U.S. patent application number 12/016783 was filed with the patent office on 2008-05-29 for identification of soybeans having resistance to phytophthora sojae.
This patent application is currently assigned to THE OHIO STATE UNIVERSITY RESEARCH FOUNDATION. Invention is credited to Kara Burnham, Anne Dorrance, Ron Fioritto, David Francis, Stuart G. Gordon, Steven St. Martin.
Application Number | 20080127361 12/016783 |
Document ID | / |
Family ID | 34886551 |
Filed Date | 2008-05-29 |
United States Patent
Application |
20080127361 |
Kind Code |
A1 |
St. Martin; Steven ; et
al. |
May 29, 2008 |
IDENTIFICATION OF SOYBEANS HAVING RESISTANCE TO PHYTOPHTHORA
SOJAE
Abstract
The invention provides soybean plants having a novel
determinant, Rps8, for resistance to Phytophthora sojae. The
invention also provides methods for identifying germplasms that are
either heterozygous or homozygous for Rps8 using marker assisted
selection. Genetic markers with known chromosomal location that are
associated with the Rps8 gene are used to confirm Rps8-derived
Phytophthora sojae resistance in germplasms. Marker assisted
selection also used when introgressing Rps8-derived soybean
Phytophthora sojae resistance into non-resistant soybean germplasm
or less resistant soybean germplasms.
Inventors: |
St. Martin; Steven;
(Columbus, OH) ; Dorrance; Anne; (Wooster, OH)
; Burnham; Kara; (Portland, OR) ; Fioritto;
Ron; (Wooster, OH) ; Francis; David; (Wooster,
OH) ; Gordon; Stuart G.; (Stow, OH) |
Correspondence
Address: |
CALFEE HALTER & GRISWOLD, LLP
800 SUPERIOR AVENUE, SUITE 1400
CLEVELAND
OH
44114
US
|
Assignee: |
THE OHIO STATE UNIVERSITY RESEARCH
FOUNDATION
Columbus
OH
|
Family ID: |
34886551 |
Appl. No.: |
12/016783 |
Filed: |
January 18, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10778018 |
Feb 12, 2004 |
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12016783 |
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10436376 |
May 12, 2003 |
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10778018 |
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60379304 |
May 10, 2002 |
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60427637 |
Nov 19, 2002 |
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Current U.S.
Class: |
800/265 ;
536/23.1; 800/301 |
Current CPC
Class: |
A01H 5/10 20130101; C12Q
2600/172 20130101; C12Q 2600/156 20130101; A01H 1/02 20130101; C12Q
1/6895 20130101; A01H 1/04 20130101 |
Class at
Publication: |
800/265 ;
536/23.1; 800/301 |
International
Class: |
A01H 1/02 20060101
A01H001/02; C07H 21/04 20060101 C07H021/04; A01H 5/00 20060101
A01H005/00 |
Goverment Interests
STATEMENT ON GOVERNMENT FUNDED RESEARCH
[0002] The present invention was made, at least in part, with
support from the United States Department of Agriculture through
Hatch Fund grants made to the Ohio Agricultural Research and
Development Center. The United States Government has certain rights
in the invention.
Claims
1. A method for introgressing Rps8-derived Phytophthora sojae
resistance into non-resistant or less resistant soybean germplasm
comprising: (A) crossing at least one Rps8 soybean plant having an
Rps8 gene with at least one non-resistant or less resistant soybean
germplasm lacking said Rps8 gene, to form a segregating population;
(B) screening soybean from said segregating population for the
presence of said Rps8 gene to determine if one or more soybean from
said segregating population contains said Rps8 gene; and (C)
selecting one or more soybean from said segregating population
containing said Rps8 gene, wherein the presence of said Rps8 gene
confers Rps8-derived Phytophthora sojae resistance to all P. sojae
pathotypes vir1a, 1b, 1c, 1d, 1k, 2, 3a, 3b, 3c, 4, 5, 6 and 7.
2. The method of claim 1, wherein the screening in step B
comprises: (i) analyzing genomic DNA of soybean from said
segregating population for the presence of a combination of
molecular markers on major linkage group F which are associated
with Rps8 locus, whereby detecting the presence of the molecular
markers provides an indication that the Rps8 gene is present in the
analyzed soybean; and (ii) confirming the presence of the Rps8 gene
in the analyzed soybean by inoculating the analyzed soybean with
one or a combination of P. sojae pathotypes vir1a, 1b, 1c, 1d, 1k,
2, 3a, 3b, 3c, 4, 5, 6 or 7 to which the soybean would be
susceptible if the soybean did not have Rps8-derived P. sojae
resistance and selecting resistant soybean, wherein the resistant
soybean from said segregating population contain said Rps8
gene.
3. The method of claim 2, wherein the molecular markers are
selected from the group consisting of Satt516, Satt595, Satt114,
Satt334, Sat.sub.--317, Sat.sub.--197, Satt510, Satt335 and
Satt144.
4. The method of claim 3, wherein the molecular markers are Satt516
and Satt114.
5. The method of claim 1, wherein the Rps8 soybean plant of step
(A) is selected from the group consisting of: PI 399073 or a
descendant thereof; HFX01-602 or a descendant thereof, OX-99128 or
a descendant thereof, OX-98317 or a descendant thereof, and
OX-99218 or a descendant thereof.
6. The method of claim 1, wherein the screening in step B comprises
inoculating the soybean from said segregating population with one
or more P. sojae pathotypes selected from the group consisting of
pathotypes vir1a, 1b, 1c, 1d, 1k, 2, 3a, 3b, 3c, 4, 5, 6 and 7 to
which the non-resistant or less resistant soybean germplasm is
susceptible and selecting resistant soybean, wherein the resistant
soybean from said segregating population contain said Rps8
gene.
7. The method of claim 6, wherein the P. sojae pathotypes are those
present in one or more P. sojae isolates selected from P. sojae
race 1, race 4, race 17, race 25 or race 30.
8. The method of claim 6, wherein the one or more P. sojae
pathotypes have virulence to Rps3a, Rps3b, or Rps3c, but not
Rps8.
9. The method of claim 1, wherein the screening in step B
comprises: (a) detecting in the soybean from said segregating
population a first nucleic acid which is genetically linked to Rps8
locus, wherein said Rps8 locus is mapped to major linkage group F
and is located between molecular markers Satt114 and Satt516; and
(b) identifying a soybean having said first nucleic acid; (c)
inoculating the identified soybean of (b) with one or a combination
of P. sojae pathotypes vir1a, 1b, 1c, 1d, 1k, 2, 3a, 3b, 3c, 4, 5,
6 or 7 to which the identified soybean would otherwise be
susceptible if the identified soybean did not have Rps8-derived P.
sojae resistance, and selecting resistant soybean; wherein the
resistant soybean from said segregating population contain said
Rps8 gene.
10. The method of claim 9, wherein the first nucleic acid is a
marker selected from the group consisting of: (a) Satt 114, Satt
516, Satt595, Satt334, Sat.sub.--317, Sat.sub.--197, Satt510,
Satt335, Satt144; and (b) a marker linked to a marker of (a).
11. The method of claim 9, wherein detecting the first nucleic acid
comprises: restriction fragment length polymorphisms ("RFLP")
analysis, single sequence repeats ("SSR") analysis, random
amplified polymorphic DNA ("RAPD") analysis, or amplified fragment
length poymorphisms (AFLPs) analysis.
12. The method of claim 1, wherein the screening in step B
comprises: (a) detecting a first locus on the genome of the soybean
from said segregating population which is genetically linked to
Rps8 locus, wherein the first locus maps to major linkage group F
and is located between molecular marker Satt114 and Satt516; and
selecting a soybean from said segregating population having said
first locus; and (b) determining that the soybean from said
segregating population that has said first locus does not have
Rps3.
13. The method of claim 12, wherein the first locus is linked to a
marker selected from the group consisting of Satt 516, Satt595,
Satt334, Sat.sub.--317, Sat.sub.--197, Satt510, Satt335, Satt 114
and Satt144.
14. The method of claim 1, further comprising the step of
determining that the at least one Rps8 soybean plant of step (A)
and the at least one non-resistant or less resistant soybean
germplasm of step (A) lack Rps3.
15. An Rps8 gene, wherein said Rps8 gene confers soybean resistance
to all Phytophthora sojae pathotypes vir1a, 1b, 1c, 1d, 1k, 2, 3a,
3b, 3c, 4, 5, 6, 7, and wherein said Rps8 gene is within an Rps8
locus that maps to a region of soybean major linkage group F that
is flanked by molecular marker Satt516 and a molecular marker
selected from the group consisting of Satt114, Satt334,
Sat.sub.--317, Satt335, Satt510, Satt144 and Sat.sub.--197.
16. The Rps8 gene of claim 15, wherein the Rps8 locus maps to a
region of soybean major linkage group F that is flanked by
molecular marker Satt516 and Satt114.
17. A method for the production of an inbred soybean cultivar
adapted for conferring, in hybrid combination with a suitable
second inbred, Rps8-derived resistance to Phytophthora sojae
pathotypes vir1a, 1b, 1c, 1d, 1k, 2, 3a, 3b, 3c, 4, 5, 6 and 7,
comprising: selecting a first donor parental line possessing the
desired Rps-8 derived Phytophthora sojae resistance said first
donor parental line having an Rps8 gene that maps to linkage group
F; crossing the first donor parental line with a second parental
line, which is high yielding in hybrid combination, to produce a
segregating plant population of genetically heterogeneous plants;
screening the segregating plant population for the Rps8 gene;
selecting plants from the segregating plant population having the
Rps8 gene; and breeding by self crossing the selected plants
containing the Rps8 gene until a line is obtained which is
homozygous for Rps8 gene to give Rps8-derived resistance to
Phytophthora sojae pathotypes vir1a, 1b, 1c, 1d, 1k, 2, 3a, 3b, 3c,
4, 5, 6 and 7 in hybrid combination.
18. The method of claim 17, wherein the first donor parental line
is selected from the group consisting of: PI 399073 or a descendant
thereof, HFX01-602 or a descendant thereof, OX-99128 or a
descendant thereof; OX-98317 or a descendant thereof; and OX-99218
or a descendant thereof.
19. A soybean plant having Rps8-derived resistance to Phytophthora
sojae produced by introgression of a soybean germplasm containing
an Rps8 gene in its genome with a soybean germplasm lacking the
Rps8 gene in its genome, wherein the presence of said Rps8 gene
confers Rps8-derived Phytophthora sojae resistance to all P. sojae
pathotypes vir1a, 1b, 1c, 1d, 1k, 2, 3a, 3b, 3c, 4, 5, 6 and 7.
20. The soybean plant of claim 19, wherein the introgression
comprises: (A) crossing a soybean germplasm containing an Rps8 gene
in its genome with a soybean germplasm lacking the Rps8 gene in its
genome, to form a segregating population; (B) screening soybean
from said segregating population for the presence of the Rps8 gene
to determine if one or more soybean from said segregating
population contains the Rps8 gene; and (C) selecting one or more
soybean from said segregating population containing the Rps8
gene.
21. The soybean plant of claim 20, wherein the screening in step B
comprises: (i) analyzing genomic DNA of soybean from said
segregating population for the presence of a combination of
molecular markers on major linkage group F which are associated
with Rps8 locus, whereby detecting the presence of the molecular
markers provides an indication that the Rps8 gene is present in the
analyzed soybean; and (ii) confirming the presence of the Rps8 gene
in the analyzed soybean by inoculating the analyzed soybean with
one or a combination of P. sojae pathotypes vir1a, 1b, 1c, 1d, 1k,
2, 3a, 3b, 3c, 4, 5, 6 or 7 to which the soybean would be
susceptible if the soybean did not have Rps8-derived P. sojae
resistance and selecting resistant soybean, wherein the resistant
soybean from said segregating population contain the Rps8 gene.
22. The soybean plant of claim 21, wherein said segregating
population is formed from crossing a soybean germplasm containing
an Rps8 gene in its genome selected from the group consisting of:
PI 399073 or a descendant thereof, HFX01-602 or a descendant
thereof; OX-99128 or a descendant thereof; OX-98317 or a descendant
thereof; and OX-99218 or a descendant thereof; with a soybean
germplasm lacking the Rps8 gene in its genome.
23. A method for producing a hybrid soybean plant line having
Rps8-derived resistance to Phytophthora sojae pathotypes vir1a, 1b,
1c, 1d, 1k, 2, 3a, 3b, 3c, 4, 5, 6 and 7, comprising: identifying
one or more molecular markers in an Rps8 soybean plant to be used
as a donor plant, wherein the one or more molecular markers are
linked to an Rps8 locus that maps to a region of linkage group F
that is flanked by molecular marker Satt516 and a molecular marker
selected from the group consisting of Satt114, Satt334,
Sat.sub.--317, Satt335, Satt510, Satt144 and Sat.sub.--197; and
introgressing Rps8-derived resistance to Phytophthora sojae into a
recipient soybean plant which is non-resistant or less resistant to
one or more P. sojae pathotypes vir1a, 1b, 1c, 1d, 1k, 2, 3a, 3b,
3c, 4, 5, 6 and 7, by performing marker assisted selection to
provide a hybrid soybean plant having Rps8-derived resistance to
Phytophthora sojae.
24. The method of claim 23, wherein said introgressing comprises:
providing F2 plants from a segregating F2 plant population derived
from selective breeding, the selective breeding including a first
cross between the donor plant and the recipient soybean plant to
yield a heterozygous F1 plant population, and self-fertilizing one
or more plants from the heterozygous F1 plant population to yield a
segregating F2 plant population; selecting F2 plants having the one
or more molecular markers in their genome; backcrossing the
selected F2 plants with plants from the recipient soybean plant to
yield backcrossed F1 generation plants, and selecting plants from
the backcrossed F1 generation plants that have the one or more
molecular markers in their genome to provide the hybrid soybean
plant having Rps8-derived resistance to Phytophthora sojae.
25. The method of claim 24, wherein the one or more molecular
markers are selected from the group consisting of: (a) Satt 114,
Satt 516, Satt595, Satt334, Sat.sub.--317, Sat.sub.--197, Satt510,
Satt335, Satt144, and (b) a marker linked to a marker of (a).
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 10/778, 018, filed Feb. 12, 2004, which is a
continuation-in-art of U.S. application Ser. No. 10/436,376, filed
May 12, 2003, which claims priority to U.S. Provisional Application
No. 60/379,304, filed May 10, 2002, U.S. Provisional Application
60/427,637, filed Nov. 19, 2002, all of which are incorporated
herein by reference in their entirety.
BACKGROUND OF THE INVENTION
[0003] This invention relates to soybean plants possessing a novel
resistance to Phytophthora sojae, which maps to a chromosomal locus
and methods for identifying and breeding these plants, the methods
involving marker assisted selection.
[0004] Soybeans, (Glycine max L. Merr) are a major cash crop and
investment commodity in North America and elsewhere. Soybean oil is
one of the most widely used edible oils, and soybeans are used
worldwide both in animal feed and in human food production.
Phytophthora root and stem rot is a devastating disease of soybean
that occurs throughout the U.S. and the world (Wrather et al., Can.
J. Plant Pathol., 2001). Phytophthora root and stem rot caused by
Phytophthora sojae is the second leading cause of yield loss in
soybean in the United States. Yield losses in 1998, due to
Phytophthora root and stem rot in top soybean producing countries
were 1149 and 92 thousand metric tons in the U.S. and Argentina,
respectively (Wrather et al., Can. J. Plant Pathol., 2001). General
resistance mechanisms against P. sojae include structural features
of the host, preformed chemical inhibitors, induced structural
barriers, hypersensitive reactions and phytoalexins (Erwin et al.,
eds, Phytophthora, 1983). Phytophthora root and stem rot was first
described in Ohio and shortly thereafter it was described in
Indiana and North Carolina (Suhovecky and Schmitthenner, 1955). The
pathogen is now referred to as Phytophthora sojae.
[0005] Resistance to Phytophthora root and stem rot is a trait
provided by multiple alleles. To date, thirteen resistance (Rps)
alleles at seven loci have been described; Rps1 (Bernard et al.,
Agron. J., 1957), Rps2 (Kilen et al., Crop Sci., 1974), Rps3
(Mueller, Phytopathology, 1978), Rps4 (Athow et al.,
Phytopathology, 1980), Rps5 (Buzzell and Anderson, Soybean Genet.
Newslett., 1981), Rps6 (Athow and Laviolette, Phytopathology,
1982), and Rps7 (Anderson and Buzzel, Plant Dis., 1992). The Rps
resistance loci are found on soybean major linkage groups (MLGs) N,
J, F, and G (Demirbas et al., Crop Science, 2001; Diers et al.,
Crop Science, 1992). Populations of P. sojae exist in many soybean
production regions that cause disease on plants with many, if not
all, of these Rps genes.
[0006] Single gene resistance has provided adequate disease
management; however, each single gene deployed in a soybean
cultivar is only effective for eight to fifteen years
(Schmitthenner, Plant Dis., 1985). Pathotypes of P. sojae,
containing virulence genes to Rps1k (the most recently deployed Rps
gene) have already been found in fields throughout the midwest
(Abney et al., 1997; Dorrance et al., 2003, Kaitany et al., 2001;
Kurle and ElAraby, 2001; Leitz et al, 2001; Schmitthenner et al.,
1994; Tang et al., 1996). Accordingly, novel resistance loci or
alleles are desirable for introduction into commercial soybean
lines to protect against yield losses caused by P. sojae.
SUMMARY
[0007] A novel method is provided for determining the presence or
absence of Phytophthora resistance in a soybean plant, soybean
seed, or soybean germplasm, as indicated by the presence or absence
of a newly-discovered resistance gene, which maps to linkage group
MLG F(referred to hereinafter as Rps8). The Rps8 locus comprises a
gene associated with resistance to Phytophthora sojae. The Rps8
gene is capable of conveying Phytophthora sojae resistance to
susceptible soybean germplasm. In accordance with the present
invention, the Rps8 gene is mapped to MLG F by genetic markers
Satt595, Satt114, Satt334, Sat.sub.--317, Sat.sub.--197, Satt510,
Satt335 and Satt144, and the Rps8 gene is located along the trait
locus between the markers. According to the method, genomic DNA
from a soybean plant, soybean seed, or soybean germplasm is
analyzed for the presence of the Rps8 gene. The presence of the
Rps8 gene is determined through the use of one or more molecular
markers linked to Rps8. According to the method, molecular
information regarding the Rps8 gene may be used to aid in the
selection of breeding plants, lines, and populations containing
Phytophthora resistance for use in introgression of this trait into
elite soybean germplasm, or germplasm of proven genetic superiority
suitable for variety release. Also according to the method,
molecular information regarding the Rps8 gene may be used to
confirm selection of Phytophthora resistance in new soybean
cultivars.
[0008] Also provided is a method for introgressing
soybeanPhytophthora sojae resistance gene Rps8 into susceptible
soybean germplasms. According to the method, nucleic acid markers
linked to Rps8 are used to select soybean plants containing the
Rps8 gene. Plants so selected have a high probability of expression
of P. sojae resistance. Plants so selected can be used in a soybean
breeding program. Through the process of introgression, the Rps8
gene is introduced from plants identified via marker assisted
selection to other plants. According to the method, agronomically
desirable plants and seeds can be produced containing the gene for
Rps8 from germplasm containing Rps8. One particular example of the
source of Rps8 resistance is the OX-99128 population, or a
descendant of this population. Similarly, the source ofPhytophthora
sojae resistance may conveniently include the OX-98317 population,
or a descendant of this population.
[0009] Also provided is a method for producing an inbred soybean
plant adapted for conferring, in hybrid combination with a suitable
second inbred, Rps8-derived resistance to Phytophthora sojae.
First, donor soybean plants containing Rps8 are selected. According
to the method, selection is accomplished via nucleic acid marker
assisted selection, as previously explained. Selected plant
material may represent, among others, an inbred line, a hybrid, a
heterogeneous population of soybean plants, or simply an individual
plant. According to techniques well known in the art of plant
breeding, this Rps8-donor parental line is crossed with a second
parental line. Preferably, the second parental line is high
yielding. This cross produces a segregating plant population
composed of genetically heterogeneous plants. Plants of the
segregating plant population are screened for the locus Rps8. Those
plants having Rps8 are selected for further breeding until a line
is obtained which is homozygous for resistance to Phytophthora
sojae at Rps8. This further breeding may include, among other
techniques, additional crosses with other lines, hybrids,
backcrossing, or self-crossing. The result is an inbred line of
soybean plants that are resistant toPhytophthora sojae in
combination with other desirable traits from one or more other
inbred lines.
[0010] Also provided is soybean germplasm designated HFX01-602
(also referred to as OX-01602). The parental lineage for HFX01-602
is shown in FIG. 2. This seed has ATCC accession number PTA-5190.
HFX01-602 was produced by introgressing an Rps8 gene from a
germplasm having Rps8-derived P. sojae resistance into
non-resistant or less resistant soybean germplasm. The invention
includes all HFX01-602 progeny that contain the locus Rps8 and
exhibit Phytophthora sojae resistance. Also provided are
populations of soybean plants, seed, tissue cultures, variants, and
mutants that are produced from HFX01-602 Rps8-containing
germplasm.
[0011] Also provided is soybean germplasm designated OX-98317. This
seed has ATCC accession number ______. OX-98317 was produced by
introgressing disease resistance, identified via Rps8, a novel
locus for Phytophthora sojae resistance originally found in Korean
PI 399073, into non-resistant or less resistant soybean germplasm.
The germplasm includes all OX-98317 progeny that contain the locus
Rps8 and exhibit Phytophthora sojae resistance. Also provided are
populations of soybean plants, seed, tissue cultures, variants, and
mutants that are produced from OX-98317 Rps8-containing
germplasm.
[0012] Also provided is soybean germplasm designated OX-99218. This
seed has ATCC accession number ______. OX-99218 was produced by
introgressing disease resistance, identified via Rps8, a novel
locus for Phytophthora sojae resistance originally found in Korean
PI 399073, into non-resistant or less resistant soybean germplasm.
The germplasm includes all OX-99218 progeny that contain the gene
Rps8 and exhibit Phytophthora sojae resistance. Also provided are
populations of soybean plants, seed, tissue cultures, variants, and
mutants that are produced from OX-98218 Rps8-containing
germplasm.
[0013] Also provided is soybean germplasm designated OX-99128. This
seed has ATCC accession number ______. OX-99128 was produced by
introgressing disease resistance, identified via Rps8, a novel
locus for Phytophthora sojae resistance originally found in Korean
PI 399073, into non-resistant or less resistant soybean germplasm.
The germplasm includes all OX-99128 progeny that contain the locus
Rps8 and exhibit Phytophthora sojae resistance. Also provided are
populations of soybean plants, seed, tissue cultures, variants, and
mutants that are produced from OX-99128 Rps8-containing
germplasm.
BRIEF DESCRIPTION OF THE DRAWING
[0014] FIG. 1 shows a genetic linkage map of MLG F of soybean. The
genetic linkage map was constructed using Joinmap 3.0 linkage
analysis software with molecular marker data (Van Ooijen and
Voorrips, 2001). Distances between markers were assigned in
centimorgans, shown to the left of the chromosome. Simple Sequence
Repeat (SSR) markers and Rps8 are shown to the right of the
chromosome. The determination of linkage groups was done with a
log-likelihood (LOD) threshold of 3.0. The calculation of linkage
maps was performed using all pairwise recombination estimates
smaller than 0.45 and a LOD score larger than 0.05. Kosambi's
mapping function was used.
DETAILED DESCRIPTION
Definitions
[0015] "Allele" is any of one or more alternative forms of a gene,
all of which alleles relate to one trait or characteristic. In a
diploid cell or organism, the two alleles of a given gene occupy
corresponding loci on a pair of homologous chromosomes.
"Backcrossing" is a process through which a breeder repeatedly
crosses hybrid progeny back to one of the parents, for example, a
first generation hybrid F.sub.1 with one of the parental genotypes
of the F.sub.1 hybrid. "Cultivar" and "variety" are used
synonymously and mean a group of plants within a species (e.g.,
Glycine max) which share certain constant characters that separate
them from the typical form and from other possible varieties within
that species. While possessing at least one distinctive trait, a
variety is also characterized by a substantial amount of overall
variation between individuals within the variety, based primarily
on the Mendelian segregation of traits among the progeny of
succeeding generations.
[0016] "Gene" means a specific sequence of nucleotides in DNA that
is located in the germplasm, usually on a chromosome, and that is
the functional unit of inheritance controlling the transmission and
expression of one or more traits by specifying the structure of a
particular polypeptide or controlling the function of other genetic
material.
[0017] "Germplasm" means the genetic material with its specific
molecular and chemical makeup that comprises the physical
foundation of the hereditary qualities of an organism. As used
herein, germplasm includes living tissue from which new plants may
be grown; or, another plant part, such as leaf, stem, pollen, or
cells, that may be cultured into a whole plant.
[0018] "Hybrid plant" means a plant offspring produced by crossing
two genetically dissimilar parent plants.
[0019] "Inbred plant" means a member of an inbred plant strain that
has been highly inbred so that all members of the strain are
genetically identical, with the exception of sexual
differences.
[0020] "Introgression" means the entry or introduction by
hybridization of a gene or trait locus from the genome of one plant
into the genome of another plant that lacks such gene or trait
locus.
[0021] "Line" or "strain," as distinguished from a "variety," means
a group of plants which display less variation between individuals,
generally (although not exclusively) by virtue of several
generations of self-pollination, and includes a group of plants
which carry a gene or locus for a particular trait, specifically
the Rps8-derived Phytophthora sojae resistance trait as disclosed
herein.
[0022] "Linkage group" means an identified chromosomal region
containing genetic material that expresses a desired trait.
[0023] "Locus" means a chromosomal region where a polymorphic
nucleic acid or trait determinant or gene is located.
[0024] "Polymorphism" means a change or difference between two
related nucleic acids. A "nucleotide polymorphism" refers to a
nucleotide which is different in one sequence when compared to a
related sequence when the two nucleic acids are aligned for maximal
correspondence. A "genetic nucleotide polymorphism" refers to a
nucleotide which is different in one sequence when compared to a
related sequence when the two nucleic acids are aligned for maximal
correspondence, where the two nucleic acids are genetically
related, i.e., homologous, e.g., where the nucleic acids are
isolated from different strains of a soybean plant, or from
different alleles of a single strain, or the like.
[0025] "Marker assisted selection" means the process of selecting a
desired trait or desired traits in a plant or plants by detecting
one or more nucleic acids from the plant, where the nucleic acid is
associated with the desired trait.
[0026] "Plant" means plant cells, plant protoplast, plant cell or
tissue culture from which soybean plants can be regenerated, plant
calli, plant clumps and plant cells that are intact in plants or
parts of plants, such as seeds, pods, flowers, cotyledons, leaves,
stems, buds, roots, root tips and the like.
[0027] "Probe" means an oligonucleotide or short fragment of DNA
designed to be sufficiently complementary to a sequence in a
denatured nucleic acid to be probed and to be bound under selected
stringency conditions.
[0028] "Qualitative trait" means a trait which is controlled by a
single dominant gene and which segregates according to normal
Mendelian genetic principles.
[0029] "Rps8-derived resistance" means P. sojae resistance in a soy
germplasm that is provided by the heterozygous or homozygous
expression of the gene within the Rps8 locus mapped to MLG F.
[0030] "Rps8 phenotype" means resistance to P. sojae by soybean
germplasm, as demonstrated by resistance to P. sojae after
inoculation with same according to the methods described
herein.
[0031] "Rps8 soybean plant" means a plant having resistance to P.
sojae that is derived from the presence and expression of at least
one Rps8 gene, or that is shown to have an Rps8 gene at the Rps8
locus described herein.
[0032] "Self-crossing" is a process through which a breeder crosses
hybrid progeny with one another, for example, a second generation
hybrid F.sub.2 with itself to yield progeny designated
F.sub.2:3.
[0033] As used herein, the terms "segregate, " "segregants,"
"co-segregate," "hybrid," "crossing," and "selfing" refer to their
conventional meanings as understood in the art (see, for instance,
Briggs, F. N. and Knowles, P. F. and, Introduction to Plant
Breeding (Reinhold Publication Corp., New York, N.Y., 1967).
Rps8 Gene and Rps8-Derived P. sojae Resistance
[0034] The plant introduction PI 399.073 is the only known soybean
cultivar to have Rps8-derived resistance to Phytophthora sojae.
However, PI 399.073 has poor agronomic traits, necessitating the
introgression of its Rps8-derived resistance into other soybean
germplasms having more desirable traits. Conventional breeding with
PI 399.073, as described below, produced several germplasms which
are more agronomically desirable and can be used as a source of
Rps8-derived Phytophthora sojae resistance in future soybean
breeding.
[0035] The locus of the Rps8 gene has been mapped according to the
methods provided herein using nucleic acid markers, and is further
defined by the association of Rps8 with particular recognized
linkage groups. Rps8 is located on major linkage group (MLG) F of
the soybean map (See Figure One). Rps8 is identified and localized
using traditional crossing populations and disease assays combined
with simple sequence repeat (SSR) molecular markers Satt595,
Satt114, Satt334, Sat.sub.--317, Sat.sub.--197, Satt510, Satt335
and Satt144.
Identification of Phytophthora sojae Resistance Trait Loci
[0036] It is of particular importance both to the soybean breeder
and to farmers who grow and sell soybeans as a cash crop to
identify the trait loci for resistance to the various pathotypes of
Phytophthora sojae. With information about such trait loci, soybean
breeders are better equipped to breed Phytophthora resistant
soybeans which are agronomically desirable (i.e., they also possess
other genotypic and phenotypic characteristics required for
commercial soybean lines).
[0037] Soybean germplasm from plant introductions (PIs) from South
Korea serve as potential new sources of resistance loci (Dorrance
and Schmitthenner, 2000). Soybean germplasm from South Korea has
previously been the source of Rps alleles, such as Rps3a from
PI86972-1 (Mueller et al., 1978). Although these South Korean plant
introductions are generally considered agronomically poor, some of
the alleles that they carry are valuable and can be moved into more
suitable backgrounds for U.S. cultivars through selective breeding
techniques, in particular the breeding procedure known as
introgression.
Soybean Breeding and Selection Methods
[0038] Soybean varieties possessing specific, desirable traits may
be developed by traditional plant breeding techniques. Two
cultivars or germplasms are typically selected for particular
traits and then interbred, one being employed as male and the other
as female. After the first cross, the F.sub.2 generation plants are
normally screened for the traits of interest. Seeds are saved from
the F.sub.2 plants selected and subsequent generations are grown
up, again selecting desirable plants from each generation.
[0039] The desired phenotype of the novel Rps8 gene is transferred
to other wild-type or commercial soybean germplasms by conventional
plant breeding methods to achieve a new germplasm line combining
these desired phenotypes with other valuable agronomic traits such
as, for example, seed size, protein content, and herbicide
resistance. The desired wild-type soybean, commercial cultivar, or
hybrid thereof is crossed by conventional plant breeding methods
with a soybean plant having the Rps8 gene and phenotype. Breeding
methods used in accordance with the present invention include, for
example, methods described in Knowles, P. F. and F. N. Briggs,
Introduction to Plant Breeding (Reinhold Publication Corp., New
York, N.Y., 1967), incorporated herein by reference, or any like
methods known in the art. Hybrid progeny exhibiting the Rps8
phenotype are selected.
[0040] The Rps8 phenotype is controlled by a single dominant gene,
which segregates according to normal Mendelian genetic principles.
Conventional plant breeding techniques can therefore be used to
introduce the phenotype into any soybean variety. Thus, by
conventional plant breeding techniques, the ordinary artisan can
cross an Rps8 soybean plant with any other soybean germplasm having
specific desirable traits to produce a soybean line possessing the
combination of desired phenotypes. The present invention is
exemplified by its application to soybean (Glycine max); however,
its operating principles may be applied to other species of
soybean. The invention is not limited to any particular soybean
cultivar, but may be applied generally to any plant variety of the
genus Glycine, whether wild, domestic or hybrids of the two.
Although the instant invention is applicable to all soybean
varieties, the breeding and selection methods are preferably
carried out by crossing soybean lines which contain the Rps8 gene
with varieties possessing other valuable agronomic traits. Soybean
varieties are widely available in commerce from several
manufacturers, providing progenitor strains having a broad range of
agronomically desirable traits.
Introgression
[0041] Selective breeding techniques for introgressing one or more
desired traits from one soybean plant line into another plant line
having other desired traits have several advantages over modem
genetic engineering techniques. While a great deal of emphasis is
usually placed on the strategy of introducing characteristics into
plants via genetic engineering techniques, the prospects for the
general use of these techniques for plant improvement are greatly
limited by the realization that very few genes corresponding to
plant traits of interest have been identified. The use of direct
gene transfer in manipulating these traits is therefore difficult
due to problems in pinpointing and then cloning those individual
loci which contribute predominantly to the expression of the
trait.
[0042] The introgression of traits from one germplasm to another
conventionally involves the identification of germplasms having
favorable genotypes in a segregating generation followed by
repeated backcrossing to commercially acceptable cultivars. This
procedure is feasible for simply inherited qualitative traits, when
one or only a few genes control a trait. As the number of genes
controlling a trait increases, screening the number of F2
segregants required to identify at least one individual which
represents the ideal (homozygous) genotype quickly becomes
prohibitive. For example, with one gene and two alleles of equal
frequency, the probability of recovering a desirable genotype on
the F2 generation is 1/4. However, if the number of genes is
increased to 5 or 10, the probability of recovering an ideal
genotype in the F2 population is reduced to approximately one in
one thousand and one in one million, respectively. Thus, to
identify desirable segregants, one must either reduce the number of
segregants needed or have available very efficient screening
procedures. Marker assisted selection is an efficient screening
procedure for expediting introgression, whether there is a single
or multiple genes that define a desired trait.
Identification and Selection Based on Markers
[0043] The ability to characterize an organism, such as a soybean
plant, by its genome is possible because of the inherent
variability of genetic information. Although DNA sequences which
code for necessary proteins are well conserved across a species,
there are regions of DNA which are non-coding or code for portions
of proteins which do not have critical functions and therefore,
absolute conservation of nucleic acid sequence is not strongly
selected for. These variable regions are identified by genetic
markers.
[0044] Genetic markers can be detected by amplification of specific
DNA sequences or amplicons which correspond to unique regions of
the genome. Use of sequence-specific PCR primers allows
confirmation of either or both the presence or location of a DNA
sequence in the genome of the subject being tested. Polymorphisms
which are represented by unique and different sequences are most
useful as markers because they permit discrimination using a
variety of genotyping procedures, as set forth herein. Various
genetic markers include, but are not limited to, markers based on
protein sequence, isozymes; hybridization, restriction fragment
length polymorphisms ("RFLP"); and polymerase chain reaction,
single sequence repeats ("SSR,") or microsattelites, or short
tandem repeats, random amplified polymorphic DNA ("RAPD") and
amplified fragment length poymorphisms (AFLPs).
[0045] Through the combination of introgression and indirect
selection procedures, plant breeders are able to increase
efficiency in the testing of traits which are difficult or
expensive to evaluate. Genetic markers closely linked to important
genes may be used to indirectly select for favorable alleles more
efficiently than direct phenotypic selection (Lande and Thompson,
Genetics 124:543-546, 1990). In the past, numerous inoculations
with different pathotypes of P. sojae were needed to confirm the
presence of a new resistance allele in a number of soybean crosses.
Mapping new alleles was also difficult due to the limited number of
classical gene markers. DNA marker technology, especially SSR, has
eliminated the need for numerous pathotype inoculation screenings
and has expedited the process of mapping a new gene with more
precision.
[0046] Using marker assisted selection, a DNA sample from soybean
plants is required. The sample is amplified using conventional
techniques to provide a set of differentially amplified nucleic
acids in the mixture. At least one of the differentially amplified
nucleic acids is mapped to a unique genetic polymorphism, thereby
providing a marker for the polymorphism. Typically, more than one
differentially amplified nucleic acid is mapped, thereby providing
a set of markers. The set can be of any size, although more
information is provided by larger sets. Typical set sizes are from
about 1-100 markers, generally about 1-5 markers. In one approach,
the method includes hybridizing a probe nucleic acid to a mixture
of DNA amplified from a biological source of DNA comprising the
polymorphism, thereby identifying the polymorphism in the
biological source of DNA. The probe nucleic acid is hybridized
under stringent conditions to a target nucleic acid comprising the
polymorphism.
[0047] Marker assisted selection involves crossing a parent plant
having a desired allelic trait, for example, P. sojae resistance,
with a second parent plant in order to create an F1 plant
population. Heterozygous plants from the F1 population are
self-fertilized, or "selfed," to create a segregating F2 plant
population exhibiting expression of the qualitative trait of
interest, e.g., P. sojae resistance. Following preparation a marker
(such as SSR, RFLP, RAPD, or isozyme) is randomly chosen, or
alternatively, selected from a genetic linkage map, and evaluated
on the population. Using other markers, the degree of association
between the trait of interest and each particular marker is
determined. In this manner, the marker(s) having the strongest
association with the trait of interest can be determined and
utilized, for example, in a breeding program to select plants
having P. sojae resistance. One or a combination of markers can be
used to identify or confirm the presence of an associated trait
locus, such as Rps8. Combinations of markers, such as at least two
markers that are known by mapping to bracket or flank the trait
locus of interest can be used effectively to identify or confirm
the presence of the trait locus in hybrid introgressed germplasms.
Particularly where one or more markers are not strongly associated
with the trait of interest, use of multiple, and particularly
flanking markers, will increase the probability of positively
confirming the isolation or presence of the trait locus of
interest.
Probes, Techniques and Conditions for Molecular Marker Analysis
[0048] Probes for use in marker assisted selection can be acquired
commercially or can be made using known sequence information or
information acquired by providing first and second samples of
amplified DNA, comparing the first and second samples of amplified
DNA to identify differentially amplified DNAs. The differentially
amplified DNA can be isolated and mapped. Typically, at least a
portion of the genetically mapped isolated DNA is sequenced to
identify associated polymorphisms. Oligonucleotide probes can then
be prepared comprising a portion of the sequenced region. Preferred
probes uniquely map to single sites in a haploid genomic DNA of a
plant or animal, or to cDNA. Many probes are commercially available
for use in marker assisted selection using a broad range of known
markers.
[0049] A labeled probe is exposed to amplified mixtures of DNA in a
biological sample and is assessed for binding. For example, a
marker comprising a polymorphic nucleic acid can be detected by
allele-specific hybridization of a probe to the region of the
marker comprising the polymorphic nucleic acid. Similarly, a marker
can be detected by Southern analysis, northern analysis, in situ
analysis, or the like.
[0050] Hybridization of probes to amplified mixtures of DNA (e.g.,
DNA amplified by AFLP techniques) is a preferred assay format.
"Hybridization" is used to denote the pairing of complementary
nucleotide sequences to produce a DNA-DNA hybrid a DNA-RNA hybrid
or an RNA-RNA hybrid. Complementary base sequences are those
sequences that are related by the well-known base-pairing rules. In
DNA, A pairs with T, and C pairs with G. In RNA, U pairs with A,
and C pairs with G. Two single-stranded nucleic acids "hybridize"
when they form a double-stranded duplex. The region of
double-strandedness can include the full-length of one or both of
the single-stranded nucleic acids, or all of one single stranded
nucleic acid and a subsequence of the other single stranded nucleic
acid, or the region of double-strandedness can include a
subsequence of each nucleic acid.
[0051] "Stringent hybridization conditions" in the context of
nucleic acid hybridization are sequence dependent and are different
under different environmental parameters. An extensive guide to the
hybridization of nucleic acids is found in Tijssen (1993)
Laboratory Techniques in Biochemistry and Molecular
Biology-Hybridization with Nucleic Acid Probes, part I chapter 2,
"Overview of principles of hybridization and the strategy of
nucleic acid probe assays" Elsevier, N.Y. Generally, stringent
conditions are selected to be about 5.degree. C. lower than the
thermal melting point (T.sub.m) for the specific sequence at a
defined ionic strength and pH. The T.sub.m is the temperature
(under defined ionic strength and pH) at which 50% of the target
sequence hybridizes to a perfectly matched probe. Highly stringent
conditions are selected to be equal to the T.sub.m point for a
particular probe. Sometimes the term "T.sub.d"is used to define the
temperature at which at least half of the probe dissociates from a
perfectly matched target nucleic acid. In any case, a variety of
estimation techniques for estimating the T.sub.m or T.sub.d are
available, and generally described in Tijssen, id. Typically, G-C
base pairs in a duplex are estimated to contribute about 3.degree.
C. to the T.sub.m, while A-T base pairs are estimated to contribute
about 2.degree. C., up to a theoretical maximum of about
80-100.degree. C. However, more sophisticated models of T.sub.m and
T.sub.d are available and appropriate in which G-C stacking
interactions, solvent effects, the desired assay temperature and
the like are taken into account.
[0052] An example of stringent hybridization conditions for
hybridization of complementary nucleic acids that have more than
100 complementary residues on a filter in a Southern or northern
blot is 50% formalin with 1 mg of heparin at 42.degree. C., with
the hybridization being carried out overnight. An example of
stringent wash conditions for a Southern blot of such nucleic acids
is a 0.2 times SSC wash at 65.degree. C. for 15 minutes (see,
Sambrook, supra for a description of SSC buffer). Often the high
stringency wash is preceded by a low stringency wash to remove
background probe signal. An example low stringency wash is 2 times
SSC at 40.degree. C. for 15 minutes.
Introgressed Soybean Lines Having Rps8-Derived P. Sojae resistance:
Description of HFX01-602 Soybean Germplasm with Rps8 Resistance to
Phytophthora sojae
[0053] HFX01-602 (also referred to as OX01-602) germplasm was
developed as an F.sub.3:4 population from the cross of Kottman x
OX-99393. OX-99393 is from the cross OX-98317 x Kottman. OX-98317
is from the cross PI 399.073 x NK S19-90. Crosses were made without
selection. S 19-90 carries the Rps1c resistance gene. Kottman was
developed at the Ohio Agricultural Research and Development Center
as an F4-derived line from the cross HS88-7363 x HS88-4988. The
female parent HS88-7363 is the F2-derived line from which `General`
was selected and derives from Voris `311` x `Resnik`. The male
parent, HS88-4988, is from `Winchester` x A83-271027. The line
A83-271027 is from Northrup King `S1492` x Asgrow `A3127`. Kottman
is an indeterminate cultivar of maturity group III with white
flowers, light tawny pubescence, and tan pods. Seeds are dull
yellow with black hila. Kottman has both the Rps1k and the Rps3
genes for resistance to Phytophthora rot. Kottman is susceptible to
brown stem rot, soybean cyst nematode, and sclerotinia white mold.
In Ohio tests, seeds of Kottman had a mean content of 43.2% protein
and 20.1% oil, in comparison with 43.3% protein and 19.9% oil for
General. Kottman was released because of its high yield in relation
to cultivars of similar maturity and its resistance to Phytophthora
rot. OX-99393 and HFX01-602 have the novel resistance locus
containing Rps8 derived from Korean PI 399073.
[0054] The HFX01-602 germplasm includes seeds. 2500 HFX01-602
F.sub.2:3 seeds were deposited with the American Type Culture
Collection. The population of seeds was obtained from bulking the
seeds produced by F.sub.2 progeny. The genotype of the seed
population is approximately 25% Rps8/Rps8, approximately 50%
Rps8/sus, and approximately 25% sus/sus.
Description of OX-99218 Soybean Germplasm with Rps8 resistance to
Phytophthora sojae
[0055] OX-99218 was developed as an F.sub.1 derived population of
bulk F.sub.4 seed from the cross of A97-873014 x OX-98317. OX-98317
was developed from the cross of Korean PI 399073 x S19-90. S 19-90
carries the Rps1c resistance gene. A97-873014 does not have any
known Phytophthora resistance genes. OX-98317 has the novel
resistance gene Rps8 derived from Korean PI 399073. The F1 (hybrid)
plant was produced by crossing, while the next three generations
were produced by self crossing. "F1-derived" indicates that there
was no further selection of single plants after the F1 generation.
That is, the F2 seeds were collected from the original F1 (hybrid)
plant. These were planted and the whole plot was harvested in bulk
to give F3 seeds. A portion of these seeds was then planted and
again harvested to provide the F4 seeds. Soybean is naturally
self-fertilized, so each plant from the F1 onward produced its own
seeds by self crossing.
[0056] Table 1 provides OX-99218 Phenotypic Data. Plants were
inoculated with pathotype OH30 (vir 1a, 1b, 1k, 2, 3a, 4, 5, 6, 7)
to test for segregation of a single resistance gene, "Rps8." Plants
were also inoculated with pathotype OH4 (vir 1a, 1c, 7) to
determine if the Rps1c was still present from the S19-90. Similar
segregation ratios for Race 30 and Race 4 indicate only Rps8 was
present.
TABLE-US-00001 TABLE 1 Resistant or Suscep- segregating tible Test
.chi.2 Cross Pathotype plants plants ratio probability A97-873014
.times. OH30 15 17 9:7 0.5-0.25 OX-98317 A97-873014 .times. OH4 7
12 9:7 0.1-0.05 OX-98317
[0057] Table 2 provides OX-99218 Genotypic Data--Plants inoculated
with OH30 were tested with a molecular marker linked to the "Rps8
".
TABLE-US-00002 TABLE 2 Sus- Resistant Segregating ceptible Test
.chi.2 Cross plants Plants plants ratio probability A97-873014
.times. 10 5 17 7:2:7 0.05-0.025 OX-98317
Description of OX-99128 Soybean Germplasm with Rps8 Resistance to
Phytophthora sojae
[0058] OX-99128 was developed as an F.sub.1 derived population of
bulk F.sub.4 seed from the cross of Darby X OX-98317. OX-98317 was
developed from the cross Korean PI 399073 x S19-90. S 19-90 carries
the Rps1c resistance gene. Darby contains the resistance gene
Rps1k. OX-98317 has the novel Rps8 resistance locus derived from PI
399073. A pathotype of Phytophthora sojae that has a susceptible
interaction with Rps1k was used to inoculate plants from this cross
in order to observe only the effect of "Rps8 ".
[0059] Table 3 provides OX-99128 Phenotypic Data--Plants were
inoculated with pathotype OH30 (vir 1a, 1b, 1k, 2, 3a, 4, 5, 6, 7)
to test for segregation of a single resistance gene. Plants were
also inoculated with pathotype OH4 (vir 1a, 1c, 7).
TABLE-US-00003 TABLE 3 Resistant or segregating Susceptible Test
.chi.2 Cross Pathotype plants plants ratio probability Darby
.times. OH30 12 19 9:7 0.05-0.025 OX-98317 Darby .times. OH4 20 0
13:3 0.05-0.025 OX-98317
[0060] Table 4 provides OX-99128 Genotypic Data--Plants inoculated
with OH30 were tested with a molecular marker linked to the "Rps8
".
TABLE-US-00004 TABLE 4 Resistant Segregating Susceptible Test
.chi.2 Cross plants Plants plants ratio probability Darby .times. 8
4 17 7:2:7 0.25-0.10 OX-98317
Germplasm Deposit Information
[0061] A deposit of 2500 viable seeds of the inbred soybean
germplasm designated HFX01-602 has been made with the American Type
Culture Collection ("ATCC"), 10801 University Blvd., Manassas, Va.
on May 9, 2003. Those deposited seeds have been assigned ATCC
Accession No. PTA-5190. The deposit was made in accordance with the
terms and provisions of the Budapest Treaty. All restrictions on
the availability to the public of the materials so deposited will
be irrevocably removed upon the granting of the patent. The
germplasm will be maintained for a term of at least thirty (30)
years and at least five (5) years after the most recent request for
the furnishing of a sample of the deposit is received by the
depository, and at least beyond the enforceable life of the
patent(s) for which the deposit was made, whichever is longer. The
germplasm will be replaced if it becomes non-viable during that
period. Additionally, the deposit has satisfied all the
requirements of 37 C.F.R. .sctn..sctn. 1.801-1.809, including a
mechanism for providing an indication of the viability of the
sample. The deposit does not constitute a waiver of any rights that
may be granted under this or any other patent application or under
the Plant Variety Protection Act (7 U.S.C. 2321), or any other
applicable treaty, law or regulation. This deposit was made to
further exemplify the invention and is not intended to any way
limit the scope of the invention.
EXAMPLES
Example 1
Phenotypic Evaluation of Rps8-derived Phytophthora sojae
Resistance
[0062] Korean plant introductions (PI399073) were obtained from the
USDA Soybean Germplasm Collection in Urbana, Ill. All other plant
material was obtained from the Ohio Agricultural Research
Development Center (OARDC) soybean breeding program. The crosses
used for mapping the new Rps8 allele were Williams (rps) X
PI399073, S 19-90 (Rps1c) X PI399073, and Williams (rps) X
PI399073. The F.sub.1 plants from these crosses were selfed to
produce populations of approximately 40 to 60 and 143 F.sub.2
plants for each cross. The F.sub.2 plants were then selfed and each
plant was thrashed individually to yield seed for F.sub.2:3
families.
[0063] Isolates of P. sojae pathotypes were maintained at the
Department of Plant Pathology, OARDC. All pathotypes used in this
study were collected in Ohio. Three isolates of P. sojae were used
in this study with the following pathotypes OH1 (vir 7), OH17 (vir
1b, 1d, 3a, 3b, 3c, 4, 5, 6, 7), and OH25 (vir 1a, 1b, 1c, 1k, 7).
Differential checks, Williams (universal suscept); Harlon (Rps1a),
Harosoy 13XX (Rps1b), Williams 79 (Rps1c); PI103091 (Rps1d);
Williams 82 (Rps1k); L76-1988 (Rps2); L83-570 (Rps3); PRX 146-36
(Rps3b); PRX 145-48 (Rps3c); L85-2352 (Rps4); L85-3059 (Rps5);
Harosoy 62XX (Rps6) and Harosoy (Rps7), were included in all tests
to ensure that the P. sojae isolate used elicited the appropriate
reaction.
[0064] Ten individual F3 seedlings per F2:3 family were inoculated
in the laboratory using a modification of the hypocotyl inoculation
technique. Inoculum was prepared by growing the P. sojae isolates
for one week on lima bean agar (50 g lima beans, 12 g agar per
liter). Seeds were placed between germination papers wetted with
water, rolled up, and stored in the dark in plastic containers. The
plastic containers had wire mesh in the bottom to allow for water
to drain from the papers. After one week of growth, papers were
unrolled and the seedlings were inoculated using a hypodermic
syringe. The syringe was filled with colonized agar from a plate of
P. sojae, and then the agar was forced through the syringe to
create a slurry. The slurry was placed back into the syringe.
Seedlings were inoculated by scratching the hypocotyl with the
needle of the hypodermic syringe and placing the agar/mycelium
mixture onto the wound. Reactions were recorded as R (resistant;
seedlings alive) or S (susceptible, seedlings dead with brown
hypocotyls) after 10 days. Each F2:3 family had 10 to 25 plants
scored, either all R, all S, or a combination of both. The 10
scores were used to develop a single classification, R, S, or H
(heterogeneous), for each F2 plant from which an F2:3 was
derived.
[0065] Chi-square analyses were performed on the phenotypic data to
test if a 3:1 resistant to susceptible ratio was present. F2 plants
were scored as R, S, or H based on the results of the hypocotyl
inoculation of the F2:3 families. For this analysis all R and H
scores were grouped together.
[0066] The cross of Williams X PI399073 resulted in F2:3 families
that fit a 3:1 resistant to susceptible phenotypic ratio in both
populations. Forty-five individual F3 seedlings from 143 F2:3
families were inoculated with P. sojae pathotype vir 7 (OH-race 1)
and after 10 days 109 F2:F3 families were scored as resistant and
34 were scored as susceptible (.chi.2=0.9) (Table 5). In order to
confirm this result, 15 additional F3 seedlings from each F2 plant
were inoculated with pathotype Race 25. A 3:1 ratio was observed
again using this second pathotype, and the same F2:3 families were
susceptible in both tests.
[0067] Table 5 provides a summary of phenotypic ratios of 143 F2:3
families tested with different pathotypes of P. sojae from the
Williams X PI399073 cross.
TABLE-US-00005 TABLE 5 Resistant or Sus- Patho- Segregating
ceptible Test .chi.2 .chi.2 Cross type Lines Lines Ratio value
probability Williams .times. OH1 109 34 3:1 0.9 0.75-0.50 PI399073
(vir 7)
Example 2
Simple Sequence Repeat (SSR) DNA Length Polymorphism Markers
Indicating Association of SSR Marker Satt595, Satt114, Satt334,
Sat-317, Sat-197, Satt510, Satt335 and Satt144 with Phytophthora
sojae Resistance in PI399073 thus Placing the Novel Trait Locus for
Rps8 on Major Linkage group (MLG) F.
[0068] The cross used for analyzing SSR marker association was
Williams (Rps) X PI 399073. The F.sub.1 plants from this cross were
selfed to produce a population of approximately 150 F.sub.2 plants.
The F.sub.2 plants were then selfed and each plant was thrashed
individually to yield seed for F.sub.2:3 families.
[0069] Whole plants from individual F3 seedlings were bulked from
each F2:3 family, and DNA was extracted as previously described
(Saghai-Maroof et al., 1984). SSR primer pairs (Research Genetics
Inc., Huntsville, Ala.), polymorphic for the parents in each cross,
were used to test the F2:3 progeny. PCR reactions were performed as
recommended by the manufacturers in a total of 20 .mu.l containing
30 ng of genomic DNA. Amplified PCR products were resolved on 5%
high-resolution agarose gels (Amresco, Solon, Ohio) and stained
with ethidium bromide for visualization of the DNA products.
Reactions were scored as 2 (homozygous for PI parent allele), 1
(homozygous for susceptible parent allele), or 3
(heterozygous).
[0070] Thirty-eight SSR markers were polymorphic between Williams
and PI399073. These markers were then tested on 143 F2:3 families.
The genotypic data from these markers was then tested with the
phenotypic data from inoculations in a single marker-trait analysis
using PROC GLM (SAS institute, 1988). The results of the ANOVA
indicated that the markers Satt595, Satt114, Satt334,
Sat.sub.--317, Sat.sub.--197, Satt510, Satt335 and Satt144 on MLG F
were significantly associated with the resistance phenotype
(P<0.009). The markers on other MLGs did not show significant
associations, indicating that the new resistance allele must be on
MLG F (Table 6). (Soybase web site http://129.186.26.94).
Table 6 provides SSR markers used to identify markers associated
with the resistance to Phytophthora sojae found in soybean
PI399073. The significance values are from an analysis of variance
(ANOVA) used to determine if the marker data was significantly
associated with the phenotype following inoculations. The analysis
for markers on MLGs A2 and F was performed in a population of 143
F2:3 lines. For all other linkage groups the analysis was performed
on a subset of 94 F2:3 lines.
TABLE-US-00006 TABLE 6 Significance Significance Marker MLG level
Marker MLG level Satt252 F 0.38 Satt187 A2 0.8 Satt516 F 0.05
Satt233 A2 0.98 Satt425 F 0.55 Satt228 A2 0.64 Satt595 F 0.009
Satt191 G 0.72 Satt114 F <0.0001 Satt394 G 0.66 Satt334 F
<0.0001 Satt199 G 0.17 Sat_317 F <0.0001 Satt485 N 0.79
Sat_197 F 0.004 Sat_091 N 0.93 Satt510 F <0.0001 Satt545 A1 0.98
Satt335 F <0.0001 Satt182 L 0.84 Satt144 F 0.001 Satt243 O 0.86
Satt470 A2 0.44 Satt231 E 0.91 Satt538 A2 0.32 Satt380 J 0.11
Satt329 A2 0.24 Satt440 I 0.8 Sat_310 A2 0.09 Satt387 N 0.14
Sat_294 A2 0.51 Satt216 D1b + W 0.17 Sat_347 A2 0.14 Satt509 B1
0.34 Sat_232 A2 0.17 Satt267 D1a + Q 0.12
Table 7 provides a summary of the segregation and chi-square
analysis of molecular markers on MLG F associated with resistance
in F2:3 families of the cross Willams X PI399073. All markers
listed are segregating as expected for a single Mendelian locus.
WW=homozygous Williams allele, PP=homozygous PI399073 allele and
WP=heterozygous. ns=not significant departure from the chi-square
distribution.
TABLE-US-00007 TABLE 7 SSR Degrees of Marker WW WP PP X.sup.2 Value
freedom Satt516 33 73 22 4.4.sup.ns 2 Satt114 36 76 32 0.7.sup.ns 2
Satt334 35 74 33 0.3.sup.ns 2 Sat_317 34 67 42 1.5.sup.ns 2 Satt335
19 32 23 1.8.sup.ns 2 Satt510 25 54 37 3.0.sup.ns 2 Satt144 29 52
30 0.5.sup.ns 2 Sat_197 35 67 40 0.8.sup.ns 2
Example 4
Linkage Map of MLG F of Soybean Using SSR Markers Providing
Placement of Novel Trait Locus for Rps8 in Relation to Rps1-Rps7 on
the Composite Soybean Genetic Map.
[0071] The crosses used for mapping the new allele were Williams
(Rps) X PI399073 and S 19-90 (Rps1c) X PI399073. The .sub.F1 plants
from these crosses were selfed to produce populations of
approximately 150 and 60 F2 plants for each cross, respectively.
The F2 plants were then selfed and each plant was thrashed
individually to yield seed for F2:3 families.
[0072] Analysis with SSR markers was performed as previously
described.
[0073] Chi-square analysis was used for each DNA marker to test
whether the F2:3 families fit the expected 1:2:1 ratio. DNA marker
data that fit the expected ratio was then used in an ANOVA to
determine if the marker data was significantly associated with
resistance. ANOVAs were conducted using the GLM procedure in SAS
(SAS institute, 1988). Once an SSR marker was found that was
significantly associated with resistance, more SSR markers were
tested from that area of the linkage group. Joinmap (Van Ooijen and
Voorips, 2001) was then used to determine the order of the marker
loci in the region of interest and the distances between them.
Linkage group designations were made using mapped loci from the
composite genetic linkage map (Cregan et al., 1999) and maintained
on the Soybase web site (http://129.186.26.94 and http
://soybase.ncgr.org/).
Example 5
Comparison of MLG F Linkage Map Between Two Different PI399073
Derived Hybrids (Williams x PI399073 and S 19-90 x PI399073)
Confirming the Relative Placement of the Novel Locus for Rps8.
[0074] In the past, numerous inoculations with different pathotypes
of P. sojae were needed to confirm the presence of a new resistance
allele in a number of soybean crosses. Mapping new alleles was also
difficult due to the limited number of classical gene markers. SSR
DNA marker technology has expedited this process of mapping a new
gene with more precision. An additional cross with S 19-90 was used
to confirm the presence of a new gene in contrast to earlier
techniques that required developing multiple populations to test
for allelism. The significant association with resistance of SSR
markers from MLG F in the S 19-90 population provides confirmatory
evidence of the location of Rps8.
[0075] S 19-90 was chosen to incorporate Sclerotinia stem rot
resistance from S 19-90 as a step towards variety development. S
19-90 contains Rps1c so expectations for segregation ratios would
be different from one locus to two loci.
[0076] The same procedures were used to map the new resistance gene
in the cross S 19-90 X PI399073. First, ten F3 seedlings from each
of 54 F2:3 families were inoculated with pathotype vir 7 (OH-race
1). The results of this inoculation fit a 15:1 ratio, which was
expected due to the presence of both the new gene and Rps1c.
Second, ten F3 seedlings were inoculated with vir 1a, 1b, 1c, 1k, 7
(OH-race 25). This inoculation also fit a 3:1 ratio, 38 F2:3
families that were scored as resistant and 16 were scored as
susceptible (.chi.2=0.61).
[0077] The SSR markers on MLG F found to be polymorphic in the
first cross were tested on the parents of the second cross S 19-90
X PI399073. Satt114 and Satt334 were also polymorphic for this
cross. Other SSR markers from MLG F were tested with S 19-90 and
PI399073 and Sat.sub.--229 was found to be polymorphic as well.
[0078] All of the polymorphic markers for S 19-90 and PI399073 were
used in single-factor ANOVAs. The markers Satt114, Satt334 and
Sat.sub.--229 on MLG F were associated with the resistance
phenotype in the S 19-90 population (P<0.05).
Example 6
Introgression to Produce HFX01-602
[0079] F.sub.1 plants of HFX01-602 were grown in the OARDC
greenhouse at Wooster, Ohio and tested for the presence of Rps8
from PI 399.073. Remnant seed from plants that were segregating or
homozygous for Rps8 were bulked to create seeds deposited as
HFX01-602.
[0080] F.sub.3 seed were single-plant harvested from four plants.
Seed of all of the plants were evaluated for the presence for Rps8
by inoculation with a Phytophthora sojae isolate with following
pathotype (1a, 1b, 1c, 1k, 3a, 3c, 4, 5, 6, 7). Kottman has genes
Rps1k and Rps3a with high levels of partial resistance and NK
S19-90 has Rps1c. No P. sojae isolate is currently known that can
differentially kill plants with Rps8 in a consistent fashion, so
there is no means to identify if these other Rps genes are present
or segregating in these lines.
[0081] Table 8 shows Phenotypic analysis of F2:3 lines of OXO01-602
soybean lines for resistance to a P. sojae pathotype (vir 1a, 1b,
1c, 1k, 3a, 3c, 4, 5, 6, 7) for the number of lines that are
homozygous resistant compared to the number of lines that are
segregating and homozygous susceptible.
TABLE-US-00008 TABLE 8 OX01-602 F.sub.1 plant F.sub.2 plant
Rps8Rps8:Total 3 1 5:39 3 3 (no molecular data) 4:34 4 1 None 4 2
17:41
* * * * *
References