U.S. patent application number 17/531046 was filed with the patent office on 2022-03-03 for markers associated with soybean rust resistance and methods of use therefor.
This patent application is currently assigned to Syngenta Participations AG. The applicant listed for this patent is Syngenta Participations AG. Invention is credited to Glenn R. Bowers, Becky Welsh Breitinger, Nanda Chakraborty, Ju-Kyung Yu.
Application Number | 20220061244 17/531046 |
Document ID | / |
Family ID | 1000005970888 |
Filed Date | 2022-03-03 |
United States Patent
Application |
20220061244 |
Kind Code |
A1 |
Bowers; Glenn R. ; et
al. |
March 3, 2022 |
MARKERS ASSOCIATED WITH SOYBEAN RUST RESISTANCE AND METHODS OF USE
THEREFOR
Abstract
Methods for conveying soybean rust (SBR) resistance into
non-resistant soybean germplasm are provided. In some embodiments,
the methods include introgressing SBR resistance into a
non-resistant soybean using one or more nucleic acid markers for
marker-assisted breeding among soybean lines to be used in a
soybean breeding program, wherein the markers are linked to and/or
associated with SBR resistance. Also provided are single nucleotide
polymorphisms (SNPs) associated with resistance to SBR; soybean
plants, seeds, and tissue cultures produced by any of the disclosed
methods; seed produced by the disclosed soybean plants; and
compositions including amplification primer pairs capable of
initiating DNA polymerization by a DNA polymerase on soybean
nucleic acid templates to generate soybean marker amplicons. 15
Inventors: |
Bowers; Glenn R.; (Bay,
AR) ; Yu; Ju-Kyung; (Slater, IA) ; Breitinger;
Becky Welsh; (Research Triangle Park, NC) ;
Chakraborty; Nanda; (Research Triangle Park, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Syngenta Participations AG |
Basel |
|
CH |
|
|
Assignee: |
Syngenta Participations AG
Basel
CH
|
Family ID: |
1000005970888 |
Appl. No.: |
17/531046 |
Filed: |
November 19, 2021 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
16838456 |
Apr 2, 2020 |
11206776 |
|
|
17531046 |
|
|
|
|
16053639 |
Aug 2, 2018 |
10624284 |
|
|
16838456 |
|
|
|
|
14872443 |
Oct 1, 2015 |
10070602 |
|
|
16053639 |
|
|
|
|
12690782 |
Jan 20, 2010 |
|
|
|
14872443 |
|
|
|
|
PCT/US2009/051003 |
Jul 17, 2009 |
|
|
|
12690782 |
|
|
|
|
61153495 |
Feb 18, 2009 |
|
|
|
61081989 |
Jul 18, 2008 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A01H 1/04 20130101; Y10T
436/143333 20150115; C12Q 2600/13 20130101; C12N 15/8282 20130101;
C12Q 1/6895 20130101; A01H 6/542 20180501; C12Q 2600/156 20130101;
A01H 5/10 20130101 |
International
Class: |
A01H 1/04 20060101
A01H001/04; A01H 5/10 20060101 A01H005/10; C12N 15/82 20060101
C12N015/82; C12Q 1/6895 20060101 C12Q001/6895; A01H 6/54 20060101
A01H006/54 |
Claims
1. A method for conveying resistance to soybean rust (SBR) into
non-resistant soybean germplasm comprising introgressing SBR
resistance into a non-resistant soybean using one or more nucleic
acid markers for marker-assisted breeding among soybean lines to be
used in a soybean breeding program, wherein the markers are linked
to an SBR resistance locus selected from the group consisting of
Rpp1, Rpp2, Rpp3, Rpp4, and Rpp5.
2. The method of claim 1, wherein the one or more nucleic acid
markers are selected from the group consisting of SEQ ID NOs: 1-13,
and informative fragments thereof.
3. The method of claim 1, wherein the marker-assisted breeding
comprises single nucleotide polymorphism (SNP) analysis.
4. The method of claim 1, further comprising screening an
introgressed soybean plant, or a cell or tissue thereof, for SBR
resistance.
5. A method for reliably and predictably introgressing soybean rust
(SBR) resistance into non-resistant soybean germplasm comprising
using one or more nucleic acid markers for marker-assisted breeding
among soybean lines to be used in a soybean breeding program,
wherein the nucleic acid markers are selected from the group
consisting of SEQ ID NOs: 1-13, and informative fragments thereof,
and introgressing the resistance into the non-resistant soybean
germplasm.
6. The method of claim 5, wherein the marker-assisted breeding
comprises single nucleotide polymorphism (SNP) analysis.
7. The method of claim 5, further comprising screening an
introgressed soybean for SBR resistance.
8. A method for producing an inbred soybean plant adapted for
conferring, in hybrid combination with a suitable second inbred,
resistance to soybean rust (SBR), the method comprising: (a)
selecting a first donor parental line possessing a desired SBR
resistance and having at least one of the resistant loci selected
from a locus mapping to Rppl and mapped by one or more of the
markers SEQ ID NOs: 1-3; a locus mapping to Rpp2 and mapped by one
or more of the markers SEQ ID NOs: 4-6; a locus mapping to Rpp3 and
mapped by one or more of the markers SEQ ID NOs: 7 and 8; a locus
mapping to Rpp4 and mapped by one or more of the markers SEQ ID
NOs: 9 and 10; and a locus mapping to Rpp5 and mapped by one or
more of the markers SEQ ID NOs: 11-13; (b) crossing the first donor
parent line with a second parental line in hybrid combination to
produce a segregating plant population; (c) screening the
segregating plant population for identified chromosomal loci of one
or more genes associated with the resistance to SBR; and (d)
selecting plants from the population having the identified
chromosomal loci for further screening until a line is obtained
which is homozygous for resistance to SBR at sufficient loci to
give resistance to SBR in hybrid combination.
9. A method for selecting an soybean rust (SBR) resistant soybean
plant, the method comprising: (a) genotyping one or more soybean
plants with respect to one or more single nucleotide polymorphisms
(SNPs), wherein the one or more SNPs correspond to one or more
molecular markers selected from the group consisting of SEQ ID NOs:
1-13, and informative fragments thereof; and (b) selecting a
soybean plant that includes at least one resistance allele
associated with the SNPs, thereby selecting an SBR resistant
soybean plant.
10. The method of claim 9, wherein the at least one resistance
allele is associated with an allele having an A at nucleotide 428
of SEQ ID NO: 1; a T at position 895 of SEQ ID NO: 2; a G at
position 932 of SEQ ID NO: 2; a T at position 57 of SEQ ID NO: 3; a
G at position 213 of SEQ ID NO: 4; a G at position 441 of SEQ ID
NO: 4; an A at position 70 of SEQ ID NO: 5; a T at position 348 of
SEQ ID NO: 5; an A at position 715 of SEQ ID NO: 6; a C at position
377 of SEQ ID NO: 7; a Tat position 100 of SEQ ID NO: 8; a G at
position 113 of SEQ ID NO: 8; a T at position 147 of SEQ ID NO: 9;
a C at position 205 of SEQ ID NO: 10; an A at position 102 at SEQ
ID NO: 11; A Tat position 159 of SEQ ID NO: 12; and/or a G at
position 357 of SEQ ID NO: 13.
11. A soybean rust (SBR) resistant soybean plant selected using the
method of claim 9, or a cell, tissue culture, seed thereof.
12. A method for selecting an soybean rust (SBR) resistant soybean
plant, the method comprising: (a) isolating one or more nucleic
acids from a plurality of soybean plants; (b) detecting in said
isolated nucleic acids the presence of one or more marker molecules
associated with SBR resistance, wherein said marker molecule is
selected from the group consisting of SEQ ID NOs: 1-13, informative
fragments thereof, and any marker molecule mapped within 1, 2, 3,
4, 5, 6, 7, 8, 9, or 10 centiMorgans or less from said marker
molecules; and (c) selecting a soybean plant comprising said one or
more marker molecules, thereby selecting an SBR resistant soybean
plant.
13. The method of claim 12, wherein said one or more marker
molecules comprises an A at nucleotide 428 of SEQ ID NO: 1; a Tat
position 895 of SEQ ID NO: 2; a G at position 932 of SEQ ID NO: 2;
a Tat position 57 of SEQ ID NO: 3; a G at position 213 of SEQ ID
NO: 4; a G at position 441 of SEQ ID NO: 4; an A at position 70 of
SEQ ID NO: 5; a T at position 348 of SEQ ID NO: 5; an A at position
715 of SEQ ID NO: 6; a Cat position 377 of SEQ ID NO: 7; a Tat
position 100 of SEQ ID NO: 8; a G at position 113 of SEQ ID NO: 8;
a Tat position 147 of SEQ ID NO: 9; a C at position 205 of SEQ ID
NO: 10; an A at position 102 at SEQ ID NO: 11; A Tat position 159
of SEQ ID NO: 12; and/or a G at position 357 of SEQ ID NO: 13.
14. A method for producing seeds that result in soybean plants
resistant to soybean rust (SBR), the method comprising: (a)
providing a Glycine max plant which contains one or more alleles
that confer resistance to SBR, which alleles are characterized one
or more of five loci Rpp1-Rpp5, wherein: (i) Rppl is defined by the
following markers: (1) a marker of about 459 by as set forth in SEQ
ID NO: 1; (2) a marker of about 1101 by as set forth in SEQ ID NO:
2; and (3) a marker of about 443 by as set forth in SEQ ID NO: 3;
or any part of a DNA sequence as in SEQ ID NOs: 82-84 linked within
1, 2, 5, or 10 cM to at least one of the markers of (1)-(3)
conferring resistance to SBR; (ii) Rpp2 is defined by the following
markers: (4) a marker of about 471 by as set forth in SEQ ID NO: 4;
(5) a marker of about 489 by as set forth in SEQ ID NO: 5; and (6)
a marker of about 794 by as set forth in SEQ ID NO: 6; or any part
of a DNA sequence as in SEQ ID NOs: 85-87 linked within 1, 2, 5, or
10 cM to at least one of the markers of (4)-(6) conferring
resistance to SBR; (iii) Rpp3 is defined by the following markers:
(7) a marker of about 568 by as set forth in SEQ ID NO: 7; and (8)
a marker of about 503 by as set forth in SEQ ID NO: 8; or any part
of a DNA sequence as in SEQ ID NOs: 88 and 89 linked within 1, 2,
5, or cM to at least one of the markers of (7) and (8) conferring
resistance to SBR; (iv) Rpp4 is defined by the following markers:
(9) a marker of about 769 by as set forth in SEQ ID NO: 9; and (10)
a marker of about 513 by as set forth in SEQ ID NO: 10; or any part
of a DNA sequence as in SEQ ID NOs: 90 and 91 linked within 1, 2,
5, or 10 cM to at least one of the markers of (9) and (10)
conferring resistance to SBR; and (v) Rpp5 is defined by the
following markers: (11) a marker of about 281 by as set forth in
SEQ ID NO: 11; (12) a marker of about 948 by as set forth in SEQ ID
NO: 12; and (13) a marker of about 485 by as set forth in SEQ ID
NO: 13; or any part of a DNA sequence as in SEQ ID NOs: 92-94
linked within 1, 2, 5, or 10 cM to at least one of the markers of
(11)-(13) conferring resistance to SBR; (b) crossing the Glycine
max plant provided in step (a) with Glycine max culture breeding
material; and (c) collecting seeds resulting from the cross in step
(b) that result in soybean plants which are resistant to SBR.
15. The method of claim 14, further comprising detecting at least
one allelic form of a polymorphic simple sequence repeat (SSR) or a
single nucleotide polymorphism (SNP) associated with at least one
of the one or more alleles that confer resistance to SBR.
16. The method of claim 15, wherein the detecting comprises
amplifying the marker locus or a portion of the marker locus and
detecting the resulting amplified marker amplicon.
17. The method of claim 16, wherein the amplifying comprises: (a)
admixing an amplification primer or amplification primer pair with
a nucleic acid isolated from the first Glycine max plant or
germplasm, wherein the primer or primer pair is complementary or
partially complementary to at least a portion of the marker locus,
and is capable of initiating DNA polymerization by a DNA polymerase
using the soybean nucleic acid as a template; and (b) extending the
primer or primer pair in a DNA polymerization reaction comprising a
DNA polymerase and a template nucleic acid to generate at least one
amplicon.
18. The method of claim 17, wherein the nucleic acid is selected
from DNA and RNA.
19. The method of claim 16, wherein the amplifying comprises
employing a polymerase chain reaction (PCR) or ligase chain
reaction (LCR) using a nucleic acid isolated from the first soybean
plant or germplasm as a template in the PCR or LCR.
20. An improved soybean plant, seed, or tissue culture produced by
the method of claim 1.
21. An introgressed Glycine max plant or germplasm produced by the
method of claim 8.
22. A composition comprising an amplification primer pair capable
of initiating DNA polymerization by a DNA polymerase on a Glycine
max nucleic acid template to generate a Glycine max marker
amplicon, wherein the Glycine max marker amplicon corresponds to
Glycine max marker comprising a nucleotide sequence of any of SEQ
ID NOs: 1-13.
23. The composition of claim 22, wherein said amplification primer
pair comprises the nucleotide sequences of SEQ ID NOs: 14 and 15;
SEQ ID NOs: 18 and 19; SEQ ID NOs: 22 and 23; SEQ ID NOs: 26 and
27; SEQ ID NOs: 30 and 31; SEQ ID NOs: 34 and 35; SEQ ID NOs: 38
and 39; SEQ ID NOs: 42 and 43; SEQ ID NOs: 46 and 47; SEQ ID NOs:
50 and 51; SEQ ID NOs: 54 and 55; SEQ ID NOs: 58 and 59; SEQ ID
NOs: 62 and 63; SEQ ID NOs: 66 and 67; SEQ ID NOs: 70 and 71; SEQ
ID NOs: 74 and 75; and SEQ ID NOs: 78 and 79.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The following application is a continuation application of
U.S. patent application Ser. No. 16/838456 filed Apr. 2, 2020,
which is a continuation of U.S. patent application Ser. No.
16/053,639 filed Aug. 2, 2018 (now U.S. Pat. No. 10,624,284), which
is a continuation of U.S. patent application Ser. No. 14/872,443
filed on Oct. 1, 2015 which is a divisional application of U.S.
patent application Ser. No. 12/690,782 filed on Jan. 20, 2010,
which itself claims the benefit of International Patent Application
No. PCT/US2009/051003 filed on Jul. 17, 2009 which claims the
benefit of U.S. Provisional Patent Application Ser. Nos. 61/081,989
and 61/153,495, filed Jul. 18, 2008 and Feb. 18, 2009,
respectively. The disclosure of each of which is incorporated
herein by reference in its entirety.
STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING
[0002] A Sequence Listing in ASCII text format, submitted under 37
C.F.R. .sctn.1.821, entitled
"72001-US-C1_SeqList_CON_US_ST25_corrected", 35 kilobytes in size,
generated on Feb. 15, 2019, which is a corrected version of
"72001-US-REG-D-NAT-1_SeqList_DIV_US_ST25", 34.2 kilobytes in size,
generated on Sep. 1, 2015 and filed via EFS-Web, is provided in
lieu of a paper copy. This Sequence Listing is hereby incorporated
by reference into the specification for its disclosures.
TECHNICAL FIELD
[0003] The presently disclosed subject matter relates to markers
associated with soybean rust (SBR) resistance and methods of use
therefor. More particularly, the presently disclosed subject matter
relates to screening soybean lines for resistance to SBR and for
producing soybean lines with improved resistance to SBR, the
methods involving genetic marker analysis.
BACKGROUND
[0004] Plant pathogens are known to cause massive damage to
important crops, resulting in significant agricultural losses with
widespread consequences for both the food supply and other
industries that rely on plant materials. As such, there is a long
felt need to reduce the incidence and/or impact of agricultural
pests on crop production.
[0005] Soybean rust (SBR), which is caused by the obligate fungal
pathogen Phakopsora pachyrhizi H. Sydow & Sydow, was first
reported in Japan in 1902. By 1934, the pathogen was reported in
several other Asian countries and in Australia. More recently, P.
pachyrhizi infection has been reported in Africa, and has spread
rapidly through the African continent.
[0006] In November 2004, P. pachyrhizi was first reported in the
continental U.S., and the pathogen has now been reported in more
than 300 U.S. counties, in Canada, and in Mexico. In 2007,
approximately 0.5 million hectares of soybean were sprayed for SBR
control in the U.S.
[0007] SBR has the potential to cause significant yield losses in
the U.S., as indicated by fungicide trials in Georgia and Florida
that reported yield losses of 30 to 33% in untreated control plots.
In Brazil, the total yield loss in the 2006-2007 growing season due
to SBR was estimated to be over U.S. S2.26 billion with an average
of 2.3 fungicide applications required per season. Yield losses up
to 80% have been reported due to severe outbreaks of SBR, which
result in early leaf drop that inhibits pod set. Consistent
economic losses in Brazil over the last several years due to severe
SBR outbreaks have raised concerns regarding the potential impact
of this disease in the United States. Soybean cultivars currently
available commercially in the United States are all susceptible to
SBR to some degree, and fungicide applications are currently
employed to control the disease.
[0008] Therefore, soybean rust resistant cultivars are needed to
reduce fungicide costs and yield losses due to SBR.
SUMMARY
[0009] The presently disclosed subject matter provides methods for
conveying resistance to soybean rust (SBR) into non-resistant
soybean germplasm. In some embodiments, the methods comprise
introgressing SBR resistance into a non-resistant soybean using one
or more nucleic acid markers for marker-assisted breeding among
soybean lines to be used in a soybean breeding program, wherein the
markers are linked to an SBR resistance locus selected from the
group consisting of Rpp1, Rpp2, Rpp3, Rpp4, and Rpp5.
[0010] The presently disclosed subject matter also provides methods
for reliably and predictably introgressing soybean rust (SBR)
resistance into non-resistant soybean germplasm. In some
embodiments, the methods comprise employing one or more nucleic
acid markers for marker-assisted breeding among soybean lines to be
used in a soybean breeding program.
[0011] The presently disclosed subject matter also provides methods
for producing a soybean plant adapted for conferring resistance to
soybean rust (SBR). In some embodiments, the methods comprise (a)
selecting a first donor parental line possessing a desired SBR
resistance and having at least one of the resistant loci selected
from a locus mapping to Rpp1 and mapped by one or more of the
markers SEQ ID NOs: 1-3; a locus mapping to Rpp2 and mapped by one
or more of the markers SEQ ID NOs: 4-6; a locus mapping to Rpp3 and
mapped by one or more of the markers SEQ ID NOs: 7 and 8; a locus
mapping to Rpp4 and mapped by one or more of the markers SEQ ID
NOs: 9 and 10; and a locus mapping to Rpp5 and mapped one or more
markers SEQ ID NOs: 11-13; (b) crossing the first donor parent line
with a second parental line in hybrid combination to produce a
segregating plant population; (c) screening the segregating plant
population for identified chromosomal loci of one or more genes
associated with the resistance to SBR; and (d) selecting plants
from the population having the identified chromosomal loci for
further screening until a line is obtained which is homozygous for
resistance to SBR at sufficient loci to give resistance to SBR.
[0012] The presently disclosed subject matter also provides methods
for selecting a soybean rust (SBR) resistant soybean plant. In some
embodiments, the methods comprise (a) genotyping one or more
soybean plants with respect to one or more single nucleotide
polymorphisms (SNPs); and (b) selecting a soybean plant that
includes at least one resistance allele associated with the SNPs,
thereby selecting an SBR resistant soybean plant.
[0013] In some embodiments, the presently disclosed methods
comprise (a) isolating one or more nucleic acids from a plurality
of soybean plants; (b) detecting in said isolated nucleic acids the
presence of one or more marker molecules associated with SBR
resistance, wherein said marker molecule is selected from the group
consisting of SEQ ID NOs: 1-13; and (c) selecting a soybean plant
comprising said one or more marker molecules, thereby selecting an
SBR resistant soybean plant.
[0014] In some embodiments, the one or more nucleic acid markers
are selected from the group consisting of SEQ ID NOs: 1-13, and
informative fragments thereof. In some embodiments, the methods and
compositions of the presently disclosed subject matter employ a
marker molecule mapped within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10
centiMorgans or less from a marker molecule selected from the group
consisting of SEQ ID NOs: 1-13.
[0015] In some embodiments, the marker-assisted breeding comprises
single nucleotide polymorphism (SNP) analysis. In some embodiments,
the methods further comprise screening an introgressed soybean
plant, or a cell or tissue thereof, for SBR resistance In some
embodiments of the presently disclosed methods, the at least one
resistance allele is associated with an allele having an A at
nucleotide 428 of SEQ ID NO: 1; a Tat position 895 of SEQ ID NO: 2;
a G at position 932 of SEQ ID NO: 2; a T at position 57 of SEQ ID
NO: 3; a G at position 213 of SEQ ID NO: 4; a G at position 441 of
SEQ ID NO: 4; an A at position 70 of SEQ ID NO: 5; a T at position
348 of SEQ ID NO: 5; an A at position 715 of SEQ ID NO: 6; a C at
position 377 of SEQ ID NO: 7; a T at position 100 of SEQ ID NO: 8;
a G at position 113 of SEQ ID NO: 8; a T at position 147 of SEQ ID
NO: 9; a C at position 205 of SEQ ID NO: 10; an A at position 102
at SEQ ID NO: 11; A Tat position 159 of SEQ ID NO: 12; and/or a G
at position 357 of SEQ ID NO: 13.
[0016] The presently disclosed subject matter also provides soybean
rust (SBR) resistant soybean plants, parts thereof (including but
not limited to pollen, ovule, leaf, embryo, root, root tip, anther,
flower, fruit, stem, shoot, seed; rootstock, protoplast, and
callus), and progeny thereof, selected using the disclosed
methods.
[0017] Thus, it is an object of the presently disclosed subject
matter to provide methods for conveying SBR resistance into
non-resistant soybean germplasm, which object is achieved in whole
or in part by the presently disclosed subject matter.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIGS. 1A-1E are genetic linkage maps depicting linkage
groups and showing relative positions of various markers linked to
Rpp1-Rpp5, respectively.
BRIEF DESCRIPTION OF THE SEQUENCE LISTING
[0019] SEQ ID NOs: 1-13 are nucleotide sequences of the soybean
genome comprising single nucleotide polymorphisms (SNPs) identified
as being associated with the Rppl gene, the Rpp2 gene, the Rpp3
gene, the Rpp4 gene, and/or the Rpp5 gene as set forth in Table
1.
[0020] SEQ ID NOs: 14-81 are nucleotide sequences of
oligonucleotide primers that can be employed to amplify and/or
otherwise assay a subsequence of the soybean genome that is
associated with the Rpp1-Rpp5 loci as also set forth in Table
1.
TABLE-US-00001 TABLE 1 Amplification and Detection Primers Genomic
Amplification Detection Rpp Subsequence Primer SEQ Primers SEQ Gene
SEQ ID NO: ID NOs. ID NOs. 1 1 14 and 15 16 and 17 1 2 18 and 19 20
and 21 1 2 22 and 23 24 and 25 1 3 26 and 27 28 and 29 2 4 30 and
31 32 and 33 2 4 34 and 35 36 and 37 2 5 38 and 39 40 and 41 2 5 42
and 43 44 and 45 2 6 46 and 47 48 and 49 3 7 50 and 51 53 and 53 3
8 54 and 55 56 and 57 3 8 58 and 59 60 and 61 4 9 62 and 63 64 and
65 4 10 66 and 67 68 and 69 5 11 70 and 71 72 and 73 5 12 74 and 75
76 and 77 5 13 78 and 79 80 and 81
[0021] SEQ ID NOs: 82-94 corresponds to subsequences of the
preliminary assembly of the soybean (Glycine max) genomic sequence
present in the Phytozyme Database, which correspond to SEQ ID NOs:
1-13 as set forth in Table 2.
TABLE-US-00002 TABLE 2 Locations of SEQ ID NOs: 1-13 in the
Phytozyme Database SEQ Relevant Nucleotide Positions ID Nucleo- in
Phytozyme Percent NO: tides Database Identity 1 1-459
60469363-60469821 99.8 2 1-1101 60577757-60578858 99.1 3 23-424
60291869-60292270 98.8 4 1-470 27963429-27963894 99.6 5 1-489
30065112-30065600 99.4 6 1-794 30330727-30331520 98.7 7 1-568
46665732-46666299 99.3 8 1-503 41676695-41677197 98.8 9 1-769
56338383-56339144 97.9 10 1-513 55961511-55962023 99.6 11 1-281
33395967-33396246 99.3 12 36-916 33200392-33201274 97.4 13 5-479
35318068-35318542 99.4
DETAILED DESCRIPTION
[0022] The presently disclosed subject matter relates at least in
part to the identification of several SNPs associated with SBR
resistance in Glycine sp. Thus, provided herein are methods of
conveying SBR resistance into non-resistant soybean germplasm,
which employ one or more of the identified SNPs in various
approaches.
[0023] All references listed below, as well as all references cited
in the instant disclosure, including but not limited to all
patents, patent applications and publications thereof, scientific
journal articles, and database entries (e.g., GENBANK.RTM. database
entries and all annotations available therein) are incorporated
herein by reference in their entireties to the extent that they
supplement, explain, provide a background for, or teach
methodology, techniques, and/or compositions employed herein.
I. Definitions
[0024] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood to one of
ordinary skill in the art to which the presently disclosed subject
matter belongs.
[0025] Following long-standing patent law convention, the articles
"a", "an", and "the" refer to "one or more" when used in this
application, including in the claims. For example, the phrase "a
marker" refers to one or more markers. Similarly, the phrase "at
least one", when employed herein to refer to an entity, refers to,
for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40,
45, 50, 75, 100, or more of that entity, including but not limited
to whole number values between 1 and 100 and greater than 100.
[0026] Unless otherwise indicated, all numbers expressing
quantities of ingredients, reaction conditions, and so forth used
in the specification and claims are to be understood as being
modified in all instances by the term "about". Accordingly, unless
indicated to the contrary, the numerical parameters set forth in
this specification and attached claims are approximations that can
vary depending upon the desired properties sought to be obtained by
the presently disclosed subject matter.
[0027] As used herein, the term "about," when referring to a value
or to an amount of mass, weight, time, volume, concentration or
percentage is meant to encompass variations of in some embodiments
.+-.20%, in some embodiments .+-.10%, in some embodiments .+-.5%,
in some embodiments .+-.1%, in some embodiments .+-.0.5%, and in
some embodiments .+-.0.1% from the specified amount, as such
variations are appropriate to perform the disclosed method.
[0028] As used herein, the term "allele" refers to any of one or
more alternative forms of a gene, all of which relate to at least
one trait or characteristic. In a diploid cell, two alleles of a
given gene occupy corresponding loci on a pair of homologous
chromosomes, although one of ordinary skill in the art understands
that the alleles in any particular individual do not necessarily
represent all of the alleles that are present in the species. Since
the presently disclosed subject matter relates to SNPs, it is in
some instances more accurate to refer to a "haplotype" (i.e., an
allele of a chromosomal segment) instead of "allele". However, in
such instances, the term "allele" should be understood to comprise
the term "haplotype".
[0029] As used herein, the phrase "associated with" refers to a
recognizable and/or assayable relationship between two entities.
For example, a trait, locus, QTL, SNP, gene, marker, phenotype,
etc. is "associated with resistance" if the presence or absence of
the trait, locus, QTL, SNP, gene, marker, phenotype, etc.,
influences an extent or degree of resistance (e.g., resistance to
SBR). In some embodiments, an allele associated with resistance to
SBR comprises an allele having an A at nucleotide 428 of SEQ ID NO:
1; a T at position 895 of SEQ ID NO: 2; a G at position 932 of SEQ
ID NO: 2; a T at position 57 of SEQ ID NO: 3; a G at position 213
of SEQ ID NO: 4; a G at position 441 of SEQ ID NO: 4; an A at
position 70 of SEQ ID NO: 5; a T at position 348 of SEQ ID NO: 5;
an A at position 715 of SEQ ID NO: 6; a C at position 377 of
[0030] SEQ ID NO: 7; a T at position 100 of SEQ ID NO: 8; a G at
position 113 of SEQ ID NO: 8; a T at position 147 of SEQ ID NO: 9;
a C at position 205 of SEQ ID NO: 10; an A at position 102 at SEQ
ID NO: 11; A T at position 159 of SEQ ID NO: 12; and/or a G at
position 357 of SEQ ID NO: 13.
[0031] As used herein, the term "backcross", and grammatical
variants thereof, refers to a process in which a breeder crosses a
progeny individual back to one of its parents, for example, a first
generation hybrid F1 with one of the parental genotypes of the Fl
hybrid. In some embodiments, a backcross is performed repeatedly,
with a progeny individual of one backcross being itself backcrossed
to the same parental genotype. The term "chromosome" is used herein
in its art-recognized meaning of the self-replicating genetic
structure in the cellular nucleus containing the cellular DNA and
bearing in its nucleotide sequence the linear array of genes.
[0032] As used herein, the terms "cultivar" and "variety" refer to
a group of similar plants that by structural or genetic features
and/or performance can be distinguished from other varieties within
the same species.
[0033] As used herein, the term "gene" refers to a hereditary unit
including a sequence of DNA that occupies a specific location on a
chromosome and that contains the genetic instruction for a
particular characteristics or trait in an organism.
[0034] As used herein, the term "hybrid" in the context of plant
breeding refers to a plant that is the offspring of genetically
dissimilar parents produced by crossing plants of different lines
or breeds or species, including but not limited to the cross
between two inbred lines.
[0035] As used herein, the term "inbred" refers to a substantially
homozygous individual or line.
[0036] As used herein, the phrase "informative fragment" refers to
a nucleic acid molecule and/or its nucleotide sequence that allows
for the proper identification of which allele of an allele set
(e.g., an SNP) the nucleic acid molecule and/or the nucleotide
sequence corresponds to. For example, whereas the SNP that
corresponds to SEQ ID NO: 1 relates to an "A" or a "G" at position
428, an "informative fragment" of SEQ ID NO: 1 would be any
sequence that comprises position 428 of SEQ ID NO: 1, thereby
allowing the nucleotide that is present in that position to be
determined.
[0037] As used herein, the terms "introgression", "introgressed",
and "introgressing" refer to both a natural and artificial process
whereby genomic regions of one species, variety, or cultivar are
moved into the genome of another species, variety, or cultivar, by
crossing those species. The process can optionally be completed by
backcrossing to the recurrent parent.
[0038] As used herein, the term "linkage" refers to a phenomenon
wherein alleles on the same chromosome tend to be transmitted
together more often than expected by chance if their transmission
was independent. Thus, in some embodiments two alleles on the same
chromosome are said to be "linked" when they segregate from each
other in the next generation less than 50% of the time, less than
25% of the time, less than 20% of the time, less than 15% of the
time, less than 10% of the time, less than 5% of the time, less
than 4% of the time, less than 3% of the time, less than 2% of the
time, or less than 1% of the time. Thus, two loci are linked if
they are within 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1 centiMorgans
(cM) of each other. For example, in some embodiments an SNP is
linked to a marker if it is within 50, 25, 20, 15, 10, 9, 8, 7, 6,
5, 4, 3, 2, or 1 cM of the marker.
[0039] As used herein, the phrase "linkage group" refers to all of
the genes or genetic traits that are located on the same
chromosome. Within the linkage group, those loci that are close
enough together can exhibit linkage in genetic crosses. Since the
probability of crossover increases with the physical distance
between loci on a chromosome, loci for which the locations are far
removed from each other within a linkage group might not exhibit
any detectable linkage in direct genetic tests. The term "linkage
group" is mostly used to refer to genetic loci that exhibit linked
behavior in genetic systems where chromosomal assignments have not
yet been made. Thus, the term "linkage group" is synonymous with
the physical entity of a chromosome, although one of ordinary skill
in the art will understand that a linkage group can also be defined
as corresponding to a region of (i.e., less than the entirety) of a
given chromosome.
[0040] As used herein, the term "locus" refers to a position that a
given gene or a regulatory sequence occupies on a chromosome of a
given species. As used herein, the term "marker" refers to an
identifiable position on a chromosome the inheritance of which can
be monitored. In some embodiments, a marker comprises a known or
detectable nucleic acid sequence.
[0041] In some embodiments, a marker corresponds to an
amplification product generated by amplifying a Glysine sp. nucleic
acid with two oligonucleotide primers, for example, by the
polymerase chain reaction (PCR). As used herein, the phrase
"corresponds to an amplification product" in the context of a
marker refers to a marker that has a nucleotide sequence that is
the same (allowing for mutations introduced by the amplification
reaction itself) as an amplification product that is generated by
amplifying Glysine sp. genomic DNA with a particular set of
primers. In some embodiments, the amplifying is by PCR, and the
primers are PCR primers that are designed to hybridize to opposite
strands of the Glysine sp. genomic DNA in order to amplify a
Glysine sp. genomic DNA sequence present between the sequences to
which the PCR primers hybridize in the Glysine sp. genomic DNA. In
some embodiments, a marker that "corresponds to" an amplified
fragment is a marker that has the same sequence of one of the
strands of the amplified fragment.
[0042] As used herein, the term "soybean" refers to a plant, or a
part thereof, of the genus Glycine including, but not limited to
Glycine max.
[0043] As used herein, the phrase "soybean-specific DNA sequence"
refers to a polynucleotide sequence having a nucleotide sequence
homology of in some embodiments more than 50%, in some embodiments
more than 60%, in some embodiments more than 70%, in some
embodiments more than 80%, in some embodiments more than 85%, in
some embodiments more than 90%, in some embodiments more than 92%,
in some embodiments more than 95%, in some embodiments more than
96%, in some embodiments more than 97%, in some embodiments more
than 98%, and in some embodiments more than 99% with a sequence of
the genome of the species Glycine that shows the greatest
similarity to it. In the case of markers for any of the Rpp genes,
a "soybean-specific DNA sequence" can comprise a part of the DNA
sequence of a soybean genome that flanks and/or is a part of an Rpp
gene sequence.
[0044] As used herein, the phrase "molecular marker" refers to an
indicator that is used in methods for visualizing differences in
characteristics of nucleic acid sequences.
[0045] Examples of such indicators are restriction fragment length
polymorphism (RFLP) markers, amplified fragment length polymorphism
(AFLP) markers, single nucleotide polymorphisms (SNPs), insertion
and deletion mutations (INDEL), microsatellite markers (SSRs),
sequence-characterized amplified regions (SCARs), cleaved amplified
polymorphic sequence (CAPS) markers or isozyme markers or
combinations of the markers described herein which defines a
specific genetic and chromosomal location. A molecular marker
"linked to" or "associated with" an Rpp gene as defined herein can
thus refer to SNPs, insertion mutations, as well as more usual AFLP
markers or any other type of marker used in the field.
[0046] As used herein, the phrase "nucleotide sequence homology"
refers to the presence of homology between two polynucleotides.
Polynucleotides have "homologous" sequences if the sequence of
nucleotides in the two sequences is the same when aligned for
maximum correspondence. The "percentage of sequence homology" for
polynucleotides, such as 50, 60, 70, 80, 90, 95, 98, 99 or 100
percent sequence homology, can be determined by comparing two
optimally aligned sequences over a comparison window (e.g., about
20-200 contiguous nucleotides), wherein the portion of the
polynucleotide sequence in the comparison window can include
additions or deletions (i.e., gaps) as compared to a reference
sequence for optimal alignment of the two sequences. Optimal
alignment of sequences for comparison can be conducted by
computerized implementations of known algorithms, or by visual
inspection. Readily available sequence comparison and multiple
sequence alignment algorithms are, respectively, the Basic Local
Alignment Search Tool (BLAST.RTM.; Altschul et al. (1990) J Mol
Biol 215:403-10; Altschul et al. (1997) Nucleic Acids Res
25:3389-3402) and ClustalX (Chenna et al. (2003) Nucleic Acids Res
31:3497-3500) programs, both available on the Internet. Other
suitable programs include, but are not limited to, GAP, BestFit,
PlotSimilarity, and FASTA, which are part of the Accelrys GCG
Package available from Accelrys Software, Inc. of San Diego,
Calif., United States of America.
[0047] As used herein, the term "offspring" plant refers to any
plant resulting as progeny from a vegetative or sexual reproduction
from one or more parent plants or descendants thereof. For
instance, an offspring plant can be obtained by cloning or selfing
of a parent plant or by crossing two parent plants and include
selfings as well as the F1 or F2 or still further generations. An
F1 is a first-generation offspring produced from parents at least
one of which is used for the first time as donor of a trait, while
offspring of second generation (F2) or subsequent generations (F3,
F4, and the like) are specimens produced from selfings or crossings
of F1s, F2s and the like. An F1 can thus be (and in some
embodiments is) a hybrid resulting from a cross between two true
breeding parents (the phrase "true-breeding" refers to an
individual that is homozygous for one or more traits), while an F2
can be (and in some embodiments is) an offspring resulting from
self-pollination of the F1 hybrids.
[0048] As used herein, the term "phenotype" refers to a detectable
characteristic of a cell or organism, which characteristics are at
least partially a manifestation of gene expression.
[0049] As used herein, the phrase "plant part" refers to a part of
a plant, including single cells and cell tissues such as plant
cells that are intact in plants, cell clumps, and tissue cultures
from which plants can be regenerated. Examples of plant parts
include, but are not limited to, single cells and tissues from
pollen, ovules, leaves, embryos, roots, root tips, anthers,
flowers, fruits, stems, shoots, and seeds; as well as scions,
rootstocks, protoplasts, calli, and the like.
[0050] As used herein, the term "population" refers to a
genetically heterogeneous collection of plants sharing a common
genetic derivation.
[0051] As used herein, the term "primer" refers to an
oligonucleotide which is capable of annealing to a nucleic acid
target and serving as a point of initiation of DNA synthesis when
placed under conditions in which synthesis of a primer extension
product is induced (e.g., in the presence of nucleotides and an
agent for polymerization such as DNA polymerase and at a suitable
temperature and pH). A primer (in some embodiments an extension
primer and in some embodiments an amplification primer) is in some
embodiments single stranded for maximum efficiency in extension
and/or amplification. In some embodiments, the primer is an
oligodeoxyribonucleotide. A primer is typically sufficiently long
to prime the synthesis of extension and/or amplification products
in the presence of the agent for polymerization. The minimum
lengths of the primers can depend on many factors, including, but
not limited to temperature and composition (A/T vs. G/C content) of
the primer.
[0052] In the context of amplification primers, these are typically
provided as a pair of bi-directional primers consisting of one
forward and one reverse primer or provided as a pair of forward
primers as commonly used in the art of DNA amplification such as in
PCR amplification.
[0053] As such, it will be understood that the term "primer", as
used herein, can refer to more than one primer, particularly in the
case where there is some ambiguity in the information regarding the
terminal sequence(s) of the target region to be amplified. Hence, a
"primer" can include a collection of primer oligonucleotides
containing sequences representing the possible variations in the
sequence or includes nucleotides which allow a typical base
pairing.
[0054] Primers can be prepared by any suitable method. Methods for
preparing oligonucleotides of specific sequence are known in the
art, and include, for example, cloning and restriction of
appropriate sequences and direct chemical synthesis. Chemical
synthesis methods can include, for example, the phospho di- or
tri-ester method, the diethylphosphoramidate method and the solid
support method disclosed in U.S. Pat. No. 4,458,066.
[0055] Primers can be labeled, if desired, by incorporating
detectable moieties by for instance spectroscopic, fluorescence,
photochemical, biochemical, immunochemical, or chemical
moieties.
[0056] The PCR method is well described in handbooks and known to
the skilled person. After amplification by PCR, target
polynucleotides can be detected by hybridization with a probe
polynucleotide which forms a stable hybrid with that of the target
sequence under stringent to moderately stringent hybridization and
wash conditions. If it is expected that the probes are essentially
completely complementary (i.e., about 99% or greater) to the target
sequence, stringent conditions can be used. If some mismatching is
expected, for example if variant strains are expected with the
result that the probe will not be completely complementary, the
stringency of hybridization can be reduced. In some embodiments,
conditions are chosen to rule out non-specific/adventitious
binding. Conditions that affect hybridization, and that select
against non-specific binding are known in the art, and are
described in, for example, Sambrook & Russell (2001). Molecular
Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., United States of
America. Generally, lower salt concentration and higher temperature
hybridization and/or washes increase the stringency of
hybridization conditions. Continuing, the term "probe" refers to a
single-stranded oligonucleotide sequence that will form a
hydrogen-bonded duplex with a complementary sequence in a target
nucleic acid sequence analyte or its cDNA derivative.
[0057] As used herein, the terms "Rpp1","Rpp2","Rpp3","Rpp4", and
"Rpp5" refer to loci that have been associated with SBR resistance
as defined by the markers defined herein. For the purposes of the
instant disclosure, these loci are said to be present on Glycine
linkage groups G, J, C2, G, and N, and linked to the markers
depicted in FIGS. 1A-1E, respectively.
[0058] As used herein, the term "quantitative trait locus" (QTL;
plural quantitative trait loci; QTLs) refers to a genetic locus (or
loci) that controls to some degree a numerically representable
trait that, in some embodiments, is continuously distributed. As
such, the term QTL is used herein in its art-recognized meaning to
refer to a chromosomal region containing alleles (e.g., in the form
of genes or regulatory sequences) associated with the expression of
a quantitative phenotypic trait. Thus, a QTL "associated with"
resistance to SBR refers to one or more regions located on one or
more chromosomes and/or in one or more linkage groups that includes
at least one gene the expression of which influences a level of
resistance and/or at least one regulatory region that controls the
expression of one or more genes involved in resistance to SBR. QTLs
can be defined by indicating their genetic location in the genome
of a specific Glysine sp. accession using one or more molecular
genomic markers. One or more markers, in turn, indicate a specific
locus. Distances between loci are usually measured by the frequency
of crossovers between loci on the same chromosome. The farther
apart two loci are, the more likely that a crossover will occur
between them. Conversely, if two loci are close together, a
crossover is less likely to occur between them. Typically, one
centiMorgan (cM) is equal to 1% recombination between loci. When a
QTL can be indicated by multiple markers, the genetic distance
between the end-point markers is indicative of the size of the
QTL.
[0059] As used herein, the term "regenerate", and grammatical
variants thereof, refers to the production of a plant from tissue
culture.
[0060] As used herein, the term "resistant" and "resistance"
encompass both partial and full resistance to infection (e.g.,
infection by a pathogen that causes SBR). A susceptible plant can
either be non-resistant or have lower levels of resistance to
infection relative to a resistant plant. The term is used to
include such separately identifiable forms of resistance as "full
resistance", "immunity", "intermediate resistance", "partial
resistance", and "hypersensitivity".
[0061] As used herein, the phrase "stringent hybridization
conditions" refers to conditions under which a polynucleotide
hybridizes to its target subsequence, typically in a complex
mixture of nucleic acids, but to essentially no other sequences.
Stringent conditions are sequence-dependent and can be different
under different circumstances. Exemplary guidelines for the
hybridization of nucleic acids can be found in Tijssen (1993) in
Laboratory Techniques in Biochemistry and Molecular Biology,
Elsevier, New York, New York, United States of America; Ausubel et
al. (1999) Short Protocols in Molecular Biology Wiley, New York,
N.Y., United States of America; and Sambrook & Russell, 2001
(supra). Generally, stringent conditions are selected to be about
5-10.degree. C. lower than the thermal melting point (Tm) for the
specific sequence at a defined ionic strength pH. The Tm is the
temperature (under defined ionic strength, pH, and nucleic acid
concentration) at which 50% of the probes complementary to the
target hybridize to the target sequence at equilibrium (as the
target sequences are present in excess, at Tm, 50% of the probes
are occupied at equilibrium). Stringent conditions are those in
which the salt concentration is less than about 1.0 M sodium ion,
typically about 0.01 to 1.0 M sodium ion concentration (or other
salts) at pH 7.0 to 8.3 and the temperature is at least about
30.degree. C. for short probes (e.g., 10 to 50 nucleotides) and at
least about 60.degree. C. for long probes (e.g., greater than 50
nucleotides). Stringent conditions can also be achieved with the
addition of destabilizing agents such as formamide Exemplary
stringent hybridization conditions include: 50% formamide, 5x SSC,
and 1% SDS, incubating at 42.degree. C.; or 5.times. SSC, 1% SDS,
incubating at 65.degree. C.; with one or more washes in 0.2.times.
SSC and 0.1% SDS at 65.degree. C. For PCR, a temperature of about
36.degree. C. is typical for low stringency amplification, although
annealing temperatures can vary between about 32.degree. C. and
48.degree. C. (or higher) depending on primer length.
[0062] As used herein, the term "susceptible" refers to a plant
having no resistance to the disease resulting in the plant being
affected by the disease, resulting in disease symptoms. The term
"susceptible" is therefore equivalent to "non-resistant".
Alternatively, the term "susceptible" can be employed in a relative
context, in which one plant is considered "susceptible" because it
is less resistant to a particular pathogen than is a second plant
(which in the context of these terms in a relative usage, would be
referred to as the "resistant" plant").
II. General Considerations
[0063] Previous studies on host resistance to P. pachyrhizi have
resulted in the identification of the four major resistance genes,
Rpp1, Rpp2, Rpp3, and Rpp4, in soybean accessions PI 200492, PI
230970, PI 462312, and PI 459025B, respectively. Three of these
genes (Rpp2, Rpp3, and Rpp4) confer a resistant reddish-brown (RB)
colored lesion as opposed to the susceptible tan (TAN) colored
lesion. Rpp1, on the other hand, confers resistance to some rust
isolates. These four genes have been mapped on linkage groups (LGs)
G, J, C2, and G, respectively. An RB lesion type resistance gene
Rpp?(Hyuuga) from the Japanese cultivar "Hyuuga" has been mapped by
to the same region on LG C2 as Rpp3. A fifth gene, Rpp5, was
recently identified from PI200456 and mapped on LG N. With the
availability of the 7X sequence coverage of the soybean genome made
possible by efforts of the U. S. Department of Energy-Joint Genome
Institute (DOE-JGI), Rpp1 has been fine mapped to a 23 kb region on
scaffold 21 of LG G, and several markers close to this gene have
been identified (Hyten et al. (2008) Theor Appl Genet
116:945-952).
[0064] Resistance conferred by Rpp1, Rpp2, Rpp3, Rpp4, and Rpp5 can
be race-specific, and can be overcome by various P. pachyrhizi
isolates. For example, resistance in soybean lines carrying either
Rpp1 or Rpp3 genes failed in Brazil within two years of the
establishment of the disease.
[0065] The presently disclosed subject matter provides in some
embodiments soybean varieties that are resistant to SBR, methods
for identifying soybean plants that carry desirable resistance
genes, and methods for introducing such desirable resistance genes
into soybeans.
III. Plant Breeding
[0066] The presently disclosed subject matter provides for better
models for marker-assisted breeding (MAB). The presently disclosed
subject matter therefore relates to methods of plant breeding and
to methods to select plants, in particular soybean plants,
particularly cultivated soybean plants as breeder plants for use in
breeding programs or cultivated soybean plants for having desired
genotypic or potential phenotypic properties, in particular related
to producing valuable soybeans, also referred to herein as
commercially valuable plants. Herein, a cultivated plant is defined
as a plant being purposely selected or having been derived from a
plant having been purposely selected in agricultural or
horticultural practice for having desired genotypic or potential
phenotypic properties, for example a plant obtained by
inbreeding.
[0067] The presently disclosed subject matter thus also provides
methods for selecting a plant of the genus Glycine exhibiting
resistance towards SBR comprising detecting in the plant the
presence of one or more resistance alleles as defined herein. In an
exemplary embodiment of the presently disclosed methods for
selecting such a plant, the method comprises providing a sample of
genomic DNA from a soybean plant; and (b) detecting in the sample
of genomic DNA at least one molecular marker associated with
resistance to SBR. In some embodiments, the detecting can comprise
detecting one or more SNPs that are associated with resistance to
SBR.
[0068] The providing of a sample of genomic DNA from a soybean
plant can be performed by standard DNA isolation methods well known
in the art.
[0069] The detecting of a molecular marker can in some embodiments
comprise the use of one or more sets of primer pairs that can be
used to produce one or more amplification products that are
suitable markers for one of the SNPs. Such a set of primers can
comprise, in some embodiments, nucleotide sequences as set forth in
SEQ ID NOs: 14-81.
[0070] In some embodiments, the detecting of a molecular marker can
comprise the use of a nucleic acid probe having a base sequence
that is substantially complementary to the nucleic acid sequence
defining the molecular marker and which nucleic acid probe
specifically hybridizes under stringent conditions with a nucleic
acid sequence defining the molecular marker. A suitable nucleic
acid probe can for instance be a single strand of the amplification
product corresponding to the marker. In some embodiments, the
detecting of a molecular marker is designed to discriminate whether
a particular allele of an SNP is present or absent in a particular
plant.
[0071] The presently disclosed methods can also include detecting
an amplified DNA fragment associated with the presence of a
particular allele of an SNP. In some embodiments, the amplified
fragment associated with a particular allele of an SNP has a
predicted length or nucleic acid sequence, and detecting an
amplified DNA fragment having the predicted length or the predicted
nucleic acid sequence is performed such that the amplified DNA
fragment has a length that corresponds (plus or minus a few bases;
e.g., a length of one, two or three bases more or less) to the
expected length as based on a similar reaction with the same
primers with the DNA from the plant in which the marker was first
detected or the nucleic acid sequence that corresponds (i.e., has a
homology of in some embodiments more than 80%, in some embodiments
more than 90%, in some embodiments more than 95%, in some
embodiments more than 97%, and in some embodiments more than 99%)
to the expected sequence as based on the sequence of the marker
associated with that SNP in the plant in which the marker was first
detected. Upon a review of the instant disclosure, one of ordinary
skill in the art would appreciate that markers (e.g., SNP alleles)
that are absent in resistant plants, while they were present in the
susceptible parent(s) (so-called trans-markers), can also be useful
in assays for detecting resistance among offspring plants.
[0072] The detecting of an amplified DNA fragment having the
predicted length or the predicted nucleic acid sequence can be
performed by any of a number or techniques, including but not
limited to standard gel-electrophoresis techniques or by using
automated DNA sequencers. The methods are not described here in
detail as they are well known to the skilled person, although
exemplary approaches are set forth in the EXAMPLES.
IV. Molecular Markers and SNPs
[0073] Molecular markers are used for the visualization of
differences in nucleic acid sequences. This visualization can be
due to DNA-DNA hybridization techniques after digestion with a
restriction enzyme (e.g., an RFLP) and/or due to techniques using
the polymerase chain reaction (e.g., STS, SSR/microsatellites,
AFLP, and the like). In some embodiments, all differences between
two parental genotypes segregate in a mapping population based on
the cross of these parental genotypes. The segregation of the
different markers can be compared and recombination frequencies can
be calculated. Methods for mapping markers in plants are disclosed
in, for example, Glick & Thompson (1993) Methods in Plant
Molecular Biology and Biotechnology, CRC Press, Boca Raton, Fla.,
United States of America; Zietkiewicz et al. (1994) Genomics
20:176-183.
[0074] The recombination frequencies of molecular markers on
different chromosomes and/or in different linkage groups are
generally 50%. Between molecular markers located on the same
chromosome or in the same linkage group, the recombination
frequency generally depends on the distance between the markers. A
low recombination frequency typically corresponds to a low genetic
distance between markers on a chromosome. Comparing all
recombination frequencies results in the most logical order of the
molecular markers on the chromosomes or in the linkage groups. This
most logical order can be depicted in a linkage map. A group of
adjacent or contiguous markers on the linkage map that is
associated with an increased level of resistance to a disease;
e.g., to a reduced incidence of acquiring the disease upon
infectious contact with the disease agent and/or a reduced lesion
growth rate upon establishment of infection, can provide the
position of a locus associated with resistance to that disease.
[0075] The markers disclosed herein can be used in various aspects
of the presently disclosed subject matter as set forth hereinbelow.
Aspects of the presently disclosed subject matter are not to be
limited to the use of the markers identified herein, however. It is
stressed that the aspects can also make use of markers not
explicitly disclosed herein or even yet to be identified. Other
than the genetic unit "gene", on which the phenotypic expression
depends on a large number of factors that cannot be predicted, the
genetic unit "QTL" denotes a region of the genome that is directly
related to a phenotypic quantifiable trait.
[0076] The markers provided by the presently disclosed subject
matter can be used for detecting the presence of one or more SBR
resistance alleles of the presently disclosed subject matter in a
suspected SBR-resistant soybean plant, and can therefore be used in
methods involving marker-assisted breeding and selection of SBR
resistant soybean plants. In some embodiments, detecting the
presence of a particular allele of an SNP of the presently
disclosed subject matter is performed with at least one of the
markers for the resistance loci defined herein. The presently
disclosed subject matter therefore relates in another aspect to a
method for detecting the presence of a particular allele associated
with SBR resistance, comprising detecting the presence of a nucleic
acid sequence of the SNP in a suspected SBR-resistant soybean
plant, which presence can be detected by the use of the disclosed
markers and oligonucleotides.
[0077] The nucleotide sequence of an SNP of the presently disclosed
subject matter can for instance be resolved by determining the
nucleotide sequence of one or more markers associated with the SNP
and designing internal primers for the marker sequences that can be
used to determine which allele of the SNP is present in the
plant.
[0078] In embodiments of such methods for detecting the presence of
an SNP in a suspected SBR-resistant soybean plant, the method can
also comprise providing a oligonucleotide or polynucleotide capable
of hybridizing under stringent hybridization conditions to a
particular nucleic acid sequence of an SNP, in some embodiments
selected from the SNPs disclosed herein, contacting the
oligonucleotide or polynucleotide with genomic nucleic acid (or a
fragment thereof, including, but not limited to a restriction
fragment thereof) of a suspected SBR-resistant soybean plant, and
determining the presence of specific hybridization of the
oligonucleotide or polynucleotide to the genomic nucleic acid (or
the fragment thereof).
[0079] In some embodiments, the method is performed on a nucleic
acid sample obtained from the suspected SBR-resistant soybean
plant, although in situ hybridization methods can also be employed.
Alternatively, one of ordinary skill in the art can design specific
hybridization probes or oligonucleotides capable of hybridizing
under stringent hybridization conditions to the nucleic acid
sequence of the allele associated with SBR resistance and can use
such hybridization probes in methods for detecting the presence of
an SNP allele disclosed herein in a suspected SBR-resistant soybean
plant.
V. Production of SBR-Resistant Soybean Plants
[0080] The presently disclosed subject matter also relates to
methods for producing an SBR-resistant soybean plant comprising
performing a method for detecting the presence of an allele
associated with resistance to SBR in a donor soybean plant
according to the presently disclosed subject matter as described
above, and transferring a nucleic acid sequence comprising at least
one allele thus detected, or an SBR resistance-conferring part
thereof, from the donor plant to an SBR-susceptible recipient
soybean plant. The transfer of the nucleic acid sequence can be
performed by any of the methods described herein.
[0081] An exemplary embodiment of such a method comprises the
transfer by introgression of the nucleic acid sequence from an
SBR-resistant donor soybean plant into an SBR-susceptible recipient
soybean plant by crossing the plants. This transfer can thus
suitably be accomplished by using traditional breeding techniques.
SBR-resistance loci are introgressed in some embodiments into
commercial soybean varieties using marker-assisted selection (MAS)
or marker-assisted breeding (MAB). MAS and MAB involves the use of
one or more of the molecular markers for the identification and
selection of those offspring plants that contain one or more of the
genes that encode for the desired trait. In the context of the
presently disclosed subject matter, such identification and
selection is based on selection of SNP alleles of the presently
disclosed subject matter or markers associated therewith. MAB can
also be used to develop near-isogenic lines (NIL) harboring the QTL
of interest, allowing a more detailed study of each QTL effect and
is also an effective method for development of backcross inbred
line (BIL) populations. Soybean plants developed according to these
embodiments can advantageously derive a majority of their traits
from the recipient plant, and derive SBR resistance from the donor
plant.
[0082] As discussed hereinabove, traditional breeding techniques
can be used to introgress a nucleic acid sequence encoding SBR
resistance into an SBR-susceptible recipient soybean plant. In some
embodiments, a donor soybean plant that exhibits resistance to SBR
and comprising a nucleic acid sequence encoding SBR resistance is
crossed with an SBR-susceptible recipient soybean plant that in
some embodiments exhibits commercially desirable characteristics,
such as, but not limited to, disease resistance, insect resistance,
valuable nutritional characteristics, and the like. The resulting
plant population (representing the F1 hybrids) is then
self-pollinated and set seeds (F2 seeds). The F2 plants grown from
the F2 seeds are then screened for resistance to SBR. The
population can be screened in a number of different ways.
[0083] First, the population can be screened using a traditional
disease screen. Such disease screens are known in the art. In some
embodiments, a quantitative bioassay is used. Second,
marker-assisted breeding can be performed using one or more of the
herein-described molecular markers to identify those progeny that
comprise a nucleic acid sequence encoding for SBR resistance. Other
methods, referred to hereinabove by methods for detecting the
presence of an allele associated with SBR resistance, can be used.
Also, marker-assisted breeding can be used to confirm the results
obtained from the quantitative bioassays, and therefore, several
methods can also be used in combination.
[0084] Inbred SBR-resistant soybean plant lines can be developed
using the techniques of recurrent selection and backcrossing,
selfing, and/or dihaploids, or any other technique used to make
parental lines. In a method of recurrent selection and
backcrossing, SBR resistance can be introgressed into a target
recipient plant (the recurrent parent) by crossing the recurrent
parent with a first donor plant, which differs from the recurrent
parent and is referred to herein as the "non-recurrent parent". The
recurrent parent is a plant that is non-resistant or has a low
level of resistance to SBR and possesses commercially desirable
characteristics, such as, but not limited to (additional) disease
resistance, insect resistance, valuable nutritional
characteristics, and the like. In some embodiments, the
non-recurrent parent exhibits SBR resistance and comprises a
nucleic acid sequence that encodes SBR resistance. The
non-recurrent parent can be any plant variety or inbred line that
is cross-fertile with the recurrent parent.
[0085] The progeny resulting from a cross between the recurrent
parent and non-recurrent parent are backcrossed to the recurrent
parent. The resulting plant population is then screened for the
desired characteristics, which screening can occur in a number of
different ways. For instance, the population can be screened using
phenotypic pathology screens or quantitative bioassays as known in
the art. Alternatively, instead of using bioassays, MAB can be
performed using one or more of the hereinbefore described molecular
markers, hybridization probes, or polynucleotides to identify those
progeny that comprise a nucleic acid sequence encoding SBR
resistance. Also, MAB can be used to confirm the results obtained
from the quantitative bioassays. In some embodiments, the markers
defined herein are suitable to select proper offspring plants by
genotypic screening.
[0086] Following screening, the F1 hybrid plants that exhibit an
SBR-resistant phenotype or, in some embodiments, genotype and thus
comprise the requisite nucleic acid sequence encoding SBR
resistance, are then selected and backcrossed to the recurrent
parent for a number of generations in order to allow for the
soybean plant to become increasingly inbred. This process can be
performed for two, three, four, five, six, seven, eight, or more
generations. In principle, the progeny resulting from the process
of crossing the recurrent parent with the SBR-resistant
non-recurrent parent are heterozygous for one or more genes that
encode SBR resistance.
[0087] In general, a method of introducing a desired trait into a
hybrid soybean variety can comprise: [0088] (a) crossing an inbred
soybean parent with another soybean plant that comprises one or
more desired traits, to produce F1 progeny plants, wherein the
desired trait is SBR resistance; [0089] (b) selecting the F1
progeny plants that have the desired trait to produce selected F1
progeny plants, in some embodiments using molecular markers as
defined herein; [0090] (c) backcrossing the selected progeny plants
with the inbred soybean parent plant to produce backcross progeny
plants; [0091] (d) selecting for backcross progeny plants that have
the desired trait and morphological and physiological
characteristics of the inbred soybean parent plant, wherein the
selection comprises the isolation of genomic DNA and testing the
DNA for the presence of at least one molecular marker for SBR
resistance, in some embodiments as described herein; [0092] (e)
repeating steps (c) and (d) two or more times in succession to
produce selected third or higher backcross progeny plants; [0093]
(f) optionally selfing selected backcross progeny in order to
identify homozygous plants; and [0094] (g) crossing at least one of
the backcross progeny or selfed plants with another soybean parent
plant to generate a hybrid soybean variety with the desired trait
and all of the morphological and physiological characteristics of
hybrid soybean variety when grown in the same environmental
conditions.
[0095] As indicated, the last backcross generation can be selfed in
order to provide for homozygous pure breeding (inbred) progeny for
SBR resistance. Thus, the result of recurrent selection,
backcrossing, and selfing is the production of lines that are
genetically homogenous for the genes associated with SBR
resistance, and in some embodiments as well as for other genes
associated with traits of commercial interest.
VI. SBR-Resistant Soybean Plants and Seeds
[0096] The development of a hybrid soybean variety in a soybean
plant breeding program can, in some embodiments, involve three
steps: (1) the selection of plants from various germplasm pools for
initial breeding crosses; (2) the selfing of the selected plants
from the breeding crosses for several generations to produce a
series of inbred lines, which, individually breed true and are
highly uniform; and (3) crossing a selected variety with an
different variety to produce the hybrid progeny (F1). After a
sufficient amount of inbreeding successive filial generations will
merely serve to increase seed of the developed inbred. In some
embodiments, an inbred line comprises homozygous alleles at about
95% or more of its loci.
[0097] A SBR-resistant soybean plant, or a part thereof, obtainable
by a method of the presently disclosed subject matter is an aspect
of the presently disclosed subject matter.
[0098] The SBR-resistant soybean plants of the presently disclosed
subject matter, or part thereof, can be heterozygous or homozygous
for the resistance traits (in some embodiments, homozygous).
Although the SBR resistance loci of the presently disclosed subject
matter, as well as resistance-conferring parts thereof, can be
transferred to any plant in order to provide for an SBR-resistant
plant, the methods and plants of the presently disclosed subject
matter are in some embodiments related to plants of the genus
Glycine.
[0099] The SBR-resistant soybean lines described herein can be used
in additional crossings to create SBR-resistant plants. For
example, a first SBR-resistant soybean plant of the presently
disclosed subject matter can be crossed with a second soybean plant
possessing commercially desirable traits such as, but not limited
to, disease resistance, insect resistance, desirable nutritional
characteristics, and the like. In some embodiments, this second
soybean line is itself SBR-resistant. In some embodiments, this
line is heterozygous or homozygous for one or more of the disclosed
SBR resistance loci, in order for one or more of these traits to be
expressed in the hybrid offspring plants.
[0100] Another aspect of the presently disclosed subject matter
relates to a method of producing seeds that can be grown into
SBR-resistant soybean plants. In some embodiments, the method
comprises providing a SBR-resistant soybean plant of the presently
disclosed subject matter, crossing the SBR-resistant plant with
another soybean plant, and collecting seeds resulting from the
cross, which when planted, produce SBR-resistant soybean
plants.
[0101] In some embodiments, the method comprises providing a
SBR-resistant soybean plant of the presently disclosed subject
matter, crossing the SBR-resistant plant with a soybean plant,
collecting seeds resulting from the cross, regenerating the seeds
into plants, selecting SBR-resistant plants by any of the methods
described herein, self-pollinating the selected plants for a
sufficient number of generations to obtain plants that are fixed
for an allele associated with SBR-resistance in the plants,
backcrossing the plants thus produced with soybean plants having
desirable phenotypic traits for a sufficient number of generations
to obtain soybean plants that are SBR-resistant and have desirable
phenotypic traits, and collecting the seeds produced from the
plants resulting from the last backcross, which when planted,
produce soybean plants which are SBR-resistant.
VII. Other Applications
[0102] With the use of these SNPs for breeding new soybean lines, a
system for developing germplasm that has more than one mode of
action against the fungus is made possible. The use of this dual
mode of action will assist in inhibiting the fungus from developing
resistance. Additionally, a regime for inhibiting fungal resistance
can include a number of different modes of action. The different
modes of action in an inhibiting fungus method could be through
germplasm or seed treatments or chemical applications. A system for
inhibiting fungal resistance includes germplasm with one or more
vertical resistant traits, and/or germplasm with one or more
horizontal tolerance traits with the possible options of including
a seed treatment that is active against the fungus, and/or an
antifungal spray. Antifungal sprays or treatments can include known
antifungal compounds for this fungus but can also include
glufosinate or glyphosate sprays, separate or in combination which
also have an antifungal affect.
[0103] The regime for inhibiting antifungal resistance is very
important for the continued effectiveness of germplasm resistance
and chemical activity. There are areas in which soybeans are
presently produced which have fungal isolates that are no longer
negatively affected by at least two of the known Rpp resistance
alleles. The ability of these fungal isolates to evolve so quickly
could render entire soybean growing areas unprofitable unless the
embodiments of the presently disclosed subject matter that provide
soybean seeds which are protected by the SNP selected traits for a
dual mode of action plus the optional seed treatment, or chemical
applications is implemented.
EXAMPLES
[0104] The following Examples provide illustrative embodiments. 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 scope of the presently claimed subject matter.
Example 1
SNP Analysis
[0105] There are SSR markers which are associated with soybean rust
resistant qualitative and quantitative genes Rpp1, Rpp2, Rpp3,
Rpp4, and Rpp5. This SSR information was employed to identify SNP
markers that map to the regions of qualitative genes and
quantitative trait loci (QTL) regions defined for each of the Rpp1,
Rpp2, Rpp3, Rpp4, and Rpp5 genes. The identified SNPs were
validated on soybean rust resistant and susceptible lines. Analyses
indicated that these SNPs mapped more closely and showed better
associations to the Rpp gene than did the SSRs. The information of
validated SNPs for soybean rust is new and is used for appropriate
breeding programs.
Example 2
SNP Genotyping Data
[0106] Molecular markers were identified for the Rpp1, Rpp2, Rpp3,
Rpp4, and Rpp5 genes, and the resistance gene first identified in
the Japanese cultivar, Hyuuga. Hyten et al., 2007 used PI200492 to
map Rppl to linkage group (LG) G between the SSR markers Sct_187
and Sat_064. Rpp2 was mapped in PI230970 to the region on LG J
between Sat_255 and Satt620. Rpp4 was mapped in PI459025 on LG G
between SSR Satt288 and RFLP marker A885_1 (Silva et al., 2008,
supra). The cultivar Hyuuga (PI506764) was once thought to contain
Rpp3 (the resistance gene contained in PIs 462312 and 459025B), but
it is now believed that there are 2 separate rust resistance genes
located near each other on LG C2, flanked by SSRs Satt460 and
Sat_263. Rpp5 was recently identified in several lines (PI200487,
PI200526, PI471904, PI200456) by Garcia et al., 2008, and mapped to
LG N in a region flanked by the SSRs Sat_275 and Sat_280. The
approximate positions of these genes and various markers are
depicted in FIGS. 1A-1E.
[0107] The soybean linkage map developed by scientists at the USDA
and made publicly available in 2006 can be found through the
website of the United States Department of Agriculture (USDA) and
is discussed in Choi et al. (2007) Genetics 176:685-696. This map
was used to locate the flanking SSRs associated with each rust
resistance gene and to select SNPs that were mapped within and
close to each region. The polymorphism information and genomic
sequence on either side of the SNP was used to design PCR-based
assays to detect each allele. The sequences with the SNP indicated
were either submitted to the Applied Biosystems Inc. (ABI; Foster
City, Calif., United States of America) Assay-by-Design service for
creation of custom TAQMAN.RTM. (Applied Biosystems Inc., Foster
City, Calif., United States of America) based assays, or assays
were manually designed using the ABI software PRIMER EXPRESS.RTM..
Similarly, TAQMAN.RTM. assays can be designed using software
available from Biosearch Technologies (Novato, Calif., United
States of America).
[0108] A goal of the SNP assay was to be able to determine which
polymorphism(s), or allele(s), is/are present in the genome of any
given soybean line, and ultimately to permit the selection of
preferred allele(s) (i.e., rust resistant gene(s)), in a
marker-assisted breeding program. A total of 17 SNPs were
identified; four for Rpp1, five for Rpp2, three for Rpp?(Hyuuga),
two for Rpp4, and three for Rpp5. A total of 18 screening panel
DNAs isolated from resistant lines (PI547875; PI200492; PI594538A;
PI368039; PI547878; PI230970; PI224270; PI462312; PI578457A;
PI518772; PI628932; PI506764; PI547879; PI459025B; PI200456;
PI200526; PI200487; and PI471904) were used for the assays.
Example 3
Allelic Discrimination Assays
[0109] In allelic discrimination assays, a PCR assay included a
forward and reverse primer and a specific, fluorescent, dye-labeled
probe for each of two alleles for a given SNP. The probes contained
different fluorescent reporter dyes (VIC.RTM.; Applied Biosystems,
Inc., Foster City, Calif., United States of America; and
6-carboxyfluorescein-aminohexyl amidite (FAM), or
N-TET-6-Aminohexanol (TET) and FAM) to differentiate the
amplification of each allele. A non-fluorescent quencher on each
probe suppressed the fluorescence until amplification by PCR.
During PCR, each probe annealed specifically to complementary
sequences between the forward and reverse primer sites. Taq
polymerase then cleaved the probes that were hybridized to each
allele. Cleavage separated the reporter dye from the quencher,
which resulted in increased fluorescence by the reporter dye.
[0110] Thus, the fluorescent signals generated by PCR amplification
indicated that one or both alleles were present in the sample. In
addition to the non-fluorescent quencher, the probes also contained
a minor groove binder at the 3' end which resulted in increased
melting temperatures (Tm), thereby allowing high specificity with
the use of shorter oligos. These probes therefore exhibited greater
Tm differences when hybridized to matched and mismatched templates,
which provided more accurate allelic discriminations. Probes of
this type were manufactured at either ABI (MGB.TM. quencher) or
Biosearch Technologies (BHQPLUS.TM. quencher). At the end of PCR
thermal cycling, fluorescence of the two reporter dyes was measured
on an ABI 7900. An increase in fluorescence for one dye only
indicated homozygosity for the corresponding allele. An increase in
both fluorescent signals indicated heterozygosity.
[0111] Exemplary starting lines and haplotypes determined using
this method are presented in Tables 3-8. In Tables 3-8, "H"
indicates that the line was heterozygous at that position, and "-"
indicates that the nucleotide at that position was not
determined.
TABLE-US-00003 TABLE 3 SNP Screening Panel Source Line Name Origin
of Seed Resistance PI547875 L85-2378 Developed in GRIN.sup.1 Rpp1
Illinois, USA PI200492 GRIN Rpp1 PI594538A GRIN Rpp1-b PI368039
Tainung GRIN Rpp1 No. 4 PI547878 L86-1752 Developed in GRIN Rpp2
Illinois, USA PI230970 GRIN Rpp2 PI224270 GRIN Rpp2 PI462312 Ankur
GRIN Rpp3 PI578457A GRIN Rpp3 PI 518772 GRIN Rpp3 PI 628932 GRIN
Rpp3 PI506764 GRIN Rpp? (Hyuuga) PI547879 L87-0482 Developed in
GRIN Rpp4 Illinois, USA PI459025B (Bing nan) GRIN Rpp4 PI200456
GRIN Rpp5 PI200526 GRIN Rpp5 PI200487 Kinoshita GRIN Rpp5 PI471904
L85-2378 Developed in GRIN Rpp5 Illinois, USA .sup.1Germplasm
Resources Information Network, Agricultural Research Service,
United States Department of Agriculture, Beltsville, Maryland,
United States of America.
TABLE-US-00004 TABLE 4 SNP Genotyping Data-Detailed for Rpp1 SEQ ID
SEQ ID SEQ ID SEQ ID NO: 1 NO: 2a NO: 2b NO: 3 Linkage Group G G G
G SPIRIT Map Position (cM) Material 100.921 101.849 101.849 103.63
ID ABBRC LOCUS Rpp1 Rpp1 Rpp1 Rpp1 PI547875 L85-2378 Rpp1 A G T T
PI200492 Rpp1 A G T T PI594538A Rpp1-b A G T T PI368039 Tainung
Rpp1 A G T T No. 4 PI547878 L86-1752 Rpp2 G A C C PI230970 Rpp2 G G
C C PI224270 Rpp2 G G C C PI462312 Ankur Rpp3 G A C C PI578457A
Rpp3 A -- T C PI 518772 Rpp3 G A C C PI 628932 Rpp3 G A C C
PI506764 Rpp? G G C C (Hyuuga) PI547879 L87-0482 Rpp4 G A C C
PI459025B (Bing nan) Rpp4 G G T C PI200456 Rpp5 G G T C PI200526
Rpp5 H G T C PI200487 Kinoshita Rpp5 A G T C PI471904 Rpp5 A G T
C
TABLE-US-00005 TABLE 5 SNP Genotyping Data-Detailed for Rpp2 SEQ ID
SEQ ID SEQ ID SEQ ID SEQ ID NO: 4a NO: 4b NO: 5a NO: 5b NO: 6
Linkage Group J J J J J SPIRIT Map Position (cM) Material 49.858
49.858 57.697 57.697 60.927 ID ABBRC LOCUS Rpp2 Rpp2 Rpp2 Rpp2 Rpp2
PI547875 L85-2378 Rpp1 G G C T G PI200492 Rpp1 G G C T A PI594538A
Rpp1-b G G C T A PI368039 Tainung Rpp1 G G C T A No. 4 PI547878
L86-1752 Rpp2 G G C T G PI230970 Rpp2 G G A T A PI224270 Rpp2 G G A
A A PI462312 Ankur Rpp3 G G C T H PI578457A Rpp3 G G C T A PI
518772 Rpp3 G G H T A PI 628932 Rpp3 G G C T H PI506764 Rpp? G G A
A A (Hyuuga) PI547879 L87-0482 Rpp4 G G C T G PI459025B (Bing nan)
Rpp4 G G C T A PI200456 Rpp5 G G A T G PI200526 Rpp5 G G H H A
PI200487 Kinoshita Rpp5 G G A A A PI471904 Rpp5 G G A A A
TABLE-US-00006 TABLE 6 SNP Genotyping Data-Detailed for Rpp3 SEQ ID
SEQ ID SEQ ID NO: 7 NO: 8a NO: 8b Linkage Group C2 C2 C2 Map
Position (cM) 115.71 117.9 117.9 Rpp3 Rpp3 Rpp3 SPIRIT and Rpp? and
Rpp? and Rpp? Material ID ABBRC LOCUS (Hyuuga) (Hyuuga) (Hyuuga)
PI547875 L85-2378 Rpp1 T T G PI200492 Rpp1 C T G PI594538A Rpp1-b C
T G PI368039 Tainung Rpp1 C T G No. 4 PI547878 L86-1752 Rpp2 T T G
PI230970 Rpp2 C T G PI224270 Rpp2 C C A PI462312 Ankur Rpp3 C T G
PI578457A Rpp3 C T G PI 518772 Rpp3 T C A PI 628932 Rpp3 C T G
PI506764 Rpp? C C A (Hyuuga) PI547879 L87-0482 Rpp4 T T G PI459025B
(Bing nan) Rpp4 C T G PI200456 Rpp5 C T G PI200526 Rpp5 C H G
PI200487 Kinoshita Rpp5 C T G PI471904 Rpp5 C T G
TABLE-US-00007 TABLE 7 SNP Genotyping Data-Detailed for Rpp4 SEQ ID
SEQ ID NO: 9 NO: 10 Linkage Group G G SPIRIT Map Position (cM)
Material 75.83 76.246 ID ABBRC LOCUS Rpp4 Rpp4 PI547875 L85-2378
Rpp1 C A PI200492 Rpp1 T C PI594538A Rpp1-b T C PI368039 Tainung
Rpp1 T C No. 4 PI547878 L86-1752 Rpp2 C A PI230970 Rpp2 T C
PI224270 Rpp2 C A PI462312 Ankur Rpp3 T A PI578457A Rpp3 T C PI
518772 Rpp3 T C PI 628932 Rpp3 C A PI506764 Rpp? C C (Hyuuga)
PI547879 L87-0482 Rpp4 T C PI459025B (Bing nan) Rpp4 T C PI200456
Rpp5 T A PI200526 Rpp5 C H PI200487 Kinoshita Rpp5 T C PI471904
Rpp5 T C
TABLE-US-00008 TABLE 8 SNP Genotyping Data-Detailed for Rpp5 SEQ ID
SEQ ID SEQ ID NO: 11 NO: 12 NO: 13 Linkage Group N N N SPIRIT Map
Position (cM) Material 34.26 34.27 38.8 ID ABBRC LOCUS Rpp5 Rpp5
Rpp5 PI547875 L85-2378 Rpp1 A T A PI200492 Rpp1 G A G PI594538A
Rpp1-b A T G PI368039 Tainung Rpp1 A T G No. 4 PI547878 L86-1752
Rpp2 A T G PI230970 Rpp2 A T A PI224270 Rpp2 A T G PI462312 Ankur
Rpp3 G A G PI578457A Rpp3 -- H G PI 518772 Rpp3 H H H PI 628932
Rpp3 A T A PI506764 Rpp? A T G (Hyuuga) PI547879 L87-0482 Rpp4 A T
G PI459025B (Bing nan) Rpp4 A T A PI200456 Rpp5 A T G PI200526 Rpp5
G H G PI200487 Kinoshita Rpp5 A T G PI471904 Rpp5 A T A
Example 4
TAQMAN.RTM. Validation
[0112] To validate TAQMAN.RTM. allelic discrimination assays for
association with disease resistance or tolerance, plants were
selected based on their known phenotypic status and compared to the
genotype at the specific SNP location. DNA isolated from leaf
tissue of seedlings 7-10 days after planting was diluted in TE
buffer and stored at 4.degree. C. until used in PCR reactions as
described below.
[0113] PCR was set up in 5 .mu.l final volumes according to the
following formula:
TABLE-US-00009 Stock Per For 96 Final concen- rxn samples concen-
Reagent tration (.mu.l) (.mu.l) tration 2X Master Mix* 2X 2.5
296.88 1X AbD primer/probe 40x .0625 6 0.5x mix (80x) PCR-quality
H2O -- 2.44 234.24 -- DNA (dried in 4.5 ng/.mu.l 4 -- 3.6 ng/.mu.l
384) (18 ng) Final Volume (.mu.l) 5.00 357.44 *The Master Mix was
JUMPSTART .TM. Taq READYMIX .TM. (Sigma Catalogue No. 2893; Sigma
Chemical Co., St. Louis, Missouri, United States of America), a
premix of all the components, including nucleotides and Taq
polymerase (but not primers and/or probes) necessary to perform a
PCT reaction. Before use, 1375 .mu.l of 1.0M MgCl.sub.2 (Sigma
Catalogue No. M1028) and 250 .mu.l of 300 .mu.M Sulforhodamine 101
(Sigma Catalogue No. S7635; ROX) were added to a 125 mL bottle of
JUMPSTART .TM. Taq READYMIX .TM..
[0114] PCR plates were placed in an ABI 9700 thermal cycler and the
following program was run: an initial denaturation of 50.degree. C.
for 2 minutes followed by 95.degree. C. for 10 minutes; 40 cycles
of 95.degree. C. for 15 seconds/60.degree. C. for 1 minute; and a
final elongation of 72.degree. C. for 5 minutes. After the cycling,
the samples were incubated at 4.degree. C. until needed.
[0115] The ABI 7900 Sequence Detection System (or TAQMAN.RTM.) was
used to visualize the results of an allelic discrimination (SNP)
assay. Using the Sequence Detection System (SDS) software, allele
calls were made based on the fluorescence for the two dyes measured
in each sample.
[0116] It will be understood that various details of the presently
disclosed subject matter may be changed without departing from the
scope of the presently disclosed subject matter. Furthermore, the
foregoing description is for the purpose of illustration only, and
not for the purpose of limitation.
Sequence CWU 1
1
941459DNAGlycine maxmisc_feature(428)..(428)r is a or g 1ttttgatcca
aaacaaagct gaaaagaaag gggacaggta tgaagcaatc ttcagcttct 60actttggaga
ctatggtcac atagcagtgc agggacctta cctgacctat gaggacacat
120atttggctgt gactggtggg tctggcatat ttgagggtgt taaaggtcaa
gtgaagctgc 180gtcagattgt gtatcctttc aagattttgt acacatttta
tctaaagggt atcaaggatt 240tgcctcagga gcttcttgtc aagactgttg
agccaattcc atctgttgaa ccttcccctg 300ctgctaaggc ccttgagccc
aatgctacca ttgctggctt caccgactaa ttcatcaact 360ttttttgtat
ttgctttggc ctttgtagta gtatgattta agttactgaa taataataac
420aagtgggrac tatgatgggt tttgtagtgg tggagtttc 45921101DNAGlycine
maxmisc_feature(255)..(932)r is a or gmisc_feature(267)..(267)s is
c or g 2ttacaccaga atcatggcca ccaatcaaat acccctatca tcttattaca
gaaaagggga 60taagagaagg ggaaaaaaaa tctctagaaa ctgaagaatc aaggtttggt
tcaaaaactg 120tgcagttgcg ttagcatgtg aacaagctca tcactagaag
gcctatctgc aggcatatca 180gagaggcatg cagcagcaat cctaaccgcc
attagcatct catcttcctc accttcttcc 240cctaacatgc tcttrtctag
agcttcstgc gcctcgcccg cttgctgcaa gtgtctcagc 300caacatccca
aacttccccc actggctgct tctccaaaga atggatctgt aggatcctta
360ccagttaaca aaacacctag tatcatgcca aaactaaaga tgtcactttt
gtcagtgtac 420ctgctgaaat tgccaaaaaa caccattcaa tctgaatacc
atgaagctcc acatatacaa 480gaatggaatg aagttaaaat aaacatctta
ttggcaatac catacccata tatgcaatgc 540catcaatcaa gcaatctttt
attgttatta cttattgcta ttgatattga tgcatccaga 600tatacaccac
tgaaaaacta gaagtattcc aagttaaatt aggaaaaaaa acctttgtca
660attattatta taatttagtt gtggtctcac tcaccaatct aggtttagta
gtttgcagca 720tgtgaactat aaactatatt attcatttgg accagactta
gtgccaactg cctaaggtct 780aaaacttgac atcaggaagt aatgggtagt
tacaaaaaca aatagcaaag tctttattaa 840ataataccaa attcaatcag
caaatgatac aatgacaata cagattacag aatartaatt 900tcgtttcaaa
acaagagtct ttcattaggg trctacaaag ggggcaaaaa accaatgagg
960tgtggagctt ttgaaaaggt aacgaatcaa acaaattccc aatatcaacc
acctaattag 1020cctattggtg taaaaaagtg aaagaacaga aacagactaa
gggaaaacac tagtgaatga 1080gtatgattga aatttgtctt a
11013433DNAGlycine maxmisc_feature(1)..(433)r is a or
gmisc_feature(1)..(433)y is c or tmisc_feature(1)..(433)n is a or g
or c or t 3ttgggagcca acctgagggg actcagttac gcaagttttg aagctgttgg
ttatgayttt 60aatgacatgg ctctaggtga acttgtggaa aggrttttrg cttgcttttg
tccagcagag 120ttttctgttg ctttgcacat tgacatgcat ggtgagaaac
ttaataaatt tcccttrgac 180aycaaaggat actactgtgg tgagaggagc
actgaagagc ttggagtagg tggtgcagtt 240gtgtaccaaa catttgttca
aggctgcgat ggtagtgcat ctcctaggtc tattctgaaa 300tgctgctgga
gtgaggacga gaacgaagat gaagtgaggg agatataagt acttctggtt
360tttatgttct gttgttattt atctttactg tcgagtgtgt tattacctaa
ataaataaat 420atggngggtc gca 4334471DNAGlycine
maxmisc_feature(1)..(471)r is a or g 4ccctatcctc aaatacaagt
ccctgatctc atcataccta agttgttaaa gaaaatctag 60cccattccat ataaatctta
ttaatacctt actccaggac ctgaaagaca acctacccaa 120ctatcattaa
acaatttagt ctataccacc tatgcccaaa tagatataga tatatagaaa
180caaaccaggt ggaagaggaa taacagagaa gtrtataaaa taaatatgca
acttcagact 240tgtatgctca agccgcaagc ttatatttga agatgtctat
cccacaattc cagatccacc 300taaacaagaa taaaaatcca caaaacatta
gaagcgaatt cgctgctgca aattagaata 360taaaattctt aaaaataatc
cttaccaaaa gattcatcac attaataata gtatccatgc 420ccatgtgagc
catcaccccc rtaataagat gaatatgaac ctctaccata a 4715489DNAGlycine
maxmisc_feature(1)..(489)m is a or cmisc_feature(1)..(489)w is a or
t 5atctcaatct gctttgacag atagagagaa ggagaatctt gacaacttag
ttgacaagga 60gtgtgcaacm acagagggta tgatatcgag ccagctcaag ttgaagtctt
ctttgtcatc 120caaagcgtct tctcatcaaa gcatggacaa acatgcaatc
cttcgacgta tcaggcaacg 180caggtttaat aacaaagcca aaaccgctct
ggaagctctc ataggcacct cagaagccaa 240caataccggc actgcccaag
aacagaagtg gcttcaactg ggtgactctt tctcatctcc 300ttaactggaa
acacaatctc cccccagttt gcaacccatc tataaatwcc ctctttagtt
360tccttcaccc aacaaagtca tcttttccat acgcttgcat ggctaggaaa
ggaaatccaa 420cccgatcttt atcggcatgc ttaagaaatc ccactatgta
tctattaact gtagtagacc 480tggtcggtt 4896794DNAGlycine
maxmisc_feature(1)..(794)r is a or gmisc_feature(1)..(794)w is a or
t 6ccaaccacgc tgattcttta ggccatcagt caacccttta ctctggaaac
tgctaaggaa 60tataaacctt gaaccagaca tgtcccctga agagtagaaa aatatcatgt
ttcagcagca 120tggttcaaga aaatttgttc atgtaggcaa aattgatttt
gaaagaaatt gattatgcaa 180aattaatgtt ggttaagatt aattttaaag
taatgtgatt tatgtttgaa tattttttat 240cgtagcaact cagtataact
tttttaattc aatgcaaaaa tattcaaagt tattttgact 300caatcaattc
tagattcaat atcaattcct catcattgaa acaaatccca acatgtattt
360aaaatcacga tagttgatat ttcaacgtaa tttcagaaaa atcaacataa
aactaaccac 420gcatcactta atgcgtgcta gggtgagcat taaaattgat
ttcaaataaa attaattttc 480taaaattggt tttggtaaaa attgattttg
aagtgacatg agttatgttt gaacgttttt 540tattctaaaa gttaataaca
aatttcaawt tttttttatc caaagcaaaa gtcatctaaa 600agttatttga
actcgaaatc aaatttaaac tcaaaatcaa ttccttaatg tgaaattaaa
660catgtgtcca tttacctcaa atcatgttat ccgatatttc aacaagactt
taaaraatcc 720tacgtgaaac caaacatgca cttactagaa taagcagagt
gaatagcaga aatacccaga 780ttgtaagctg caga 7947568DNAGlycine
maxmisc_feature(1)..(568)r is a or gmisc_feature(1)..(568)w is a or
t 7gttttacaca gttgcacaaa gtcataaact tcagtgacca ttttttatat
tagcctggca 60gtaagatggc tatttatttc tgagtgtagt ttgtactgat ttaatggttt
tattatatgc 120ctagactttt ctttgatatc aatatcttat gtcatcttct
cttggttgtg gatggtattt 180ttcttctgtg gtgtawtttg ttaaaccaat
gatattgtat attaattact tttgtaattt 240tgatctagta atctattttc
atggctattt taaaatattg tagtgggttg aggggaaaat 300atttagggaa
attatttagg ttgactgtgc cttgcctttt aggaatgcat aacgactttt
360tttcttctaa cagttgratt atatacttag ctttgtaatt aattttgttt
arttattcaa 420ccgatttttt tcccacttta ttagcattta tcatgatctg
gttccttcac tatattttgt 480ttctgctagt gccaattttt tacctcaaga
ctttactttt gtagcttttg tgctgtttat 540ttgtttctgt ggatttgtct cgattcat
5688503DNAGlycine maxmisc_feature(1)..(503)r is a or
gmisc_feature(1)..(503)w is a or tmisc_feature(1)..(503)y is c or t
8ccgagacgrg tgaggaaatc ctcgaggtgg gtcaaggacc cctcgtgctt gcttgggtcg
60aacatggagg caacgagagt acaaaatgtt agggacacar taagctggtt ttygaggaac
120tagtacctcc agcaattcaa taaagaagaa gaagaacaaa gagggaaaga
gtgaggtaat 180tggaaatttc attcatgcct tgcttgttat ttacattagt
atttataaac ttcctagtaa 240cagaaagatc ctaggaatat ttgcccataa
caaaatttgt acacattgtc ccctaggagc 300agacaatatc aaccaaggaa
attctagaag gtagcctcag ctggtctcac ccwtcttcca 360aaaacgtact
ctttraggta agaaggtttt gtaattcttc tttcgaactt agcttcttcc
420tgtaccccct catttgcttt tgtgacccct gcccttgcct ctgtttgtaa
gctcgtatca 480acatrtttcc gatacagatg cag 5039769DNAGlycine
maxmisc_feature(1)..(769)m is a or cmisc_feature(1)..(769)r is a or
gmisc_feature(1)..(769)s is c or gmisc_feature(1)..(769)w is a or
tmisc_feature(1)..(769)y is c or t 9aacagagttg gctataacaa
ggttttggat aatgccaagc ccatgaatga tccagatgga 60aggcactttt catcttttac
ttacctgagg cttagttcac ttctcatgga acgacaaaac 120ttcatagagt
ttgaaagatt tgtcaaraga atgcatggta agttgatgaa gaaaatttta
180aatgtatcty tagcctctaa ttgatttatg aaaatatcta ttaaggaaag
aaaatcatcc 240ctttatctgg ccttgattga atgaatgctc acgtgtcatc
tgraaccttc ttactccaca 300gtcccacact cagtagggta aatgggtaat
carcaaaacc ctgaagaccc aaatttcttt 360cagaggcatg gttcagcaac
tgtactaata tcatattgaa ctttggattc cattattcgm 420tcccactgat
agtgttcttt gatccacctw gcactagtct ttttatctga ttggaatcat
480tttataaata attgaacacg aaasgagtat taatgtcagt tttaagaggt
gtacttctct 540tggagagaaa aacactgaaa ctttaagtag ctgcaaaagt
tgttgctatc ttgtgggaca 600ttgattatat tagggttgat gactgtgtca
tttctcattg caggggaagc tgttcttgat 660cttcaggtat aaccggtggg
aaacaaagtc ttgttgaatt tgtatttata agtcccctga 720caaaatggtt
gggggattct gttatagaat aggagaaata ggattgtta 76910513DNAGlycine
maxmisc_feature(1)..(513)m is a or cmisc_feature(1)..(513)r is a or
g 10gagaaagaga agtattgcgc atcatgccca aatgtggcca cacttttcat
ctttcttgca 60ttgatatatg gctgaggaaa caatccacct gtccagtatg ccgtctgccg
ttgaaaaact 120cttccgaaac gaaacatgtg agacctgtga catttaccat
gagccaatcc cttgacgagt 180ctcacacatc agacagaaac gatgmtattg
agagatatgt tgaacctaca cctactgcag 240ccagtaactc tttacaacca
acttcaggag aacaagaagc aagrcaatga tcttagagaa 300ctaaaggggt
tgttctgctc aaaaagagaa gaatgtagaa tttctgcttc tatagaggaa
360tgcttctaat tatagattgg attcaaattc tttgtctgta atatggcctt
catattcact 420tggtggtgta aatatgtttc cttttgtagc atatgcgggc
caaggttttg gtggaatttc 480ttgcataccg atttgaagtt cttttgtcta tgg
51311281DNAGlycine maxmisc_feature(1)..(281)y is c or t
11atccaccaac accacttcac caccaaagcc tagtggagct gtaactgttt ccagtggcat
60tggccttttg gtggggtctt tctttgtgtc agcaattatg tytggtttca tgggctaggg
120aagaggcagt gctgtttttt tttttattct cctttttatt aatatgttat
tgttttgctg 180agagtttgtg tgaatttcat catttaatcc agatgtatat
ctaatcttga tatgtatctc 240cttttaatat ttaaaaatga cacatttctt
tagctgcttc c 28112948DNAGlycine maxmisc_feature(1)..(948)w is a or
tmisc_feature(1)..(948)n is a or g or c or t 12tgaatttttg
ttatgagagc atctccattt taaagagatt tcgtttctgg gtatcagaaa 60acacctccct
tccgattaga tttctatatt ttgcctagac gtcaaatgca cgatgaattc
120tttgtatgat gtttgaaaat tgaagtcttc tgtaatacwt gattatcaac
ctcaaatttt 180gatgcgacaa caagttatgc tttgtgaant tgttattgat
gattgtatgg tgtgatttgg 240tttcaggttt gaaacttgtt actgtggacc
gtccttttgc tgagaagcac tatgctgatt 300tgtctgccaa gcctttcttt
agtggtttgg tggattacat tatctctggc cctgttgttg 360ccatgatctg
ggagggcaag aatgttgtta caactggccg aaagattatn ggagccacaa
420acccagctca atctgagcct ggaactatcc gtggtgactt tgccattgac
attggaaggt 480atagacttgt tttcatcatc gttcaagcaa gtttacgttt
tcaaatcttt tgttttaaca 540catgaatatg aatctgtttt cattgttgtt
catgtttcta atctagtttg gtggatgtta 600ggttttcatt nttgttgttt
gattttcaat ttctcccgaa tttttttctt atttacaaca 660ttggtttcca
acgttctgtg ttttagagaa aagagtgttg tgctcaacca attctgcgaa
720tattgcaata tgttcatgac atcatcaatt aagcaagatg ataacactgt
tgagtacatc 780ataatcaagg gagtggaatc caacaaatcc atgttctgta
tcttgtattc atgaaaatca 840aaattgttgg ggtagtcaat gtttatcaac
attaaaaggt agtgtgnatc tanatactta 900ttatttncaa ttctataaag
gatttgaagg gaactttgaa tttttttt 94813485DNAGlycine
maxmisc_feature(1)..(485)r is a or g 13ttctatgacc aacctaccgc
ctgtgggtaa agctaaacta agttctcaca ggtgaggtga 60gttcaccaac cacggccgcc
agaatttcca gatccccatc cgttccctga tgctgctttc 120ttgggggcag
atccccagcc agagcttcct tgttcaccat cacttggacc agcaccacca
180ctcgcttgtc ccccccagcc accattgtca ctgttactag ctccacctcc
tccccctccc 240caaccactac tgcctccacc gcttccacca ccaccacccc
aaccaccagg aaaagcttcc 300cttccaggag aattctgaac cttggcccct
ggaaaattgc ttaaaccatc atccgartcc 360tttgtattat tagaacccca
tctgccccca taaccagagt cctgcctttc attattggag 420ttgtctcctc
tattgttata agaacctcga ccacgaccac gtacacgccc ttttccaccc 480accga
4851420DNAGlycine max 14aggatttgcc tcaggagctt 201522DNAGlycine max
15gaaactccac cactacaaaa cc 221616DNAGlycine max 16agtggggact atgatg
161717DNAGlycine max 17agtgggaact atgatgg 171831DNAGlycine max
18caatcagcaa atgatacaat gacaatacag a 311922DNAGlycine max
19agctccacac ctcattggtt tt 222019DNAGlycine max 20tttcattagg
gtgctacaa 192121DNAGlycine max 21tctttcatta gggtactaca a
212222DNAGlycine max 22caaaagctcc acacctcatt gg 222325DNAGlycine
max 23ttgacatcag gaagtaatgg gtagt 252422DNAGlycine max 24ttgaaacgaa
attattattc tg 222521DNAGlycine max 25tgttttgaaa cgaaattact a
212623DNAGlycine max 26gggactcagt tacgcaagtt ttg 232721DNAGlycine
max 27gtgctcctct caccacagta g 212819DNAGlycine max 28aagctgttgg
ttatgattt 192918DNAGlycine max 29agctgttggt tatgactt
183020DNAGlycine max 30atcccacaat tccagatcca 203126DNAGlycine max
31ttatggtaga ggttcatatt catctt 263216DNAGlycine max 32agccatcacc
cccgta 163316DNAGlycine max 33agccatcacc cccata 163423DNAGlycine
max 34agtctatacc acctatgccc aaa 233522DNAGlycine max 35ggcttgagca
tacaagtctg aa 223620DNAGlycine max 36aggaataaca gagaagtgta
203722DNAGlycine max 37aggaataaca gagaagtata ta 223825DNAGlycine
max 38agagagaagg agaatcttga caact 253922DNAGlycine max 39acgtcgaagg
attgcatgtt tg 224016DNAGlycine max 40aggagtgtgc aaccac
164117DNAGlycine max 41aggagtgtgc aacaaca 174220DNAGlycine max
42ccccagtttg caacccatct 204323DNAGlycine max 43ccatgcaagc
gtatggaaaa gat 234417DNAGlycine max 44ctaaagaggg aatttat
174520DNAGlycine max 45aaactaaaga gggtatttat 204629DNAGlycine max
46gtgtccattt acctcaaatc atgttatcc 294724DNAGlycine max 47gtaagtgcat
gtttggtttc acgt 244818DNAGlycine max 48aagactttaa agaatcct
184919DNAGlycine max 49caagacttta aaaaatcct 195022DNAGlycine max
50taaatgctaa taaagtggga aa 225116DNAGlycine max 51aggttgactg tgcctt
165221DNAGlycine max 52agctaagtat ataatccaac t 215322DNAGlycine max
53agctaagtat ataattcaac tg 225425DNAGlycine max 54ggaggcaacg
agagtacaaa atgtt 255525DNAGlycine max 55tgaattgctg gaggtactag ttcct
255616DNAGlycine max 56cagcttactg tgtccc 165717DNAGlycine max
57ccagcttatt gtgtccc 175822DNAGlycine max 58gaacatggag gcaacgagag
ta 225929DNAGlycine max 59ttcttcttta ttgaattgct ggaggtact
296016DNAGlycine max 60ttcctcaaaa accagc 166115DNAGlycine max
61tcctcgaaaa ccagc 156223DNAGlycine max 62tttcataaat caattagagg cta
236319DNAGlycine max 63gaggcttagt tcacttctc 196416DNAGlycine max
64accatgcatt ctcttg 166517DNAGlycine max 65accatgcatt cttttga
176622DNAGlycine max 66tcccttgacg agtctcacac at 226724DNAGlycine
max 67ttgcttcttg ttctcctgaa gttg 246818DNAGlycine max 68cagacagaaa
cgatgata 186917DNAGlycine max 69cagaaacgat gctattg 177019DNAGlycine
max 70gtggcattgg ccttttggt 197119DNAGlycine max 71cactgcctct
tccctagcc 197218DNAGlycine max 72catgaaacca aacataat
187318DNAGlycine max 73catgaaacca gacataat 187429DNAGlycine max
74gcacgatgaa ttctttgtat gatgtttga 297525DNAGlycine max 75aaagcataac
ttgttgtcgc atcaa 257620DNAGlycine max 76ttctgtaata cttgattatc
207720DNAGlycine max 77ttctgtaata catgattatc 207823DNAGlycine max
78tcccttccag gagaattctg aac 237923DNAGlycine max 79tgaaaggcag
gactctggtt atg 238017DNAGlycine max 80aaccatcatc cgaatcc
178116DNAGlycine max 81taaaccatca tccgag 1682459DNAGlycine max
82ttttgatcca aaacaaagct gaaaagaaag gggacaggta tgaagcaatc ttcagcttct
60actttggaga ctatggtcac atagcagtgc agggacctta cctgacctat gaggacacat
120atttggctgt gactggtggg tctggcatat ttgagggtgt taaaggtcaa
gtgaagctgc 180gtcagattgt
gtatcctttc aagattttgt acacatttta tctaaagggt atcaaggatt
240tgcctcagga gcttcttgtc aagactgttg agccaattcc atctgttgaa
ccttcccctg 300ctgctaaggc ccttgagccc aatgctacca ttgctggctt
caccgactaa ttcatcaact 360ttttttgtat ttgctttggc ctttgtagta
gtatgattta agttactgaa taataataac 420aagtggggac tatgatgggt
tttgtagtgg tggagtttc 459831102DNAGlycine max 83ttacaccaga
atcatggcca ccaatcaaat acccctatca tcttattaca gaaaagggga 60taagagaagg
ggaaaaaaaa tctctagaaa ctgaagaatc aaggtttggt tcaaaaactg
120tgcagttgcg ttagcatgtg aacaagctca tcactagaag gcctatctgc
aggcatatca 180gagaggcatg cagcagcaat cctaaccgcc attagcatct
catcttcctc accttcttcc 240cctaacatgc tcttatctag agcttcgtgc
gcctcgcccg cttgctgcaa gtgtctcagc 300caacatccca aacttccccc
actggctgct tctccaaaga atggatctgt aggatcctta 360ccagttaaca
aaacacctag tatcatgcca aaactaaaga tgtcactttt gtcagtgtac
420ctgctgaaat tgccaaaaaa caccattcaa tctgaatacc atgaagctcc
acatatacaa 480gaatggaatg aagttaaaat aaacatctta ttggcaatac
catacccata tatgcaatgc 540catcaatcaa gcaatctttt attgttatta
cttattgcta ttgatattga tgcatccaaa 600tatacaccac tgaaaaacta
gaagtattcc aagttaaatt aggaaaaaaa acctttgtca 660attattatta
taatttagtt gtggtctcac tcaccaatct aggtttagta gtttgcagca
720tgtgaactat aaactatatt attcatttgg accagactta gtgccaactg
cctaaggtct 780aaaacttgac atcaggaagt aatgggtagt tacaaaaaca
aatagcaaag tctttattaa 840ataataccaa attcaatcag caaatgatac
aatgacaata cagattacag aatagtaatt 900tcgtttcaaa acaagagtct
ttcattaggg tactacaaag ggggcaaaaa accaatgagg 960tgtggagctt
ttgaaaaggt aacgaatcaa acaaattccc aatatcaacc acctaattac
1020cctattggtg taaaaaagtg aaagaacaga aacagaccaa gttcaaaaca
ctagtgaatg 1080agtatgattg aaatttgtct ta 110284402DNAGlycine max
84tcagttacgc aagttttgaa gctgttggtt atgactttaa tgacatggct ctaggtgaac
60ttgtggaaag gattttagct tgcttttgtc cagcagagtt ttctgttgct ttgcacattg
120acatgcatgg tgagaaactt aataaatttc ccttagacat caaaggatac
tactgtggtg 180agaggagcac tgaagagctt ggagtaggtg gtgcagttgt
gtaccaaaca tttgttcaag 240gctgcgatgg tagtgcatct cctaggtcta
ttctgaaatg ctgctggagt gaggacgaga 300acgaagatga agtgagggag
atataagtac ttctggtttt tatgttctgt tgttatttat 360ctttactgtc
gagtgtgtta ttacctaaat aaataaatat gg 40285466DNAGlycine max
85atcctcaaat acaagtccct gatctcatca tacctaagtt gttaaagaaa atctagccca
60ttccatataa atcttattaa taccttactc caggacctga aagacaacct acccaactat
120cattaaacaa tttagtctat accacctatg cccaaataga tatagatata
tagaaacaaa 180ccaggtggaa gaggaataac agagaagtgt ataaaataaa
tatgcaactt cagacttgta 240tgctcaagcc gcaagcttat atttgaagat
gtctatccca caattccaga tccacctaaa 300caagaataaa aatccacaaa
acattagaag cgaattcgct gctgcaaatt agaatataaa 360attcttaaaa
ataatcctta ccaaaagatt catcacatta ataatagtat ccatgcccat
420gtgagccatc acccccgtaa taagatgaat atgaacctct accata
46686489DNAGlycine max 86atctcaatct gctttgacag atagagagaa
ggagaatctt gacaacttag ttgacaagga 60gtgtgcaacc acagagggta tgatatcgag
ccagctcaag ttgaagtctt ctttgtcatc 120caaagcgtct tctcatcaaa
gcatggacaa acatgcaatc cttcgacgta tcaggcaacg 180caggtttaat
aacaaagcca aaaccgctct ggaagctctc ataggcacct cagaagccaa
240caataccggc actgcccaag aacagaagtg gcttcaactg ggtgactctt
tctcatctcc 300ttaactggaa acacaatctc cccccagttt gcaacccatc
tataaatacc ctctttagtt 360tccttcaccc aacaaagtca tcttttccat
acgcttgcat ggctaggaaa ggaaatccaa 420cccgatcttt atcggcatgc
ttaagaaatc ccactatgta tctattaact gtagtagacc 480ttgtcggtt
48987794DNAGlycine max 87ccaaccacgc tgattcttta ggccatcagt
caacaaatta ctctggaaac tgctaaggaa 60tataaacctt gaaccagaca tgtcccctga
agagtagaaa aatatcatgt ttcagcagca 120tggttcaaga aaatttgttc
atgtaggcaa aattgatttt gaaagaaatt gattatgcaa 180aattaatgtt
ggttaagatt aattttaaag taatgtgatt tatgtttgaa tattttttat
240cgtagcaact cagtataact tttttaattc aatgcaaaaa tattcaaagt
tattttgact 300caatcaattc tagattcaat atcaattcct catcattgaa
acaaatgcca acatgtattt 360aaaatcacga tagttgatat ttcaacgtaa
ttcaagaaaa ttcaacataa aactaaacac 420gcatcactta atgcgtgcta
gggtgagcat taaaattgat ttcaaataaa attaattttc 480taaaattggt
tttggtaaaa attgattttg aagtgacatg agttatgttt gaacgttttt
540tattctaaaa gttaataaca aatttcaaat tttttttatc caaagcaaaa
gtcatctaaa 600agttatttga actcgaaatc aaatttaaac tcaaaatcaa
ttccttaatg tgaaattaaa 660catgtgtcca tttacctcaa atcatgttat
ccgatatttc aacaagactt taaagaatcc 720tacgtgaaac caaacatgca
cttactagaa taagcagagt gaatagcaga aatacccaga 780ttgtaagctg caga
79488568DNAGlycine max 88atgaatagag acaaatccac agaaacaaat
aaacagcaca aaagctacaa aagtaaagtc 60ttgaggtaaa aaattggcac tagcagaaac
aaaatatagt gaaggaacca gatcatgata 120aatgctaata aagtgggaaa
aaaatcggtt gaataattaa acaaaattaa ttacaaagct 180aagtatataa
ttcaactgtt agaagaaaaa aagtcgttat gcattcctaa aaggcaaggc
240acagtcaacc taaataattt ccctaaatat tttcccctca acccactaca
atattttaaa 300atagccatga aaatagatta ctagatcaaa attacaaaag
taattaatat acaatatcat 360tggtttaaca aattacacca cagaagaaaa
ataccatcca caaccaagag aagatgacat 420aagatattga tatcaaagaa
aagtctaggc atataataaa accattaaat cagtacaaac 480tacactcaga
aataaatagc catcttactg ccaggctaat ataaaaaatg gtcactgaag
540tttatgactt tgtgcaactg tgtaaaac 56889503DNAGlycine max
89ccgagacgag tgaggaaatc ctcgaggtgg gtcaaggacc cctcgtgctt gcttgggtcg
60aacatggagg caacgagagt acaaaatgtt agggacacaa taagctggtt ttcgaggaac
120tagtacctcc agcaattcaa taaagaagaa gaagaacaaa gagggaaaga
gtgaggtaat 180tggaaatttc attcatgcct tgcttgttat ttacattagt
atttataaac ttcctagtaa 240cagaaagatc ctaggaatat ttgcccataa
caaaatttgt acacattgtc ccctaggagc 300agacaatatc aaccaaggaa
attctagaag gtagcctcag ctggtctcac ccttcttcca 360aaaacgtact
ctttgaggta agaaggtttt gtaattcttc tttcgaactt agcttcttcc
420tgtaccccct catttgcttt tgtgacccct gcccttgcct ctgtttgtaa
gctcgtatca 480acatgtttcc gatacagatg cag 50390762DNAGlycine max
90aacagagttg gctataacaa ggttttggat aatgccaagc ccatgaatga tccagatgga
60aggcactttt catcttttac ttacctgagg cttagttcac ttctcatgga acgacaaaac
120ttcatagagt ttgaaagatt tgtcaagaga atgcatggta agttgatgaa
gaaaatttta 180aatgtatctc tagcctctaa ttgatttatg aaaatatcta
ttaaggaaag aaaatcatcc 240ctttatctgg ccttgattga atgaatgctc
acgtgtcatc tgaaaccttc ttactccaca 300gtcccacact cagtagggta
atcagcaaaa ccctgaagac ccaaatttct ttcagaggca 360tggttcagca
actgtactaa tatcatattg aactttggat tccattattc gctcccactg
420atagtgttct ttgatccacc tagcactagt ctttttatct gattggaatc
attttataaa 480taattgaaca cgaaaggagt attaatgtca gttttaagag
gtgtacttct cttggagaga 540aaaacactga aactttaagt agctgcaaaa
gttgttgcta tcttgtggga cattgattat 600attagggttg atgactgtgt
catttctcat tgcaggggaa gctgttcttg atcttcaggt 660ataaccggtg
ggaaacaaag tcttgttgaa tttgtattta taagtcccct gacaaaatgg
720ttgggggtat tctgttatag aataggagaa ataggattgt ta
76291513DNAGlycine max 91gagaaagaga agtattgcgc atcatgccca
aatgtggcca cacttttcat ctttcttgca 60ttgatatatg gctgaggaaa caatccacct
gtccagtatg ccgtctgccg ttgaaaaact 120cttccgaaac gaaacatgtg
agacctgtga catttaccat gagccaatcc cttgacgagt 180ctcacacatc
agacagaaac gatgatattg agagatatgt tgaacctaca cctactgcag
240ccagtaactc tttacaacca acttcaggag aacaagaagc aaggcaatga
tcttagagaa 300ctaaaggggt tgttctgctc aaaaagagaa gaatgtagaa
tttctgcttc tatagaggaa 360tgcttctaat tatagattgg attcaaattc
tttgtctgta atatggcctt catattcact 420tggtggtgta aatatgtttc
cttttgtagc atatgcgggc caaggttttg gtggaatttc 480ttgcataccg
atttgaagtt cttttgtcta tgg 51392280DNAGlycine max 92atccaccaac
accacttcac caccaaagcc tagtggagct gtaactgttt ccagtggcat 60tggccttttg
gtggggtctt tctttgtgtc agcaattatg tttggtttca tgggctaggg
120aagaggcagt gctgtttttt ttttattctc ctttttatta atatgttatt
gttttgctga 180gagtttgtgt gaatttcatc atttaatcca gatgtatatc
taatcttgat atgtatctcc 240ttttaatatt taaaaatgac acatttcttt
agctgcttcc 28093883DNAGlycine max 93agatttcgtt tctgggtatc
agaaaacacc taggttccga ttagatttct atattttgcc 60tagacgtcaa atgcacgatg
aattctttgt atgatgtttg aaaattgaag tcttctgtaa 120tacttgatta
tcaacctcaa attttgatgc gacaacaagt tatgctttgt gaagttgtta
180ttgatgattg tatggtgtga tttggtttca ggtttgaaac ttgttactgt
ggaccgtcct 240tttgctgaga agcactatgc tgatttgtct gccaagcctt
tctttagtgg tttggtggat 300tacattatct ctggccctgt tgttgccatg
atctgggagg gcaagaatgt tgttacaact 360ggccgaaaga ttatcggagc
cacaaaccca gctcaatctg agcctggaac tatccgtggt 420gactttgcca
ttgacattgg aaggtataga cttgttttca tcatcgttca agcaagttta
480cgttttcaaa tcttttgttt taacacatga atatgaatct gttttcattg
ttgttcatgt 540ttctaatcta gtttggtgga tgttaggttt tcatttttgt
tgtttgattt tcaatttctc 600ccgaattttt ttcttattta caacattggt
ttccaacgtt ctgtgtttta gagaaaagag 660tgttgtgctc aaccaattct
gcgaatattg caatatgttc atgacatcat caattaagca 720agatgataac
actgttgagt acatcataat caaggcagtg gaatccaaac aaatccatgt
780tctgtatctt tgtattcatg aaaatcaaaa attgttgttt tagtcaatgt
ttatcaacat 840tgctcttagt gtggatctag atacttatta tttacaattc tat
88394475DNAGlycine max 94atgaccaacc taccgcctgt gggtaaagct
aaactaagtt ctcacaggtg aggtgagttc 60accaaccacg gccgccagaa tttccagatc
cccatccgtt ccctgatgct gctttcttgg 120gggcagatcc ccagccagag
cttccttgtt caccatcact tggaccagca ccaccactcg 180cttgtccccc
ccagccacca ttgtcactgt tactagctcc acctcctccc cctccccaac
240cactactgcc tccaccgctt ccaccaccac caccccaacc accaggaaaa
gcttcccttc 300caggagaatt ctgaaccttg gcccctggaa aattgcttaa
accatcatcc gagtcctttg 360tattattaga accccatctg cccccataac
cagagtcctg cctttcatta ttggagttgt 420ctcctctatt gttataagaa
cctcgaccac gaccacgtcc acgccctctt ccacc 475
* * * * *