter region fragment is
compared to an amount of reporter gene expression to a control
construct comprising the reporter gene in the absence of a promoter
region fragment. A promoter region fragment is identified as having
promoter activity when there is significant increase in an amount
of reporter gene expression in a test construct as compared to a
control construct. The term "significant increase", as used herein,
refers to an quantified change in a measurable quality that is
larger than the margin of error inherent in the measurement
technique, preferably an increase by about 2-fold or greater
relative to a control measurement, more preferably an increase by
about 5-fold or greater, and most preferably an increase by about
10-fold or greater.
[0124] A representative SCN/SDS resistance gene promoter, the rhg1
promoter, is set forth as SEQ ID NO:15. The rhg1 promoter is useful
for directing gene expression of heterologous sequences in vivo or
in assays to identify modulators of rhg1 expression, described
further herein below.
[0125] The present invention further provides an isolated SCN/SDS
resistance gene promoter region, or functional portion thereof,
comprising an about 90 kb fragment of soybean genomic clone 73P6
between BamHI restriction sites and 21d9 between HinDIII
restriction site. The genomic clone is available from the Forrest
BAC library described in Meksem et al (2000), Theor Appl Genet. 101
5/6: 747-755, available through Southern Illinois
University-Carbondale (Carbondale, Ill.), Texas A&M University
BAC center (College Station, Tex.), and Research Genetics
(Huntsville, Ala.). An isolated SCN/SDS resistance gene promoter
region, or functional portion thereof, comprising an about 4.5 kb
fragment of soybean genomic clone 21d9A2 8F8 between EcoRI
restriction sites is also disclosed.
[0126] III.E. Chimeric Genes
[0127] The present invention also encompasses chimeric genes
comprising the disclosed SCN/SDS resistance gene sequences. The
term "chimeric gene", as used herein, refers to an SCN/SDS
resistance gene promoter region operably linked to an open reading
frame, wherein the nucleotide sequence created is not naturally
occurring. In this regard, the open reading frame is also described
as a "heterologous sequence". The term "chimeric gene" also
encompasses a promoter region operably linked to an SCN/SDS
resistance gene coding sequence, a nucleotide sequence producing an
antisense RNA molecule, a RNA molecule having tertiary structure,
such as a hairpin structure, or a double-stranded RNA molecule.
[0128] The term "operably linked", as used herein, refers to a
promoter region that is connected to a nucleotide sequence in such
a way that the transcription of that nucleotide sequence is
controlled and regulated by that promoter region. Techniques for
operatively linking a promoter region to a nucleotide sequence are
well known in the art.
[0129] The terms "heterologous gene", "heterologous DNA sequence",
"heterologous nucleotide sequence", "exogenous nucleic acid
molecule", or "exogenous DNA segment", as used herein, each refer
to a sequence that originates from a source foreign to an intended
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, for example by mutagenesis or by isolation from native
cis-regulatory sequences. The terms also include non-naturally
occurring multiple copies of a naturally occurring nucleotide
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 wherein the element is
not ordinarily found.
IV. Polypeptide Sequences of SCN/SDS Resistance Proteins
[0130] The polypeptides provided by the present invention include
the isolated polypeptide of SEQ ID NO:14, fusion proteins
comprising SCN/SDS resistance gene amino acid sequences,
biologically functional analogs, and polypeptides that cross-react
with an antibody that specifically recognizes an SCN/SDS resistance
gene polypeptide.
[0131] The term "isolated", as used in the context of a
polypeptide, indicates that the polypeptide exists apart from its
native environment and is not a product of nature. An isolated
polypeptide can exist in a purified form or can exist in a
non-native environment such as, for example, in a transgenic host
cell.
[0132] The term "purified", when applied to a polypeptide, denotes
that the polypeptide is essentially free of other cellular
components with which it is associated in the natural state.
Preferably, a polypeptide is a homogeneous solid or aqueous
solution. Purity and homogeneity are typically determined using
analytical chemistry techniques such as polyacrylamide gel
electrophoresis or high performance liquid chromatography. A
polypeptide that is the predominant species present in a
preparation is substantially purified. The term "purified" denotes
that a polypeptide gives rise to essentially one band in an
electrophoretic gel. Particularly, it means that the polypeptide is
at least about 50% pure, more preferably at least about 85% pure,
and most preferably at least about 99% pure.
[0133] The term "substantially identical" in the context of two or
more polypeptides sequences is measured by (a) polypeptide
sequences having about 35%, or 45%, or preferably from 45-55%, or
more preferably 55-65%, or most preferably 65% or greater amino
acids that are identical or functionally equivalent. Percent
"identity" and methods for determining identity are defined herein
under the heading Nucleotide and Amino Acid Sequence
Comparisons.
[0134] Substantially identical polypeptides also encompass two or
more polypeptides sharing a conserved three-dimensional structure.
Computational methods can be used to compare structural
representations, and structural superpositions can be generated and
easily tuned to identify similarities around important active sites
or ligand binding sites. See Henikoff et al. (2000) Electrophoresis
21(9):1700-1706; Huang et al. (2000) Pac Symp Biocomput 230-241;
Saqi et al., 1999; and Barton (1998) Acta Crystallogr D Biol
Crystallogr 54:1139-1146.
[0135] The term "functionally equivalent" in the context of amino
acid sequences is well known in the art and is based on the
relative similarity of the amino acid side-chain substituents. See
Henikoff and Henikoff (2000) Adv Protein Chem 54:73-97. Relevant
factors for consideration include side-chain hydrophobicity,
hydrophilicity, charge, and size. For example, arginine, lysine,
and histidine are all positively charged residues; that alanine,
glycine, and serine are all of similar size; and that
phenylalanine, tryptophan, and tyrosine all have a generally
similar shape. By this analysis, described further herein below,
arginine, lysine, and histidine; alanine, glycine, and serine; and
phenylalanine, tryptophan, and tyrosine; are defined herein as
biologically functional equivalents.
[0136] In making biologically functional equivalent amino acid
substitutions, the hydropathic index of amino acids can be
considered. Each amino acid has been assigned a hydropathic index
on the basis of their hydrophobicity and charge characteristics,
these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);
phenylalanine (+2.8); cysteine (+2.5); methionine (+1.9); alanine
(+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan
(-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2);
glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine
(-3.5); lysine (-3.9); and arginine (-4.5).
[0137] The importance of the hydropathic amino acid index in
conferring interactive biological function on a protein is
generally understood in the art (Kyte et al. (1982) J Mol Biol
157:105.). It is known that certain amino acids can be substituted
for other amino acids having a similar hydropathic index or score
and still retain a similar biological activity. In making changes
based upon the hydropathic index, the substitution of amino acids
whose hydropathic indices are within .+-.2 of the original value is
preferred, those which are within .+-.1 of the original value are
particularly preferred, and those within .+-.0.5 of the original
value are even more particularly preferred.
[0138] It is also understood in the art that the substitution of
like amino acids can be made effectively on the basis of
hydrophilicity. U.S. Pat. No. 4,554,101 states that the greatest
local average hydrophilicity of a protein, as governed by the
hydrophilicity of its adjacent amino acids, correlates with its
immunogenicity and antigenicity, i.e. with a biological property of
the protein. It is understood that an amino acid can be substituted
for another having a similar hydrophilicity value and still obtain
a biologically equivalent protein.
[0139] As detailed in U.S. Pat. No. 4,554,101, the following
hydrophilicity values have been assigned to amino acid residues:
arginine (+3.0); lysine (+3.0); aspartate (+3.0.+-.1); glutamate
(+3.0.+-.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);
glycine (0); threonine (-0.4); proline (-0.5.+-.1); alanine (-0.5);
histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine
(-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3);
phenylalanine (-2.5); tryptophan (-3.4).
[0140] In making changes based upon similar hydrophilicity values,
the substitution of amino acids whose hydrophilicity values are
within .+-.2 of the original value is preferred, those which are
within .+-.1 of the original value are particularly preferred, and
those within .+-.0.5 of the original value are even more
particularly preferred.
[0141] The present invention also encompasses SCN/SDS resistance
gene polypeptide fragments or functional portions of an SCN/SDS
resistance gene polypeptide. Such functional portion need not
comprise all or substantially all of the amino acid sequence of a
native resistance gene product. The term "functional" includes any
biological activity or feature of SCN/SDS resistance gene,
including immunogenicity.
[0142] The present invention also includes longer sequences
comprising an SCN/SDS resistance gene polypeptide, or portion
thereof. For example, one or more amino acids can be added to the
N-terminal or C-terminal of an SCN/SDS resistance gene polypeptide.
Fusion proteins comprising SCN/SDS resistance gene polypeptide
sequences are also provided within the scope of the present
invention. Methods of preparing such proteins are known in the
art.
[0143] The present invention also encompasses functional analogs of
an
[0144] SCN/SDS resistance gene polypeptide. Functional analogs
share at least one biological function with an SCN/SDS resistance
gene polypeptide. An exemplary function is immunogenicity. In the
context of amino acid sequence, biologically functional analogs, as
used herein, are peptides in which certain, but not most or all, of
the amino acids can be substituted. Functional analogs can be
created at the level of the corresponding nucleic acid molecule,
altering such sequence to encode desired amino acid changes. In one
embodiment, changes can be introduced to improve the antigenicity
of the protein. In another embodiment, an SCN/SDS resistance gene
polypeptide sequence is varied so as to assess the activity of a
mutant SCN/SDS resistance gene polypeptide. In still another
embodiment, amino acid changes can be made to improve the stability
of the polypeptide.
[0145] Isolated polypeptides and recombinantly produced
polypeptides can be purified and characterized using a variety of
standard techniques that are well known to the skilled artisan. See
e.g. Ausubel et al. (1992); Bodanszky et al., 1976; and Zimmer et
al. (1993) Peptides, pp. 393B394, ESCOM Science Publishers, B.
V.
V. Nucleotide and Amino Acid Sequence Comparisons
[0146] The terms "identical" or percent "identity" in the context
of two or more nucleotide or polypeptide sequences, refer to two or
more sequences or subsequences that are the same or have a
specified percentage of amino acid residues or nucleotides that are
the same, when compared and aligned for maximum correspondence, as
measured using one of the sequence comparison algorithms disclosed
herein or by visual inspection.
[0147] The term "substantially identical" in regards to a
nucleotide or polypeptide sequence means that a particular sequence
varies from the sequence of a naturally occurring sequence by one
or more deletions, substitutions, or additions, the net effect of
which is to retain at least some of biological activity of the
natural gene, gene product, or sequence. Such sequences include
"mutant" sequences, or sequences wherein the biological activity is
altered to some degree but retains at least some of the original
biological activity. The term "naturally occurring", as used
herein, is used to describe a composition that can be found in
nature as distinct from being artificially produced by man. For
example, a protein or nucleotide sequence present in an organism,
which can be isolated from a source in nature and which has not
been intentionally modified by man in the laboratory, is naturally
occurring.
[0148] For sequence comparison, typically one sequence is regarded
as a reference sequence to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are entered into a computer program, subsequence coordinates are
designated if necessary, and sequence algorithm program parameters
are selected. The sequence comparison algorithm then calculates the
percent sequence identity for the designated test sequence(s)
relative to the reference sequence, based on the selected program
parameters.
[0149] Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith &
Waterman (1981) Adv Appl Math 2:482, by the homology alignment
algorithm of Needleman & Wunsch (1970) J Mol Biol 48:443, by
the search for similarity method of Pearson & Lipman (1988)
Proc Natl Acad Sci USA 85:2444, by computerized implementations of
these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package, Genetics Computer Group, Madison, Wis.),
or by visual inspection. See generally, Ausubel et al. (1992).
[0150] A preferred algorithm for determining percent sequence
identity and sequence similarity is the BLAST algorithm, which is
described in Altschul et al. (1990) J Mol Biol 215: 403-410.
Software for performing BLAST analyses is publicly available
through the National Center for Biotechnology Information
(http://www.ncbi.nlm.nih.gov/). This algorithm involves first
identifying high scoring sequence pairs (HSPs) by identifying short
words of length W in the query sequence, which either match or
satisfy some positive-valued threshold score T when aligned with a
word of the same length in a database sequence. T is referred to as
the neighborhood word score threshold. These initial neighborhood
word hits act as seeds for initiating searches to find longer HSPs
containing them. The word hits are then extended in both directions
along each sequence for as far as the cumulative alignment score
can be increased. Cumulative scores are calculated using, for
nucleotide sequences, the parameters M (reward score for a pair of
matching residues; always >0) and N (penalty score for
mismatching residues; always <0). For amino acid sequences, a
scoring matrix is used to calculate the cumulative score. Extension
of the word hits in each direction are halted when the cumulative
alignment score falls off by the quantity X from its maximum
achieved value, the cumulative score goes to zero or below due to
the accumulation of one or more negative-scoring residue
alignments, or the end of either sequence is reached. The BLAST
algorithm parameters W, T, and X determine the sensitivity and
speed of the alignment. The BLASTN program (for nucleotide
sequences) uses as defaults a wordlength W=11, an expectation E=10,
a cutoff of 100, M=5, N=-4, and a comparison of both strands. For
amino acid sequences, the BLASTP program uses as defaults a
wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62
scoring matrix. See Henikoff and Henikoff (1989) Proc Natl Acad Sci
USA 89:10915.
[0151] In addition to calculating percent sequence identity, the
BLAST algorithm also performs a statistical analysis of the
similarity between two sequences. See e.g., Karlin and Altschul
(1993) Proc Natl Acad Sci USA 90:5873-5887. One measure of
similarity provided by the BLAST algorithm is the smallest sum
probability (P(N)), which provides an indication of the probability
by which a match between two nucleotide or amino acid sequences
would occur by chance. For example, a test nucleic acid sequence is
considered similar to a reference sequence if the smallest sum
probability in a comparison of the test nucleic acid sequence to
the reference nucleic acid sequence is less than about 0.1, more
preferably less than about 0.01, and most preferably less than
about 0.001.
VI. Method for Detecting a Nucleic Acid Molecule Associated with
SCN/SDS Resistance
[0152] In another aspect of the invention, a method is provided for
detecting a nucleic acid molecule that encodes an SCN/SDS
resistance polypeptide. Such methods can be used to detect SCN/SDS
resistance gene variants and related resistance gene sequences. The
disclosed methods facilitate genotyping, cloning, gene mapping, and
gene expression studies.
[0153] VI.A. Genetic Variants
[0154] In one embodiment, genetic assays based on nucleic acid
molecules of the present invention can be used to screen for
genetic variants by a number of PCR-based techniques, including
single-strand conformation polymorphism (SSCP) analysis (Orita et
al. (1989) Proc Natl Acad Sci USA 86(8):2766-2770),
SSCP/heteroduplex analysis, enzyme mismatch cleavage, direct
sequence analysis of amplified exons (Kestila et al. (1998) Mol
Cell 1(4):575-582; Yuan et al. (1990) Hum Mutat 14(5):440-446),
allele-specific hybridization (Stoneking et al. (1991) Am J Hum
Genet 48(2):370-82), and restriction analysis of amplified genomic
DNA containing the specific mutation. Automated methods can also be
applied to large-scale characterization of single nucleotide
polymorphisms (Brookes (1999) Gene 234(2):177-186; Wang et al.
(1998) Science 280(5366):1077-82). Preferred detection methods are
non-electrophoretic, including, for example, the TaqMan.TM. allelic
discrimination assay, PCR-OLA, molecular beacons, padlock probes,
and well fluorescence. See Landegren et al. (1998) Genome Res
8:769-776.
[0155] In a preferred embodiment, genetic markers for SCN/SDS
resistance disclosed herein are used in a PCR-based genotyping
assay, preferably, a TaqMan.TM. assay as disclosed in Example 6.
The TaqMan.TM. allelic discrimination assay is based on the 5'
nuclease activity of Taq polymerase and detection of a fluorescent
reporter during or after PCR reactions (Livak et al. (1995) PCR
Meth and Applic 4:357-362; Livak et al. (1995) Nat Genet.
9:341-342). Each TaqMan.TM. probe consists of a 25-35 base
oligonucleotide complementary to one of two alleles with a 3'
quencher dye attached (6-carboxy-N,N,N'5N' tetrachlorofluorescein;
TAMRA). The oligomer complimentary to allele 1 is linked covalently
to a 5' reporter dye (6-carboxy-4,7,2',7', tetrachlorofluorescenin;
TET) while allele 2 is linked to a dye that fluoresces at a
distinct wavelength (6-carboxyfluorescein; FAM). PCR directed by
flanking oligomers of 18-20 bases causes degradation during the
extension phase of the oligomer that hybridizes most efficiently to
the polymorphic site(s) in the sample. Adaptations can make the
assay chemistry suitable for multiplexing (Nasarabadi et al. (1999)
BioTechniques 27:1116-1117) and miniaturization (Kalinina et al.
(1997) Nucl Acids Res 25:1999-2004) to reduce cost and increase
throughput.
[0156] The present invention discloses sequences suitable for use
with the TaqMan.TM. method for genotyping SCN/SDS resistance,
further disclosed in Example 6. As one example, the TaqMan.TM.
assay was used to distinguish between two insertion polymorphisms
in alleles of an AFLP marker that is located about 50 kbp from the
Rhg4 gene (FIG. 4). Genomic DNA samples were analyzed using the
TaqMan.TM. PCR protocol (Livak et al., 1995a, 1995b). Using the raw
fluorescence signals of the reporter dyes FAM and TET from the "dye
component" field of the sequence detection software, two grouping
methods were performed. Each method detected four distinct
populations (FIG. 5). The four populations could be assigned
according to the FAM:TET ratio based on where the heterogeneous
class cut-off was placed.
[0157] For the TaqMan.TM. selection, two grouping methods were
arbitrarily selected to attempt to accurately separate
heterogeneous lines, from homogeneous lines at each allele. For
grouping method 1 (Taqman.TM. 1) a stringent cut-off was used to
reduce the number called as potentially heterogeneous. Fluorophore
ratios were as follows; no amplification (FAM and TET both less
than 6 units); allele 1 homozygous (FAM less than 7, TET greater
than 7); allele 2 homozygous (FAM greater than 10, TET less than
5); and heterogeneous for allele 1 and allele 2 (FAM greater than
7, TET 5-8). For TaqMan.TM. selection grouping method 2 (TaqMan.TM.
2), a lower stringency cut-off value was used to increase the
number called as potentially heterogeneous. Ratios were: no
amplification (FAM and TET both less than 6 units); allele 1
homozygous (FAM less than 5, TET greater than 7); allele 2
homozygous (FAM greater than 10, TET less than 5); and
heterogeneous for allele 1 and allele 2 (FAM greater than 5, TET
5-9).
[0158] Based on the FI of the ExF RIL population, the 86 selected
individuals were classified into 3 classes: 15 resistant, 60
susceptible and 11 segregating lines. TaqMan.TM. analysis of 86
individuals from the RILs by method 1 (high stringency) shows a
strong agreement between allele 1 and susceptibility to SCN (56
from the 60 susceptible lines were allele 1 type). However, there
was lesser agreement between allele 2 and resistance to SCN (only
15 lines from the 23 lines showing the presence of allele 2 were
resistant by phenotype) due to the segregation of rhg1, the second
gene necessary for resistance to SCN in Forrest. Of the 11 lines
known to be heterogeneous for the resistance to SCN phenotype, five
should segregate at Rhg4. TaqMan.TM. method 1 identified one among
the five classified as heterogenous (the 5 include 4
miss-classified lines, see below). TaqMan.TM. method 2 identified
all five among the 11 classified as heterogenous, however the 11
include 6 miss-classified lines.
[0159] To validate the specificity of TaqMan.TM. genotyping,
samples of each of the RILs classified by the TaqMan.TM. method
(FIG. 5) were re-scored by PCR and gel electrophoresis (FIG. 6)
according to methods described in Example 7. The classifications
produced by the two methods agreed with Taqman.TM. assay 1 most
closely but with eight exceptions. The miss-scores were as follows
(annotated as RIL#; FI phenotype; allele with TaqMan.TM. grouping
method 2; allele with TaqMan.TM. grouping method 1; allele by gel
marker score): 4;S;H;H;S: 21;R;H;H;R: 32;R;H;H;R: 44;S;S;S;H:
51;S;S;S;H: 59;R;H;H;R: 63,S;S;S;R: 78;R;H;H;R.
[0160] The majority of disagreements resulted from resistant lines
that were scored as heterogeneous by TaqMan.TM. but not gel
electrophoresis or phenotype (4 of 8) and phenotypically
susceptible lines that were scored incorrectly by gel
electrophoresis (3 of 8). One genotype (RIL84) was miss-scored
relative to phenotype (84SRRR) by all the allele genotyping methods
and may represent a recombination event between A2D8 and Rhg4.
[0161] The genoytpe and phenotype were generally in close agreement
among the eighty six genomic DNA samples analyzed using the
TaqMan.TM. PCR protocol. The lesser agreement between Allele 2 and
resistance to SCN (15 of 23) was shown to be due to the segregation
of rhg1, by scoring of the BARC-Satt 309 marker (Meksem et al.,
1999). The bias toward a higher frequency of allele 1 is caused by
sampling error (Chang et al., 1997). The accuracy of genotyping was
high by the TaqMan.TM. assay and was better than one pass gel
electrophoresis (Prabhu et al., 1999). Even compared to a highly
optimized gel electrophoresis assay reported herein the assays were
not significantly different in accuracy for detecting the genotypes
within the F.sub.5 derived RILs in a single pass assay. Exactly 78
of the 86 tested with both, TaqMan.TM. and gel electrophoresis
results agreed. There were 5 errors with Taqman.TM. (94% accurate)
and 3 errors with gel electrophoresis (96% accurate) judged by
replicated genotyping (not shown) and the phenotype. Low
frequencies of error are important to the accurate selection of
resistance (Cregan et al., 1999a; Prabhu et al., 1999) and in the
generation of accurate genetic maps (Cregan et al., 1999b).
[0162] VI.B. Cloning of SCN/SDS Resistance Genes and Related
Genes
[0163] The nucleic acids of the present invention can be used to
clone genes and genomic DNA comprising the sequences.
Alternatively, the nucleic acids of the present invention can be
used to clone genes and genomic DNA of related sequences. For this
purpose, representative probes, hybridization conditions, and PCR
primers are described in the section entitled Nucleotide Sequences
of SCN/SDS Resistance Genes and Associated Markers herein above and
in Examples 4 and 5. Preferably, the nucleic acids used for this
method comprise sequences set forth as any one of SEQ ID NOs:13,
15-114, more preferably SEQ ID NOs: 13 and 16-19.
[0164] In another embodiment, the present invention provides a
method of positional cloning of genes and other sequences located
adjacent or near the disclosed sequences within the soybean genome.
The method comprises: (a) identifying a first nucleic acid
genetically linked to a SCN/SDS resistance locus; and (b) cloning
the first nucleic acid. Optionally, the first nucleic acid can
comprise the rhg1 and SDS locus or the Rhg4 locus. Preferably, the
SCN/SDS resistance locus corresponds to a nucleic acid selected
from any one of SEQ ID NOs:13 and 16-19.
[0165] Positional cloning first involves creating a physical map of
a contig (contiguous overlapping of cloned DNA inserts), in the
genomic region encompassing one or more marker loci and the target
gene. The target gene is then identified and isolated within one or
more clones residing in the contig. The cloned gene can be used
according to any suitable method known in the art, including, for
example, genetic studies, transformation, and the development of
novel phenotypes.
[0166] Mapped SCN, SDS, or SCN and SDS markers, especially those
most closely linked to SCN/SDS resistance can be used to identify
homologous clones from soybean genomic libraries, including, for
example, soybean genomic libraries made in bacterial artificial
chromosomes (BAC), yeast artificial chromosomes (YAC), or P1
bacteriophage. These types of vectors are preferred for positional
cloning because they have the capacity to carry larger DNA inserts
than possible with other vector technologies. These larger DNA
inserts allow the researcher to move physically farther along the
chromosome by identifying overlapping clones. Exemplary libraries
available for positional cloning efforts in soybean include those
described by Meksem et al., 2000; Kanazin et al. (1996) Proc Natl
Acad Sci USA 93(21):11746-11750; Zhu et al. (1996) Mol Gen Genet.
252:483-488. Exemplary hybridization methods are disclosed in
Examples 4 and 5.
[0167] Mapped SCN, SDS, or SCN and SDS markers can be used as DNA
probes to hybridize and select homologous genomic clones from such
libraries. Alternatively, the DNA of mapped marker clones are
sequenced to design PCR primers that amplify and therefore identify
homologous genomic clones from such libraries. Either method is
used to identify large-insert soybean clones that is then used to
start or finish a contig constructed in chromosome walking to clone
an SCN, SDS, or SCN and SDS resistance QTL.
[0168] As examples, the positional cloning strategy was
successfully used to clone the cystic fibrosis gene in humans
(Rommens et al. (1989) Science 245:1059-1065), an omega-3
desaturase gene in Arabidopsis (Arondel et al. (1992) Science
258:1353-1355), a protein kinase gene (Pto) conferring fungal
resistance in tomato (Martin et al. (1993) Science 262:1432-1436),
a YAC clone containing the jointless gene that suppresses
abscission of flowers and fruit in tomato (Zhang et al. (1994) Mol
Gen Genet. 244:613-621), and sequences comprising the rhg1 and Rhg4
genes, disclosed herein.
[0169] VI.C. Mapping Methods
[0170] The isolated and purified polynucleotide sequences disclosed
herein can also be used in a variety of applications pertaining to
mapping SCN and SDS resistance. For example, the isolated
polynucleotides disclosed herein are useful in studies of genome
organization; in gene structure and organization experiments; in
BAC-FISH experiments; in chromosome painting techniques; and in
chromosome manipulation.
[0171] Thus, in accordance with the present invention, the nucleic
acid sequences which encode SCN/SDS resistance polypeptides can
also be used to generate hybridization probes which are useful for
mapping naturally occurring genomic sequences and/or resistance
loci. The sequences can be mapped to a particular chromosome or to
a specific region of the chromosome using well-known techniques.
Such techniques include FISH, FACS, or artificial chromosome
constructions, such as yeast artificial chromosomes, bacterial
artificial chromosomes, bacterial P1 constructions or single
chromosome cDNA libraries as reviewed in Price (1993) Blood Rev
7:127-134, and Trask (1991) Trends Genet. 7:149-154.
[0172] FISH (as described in Verma et al. (1988) Human Chromosomes:
A Manual of Basic Techniques, Pergamon Press, New York, N.Y.) can
be correlated with other physical chromosome mapping techniques and
genetic map data. Examples of genetic map data can be found in the
1994 Genome Issue of Science (265:1981f). Correlation between the
location of the gene encoding SCN, SDS, or both SCN and SDS
resistance on a physical chromosomal map and another resistance
characteristic, or lack thereof, can help delimit the region of DNA
associated with that genetic characteristic. The nucleotide
sequences of the subject invention can be used to detect
differences in gene sequences between normal, carrier, or
susceptible individuals.
[0173] In situ hybridization of chromosomal preparations and
physical mapping techniques such as linkage analysis and
chromosomal painting using established chromosomal markers can be
used for extending genetic maps. Often the placement of a gene on
the chromosome of another plant species, such as tomato species or
other soybean species, reveals associated markers also found in
other plants such as soybeans even if the number or arm of a
particular chromosome is not known. New sequences can be assigned
to chromosomal arms, or parts thereof, by physical mapping. This
provides valuable information to investigators searching for
resistance or other genes using positional cloning or other gene
discovery techniques. Once the resistance or other gene has been
crudely localized by genetic linkage to a particular genomic
region, any sequences mapping to that area can represent associated
or regulatory genes for further investigation. The nucleotide
sequences of the present invention can thus also be used to detect
differences in the chromosomal location due to translocation,
inversion, etc. among normal, carrier, or susceptible individuals,
and to detect gene regulatory sequences (e.g. promoters).
[0174] Hybridization of the subject DNAs to reference chromosomes
can also be performed to give information on relative copy numbers
of sequences. Normalization is required to obtain absolute copy
number information. One convenient method to do this is to
hybridize a probe, for example a cosmid specific to some single
locus in the normal haploid genome, to the interphase nuclei of the
subject cell or cell population(s) (or those of an equivalent cell
or representative cells therefrom, respectively). Quantiation of
the hybridization signals in a representative population of such
nuclei gives the absolute sequence copy number at that location.
Given that information at one locus, the intensity (ratio)
information from the hybridization of the subject DNA(s) to the
reference condensed chromosomes gives the absolute copy number over
the rest of the genome. In practice, use of more than one reference
locus can be desirable. In this case, the best fit of the intensity
(ratio) data through the reference loci can give a more accurate
determination of absolute sequence copy number over the rest of the
genome.
[0175] Thus, the methods of the present invention can provide
information on the absolute copy numbers of substantially all RNA
or DNA sequences in subject cell(s) or cell population(s) as a
function of the location of those sequences in a reference genome.
Additionally, chromosome painting probes can be prepared using the
markers and sequence data herein disclosed. Hybridization with one
or more of such probes indicates the absolute copy numbers of the
sequences to which the probes bind.
[0176] Further, when the subject nucleic acid sequences are DNA,
the reference copy numbers can be determined by Southern analysis.
When the subject nucleic acid sequences are RNA, the reference copy
numbers can be determined by Northern analysis.
[0177] VI.D. Assays Kits
[0178] In another aspect, the present invention provides assay kits
for detecting the presence, in biological samples, of a
polynucleotide that encodes a polypeptide of the present invention
or of a chromosome bearing a gene or locus of the present
invention, the kits comprising a first container that contains a
second polynucleotide identical or complementary to a segment of at
least 10 contiguous nucleotide bases of, as a preferred example,
any of SEQ ID NOs:13 and 16-19.
VII. Recombinant Expression B Expression Cassettes
[0179] The term "expression cassette" as used herein means a DNA
sequence capable of directing expression of a particular nucleotide
sequence in an appropriate host cell, comprising a promoter
operably linked to the nucleotide sequence of interest which is
operably linked to termination signals. It also typically comprises
sequences required for proper translation of the nucleotide
sequence. The expression cassette comprising the nucleotide
sequence of interest can be chimeric. The expression cassette can
also be one which is naturally occurring but has been obtained in a
recombinant form useful for heterologous expression. The expression
cassettes can also comprise any further sequences required or
selected for the expression of the transgene. Such sequences
include, but are not restricted to, transcription terminators,
extraneous sequences to enhance expression such as introns, vital
sequences, and sequences intended for the targeting of the gene
product to specific organelles and cell compartments.
[0180] VII.A. Promoters
[0181] The expression of the nucleotide sequence in the expression
cassette can be under the control of a constitutive promoter or an
inducible promoter which initiates transcription only when the host
cell is exposed to some particular external stimulus. For bacterial
production of a SCN/SDS resistance polypeptide, exemplary promoters
include Simian virus 40 early promoter, a long terminal repeat
promoter from retrovirus, an actin promoter, a heat shock promoter,
and a metallothionein protein. For in vivo production of a SCN/SDS
resistance polypeptide in plants, exemplary constituitve promoters
are derived from the CaMV 35S, rice actin, and maize ubiquitin
genes, each described herein below. Exemplary inducible promoters
for this purpose include the chemicaly inducible PR-1a promoter and
a wound-inducible promoter, also described herein below.
[0182] Selected promoters can direct expression in specific cell
types (such as leaf epidermal cells, mesophyll cells, root cortex
cells) or in specific tissues or organs (roots, leaves or flowers,
for example). Exemplary tissue-specific promoters include
well-characterized root-, pith-, and leaf-specific promoters, each
described herein below.
[0183] Depending upon the host cell system utilized, any one of a
number of suitable promoters can be used. Promoter selection can be
based on expression profile and expression level. The following are
non-limiting examples of promoters that can be used in the
expression cassettes.
[0184] VII.A.1.Constituitive Expression
[0185] 35S Promoter. The CaMV 35S promoter can be used to drive
constituitive gene expression. Construction of the plasmid pCGN1761
is described in the published patent application EP 0 392 225,
which is hereby incorporated by reference. pCGN1761 contains the
"double" CaMV 35S promoter and the tml transcriptional terminator
with a unique EcoRI site between the promoter and the terminator
and has a pUC-type backbone. A derivative of pCGN1761 is
constructed which has a modified polylinker which includes NotI and
XhoI sites in addition to the existing EcoRI site. This derivative
is designated pCGN1761ENX. pCGN1761ENX is useful for the cloning of
cDNA sequences or gene sequences (including microbial ORF
sequences) within its polylinker for the purpose of their
expression under the control of the 35S promoter in transgenic
plants. The entire 35S promoter-gene sequence-tml terminator
cassette of such a construction can be excised by HindIII, SphI,
SalI, and XbaI sites 5' to the promoter and XbaI, BamHI and BgII
sites 3' to the terminator for transfer to transformation vectors
such as those described below. Furthermore, the double 35S promoter
fragment can be removed by 5' excision with HindIII, SphI, SalI,
XbaI, or PstI, and 3' excision with any of the polylinker
restriction sites (EcoRI, NotI or XhoI) for replacement with
another promoter.
[0186] Actin Promoter. Several isoforms of actin are known to be
expressed in most cell types and consequently the actin promoter is
a good choice for a constitutive promoter. In particular, the
promoter from the rice ActI gene has been cloned and characterized
(McElroy et al. (1990) Plant Cell 2:163-171). A 1.3 kb fragment of
the promoter was found to contain all the regulatory elements
required for expression in rice protoplasts. Furthermore, numerous
expression vectors based on the ActI promoter have been constructed
specifically for use in monocotyledons (McElroy et al. (1991) Mol
Gen Genet. 231:150-160). These incorporate the ActI-intron 1, AdhI
5' flanking sequence and AdhI-intron 1 (from the maize alcohol
dehydrogenase gene) and sequence from the CaMV 35S promoter.
Vectors showing highest expression were fusions of 35S and ActI
intron or the ActI 5' flanking sequence and the ActI intron.
Optimization of sequences around the initiating ATG (of the GUS
reporter gene) also enhanced expression. The promoter expression
cassettes described by McElroy et al. (1991) can be easily modified
for gene expression and are particularly suitable for use in
monocotyledonous hosts. For example, promoter-containing fragments
is removed from the McElroy constructions and used to replace the
double 35S promoter in pCGN1761ENX, which is then available for the
insertion of specific gene sequences. The fusion genes thus
constructed can then be transferred to appropriate transformation
vectors. In a separate report, the rice ActI promoter with its
first intron has also been found to direct high expression in
cultured barley cells (Chibbar et al. (1993) Plant Cell Rep
12:506-509).
[0187] Ubiquitin Promoter. Ubiquitin is another gene product known
to accumulate in many cell types and its promoter has been cloned
from several species for use in transgenic plants (e.g.
sunflower--Binet et al. (1991) Plant Science 79: 87-94 and
maize--Christensen et al. (1989) Plant Molec Biol 12:619-632). The
maize ubiquitin promoter has been developed in transgenic monocot
systems and its sequence and vectors constructed for monocot
transformation are disclosed in the patent publication EP 0 342 926
which is herein incorporated by reference. Taylor et al. (1993)
Plant Cell Rep 12:491-495 describe a vector (pAHC25) that comprises
the maize ubiquitin promoter and first intron and its high activity
in cell suspensions of numerous monocotyledons when introduced via
microprojectile bombardment. The ubiquitin promoter is suitable for
gene expression in transgenic plants, especially monocotyledons.
Suitable vectors are derivatives of pAHC25 or any of the
transformation vectors described in this application, modified by
the introduction of the appropriate ubiquitin promoter and/or
intron sequences.
[0188] VII.A.2. Inducible Expression
[0189] Chemically Inducible PR-1a Promoter. The double 35S promoter
in pCGN1761ENX can be replaced with any other promoter of choice
which will result in suitably high expression levels. By way of
example, one of the chemically regulatable promoters described in
U.S. Pat. No. 5,614,395 can replace the double 35S promoter. The
promoter of choice is preferably excised from its source by
restriction enzymes, but can alternatively be PCR-amplified using
primers that carry appropriate terminal restriction sites. Should
PCR-amplification be undertaken, then the promoter should be
re-sequenced to check for amplification errors after the cloning of
the amplified promoter in the target vector. The chemical/pathogen
regulated tobacco PR-1a promoter is cleaved from plasmid pCIB1004
(for construction, see EP 0 332 104, which is hereby incorporated
by reference) and transferred to plasmid pCGN 1761 ENX (Uknes et
al. (1992) The Plant Cell 4:645-656).
[0190] pCIB1004 is cleaved with NcoI and the resultant 3' overhang
of the linearized fragment is rendered blunt by treatment with 14
DNA polymerase. The fragment is then cleaved with HindIII and the
resultant PR-1a promoter-containing fragment is gel purified and
cloned into pCGN1761ENX from which the double 35S promoter has been
removed. This is done by cleavage with XhoI and blunting with T4
polymerase, followed by cleavage with HindIII and isolation of the
larger vector-terminator containing fragment into which the
pCIB1004 promoter fragment is cloned. This generates a pCGN1761ENX
derivative with the PR-1a promoter and the tml terminator and an
intervening polylinker with unique EcoRI and NotI sites. The
selected coding sequence can be inserted into this vector, and the
fusion products (i.e. promoter-gene-terminator) can subsequently be
transferred to any selected transformation vector, including those
described below. Various chemical regulators can be employed to
induce expression of the selected coding sequence in the plants
transformed according to the present invention, including the
benzothiadiazole, isonicotinic acid, and salicylic acid compounds
disclosed in U.S. Pat. Nos. 5,523,311 and 5,614,395, herein
incorporated by reference.
[0191] Wound-Inducible Promoters. Wound-inducible promoters can
also be suitable for gene expression. Numerous such promoters have
been described (e.g. Xu et al. (1993) Plant Molec Biol 22:573-588;
Logemann et al. (1989) Plant Cell 1:151-158; Rohrmeier & Lehle
(1993) Plant Molec Biol 22:783-792; Firek et al. (1993) Plant Molec
Biol 22:129-142; Warner et al. (1993) Plant J 3:191-201) and all
are suitable for use with the instant invention. Logemann et al.
(1989) describe the 5' upstream sequences of the dicotyledonous
potato wunI gene. Xu et al. (1993) show that a wound-inducible
promoter from the dicotyledon potato (pin2) is active in the
monocotyledon rice. Further, Rohrmeier & Lehle (1993) describe
the cloning of the maize WipI cDNA which is wound induced and which
can be used to isolate the cognate promoter using standard
techniques. Similarly, Firek et al. (1993) and Warner et al. (1993)
have described a wound-induced gene from the monocotyledon
Asparagus officinalis, which is expressed at local wound and
pathogen invasion sites. Using cloning techniques well known in the
art, these promoters can be transferred to suitable vectors, fused
to the genes pertaining to this invention, and used to express
these genes at the sites of plant wounding.
[0192] VII.A.3. Tissue-Specific Expression
[0193] Root Promoter. Another pattern of gene expression is root
expression. A suitable root promoter is described by de Framond
(1991) FEBS 290:103-106 and also in the published patent
application EP 0 452 269, which is herein incorporated by
reference. This promoter is transferred to a suitable vector such
as pCGN1761ENX for the insertion of a selected gene and subsequent
transfer of the entire promoter-gene-terminator cassette to a
transformation vector of interest.
[0194] Pith Promoter. International Publication No. WO 93/07278,
which is herein incorporated by reference, describes the isolation
of the maize trpA gene, which is preferentially expressed in pith
cells. The gene sequence and promoter extending up to -1726 bp from
the start of transcription are presented. Using standard molecular
biological techniques, this promoter, or parts thereof, can be
transferred to a vector such as pCGN1761 where it can replace the
35S promoter and be used to drive the expression of a foreign gene
in a pith-preferred manner. In fact, fragments containing the
pith-preferred promoter or parts thereof can be transferred to any
vector and modified for utility in transgenic plants.
[0195] Leaf Promoter. A maize gene encoding phosphoenol carboxylase
(PEPC) has been described by Hudspeth & Grula (1989) Plant
Molec Biol 12:579-589. Using standard molecular biological
techniques the promoter for this gene can be used to drive the
expression of any gene in a leaf-specific manner in transgenic
plants.
[0196] VII.B. Transcriptional Terminators
[0197] A variety of transcriptional terminators are available for
use in expression cassettes. These are responsible for the
termination of transcription beyond the transgene and its correct
polyadenylation. Appropriate transcriptional terminators are those
that are known to function in plants and include the CaMV 35S
terminator, the tml terminator, the nopaline synthase terminator
and the pea rbcS E9 terminator. These can be used in both
monocotyledons and dicotyledons.
[0198] VII.C. Sequences for the Enhancement or Regulation of
Expression
[0199] Numerous sequences have been found to enhance gene
expression from within the transcriptional unit and these sequences
can be used in conjunction with the genes of this invention to
increase their expression in transgenic plants.
[0200] If desired, modifications around the cloning sites can be
made by the introduction of sequences that can enhance translation.
This is particularly useful when overexpression is desired. For
example, pCGN1761ENX can be modified by optimization of the
translational initiation site as disclosed in U.S. Pat. No.
5,639,949, incorporated herein by reference.
[0201] Various intron sequences have been shown to enhance
expression, particularly in monocotyledonous cells. For example,
the introns of the maize AdhI gene have been found to significantly
enhance the expression of the wild-type gene under its cognate
promoter when introduced into maize cells. Intron 1 was found to be
particularly effective and enhanced expression in fusion constructs
with the chloramphenicol acetyltransferase gene (Callis et al.
(1987) Genes Develop 1:1183-1200). In the same experimental system,
the intron from the maize bronze1 gene had a similar effect in
enhancing expression. Intron sequences have been routinely
incorporated into plant transformation vectors, typically within
the non-translated leader.
[0202] A number of non-translated leader sequences derived from
viruses are also known to enhance expression, and these are
particularly effective in dicotyledonous cells. Specifically,
leader sequences from Tobacco Mosaic Virus (TMV, the "W-sequence"),
Maize Chlorotic Mottle Virus (MCMV), and Alfalfa Mosaic Virus (AMV)
have been shown to be effective in enhancing expression (e.g.
Gallie et al. (1987) Nucl Acids Res 15:8693-8711; Skuzeski et al.
(1990) Plant Molec Biol 15:65-79).
[0203] VII.D. Targeting of the Gene Product Within the Cell
[0204] Various mechanisms for targeting gene products are known to
exist in plants and the sequences controlling the functioning of
these mechanisms have been characterized in some detail. For
example, the targeting of gene products to the chloroplast is
controlled by a signal sequence found at the amino terminal end of
various proteins which is cleaved during chloroplast import to
yield the mature protein (e.g. Comai et al. (1988) J Biol Chem
263:15104-15109). These signal sequences can be fused to
heterologous gene products to effect the import of heterologous
products into the chloroplast (van den Broeck et al. (1985) Nature
313:358-363). DNA encoding for appropriate signal sequences can be
isolated from the 5' end of the cDNAs encoding the RUBISCO protein,
the CAB protein, the EPSP synthase enzyme, the GS2 protein and many
other proteins which are known to be chloroplast localized. See
also, U.S. Pat. No. 5,639,949, herein incorporated by
reference.
[0205] Other gene products are localized to other organelles such
as the mitochondrion and the peroxisome (e.g. Unger et al. (1989)
Plant Molec Biol 13:411-418). The cDNAs encoding these products can
also be manipulated to effect the targeting of heterologous gene
products to these organelles. Examples of such sequences are the
nuclear-encoded ATPases and specific aspartate amino transferase
isoforms for mitochondria. Targeting cellular protein bodies has
been described by Rogers et al. (1989) Proc Natl Acad Sci USA
82:6512-6516).
[0206] In addition, sequences have been characterized which cause
the targeting of gene products to other cell compartments. Amino
terminal sequences are responsible for targeting to the ER, the
apoplast, and extracellular secretion from aleurone cells (Koehler
& Ho (1990) Plant Cell 2:769-783). Additionally, amino terminal
sequences in conjunction with carboxy terminal sequences are
responsible for vacuolar targeting of gene products (Shinshi et al.
(1990) Plant Molec Biol 14:357-368).
[0207] By the fusion of the appropriate targeting sequences
described above to transgene sequences of interest, it is possible
to direct the transgene product to any organelle or cell
compartment. For chloroplast targeting, for example, the
chloroplast signal sequence from the RUBISCO gene, the CAB gene,
the EPSP synthase gene, or the GS2 gene is fused in frame to the
amino terminal ATG of the transgene. The signal sequence selected
should include the known cleavage site, and the fusion constructed
should take into account any amino acids after the cleavage site
which are required for cleavage. In some cases this requirement can
be fulfilled by the addition of a small number of amino acids
between the cleavage site and the transgene ATG or, alternatively,
replacement of some amino acids within the transgene sequence.
Fusions constructed for chloroplast import can be tested for
efficacy of chloroplast uptake by in vitro translation of in vitro
transcribed constructions followed by in vitro chloroplast uptake
using techniques described by Bartlett et al. (1982) in Methods in
Chloroplast Molecular Biology, Edelmann et al. (Eds.), pp
1081-1091, Elsevier and Wasmann et al. (1986) Mol Gen Genet.
205:446-453.
[0208] These construction techniques are well known in the art and
are equally applicable to mitochondria and peroxisomes.
[0209] The above-described mechanisms for cellular targeting can be
utilized not only in conjunction with their cognate promoters, but
also in conjunction with heterologous promoters so as to effect a
specific cell-targeting goal under the transcriptional regulation
of a promoter that has an expression pattern different to that of
the promoter from which the targeting signal derives.
VIII. Recombinant Expression Vectors
[0210] Suitable expression vectors which can be used include, but
are not limited to, the following vectors or their derivatives:
human or animal viruses such as vaccinia virus or adenovirus, yeast
vectors, bacteriophage vectors (e.g., lambda phage), and plasmid
and cosmid DNA vectors.
[0211] Numerous vectors available for plant transformation are
known to those of ordinary skill in the plant transformation arts,
and the genes pertinent to this invention can be used with any such
vectors. Exemplary vectors include pCIB200, pCIB2001, pCIB10,
pCIB3064, pSOG19, and pSOG35, each described herein below. The
selection of vector will depend upon the preferred transformation
technique and the target species for transformation.
[0212] VIII.A. Agrobacterium Transformation Vectors.
[0213] Many vectors are available for transformation using
Agrobacterium tumefaciens. These typically carry at least one T-DNA
border sequence and include vectors such as pBIN19 (Bevan (1984)
Nucl Acids Res 12:8711-8721) and pXYZ. Below, the construction of
two typical vectors suitable for Agrobacterium transformation is
described.
[0214] pCIB200 and pCIB2001. The binary vectors pcIB200 and
pCIB2001 are used for the construction of recombinant vectors for
use with Agrobacterium and are constructed in the following manner.
pTJS75kan is created by Narl digestion of pTJS75 (Schmidhauser
& Helinski (1985) J Bacteriol 164:446-455) allowing excision of
the tetracycline-resistance gene, followed by insertion of an AccI
fragment from pUC4K carrying an NPTII (Messing & Vierra (1982)
Gene 19:259-268; Bevan et al. (1983) Nature 304:184-187; McBride et
al. (1990) Plant Molecular Biology 14:266-276). XhoI linkers are
ligated to the EcoRV fragment of PCIB7 which contains the left and
right T-DNA borders, a plant selectable nos/nptII chimeric gene and
the pUC polylinker (Rothstein et al. (1987) Gene 53:153-161), and
the XhoI-digested fragment are cloned into SalI-digested pTJS75kan
to create pCIB200 (see also EP 0 332 104, herein incorporated by
reference).
[0215] pCIB200 contains the following unique polylinker restriction
sites: EcoRI, SstI, KpnI, BgIII, XbaI, and SalI. pCIB2001 is a
derivative of pCIB200 created by the insertion into the polylinker
of additional restriction sites. Unique restriction sites in the
polylinker of pCIB2001 are EcoRI, SstI, KpnI, BgIII, XbaI, SalI,
MluI, BclI, AvrlI, ApaI, HpaI, and StuI. pCIB2001, in addition to
containing these unique restriction sites also has plant and
bacterial kanamycin selection, left and right T-DNA borders for
Agrobacterium-mediated transformation, the RK2-derived trfA
function for mobilization between E. coli and other hosts, and the
OriT and OriV functions also from RK2. The pCIB 2001 polylinker is
suitable for the cloning of plant expression cassettes containing
their own regulatory signals.
[0216] pCIB10 and Hygromycin Selection Derivatives thereof. The
binary vector pCIB10 contains a gene encoding kanamycin resistance
for selection in plants and T-DNA right and left border sequences
and incorporates sequences from the wide host-range plasmid pRK252
allowing it to replicate in both E. coli and Agrobacterium. Its
construction is described by Rothstein et al. (1987). Various
derivatives of pCIB10 are constructed which incorporate the gene
for hygromycin B phosphotransferase described by Gritz et al.
(1983) Gene 25:179-188. These derivatives enable selection of
transgenic plant cells on hygromycin only (pCIB743), or hygromycin
and kanamycin (pCIB715, pCIB717).
[0217] VIII.B. Other Plant Transformation Vectors
[0218] Transformation without the use of Agrobacterium tumefaciens
circumvents the requirement for T-DNA sequences in the chosen
transformation vector and consequently vectors lacking these
sequences can be utilized in addition to vectors such as the ones
described above which contain T-DNA sequences. Transformation
techniques that do not rely on Agrobacterium include transformation
via particle bombardment, protoplast uptake (e.g. PEG and
electroporation) and microinjection. The choice of vector depends
largely on the preferred selection for the species being
transformed. Below, the construction of typical vectors suitable
for non-Agrobacterium transformation is described.
[0219] pCIB3064. pCIB3064 is a pUC-derived vector suitable for
direct gene transfer techniques in combination with selection by
the herbicide basta (or phosphinothricin). The plasmid pCIB246
comprises the CaMV 35S promoter in operational fusion to the E.
coli GUS gene and the CaMV 35S transcriptional terminator and is
described in the Internation Publication No. WO 93/07278. The 35S
promoter of this vector contains two ATG sequences 5' of the start
site. These sites are mutated using standard PCR techniques in such
a way as to remove the ATGs and generate the restriction sites SspI
and PvulI. The new restriction sites are 96 and 37 bp away from the
unique SalI site and 101 and 42 bp away from the actual start site.
The resultant derivative of pCIB246 is designated pCIB3025.
[0220] The GUS gene is then excised from pCIB3025 bp digestion with
SalI and SacI, the termini rendered blunt and religated to generate
plasmid pCIB3060. The plasmid pJIT82 is obtained from the John
Innes Centre, Norwich and the a 400 bp SmaI fragment containing the
bar gene from Streptomyces viridochromogenes is excised and
inserted into the HpaI site of pCIB3060 (Thompson et al. (1987)
EMBO J. 6:2519-2523). This generated pCIB3064, which comprises the
bar gene under the control of the CaMV 35S promoter and terminator
for herbicide selection, a gene for ampicillin resistance (for
selection in E. coli) and a polylinker with the unique sites SphI,
PstI, HindIII, and BamHI. This vector is suitable for the cloning
of plant expression cassettes containing their own regulatory
signals.
[0221] pSOG19 and pSOG35. pSOG35 is a transformation vector that
utilizes the E. coli gene dihydrofolate reductase (DFR) as a
selectable marker conferring resistance to methotrexate. PCR is
used to amplify the 35S promoter (-800 bp), intron 6 from the maize
Adh1 gene (-550 bp) and 18 bp of the GUS untranslated leader
sequence from pSOG10. A 250-bp fragment encoding the E. coli
dihydrofolate reductase type II gene is also amplified by PCR and
these two PCR fragments are assembled with a SacI-PstI fragment
from pB1221 (Clontech, Palo Alto, Calif.) which comprises the pUC19
vector backbone and the nopaline synthase terminator. Assembly of
these fragments generates pSOG19 which contains the 35S promoter in
fusion with the intron 6 sequence, the GUS leader, the DHFR gene
and the nopaline synthase terminator. Replacement of the GUS leader
in pSOG19 with the leader sequence from Maize Chlorotic Mottle
Virus (MCMV) generates the vector pSOG35. pSOG19 and pSOG35 carry
the pUC gene for ampicillin resistance and have HindIII, SphI, PstI
and EcoRI sites available for the cloning of foreign
substances.
[0222] VIII.C. Selectable Markers
[0223] For certain target species, different antibiotic or
herbicide selection markers can be preferred. Selection markers
used routinely in transformation include the nptII gene, which
confers resistance to kanamycin and related antibiotics (Messing
& Vierra (1982) Gene 19:259-268; Bevan et al., 1983), the bar
gene, which confers resistance to the herbicide phosphinothricin
(White et al. (1990) Nucl Acids Res 18:1062; Spencer et al. (1990)
Theor Appl Genet. 79:625-631), the hph gene, which confers
resistance to the antibiotic hygromycin (Blochlinger &
Diggelmann (1984) Mol Cell Biol 4:2929-2931), the dhfr gene, which
confers resistance to methatrexate (Bourouis et al., (1983) EMBO J.
2(7):1099-1104), and the EPSPS gene, which confers resistance to
glyphosate (U.S. Pat. Nos. 4,940,935 and 5,188,642).
IX. Recombinant Expression in Host Cells
[0224] The term "host cell", as used herein, refers to a cell into
which a heterologous nucleic acid molecule has been introduced.
Transformed cells, tissues, or organisms are understood to
encompass not only the end product of a transformation process, but
also transgenic progeny thereof. A host cell strain can be chosen
which modulates the expression of the inserted sequences, or
modifies and processes the gene product in the specific fashion
desired. For example, different host cells have characteristic and
specific mechanisms for the translational and post-translational
processing and modification (e.g., glycosylation, phosphorylation
of proteins). Appropriate cell lines or host systems can be chosen
to ensure the desired modification and processing of the foreign
protein expressed. Expression in a bacterial system can be used to
produce a non-glycosylated core protein product. Expression in
yeast will produce a glycosylated product. Expression in plant
cells can be used to ensure "native" glycosylation of a
heterologous protein.
[0225] The present invention provides methods for recombinant
expression of SCN/SDS resistance genes in plants by the
construction of transgenic plants. The phrase "a plant, or parts
thereof" as used herein shall mean an entire plant; and shall mean
the individual parts thereof, including but not limited to seeds,
leaves, stems, and roots, as well as plant tissue cultures.
Transgenic plants of the present invention are understood to
encompass not only the end product of a transformation method, but
also transgenic progeny thereof. The term "converted plant" as used
herein shall mean any plant (1) having resistance to SDS or
resistance to SCN and (2) and was derived by genetic selection
employing RFLP, RADP, AFLP, or microsatellite (SSR) data for at
least one of the loci herein defined.
[0226] Preferably, the plant is a soybean plant. However, disease
resistance can be conferred to a wide variety of plant cells,
including those of gymnosperms, monocots, and dicots. Although the
gene can be inserted into any plant cell falling within these broad
classes, it is particularly useful in crop plant cells, such as
rice, wheat, barley, rye, corn, potato, carrot, sweet potato, sugar
beet, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli,
turnip, radish, spinach, asparagus, onion, garlic, eggplant,
pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple,
pear, quince, melon, plum, cherry, peach, nectarine, apricot,
strawberry, grape, raspberry, blackberry, pineapple, avocado,
papaya, mango, banana, tobacco, tomato, sorghum and sugarcane.
X. Recombinant Expression--Transfection and Transformation
Methods
[0227] Expression constructs are transfected into a host cell by a
standard method suitable for the selected host, including
electroporation, calcium phosphate precipitation, DEAE-Dextran
transfection, liposome-mediated transfection, infection using a
retrovirus, transposon-mediated transfer, and particle bombardment
techniques. The SCN/SDS resistance gene-encoding nucleotide
sequence carried in the expression construct can be stably
integrated into the genome of the host or it can be present as an
extrachromosomal molecule. Below are descriptions of representative
techniques for transforming both dicotyledonous and
monocotyledonous plants.
[0228] X.A. Transformation of Dicotyledons
[0229] Transformation techniques for dicotyledons are well known in
the art and include Agrobacterium-based techniques and techniques
that do not require Agrobacterium. Non-Agrobacterium techniques
involve the uptake of exogenous genetic material directly by
protoplasts or cells. This can be accomplished by PEG or
electroporation mediated uptake, particle bombardment-mediated
delivery, or microinjection. Examples of these techniques are
described by Paszkowski et al. (1984) EMBO J. 3:2717-2722; Potrykus
et al. (1985) Mol Gen Genet. 199:169-177; Reich et al. (1986)
Biotechnology 4:1001-1004; and Klein et al. (1987) Nature
327:70-73. In each case the transformed cells are regenerated to
whole plants using standard techniques known in the art.
[0230] Agrobacterium-mediated transformation is a preferred
technique for transformation of dicotyledons because of its high
efficiency of transformation and its broad utility with many
different species. Agrobacterium transformation typically involves
the transfer of the binary vector carrying the foreign DNA of
interest (e.g. pCIB200 or pCIB2001) to an appropriate Agrobacterium
strain, which can depend of the complement of vir genes carried by
the host Agrobacterium strain either on a co-resident Ti plasmid or
chromosomally (e.g. strain CIB542 for pCIB200 and pCIB2001 (Uknes
et al. (1993) Plant Cell 5:159-169). The transfer of the
recombinant binary vector to Agrobacterium is accomplished by a
triparental mating procedure using E. coli carrying the recombinant
binary vector, a helper E. coli strain which carries a plasmid such
as pRK2013 and which is able to mobilize the recombinant binary
vector to the target Agrobacterium strain. Alternatively, the
recombinant binary vector can be transferred to Agrobacterium by
DNA transformation (Hofgen & Willmitzer (1988) Nucl Acids Res
16:9877).
[0231] Transformation of the target plant species by recombinant
Agrobacterium usually involves co-cultivation of the Agrobacterium
with explants from the plant and follows protocols well known in
the art. Transformed tissue is regenerated on selectable medium
carrying the antibiotic or herbicide resistance marker present
between the binary plasmid T-DNA borders.
[0232] Another approach to transforming plant cells with a gene
involves propelling inert or biologically active particles at plant
tissues and cells. This technique is disclosed in U.S. Pat. Nos.
4,945,050, 5,036,006, and 5,100,792. Generally, this procedure
involves propelling inert or biologically active particles at the
cells under conditions effective to penetrate the outer surface of
the cell and afford incorporation within the interior thereof. When
inert particles are utilized, the vector can be introduced into the
cell by coating the particles with the vector containing the
desired gene. Alternatively, the target cell can be surrounded by
the vector so that the vector is carried into the cell by the wake
of the particle. Biologically active particles (e.g., dried yeast
cells, dried bacterium or a bacteriophage, each containing DNA
sought to be introduced) can also be propelled into plant cell
tissue.
[0233] X.B. Transformation of Monocotyledons
[0234] Transformation of most monocotyledon species has now also
become routine. Preferred techniques include direct gene transfer
into protoplasts using PEG or electroporation techniques, and
particle bombardment into callus tissue. Transformations can be
undertaken with a single DNA species or multiple DNA species (i.e.
co-transformation) and both these techniques are suitable for use
with this invention. Co-transformation can have the advantage of
avoiding complete vector construction and of generating transgenic
plants with unlinked loci for the gene of interest and the
selectable marker, enabling the removal of the selectable marker in
subsequent generations, should this be regarded desirable. However,
a disadvantage of the use of co-transformation is the less than
100% frequency with which separate DNA species are integrated into
the genome (Schocher et al. (1986) Biotechnology 4:1093-1096).
[0235] Patent Application Nos. EP 0 292 435, EP 0 392 225, and
International Publication No. WO 93/07278 describe techniques for
the preparation of callus and protoplasts from an elite inbred line
of maize, transformation of protoplasts using PEG or
electroporation, and the regeneration of maize plants from
transformed protoplasts. Gordon-Kamm et al. (1990) Plant Cell
2:603-618 and Fromm et al. (1990) Biotechnology 8:833-839 have
published techniques for transformation of A188-derived maize line
using particle bombardment. Furthermore, International Publication
No. WO 93/07278 and Koziel et al. (1993) Biotechnology 11:194-200
describe techniques for the transformation of elite inbred lines of
maize by particle bombardment. This technique utilizes immature
maize embryos of 1.5-2.5 mm length excised from a maize ear 14-15
days after pollination and a PDS-1000He BIOLISTICS.RTM. device for
bombardment.
[0236] Transformation of rice can also be undertaken by direct gene
transfer techniques utilizing protoplasts or particle bombardment.
Protoplast-mediated transformation has been described for
Japonica-types and Indica-types (Zhang et al. (1988) Plant Cell Rep
7:379-384; Shimamoto et al. (1989) Nature 338:274-277; Datta et al.
(1990) Biotechnology 8:736-740). Both types are also routinely
transformable using particle bombardment (Christou et al. (1991)
Biotechnology 9:957-962). Furthermore, Internation Publication
Number WO 93/21335 describes techniques for the transformation of
rice via electroporation. Patent Application EP 0 332 581 describes
techniques for the generation, transformation and regeneration of
Pooideae protoplasts. These techniques allow the transformation of
Dactylis and wheat. Furthermore, wheat transformation has been
described by Vasil et al. (1992) Biotechnology 10:667-674 using
particle bombardment into cells of type C long-term regenerable
callus, and also by Vasil et al. (1993) Biotechnology 11:1553-1558
and Weeks et al. (1993) Plant Physiol 102:1077-1084 using particle
bombardment of immature embryos and immature embryo-derived callus.
A preferred technique for wheat transformation, however, involves
the transformation of wheat by particle bombardment of immature
embryos and includes either a high sucrose or a high maltose step
prior to gene delivery. Prior to bombardment, any number of embryos
(0.75-1 mm in length) are plated onto MS medium with 3% sucrose
(Murashiga & Skoog (1962) Physiologia Plantarum 15:473-497) and
3 mg/l 2,4-D for induction of somatic embryos, which is allowed to
proceed in the dark. On the chosen day for bombardment, embryos are
removed from the induction medium and placed onto the osmoticum
(i.e. induction medium with sucrose or maltose added at the desired
concentration, typically 15%). The embryos are allowed to
plasmolyze for 2-3 h and are then bombarded. Twenty embryos per
target plate is typical, although not critical.
[0237] An appropriate gene-carrying plasmid (such as pCIB3064 or
pSG35) is precipitated onto micrometer size gold particles using
standard procedures. Each plate of embryos is shot with the DuPont
BIOLISTICS.RTM. helium device using a burst pressure of about 1000
psi using a standard 80 mesh screen. After bombardment, the embryos
are placed back into the dark to recover for about 24 hours (still
on osmoticum). After 24 hours, the embryos are removed from the
osmoticum and placed back onto induction medium where they stay for
about a month before regeneration. Approximately one month later
the embryo explants with developing embryogenic callus are
transferred to regeneration medium (MS+1 mg/liter NAA, 5 mg/liter
GA), further containing the appropriate selection agent (10 mg/l
basta in the case of pCIB3064 and 2 mg/l methotrexate in the case
of pSOG35). After approximately one month, developed shoots are
transferred to larger sterile containers known as "GA7s" which
contain half-strength MS, 2% sucrose, and the same concentration of
selection agent.
[0238] More recently, tranformation of monocotyledons using
Agrobacterium has been described. See WO 94/00977 and U.S. Pat. No.
5,591,616, both of which are incorporated herein by reference.
XI. Antibodies
[0239] The present invention also provides an antibody
immunoreactive with an SCN/SDS resistance polypeptide. The term
"antibody" indicates an immunoglobulin protein, or functional
portion thereof, including a polyclonal antibody, a monoclonal
antibody, a chimeric antibody, a single chain antibody, Fab
fragments, and an Fab expression library. "Functional portion"
refers to the part of the protein that binds a molecule of
interest. In a preferred embodiment, an antibody of the invention
is a monoclonal antibody. Techniques for preparing and
characterizing antibodies are well known in the art (See, e.g.,
Harlow and Lane (1988). A monoclonal antibody of the present
invention can be readily prepared through use of well-known
techniques such as the hybridoma techniques exemplified in U.S.
Pat. No. 4,196,265 and the phage-displayed techniques disclosed in
U.S. Pat. No. 5,260,203.
[0240] The phrase "specifically (or selectively) binds to an
antibody", or "specifically (or selectively) immunoreactive with",
when referring to a protein or peptide, refers to a binding
reaction which is determinative of the presence of the protein in a
heterogeneous population of proteins and other biological
materials. Thus, under designated immunoassay conditions, the
specified antibodies bind to a particular protein and do not show
significant binding to other proteins present in the sample.
Specific binding to an antibody under such conditions can require
an antibody that is selected for its specificity for a particular
protein. For example, antibodies raised to a protein with an amino
acid sequence encoded by the nucleic acid sequence of SEQ ID No:13
can be selected to obtain antibodies specifically immunoreactive
with that protein and not with unrelated proteins.
[0241] The use of a molecular cloning approach to generate
antibodies, particularly monoclonal antibodies, and more
particularly single chain monoclonal antibodies, are also provided.
The production of single chain antibodies has been described in the
art. See, e.g., U.S. Pat. No. 5,260,203. For this approach,
combinatorial immunoglobulin phagemid libraries are prepared from
RNA isolated from the spleen of the immunized animal, and phagemids
expressing appropriate antibodies are selected by panning on
endothelial tissue. The advantages of this approach over
conventional hybridoma techniques are that approximately 10.sup.4
times as many antibodies can be produced and screened in a single
round, and that new specificities are generated by heavy (H) and
light (L) chain combinations in a single chain, which further
increases the chance of finding appropriate antibodies. Thus, an
antibody of the present invention, or a "derivative" of an antibody
of the present invention, pertains to a single polypeptide chain
binding molecule which has binding specificity and affinity
substantially similar to the binding specificity and affinity of
the light and heavy chain aggregate variable region of an antibody
described herein.
[0242] The term "immunochemical reaction", as used herein, refers
to any of a variety of immunoassay formats used to detect
antibodies specifically bound to a particular protein, including
but not limited to, competitive and non-competitive assay systems
using techniques such as radioimmunoassays, ELISA (enzyme linked
immunosorbent assay), "sandwich" immunoassays, immunoradiometric
assays, gel diffusion precipitin reactions, immunodiffusion assays,
in situ immunoassays (e.g., using colloidal gold, enzyme or
radioisotope labels), western blots, precipitation reactions,
agglutination assays (e.g., gel agglutination assays,
hemagglutination assays), complement fixation assays,
immunofluorescence assays, protein A assays, and
immunoelectrophoresis assays, etc. See Harlow and Lane (1988) for a
description of immunoassay formats and conditions.
XII. Method for Detecting a SCN/SDS Resistance Polypeptide
[0243] In another aspect of the invention, a method is provided for
detecting a level of SCN/SDS resistance polypeptide using an
antibody that specifically recognizes a SCN/SDS resistance
polypeptide, or portion thereof. In a preferred embodiment,
biological samples from an experimental plant and a control plant
are obtained, and SCN/SDS resistance polypeptide is detected in
each sample by immunochemical reaction with the SCN/SDS resistance
polypeptide antibody. More preferably, the antibody recognizes
amino acids of SEQ ID NO:14 and is prepared according to a method
of the present invention for producing such an antibody.
[0244] In one embodiment, a SCN/SDS resistance polypeptide antibody
is used to screen a biological sample for the presence of a SCN/SDS
resistance polypeptide. A biological sample to be screened can be a
biological fluid such as extracellular or intracellular fluid, or a
cell or tissue extract or homogenate. A biological sample can also
be an isolated cell (e.g., in culture) or a collection of cells
such as in a tissue sample. A tissue sample can be suspended in a
liquid medium or fixed onto a solid support such as a microscope
slide. In accordance with a screening assay method, a biological
sample is exposed to an antibody immunoreactive with an SCN/SDS
resistance polypeptide whose presence is being assayed, and the
formation of antibody-polypeptide complexes is detected. Techniques
for detecting such antibody-antigen conjugates or complexes are
well known in the art and include but are not limited to
centrifugation, affinity chromatography and the like, and binding
of a labeled secondary antibody to the antibody-candidate receptor
complex.
XIII. Identification of Modulators of SCN/SDS Resistance
[0245] The present invention further discloses a method for
identifying a compound that modulates SCN/SDS resistance. As used
herein, the terms "candidate substance" and "candidate compound"
are used interchangeably and refer to a substance that is believed
to interact with another moiety, wherein a biological activity is
modulated. For example, a representative candidate compound is
believed to interact with a complete, or a fragment of, a SCN/SDS
resistance polypeptide, and which can be subsequently evaluated for
such an interaction. Exemplary candidate compounds that can be
investigated using the methods of the present invention include,
but are not restricted to, compounds that confer SCN/SDS
resistance, viral epitopes, peptides, enzymes, enzyme substrates,
co-factors, lectins, sugars, oligonucleotides or nucleic acids,
oligosaccharides, proteins, chemical compounds small molecules, and
monoclonal antibodies. A candidate compound to be tested by these
methods can be a purified molecule, a homogenous sample, or a
mixture of molecules or compounds.
[0246] As used herein, the term "modulate" means an increase,
decrease, or other alteration of any or all chemical and biological
activities or properties of a wild-type SCN/SDS resistance
polypeptide, preferably a SCN/SDS resistance polypeptide of SEQ ID
NO:14. Preferably, a SCN/SDS resistance modulator is an agonist of
SCN/SDS resistance protein activity. As used herein, the term
"agonist" means a substance that supplements or potentiates the
biological activity of a functional SCN/SDS resistance protein.
[0247] In accordance with the present invention there is also
provided a rapid and high throughput screening method that relies
on the methods described above. This screening method comprises
separately contacting each compound with a plurality of
substantially identical samples. In such a screening method the
plurality of samples preferably comprises more than about 10.sup.4
samples, or more preferably comprises more than about
5.times.10.sup.4 samples. In an alternative high-throughput
strategy, each sample can be contacted with a plurality of
candidate compounds.
[0248] XIII.A. Methods for Identifying Modulators of SCN/SDS
Resistance Gene Expression
[0249] The nucleic acid sequences of the present invention can be
used to identify regulators of SCN/SDS resistance polypeptide gene
expression. Several molecular cloning strategies can be used to
identify substances that specifically bind SCN/SDS resistance
polypeptide cis-regulatory elements. A preferred promoter region to
be used in such assays is an SCN/SDS resistance polypeptide
promoter region from soybean, more preferably the promoter region
includes some or all amino acids of SEQ ID NO:14.
[0250] In one embodiment, a cDNA library in an expression vector,
such as the lambda-gt11 vector, can be screened for cDNA clones
that encode an SCN/SDS resistance polypeptide regulatory element
DNA-binding activity by probing the library with a labeled SCN/SDS
resistance polypeptide DNA fragment, or synthetic oligonucleotide
(Singh et al. (1989) Biotechniques 7:252-261). Preferably the
nucleotide sequence selected as a probe has already been
demonstrated as a protein binding site using a protein-DNA binding
assay described above.
[0251] In another embodiment, transcriptional regulatory proteins
are identified using the yeast one-hybrid system (Luo et al. (1996)
Biotechniques 20(4):564-568; Vidal et al. (1996) Proc Natl Acad Sci
USA 93(19):10315-10320; Li and Herskowitz (1993) Science
262:1870-1874). In this case, a cis-regulatory element of a SCN/SDS
resistance gene is operably fused as an upstream activating
sequence (UAS) to one, or typically more, yeast reporter genes such
as the lacZ gene, the URA3 gene, the LEU2 gene, the HIS3 gene, or
the LYS2 gene, and the reporter gene fusion construct(s) is
inserted into an appropriate yeast host strain. It is expected that
the reporter genes are not transcriptionally active in the
engineered yeast host strain, for lack of a transcriptional
activator protein to bind the UAS derived from the SCN/SDS
resistance gene promoter region. The engineered yeast host strain
is transformed with a library of cDNAs inserted in a yeast
activation domain fusion protein expression vector, e.g. pGAD,
where the coding regions of the cDNA inserts are fused to a
functional yeast activation domain coding segment, such as those
derived from the GAL4 or VP16 activators. Transformed yeast cells
that acquire a cDNA encoding a protein that binds a cis-regulatory
element of a SCN/SDS resistance gene can be identified based on the
concerted activation the reporter genes, either by genetic
selection for prototrophy (e.g. LEU2, HIS3, or LYS2 reporters) or
by screening with chromogenic substrates (lacZ reporter) by methods
known in the art.
[0252] The present invention also provides an in vivo assay for
discovery of modulators of SCN/SDS resistance gene expression. In
this case, a transgenic plant is made such that a transgene
comprising a SCN/SDS resistance gene promoter and a reporter gene
is expressed and a level of reporter gene expression is assayable.
Such transgenic animals can be used for the identification of
compounds that are effective in modulating SCN/SDS resistance gene
expression.
[0253] In vitro or in vivo screening approaches may survey more
than one modulatable transcriptional regulatory sequence
simultaneously.
[0254] XIII.B. Methods for Identifying Modulators of SCN/SDS
Resistance Polypeptides
[0255] According to the method, a SCN/SDS resistance polypeptide is
exposed to a plurality of candidate substances, and binding of a
candidate substance to the SCN/SDS resistance polypeptide is
assayed. A compound is selected that demonstrates specific binding
to the SCN/SDS resistance polypeptide. Preferably, the SCN/SDS
resistance polypeptide used in the binding assay of the method
includes some or all amino acids of SEQ ID NO:14.
[0256] The term "binding" refers to an affinity between two
molecules, for example, a ligand and a receptor, means a
preferential binding of one molecule for another in a mixture of
molecules. The binding of the molecules can be considered specific
if the binding affinity is about 1.times.10.sup.4 M.sup.-1 to about
1.times.10.sup.6 M.sup.-1 or greater. Binding of two molecules also
encompasses a quality or state of mutual action such that an
activity of one protein or compound on another protein is
inhibitory (in the case of an antagonist) or enhancing (in the case
of an agonist).
[0257] Several techniques can be used to detect interactions
between a protein and a chemical ligand without employing an in
vivo ligand. Representative methods include, but are not limited
to, fluorescence correlation spectroscopy, surface-enhanced laser
desorption/ionization, and biacore technology, each described
herein below. These methods are amenable to automated,
high-throughput screening.
[0258] Fluorescence Correlation Spectroscopy (FCS). FCS measures
the average diffusion rate of a fluorescent molecule within a small
sample volume (Madge et al. (1972) Phys Re Lett 29:705-708, Maiti
et al. (1997) Proc Natl Acad Sci USA, 94:11753-11757). The sample
size can be as low as 10.sup.3 fluorescent molecules and the sample
volume as low as the cytoplasm of a single bacterium. The diffusion
rate is a function of the mass of the molecule and decreases as the
mass increases. FCS can therefore be applied to protein-ligand
interaction analysis by measuring the change in mass and therefore
in diffusion rate of a molecule upon binding. In a typical
experiment, the target to be analyzed is expressed as a recombinant
protein with a sequence tag, such as a poly-histidine sequence,
inserted at the N-terminus or C-terminus. The target protein is
expressed in E. coli, yeast, or plant cells. The protein is
purified by chromatography. For example, the poly-histidine tag can
be used to bind the expressed protein to a metal chelate column
such as Ni.sup.2+ chelated on iminodiacetic acid agarose. The
protein is then labeled with a fluorescent tag such as
carboxytetramethylrhodamine or BODIPY.TM. (Molecular Probes,
Eugene, Oreg.). The protein is then exposed in solution to a
candidate compound, and its diffusion rate is determined by FCS,
using for example, instrumentation available from Carl Zeiss, Inc.
(Thornwood, N.Y.). Ligand binding is determined by changes in the
diffusion rate of the protein.
[0259] Surface-Enhanced Laser Desorption/Ionization (SELDI). SELDI
can be used in combination with a time-of-flight mass spectrometer
(TOF) to provide a means to rapidly analyze molecules retained on a
chip (Hutchens and Yip (1993) Rapid Commun Mass Spectrom
7:576-580). It can be applied to ligand-protein interaction
analysis by covalently binding the target protein on the chip and
using mass spectroscopy to analyze the small molecules that bind to
the target protein (Worrall et al. (1998) Anal Biochem 70:750-756).
In a typical experiment, the target to be analyzed is recombinantly
expressed, optionally with a tag, such as poly-histidine, to
facilitate purification and handling. The purified protein is bound
to the SELDI chip either by utilizing the poly-histidine tag or by
other interaction such as ion exchange or hydrophobic interaction.
The chip thus prepared is then exposed to a candidate compound via,
for example, a delivery system able to pipet the ligands in a
sequential manner (autosampler). The chip is then washed in buffers
of increasing stringency, for example a series of buffer solutions
containing incrementally increasing ionic strength. After each
wash, the bound material is analyzed by SELDI-TOF. Compounds that
specifically bind the target are identified by elution in high
stringency wahes.
[0260] Biacore. Biacore technology utilizes changes in the
refractive index at the surface layer upon binding of a ligand to a
protein immobilized on the layer. In this system, a collection of
small ligands is injected sequentially in a 2-5 microliter cell,
wherein the protein is immobilized within the cell. Binding is
detected by surface plasmon resonance (SPR) of laser light
refracting from the surface. In general, the refractive index
change for a given change of mass concentration at the surface
layer is practically the same for all proteins and peptides,
allowing a single method to be applicable for any protein (Liedberg
et al. (1983) Sensors Actuators 4:299-304; Malmquist (1993) Nature
361:186-187). In a typical experiment, the target protein to be
analyzed is recombinantly expressed an purified according to
standard methods. It is bound to the Biacore chip either by
utilizing a poly-histidine tag or by other interaction such as ion
exchange or hydrophobic interaction. The chip thus prepared is then
exposed to a candidate compound via the delivery system
incorporated in the instruments sold by Biacore (Uppsala, Sweden)
to pipet the ligands in a sequential manner (autosampler). The SPR
signal on the chip is recorded and changes in the refractive index
indicate an interaction between the immobilized target and the
ligand. Analysis of the signal kinetics on rate and off rate allows
the discrimination between non-specific and specific
interaction.
[0261] Rational Drug Design. Similarly, the knowledge of the
structure a native SCN/SDS resistance polypeptide provides an
approach for rational drug design. The structure of an SCN/SDS
resistance polypeptide can be determined by X-ray crystallography
or by computational algorithms that generate three-dimensional
representations. See Huang et al. (2000) and Saqi et al. (1999)
Computer models can further predict binding of a protein structure
to various substrate molecules, that can be synthesized and tested.
Additional drug design techniques are described in U.S. Pat. Nos.
5,834,228 and 5,872,011.
XIV. Modulation of SCN/SDS Resistance in a Plant
[0262] In accordance with the present invention a method of
modulating SCN/SDS resistance in a plant is also provided. The
method comprises the step of administering to the plant an
effective amount of a substance that modulates expression of an
SCN/SDS resistance activity-encoding nucleic acid molecule in the
plant to thereby modulate SCN/SDS resistance in the plant.
Preferably, the substance that modulates expression of an SCN/SDS
resistance activity-encoding nucleic acid molecule comprises a
ligand for a modulatable transcriptional regulatory sequence of an
SCN/SDS resistance activity-encoding nucleic acid molecule
identified in accordance with the methods described above. More
preferably, the plant is a soybean plant.
[0263] Particularly, provided chemical entities (e.g. small
molecule mimetics) do not naturally occur in any cell of a lower
eucaryotic organism such as yeast. More particularly, provided
chemical entities do not naturally occur in any cell, whether of a
multicellular or a unicellular organism. Even more particularly,
the provided chemical entity is not a naturally occurring molecule,
e.g. it is a chemically synthesized entity. Provided chemical
entities can be hydrophobic, polycyclic, or both, molecules, and
are typically about 500-1,000 daltons in molecular weight.
XV. Method for Providing SCN/SDS Resistance B Transgenic Plants
[0264] A "transgenic plant" is a plant that has been genetically
modified to contain and express heterologous DNA sequences, either
as regulatory RNA molecules or as proteins. As specifically
exemplified herein, a transgenic plant is genetically modified to
contain and express at least one heterologous DNA sequence operably
linked to and under the regulatory control of transcriptional
control sequences which function in plant cells or tissue or in
whole plants. As used herein, a transgenic plant also refers to
progeny of the initial transgenic plant where those progeny contain
and are capable of expressing the heterologous coding sequence
under the regulatory control of the plant-expressible transcription
control sequences described herein. Seeds containing transgenic
embryos are encompassed within this definition as are cuttings and
other plant materials for vegetative propagation of a transgenic
plant.
[0265] When plant expression of a heterologous gene or coding
sequence of interest is desired, that coding sequence is operably
linked in the sense orientation to a suitable promoter and
advantageously under the regulatory control of DNA sequences which
quantitatively regulate transcription of a downstream sequence in
plant cells or tissue or in planta, in the same orientation as the
promoter, so that a sense (i.e., functional for translational
expression) mRNA is produced. A transcription termination signal,
for example, as polyadenylation signal, functional in a plant cell
is advantageously placed downstream of the SCN/SDS resistance
coding sequence, and a selectable marker which can be expressed in
a plant, can be covalently linked to the inducible expression unit
so that after this DNA molecule is introduced into a plant cell or
tissue, its presence can be selected and plant cells or tissue not
so transformed will be killed or prevented from growing.
[0266] In the present invention, the SCN/SDS resistance coding
sequence can optionally serve as a selectable marker for
transformation of plant cells or tissue. Where constitutive gene
expression is desired, suitable plant-expressible promoters include
a native promoter (e.g. SEQ ID NO:15) of the SCN/SDS coding
sequences set forth herein as the native promoter is activated in
the presence of SCN; the 35S or 19S promoters of Cauliflower Mosaic
Virus; the nos, ocs or mas promoters of Agrobacterium tumefaciens
Ti plasmids; and others known to the art.
[0267] Indeed, a native promoter (e.g. SEQ ID NO:15) of the SCN/SDS
coding sequences set forth herein is activated in the presence of
SCN and thus can be used to produce transgenic plants in accordance
with the techniques disclosed herein. Particularly, the native
promoter can be linked to a nucleic acid encoding a polypeptide of
interest in a construct, and the construct can be used to a prepare
a transgenic plant in accordance with techniques described herein.
Other techniques are disclosed in U.S. Pat. Nos. 5,994,526 and
5,994,527, herein incorporated by reference in their entirety. The
polypeptide of interest is then expressed in the plant when the
promoter is activated, such as in the presence of SCN or other
environmental stimulus.
[0268] Where tissue-specific expression of the SCN/SDS resistance
coding sequence is desired, the skilled artisan will choose from a
number of well-known sequences to mediate that form of gene
expression as disclosed herein. Environmentally regulated promoters
are also well known in the art, and the skilled artisan can choose
from well known transcription regulatory sequences to achieve the
desired result.
[0269] A method for providing a resistance characteristic to a
plant is therefore disclosed. The method comprises introducing to
said plant a construct comprising a nucleic acid sequence encoding
an SCN/SDS resistance gene product operatively linked to a
promoter, wherein production of the SCN/SDS resistance gene product
in the plant provides a resistance characteristic to the plant. The
construct can further comprises a vector selected from the group
consisting of a plasmid vector or a viral vector. The SCN/SDS
resistance gene product comprises a protein having an amino acid
sequence as set forth as SEQ ID NO:14. The nucleic acid sequence
can be a nucleic acid sequence set forth as SEQ ID NO:13, or a
nucleic acid that is substantially similar to SEQ ID NO:13, and
which encodes an SCN/SDS resistance polypeptide.
[0270] The resistance characteristic is preferably nematode
resistance, fungal resistance or combinations thereof. More
preferably, the nematode resistance is H. glycines resistance or
root knot nematode resistance.
[0271] In an alternative embodiment, the construct further
comprises another nucleic acid molecule encoding a polypeptide that
provides an additional desired characteristic to the plant. Other
desired characteristics include yield, drought resistance, chemical
resistance (e.g. herbicide or pesticide resistance), spoilage
resistance or any or other desired characteristic as would be
apparent to one of ordinary skill in the art after review of the
disclosure of the present invention. Representative nucleic acids
sequences are described in the following U.S. patents (incorporated
herein by reference in their entirety): U.S. Pat. No. 5,948,953 to
Webb (brown rot fungus resistance); U.S. Pat. No. RE36,449 to
Lebrun et al. (herbicide resistance); U.S. Pat. No. 5,952,546 to
Bedbrook et al. (delayed ripening tomato plants); and U.S. Pat. No.
5,986,173 to Smeekens et al. (transgenic plants showing a modified
fructan pattern).
[0272] Optionally, the method further comprises monitoring an
insertion point for the construct in the plant genome; and
providing for insertion of the construct into the plant genome at a
location not associated with the resistance characteristic, the
desired characteristic, or both the resistance or the desired
characteristic.
XVI. Method for Providing SCN/SDS Resistance B Marker-Assisted
Selection and Development of a Breeding Program
[0273] The present invention relates to a novel and useful method
for introgressing, in a reliable and predictable manner, SCN/SDS
resistance into non-resistant soybean germplasm. The method
involves the genetic mapping of loci associated with SCN/SDS
resistance, definition of genetic markers that are linked with
SCN/SDS resistance, and a high-throughput PCR-based assay for
detecting such a genetic marker. Markers useful in a preferred
embodiment of the invention include the following: a locus mapping
to linkage group G and mapped by one or more of the markers set
forth SEQ ID NOs:1-6, a locus mapping to linkage group A2 and
mapped by one or more of the markers set forth as SEQ ID NOs:7-12;
or combinations thereof. Also preferably, a genetic marker used for
marker-assisted selection comprises a sequence, or portion thereof,
of any one of SEQ ID NOs:13 and 16-19, or combinations thereof.
[0274] From the sequence data found in SEQ ID NOs:1-13 and 16-19,
and from the other markers identified herein, primer pairs, as for
example, PCR primer pairs, capable of distinguishing differences
among these genotypes are developed. Simple assays for the markers
and genes use a label, such as, but not limited to, a covalently
attached chromophores, that do not need electrophoresis are
developed to increase the capacity of marker assisted selection to
help plant breeders. A preferred assay is the TaqMan.TM. assay
disclosed in Example 6. Non-destructive sampling of dried seed for
DNA preparations are developed to allow selection prior to
planting, for example, using the methods set forth in Example 9.
This enables the testing of the effectiveness of marker assisted
selection in predicting field resistance to SON and SDS.
[0275] A preferred manner for providing SCN/SDS resistance to a
plant involves providing one or more plants from a parental soybean
plant line which comprises in its genome one or more molecular
markers comprising a sequence, or portion thereof, set forth as any
one of SEQ ID NOs:1-13 and 16-19. Preferably, the parental plant is
purebreeding for one or more of the molecular markers, more
preferably the parent plant is purebreeding for molecular markers
comprising a sequence, or portion thereof, set forth as any one of
SEQ ID NOs:1-13 and 16-19. In one preferred embodiment, the
parental line is "Forrest" or a line derived therefrom.
[0276] The SCN/SDS resistance trait can be introgressed into a
recipient soybean plant line which is non-resistant or less
resistant to SCN/SDS by performing marker-assisted selection based
on the molecular markers of the present invention as set forth as
SEQ ID NOs:1-13 and 16-19.
[0277] Introgressing can be accomplished by any method known in the
art, including but not limited to single seed descent, pedigree
method, or backcrossing, each described herein below. Additional
methods for introgressing are disclosed in U.S. Pat. Nos. 5,948,953
and 6,162,967. Any suitable method can be used, the critical
feature being marker-assisted selection of a marker of the present
invention using a nucleotide sequence assay.
[0278] Single Seed Descent. According to this method, "Forrest" can
be crossed to "Essex", and the seed planted in a field. The
resulting seed (F2) is planted in the greenhouse and the resulting
seeds (F3) are harvested while keeping separate the seeds from each
plant. A random F3 seed from each of approximately 200 plants is
planted and the resulting F4 seed is harvested. The seeds from each
individual plant are again kept separate. A random F4 seed from
each of the approximately 200 plants is planted and the resulting
F5 seed is harvested. This selection process is repeated until F7
seed is harvested and identified as an inbred line. At each
generation beginning with the F3 generation, plants are screened
with soybean cyst nematodes, and plants were selected for
advancement based upon the presence of SCN resistance and other
phenotypic characteristics. Alternatively, plants are screened for
the presence of one or more of the molecular markers listed herein
using a TaqMan.TM. genotyping assay and selected for advancement
based upon the presence of one or more of the markers.
[0279] Pedigree Method. Using a SCN resistant recombinant inbred
line, produced for example by single seed descent, as a donor
source, the SCN resistant trait can be introgressed into other germ
plasm sources. To develop new germplasm, the SCN resistant
recombinant inbred line is used as one of the parents. The
resulting progenies are evaluated and selected at various locations
for a variety of traits, including SCN resistance. SCN resistance
is determined by phenotypic screening or by genotyping based upon
the presence of the molecular markers listed herein.
[0280] Backcrossing. Using a SCN resistant recombinant inbred line,
produced for example by single seed descent, as a donor source, the
SCN resistant trait is introgressed into other soybean plant lines.
The SCN resistant recombinant inbred line is crossed to a line that
demonstrates little or non SCN resistance (the recipient). The
resulting plants are crossed back to the recipient soybean plant
line that is being converted to SCN resistance. This crossing back
to the parental line that is being converted may be repeated
several times. After each round of backcrossing, plants are
selected for SCN resistance, which can be determined by either
phenotypic screening or by the selection of molecular markers
linked to SCN resistance loci. Besides selecting for SCN
resistance, the plants are also selected that most closely resemble
the original plant line being converted to SCN resistance. This
selection for the original plant line is done phenotypically or
with molecular markers.
[0281] In one specific preferred method, BC.sub.NF1 plants are
genotypically screened for the presence of one or more markers
linked to SCN resistance genomic loci. As used herein, the term
"BC.sub.NF1 plant" is intended to refer to a plant in the first
generation after a specific backcross event, the specific backcross
event being designated by the term "N", irrespective of the number
of previous backcross events employed to produce the plant. Plants
having the one or more markers present may preferably be
backcrossed with plants of the parental line or, alternatively, be
selfed, the plants resulting from either of these events also being
genotypically screened for the presence of one or more markers
linked to SCN resistance genomic loci. This procedure can be
repeated several times.
[0282] In another specific preferred method, BC.sub.NF1 plants are
selfed to produce BC.sub.NF2 seeds. BC.sub.NF2 plants are then
screened either genotypically using, for example a TaqMan.TM. assay
as disclosed in Example 6, or by phenotypic assessment of SCN
resistance. Those plants having present one or more molecular
markers linked to SCN resistance, or those plants displaying
resistance, depending upon the screening method used, are
backcrossed with plants of the parental line to produce BC.sub.NF3
seeds and plants. This procedure can be repeated several times. In
a soybean breeding program, the methods of the present invention
can be used for marker-assisted selection of the molecular markers
described herein. Genetic markers closely linked to SCN/SDS
resistance genes can be used to indirectly select for favorable
alleles more efficiently than phenotypic selection. Genetic markers
comprising SCN/SDS resistance genes, as disclosed herein, can be
used to select for SCN/SDS resistance genes with optimal efficiency
and accuracy.
[0283] Marker-assisted selection can be employed to select one or
more loci at a wide variety of population development stages in a
two-parent population, multiple parent population, or a backcross
population. Such populations are described in Fehr (1987) Breeding
Methods for Cultivar Development J. R. Wilcox (ed.) and Soybeans:
Improvement, Production, and Uses, 2nd ed.
[0284] Marker-assisted selection according to art-recognized
methods can be made, for example, step-wise, whereby the different
SCN resistance loci are selected in more than one generation; or,
as an alternative example, simultaneously, whereby all loci are
selected in the same generation. Marker-assisted selection for SCN
resistance can be done before, in conjunction with, or after
testing and selection for other traits such as seed yield, plant
height, seed type, etc. The DNA from target populations, isolated
for use in accordance with genetic marker detection, can be
obtained from any plant part, and each DNA sample can represent the
genotype of single or multiple plant individuals, including
seed.
[0285] Marker-assisted selection can also be used to confirm
previous selection for SCN resistance or susceptibility made by
challenging plants with SCNs in the field or greenhouse and scoring
the resulting phenotypes. Alternatively, plants can be analyzed by
TaqMan.TM. genotyping to determine the presence of the
above-described molecular markers, thus confirming the presence of
a genomic locus associated with SCN resistance.
[0286] As such, also provided by the present invention are methods
for determining the presence or absence of SCN resistance in a
soybean plant, or alternatively in a soybean seed. These methods
comprise analyzing genomic DNA from a plant or a seed for the
presence of one or more of the molecular markers set forth as SEQ
ID NOs:1-13 and 16-19. According to this method, the analyzing
comprises performing a TaqMan.TM. assay as disclosed in Example 6,
or any other suitable method known in the art.
[0287] The ability to distinguish heterozygotes and their derived
heterogeneous lines is important to early generation selection
(before the F.sub.5) in soybean breeding programs when within
population variability is high (Bernard et al. (1988) USDA Tech
Bull 1796; Brown et al., 1987). The lower stringency TaqMan.TM. 2
assay disclosed herein was most effective for identifying most of
the heterogeneous lines in this population. However, the cutoff
values of FAM and TET for the efficient identification of
heterogeneous lines (or heterozygous F2 lines) is likely to vary
across assays and should be set arbitrarily according to
expectations of the number of lines that are expected to contain
both alleles. The assay was used for analyzing 2,000 lines derived
from specific cultivar crosses over 3 days. A single researcher can
process 768 sample per day (8.times.96 samples) since the reading
time of the machine is 15 minutes for one 96 well plate and the
thermal cycler stage takes about 2 hours.
[0288] Table 3 shows that with genomic DNA from 94 cultivars the
standard TaqMan.TM. allelic discrimination assays and PCR assays
provided allele scores that were in good agreement with the
cultivar phenotypes (Concibidio, 1997; Bernard et al., 1988).
Cultivars, plant introductions (PI), breeding lines and germplasm
releases listed in Table 3 were parents in the SCN molecular
breeding program at Southern Illinois University-Carbondale (SIUC)
from 1997-1999. The prevalence of allele 1 was in good agreement
with allele frequencies for markers that are closely linked to Rhg4
(Cregan et al. 1999; Mathews et al. (1998) Theor Appl Genet
97:1047-1052; Mahalingam et al., 1995). Those resistant cultivars
sharing allele 1 with the susceptible lines may not require the
presence of Rhg4 for resistance to SCN or have derived their
resistance to SCN at the Rhg4 locus from alleles derived from
cultivars other than Forrest. In addition, some soybean breeders
may have been effective in separating even the most closely linked
marker from resistance genes using phenotypic selection. However,
this is probably infrequent since selection to generate the
resistance allele 2 in susceptible cultivars has not occurred
frequently. Only three cultivars with allele 2 were
susceptible.
TABLE-US-00005 TABLE 3 Resistant Susceptible Allele 2 Forrest,
Hartwig, Fayette, Pharaoh, Picket, MD93-5298 Accomac, Bedford,
Delsoy4710, Peking, Pace PI88788, PI209332, PI90763, PI437654,
Holladay LS92-1088, LS92-4173, LS94-3207, LS95-0259, LS95-0709,
LS95-1454, LS96-1631, LS90-1920, LS94-3545, S92- 1679, S92-2711A,
S94-2086, LN94-10527, A5560K1390, K1425 Allele 1 Manokin, Mustang,
Dwight, Pana, Ina, Essex, Bragg, Dunfield, Hill, CNS, PI 398680,
IA2036, IA3005, LS92-3660, Lee, Noir1, Ogden, Calhoun, LS93-0292,
LS93-0375, LS94-2435, Chesapeake, Choska, Stressland, LS96-0735,
LS96-3813, LS96-5009, Macon, Misuzudaiza, Nakasennari, LN92-10725,
GX93-1573, SS94-7546, PI 520733, PI567445B, P1567583C, SS94-4337,
S95-1908, A4138, A95-483010, PI567650B, PI 567374, PI 567650B,
M92-1645, M92-1708, M90-184111, K1423, IA3010, IA1006, TN96-58,
N96-180, K1424 LN93-11632, LN93-11945, LN95- 5417, A94-674017,
A94-774021, A96-494018, C1963, HC93- 2690, HS93-4118, K1410
[0289] Summarily, the sequences and methods disclosed herein enable
automated, high throughput, rapid genotyping of DNA polymorphisms
for selection of SCN/SDS resistance in breeding programs.
EXAMPLES
[0290] The following Examples have been included to illustrate
preferred modes of the invention. Certain aspects of the following
Examples are described in terms of techniques and procedures found
or contemplated by the present inventors to work well in the
practice of the invention. These Examples are exemplified through
the use of standard laboratory practices of the inventors. In light
of the present disclosure and the general level of skill in the
art, those of skill will appreciate that the following Examples are
intended to be exemplary only and that numerous changes,
modifications and alterations can be employed without departing
from the spirit and scope of the invention.
Example 1
Plant Material
[0291] A mapping population consisted of approximately 100
recombinant inbred lines derived at the F5 generation from a cross
of `Essex` (Smith & Camper (1973) Crop Sci 13:459) by `Forrest`
(Hartwig & Epps (1973) Crop Sci 13:287). The recombinant inbred
line (RILs) population was advanced to the F5:13 generation from
300 plants per RIL per generation (Hnetkovsky et al., 1996).
Forrest is resistant to the soybean cyst nematode (SCN) populations
classified as race 3 and Essex is susceptible to all populations of
SCN (Chang et al., 1997; Meksem et al. 1999).
Example 2
SCN Female Index (FI) Determination
[0292] The number of white female cysts was compared on each
genotype to the number of white female cysts on a susceptible
control, such as Essex, to determine the female index (FI) for each
population (Meksem et al., 1999). Seedlings were inoculated with
2000+/-25 eggs from a homogenous isolate of H. glycines. All
experiments used five single-plant replications per line. The mean
number of white female cysts on each genotype and the susceptible
control were determined and FI was calculated as the ratio of the
mean number of cysts on each genotype to the mean number of cysts
on the susceptible check.
Example 3
Characterization of New Markers for SCN/SDS Resistance
[0293] Soybean genomic DNA used for AFLP analysis was extracted and
purified using the Qiagen (Hilden, Germany) Plant Easy DNA
Extraction Kit. Primary template DNA was prepared using the
restriction enzymes EcoRI and MseI.
[0294] AFLP analysis was performed as described by Vos et al.
(1995) Nuc Acids Res 23:4407-4414 except that the streptavidin bead
selection step was omitted. PCR reactions were performed with using
primer pairs derived from each of two sets of primers. Primers
within EcoRI set all included the core sequence E: 5'-GAC TGC GTA
CCA ATT C (SEQ ID NO:115) with 1 or 3 base pair extensions. Primers
of the MseI set have the sequence M: 5'-GAT GAG TCC TGA GTA A (SEQ
ID NO:116) with 1 or 3 base pair extensions. The primer
combinations (EA and MC) and (EC and MA) were used for
pre-amplification of primary template. Three selective nucleotides
per primer were used to generate AFLP fragments from the secondary
templates. AFLP bands were labeled with .sup.33P by primer
phosphorylation, separated by electrophoresis on 4% (w/v) PAGE and
visualized by exposing X-ray film to the dried gel.
[0295] Target AFLP bands on the autoradiograph were matched to the
corresponding area in the gel and the appropriate AFLP fragment was
excised from the dried gel. The band was eluted from the gel by
incubation in 100 ml of water at 4.degree. C. for 1 hour. Sequence
isolation in bacterial clones was performed as described by Meksem
et al. (1995) Mol Gen Genet. 249:74-81 with the modification that
the pGEM-T vector (Promega, Madison, Wis.) was ligated to PCR
amplified, gel eluted DNA. DNA sequencing of clones allowed PCR
primers to be designed for each unique DNA sequence using Oligo 5.0
software (PE Biosystems, Foster City, Calif.). The PCR product was
analyzed on 4% (w/v) Metaphor7 (FMC, Rockland, Me.) agarose
gel.
[0296] AFLP markers that were dominant or co-dominant, in repulsion
and in coupling phases were used. For dominant AFLP markers, the
band of the dominant allele was cloned and sequenced. The
corresponding marker for the recessive allele was isolated by PCR
using primers designed from the dominant band sequence. For
apparently co-dominant AFLP markers, both, the coupling and
repulsion phase bands were cloned simultaneously from the
acrylamide gel.
[0297] The general strategy employed to identify the specific
sequence underlying AFLP band polymorphisms was as follows. If the
polymorphism was dominant (e.g. E.sub.ATGM.sub.CGA87) a primer pair
was designed to flank each of the unique sequences derived from the
AFLP band. Each primer pair was used to amplify genomic DNA from
both Essex and Forrest. Any primer set that revealed polymorphism
(dominant or co-dominant) between the two parents was used to
amplify members of the RIL mapping population. The primer pair that
generated a marker on the map corresponding to the map position of
the original AFLP band was inferred to be the specific marker
STS.
[0298] For some AFLP bands the above strategy was ineffective,
presumably because polymorphism was within or close to the
restriction site used for AFLP linker ligation (e.g.
E.sub.CGGM.sub.AGA116). In such cases genomic DNA from the parents
and mapping population was used in a modified AFLP protocol as
follows. The pre-amplification step was omitted and the six
selective nucleotide step was replaced by an extended highly
selective MseI primer to which we added the first 7 bases of the
sequenced band, combined with a non selective EcoRI primer E (e.g.
MseI primer M AGAGACT and EcoRI primer E). The MseI primer was
end-labeled by phosphorylating the 5' end with 5 ml [g-.sup.33P]
ATP (3000 Ci/mmol) for 30 min at 37.degree. C. with 10 units of T4
Kinase (Pharmacia, Piscataway, N.J.). Any primer set that revealed
polymorphism (dominant or co-dominant) between the two parents was
used to amplify members of the RIL mapping population. The primer
pair that generated a marker on the map corresponding to the map
position of the original AFLP band was inferred to be the specific
marker STS.
Example 4
Cloning of SCN/SDS Resistance Genes in Linkage Groups G and A2
[0299] The cloned AFLP bands of Example 3 were used to screen the
soybean Forrest BamHI or HindIII BAC libraries by PCR as described
by Meksem et al. (2000).
[0300] Both plasmid and BAC DNA was prepared using the appropriate
kit (Qiagen, Hilden, Germany). Sequence determinations were
performed by the di-deoxy chain-termination method using Advanced
Biosystems (ABI, Foster city, Calif.) "big dye" cycle sequencing
separated on ABI 377 automated DNA sequencer.
[0301] Plasmids containing clones derived from AFLP bands were
sequenced using M13 universal forward and reverse primers. Direct
BAC insert sequencing was performed as above with the following
modifications: BAC DNA was heated for 30 min at 70.degree. C., and
sheared by pippeting into a narrow gauge tip for 2 min. Two primers
designed from the target AFLP band sequence were used for
sequencing. For the E.sub.ATGM.sub.CGA87 positive BAC insert DNA,
the forward primer, named ATG4BACF (SEQ ID NO:117), was 5'
gggtttcagataaccgtggtcg 3', the reverse primer was the complementary
strand sequences of the ATG4BACF primer. The PCR conditions used
was 95.degree. C. for 10 min, then 45 cycles of 95.degree. C. for
30 sec, 55.degree. C. for 20 sec and 60.degree. C. for 4 min.
Example 5
Taqman.TM. Genotyping Assay
[0302] PCR primers and TaqMan.TM. probes were designed with the
primer express program (Perkin-Elmer/Applied Biosystems, Foster
City, Calif.) and were custom synthesized by Perkin-Elmer. Two
TaqMan.TM. probes were designed to encompass the A2D8 (FIG. 1)
insertion polymorphisms (underlined). The A2D8 SCAR was derived
from the codominant AFLP bands E.sub.CCG-M.sub.AAC417 (Essex,
allele 1, GenBank Accession No. AF286701) and
E.sub.CCG-M.sub.AAC409 (Forrest, allele 2, GenBank Accession No.
AF286700) that contain a homolog (P=2e-05) of one component (Tic22;
GenBank Accession No. AAC64606.1) of the protein import apparatus
of the chloroplast inner envelope membrane. Allele 1: 5'-TET-TTG
CAG ATA TTT TAG TTG ATT GGC C-TAMRA (SEQ ID NO:118). Allele 2:
5'-6FAM-AGT TGA TTG GCT CAA ACC ATG GCC-TAMRA (SEQ ID NO:119).
Reverse Primer:' 5' d TTG CGT GTG ATC GGT ATT AC 3' (SEQ ID
NO:120). Forward primer: 5' d T ACC TGA GTT CTC TCA AGT C 3' (SEQ
ID NO:121).
[0303] TaqMan.TM. reactions were performed essentially as the
Perkin-Elmer TaqMan.TM. PCR Reagent Kit protocol describes except
the PCR reaction was performed in 384 well plates to reduce assay
volume and cost. Briefly, each reaction contained 10 ng of the
extracted DNA, 0.025 units/ml of AmpliTaq Gold.TM.
(Perkin-Elmer/Applied Biosystems, Foster City, Calif.), 400 nM of
the forward and reverse primers (Research Genetics, Huntsville,
Ala.), 50 nM of FAM fluorescent probe and 150 nM of TET fluorescent
probe (Perkin-Elmer/Applied Biosystems, Foster City, Calif.) in
1.times.universal master mix (Perkin-Elmer/Applied Biosystems,
Foster City, Calif.). The above ratio of primers and probes was
optimized using a series of primer/probe combinations to reach a
maximal signal and the balance of the two probes by reading in an
ABI 7200 sequence detector. The TaqMan.TM. universal PCR master mix
is a premix of all the components, except primer and probes,
necessary to perform a 5' nuclease assay. The final optimized
conditions represented a two step PCR protocol, with two holds
followed by cycling, on a 384 well thermal cycler (GeneAmp PCR
System 9700, Perkin-Elmer/Applied Biosystems, Foster City, Calif.).
The two hold cycles were 50.degree. C. for 2 min and 95.degree. C.
for 10 min. The 35 cycles were at 95.degree. C. for 15 sec,
60.degree. C. for 1 min. After amplification the plates were cooled
to room temperature and samples were transferred from a 384 well
plate to a 96 well MicroAmpJ optical tray and fluorescence was
detected on an ABI PrismJ 7200 Sequence Detector
(Perkin-Elmer/Applied Biosystems, Foster City, Calif.).
[0304] The results were analyzed by allelic discrimination of the
sequence detection software (Perkin-Elmer/Applied Biosystems,
Foster City, Calif.). Two grouping methods were used to attempt to
accurately separate heterogeneous lines from homogeneous lines at
each allele. In grouping method 1 (TaqMan.TM. 1) a stringent
cut-off for FAM (>7) was used for allele 1 compared to
heterogenous scores. This served to reduce the number called as
potentially heterogeneous to about the percentage expected from the
breeding method used for RIL development (6%). Fluorophore ratios
were as follows; no amplification (FAM and TET both less than 6
units); allele 1 homozygous (FAM less than 7, TET greater than 7);
allele 2 homozygous (FAM greater than 10, TET less than 5); and
heterogeneous for allele 1 and allele 2 (FAM greater than 7, TET
5-8). For TaqMan.TM. selection grouping method 2 ratios were; no
amplification (FAM and TET both less than 6 units); allele 1
homozygous (FAM less than 5, TET greater than 7); allele 2
homozygous (FAM greater than 10, TET less than 5); and
heterogeneous for allele 1 and allele 2 (FAM greater than 5, TET
5-9). The FAM and TET signals were stable in the dark for 2 days
after PCR.
Example 6
Genotyping Assay Using Gel Electrophoresis Markers
[0305] PCR reactions were performed with DNA from the recombinant
inbred lines. The 114 and 120 base pair PCR products were generated
using the forward and reverse primers (SEQ ID NOs:120-121). The
final optimized conditions were 94.degree. C. for 10 min, then 35
cycles of 94.degree. C. for 25 sec, 56.degree. C. for 30 sec and
72.degree. C. for 60 sec. After the PCR reactions were completed,
the plates were cooled to room temperature and the PCR products
separated by electrophoresis on a 4% (w/v) agarose gel.
Example 7
Allele Distribution in Soybean Germplasm
[0306] Genotypes at A2D8 were determined from the genomic DNA of 94
cultivars that represented the parents of populations in the SIUC
soybean breeding program from 1997-1999 (Table 3). There were 38
cultivars susceptible to SCN and 56 cultivars resistant to SCN race
3. Allele 2 (R) was found in 32 of 94 cultivars tested. There were
very few susceptible genotypes with allele 2 (3 of 32) and the
majority of genotypes with allele 2 (29 of 32) were resistant to
SCN. In contrast, allele 1 (S) was found in 62 cultivars but
frequently in both resistant cultivars (27 of 56) and susceptible
cultivars (35 of 38).
Example 8
Selection of SCN/SDS Resistant Seeds
[0307] G.max L. seeds used to start cultures should be less than
six months old and have been stored in darkness at 4.degree. C.
Then, the seeds are cultured as folllows:
[0308] 1. Surface disinfect with 70% (v/v) ethanol for 2 min then
20% (v/v) bleach for 20 min. Rinse three times in sterile MS
media.
[0309] 2. Germinate the seed on MS media containing 10 g/l agar, 30
g/l sucrose but no PGRs for 3 days at 27.degree. C.
[0310] 3. Axenically remove the testa, remove the cotyledonary
notes, cut the cotyledons transversely in half and use the distal
cotyledonary halves to establish callus cultures.
[0311] To initiate callus growth, cotyledonary halves are placed on
MS medium with 30 g/l sucrose, 5 mM kinetin, 100 mg/l myoinositol,
0.5 mg/mL thiamine.cndot.HCl pH 5.7 at 27.degree. C. unless noted
below. The medium contains 5 mM indolebutyric acid as auxin. Place
cotyledonary halves in tubes containing 10 mL solidified media.
Incubate for 28 days.
[0312] To assay callus growth, pieces of callus each approximately
25 mg should be added to sterile tubes containing 10 mL media with
varying concentrations of H. glycines, F. solani or extracts
thereof. After 28 days at 28.degree. C. the explants are evaluated
for growth and growing sectors subcultured.
[0313] Cell suspensions are derived by placing 2 g of a macerated
callus in 40 mL of MS medium. The flask, a 125 mL Erlenmeyer flask,
should be capped with a foam plug. Subcultures should be made every
14 days into fresh media by allowing the cells to settle, removing
the old media by aspiration, adding twice the volume of fresh media
and splitting into two flasks.
[0314] Soybean tissue capable of regeneration to whole plants are
grown in the presence of H. glycines, F. solani or extracts
thereof. Cell lines representing mutants capable of continued
growth are regenerated and the heritability of SCN or SDS
resistance determined in these plants or their seed or tissue
derived progeny.
REFERENCES
[0315] The publications and other materials listed, below and/or
set forth in the text above to illuminate the background of the
invention, and in particular cases, to provide additional details
respecting the practice, are incorporated in their entirety herein
by reference. Materials used herein include but are not limited to
the following listed references. [0316] Adelman et al. (1983) DNA
2:183-193. [0317] Alam and Cook (1990) Anal Biochem 188:245-254.
[0318] Altschul et al. (1990) J Mol Biol 215:403-410. [0319]
Arondel et al. (1992) Science 258:1353-1355. [0320] Ausubel et al.
(1992) Current Protocols in Molecular Biology, John Wylie and Sons,
Inc., New York. [0321] Bartlett et al. (1982) in Methods in
Chloroplast Molecular Biology, Edelmann et al. (Eds.), pp
1081-1091, Elsevier. [0322] Barton (1998) Acta Crystallogr D Biol
Crystallogr 54:1139-1146. [0323] Batzer et al. (1991) Nucleic Acid
Res 19:3619-3623. [0324] Bell-Johnson et al. (1998) Soybean Genet
Newslett 25:115-118. [0325] Bernard et al. (1988) USDA Tech Bull
1796. [0326] Bevan (1984) Nucl Acids Res 12:8711-8721. [0327] Bevan
et al. (1983) Nature 304:184-187. [0328] Binet et al. (1991) Plant
Science 79: 87-94. [0329] Blochlinger & Diggelmann (1984) Mol
Cell Biol 4:2929-2931. [0330] Bourouis et al., (1983) EMBO J.
2(7):1099-1104. [0331] Bodanszky, et al. (1976) Peptide Synthesis,
John Wiley and Sons, Second Edition, New York. [0332] Brookes
(1999) Gene 234(2):177-186. [0333] Brown et al. (1987) Principles
and Practice of Nematode Control in Crops, pp 179-232, Academic
Press, Orlando Fla. [0334] Callis et al. (1987) Genes Develop
1:1183-1200. [0335] Chang et al. (1996) Crop Sci 36:965-971. [0336]
Chang et al. (1997) Crop Science 37(3):965-971. [0337] Chase et al.
(1997) Theor Appl Genet. 94:724-730. [0338] Chibbar et al. (1993)
Plant Cell Rep 12:506-509. [0339] Christou et al. (1991)
Biotechnology 9:957-962. [0340] Comai et al. (1988) J Biol Chem
263:15104-15109. [0341] Concibido (1996) Theor Appl Genet.
93:234-241. [0342] Concibido et al. (1997) Crop Sci 37:258-264.
[0343] Conner et al. (1983) Proc Natl Acad Sci USA 80:278-282.
[0344] Cregan et al. (1999a) Crop Sci 39:1464-1490. [0345] Cregan
et al. (1999b) Theor Appl Genet. 99:811-818. [0346] Cregan et al.
(1999c) Theor Appl Genet. 99:918-928. [0347] Cubitt et al. (1995)
Trends Biochem Sci 20:448-455. [0348] Datta et al. (1990)
Biotechnology 8:736-740. [0349] EP 0 292 435 [0350] EP 0 332 104
[0351] EP 0 332 581 [0352] EP 0 342 296 [0353] EP 0 392 225 [0354]
EP 0 452 269 [0355] Fehr (1987) in Soybeans: Improvement Production
and Uses 2nd Ed., J. R. Wilcox (ed.), American Society of Agronomy,
Madison, Wis. [0356] Firek et al. (1993) Plant Molec Biol
22:129-142. [0357] Fromm et al. (1990) Biotechnology 8:833-839.
[0358] Gallie et al. (1987) Nucl Acids Res 15:8693-8711. [0359]
Glover, ed. (1985) DNA Cloning: A Practical Approach, MRL Press,
Ltd., Oxford, United Kingdom. [0360] Gomez A. K. and A. A. Gomez
(1984) Statistical Procedures For Agricultural Research. 2nd ed.
John Wiley & Sons New York [0361] Gordon-Kamm et al. (1990)
Plant Cell 2:603-618. [0362] Gritz et al. (1983) Gene 25:179-188.
[0363] Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. [0364]
Hartwig and Epps (1973) Crop Science 13:287. [0365] Henikoff et al.
(2000) Electrophoresis 21(9):1700-1706. [0366] Henikoff and
Henikoff (1989) Proc Natl Acad Sci USA 89:10915. [0367] Henikoff
and Henikoff (2000) Adv Protein Chem 54:73-97. [0368] Hnetkovsky et
al. (1996) Crop Science 36(2):393-400. [0369] Hofgen &
Willmitzer (1988) Nud Acids Res 16:9877. [0370] Huang et al. (2000)
Pac Symp Biocomput 230-241. [0371] Hudspeth & Grula (1989)
Plant Molec Biol 12:579-589. [0372] Hutchens and Yip (1993) Rapid
Commun Mass Spectrom 7: 576-580. [0373] Kalinina et al. (1997) Nucl
Acids Res 25:1999-2004. [0374] Kanazin et al. (1996) Proc Natl Acad
Sci USA 93(21):11746-11750. [0375] Karlin and Altschul (1993) Proc
Natl Acad Sci USA 90:5873-87. [0376] Keim et al. (1997) Crop
Science 37:537-543. [0377] Kestila et al. (1998) Mol Cell
1(4):575-582. [0378] Klein et al. (1987) Nature 327:70-73. [0379]
Koduri and Poola (2001) Steroids 66(1):17-23. [0380] Koziel et al.
(1993) Biotechnology 11:194-200. [0381] Kyte et al. (1982) J Mol
Biol 157:105. [0382] Landers & Botstein (1989) Genetics
121:185-199. [0383] Landgren et al. (1988) Science 241:1007. [0384]
Landgren et al. (1988) Science 242:229-237. [0385] Landegren et al.
(1998) Genome Res 8:769-776. [0386] Lark et al. (1993) Theor Appl
Genet. 86:901-906. [0387] Li and Herskowitz (1993) Science
262:1870-1874. [0388] Liedberg et al. (1983) Sensors Actuators
4:299-304. [0389] Livak et al. (1995) PCR Meth and Applic
4:357-362. [0390] Livak et al. (1995) Nat Genet. 9:341-342. [0391]
Logemann et al. (1989) Plant Cell 1:151-158. [0392] Luo et al.
(1999) Plant Disease 83:1155-1159. [0393] Madge et al. (1972) Phys
Rev Lett 29:705-708. [0394] Mahalingam et al. (1995) Breed Sci
45:435-445. [0395] Mahalingham et al. (1996) Genome 39:986-998
[0396] Maiti et al. (1997) Proc Natl Acad Sci USA, 94:11753-11757.
[0397] Malmquist (1993) Nature 361:186-187. [0398] Martin et al.
(1993) Science 262:1432-1436. [0399] Mathews et al. (1998) Theor
Appl Genet. 97:1047-1052. [0400] Matthews et al. (1991) Soybean
Genetics Newsletter. [0401] McBride et al. (1990) Plant Molecular
Biology 14:266-276. [0402] McElroy et al. (1990) Plant Cell
2:163-171. [0403] McElroy et al. (1991) Mol Gen Genet. 231:150-160.
[0404] Meksem et al. (1995) Mol Gen Genet. 249:74-81. [0405] Meksem
et al. (1999) Theor Appl Genet. 99:1131-1142. [0406] Meksem et al.
(2000) Theor Appl Genet. 101:747-755. [0407] Messing & Vierra
(1982) Gene 19:259-268. [0408] Myers & Anand (1991), Euphytica
55:197-201. [0409] Nasarabadi et al. (1999) BioTechniques
27:1116-1117. [0410] Needleman & Wunsch (1970) J Mol Biol
48:443-453. [0411] Njiti et al. (1996) Crop Science 36:1165-1170.
[0412] Ochman et al. (1990) in PCR protocols: a Guide to Methods
and Applications, Innis et al. (eds.), pp. 219-227, Academic Press,
San Diego, Calif. [0413] Ohtsuka et al. (1985) J Biol Chem
260:2605-2608. [0414] Orita et al. (1989) Proc Natl Acad Sci USA
86(8):2766-2770. [0415] Paszkowski et al. (1984) EMBO J.
3:2717-2722. [0416] Paterson et al. (1990) Genetics 124:735-742.
[0417] Pearson & Lipman (1988) Proc Natl Acad Sci USA
85:24442448. [0418] Potrykus et al. (1985) Mol Gen Genet.
199:169-177. [0419] Prabhu et al. (1999) Crop Science
39(4):982-987. [0420] Price (1993) Blood Rev 7:127-134. [0421]
Rao-Arrelli et al. (1988) Crop Science 28:650-652. [0422]
Rao-Arrelli et al. (1992) Crop Science 32:862-864. [0423] Regan et
al. (2000) Anal Biochem 286(2):265-276. [0424] Reich et al. (1986)
Biotechnology 4:1001-1004. [0425] Riggs and Schmidt (1988) J
Nematol 20:392-395. [0426] Rogers et al. (1989) Proc Natl Acad Sci
USA 82:6512-6516. [0427] Rohrmeier & Lehle (1993) Plant Molec
Biol 22:783-792. [0428] Rommens et al. (1989) Science
245:1059-1065. [0429] Rose & Botstein (1983) Meth Enzymol
101:167-180. [0430] Rossolini et al. (1994) Mol Cell Probes
8:91-98. [0431] Rothstein et al. (1987) Gene 53:153-161. [0432]
Saiki et al. (1985) Bio/Technology 3:1008-1012. [0433] Sambrook et
al. eds. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, New York, N.Y. [0434]
Saqi et al. (1999) Bioinformatics 15:521-522. [0435] Sauer (1998)
Methods 14(4):381-392. [0436] Schmidhauser & Helinski (1985) J
Bacteriol 164:446-455. [0437] Schocher et al. (1986) Biotechnology
4:1093-1096. [0438] Shimamoto et al. (1989) Nature 338:274-277.
[0439] Shinshi et al. (1990) Plant Molec Biol 14:357-368. [0440]
Shoemaker et al. (1995) Crop Science 35:436-446. [0441] Silhavy et
al. (1984) Experiments with Gene Fusions, Cold Spring Harbor
Laboratory, Cold Spring Harbor, New York, N.Y. [0442] Singh et al.
(1989) Biotechniques 7:252-261. [0443] Skuzeski et al. (1990) Plant
Molec Biol 15:65-79. [0444] Smith & Waterman (1981) Adv Appl
Math 2:482. [0445] Smith & Camper (1973) Crop Science 13:459.
[0446] Spencer et al. (1990) Theor Appl Genet. 79:625-631. [0447]
Staskawicz (1995) Science 268:661-667. [0448] Stoneking et al.
(1991) Am J Hum Genet. 48(2):370-82. [0449] Thompson et al. (1987)
EMBO J. 6:2519-2523. [0450] Tijssen (1993) in Laboratory Techniques
in Biochemistry and Molecular Biology-Hybridization with Nucleic
Acid Probes, part 1 chapter 2, Elsevier, New York, N.Y. [0451]
Trask (1991) Trends Genet. 7:149-154. [0452] Uknes et al. (1992)
The Plant Cell 4:645-656. [0453] Uknes et al. (1993) The Plant Cell
5:159-169. [0454] Unger et al. (1989) Plant Molec Biol 13:411-418.
[0455] U.S. Pat. No. 4,196,265 [0456] U.S. Pat. No. 4,554,101
[0457] U.S. Pat. No. 4,940,935 [0458] U.S. Pat. No. 4,945,050
[0459] U.S. Pat. No. 5,036,006 [0460] U.S. Pat. No. 5,100,792
[0461] U.S. Pat. No. 5,188,642 [0462] U.S. Pat. No. 5,260,203
[0463] U.S. Pat. No. 5,523,311 [0464] U.S. Pat. No. 5,591,616
[0465] U.S. Pat. No. 5,614,395 [0466] U.S. Pat. No. 5,629,158
[0467] U.S. Pat. No. 5,639,949 [0468] U.S. Pat. No. 5,834,228
[0469] U.S. Pat. No. 5,872,011 [0470] U.S. Pat. No. 5,948,953
[0471] U.S. Pat. No. 5,952,546 [0472] U.S. Pat. No. 5,958,624
[0473] U.S. Pat. No. 5,986,173 [0474] U.S. Pat. No. 5,994,526
[0475] U.S. Pat. No. 5,994,527 [0476] U.S. Pat. No. 6,096,555
[0477] U.S. Pat. No. 6,162,967 [0478] U.S. Pat. No. RE36,449 [0479]
van den Broeck et al. (1985) Nature 313:358-363. [0480] Vasil et
al. (1992) Biotechnology 10:667-674. [0481] Vasil et al. (1993)
Biotechnology 11:1553-1558. [0482] Vidal et al. (1996) Proc Natl
Acad Sci USA 93(19):10315-10320. [0483] Vos et al. (1995) Nucleic
Acids Research 23:4407-4414. [0484] Wang et al. (1998) Science
280(5366):1077-82. [0485] Warner et al. (1993) Plant J 3:191-201.
[0486] Webb et al. (1995) Theor Appl Genet. 91:574-581. [0487]
Weeks et al. (1993) Plant Physiol 102:1077-1084. [0488] Weiseman et
al. (1992) Theor Appl Genet. 85:136-138 [0489] White et al. (1990)
Nucl Acids Res 18:1062. [0490] WO 93/07278 [0491] WO 93/21335
[0492] WO 94/00977 [0493] WO 97/47763 [0494] Worrall et al. (1998)
Anal Biochem 70:750-756. [0495] Wrather et al. (1995) Plant Disease
79:1076-1079. [0496] Xu et al. (1993) Plant Molec Biol 22:573-588.
[0497] Yuan et al. (1999) Hum Mutat 14(5):440-446. [0498] Zhang et
al. (1988) Plant Cell Rep 7:379-384. [0499] Zhang et al. (1994) Mol
Gen Genet. 244:613-621. [0500] Zhu et al. (1996) Mol Gen Genet.
252:483-488. [0501] Zimmer et al. (1993) Peptides pp. 393B394,
ESCOM Science Publishers, B. V. [0502] Zobrist et al. (2000)
Soybean Genet Newslett 27:10-15.
[0503] It will be understood that various details of the invention
can be changed without departing from the scope of the invention.
Furthermore, the foregoing description is for the purpose of
illustration only, and not for the purpose of limitation--the
invention being defined by the claims.
Sequence CWU 1
1
136187DNAGlycine max 1gaattcatgg tttctcttat gacattgttg ccaagtaata
ctactatata aattcagatt 60tgggtttctg ataaccgtgg tcgttaa
87292DNAGlycine max 2gaattcatgg tttctcttat cttatgacat tgttgccaag
taatactact atataaattc 60agatttgggt ttcagataac cgtggtcgtt aa
923113DNAGlycine max 3gaattcctaa tatacgagtg aatattattg taatgcttgt
aaaaaaacat gataaaatgc 60aaaaatttgg ggtgaatttt tacgacatta gtgaaaaaaa
catatccctt taa 1134135DNAGlycine max 4ttaaagggat atgttttttt
cactaatgct gtaaaaattc acccagattt ttgcattttc 60tttgaaaaaa tgtactagat
atatcatgtt tttttacaag cattacaata atattcactc 120gtatattagg aattc
1355116DNAGlycine max 5gaattccggt tatctcagac aacttttgtt tggtttggtt
atagtaaaga cacgattatc 60caggctttga gaggcataga aataattttt ttatataaaa
aaaaaagtct ctttaa 1166114DNAGlycine max 6gaatttcggt tatctcagac
aacttttgtt tggtttggtt atagtaaaga cacgattatc 60caggctttga gaggcataga
aataattttt ttatataaaa aaaagtctct ttaa 1147409DNAGlycine
maxmisc_feature(1)..(409)This sequence is derived from Glycine max
cv. 'Forrest' 7gagtaaaacc ttgcgtgtga tcggtattac agtacgcagg
gccaatcaac taaaatatct 60gcaaacgata atataattat aagaaaaaga cacactttga
gggcattttt gacttgagag 120aactcaggta tcaatctaaa agcaacgctg
ttcaccttga gctgaaacac ctggaggaga 180aagcaaagca aaccaaacgc
gagagagaaa taaagaacgg aaacagagag agagagagga 240aggaccttgt
tcaaagcaac ggggacaact ttagagccct ggcgcgcgtg ggggtcaata
300agcgtaacct ggctgaggag agcctcggcg tcgtccttgc tgaagcagaa
gaggaagagc 360acgagaccaa gagaaactcc tcggaagcaa cgggaattgg tacgcagtc
4098417DNAGlycine max 8gagtaaaacc ttgcgtgtga tcggtattac agtacgcagg
gccatggttt gagccaatca 60actaaaatat ttgcaaacga taatataatt ataagaaaaa
gactcacttt gagggcattt 120ttgacttgag agaactcagg tatcaatcta
aaagcaacgc tgttcacctt gagctgaaac 180acctggagga gaaagcaaag
caaaccaaac gcgagagaga aataaagaac ggaaacagag 240agagaggaag
gaccttgttc aaagcaacgg ggacaacttt agagccctgg cgcgcgtggg
300ggtcaataag cgtaacctgg ctgaggagag cctcggcgcc gtccttgctg
aagcagaaga 360ggaagagccc gagaccaaga gaaactcctc ggaagcaacg
ggaattggta cgcagtc 4179165DNAGlycine max 9gagtaaatga aaatcgatca
aaatcaaata atatatgctt tttttagttg tgttcaagta 60actttttttt attgaaaaaa
tcgacccaag ttgaaacaca tgtttgagaa ttgttttgtg 120catccaacgt
ttttcttgta caatcagctg tgagagggga attgg 16510164DNAGlycine max
10gagtaaatga aaatcgatca aaatcaaata atatatgctt tttttagttg ggttcaagta
60ctttttttta ttgaaaaaat cgacccaagt tgaaacacat gtttgagaat tgttttgtgc
120atccaacgtt tttcttgtac aatcagctgt gagaggggaa ttgg
16411114DNAGlycine max 11gaattcccag ctagatttgt atcaaacatg
tattgtccac aaaatgttca agcatcttag 60ggaactgcta ttcttacttc taaatttttt
attgacatcc aaagtgtgct ttaa 11412114DNAGlycine max 12gaattcccag
ccagatttgt atcaaacatg tattgtccac aaaatgttca agcatcttag 60ggaactgcta
ttcttacttc taaatttttt attgacatcc aaagtgtgct ttaa
114133106DNAGlycine maxmisc_feature(1832)..(1832)n is a, c, g, or t
13aatgggagga gtgggaaaga cagtggctat ggagcttgtt ccggaggttg ggttggaatc
60aagtgtgctc agggacaggt tattgtgatc cagcttcctt ggaagggttt gaggggtcga
120atcaccgaca aaattggcca acttcaaggc ctcaggaagc ttagtcttca
tgataaccaa 180attggtggtt caatcccttc aactttggga cttcttccca
accttagagg ggttcagtta 240ttcaacaata ggcttacagg ttccatacct
ctttctttag gtttctgcct ttgcttcaag 300tctcttgacc tcagcaacaa
cttgctcaca ggagcaatcc cttatagtct tgctaattcc 360actaagcttt
attggcttaa cttgagtttc aactccttct ctggtccttt accagctagc
420ctaactcact cattttctct cacttttctt tctcttcaaa ataacaatct
ttctggctcc 480cttcctaact cttggggtgg gaattccaag aatggcttct
ttaggcttca aaatttgatc 540ctagatcata actttttcac tggtgacgtt
cctgcttctt tgggtagctt aagagagctc 600aatgagattt cccttagtca
taataagttt agtggagcta taccaaatga aataggaacc 660ctttctaggc
ttaagacact tgacatttct aataatgcct tgaatgggaa cttgcctgct
720accctctcta atttatcctc acttacactg ctgaatgcag agaacaacct
ccttgacaat 780caaatccctc aaagtttagg tagattgcgt aatctttctg
ttctgatttt gagtagaaac 840caatttagtg gacatattcc ttcaagcatt
gcaaacattt cctcgcttag gcagcttgat 900ttgtcactga ataatttcag
tggagaaatt ccagtctcct ttgacagtca gcgcagtcta 960aatctcttca
atgtttccta caatagcctc tcaggttctg tcccccctct gcttgccaag
1020aaatttaact caagctcatt tgtgggaaat attcaactat gtgggtacag
cccttcaacc 1080ccatgtcttt cccaagctcc atcacaagga gtcattgccc
cacctcctga agtgtcaaaa 1140catcaccatc ataggaagct aagcaccaaa
gacataattc tcatagtagc aggagttctc 1200ctcgtagtcc tgattatact
ttgttgtgtc ctgcttttct gcctgatcag aaagagatca 1260acatctaggc
cgggaacggc caagccaccc gagggtagag cggccactat gaggacagaa
1320aaaggagtcc ctccagttgc tggtggtgat gttgaagcag gtggggaggc
tggagggaaa 1380ctagtccatt ttgatggacc aatggctttt acagctgatg
atctcttgtg tgcaacagct 1440gagatcatgg gaaagagcac ctatggaact
gtttataagg ctattttgga ggatggaagt 1500caagttgcag taaagagatt
gagggaaaag atcactaaag gtcatagaga atttgaatca 1560gaagtcagtg
ttctaggaaa aattagacac cccaatgttt tggctctgag ggcctattac
1620ttgggaccca aaggggaaaa gcttctgggt tttgatacat gtctaaagga
agtcttgctt 1680ctttcctaca tggaaggttc gtgtgctggt tctttcatta
aagtgttgtg tgtgctggtc 1740tttaattata atttggagtt ttaccttagt
aatctgtata attctaatcg gagaacagta 1800caaacaaaaa cacctaagga
acaacacctt anctttaata taccatatca ataaagtgaa 1860atattttctt
ggtcatcttg atgcaggggg aactgaacat tcattattgg ccacaagatt
1920aaaatagccc aagccttggc ccgggcttgt ttgccttcat tcccaggaga
acatcataca 1980tgggacctcn catccagcaa tgtgtggctt gatgaaaaac
aaatgctaaa attcagattt 2040tggtcttttt cgggttgatg tcaactgctg
ctaattccaa cgtgatagct acagctggag 2100cattggatac cgggcacctg
agctctcaaa gctcaagaaa gcaaacacta aaactgatat 2160ctacagtctt
ggtgttatct tgttagaact cctaacgagg aaatcacctg gggtgtctat
2220gaatggacta gatttgcctc agtgggttgc ctcagttgtc aaagaggagt
ggacaaatga 2280ggtttttgat gcagacttga tgagagatgc atccacagtt
ggcgacgagt tgctaaacac 2340gttgaagctc gctttgcact gtgttgatcc
ttctccatca gcacgaccag aagttcatca 2400agttctccag cagctgaaga
gattagacca gagagatcag tcacagccag tcccggggac 2460gatatcgtat
agcacaaatt ttgcattgat ttttttgtgc caaatgtagt aggcctacta
2520tatatatgtt ctatgattct ttcattctta tattattttt gcctgtttga
atgcttgaat 2580ttgtacatac tcatactaca ataaggtgta gttctggtta
attttacctc tacctcaaag 2640ctggggtgta attctgtttc ctccaaggca
cataatagtt gaaaatagtt ctcaggagca 2700ttcattgttt attctgcaag
attctctttc acggctgcta tcttctatgc atgccctgcc 2760cataaatgca
ttatgaagaa ttgtaacggc tgtgtttttg gacttcttca aaaagtttat
2820gttattgcca ggtgtatata tcaacatgtt ttaaagattt tcaaacaatc
aggttttaga 2880tgtgggtttg catgcatgag attggactag tgcgcttgat
gtagtataaa atataaattg 2940tccaatcaag caccctctac atgtccaaat
aatgggcctt atgaaactta attttttaat 3000tacaaactac agtaatcttt
ttgaataaag atttacaaat tacaacngac atgtgaagcn 3060gcatctttna
ttgncaatct ttcaagttac tctattattt tctgcn 310614830PRTGlycine
maxmisc_feature(611)..(611)Xaa can be any naturally occurring amino
acid 14Asn Gly Arg Ser Gly Lys Asp Ser Gly Tyr Gly Ala Cys Ser Gly
Gly1 5 10 15Trp Val Gly Ile Lys Cys Ala Gln Gly Gln Val Ile Val Ile
Gln Leu 20 25 30Pro Trp Lys Gly Leu Arg Gly Arg Ile Thr Asp Lys Ile
Gly Gln Leu 35 40 45Gln Gly Leu Arg Lys Leu Ser Leu His Asp Asn Gln
Ile Gly Gly Ser 50 55 60Ile Pro Ser Thr Leu Gly Leu Leu Pro Asn Leu
Arg Gly Val Gln Leu65 70 75 80Phe Asn Asn Arg Leu Thr Gly Ser Ile
Pro Leu Ser Leu Gly Phe Cys 85 90 95Pro Leu Leu Gln Ser Leu Asp Leu
Ser Asn Asn Leu Leu Thr Gly Ala 100 105 110Ile Pro Tyr Ser Leu Ala
Asn Ser Thr Lys Leu Tyr Trp Leu Asn Leu 115 120 125Ser Phe Asn Ser
Phe Ser Gly Pro Leu Pro Ala Ser Leu Thr His Ser 130 135 140Phe Ser
Leu Thr Phe Leu Ser Leu Gln Asn Asn Asn Leu Ser Gly Ser145 150 155
160Leu Pro Asn Ser Trp Gly Gly Asn Ser Lys Asn Gly Phe Phe Arg Leu
165 170 175Gln Asn Leu Ile Leu Asp His Asn Phe Phe Thr Gly Asp Val
Pro Ala 180 185 190Ser Leu Gly Ser Leu Arg Glu Leu Asn Glu Ile Ser
Leu Ser His Asn 195 200 205Lys Phe Ser Gly Ala Ile Pro Asn Glu Ile
Gly Thr Leu Ser Arg Leu 210 215 220Lys Thr Leu Asp Ile Ser Asn Asn
Ala Leu Asn Gly Asn Leu Pro Ala225 230 235 240Thr Leu Ser Asn Leu
Ser Ser Leu Thr Leu Leu Asn Ala Glu Asn Asn 245 250 255Leu Leu Asp
Asn Gln Ile Pro Gln Ser Leu Gly Arg Leu Arg Asn Leu 260 265 270Ser
Val Leu Ile Leu Ser Arg Asn Gln Phe Ser Gly His Ile Pro Ser 275 280
285Ser Ile Ala Asn Ile Ser Ser Leu Arg Gln Leu Asp Leu Ser Leu Asn
290 295 300Asn Phe Ser Gly Glu Ile Pro Val Ser Phe Asp Ser Gln Arg
Ser Leu305 310 315 320Asn Leu Ser Asn Val Ser Tyr Asn Ser Leu Ser
Gly Ser Val Pro Pro 325 330 335Leu Leu Ala Lys Lys Phe Asn Ser Ser
Ser Phe Val Gly Asn Ile Gln 340 345 350Leu Cys Gly Tyr Ser Pro Ser
Thr Pro Cys Leu Ser Gln Ala Pro Ser 355 360 365Gln Gly Val Ile Ala
Pro Pro Pro Glu Val Ser Lys His His His His 370 375 380Arg Lys Leu
Ser Thr Lys Asp Ile Ile Leu Ile Val Ala Gly Val Leu385 390 395
400Leu Val Val Leu Ile Ile Leu Cys Cys Val Leu Leu Phe Cys Leu Ile
405 410 415Arg Lys Arg Ser Thr Ser Lys Ala Gly Asn Gly Gln Ala Thr
Glu Gly 420 425 430Arg Ala Ala Thr Met Arg Thr Glu Lys Gly Val Pro
Pro Val Ala Gly 435 440 445Gly Asp Val Glu Ala Gly Gly Glu Ala Gly
Gly Lys Leu Val His Phe 450 455 460Asp Gly Pro Met Ala Phe Thr Ala
Asp Asp Leu Leu Cys Ala Thr Ala465 470 475 480Glu Ile Met Gly Lys
Ser Thr Tyr Gly Thr Val Tyr Lys Ala Ile Leu 485 490 495Glu Asp Gly
Ser Gln Val Ala Val Lys Arg Leu Arg Glu Lys Ile Thr 500 505 510Lys
Gly His Arg Glu Phe Glu Ser Glu Val Ser Val Leu Gly Lys Ile 515 520
525Arg His Pro Asn Gly Leu Ala Leu Arg Ala Tyr Tyr Leu Gly Pro Lys
530 535 540Gly Glu Lys Leu Leu Val Phe Asp Tyr Met Ser Lys Gly Gly
Leu Leu545 550 555 560Leu Phe Tyr Met Glu Gly Ser Cys Ala Gly Ser
Phe Ile Lys Val Leu 565 570 575Cys Val Leu Val Phe Asn Tyr Asn Leu
Glu Phe Tyr Leu Ser Asn Leu 580 585 590Tyr Asn Ser Asn Arg Arg Thr
Val Gln Thr Lys Thr Pro Lys Glu Gln 595 600 605His Leu Xaa Phe Asn
Ile Pro Tyr Gln Xaa Ser Glu Ile Phe Ser Trp 610 615 620Ser Ser Xaa
Cys Arg Gly Asn Xaa Thr Phe Ile Ile Gly His Lys Met625 630 635
640Lys Ile Xaa Gln Asp Leu Ala Val Ala Cys Ser Pro Ser Phe Pro Glu
645 650 655Thr Ser Tyr Met Asp Leu Xaa Ser Ser Asn Val Cys Xaa Xaa
Asn Xaa 660 665 670Met Leu Lys Leu Gln Phe Trp Ser Phe Ser Val Asp
Val Asn Cys Cys 675 680 685Xaa Phe Gln Arg Asp Ser Tyr Ser Trp Ser
Ile Gly Ile Pro Gly Thr 690 695 700Xaa Ala Leu Lys Ala Gln Glu Ser
Lys His Xaa Asn Xaa Tyr Leu Gln705 710 715 720Ser Trp Cys Tyr Leu
Val Arg Thr Pro Asn Glu Glu Ile Thr Trp Gly 725 730 735Val Tyr Glu
Trp Thr Arg Phe Ala Ser Val Gly Cys Leu Ser Cys Gln 740 745 750Arg
Gly Val Asp Lys Xaa Gly Phe Xaa Cys Arg Leu Asp Glu Arg Cys 755 760
765Ile His Ser Trp Arg Arg Val Ala Lys His Val Glu Ala Arg Phe Ala
770 775 780Leu Cys Xaa Ser Phe Ser Ile Ser Thr Thr Arg Ser Ser Ser
Ser Ser785 790 795 800Pro Ala Ala Gly Arg Asp Xaa Thr Arg Glu Ile
Ser His Ser Gln Ser 805 810 815His Leu Pro Gly Arg Pro Leu Glu Pro
Tyr Ser Glu Ser Tyr 820 825 83015726DNAGlycine
maxmisc_feature(1)..(726)promoter region 15gaatacgaat tccattttcg
cgacagtagc tcagaatagg ttcatactcc tgccatcttt 60gaggcggnca atgcaacgtg
taagacttca aggtgtctcc atctatcctg ccatgaaagt 120caagtttcag
gacaagtaat gcagaattat ggaaaagcaa tctgactaag acaaaagagc
180ttcagagatt aacagaaaat agtgagccag aaaaaagatt gcgagacaga
aattggtcgc 240caacaaaaag ttgtctcttt tataattttt aattgaaatt
ttcttaattt agctaacatg 300acttcctacg gccacaattg cgtttgcaga
cacttaaaaa acttgatgtt gcagcaaaaa 360tcacgtttta tttattattg
atgtcaatta tttaacagtt ttatgttagg tttaataaca 420gtaggttgat
gcaagaggct aaacattaat cagaaattga aaggcagggn tattacttct
480tatccatata ctgattgagc gggtcctgaa gaatagcggg aaaaacttca
agcgccagag 540acaatagttt tttcttttca aacagcgcct atgcaaattc
ttccaatctc aagcttcaat 600tcctatcgtc tcgaaccgga cttgntctgn
ttnacctaaa tccccactcg gcattnatna 660acttntcccc actttccttt
ntctttccta tcgccaccgg tcttctatnc ccgcccgtcg 720naatct
72616649DNAGlycine maxmisc_feature(1)..(649)partial cDNA
16aggagtggga aagacagtgg ctatggagct tgttccggag gttgggttgg aatcaagtgt
60gctcagggac aggttattgt gatccagctt ccttggaagg gtttgagggg tcgaatcacc
120gacaaaattg gccaacttca aggcctcagg aagcttagtc ttcatgataa
ccaaattggt 180ggttcaatcc cttcaacttt gggacttctt cccaacctta
gaggggttca gttattcaac 240aataggctta caggttccat acctctttct
ttaggtttct gccctttgct tcagtctctt 300gacctcagca acaacttgct
cacaggagca atcccttata gtcttgctaa ttccactaag 360ctttattggc
ttaacttgag tttcaactcc ttctctggtc ctttaccagc tagcctaact
420cactcatttt ctctcacttt tctttctctt caaaataaca atctttctgg
ctcccttcct 480aactcttggg gtgggaattc caagaatggc ttctttaggc
ttcaaaattt gatcctagat 540cataactttt tcactggtga cgttcctgct
tctttgggta gcttaagaga gctcaatgag 600aattccctta agcataataa
ggttagggga gctatcccaa atgaaatnt 64917558DNAGlycine
maxmisc_feature(1)..(558)partial cDNA 17aggantgggn aagacantgg
ctattttagc tttggtcccg gagggtgggt tggaatcaan 60tgngctcaag gacaaggtat
tgtgaaccaa cttnctttga aaggnttgag ggggcgaaac 120acccacaaaa
atgggcaact tnaaagnctc angaagctta atcttnatga aaaccaaaat
180ggggggtcaa anccntcaac ttttggactt ctttccaacc ttagaggggg
tcaattattc 240aacaataggn ttacagggtc catacctctt tctttaaggt
tctgcccttt gnttcagnct 300cttgacctca acaacaactt gctnacagga
agcaatccct tatagtcttg ctaattccac 360taagctttat tggcttaact
ttgagnttca actnctttct ntgggncttt accaactagn 420ctaactcact
cattttctct cacttttttt tntntttaaa aaaacaaaca tttntngntt
480cccttctnac tcntgggggg gggaaaaaca annaaaggnt tctttaggnt
tcaaaaaatg 540atcctanaac ataacttt 55818794DNAGlycine
maxmisc_feature(1)..(794)partial cDNA 18aatgggagga gtgggaaaga
cagtggctat ggagcttgtt ccggaggttg ggttggaatc 60aagtgtgctc agggacaggt
tattgtgatc cagcttcctt ggaagggttt gaggggtcga 120atcaccgaca
aaattggcca acttcaaggc ctcaggaagc ttagtcttca tgataaccaa
180attggtggtt caatcccttc aactttggga cttcttccca accttagagg
ggttcagtta 240ttcaacaata ggcttacagg ttccatacct ctttctttag
gtttctgccc tttgcttcag 300tctcttgacc tcagcaacaa cttgctcaca
ggagcaatcc cttatagtct tgctaattcc 360actaagcttt attggcttaa
cttgagtttc aactccttct ctggccttta ccagctagcc 420taactcactc
attttctctc acttttcttt ctcttcaaaa taacaatctt tctggctccc
480ttcctaactc ttggggnggg aatttcaaga atggcttctt taggcttcaa
aatttgatcc 540tagatcataa ctttttnctg gtgacgttcc tgcttctttg
ggtagcttaa gagagcccna 600tgagaattcc cttagtcatn ataagnttag
tggagctttc caantgaaat anggacccct 660tntaggctta aacactngnc
attctaataa tgccttgaat gggaacctcc ctgttccctc 720tttanttatc
tcccttncnc ngctggangc cagaccaccn cntgncaatn aatccctcaa
780agttaggtac atcg 79419781DNAGlycine
maxmisc_feature(1)..(781)partial cDNA 19ggaggagtgg gaaagacagt
ggctatggag cttgttccgg aggttgggtt ggaatcaagt 60gtgctcaggg acaggttatt
gtgatccagc ttccttggaa ggggtttgag gggtcgaatc 120accgacaaaa
ttggccaact tcaaggcctc aggaagctta gtcttcatga taaccaaatt
180ggtggtcaat cccttcaact ttgggacttc ttccaacctt agaggggttc
aagttattca 240acaataggct tacaggttcc atacctcttt ctttaggttt
ctgccctttg cttcaagtct 300cttgacctca gcaacaactt gctcacagga
gcaatccctt atagtcttgc taattccact 360aagctttatt ggcttaactt
gagtttcaac tncttctctg gncctttacc agctagccta 420actcactcat
tttctctcac ttttctttct cttcaaaaaa acaaactttc tgggtccttt
480ctactcttgg ggggggaatt ccagaatggn ttctttaggg ttnaaaattg
atcctagaca 540tactttttac tggggacgtc ctgcttcttt ggnagcttaa
agagctcaat gagattncct 600tagcataata agttaggggg gctttnccaa
agnaatagga ncctttntag ggttaaaaac 660ctggcatttt taaaatgcct
tgaangggac ttgnccgctn cccctntaat tatccncctt 720acnccgntgg
anggagagaa aanccccttg caaanaaaac cctcaaaggt tagggngatc 780g
78120861DNAGlycine maxmisc_feature(670)..(670)n is a, c, g, or t
20gaatgggagg agtgggaaag acagtggcta tggagcttgt tccggaggtt gggttggaat
60caagtgtgct cagggacagg ttattgtgat ccagcttcct tggaagggtt tgaggggtcg
120aatcaccgac aaaattggcc
aacttcaagg cctcaggaag cttagtcttc atgataacca 180aattggtggt
tcaatccctt caactttggg acttcttccc aaccttagag gggttcagtt
240attcaacaat aggcttacag gttccatacc tctttcttta ggtttctgcc
ctttgcttca 300gtctcttgac ctcagcaaca acttgctcac aggagcaatc
ccttatagtc ttgctaattc 360cactaagctt tattggctta acttgagttt
caactccttc tctggtcctt taccagctag 420cctaactcac tcattttctc
tcacttttct ttctcttcaa aataacaatc tttctggctc 480ccttcctaac
tcttggggtg ggaattccaa gaatggcttc tttaggcttc aaaatttgat
540cctagatcat aactttttca ctggtgacgt tcctgcttct ttgggtagct
taagagagct 600caatgagatt tcccttagtc ataataaagt ttaatggagc
tataccaaat gaaataggaa 660ccctttctan gcttaaacac ttgacatttn
taataatgnc ttgaatggga acttgcctgc 720taccctctnt aattatcctn
cttacactgn tgaatgcaaa aaacaacctc ttgcaataaa 780tcccttaaan
ttangnnaat gggaaanttn tttntgattt gagtnaaacc aattaatggc
840atattnttta acatttaaan t 86121761DNAGlycine
maxmisc_feature(442)..(442)n is a, c, g, or t 21gaatgggagg
agtgggaaag acatggggtt gaagggctgt acccacatag ttgaatattt 60cccacaaatg
agcttgagtt aaatttcttg gcaagcagag gggggacaga acctgagagg
120ctattgtagg aaacattgaa gggatttaga ctgcgctgac tgtcaaagga
gactggaatt 180tctccactga aattattcag tgacaaatca agctgcctaa
gcgaggaaat gtttgcaatg 240cttgaaggaa tatgtccact aaattggttt
ctactcaaaa tcagaacaga aagattacgc 300aatctaccta aactttgagg
gatttgattg tcaaggaggt tgttctctgc attcagcagt 360gtaagtgagg
ataaattaga gagggtagca ggcaagttcc cattcaaggc attattagaa
420atgtcaagtg tcttaagcct anaaagggtt cctatttcat ttggtatagc
tccctaaact 480tattatgact aagggaaatc tnattgagct ctnttaactc
ccaaagaaca ggacgtncca 540gtgaaaaagt atnatctagg atcaaatttg
aacctaaaaa gcattttgga tccccccaaa 600gtaggaagga gcanaagatg
tntttnaaaa anaaatanaa aatatagtag tactgtaagc 660naaaaggtga
ctaatagcat aantatgata caaattagga tttcttanaa ttttttnnaa
720aatnnnangn aaccaaaaaa gngacntncn tttnaanacc c 76122856DNAGlycine
maxmisc_feature(462)..(462)n is a, c, g, or t 22aatgggagga
gtgggaaaga cagtggctat ggagcttgtt ccggaggttg ggttggaatc 60aagtgtgctc
agggacaggt tattgtgatc cagcttcctt ggaagggttt gaggggtcga
120atcaccgaca aaattggcca acttcaaggc ctcaggaagc ttagtcttca
tgataaccaa 180attggtggtt caatcccttc aactttggga cttcttccca
accttagagg ggttcagtta 240ttcaacaata ggcttacagg ttccatacct
ctttctttag gtttctgcct ttgcttcaag 300tctcttgacc tcagcaacaa
cttgctcaca ggagcaatcc cttatagtct tgctaattcc 360actaagcttt
attggcttaa cttgagtttc aactccttct ctggtccttt accagctagc
420ctaactcact cattttctct cacttttctt tctcttcaaa anaacaatct
ttctggctcc 480cttcctaact cttggggtgg gaattccaag aatggcttct
ttaggcttca aaaattgatc 540ctagaacata acttttttac tggtgacgtt
cctgcttttt ttggtaggct taaaganaag 600ccaatgagaa tttccttagt
catnataaag ttaaggggag cttttnccaa atgaaaaaag 660gaaccctttn
taggcttaaa nanacttgac aatttntaat aatgcccttg aatngggaac
720ttgcctgcta ccccctttaa tttatcctac ttaccctgnt ngaaggcaaa
naacaacccc 780tttgcaataa aaacccnaaa gttaagggga angnggnact
ttntntctnn tttngggnaa 840accanttann ggcnct 85623826DNAGlycine
maxmisc_feature(494)..(494)n is a, c, g, or t 23gaatgggagg
agtgggaaag acatggggtt gaagggctgt acccacatag ttgaatattt 60cccacaaatg
agcttgagtt aaatttcttg gcaagcagag gggggacaga acctgagagg
120ctattgtagg aaacattgaa gggatttaga ctgcgctgac tgtcaaagga
gactggaatt 180tctccactga aattattcag tgacaaatca agctgcctaa
gcgaggaaat gtttgcaatg 240cttgaaggaa tatgtccact aaattggttt
ctactcaaaa tcagaacaga aagattacgc 300aatctaccta aactttgagg
gatttgattg tcaaggaggt tgttctctgc attcagcagt 360gtaagtgagg
ataaattaga gagggtagca ggcaagttcc cattcaaggc attattagaa
420atgtcaagtg tcttaagcct agaaagggtt cctatttcat ttggtatagc
ttcactaaac 480ttattatgac taanggaaat ctcattgagc tctcttaagc
tacccaaaga agcaggaacc 540gtcaccagtg aaaaaagtta tgatctagga
tcaaattttg aacctaaaaa accattcttg 600gaattccacc ccaagaatta
ggaagggagc canaaagatt gttattttga aaaaaaaaga 660aaagtgagaa
aaaatgagtg agttaggctt actggtaaaa ggaccaaaaa aaggantttg
720aaactnaaan ttaanccaat aaaacttaat ggnaataaca aanactttta
nggaattctc 780ttttnaacaa attnttnctt angncaaaaa anttaancaa aggnct
82624571DNAGlycine maxmisc_feature(439)..(439)n is a, c, g, or t
24tgggactggc tgtgactgat ctctctggtc taatctcttc cagctgctgg agaacttgat
60gaacttctgg tcgtgctgat ggagaaggat caacacagtg caaagcgagc ttcaacgtgt
120ttagcaactc gtcgccaact gtggatgcat ctctcatcaa gtctgcatca
aaaacctcat 180ttgtccactc ctctttgaca actgaggcaa cccactgagg
caaatctagt ccattcatag 240acaccccagg tgatttcctc gttaggagtt
ctaacaagat aacaccaaga ctgtagatat 300cagttttagt gtttgctttc
ttgagctttg agagctcagg tgcccggtat cccaatgctt 360cagctgtagc
tatcacgttg gaattagcag cagttgacat caaccgagaa agaccaaaat
420ctgcaatttt agcatttgna ttctcattaa acaacacaat gntggatgng
anggtnccat 480ggatgaaggt cttctnggna agnaagnaaa acaaagcacc
gggccaaggn ttgggctaat 540ttcaaccttg gggggcaaac naanaaatgt t
57125727DNAGlycine maxmisc_feature(685)..(685)n is a, c, g, or t
25ttacaactag tgttatcgga gaatgaaaaa ttgaagaata ataagttcag ctataataaa
60ctcgagggag gaaaaacaaa gaaattcatg ataaatagat ataacttatt aaatttaagg
120ggtgtatttg cacaccctga attatagaga ttcttatatc tttgagaaaa
taattaaatt 180gggaaaaaag agataatgac tgattgagat ttgcctcaga
attgttcgtt ttaatattgg 240tacgaatcta atggttttat cctgaaagat
gctcacaagt attgagggac taataaattg 300tttataaact actactaaat
gagatgagac tttaaggtgt actgaagcaa tatcatttaa 360aaaatgacta
ctcgtatttg tgttgagaaa atttattttc aatgaaaaga aaatatatac
420atataagata aagtaattaa cataaccgaa aggaaataaa atgcaacatt
ataaaaacta 480caactatata aatgatatat acaactccta gcacatgcat
tggattgtga attaattaaa 540atgttgtatg gatggtaaaa attcaaaact
aaacccccca caatttaagt gacacagaat 600ataattagcg gtggtctttt
tacagaaacg acgagaacaa aggtgtcaaa ggaaaggaga 660tggatgcatg
tggtatgagc tcatncaatt ccaacctgtt gtggaccaaa gccgaagtcc 720ttgacnn
72726560DNAGlycine maxmisc_feature(8)..(8)n is a, c, g, or t
26attacgcnag ctctatacga ctcactatag ggagacaagc ttgcatgcct gcaggtcgac
60tctagaggat ccccgggtac cgagctcgaa ttcccaatgc cagagcttcc ctatcgtggg
120ccccacctat gaagaataca cccacgttga aatacatgtt gttgttgttg
gacgcgccca 180gccgagagtg ccggtccacg agtatcccca acgtgcatgg
cgcatgcgct tgaaacctag 240tattcatctt cctgatggag gcagccacgt
gtccgacaag gtcaatgttg ccgttttcgt 300gaaaagggat gataatgaaa
ggcaccatat tgtcttgggc gaggttgaaa atggcgtcgt 360gcatgctctt
gtaaggtgcc acgttgatgt agggaagaac cttgactggc ccacttgagt
420tgttggagta gttttcgaag gcttgcatga tgtggttggt gttggggtaa
ttcacagaca 480agaattttct gngacccgtg tctatgtttt atgggaagga
gaatgggtgc cttttcccca 540cnagctngat naggnggact 56027630DNAGlycine
maxmisc_feature(616)..(616)n is a, c, g, or t 27actgcatgca
tgcaagcaaa tttaacttta cacaacacac caccagagtg taagctgttt 60cataaaaaat
gattgtttcg ggctttcgga tcacaaggct tgtttagtat tcggtaagaa
120agaaagaaat aggtgataaa taaagtggat agaaacataa aagaaaggaa
taaagtaatg 180aaaataaggg agaagtagaa taatggaaat agataagaaa
tagaatggat tcgatagtat 240atctagttta agagaaataa gaaaaaataa
gaacaagaaa aaaaattgca ttttaattta 300ttatttgtac tgtatcgatg
attggcacga gattataagt tttttttttc gtgtttaccg 360ttgaaggatt
atatatcata ccatttgttt gtcaaccaac acggaacttt aagtctcttg
420atgttcaaaa gcacttaaaa ctaaggaatt ttacatcata ttagtcgtct
gtagactgat 480acaggatttt aagcctatat atctagcatt gatccggttg
gcaatcaata tcacattaat 540gatcggtaaa ccattcatat aacccctttg
attggtcaag aaatggcttt atgaatccca 600ggattgagcc cagaancagg
ngatactagn 63028756DNAGlycine maxmisc_feature(103)..(103)n is a, c,
g, or t 28attggcttaa cttgagtttc aactccttct ctggtccttt accagctagc
ctaactcact 60cattttctct cacttttctt tctcttcaaa ataacaatct ttntggctcc
cttnctaact 120gtgggggggg gaatancaag ggnggcttta ggctgcaaaa
tttgatccta gatcataact 180ttttcactgg tgacgttcct gcttctttgg
gtagcttaag agagctcaat gagatttccc 240ttagtcataa taagtttagt
ggagctatac caaatgaaat aggaaccctt tctaggctta 300agacacttga
catttctaat aatgccttga atgggaactt gcctgctacc ctctctaatt
360tatcctcact tacactgctg aatgcagaga acaacctcct tgacaatcaa
atccctcaaa 420gtttaggtag attgcgtact ctttcctgtt ccgattttga
gtagaaacca atttagtgga 480catattcctt caagcatngc nnacatttcc
tcgcttaggc agcttgattg tcactgaata 540atttcaggtg gagaaattnc
agtctncttt gacagtcagc gcagtctaaa tcttcttcaa 600tggttnctac
aataggcctc tcagggtctg gccccccttt gnttggccaa ggaaanttaa
660cttaagctta tttggngggg aaanattcaa ctatgggggg acncggccct
ttaaacccca 720gggnttttcc caggttcctt ccaagggngc anttgt
75629566DNAGlycine maxmisc_feature(554)..(554)n is a, c, g, or t
29gacccttgtt ctatagaacc gaattcgagc tcggtacccg gggatcctct agagtcgacc
60tgcaggcatg caagcttatt attactacta ctacttatct tcactccacc acactgtgtc
120actaaaaccg gaaccatccc catacaaaat tctactgaag acaacatatc
ccccaatatt 180cccaatgcat cagcgttctc catgaaagtt gtcatttctt
ttccattcaa agatccatca 240ttgtggcgcc ttcccaccat cacaagatca
tagtttcctt ccaaactatg cactgcttcc 300aacacctcca ccccatcgtc
caccgtaatc tcgtaccaac aaacgttacc aatgccatat 360ttcatgctct
tgaactcgtc aattaacccc tcgtccaaca tggtatcttc ctcttcctct
420tcacgctctt ctcttgcaaa ataattttac aaccacacgg tttcttggtc
acgataacaa 480acctaaacaa gctaccctcg tatctgcacg ctccgcattc
gaattcccaa tgccagagct 540tccctatcgg gggncccacc tatgaa
56630673DNAGlycine maxmisc_feature(421)..(421)n is a, c, g, or t
30gggactggct gtgactgatc tctctggtct aatctcttcc agctgctgga gaacttgatg
60aacttctggt cgtgctgatg gagaaggatc aacacagtgc aaagcgagct tcaacgtgtt
120tagcaactcg tcgccaactg tggatgcatc tctcatcaag tctgcatcaa
aaacctcatt 180tgtccactcc tctttgacaa ctgaggcaac ccactgaggc
aaatctagtc cattcataga 240caccccaggt gatttcctcg ttaggagttc
taacaagata acaccaagac tgtagatatc 300agttttagtg tttgctttct
tgagcttttg agaagctcag gtgcccggta tcccaaatgc 360ttccagctgt
agcttatcac cgttgggaat taagcagcaa gttggacatt caacccggag
420naaaagaccc aaaaattttg caaattttta agcaatttng gnanttcttn
aatcaaggcc 480aaccaccaat tggnttggga atggtggaag ggtttcccca
atggtaattg gaagggtttc 540ttccctnggg gaaaatggaa aggggcaana
aaacaaaggc ccaacngggg ccccaaaggt 600nttttggggg ccttattttt
tncnaatncc ctttggnngg ggncccaaat tcnaaantgg 660aaattggntt tnn
67331736DNAGlycine maxmisc_feature(5)..(6)n is a, c, g, or t
31gttgnntagn tgcactatag aatncgaatt caatttaaac atttttaatt ttttgtcttt
60gtattctatt ttttcataaa ttctaatctt gctaataatt tcaattcata ttaagatcgg
120taaatagaaa atctagaaaa aaaaacaaaa aaagtatttt tttttcattg
attttatttt 180caattgattt gtcactaaca aactgattcc tcttaaatct
cacaaaagta catgtcgata 240taaatatgag attataaatt catgatatct
attttcgatt tttacatata atgttttttt 300tatctttttt agttcctaat
aagcattttt aaatgtctta tgttcctact ttgcatatca 360gggacccatt
aatgggacga ggtcactgcg agcatgaaca acgtgtcttt cgtctcccga
420acaacgtgcc atcttgcagg ctcaccacct cggaatccct ggagtggtca
ccactgattt 480tccggggaaa gcccgccggt gaaagtttga ttacaccggc
aatgtgagcc ggtcgctgtg 540gcaaccctgg tnccgggaca aangcacacc
aagttgnaan tttgggtccg aggggngcca 600naattggggt tgcanggata
ctaagcnntt ggnnacttnc ctggnnaacc cacccctaat 660nccatntttc
aatggggnac cnaatttctt acaattggnt gcaananggg nttttngggn
720aacctttnna ccccca 73632566DNAGlycine maxmisc_feature(5)..(6)n is
a, c, g, or t 32gaccnnagac gctactatag ggagacaagc tattcgaagg
ggaactgaga acgatccaaa 60gcactccaag aaacagagag tttcacattg tttgttgtgt
acataatgaa gcaaacgtgc 120gtggcatcac tgccttatta gaagagtgca
acccagtgca agagagcccc atatgcgtct 180acgcagtcca ccttatcgag
ctcgtgggga aaagtgcacc cattctcctt cccataaaac 240atagacacgg
tcgcagaaaa ttcttgtctg tgaattaccc caacaccaac cacatcatgc
300aagccttcga aaactactcc aacaactcaa gtgggccagt caaggttctt
ccctacatca 360acgtggcacc ttacaagagc atgcacgacg ccattttcaa
cctcgcccaa gacaatatgg 420tgcctttcat tatcatccct tttcacgaaa
acggcaacat tgaccttgtc ggacacgtgg 480ctgcctccat caggaagatg
aatactaggt ttcaagcgca tgcgccatgc cgttggggat 540actcgnggcc
ggnactctng gtgggn 56633614DNAGlycine maxmisc_feature(138)..(138)n
is a, c, g, or t 33acaacaagca acgaacagct tttaacctta aactaggcaa
atgccaatat taaacaacaa 60ataattaaaa ttgtaaggct ggtcgagtat aaattaaaca
aaaggccctc tattcaaacc 120ttcatatatc atacctgntt ttaattaacg
cggactactt tttcatataa aaaaaagatc 180attagaggat taatttaaag
cgntttagtt tttaattacc aaagagtata attattatta 240ggcgctttgg
cccacaatca atcacctaaa caagaaaaag aaaaagaaaa aaaaaggcaa
300attggactaa tgcaaaagtg gcacaatctt tgncttgaac tctttaatta
gcaacaaatn 360atactcttct gcacaaatca caagaatacc ttacatgaaa
agaatggnaa tntgacgggt 420tacattaaat tatatgcagg tttctgcagg
gaatcaattn tcaagaattt aagggggggt 480gggaattttc aatagctagc
ttgactagca aagggaaaga ataaaggnaa aangcttctt 540ggctnggcct
tttggganng gnatcctttt ngctaaaccg gaaanggnta tangaatggg
600aaaggagana atcg 61434602DNAGlycine maxmisc_feature(509)..(509)n
is a, c, g, or t 34aggctagctg gtaaaggacc agagaaggat ttgaaactca
agttaagcca ataaagctta 60gtggaattag caagactata agggattgct cctgtgagca
agttgttgct gaggtcaaga 120gactgaagca aagggcagaa acctaaagaa
agaggtatgg aacctgtaag cctattgttg 180aataactgaa cccctctaag
gttgggaaga agtcccaaag ttgaagggat tgaaccacca 240atttggttat
catgaagact aagcttcctg aggccttgaa gttggccaat tttggcggtg
300attcgacccc tcaaaccctt ccaaggaagc tggatcacaa taacctgtcc
ctgagcacac 360ttgattccaa cccaacctcc ggaacaagct ccatagccac
tggcattcca gctcccgcaa 420gaacccttct ggatcagcca actcttgctt
gaaagcttat cacatgtacc tctctacaga 480taggagggtg cttcttccct
ttcactggnc tacctcttcg ggaataagcc acctaatgag 540aaagaaagan
ctgggatagc taactctaca tagnctcaag gcnagagata attagggaaa 600ng
60235644DNAGlycine maxmisc_feature(36)..(36)n is a, c, g, or t
35ggaattttga agagaagtaa agtgagagaa aatgantgan nnaggctagc tggtaaagga
60ccagagaagg atttgaaact caagttaagc caataaagct tagtggaatt agcaagacta
120taagggattg ctcctgtgag caagttgttg ctgaggtcaa gagactgaag
caaagggcag 180aaacctaaag aaagaggtat ggaacctgta agcctattgt
tgaataactg aacccctcta 240aggttgggaa gaagtcccaa agttgaaggg
attgaaccac caatttggtt atcatgaaga 300ctaagcttcc tgaggccttg
aagttggcca attttggcgg tgattcgacc cctcaaaccc 360ttccaaggaa
gctggatcac aataacctgt ccctgagcac acttgattcc aacccaacct
420ccggaacaag ctccatagcc actggcattc cagctcccgc aagaaccctt
ctggatcagc 480caactcttgc ttgaaagctt atcacatgta cctctctaca
gataggaggg tgcttcttcc 540ctttcactgg nctacctctt cgggaataag
ccacctaatg agaaagaaag anctgggata 600gctaactcta catagnctca
aggcnagaga taattaggga aang 64436748DNAGlycine
maxmisc_feature(625)..(625)n is a, c, g, or t 36attggcttaa
cttgagtttc aactccttct ctggtccttt accagctagc ctaactcact 60cattttctct
cacttttctt tctcttcaaa ataacaatct ttctggctcc cttcctaact
120cttggggtgg gaattccaag aatggcttct ttaggcttca aaatttgatc
ctagatcata 180actttttcac tggtgacgtt cctgcttctt tgggtagctt
aagagagctc aatgagattt 240cccttagtca taataagttt aatggagctg
taccaaatga aataggaacc ctttctaggc 300ttaagacact tgacatttct
aataatgcct tgaatgggaa cttgcctgct accctctcta 360atttatcctc
acttacactg ctgaatgcag agaacaacct ccttgacaat caaatccctc
420aaagtttagg tagattgcgt aatctttctg ttctgatttt gggtagaaac
caatttagtg 480gacatattcc ttcaagcatt gcaaacattt cctcgcttag
gcagcttgat ttgcactgaa 540taatttcagt ggagaaattc cagtctcctt
tgacagtcaa gcgcaagtct aaatctcttc 600aatgtttcct acaatagcct
ctcanggtct gncccccctc tgcttgccaa gaaatttaac 660tcaagctcat
ttgtgggaaa tattcaacta tgtgggacag nccttcaacc ccatgttttn
720ccaagcttca tacaaggagc atggccct 74837563DNAGlycine max
37ctggctgtga ctgatctctc tggtctaatc tcttccagct gctggagaac ttgatgaact
60tctggtcgtg ctgatggaga aggatcaaca cagtgcaaag cgagcttcaa cgtgtttagc
120aactcgtcgc caactgtgga tgcatctctc atcaagtctg catcaaaaac
ctcatttgtc 180cactcctctt tgacaactga ggcaacccac tgaggcaaat
ctagtccatt catagacacc 240ccaggtgatt tcctcgttag gagttctaac
aagataacac caagactgta gatatcagtt 300ttagtgtttg ctttcttgag
ctttgagagc tcaggtgccc ggtatcccaa tgctccagct 360gtagctatca
cgttggaatt agcagcagtt gacatcaacc cgagaaagac caaaatctgc
420aattttagca tttgtattct catcaagcaa cacattgctg gatgtgaggt
tcccatgtat 480gatgttctcc tgggaatgaa ggcagaacaa gccacggcca
agcttggcta tttcatcctt 540gtggccaatc aatgaatggt cat
56338623DNAGlycine maxmisc_feature(507)..(507)n is a, c, g, or t
38gattttgcac atctacttga gtaggcttca catgattccg tgtattactt ttattttggt
60atatatacca tgtggagtat agtatcactt tttgtcctac aaccacattt tatgagactt
120gcattttatg tgacatgaac ataaaaaata atgaaaaaga aaatgtcaca
tatatatgat 180acaatctttt taaaagtcaa tttgaataat ttttcatcag
gaggaaaaag aagagagaaa 240atgaattaag tttcttctaa aaattaaaat
caacttataa aaagaaaaaa ctttaatgaa 300aaaaattcaa aaagaaaaag
aataaaatga tcaatagcct ttaggtttaa gcacaaggtg 360aatccaaata
aagaccccaa aagatagtac agaacccaac aatggtaaaa tctagaaata
420tacatgtaaa gactgcattt atagaccatc atgactagca aatgcttaaa
ggcacataga 480tgaattaatc tatgcaacaa aatctgnccc aagttttttt
tangcaagga aaatcatatc 540attttattaa ggataactga gaggaccaat
ggtgtaatca attgaaatca tgcgaggctt 600acatgaaatc tgtcaccaag tac
62339785DNAGlycine maxmisc_feature(80)..(80)n is a, c, g, or t
39caattaggaa ataaatatat tgaaaagaat tggtagtcag ttcaatgaaa gtgaggtcct
60caaacaactt gatgcagcan ctgtatgata caaaatatat taataactac accagcagaa
120aaatataggt caatctatat ttgggaacca aataatattt aatttgtatc
tgatagactc 180aagaaattat aactaatttg gaagaaatgg atacctagta
ttattaaaac accaaaacac 240agggcagatt atagtagcta aagaggaaga
agctaactag tcaaagtgtc acactattca 300acactacaaa ggaccaatcc
ccttttagag agcctgacct ttctcaccca agagctaccc 360aagagaatac
acaccctctc ctccatatcc cctcccatat aacacaatcc tcaccaacta
420agcacctacc tgacaattcc ctcctaacca actctctgct catcagggtt
gattctcttc 480tctttccaag actttgggct tttgttttga ctaagccaaa
tttctatctg ctggcctggt
540ccaacagtat cttttacaga caagtttaca aaatattcgt atttgttaga
atttattgat 600attcctatta tggtccccac tgtgtgcaaa catttagaaa
ctaatattac aattaacagt 660ttttggtgaa tgcagcaaaa ctaaatatat
ttgatataga aatcaacaaa ctgaaaaatt 720atatngcaag gncaattgga
aaagaaaatt gatacccctt ttgnggnaat aaatatantg 780nntac
78540640DNAGlycine maxmisc_feature(411)..(411)n is a, c, g, or t
40tggaaggcgt ccttcaattc aatcacaaag tctaaatcaa agacgagggg gctgaaatca
60tgggggacat tgacaacgta aggtaaccac taattaatta accactaata ttatcccatt
120aatatcccat taagagataa tacatataga gccaataaat aagcatctta
acaagacaaa 180taaattatcc attattcagc ttatgcccat ggtggtatta
gaagtttagg aaaaaaaaat 240tcatcatttg gcaattttgg gctcattagc
ttgaattggt tacaaggtgt ggtatggact 300tttttctttt cttttctcta
aattcttcct tctatgatat acttttggtc aacttaaact 360caatttctta
tagctcaata ttttggattt agattggaaa tatctaaaag ncacttaaat
420tttatattta caaaaaaaaa aaaagcatcg ntctttttct ttttataaca
aagggggatc 480aaaatcactc tttttatgaa tccgcattat ccttnataat
aattaacctc cactgggatt 540taaagggnga ttaattaaat ccggaggcca
tggaaggata tgggggaacc taatctaaaa 600ntncatcctc aaccctaang
ggaaaataaa ggaatngggg 64041808DNAGlycine maxmisc_feature(83)..(83)n
is a, c, g, or t 41cttttgacac tatgaatacg aattcaaata ttaaatattt
ttattttttg tctttgtatt 60ctattttttc ataaattcta atnttgctaa taatttcaat
tcatattaag atcggtaaat 120agaaaatcta gaaaaaaaaa caaaaaaagt
attttttttt cattgatttt attttcaatt 180gatttgtcac taacaaactg
attcctctta aatctcacaa aagtacatgt cgatataaat 240atgagattat
aaattcatga tatctatttt cgatttttac atataatgtt ttttttatct
300tttttagttc ctaataagca tttttaaatg tcttatgttc ctactttgca
tatcagggac 360ccattaatgg gacgaggttc actgcgagca tgaacaacgt
gtctttcgtt ctcccgaaca 420acgtgtccat cttgcaggct caccacctcg
gaatccctgg agtgttcacc actgattttc 480cggggaagcc gccggtgaag
tttgattaca ccggcaatgt gagccgttcg ctgtggcaac 540ctgttcccgg
gacaaaggca cacaagttga agtttgggtc cgagggtgca gattgtgttg
600caggatacta gcattgtcac tcctgagaac caccctatcc atcttcatgg
gtcgatttct 660acattgttgc agagggtttc gggaacttcg acccaaagaa
agatccgcga aattcaacct 720tggtggatcc cctttgaaaa acacagtggc
tggcctgtaa atggatgggc aagtattcga 780tttgggggct gataacccna gtaaatnt
80842605DNAGlycine maxmisc_feature(168)..(168)n is a, c, g, or t
42ctcccgggtc ccaagtaata ggcccctcag agccaaaaca ttgggggggc taatttttcc
60tagaacactg acttctgatt caaattctct atgaccttta gtgatctttt ccctcaatct
120ctttactgca acttgacttc catcctccaa aatagcctta taaacagntc
cataggtgct 180ctttcccatg atctcagctg gtgcacacaa gagatcatca
gctgtaaaag ccattggtcc 240atcaaaatgg actagtttcc ctccagcctc
cccacctgct tcaacatcac caccagcaac 300tggagggact cctttttctg
cctcatagtg gccgctctac cctcggtggc ttggccgntc 360ccggccttag
atgntgatct ctttctgatc aggcagaaaa gcaggacaca acaaagnata
420atcaggacta cgaggagaac tcctgctact atgagaatta tgnctttggg
gcttagcttc 480ctatgatggg gatggttnga cacttcanga gggggggcaa
tgactccctg gganggagct 540tgggaaagac atgggggtga aggnctgnac
ccacataggn gaaaaattcc cacaaangag 600cnngn 60543275DNAGlycine
maxmisc_feature(225)..(225)n is a, c, g, or t 43ctgaacggaa
gtgactgcgt ttgtgtcggt tgtaagcagg gagtggaggc attataggtc 60tcggttttgc
tctttactcc tttggcacga tggtgagaat gcttattgtg gtgattcggt
120gatttgtatt cgagtatggc ggttgtagtg gtgttgtcga aggcagcgtt
ttgggcggat 180tggtacgcac gcgccgccat gtagtagcgg gaaggtggct
ggtcnccggt gattaagacg 240tcggcggttt gcccggggcc cactatgagg acttt
27544632DNAGlycine maxmisc_feature(574)..(574)n is a, c, g, or t
44tgtatataat taaaatgagt ttaatattta tgtattaata gtataaaatt tatcatacat
60gatgaatggt gaaattttga attatgatta aataattata taaaaaaatt tacatgatga
120atgaataact ttttttttct caattaaaat tatgatcctt tgtcgatatg
ttttactgtg 180tcgacctttt ttttcggggg agaggggacc agtaggagaa
gtagtattta gtaaaagaag 240ggagagagaa gttgacttat cctttaatta
gtttagagaa aattagacga gaaggaaaaa 300aaataggcga aagtcacttt
ttctttctat ctctaccaag aatgttgatg aaaaagtggg 360gagcagaatt
ttaaattttt attttcatat ttatccttct ccacattttt ggtttcttcc
420atttttttat aaaatgattt attttagggc ataggtaact tttcaatttt
tttcattcta 480ttcgatcaaa taaatagaaa aataatttac ttttctttct
tttaaccttt ttcatatttc 540tctcataacg accacttatt aattacctct
tttnccccac tttttgctat ncaaatctat 600ctttgaattt cttccttttc
attttggtct cn 63245650DNAGlycine maxmisc_feature(573)..(573)n is a,
c, g, or t 45ttcacagaca tagcaaaatt ctgaagtaag aagcaagttc acgtgtgatg
gcgaaaccca 60ttatagaata tgttagactg aaaggtaaca aattaaaata tgttttattg
cagaaaccat 120aaactaataa accttttggg tagatagaaa agtgataaat
catacataat aataactgaa 180atactcagct tttaatcaat ttaattcaat
atatatctat ttttgaattt ttcaaagaga 240tgcttagcta gggaggaaac
ctaatttagt ataaaaaaaa gaaacaaatt aaaaacataa 300attgccattg
aatgcctctt aaaatattcc gatccattga tgtctacata ataatatata
360ttattgatat aataaccgat tgaataaaat ggatatacct attacgtaat
agcagatttg 420tctacgcaaa agagacagtc aaaggtgcta attagaaatt
aatcgcccca taataaaatt 480ctaaaccttt gaaaagataa atcaattctc
aaaaagattt attttactta tctcagtacc 540atgcaccatg gatcatctta
ctggtctggt tangaatttt caaagctacg ccacaaattg 600aaattgggct
aaaaatcaaa catgcatggt gtcacaacta tattactagt 65046628DNAGlycine
maxmisc_feature(38)..(39)n is a, c, g, or t 46gaatgcacat tttataaacg
tgttgatcct ctccccgnng ggggaccaat taataaggta 60ccctgttgcc cctaggggac
attggatggc catcagatgg tgcatataca caccaaagtt 120tatacagcat
tatagtgact ttcaacctcc tcactccgag gtccccatat attctctcta
180ttgaacttgt aaagactaat gaacttatga agactatcac tgaaacccac
tatggaagcc 240ccagtagtaa aatggncatg catgctcacc aaaagtttat
acagcattat agcgacatac 300gacctcactc ccaggnccac atgctctatn
gaacttctaa agctatctcn gaaccctatt 360atagcttcat gagggtaaca
tgcattttag cgacttagaa aactacatat cattgagcgt 420gatcnttaag
aaggcctcat tttgacacaa aagaacatga tggatttgcc tttatattcg
480gttactaacc ttgatagcta ttttggncag agagaaaaat attgacatgc
ccgnggaatc 540aaaaggtaga taatnattaa agagataaag aactatcccc
ttgctagggg naaaaaaaaa 600ntatatccct atttaaataa aanccatc
62847736DNAGlycine maxmisc_feature(696)..(696)n is a, c, g, or t
47tggtgtatat aattaaaatg agtttaatat ttatgtatta atagtataaa atttatcata
60catgatgaat ggtgaaattt tgaattatga ttaaataatt atataaaaaa atttacatga
120tgaatgaata actttttttt tctcaattaa aattatgatc ctttgtcgat
atgttttact 180gtgtcgacct tttttttcgg gggagagggg accagtagga
gaagtagtat ttagtaaaag 240aagggagaga gaagttgact tatcctttaa
ttagtttaga gaaaattaga cgagaaggaa 300aaaaaatagg cgaaagtcac
tttttctttc tatctctacc aagaatgttg atgaaaaagt 360ggggagcaga
attttaaatt tttattttca tatttatcct tctccacatt tttgttttct
420tccatttttt tataaaatga tttattttag ggcatagtta acttttcaat
ttttttcatt 480tctattcgat caaataaata gaaaaataat ttacttttct
ttcttttaac cttttcatat 540ttctctcata acgaacaact tattaattta
cctcttttcc cccacttttg tctatccaaa 600ttctatcttt gaattttctt
ccttttcatt ttggttctca acccaaataa agaagaacga 660gtttggataa
atcataaagg ttatataccc tataantgga agaacattta aatggtccaa
720ngggccttaa aattct 73648695DNAGlycine
maxmisc_feature(471)..(471)n is a, c, g, or t 48atgccagagc
tttccttatc gtggccccac ctatgaagaa tacacccacg ttgaaataca 60tgttgttgtt
gttggacgcg cccagcccga gagtgccggt ccacgagtat ccccaacgtg
120catggcgcat gcgcttgaaa cctagtattc atcttcctga tggaggcagc
cacgtgtccg 180acaaggtcaa tgttgccgtt ttcgtgaaaa gggatgataa
tgaaaggcac catattgtct 240tgggcgaggt tgaaaatggc gtcgtgcatg
ctcttgtaag gtgccacgtt gatgtaggga 300agaaccttga ctggcccact
tgagttgttg gagtagtttt cgaaggcttg catgatgtgg 360ttggtgttgg
ggtaattcac agacaagaat tttctgcgac cgtgtctatg ttttatggga
420aggagaatgg gtgcactttt cccacgagct cgataaaggt ggactgcgta
naccatatgg 480gctctnttgc actgggttgc actcttctaa taanggcagn
gatgccncnc nccgtttgct 540tnattatgta cncaacaaac aatgngaaac
tctctgnttn ttgggagngc tttggatcgn 600tctcanntnc ccttnnaata
anctttntnn gngnacttnn agggcgangc ttnnncnata 660tgntaaccaa
gggngntacn annnnnggnt ntaan 69549625DNAGlycine
maxmisc_feature(401)..(401)n is a, c, g, or t 49tttcccacaa
tctttaatct tgctaataat ttcaattcat attaagatcg gaaaatagaa 60aatctataaa
aaaaaacaaa aaaagtattt ttttttcatt gattttattt tcaattgatt
120tgtcactaac aaactgattc ctcttaaatc tcacaaaagt acatgtcgat
ataaatatga 180gattataaat tcatgatatc tattttcgat ttttacatat
aatgtttttt ttatcttttt 240tagttcctaa taagcatttt taaatggctt
atgttcctac tttgcatatc agggacccat 300taatgggacg aggttcactg
cgagcatgaa caacgtggct ttcgttctcc cgaacaacgt 360gtccatcttg
caggctcacc acctcggaat ccctggagtg ntcaccactg attttccggg
420gaagccgccg gtgaagttng attacacccg gcaatgtgag ccgntcgctg
gggcaacctg 480ntcccgggac aaaggcacac aagttgaagt ttgggtcgag
ggngcagatt ggggntgcan 540gatactagca ttgcactcct gagaaccacc
ctatccatct tcatggggac caattctaca 600ttggtgcaga nggttccggg aacnc
62550621DNAGlycine max 50actggtgtac gatttagtgt tactagctat
cccatgtaat aaatatataa atcttgaatc 60acaaggaatg atgcaatata tggttcctct
aatagtaagt tatcccacca aatctgaata 120taattaagaa gttgtattcg
tctgaatgtt gtgtctaaaa gggttgattg atgaatgatg 180gctacatgtg
agagtttgat aacaacagct agctagccat tagccaagcc actaactaga
240cattagtttt ggttggttgt cagacaaacc gttagacctg agaacgaaag
cgtattaaac 300aaaagatgat atgtagactt ttaatataaa aagagatgga
gaaaccaaat tgagatttga 360taggtgaact ataaatcatg acagtgcatt
agacaagttg gtagagtttg ttactaactc 420atcagattct taagaaaggc
aaaaatagaa actacaccac atgtcgctag cgataacgtg 480caatttataa
ataaataatg gcttcatttt catggttagt tataaattaa tgggtcacaa
540ttcttaattt attaggaacg tatacttcat tttgagagtg tataaagttg
gaagaagaaa 600agggatatag aaagaataaa a 62151480DNAGlycine
maxmisc_feature(10)..(10)n is a, c, g, or t 51aagctccgcn cggggaacct
nnagagtcta cctgaatccc caagntngaa cgaatacttg 60ccaacacaaa tacgggcgat
gggaaacatc tgaagaccgc tccaaagcgc cncatactaa 120attgnnagga
aaatttatat ctgacctttc atgggtgggg ggtgcatctg ctataaggaa
180gggttcattc tgggcaagat ctgtggaaaa caatattggg gatcaaattt
tagggagtga 240tgctacaacc tcttcattat acatggattc tgaaataagt
ggtgtgaact ttaaagtgaa 300cgaagacggc atgcaaatgc ctggtattca
tctagttgat ttatttgaga ctgacaccaa 360tacaagcggc gataaacatg
attcccacta tgatgaagng ccatcatctt atgggtttga 420gggcttacga
cgatccaaac gtaggaacat acaacctgaa ccgntactct gattggggga
48052480DNAGlycine maxmisc_feature(10)..(10)n is a, c, g, or t
52aagctccgcn cggggaacct nnagagtcta cctgaatccc caagntngaa cgaatacttg
60ccaacacaaa tacgggcgat gggaaacatc tgaagaccgc tccaaagcgc cncatactaa
120attgnnagga aaatttatat ctgacctttc atgggtgggg ggtgcatctg
ctataaggaa 180gggttcattc tgggcaagat ctgtggaaaa caatattggg
gatcaaattt tagggagtga 240tgctacaacc tcttcattat acatggattc
tgaaataagt ggtgtgaact ttaaagtgaa 300cgaagacggc atgcaaatgc
ctggtattca tctagttgat ttatttgaga ctgacaccaa 360tacaagcggc
gataaacatg attcccacta tgatgaagng ccatcatctt atgggtttga
420gggcttacga cgatccaaac gtaggaacat acaacctgaa ccgntactct
gattggggga 48053736DNAGlycine maxmisc_feature(633)..(633)n is a, c,
g, or t 53aatttattta gttgatataa ccactttcaa aaatctgact tacaagactc
tttagaattc 60ataatagtga cacttgatta agttagatta gactttataa aacacgagtt
tgattttttt 120tttaataata attaaggttc tagcttatat atattatata
gttgatatag actactttca 180aaagtctgac ttaaaagtct ctttagtata
cataataata taacctttta atttagttaa 240aaaatttgtc cctaaataaa
ttaataaatc caaacttata tacaagttaa taggcttaag 300tcttaaaaaa
ataatatata tatatatata taaagcatta aaacatttca atgaaaacaa
360tataataata ataataataa atatattatt gttattaatt catagatttt
attattacta 420ttatagaata atttgtgtgt atatatataa atatatagag
agagagaggg tcattttata 480tgagtgagaa aatttaaata ttattatgaa
ttttcaaaat taaaatcaca tgccatatga 540ttttcttaaa aaattacgta
actttttttt ttacaaaagt aatcatatgg ttttaaaaac 600taatttaaat
aacttatata taactatatc agntaaaatt ngggtcataa aataagtata
660tcagntattt tacaaaaatt ataagtnttc ataaataaat accaaatgat
agtcccaggn 720gatgggncag cttnng 73654642DNAGlycine
maxmisc_feature(573)..(573)n is a, c, g, or t 54ttaactttac
acaacacacc accagagtgt aagctgtttc ataaaaaatg attgtttcgg 60gctttcggat
cacaaggctt gtttagtatt cggtaagaaa gaaagaaata ggtgataaat
120aaagtggata gaaacataaa agaaaggaat aaagtaatga aaataaggga
gaagtagaat 180aatggaaata gataagaaat agaatggatt cgatagtata
tctagtttaa gagaaataag 240aaaaaataag aacaagaaaa aaaattgcat
tttaatttat tatttgtact gtatcgatga 300ttggcacgag attataagtt
ttttttttcg tgtttacgtt gaaggattat atatcatacc 360atttgtttgt
caaccaacac ggaactttaa gtctcttgat gttcaaaagc acttaaaact
420aaggaatttt acatcatatt agtcgctgta gactgataca ggattttaag
cctatatatc 480tagcattgat cgggtgtcaa tcaatatcac attaatgatc
ggtaaaccat tcatataacc 540cctttgattg gtcaagaaat ggctttatga
atncccagga ttgagcccag aagacaggtg 600atactaggtt caattcatgg
ttttaggata ggctcgtaaa cc 64255659DNAGlycine
maxmisc_feature(362)..(362)n is a, c, g, or t 55aaaaggacct
aaaagcaaaa agaaaattga gtatccttag gaattaaaaa tattccaata 60aaaataaaat
aaagatccaa atgatagtgg gataaccgaa gaggaatgtc tttcaaccac
120tgcctgaccg ccaccactgc caacagccta gtatcaaccg aatccacata
taccaacaat 180cttcagacaa acacttctaa gttggtgctg aagagacaat
atctcatggg tagatcaaat 240taagagtgct accaataaca aaatcgggat
catttgacta acaaacagtt atgtgcattg 300gatgttctac catagtacat
tgctttatgt gaaattcttt taattattca atattgacat 360gntcttatat
atatatatat atatatatat atatatatat atatacgagg gattgnatta
420tctctgaaaa aagattttat cataaaatca taatgatttc tcataatgna
tctttacatt 480ttaaaggtag ataaataaaa ttgatttaaa tnggnagata
taattaaaat acataattaa 540tatgactttt aaccaaattg atatataaac
acttaaaaaa aagttcatga acgnccgggg 600ngnattggnt gggncaaaaa
aaaattaata ctatcaacct aattaaaaat tatttatan 65956805DNAGlycine
maxmisc_feature(610)..(610)n is a, c, g, or t 56ccaatgccag
agcttcccta tcgtgggccc cacctatgaa gaatacaccc acgttgaaat 60acatgttgtt
gttgttggac gcgcccagcc gagagtgccg gtccacgagt atccccaacg
120tgcatggcgc atgcgcttga aacctagtat tcatcttcct gatggaggca
gccacgtgtc 180cgacaaggtc aatgttgccg ttttcgtgaa aagggatgat
aatgaaaggc accatattgt 240cttgggcgag gttgaaaatg gcgtcgtgca
tgctcttgta aggtgccacg ttgatgtagg 300gaagaacctt gactggccca
cttgagttgt tggagtagtt ttcgaaggct tgcatgatgt 360ggttggtgtt
ggggtaattc acagacaaga attttctgcg accgtgtcta tgttttatgg
420gaaggagaat gggtgcactt ttccccacga gctcgataag gtggactgcg
tagacgcata 480tggggctctc ttgcactggg ttgcactctt ctaataaggc
agtgatgcca cgcacgtttg 540ctttcattat gtacacaaca aaacaatgtg
aaaactctct gtttcttgga ggtgctttgg 600atcgttctcn agttcccctt
cgaataagct ttctgcgtgn tacttcnagg ggcnnatgct 660ttgtaccaat
atgnttancc caagggngnt tnccattncn ggtctttact accacnacat
720aacacccnat tnnttgaann gnanccnatc caacntctac naaancgtna
tcaatnacnt 780tnnattngat ttganncact ggccn 80557632DNAGlycine max
57tttagaattc ataatagtga cacttgatta agttagatta gactttataa aacacgagtt
60tgattttttt tttaataata attaaggttc tagcttatat atattatata gttgatatag
120actactttca aaagtctgac ttaaaagtct ctttagtata cataataata
taacctttta 180atttagttaa aaaatttgtc cctaaataaa ttaataaatc
caaacttata tacaagttaa 240taggcttaag tcttaaaaaa ataatatata
tatatatata taaagcatta aaacatttca 300atgaaaacaa tataataata
ataataataa atatattatt gttattaatt catagatttt 360attattacta
ttatagaata atttgtgtgt atatatataa atatatagag agagagaggg
420tcattttata tgagtgagaa aatttaaata ttattatgaa ttttcaaaat
taaaatcaca 480tgccatatga ttttcttaaa aaattacgta actttttttt
ttacaaaagt aatcatatgg 540ttttaaaaac taatttaaat aacttatata
taactatatc agttaaattt ggttcataaa 600ataagtatat cagttatttt
acaaaattat aa 63258437DNAGlycine maxmisc_feature(14)..(14)n is a,
c, g, or t 58cttttgacac tatngaatac gaattcgaat gtcggagcgt gcagatacga
gggtgagctt 60gtttaggttt gttatcgtga acaagaaacc gtgtggttgt aaaattattt
tgacaagaga 120agagcgtgaa gaggaagagg aagataccat gttggacgag
gggttaattg acgagttcaa 180gagcatgaaa tatggcattg gtaacgtttg
ttggtacgag attacggtgg acgatggggt 240ggaggtgttg gaagcagtgc
atagtttgga aggaaactat gatcttgtga tggtgggaag 300gcgccacaat
gatggatctt tgaatggaaa agaaatgaca actttcatgg agaacgctga
360tgcattggga atattgtggg atatgttccc ttcncccanc ntgnntggcn
tngttccgct 420tttttcgnct ntnngcc 43759681DNAGlycine
maxmisc_feature(3)..(3)n is a, c, g, or t 59ggnttcttta gggcttcaaa
atttgatcct agatcataac ttttttcact ggtgacgttc 60ctgcttcttt gggtagctta
agagagctca atgagatttc ccttagtcat aataagttta 120gtggagctat
accaaatgaa ataggaaccc tttctaggct taagacactt gacatttcta
180ataatgcctt gaatgggaac ttgcctgcta ccctctctaa tttatcctca
cttacactgc 240tgaatgcaga gaacaacctc cttgacaatc aaatccctca
aagtttaggt agattgcgta 300atctttctgt tctgattttg agtagaaacc
aatttagtgg acatattcct tcaagcattg 360caaacatttc ctcgcttagg
cagcttgatt tgcactgaat aatttcagtg gagaaattcc 420agtctccttt
gacagtcaag cgcagctaaa tctcttcaat ggttcctaca atagcctctc
480agggtctgcc cccctctgct tggcaagaaa tttaactcaa gctcatttgt
gggaaatatt 540caactatgtg gggtacagcc ttcaacccca tggctttcca
agctncatca caagggggca 600ttggccccct cctgagnggc aaacatcacc
atcataggaa gctaacccca aagacataat 660tctcatagta nccaggaggt n
68160644DNAGlycine maxmisc_feature(635)..(635)n is a, c, g, or t
60acaacaagca acgaacagct tttaacctta aactaggcta atgccaatat taaagaagaa
60ataattaaaa ttgtaaggct ggtcgtgtat aaattaaaca aaaggccctc tattcaaacc
120ttcatatatc atacctgttt ttaattaacg cggactactt tttcatataa
aaaaaagatc 180attagaggat taatttaaag cgttttagtt tttaattacc
aaagagtata attattatta 240ggcgctttgt cccacaatca atcacctaaa
caagaaaaag aaaaagaaaa aaaaagtcaa 300attggactaa tgcaaaagtg
gcacaatctt tgtcttgaac tctttaatta gcaacaaatt 360atactcttct
gcacaaatca caagaatacc ttacatgaaa agaatggtaa tttgacgggt
420tacattaaat tatatgcagt tttctgcagg taattaattt tcaagaattt
aagggtgggt 480ggtaattttc aatagctagc ttgactagca aaggaaagaa
taaaggtaaa atgcttcttg 540gtttggcctt ttggattggt atactttttg
ctaaacggaa atggttatat gaatggtaaa
600ggagataaat tggtacatag ctaaaatggt atagncttaa tccn
64461678DNAGlycine max 61aaattcattt aacttctcta atttttaaat
cgatcaaatt tggtttttca atctaaaata 60taagaaacta tattttgtga tgggtttaaa
atcgacatta agtgttctta atctaccaca 120aaaagcacat ttccaaaaaa
ataaattaat tttaaaaatt ataagatcaa attgaatcaa 180ttttaaaaat
taaaatatta aattgaaaaa aaaaataaag gatcaaattg aacataaata
240ataaatttga ggattaaaaa actaatttaa cctttaattt tttctcactt
atattaatat 300taaaaaatta tattgatttt cctaataact ccttatctca
attaaaattt ccaaaaatta 360attctagcat cttcaaacac tactcaccat
gaaagttcat cacaaccatc tttctttctc 420ttttctctac atcatgtttt
cgcttcgcaa actttattgt gttcctagtc ttagacgtct 480gataatcttc
cacaagtatt gaactataac acttattgga cttgcaccgg taatagctaa
540caccaaatga gacgtgcact tgacttttat atcactaaga aaatttcaac
acattgacca 600agattagctc catcttgctt taacacttgg ttgactagtc
acttaagtgc aacaaccact 660ttgatatcat tgggtgga 67862571DNAGlycine
maxmisc_feature(534)..(534)n is a, c, g, or t 62tcttttaaga
ccattcgaca ctatagaata cgaattccat aattaacaat aaagtcatct 60tctattatat
attttttctt cttaaattac atgatagtat ttcatcatta tttgacaata
120atgatatttt tatctcataa atattatttt gttttaaaaa tattcatagc
acacacgagt 180tttttatatc aacaaagagg tatcacttca gttggtcaat
ttggtctaac ttttagacaa 240tgtcgtatag ttgaattgaa ttggaatttg
gcagtatata ttttactttt tgccccctta 300ttttcaatca aattagagta
gacgcctcgt attattggca tacatggata ttggatcggc 360acctgtgttt
cagacctgag tcacatctga ctcggatcga ttttatctta catgaaaatt
420ccaaaataat gaaagatatg gcaattggca ccatgtaact ctatggacac
caatgcttca 480ccgtagagct ctaaatttcg aggccttcta tatatagctt
tgcgtgacta tgtnaaatta 540ntcaatatcn tnttaatttt tttgnggccc c
57163856DNAGlycine maxmisc_feature(723)..(723)n is a, c, g, or t
63aattagttgt cttgtttatt cattaccttt tcaatttttt taatcaccat aattaaggcc
60tttcgaatcc ctttaagtga taaaagaaac gtgcaattat gcgaacaaat aaattttcgt
120tatgttacta tttagtcaag gaggaaaaaa aagtgataag ggaagaaaca
agggatattt 180cctgttataa caaacttaaa atggcgacta ttttgacgac
attgcaaata ctcatagtac 240gatataaatt ttgaatttaa tatacaatga
ataggcatat tcattttcta ccccaaaaaa 300gcatactcat ttatgtacat
ttaattttct ctccatagag gaattaatgt acaaccatgc 360ataagggatg
agcgaaaggg acagattatt gcaatccaga agcatccaag gaaagttgga
420taaacaaatc aattaatata tataaaaaaa aaacaaaaat gctcctagta
gaagattaaa 480ggaagagttg gctatatatg gcaaaccttt tctaactggt
ttaccctctt ctcatcaccc 540gcattgcatc accaatacgg gaacttttcc
cattacaaaa ctcattggaa gccaacatat 600cccccaaaat tccactggat
ctgcattgtc catgaaattt gacatttctt cttctacaaa 660attcccatgc
tatgtcgttt tccaccatcc taggtcatag tccttcttca ttccccgaat
720cgnttcacac ttgtatgcaa tcttccaccc cagcctcatg ggaaacaccg
ntaacactat 780cactctaata tcattcttgg cataaactca tctataaacc
tctcgnccac gggctcttta 840aattctcatc ttnttn 85664639DNAGlycine
maxmisc_feature(625)..(625)n is a, c, g, or t 64tcccctttgg
gtcccaagta ataggccctc agagccaaaa cattggggtg tctaattttt 60cctagaacac
tgacttctga ttcaaattct ctatgacctt tagtgatctt ttccctcaat
120ctctttactg caacttgact tccatcctcc aaaatagcct tataaacagt
tccataggtg 180ctctttccca tgatctcagc tgttgcacac aagagatcat
cagctgtaaa agccattggt 240ccatcaaaat ggactagttt ccctccagcc
tccccacctg cttcaacatc accaccagca 300actggaggga ctcctttttc
tgtcctcata gtggccgctc taccctcggt ggcttggccg 360tcccggcctt
agatgttgat ctctttctga tcaggcagaa aagcaggaca caacaaagta
420taatcaggac tacgaggaga actcctgcta ctatgagaat tatgtctttg
ggcttagctt 480ctatgatggt gatggtttga cacttcagga ggtggggcaa
tgactccttg tgatggagct 540tgggaaagac atggggttga agggctggac
ccacatagtt gaatatttcc acaaatgagc 600ttgagttaaa attcttggca
agcananggg ggacagaan 63965495DNAGlycine maxmisc_feature(43)..(43)n
is a, c, g, or t 65ttcccaatgc cggagcttcc ctatcgtggg ccccacctat
gangaataca ccctcgaatg 60aaatacatgt tgttgntgnt ggacgcgccc agccgagagt
gccggtccac tagtatcccc 120aacgtgcatg gcgcatgcgc ttgaaaccta
gtattcatct tcctgatgga ggcagccacg 180tgtccgacaa ggtcaatgtt
gccgttttcg tgaaaaggga tgataatgaa aggcaccata 240ttgtcttggg
cgaggttgaa aatggcgtcg tgcatgctct tgtaaggtgc cacgttgatg
300tagggaagaa ccttgactgg cccacttgag ttgttggagt agttttcgaa
ggcttgcatg 360atgtggttgg tgttggggta attcacagac aagaattttc
tgcgaccggg tctatgtttt 420atgggaagga gaatgggtgc acttttccca
cgagctcnat aagggggact gcntanacnc 480atatggggct ctctt
49566480DNAGlycine maxmisc_feature(2)..(2)n is a, c, g, or t
66cnttcttaga atcgaattct ttggtatcag aacatatcag tcatttttaa agaataagaa
60attaaattag acttaatttt taagagtatg gattaaaatg taaaatttgt ggggattata
120aacataaata agtaattttt cctatatgag acatttattg aaatcttaag
ataagatacg 180tacatgcaaa ttaaattgat gcatgataat agaattaggt
gaatagtcca atacctgaca 240cctctttggt ccgaagtttt tggggcactt
cttgatacct aaacccacag tgaagaagag 300gctctggtca atttcagtgg
gtacttcaac ttttctaggg cttctgaagc ttttgctgaa 360ggaagtgact
gcgtttgtgt ccgttgtaag cagggagtgg aggcattata ggtttggttt
420tgttctttac tcctttggca cgatggtgag aatgcttatt gtggtgattc
ggtgatttgt 48067669DNAGlycine maxmisc_feature(486)..(486)n is a, c,
g, or t 67atgcccaaaa aatttaccta aaagcaaata aaaaagatga gtatttcttt
taaattaaaa 60atattttaat aaaaataaaa taaagatcca aatgataatg tgataaccga
agaggaatgt 120ctttcaacca ctgcctgacc gccaccactg ccaacagcct
agtatcaacc gaatccacat 180ataccaacaa tcttcagaca aacacttcta
agttggtgct gaagagacaa tatctcatgg 240gtagatcaaa ttaagagtgc
taccaataac aaaatcggga tcatttgact aacaaacagt 300tatgtgcatt
ggatgttcta ccatagtaca ttgctttatg tgaaattctt ttaattattc
360aatattgaca tgggtcttat atatatatat atatatatat atatatatat
atatatacga 420gggattgtat tatctctgaa aaaagatttt atcataaaat
cataatgatt tctcataatg 480gatctntaca ttttaaaggt agataaataa
aattgatttt aaatngggag atataattaa 540aanacataat taatatgact
tttaacaaat tgatatataa acacttaaaa aaaagntcca 600tgacgcacng
ggggnattgg tgggacaaaa aaaattatct atcactaatt aaaantatta 660taaatatan
66968486DNAGlycine maxmisc_feature(415)..(415)n is a, c, g, or t
68tggtgtatat aattaaaatg agtttaatat ttatgtatta atagtataaa atttatcata
60catgatgaat ggtgaaattt tgaattatga ttaaataatt atataaaaaa atttacatga
120tgaatgaata actttttttt tctcaattaa aattatgatc ctttgtcgat
atgttttact 180gtgtcgacct tttttttcgg gggagagggg accagtagga
gaagtagtat ttagtaaaag 240aagggagaga gaagttgact tatcctttaa
ttagtttaga gaaaattaga cgagaaggaa 300aaaaaatagg cgaaagtcac
tttttctttc tatctctacc aagaatgttg atgaaaaagt 360ggggagcaga
attttaaatt tttattttca tatttatcct tctccacatt tttgntttct
420tccatttttt tataaaanga tttattttag gcatagntaa cttttcaatt
tttttcattt 480ctattc 48669779DNAGlycine
maxmisc_feature(632)..(632)n is a, c, g, or t 69tatttgtaaa
ttgtttttta taattgaaaa gaaaataagg ttaaattatt ttcatataaa 60aaatttaatt
tgttcttata agttattttg aaaattttat taaaataagt tgaaaacaat
120ttataaataa atcataaact ataattttat aagttttctt aaatacttac
acgtatgcca 180taaaataagt tcagataaga tataaataaa ttcctccaaa
cacatcttaa atctatattt 240ttttaaaaca aactttcatc gttaaaagga
tattataata ataataataa acttcaatca 300ttaacaatta atatatgtgg
ataaaagagc attcaaaatg atattttatt agcacatgac 360aaatcacatt
actctcaagc tattttttta aactaataaa aacttacata ttatatgata
420tgatatatac tctctctata tttacacttt tttgagataa acaaggataa
aaaatgatgt 480aaatatgacc gcatataata ttatttataa tgtacggaat
gccgtttttg acattttata 540taatatatct gggggcaatt attttcttaa
ccaataatta gcaaattttt atcttgcttt 600ttctccatgg gggctaaatt
aaactaaagg gncgtaccca atccagtccc actttttttt 660aaataattnn
tttccntccc acttagnaaa ggagtntttn ggcttaaatn ggcagnncca
720ttaaccataa gcctttntgg taaggagtct taccaantaa aatggggaag gcccccccc
77970677DNAGlycine maxmisc_feature(622)..(622)n is a, c, g, or t
70ttattggctt aacttgagtt tcaactcctt ctctggtcct ttaccagcta gcctaactca
60ctcattttct ctcacttttc tttctcttca aaataacaat ctttctggct cccttcctaa
120ctcttggggt gggaattcca agaatggctt ctttaggctt caaaatttga
tcctagatca 180taactttttc actggtgacg ttcctgcttc tttgggtagc
ttaagagagc tcaatgagat 240ttcccttagt cataataagt ttagtggagc
tataccaaat gaaataggaa ccctttctag 300gcttaagaca cttgacattt
ctaataatgc cttgaatggg aacttgcctg ctaccctctc 360taatttatcc
tcacttacac tgctgaatgc agagaacaac ctccttgaca atcaaatccc
420tcaaagttta ggtagattgc gtaatctttc tgttctgatt ttgagtagaa
accaatttag 480tggacatatt ccttcaagca ttgcaaacat ttcctcgctt
aggcagcttg atttgcactg 540aataatttca gtggagaaat tccagctcct
ttgcagtcag cgcagctaaa tctcttcaat 600ggttcctaca atagcctctc
anggtctgtc ccccctctgc ttgccaagaa atttaactca 660agctcatttg tgggaat
67771571DNAGlycine maxmisc_feature(439)..(439)n is a, c, g, or t
71tgggactggc tgtgactgat ctctctggtc taatctcttc cagctgctgg agaacttgat
60gaacttctgg tcgtgctgat ggagaaggat caacacagtg caaagcgagc ttcaacgtgt
120ttagcaactc gtcgccaact gtggatgcat ctctcatcaa gtctgcatca
aaaacctcat 180ttgtccactc ctctttgaca actgaggcaa cccactgagg
caaatctagt ccattcatag 240acaccccagg tgatttcctc gttaggagtt
ctaacaagat aacaccaaga ctgtagatat 300cagttttagt gtttgctttc
ttgagctttg agagctcagg tgcccggtat cccaatgctt 360cagctgtagc
tatcacgttg gaattagcag cagttgacat caaccgagaa agaccaaaat
420ctgcaatttt agcatttgna ttctcattaa acaacacaat gntggatgng
anggtnccat 480ggatgaaggt cttctnggna agnaagnaaa acaaagcacc
gggccaaggn ttgggctaat 540ttcaaccttg gggggcaaac naanaaatgt t
57172756DNAGlycine maxmisc_feature(103)..(103)n is a, c, g, or t
72attggcttaa cttgagtttc aactccttct ctggtccttt accagctagc ctaactcact
60cattttctct cacttttctt tctcttcaaa ataacaatct ttntggctcc cttnctaact
120gtgggggggg gaatancaag ggnggcttta ggctgcaaaa tttgatccta
gatcataact 180ttttcactgg tgacgttcct gcttctttgg gtagcttaag
agagctcaat gagatttccc 240ttagtcataa taagtttagt ggagctatac
caaatgaaat aggaaccctt tctaggctta 300agacacttga catttctaat
aatgccttga atgggaactt gcctgctacc ctctctaatt 360tatcctcact
tacactgctg aatgcagaga acaacctcct tgacaatcaa atccctcaaa
420gtttaggtag attgcgtact ctttcctgtt ccgattttga gtagaaacca
atttagtgga 480catattcctt caagcatngc nnacatttcc tcgcttaggc
agcttgattg tcactgaata 540atttcaggtg gagaaattnc agtctncttt
gacagtcagc gcagtctaaa tcttcttcaa 600tggttnctac aataggcctc
tcagggtctg gccccccttt gnttggccaa ggaaanttaa 660cttaagctta
tttggngggg aaanattcaa ctatgggggg acncggccct ttaaacccca
720gggnttttcc caggttcctt ccaagggngc anttgt 75673557DNAGlycine
maxmisc_feature(233)..(233)n is a, c, g, or t 73tgtgactgat
ctctctggtc taatctcttc cagctgctgg agaacttgat gaacttctgg 60tcgtgctgat
ggagaaggat caacacagtg caaagcgagc ttcaacgtgt ttagcaactc
120gtcgccaact gtggatgcat ctctcatcaa gtctgcatca aaaacctcat
ttgtccactc 180ctctttgaca actgaggcaa cccactgagg caaatctagt
ccattcatag acnccccagg 240tgatttcntc gttaggagtt ntaacaagat
aacaccaaga ctgtagatat cagttttagt 300gtttgctttc ttgagctttg
agagttaagg gncccggant cccanngntc nagttgnagt 360tatancgttg
gaattagcag nagttgcntc aaccgaaaaa gaccaaaatc tgaattttag
420catttgtttt tcatcaagca acacattgnt ggatgngagg tcccatgtat
gatgttctcc 480tgggaatgaa ggcaaacaag cccgggccaa ggcttgggct
attttaatcc ttggtggcca 540aacaatgaaa ggttnat 55774673DNAGlycine
maxmisc_feature(421)..(421)n is a, c, g, or t 74gggactggct
gtgactgatc tctctggtct aatctcttcc agctgctgga gaacttgatg 60aacttctggt
cgtgctgatg gagaaggatc aacacagtgc aaagcgagct tcaacgtgtt
120tagcaactcg tcgccaactg tggatgcatc tctcatcaag tctgcatcaa
aaacctcatt 180tgtccactcc tctttgacaa ctgaggcaac ccactgaggc
aaatctagtc cattcataga 240caccccaggt gatttcctcg ttaggagttc
taacaagata acaccaagac tgtagatatc 300agttttagtg tttgctttct
tgagcttttg agaagctcag gtgcccggta tcccaaatgc 360ttccagctgt
agcttatcac cgttgggaat taagcagcaa gttggacatt caacccggag
420naaaagaccc aaaaattttg caaattttta agcaatttng gnanttcttn
aatcaaggcc 480aaccaccaat tggnttggga atggtggaag ggtttcccca
atggtaattg gaagggtttc 540ttccctnggg gaaaatggaa aggggcaana
aaacaaaggc ccaacngggg ccccaaaggt 600nttttggggg ccttattttt
tncnaatncc ctttggnngg ggncccaaat tcnaaantgg 660aaattggntt tnn
67375602DNAGlycine maxmisc_feature(509)..(509)n is a, c, g, or t
75aggctagctg gtaaaggacc agagaaggat ttgaaactca agttaagcca ataaagctta
60gtggaattag caagactata agggattgct cctgtgagca agttgttgct gaggtcaaga
120gactgaagca aagggcagaa acctaaagaa agaggtatgg aacctgtaag
cctattgttg 180aataactgaa cccctctaag gttgggaaga agtcccaaag
ttgaagggat tgaaccacca 240atttggttat catgaagact aagcttcctg
aggccttgaa gttggccaat tttggcggtg 300attcgacccc tcaaaccctt
ccaaggaagc tggatcacaa taacctgtcc ctgagcacac 360ttgattccaa
cccaacctcc ggaacaagct ccatagccac tggcattcca gctcccgcaa
420gaacccttct ggatcagcca actcttgctt gaaagcttat cacatgtacc
tctctacaga 480taggagggtg cttcttccct ttcactggnc tacctcttcg
ggaataagcc acctaatgag 540aaagaaagan ctgggatagc taactctaca
tagnctcaag gcnagagata attagggaaa 600ng 60276748DNAGlycine
maxmisc_feature(625)..(625)n is a, c, g, or t 76attggcttaa
cttgagtttc aactccttct ctggtccttt accagctagc ctaactcact 60cattttctct
cacttttctt tctcttcaaa ataacaatct ttctggctcc cttcctaact
120cttggggtgg gaattccaag aatggcttct ttaggcttca aaatttgatc
ctagatcata 180actttttcac tggtgacgtt cctgcttctt tgggtagctt
aagagagctc aatgagattt 240cccttagtca taataagttt aatggagctg
taccaaatga aataggaacc ctttctaggc 300ttaagacact tgacatttct
aataatgcct tgaatgggaa cttgcctgct accctctcta 360atttatcctc
acttacactg ctgaatgcag agaacaacct ccttgacaat caaatccctc
420aaagtttagg tagattgcgt aatctttctg ttctgatttt gggtagaaac
caatttagtg 480gacatattcc ttcaagcatt gcaaacattt cctcgcttag
gcagcttgat ttgcactgaa 540taatttcagt ggagaaattc cagtctcctt
tgacagtcaa gcgcaagtct aaatctcttc 600aatgtttcct acaatagcct
ctcanggtct gncccccctc tgcttgccaa gaaatttaac 660tcaagctcat
ttgtgggaaa tattcaacta tgtgggacag nccttcaacc ccatgttttn
720ccaagcttca tacaaggagc atggccct 74877563DNAGlycine max
77ctggctgtga ctgatctctc tggtctaatc tcttccagct gctggagaac ttgatgaact
60tctggtcgtg ctgatggaga aggatcaaca cagtgcaaag cgagcttcaa cgtgtttagc
120aactcgtcgc caactgtgga tgcatctctc atcaagtctg catcaaaaac
ctcatttgtc 180cactcctctt tgacaactga ggcaacccac tgaggcaaat
ctagtccatt catagacacc 240ccaggtgatt tcctcgttag gagttctaac
aagataacac caagactgta gatatcagtt 300ttagtgtttg ctttcttgag
ctttgagagc tcaggtgccc ggtatcccaa tgctccagct 360gtagctatca
cgttggaatt agcagcagtt gacatcaacc cgagaaagac caaaatctgc
420aattttagca tttgtattct catcaagcaa cacattgctg gatgtgaggt
tcccatgtat 480gatgttctcc tgggaatgaa ggcagaacaa gccacggcca
agcttggcta tttcatcctt 540gtggccaatc aatgaatggt cat
56378623DNAGlycine maxmisc_feature(507)..(507)n is a, c, g, or t
78gattttgcac atctacttga gtaggcttca catgattccg tgtattactt ttattttggt
60atatatacca tgtggagtat agtatcactt tttgtcctac aaccacattt tatgagactt
120gcattttatg tgacatgaac ataaaaaata atgaaaaaga aaatgtcaca
tatatatgat 180acaatctttt taaaagtcaa tttgaataat ttttcatcag
gaggaaaaag aagagagaaa 240atgaattaag tttcttctaa aaattaaaat
caacttataa aaagaaaaaa ctttaatgaa 300aaaaattcaa aaagaaaaag
aataaaatga tcaatagcct ttaggtttaa gcacaaggtg 360aatccaaata
aagaccccaa aagatagtac agaacccaac aatggtaaaa tctagaaata
420tacatgtaaa gactgcattt atagaccatc atgactagca aatgcttaaa
ggcacataga 480tgaattaatc tatgcaacaa aatctgnccc aagttttttt
tangcaagga aaatcatatc 540attttattaa ggataactga gaggaccaat
ggtgtaatca attgaaatca tgcgaggctt 600acatgaaatc tgtcaccaag tac
62379605DNAGlycine maxmisc_feature(168)..(168)n is a, c, g, or t
79ctcccgggtc ccaagtaata ggcccctcag agccaaaaca ttgggggggc taatttttcc
60tagaacactg acttctgatt caaattctct atgaccttta gtgatctttt ccctcaatct
120ctttactgca acttgacttc catcctccaa aatagcctta taaacagntc
cataggtgct 180ctttcccatg atctcagctg gtgcacacaa gagatcatca
gctgtaaaag ccattggtcc 240atcaaaatgg actagtttcc ctccagcctc
cccacctgct tcaacatcac caccagcaac 300tggagggact cctttttctg
cctcatagtg gccgctctac cctcggtggc ttggccgntc 360ccggccttag
atgntgatct ctttctgatc aggcagaaaa gcaggacaca acaaagnata
420atcaggacta cgaggagaac tcctgctact atgagaatta tgnctttggg
gcttagcttc 480ctatgatggg gatggttnga cacttcanga gggggggcaa
tgactccctg gganggagct 540tgggaaagac atgggggtga aggnctgnac
ccacataggn gaaaaattcc cacaaangag 600cnngn 60580711DNAGlycine
maxmisc_feature(5)..(5)n is a, c, g, or t 80ttaangncca acgactcact
atagggcgaa ttgggcccga cgtcgcatgc tcccggccgc 60catggccgcg ggattggctt
aacttgagtt tcaactcctt ctctggtcct ttaccagcta 120gcctaactca
ctcattttct ctcacttttc tttctcttcn taaaataaca atctttctgg
180ctcccttcct aactcttggg gtgggaattc caagaatggc ttctttaggc
ttcaaaattt 240gatcctagat cataactttt tcactggtga cgttcctgct
tctttgggta gcttaagaga 300gctcaatgag atttccctta gtcataataa
gtttagtgga gctataccaa atgaaatagg 360aaccctttct aggcttaaga
cacttgacat ttctaataat gccttgaatg ggaacttgcc 420tgctaccctc
tctaatttat cctcacttac actgctgaat gcagagaaca acctccttga
480caatcaaatc cctcaaagtt taggtagatt gcgtaatctt tctgttctga
ttttgagtag 540aaaccaattt agtggacata ttccttcaag cattgcaaac
atttcctcgc ttaggcagct 600tgatttgtca ctgaataatt tcagtggaga
aattccagtc tcctttgaca gtcagcgcag 660tctaaatctc ttcaatgttt
cctacaatag cctctcaggg tctgtccccc n 71181716DNAGlycine
maxmisc_feature(3)..(4)n is a, c, g, or t 81ttnntgaaaa ccctttgcta
tttaggtgac actatagaat actcaagcta tgcatccaac 60gcgttgggag ctctcccata
tggtcgacct gcaggcggcc gcactagtga ttaatacgac 120tcactatagg
gctcgagcgg ccgcccgggc aggtgggact ggctgtgact gatctctctg
180gtctaatctc ttccagctgc tggagaactt gatgaacttc tggtcgtgct
gatggagaag 240gatcaacaca gtgcaaagcg agcttcaacg tgtttagcaa
ctcgtcgcca actgtggatg 300catctctcat caagtctgca tcaaaaacct
catttgtcca ctcctctttg acaactgagg 360caacccactg aggcaaatct
agtccattca tagacacccc aggtgatttc ctcgttagga 420gttctaacaa
gataacacca agactgtaga tatcagtttt agtgtttgct ttcttgagct
480ttgagagctc aggtgcccgg tatcccaatg ctccagctgt agctatcacg
ttggaattag 540cagcagttga catcaaccga gaaagaccaa aatctgcaat
tttagcattt gtattctcat 600caagcaacac attgctggat gtgaggttcc
catgtatgat gttctcctgg gaatgaaggc 660agaacaagcc acgggccaag
tcttgggcta ttttcatcct tggtgggcca atcaan 71682713DNAGlycine
maxmisc_feature(8)..(8)n is a, c, g, or t 82ttcctaangc ctacgactcc
tatagggcga attgggcccg acgtcgcatg ctcccggccg 60ccatggccgc gggattatac
gactcactat agggctcgag cggccactat gaggacagaa 120aaaggagtcc
ctccagttgc tggtggtgat gttgaagcag gtggggaggc tggagggaaa
180ctagtccatt ttgatggacc aatggctttt acagctgatg atctcttgtg
tgcaacagct 240gagatcatgg gaaagagcac ctatggaact gtttataagg
ctattttgga ggatggaagt 300caagttgcag taaagagatt gagggaaaag
atcactaaag gtcatagaga atttgaatca 360gaagtcagtg ttctaggaaa
aattagacac cccaatgttt tggctctgag ggcctattac 420ttgggaccca
aaggggaaaa gcttctggtt tttgattaca tgtctaaagg aagtcttgct
480tctttcctac atggtaagtt tcgtgtgctg ttctttcatt aagtgttgtg
tgtgctgttc 540tttaattata atttggagtt ttaccttagt aatctgtata
attctaatcg gagaacagta 600caaacaaaaa cacctaagga acaacacctt
anctttaata taccatatca ataagtgaat 660tattttctta ttcatcttga
tgcaggtggt ggaactgaaa catttatttg atn 71383712DNAGlycine
maxmisc_feature(1)..(3)n is a, c, g, or t 83nnnctaaggc ccnttactca
ctatngggcg aattgggccc gacgtcgcat gctcccggcc 60gccatggccc gcgggattgg
cttaacttga gtttcaactc cttctctggt cctttaccag 120ctagcctaac
tcactcattt tctctcactt ttctttctct ttaaaataac aatctttctg
180gctcccttcc taactcttgg ggtgggaatt ccaagaatgg cttctttagg
cttcaaaatt 240tgatcctaga tcataacttt ttcactggtg acgttcctgc
ttctttgggt agcttaagag 300agctcaatga gatttccctt agtcataata
agtttagtgg agctatacca aatgaaatag 360gaaccctttc taggcttaag
acacttgaca tttctaataa tgccttgaat gggaacttgc 420ctgctaccct
ctctaattta tcctcactta cactgctgaa tgcagagaac aacctccttg
480acaatcaaat ccctcaaagt ttaggtagat tgcgtaatct ttctgttctg
attttgagta 540gaaaccaatt tagtggacat attccttcaa gcattgcaaa
catttcctcg cttaggcagc 600ttgatttgca ctgaataatt tcagtggaga
aattccagtc tcctttgcag tcagcgcagt 660ctaaatctct tcaatggttn
ctacaatagn ctctcagggt ctgncccccc tn 71284681DNAGlycine
maxmisc_feature(3)..(3)n is a, c, g, or t 84ggnttcttta gggcttcaaa
atttgatcct agatcataac ttttttcact ggtgacgttc 60ctgcttcttt gggtagctta
agagagctca atgagatttc ccttagtcat aataagttta 120gtggagctat
accaaatgaa ataggaaccc tttctaggct taagacactt gacatttcta
180ataatgcctt gaatgggaac ttgcctgcta ccctctctaa tttatcctca
cttacactgc 240tgaatgcaga gaacaacctc cttgacaatc aaatccctca
aagtttaggt agattgcgta 300atctttctgt tctgattttg agtagaaacc
aatttagtgg acatattcct tcaagcattg 360caaacatttc ctcgcttagg
cagcttgatt tgcactgaat aatttcagtg gagaaattcc 420agtctccttt
gacagtcaag cgcagctaaa tctcttcaat ggttcctaca atagcctctc
480agggtctgcc cccctctgct tggcaagaaa tttaactcaa gctcatttgt
gggaaatatt 540caactatgtg gggtacagcc ttcaacccca tggctttcca
agctncatca caagggggca 600ttggccccct cctgagnggc aaacatcacc
atcataggaa gctaacccca aagacataat 660tctcatagta nccaggaggt n
68185639DNAGlycine maxmisc_feature(625)..(625)n is a, c, g, or t
85tcccctttgg gtcccaagta ataggccctc agagccaaaa cattggggtg tctaattttt
60cctagaacac tgacttctga ttcaaattct ctatgacctt tagtgatctt ttccctcaat
120ctctttactg caacttgact tccatcctcc aaaatagcct tataaacagt
tccataggtg 180ctctttccca tgatctcagc tgttgcacac aagagatcat
cagctgtaaa agccattggt 240ccatcaaaat ggactagttt ccctccagcc
tccccacctg cttcaacatc accaccagca 300actggaggga ctcctttttc
tgtcctcata gtggccgctc taccctcggt ggcttggccg 360tcccggcctt
agatgttgat ctctttctga tcaggcagaa aagcaggaca caacaaagta
420taatcaggac tacgaggaga actcctgcta ctatgagaat tatgtctttg
ggcttagctt 480ctatgatggt gatggtttga cacttcagga ggtggggcaa
tgactccttg tgatggagct 540tgggaaagac atggggttga agggctggac
ccacatagtt gaatatttcc acaaatgagc 600ttgagttaaa attcttggca
agcananggg ggacagaan 63986661DNAGlycine
maxmisc_feature(537)..(537)n is a, c, g, or t 86gaaggatggt
tattttgaag agaaagaaaa gtgagagaaa atgagtgagt taggctagct 60ggtaaaggac
cagagaagga gttgaaactc aagttaagcc aataaagctt agtggaatta
120gcaagactat aagggattgc tcctgtgagc aagttgttgc tgaggtcaag
agactgaagc 180aaagggcaga aacctaaaga aagaggtatg gaacctgtaa
gcctattgtt gaataactga 240acccctctaa ggttgggaag aagtcccaaa
gttgaaggga ttgaaccacc aatttggtta 300tcatgaagac taagcttctg
aggccttgaa gttggccaat tttgtcggtg attcgacccc 360tcaaaccctt
ccaaggaagc tggatcacaa taacctgtcc ctgagcacac ttgattccaa
420cccacctccg gaacaagctc catagccact gtcattccag cttccgcaag
aacccttctg 480gatcagccaa ctcttgcttg aaaagcttat cacatgtacc
ttttacagat aggaggntgc 540ttcttccttt cactggtcta cctcttcgga
ataagccaac ctaatgagaa agaaagatct 600gngatagctn acttacatac
tnagncagag ataattantg naagcnnaag ttaaacntnt 660t 66187626DNAGlycine
maxmisc_feature(564)..(564)n is a, c, g, or t 87aattcgtggg
ctacaaagga tgaacgtaaa ctatatgcac ctccagctgg ttcaggcttc 60atatctggct
ttacttctat ctcacgcaga tcttctgttg atagtactca aaatctgtct
120attccttttg gtccaagctc atacctttct gcacaggctc gagtagttga
tgagtattct 180atgtcccaga ttatcttaca aaatgtgctt gatggagggg
tcactggtat gttaatagtt 240gtcactggtg caagccatgt tacatatgga
tctagaggaa ctggagtgcc agcaagaatt 300tcaggaaaaa tacaaaagaa
aaaccatgca gttatattac ttgaccctga aagacaattc 360attcgcagag
aaggagaagt tcctgttgct gattttttgt ggtattctgc tgcgagaccc
420tgtagtagaa attgctttga ccgtgctgag attgctcggg ttatgaatgc
tgctgggcgg 480aggcgagatg ccctcccaca ggtaaaccaa caattacagt
tactaatttg tttgactgtt 540aatcttcttg ccccatagac cctncttcca
atttttagcc ctttatgtcc tctcattcct 600agngggataa gggtttgggg gnggtg
62688627DNAGlycine max 88tgaaaaactg aaggaccaaa ttaaatctaa
aaaataaata aattaaaaga ctaaaaaata 60aatctatcca aaattaaaag gtttattctt
ggaagtaatg aaatgtattt tgactctttg 120aagaatgcat tactataatg
aaagagtagg tggagagagg ggataataaa atcccactaa 180ataacatcca
tgactatcac tataaaaaaa aatattatta ttaagataag aagaattatc
240taacttgaat aagagactac taccaaagtg agaaaaaggt cttataacat
agagtttttc 300aagtttacct ataaaacttg taataagatt tgttttccaa
ccatctaatt ttttattagt 360gtggactgca taaaaaaaat atagtaacaa
gaaactacta aattagactt tttgaactat 420tcattgtatg gctgccatga
aacctacctg cctggagggg tgggtcccac gtaagactgt 480aagagggagg
agggaagcac tagtcacaca ccggcgcacg ttagcgaggc aatgttccta
540gattgaaacg gagaaggtga ttagaggggc ggaaatctca aagcagacac
aggcaactaa 600tttatcgcct ctttcctcat tcgctta 62789782DNAGlycine
maxmisc_feature(703)..(703)n is a, c, g, or t 89cacataatta
acaataaagt catcttctat tatatatttt ttcttcttaa attacatgat 60agtatttcat
cattatttga caataatgat atttttatct cataaatatt attttgtttt
120aaaaatattc atagcacaca cgagtttttt atatcaacaa agaggtatca
cttcagttgg 180tcaatttggt ctaactttta gacaatgtcg tatagttgaa
ttgaattgga atttggcagt 240atatatttta ctttttgccc ccttattttc
aatcaaatta gagtagacgc ctcgtattat 300tggcatacat ggatattgga
tcggcacctg tgtttcagac ctgagtcaca tctgactcgg 360atcgatttta
tcttacatga aaattccaaa ataatgaaag atatggtaat tggcaccatg
420taactctatg gacaccaatg cttcacgtag agctctaaat ttgaggcctt
ctatatatag 480tttgcgtgac tatgtaaatt atcaatatca tttaattttt
ttgcgaccac gaaatatacg 540aatttattat tgaacacaaa aagtagagtg
tatattttaa gtctaggatt ttatgagagg 600caaaaataag aataacctct
tgatatattt tcttggatac actttcttta ttatatattt 660tttaataatg
gattataatt tattggaaac aatcaaatta tangggaaaa ttcattggaa
720taaaagaang aaatttaaaa aaaaatataa tttttaataa atttaagtaa
taaaaatcct 780tt 78290160DNAGlycine max 90tggttgagat gtgtataaga
gacagttgcc ccacctcctg aagtgtcaaa acatcaccat 60cataggaagc taagcaccaa
agacataatt ctcatagtag caggagttct cctcgtagtc 120ctgattatac
tttgttgtgt cctgcttttc tgcctgatca 16091779DNAGlycine
maxmisc_feature(20)..(20)n is a, c, g, or t 91tgctcccggc gcatggccgn
gggattggct taacttgagt ttcaactcct tctctggtcc 60tttaccagct agcctaactc
actcattttc tctcactttt ctttctcttc aaaataacaa 120tctttntggc
tcccttncta actgtggggg ggggaatanc aagggnggct ttaggctgca
180aaatttgatc ctagatcata actttttcac tggtgacgtt cctgcttctt
tgggtagctt 240aagagagctc aatgagattt cccttagtca taataagttt
agtggagcta taccaaatga 300aataggaacc ctttctaggc ttaagacact
tgacatttct aataatgcct tgaatgggaa 360cttgcctgct accctctcta
atttatcctc acttacactg ctgaatgcag agaacaacct 420ccttgacaat
caaatccctc aaagtttagg tagattgcgt actctttcct gttccgattt
480tgagtagaaa ccaatttagt ggacatattc cttcaagcat ngcnnacatt
tcctcgctta 540ggcagcttga ttgtcactga ataatttcag gtggagaaat
tncagtctnc tttgacagtc 600agcgcagtct aaatcttctt caatggttnc
tacaataggc ctctcagggt ctggcccccc 660tttgnttggc caaggaaant
taacttaagc ttatttggng gggaaanatt caactatggg 720gggacncggc
cctttaaacc ccagggnttt tcccaggttc cttccaaggg ngcanttgt
77992743DNAGlycine maxmisc_feature(623)..(623)n is a, c, g, or t
92ttggcttaac ttgagtttca actccttctc tggtccttta ccagctagcc taactcactc
60attttctctc acttttcttt ctcttcaaaa taacaatctt tctggctccc ttcctaactc
120ttggggtggg aattccaaga atggcttctt taggcttcaa aatttgatcc
tagatcataa 180ctttttcact ggtgacgttc ctgcttcttt gggtagctta
agagagctca atgagatttc 240ccttagtcat aataagttta gtggagctat
accaaatgaa ataggaaccc tttctaggct 300taagacactt gacatttcta
ataatgcctt gaatgggaac ttgcctgcta ccctctctaa 360tttatcctca
cttacactgc tgaatgcaga gaacaacctc cttgacaatc aaatccctca
420aagtttaggt agattgcgta atctttctgt tctgattttg agtagaaacc
aatttagtgg 480acatattcct tcaagcattg caaacatttc ctcgcttagg
cagcttgatt tgtcactgaa 540taatttcagt ggagaaattc cagtctcctt
tgacagtcag cgcagtctaa atctcttcaa 600tgtttcctac aatagcctct
cangttctgn cccccctctg cttgccaaga aattaactca 660agctcatttg
tgggaaatat tcaactatgt gggacaggcc ttcaacccca ngctttncca
720agcttcatca caaggggcat tgg 74393742DNAGlycine
maxmisc_feature(619)..(619)n is a, c, g, or t 93ttaacttgag
tttcaactcc ttctctggtc ctttaccagc tagcctaact cactcatttt 60ctctcacttt
tctttctctt caaaataaca atctttctgg ctcccttcct aactcttggg
120gtgggaattc caagaatggc ttctttaggc ttcaaaattt gatcctagat
cataactttt 180tcactggtga cgttcctgct tctttgggta gcttaagaga
gctcaatgag atttccctta 240gtcataataa gtttaatgga gctgtaccaa
atgaaatagg aaccctttct aggcttaaga 300cacttgacat ttctaataat
gccttgaatg ggaacttgcc tgctaccctc tctaatttat 360cctcacttac
actgctgaat gcagagaaca acctccttga caatcaaatc cctcaaagtt
420taggtagatt gcgtaatctt tctgttctga ttttgggtag aaaccaattt
agtggacata 480ttccttcaag cattgcaaac atttcctcgc ttaggcagct
tgatttgcac tgaataattt 540cagtggagaa attccagtct cctttgacag
tcaagcgcaa gtctaaatct cttcaatgtt 600tcctacaata gcctctcang
gtctgncccc cctctgcttg ccaagaaatt taactcaagc 660tcatttgtgg
gaaatattca actatgtggg acagnccttc aaccccatgt tttnccaagc
720ttcatacaag gagcatggcc ct 74294741DNAGlycine
maxmisc_feature(619)..(619)n is a, c, g, or t 94cttaacttga
gtttcaactc cttctctggt cctttaccag ctagcctaac tcactcattt 60tctctcactt
ttctttctct tcaaaataac aatctttctg gctcccttcc taactcttgg
120ggtgggaatt ccaagaatgg cttctttagg cttcaaaatt tgatcctaga
tcataacttt 180ttcactggtg acgttcctgc ttctttgggt agcttaagag
agctcaatga gatttccctt 240agtcataata agtttagtgg agctatacca
aatgaaatag gaaccctttc taggcttaag 300acacttgaca tttctaataa
tgccttgaat gggaacttgc ctgctaccct ctctaattta 360tcctcactta
cactgctgaa tgcagagaac aacctccttg acaatcaaat ccctcaaagt
420ttaggtagat tgcgtaatct ttctgttctg attttgagta gaaaccaatt
tagtggacat 480attccttcaa gcattgcaaa catttcctcg cttaggcagc
ttgatttgca ctgaataatt 540tcagtggaga aattccagtc tcctttgaca
gtcaagcgca gtctaaatct cttcaatgtt 600tcctacaata gcctctcang
ttctgccccc ctctgcttgc caagaaattt aactcaagct 660catttgtggg
aaatattcaa ctatgtggga caggccttca accccatgtt tttccaagct
720ccatcacaag gggcattgcc t 74195743DNAGlycine
maxmisc_feature(556)..(556)n is a, c, g, or t 95cttaacttga
gtttcaactc cttctctggt cctttaccag ctagcctaac tcactcattt 60tctctcactt
ttctttctct tcaaaataac aatctttctg gctcccttcc taactcttgg
120ggtgggaatt ccaagaatgg cttctttagg cttcaaaatt tgatcctaga
tcataacttt 180ttcactggtg acgttcctgc ttctttgggt agcttaagag
agctcaatga gatttccctt 240agtcataata agtttagtgg agctatacca
aatgaaatag gaaccctttc taggcttaag 300acacttgaca tttctaataa
tgccttgaat gggaacttgc ctgctaccct ctctaattta 360tcctcactta
cactgctgaa tgcagagaac aacctccttg acaatcaaat ccctcaaagt
420ttaggtagat tgcgtaatct ttctgttctg attttgagta gaaaccaatt
tagtggacat 480attccttcaa gcattgcaaa catttcctcg cttaggcagc
ttgatttgca ctgaataatt 540tcaaggggag aaattncagt ctcctttgac
agtcaagcgc aagtctaaat ctcttcaatg 600gttcctacaa taagcctctc
anggtctgnc ccccctctgc ttgncaagaa aattaactca 660agctcatttg
ggggaaatat tcaactatgn gggacagncc ttcaacccat gttttccaag
720ctccatacan gagcatggcc cnt 74396742DNAGlycine
maxmisc_feature(621)..(621)n is a, c, g, or t 96cttaacttga
gtttcaactc cttctctggt cctttaccag ctagcctaac tcactcattt 60tctctcactt
ttctttctct tcaaaataac aatctttctg gctcccttcc taactcttgg
120ggtgggaatt ccaagaatgg cttctttagg cttcaaaatt tgatcctaga
tcataacttt 180ttcactggtg acgttcctgc ttctttgggt agcttaagag
agctcaatga gatttccctt 240agtcataata agtttagtgg agctatacca
aatgaaatag gaaccctttc taggcttaag 300acacttgaca tttctaataa
tgccttgaat gggaacttgc ctgctaccct ctctaattta 360tcctcactta
cactgctgaa tgcagagaac aacctccttg acaatcaaat ccctcaaagt
420ttaggtagat tgcgtaatct ttctgttctg attttgagta gaaaccaatt
tagtggacat 480attccttcaa gcattgcaaa catttcctcg cttaggcagc
ttgatttgtc actgaataat 540ttcaggggga gaaattccag tctcctttga
cagtcagcgc aagtctaaat ctcttcaatg 600gttcctacaa tagcctctca
nggtctgncc cccctctgct tgncaagaaa ttaactcaag 660ctcatttgtg
ggaaatattc aactatgngg gacaggcctt caacccatgt ttttccaagc
720ttcatacaag gagtaatggc ct 74297716DNAGlycine
maxmisc_feature(399)..(399)n is a, c, g, or t 97ggacagaaaa
aggagtccct ccagttgctg gtggtgatgt tgaagcaggt ggggaggctg 60gagggaaact
agtccatttt gatggaccaa tggcttttac agctgatgat ctcttgtgtg
120caacagctga gatcatggga aagagcacct atggaactgt ttataaggct
attttggagg 180atggaagtca agttgcagta aagagattga gggaaaagat
cactaaaggt catagagaat 240ttgaatcaga agtcagtgtt ctaggaaaaa
ttagacaccc caatgttttg gctctgaggg 300cctattactt gggacccaaa
ggggaaaagc ttctggtttt tgattacatg tctaaaggaa 360gtcttgcttc
tttcctacat ggtaagtttc gtgtgctgnt ctttcattaa agtgntgggn
420gggctggtct ttaattataa tttggagttt taccttanta atctgtataa
ttctaatcgg 480agacaagtca aacaaaaacc ctaaggaaca acnccttanc
tttaatatnc catatcaata 540angngaatta ttttnttggt tcatttgatg
cnngggggng gnacntnaaa cnttnatttg 600ntgggccacn anggnnnnaa
aannncacaa ananttggnc cngnggnttn gnnntgcctt 660tantnccang
anaaacatna tacanggnan ctnncntcnn naangtnntn gttngn
71698616DNAGlycine maxmisc_feature(447)..(447)n is a, c, g, or t
98ggacagaaaa aggagtccct ccagttgctg gtggtgatgt tgaagcaggt ggggaggctg
60gagggaaact agtccatttt gatggaccaa tggcttttac agctgatgat ctcttgtgtg
120caacagctga gatcatggga aagagcacct atggaactgt ttataaggct
attttggagg 180atggaagtca agttgcagta aagagattga gggaaaagat
cactaaaggt catagagaat 240ttgaatcaga agtcagtgtt ctaggaaaaa
ttagacaccc caatgttttg gctctgaggg 300cctattactt gggacccaaa
ggggaaaagc ttctggtttt tgattacatg tctaaaggaa 360gtcttgcttc
tttcctacat ggtaagtttc gtgtgctgtt ctttcattaa gtgttgtgtg
420tgctgttctt taattataat ttggagnttt accttagtaa tctgtataat
tctaatcgga 480gaacagtcaa acaaaacacc taaggaacaa caccttagct
ttaatatcca tatcaataag 540tgaatatttt cttggtcatc ttgatgcagg
nggnggaact tgaacaatca ttgattggnc 600caccanggat gaaaat
61699532DNAGlycine max 99actggctgtg actgatctct ctggtctaat
ctcttccagc tgctggagaa cttgatgaac 60ttctggtcgt gctgatggag aaggatcaac
acagtgcaaa gcgagcttca acgtgtttag 120caactcgtcg ccaactgtgg
atgcatctct catcaagtct gcatcaaaaa cctcatttgt 180ccactcctct
ttgacaactg aggcaaccca ctgaggcaaa tctagtccat tcatagacac
240cccaggtgat ttcctcgtta ggagttctaa caagataaca ccaagactgt
agatatcagt 300tttagtgttt gctttcttga gctttgagag ctcaggtgcc
cggtatccca atgctccagc 360tgtagctatc acgttggaat tagcagcagt
tgacatcaac cgagaaagac caaaatctgc 420aattttagca tttgtattct
catcaagcaa cacattgctg gatgtgaggt tcccatgtat 480gatgttctcc
tgggaatgaa ggcggaacaa gccacgggcc aagtcttgtg ct 532100568DNAGlycine
maxmisc_feature(15)..(15)n is a, c, g, or t 100tatgaggaca
gaaanttnag tccctccagt tgctggtggt gatgttgaag caggtgggga 60ggctggaggg
aaactagtcc attttgatgg accaatggct tttacagctg atgatctctt
120gtgtgcaaca gctgagatca tgggaaagag cacctatgga actgtttata
aggctatttt 180ggaggatgga agtcaagttg cagtaaagag attgagggaa
aagatcacta aaggtcatag 240agaatttgaa tcagaagtca gtgttctagg
aaaaattaga caccccaatg ttttggctct 300gagggcctat tacttgggac
ccaaagggga aaagcttctg gtttttgatt acatgtctaa 360aggaagtctt
gcttctttcc tacatggtaa gtttcgtgtg ctgttctttc attaagtgtt
420gtgtgtgctg ttctttaatt ataatttgga gttttacctt agtaatctgt
ataattctaa 480tcggagaaca gtcaaacaaa aaccctaagg aacacacctt
actttaatat accatatcaa 540taagngaatn atttcttggt catcttga
568101678DNAGlycine maxmisc_feature(535)..(535)n is a, c, g, or t
101ggtgggactg gctgtgactg atctctctgg tctaatctct tccagctgct
ggagaacttg 60atgaacttct ggtcgtgctg atggagaagg atcaacacag tgcaaagcga
gcttcaacgt 120gtttagcaac tcgtcgccaa ctgtggatgc atctctcatc
aagtctgcat caaaaacctc 180atttgtccac tcctctttga caactgaggc
aacccactga ggcaaatcta gtccattcat 240agacacccca ggtgatttcc
tcgttaggag ttctaacaag ataacaccaa gactgtagat 300atcagtttta
gtgtttgctt tcttgagctt tgagagctca ggtgcccggt atcccaatgc
360tccagctgta gctatcacgt tggaattagc agcagttgac atcaaccgag
aaagaccaaa 420atctgcaatt ttagcatttg tattctcatc aagcaacaca
ttgctggatg tgaggttccc 480atgtatgatg ttctcctggg aatgaaggca
gaacaagcca cgggccaagt cttgngctat 540tttcatcctt ggtggccaat
caatgaatgg ttcagttnca ccacctgcat caagatgaac
600aagaaaataa ttcacttatt gatatggnat attaaaagct aaggggtggt
ccctaggggg 660tttggttgga ccggncnn 678102673DNAGlycine
maxmisc_feature(534)..(534)n is a, c, g, or t 102ggtgggactg
gctgtgactg atctctctgg tctaatctct tccagctgct ggagaacttg 60atgaacttct
ggtcgtgctg atggagaagg atcaacacag tgcaaagcga gcttcaacgt
120gtttagcaac tcgtcgccaa ctgtggatgc atctctcatc aagtctgcat
caaaaacctc 180atttgtccac tcctctttga caactgaggc aacccactga
ggcaaatcta gtccattcat 240agacacccca ggtgatttcc tcgttaggag
ttctaacaag ataacaccaa gactgtagat 300atcagtttta gtgtttgctt
tcttgagctt tgagagctca ggtgcccggt atcccaatgc 360tccagctgta
gctatcacgt tggaattagc agcagttgac atcaaccgag aaagaccaaa
420atctgcaatt ttagcatttg tattctcatc aagcaacaca ttgctggatg
tgagggtccc 480atgtatgatg ttctcctggg aatgaaggca gaacaagcca
cggccaagtc ttgngctatt 540ttcatccttg ttggccaatc aatgaatggt
tcaagttccc cacctgcatc aagatgaaca 600agaaaataat tcacttaatg
gatatggnat attaaagcta aggggtggtc cntaggggtt 660ttgggttgnc cng
673103665DNAGlycine maxmisc_feature(494)..(494)n is a, c, g, or t
103ggtgggactg gctgtgactg atctctctgg tctaatctct tccagctgct
ggagaacttg 60atgaacttct ggtcgtgctg atggagaagg atcaacacag tgcaaagcga
gcttcaacgt 120gtttagcaac tcgtcgccaa ctgtggatgc atctctcatc
aagtctgcat caaaaacctc 180atttgtccac tcctctttga caactgaggc
aacccactga ggcaaatcta gtccattcat 240agacacccca ggtgatttcc
tcgttaggag ttctaacaag ataacaccaa gactgtagat 300atcagtttta
gtgtttgctt tcttgagctt tgagagctca ggtgcccggt atcccaatgc
360tccagctgta gctatcacgt tggaattagc agcagttgac atcaaccgag
aaagaccaaa 420atctgcaatt ttagcatttg tattctcatc aagcaacaca
ttgctggatg tgagggtccc 480atgtatgatg tctnctggga atgaaggcan
aacaagccac ggccaagtct tgggctattt 540tcatccttgt ggncaatcaa
tgaatggtta anttcccccc ctgcttcaag atgaacaaga 600aaataattca
cttattggtt gggntatnaa actaaggggn gnccctaggg gnttngntgn 660ccnct
665104671DNAGlycine maxmisc_feature(534)..(534)n is a, c, g, or t
104ggtgggactg gctgtgactg atctctctgg tctaatctct tccagctgct
ggagaacttg 60atgaacttct ggtcgtgctg atggagaagg atcaacacag tgcaaagcga
gcttcaacgt 120gtttagcaac tcgtcgccaa ctgtggatgc atctctcatc
aagtctgcat caaaaacctc 180atttgtccac tcctctttga caactgaggc
aacccactga ggcaaatcta gtccattcat 240agacacccca ggtgatttcc
tcgttaggag ttctaacaag ataacaccaa gactgtagat 300atcagtttta
gtgtttgctt tcttgagctt tgagagctca ggtgcccggt atcccaatgc
360tccagctgta gctatcacgt tggaattagc agcagttgac atcaaccgag
aaagaccaaa 420atctgcaatt ttagcatttg tattctcatc aagcaacaca
ttgctggatg tgaggttccc 480atgtatgatg ttctcctggg aatgaaggca
gaacaagcca cggccaagtc ttgngctatt 540ttcatccttg gtggccaatc
aatgaatgtt tcagttccac cacctgcatc aagatgaaca 600agaaaataat
tcacttattg atatggnata ttaaagctaa ggggtggtcc ntagggggtt
660tngntggncc c 671105670DNAGlycine maxmisc_feature(443)..(443)n is
a, c, g, or t 105ggtgggactg gctgtgactg atctctctgg tctaatctct
tccagctgct ggagaacttg 60atgaacttct ggtcgtgctg atggagaagg atcaacacag
tgcaaagcga gcttcaacgt 120gtttagcaac tcgtcgccaa ctgtggatgc
atctctcatc aagtctgcat caaaaacctc 180atttgtccac tcctctttga
caactgaggc aacccactga ggcaaatcta gtccattcat 240agacacccca
ggtgatttcc tcgttaggag ttctaacaag ataacaccaa gactgtagat
300atcagtttta gtgtttgctt tcttgagctt tgagagctca ggtgcccggt
atcccaatgc 360tccagctgta gctatcacgt tggaattagc agcagttgac
atcaaccgag aaagaccaaa 420atctgcaatt ttagcatttg tantctcatc
aagcaacaca ttgctggatg tgagggtccc 480atgtatgatg tcctcctggg
aatgaaggca gaacaagcca cgggccaagt cttgggctat 540tttcatcctt
ggtgggccaa tcaatgaatg gttcaanttc ancacctgcn tcaagangaa
600caagaaaata attncntatg gnnnggatat naaactaagg ggnggnccta
ggggtntngn 660nngnccggcn 670106662DNAGlycine
maxmisc_feature(494)..(494)n is a, c, g, or t 106ggtgggactg
gctgtgactg atctctctgg tctaatctct tccagctgct ggagaacttg 60atgaacttct
ggtcgtgctg atggagaagg atcaacacag tgcaaagcga gcttcaacgt
120gtttagcaac tcgtcgccaa ctgtggatgc atctctcatc aagtctgcat
caaaaacctc 180atttgtccac tcctctttga caactgaggc aacccactga
ggcaaatcta gtccattcat 240agacacccca ggtgatttcc tcgttaggag
ttctaacaag ataacaccaa gactgtagat 300atcagtttta gtgtttgctt
tcttgagctt tgagagctca ggtgcccggt atcccaatgc 360tccagctgta
gctatcacgt tggaattagc agcagttgac atcaaccgag aaagaccaaa
420atctgcaatt ttagcatttg tattctcatc aagcaacaca ttgctggatg
tgaggttcca 480tgtatgatgt tctnctggga atgaaggcag aacaagccac
gggccaagtc ttgngctatt 540tcatccttgt gggcaatcaa tgaatgttta
anttccncac ctgcttnaga ggaccaagaa 600aanattactt attggntggg
tattaaagct aagggggggn cctaaggggn tttggnnggc 660cc
662107792DNAGlycine maxmisc_feature(258)..(258)n is a, c, g, or t
107tatttacaac tagtgttatc ggagaatgaa aaattgaaga ataataagtt
cagctataat 60aaactcgagg gaggaaaaac aaagaaattc atgataaata gatataactt
attaaattta 120aggggtgtat ttgcacaccc tgaattatag agattcttat
atctttgaga aaataattaa 180attgggaaaa aagagataat gactgattga
gatttgcctc agaattgttc gttttaatat 240tggtacgaat ctaatggntt
tatcctgaaa gatgctcaca agtattgagg gactaataaa 300ttgnttataa
actactacta aatgagatga gactttaagg ngtactgaag caatatcatt
360taaaaaatga ctactcgcat ttgngttgag aaaatttatt ttcatgaaag
naaattttnt 420ccnttttang ataaagccat ttnncttaac cnnangggga
nataaaatgg cccccnttca 480taaaaaacct accanctata taaatggatn
tataccaacc ttcctangca ccatgccatt 540gggatnggng gaattaaatt
naaaangntt gcnttggaat gggtaaaaaa ttccaaaact 600tnaacccccn
ccacaatttt agtggccacn gnaatattnn ttanccgntg gncttttttc
660caggaaaacg acccgtaacc aaanggggnn aaaagggaaa gggagatgga
ttgcntgnng 720gtntgaggct catcccnatt cccaaacatg ttngggnccc
aaaaccgaag tncccctgga 780ccatggatgn cn 792108573DNAGlycine
maxmisc_feature(432)..(432)n is a, c, g, or t 108gggactggct
gtgactgatc tctctggtct aatctcttcc agctgctgga gaacttgatg 60aacttctggt
cgtgctgatg gagaaggatc aacacagtgc aaagcgagct tcaacgtgtt
120tagcaactcg tcgccaactg tggatgcatc tctcatcaag tctgcatcaa
aaacctcatt 180tgtccactcc tctttgacaa ctgaggcaac ccactgaggc
aaatctagtc cattcataga 240caccccaggt gatttcctcg ttaggagttc
taacaagata acaccaagac tgtagatatc 300agttttagtg tttgctttct
tgagctttga gagctcaggt gcccggtatc caatgctcca 360gctgtagcta
tcacgttgga attagcagca gttgacatca acccgagaaa gaccaaaatt
420gcaatttagc anttgnattc ttatnaacaa cacaatggtt ggatgngang
gtnccaagga 480ttgangtttt ctgggaatga aaggganaaa caagccccgg
gccaaagntt ggggttattt 540tnaancctgg ngggncaaan aaangaaagg ttn
573109673DNAGlycine maxmisc_feature(421)..(421)n is a, c, g, or t
109gggactggct gtgactgatc tctctggtct aatctcttcc agctgctgga
gaacttgatg 60aacttctggt cgtgctgatg gagaaggatc aacacagtgc aaagcgagct
tcaacgtgtt 120tagcaactcg tcgccaactg tggatgcatc tctcatcaag
tctgcatcaa aaacctcatt 180tgtccactcc tctttgacaa ctgaggcaac
ccactgaggc aaatctagtc cattcataga 240caccccaggt gatttcctcg
ttaggagttc taacaagata acaccaagac tgtagatatc 300agttttagtg
tttgctttct tgagcttttg agaagctcag gtgcccggta tcccaaatgc
360ttccagctgt agcttatcac cgttgggaat taagcagcaa gttggacatt
caacccggag 420naaaagaccc aaaaattttg caaattttta agcaatttng
gnanttcttn aatcaaggcc 480aaccaccaat tggnttggga atggtggaag
ggtttcccca atggtaattg gaagggtttc 540ttccctnggg gaaaatggaa
aggggcaana aaacaaaggc ccaacngggg ccccaaaggt 600nttttggggg
ccttattttt tncnaatncc ctttggnngg ggncccaaat tcnaaantgg
660aaattggntt tnn 673110564DNAGlycine max 110actggctgtg actgatctct
ctggtctaat ctcttccagc tgctggagaa cttgatgaac 60ttctggtcgt gctgatggag
aaggatcaac acagtgcaaa gcgagcttca acgtgtttag 120caactcgtcg
ccaactgtgg atgcatctct catcaagtct gcatcaaaaa cctcatttgt
180ccactcctct ttgacaactg aggcaaccca ctgaggcaaa tctagtccat
tcatagacac 240cccaggtgat ttcctcgtta ggagttctaa caagataaca
ccaagactgt agatatcagt 300tttagtgttt gctttcttga gctttgagag
ctcaggtgcc cggtatccca atgctccagc 360tgtagctatc acgttggaat
tagcagcagt tgacatcaac ccgagaaaga ccaaaatctg 420caattttagc
atttgtattc tcatcaagca acacattgct ggatgtgagg ttcccatgta
480tgatgttctc ctgggaatga aggcagaaca agccacggcc aagcttggct
atttcatcct 540tgtggccaat caatgaatgg tcat 564111456DNAGlycine
maxmisc_feature(256)..(256)n is a, c, g, or t 111actatgagga
cagaaaaagg agtccctcca gttgctggtg gtgatgttga agcgggtggg 60gaggctggag
ggaaactagt ccattttgat ggaccaatgg cttttacagc tgatgatctc
120ttgtgtgcaa cagctgagat catgggaaag agcacctatg gaactgttta
taaggctatt 180ttggaggatg gaagtcaagt tgcagtaaag agattgaggg
aaaagatcac taaaggtcat 240agagaatttg aatcanaagt cagtgttcta
ggaaaaatta nacaccccaa tgttttggtt 300ntgaggccta ttacttggga
cccaaagggg aaaagcttnt ggtttttgat tcatgtntaa 360aggaagtctt
gcttntttcc tacatggnaa gtttcggggc tgtctttnat taanggtngg
420gngngctgnn tttaattata attnggngtt tacctt 456112592DNAGlycine
maxmisc_feature(463)..(464)n is a, c, g, or t 112actatgagga
cagaaaaagg agtccctcca gttgctggtg gtgatgttga agcaggtggg 60gaggctggag
ggaaactagt ccattttgat ggaccaatgg cttttacagc tgatgatctc
120ttgtgtgcaa cagctgagat catgggaaag agcacctatg gaactgttta
taaggctatt 180ttggaggatg gaagtcaagt tgcagtaaag agattgaggg
aaaagatcac taaaggtcat 240agagaatttg aatcagaagt cagtgttcta
ggaaaaatta gacaccccaa tgttttggct 300ctgagggcct attacttggg
acccaaaggg gaaaagcttc tggtttttga ttacatgtct 360aaaggaagtc
ttgcttcttt cctacatggt aagtttcgtg tgctgttctt tcattaagtg
420ttgggtgtgc tggtctttaa ttataatttg gagtttacct tannaatctg
gataattcta 480atcggagaac agncaaacaa aanccctaag gaacaaccct
tanctttaat atccatatca 540ataagngaan tatttcttgg tcatcttgat
gcaggggggg gnactgaaca tt 592113460DNAGlycine
maxmisc_feature(438)..(438)n is a, c, g, or t 113gggactggct
gtgactgatc tctctggtct aatctcttcc agctgctgga gaacttgatg 60aacttctggt
cgtgctgatg gagaaggatc aacacagtgc aaagcgagct tcaacgtgtt
120tagcaactcg tcgccaactg tggatgcatc tctcatcaag tctgcatcaa
aaacctcatt 180tgtccactcc tctttgacaa ctgaggcaac ccactgaggc
aaatctagtc cattcataga 240caccccaggt gatttcctcg ttaggagttc
taacaagata acaccaagac tgtagatatc 300agttttagtg tttgctttct
tgagctttga gagctcaggt gcccggtatc ccaatgcttc 360agctgtagct
atcacgttgg aattagcagc agttgacatc aaccgagaaa gaccaaaatc
420tgcaatttta gcatttgnat tctcattaaa caacacaatg 460114566DNAGlycine
maxmisc_feature(242)..(242)n is a, c, g, or t 114gggactggct
gtgactgatc tctctggtct aatctcttcc agctgctgga gaacttgatg 60aacttctggt
cgtgctgatg gagaaggatc aacacagtgc aaagcgagct tcaacgtgtt
120tagcaactcg tcgccaactg tggatgcatc tctcatcaag tctgcatcaa
aaacctcatt 180tgtccactcc tctttgacaa ctgaggcaac ccactgaggc
aaatctagtc cattcataga 240cnccccaggt gatttcntcg ttaggagttn
taacaagata acaccaagac tgtagatatc 300agttttagtg tttgctttct
tgagctttga gagttaaggg ncccggantc ccanngntcn 360agttgnagtt
atancgttgg aattagcagn agttgcntca accgaaaaag accaaaatct
420gaattttagc atttgttttt catcaagcaa cacattgntg gatgngaggt
cccatgtatg 480atgttctcct gggaatgaag gcaaacaagc ccgggccaag
gcttgggcta ttttaatcct 540tggtggccaa acaatgaaag gttnat
56611516DNAGlycine max 115gactgcgtac caattc 1611616DNAGlycine max
116gatgagtcct gagtaa 1611722DNAGlycine max 117gggtttcaga taaccgtggt
cg 2211825DNAGlycine max 118ttgcagatat tttagttgat tggcc
2511924DNAGlycine max 119agttgattgg ctcaaaccat ggcc
2412020DNAGlycine max 120ttgcgtgtga tcggtattac 2012120DNAGlycine
max 121tacctgagtt ctctcaagtc 20122252DNAGlycine
maxmisc_feature(20)..(20)n is a, c, g, or t 122gatttagact
gcgctgactn tcaaaggaga ctggaatttc tccactgaaa ttattcagtg 60acaaatcaag
ctgcctaagc gaggaaatgt ttgcaatgct tgaaggaata tgtccactaa
120attggtttct actcaaaatc agaacagaaa gattacgcaa tctacctaaa
ctttgaggga 180tttgattgtc aaggaggttg ttctctgcat tcagcagtgt
aagtgaggat aaattagaga 240gggtagcagg ca 252123199DNAGlycine max
123ttatcatcca aattaaaatt gaaaacttta atacaaatgc acattttgga
gccattcatg 60tcatctcttg gtctgagtct tatcattctg tggattgaat tcatggtttc
tcttatgaca 120ttgttgccaa gtaatactac tatataaatt cagatttggg
tttctgataa ccgtggtcgt 180taatactata tataatacc 199124213DNAGlycine
max 124ttatcatcca aattaaaatt gaaaacttta atacaaatgc acattttgga
gccattcatg 60tcatctcttg gtctgagtct tatcattctg tggattgaat tcatggtttc
tcttatctta 120tgaattcatg gtttctctta tcttatgaca ttgttgccaa
gtaatactac tatataaatt 180cagatttggg tttcagataa ccgtggtcgt taa
213125133DNAGlycine max 125ttaaagggat atgttttttt cactaatgct
gtaaaaattc acccagattt ttgcattttc 60tttgaaaaaa tgttagatat atcatgtttt
tttacaagca ttacaataat attcactcgt 120atattaggaa ttc
133126113DNAGlycine max 126ttaaagggat atgttttttt cactaatgtc
gtaaaaattc accccaaatt tttgcatttt 60atcatgtttt tttacaagca ttacaataat
attcactcgt atattaggaa ttc 113127397DNAGlycine max 127ttaaaacctt
gcgtgtgatc ggtattacag tacgcagggc caatcaacta aaatatctgc 60aaacgataat
ataattataa gaaaaagaca cactttgagg gcatttttga cttgagagaa
120ctcaggtatc aatctaaaag caacgctgtt caccttgagc tgaaacacct
ggaggagaaa 180gcaaagcaaa ccaaacgcga gagagaaata aagaacggaa
acagagagag agagaggaag 240gaccttgttc aaagcaacgg ggacaacttt
agagccctgg cgcgcgtggg ggtcaataag 300cgtaacctgg ctgaggagag
cctcggcgtc gtccttgctg aagcagaaga ggaagagcac 360gagaccaaga
gaaactcctc ggaagcaacg ggaattc 397128405DNAGlycine max 128ttaaaacctt
gcgtgtgatc ggtattacag tacgcagggc catggtttga gccaatcaac 60taaaatattt
gcaaacgata atataattat aagaaaaaga ctcactttga gggcattttt
120gacttgagag aactcaggta tcaatctaaa agcaacgctg ttcaccttga
gctgaaacac 180ctggaggaga aagcaaagca aaccaaacgc gagagagaaa
taaagaacgg aaacagagag 240agaggaagga ccttgttcaa agcaacgggg
acaactttag agccctggcg cgcgtggggg 300tcaataagcg taacctggct
gaggagagcc tcggcgccgt ccttgctgaa gcagaagagg 360aagagcccga
gaccaagaga aactcctcgg aagcaacggg aattc 405129161DNAGlycine max
129ttaaatgaaa atcgatcaaa atgaaataat atatgctttt tttagttggg
ttcaagtact 60tttttttatt gaaaaaatcg acccaagttg aaacacatgt ttgagaattg
ttttgtgcat 120ccaacgtttt tcttgtacaa tcagctgtga gaggggaatt c
161130162DNAGlycine max 130ttaaatgaaa atcgatcaaa atgaaataat
atatgctttt tttagttgtg ttcaagtaac 60ttttttttat tgaaaaaatc gacccaagtt
gaaacacatg tttgagaatt gttttgtgca 120tccaacgttt ttcttgtaca
atcagctgtg agaggggaat tc 16213118DNAGlycine max 131agggatatgt
ttttttca 1813218DNAGlycine max 132gaattcctaa tatacgag
1813321DNAGlycine max 133atctcttggt ctgagtctta t 2113425DNAGlycine
max 134tggtttctct tatgacattg ttgcc 2513526DNAGlycine max
135ttctcttatc ttatgacatt gttgcc 2613620DNAGlycine max 136tattaacgac
cacggttatc 20
* * * * *
References