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CROP SCIENCE, VOL. 48, JANUARY–FEBRUARY 2008 253
RESEARCH
PUCCINI A GRAMINIS Pers.:Pers. f. sp. tritici Eriks. & E Henn., the
causal agent of stem rust, can potentially devastate both durum
wheat (Triti c u m durum Desf.) and common wheat (T. a e s t i v u m L.)
crops throughout the world. Recently, stem rust reemerged as a
serious threat because of a new highly virulent race TTKS (com-
monly known as Ug99) (Pretorius et al., 2000). The rst outbreak
was in Uganda in 1999, and the race has also been seen in parts of
Kenya and Ethiopia (Wanyera et al., 2006). Currently, researchers
with the Global Rust Initiative (http://www.cimmyt.org) have
con rmed the existence of TTKS in Yemen in the Arabian Pen-
insula. This new race has the potential to spread from the a ected
countries and jeopardize wheat production worldwide (Expert
Panel on the Stem Rust Outbreak in Eastern Africa, 2005). The
threat of TTKS has resulted in the establishment of the Global
Rust Initiative and its recommendation to use and incorporate
multiple stem rust resistance genes in commercial cultivars as a
strateg y to provide durable resistance against stem rust.
The two hard red spring cultivars, CItr 12632 (= W1656)
and CItr 12633 (= W1657), carry the stem rust resistance gene
Sr36, derived from T. timopheevi (Allard and Shands, 1954). These
Diagnostic Microsatellite Markers for the
Detection of Stem Rust Resistance Gene Sr36
in Diverse Genetic Backgrounds of Wheat
Toi J. Tsilo,* Yue Jin, and James A. Anderson
ABSTRACT
The wheat stem rust resistance gene Sr36,
derived from Triticum timopheevi, confers a high
level of resistance against a new race (TTKS,
or commonly known as Ug99) and many other
races of Puccinia graminis f. sp. tritici. Because
Sr36-virulent races exist, breeding for durable
resistance would require pyramiding Sr36 with
other genes, a process that can be facilitated
by DNA markers. The aim of this study was to
identify and validate microsatellite markers for
the detection of Sr36 in wheat breeding pro-
grams. Two populations of 122 F2 (LMPG ×
Sr36/9*LMPG) and 112 F2 (‘Chinese Spring’
× W2691Sr36-1) were evaluated for stem rust
reaction. Both populations exhibited distorted
segregation with a preferential transmission
of the Sr36-carrying segment. Three markers,
Xstm773-2, Xgwm319, and Xwmc477, were
in complete linkage with Sr36 in the LMPG ×
Sr36/9*LMPG population. In the Chinese Spring
× W2691Sr36-1 population, Xgwm319 was 0.9
cM away from Xstm773-2, Xwmc477, and Sr36.
These codominant markers were easy to score
and diagnostic for Sr36 in a set of 76 wheat cul-
tivars and breeding lines developed in 12 coun-
tries. Together, these markers can be used in
marker-assisted selection of Sr36.
T.J. Tsilo and J.A. Anderson, Dep. of Agronomy and Plant Genetics,
411 Borlaug Hall, Univ. of Minnesota, St. Paul, MN 55108; Y. Jin,
USDA-ARS, Cereal Disease Lab., 1551 Lindig Ave., Univ. of Min-
nesota, St. Paul, MN 55108. Received 12 Apr. 2007. *Corresponding
author (tsilo001@umn.edu).
Abbreviations: CS, Chinese Spring; DH, double haploid; HR, homo-
zygous resistant; HS, homozygous susceptible; IT, infection type; PCR,
polymerase chain reaction; RFLP, restriction fragment length polymor-
phism; seg, segregating; SSR, simple sequence repeat.
Published in Crop Sci. 48:253–261 (2008).
doi: 10.2135/cropsci2007.04.0204
© Crop Science Society of America
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has been obtained by the publisher.
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254 WWW.CROPS.ORG CROP SCIENCE, VOL. 48, JANUARY–FEBRUARY 2008
cultivars served as the original sources of Sr36 in wheat
breeding programs worldwide (Roelfs, 1988a; Knott,
1989; McIntosh et al., 1995). The Sr36 gene is one of the
18 stem rust resistance genes that provide a major source
of resistance to TTKS (Singh et al., 2005; Wanyera et al.,
2006). However, none of t he 18 g ene s occ urs at a high f re-
quency in breeding materials, except Sr36. In the United
States, Sr36 provides resistance against QFCS, which was
the most predominant race in recent surveys ( Jin, 2005)
and was also found in previous surveys (McVey et al.,
1996, 2002). To some races of stem rust, Sr36 conditions
unusual (mixed) infection types (Ashagari and Rowell,
1980), which can make it di cult to distinguish cultivars
carrying this gene.
Because Sr36-virulent races exist (Knott, 1989), this
gene is best deployed when pyramided with other Sr genes
(Knott, 1988), a process that cannot easily be achieved
through the conventional phenotypic screening methods.
The drawback of classical breeding methods is that the
process of pyramiding genes in a single line can be time
consuming or impossible, especially when more than one
gene confers resistance against known races of P. graminis f.
sp. tritici; hence, it becomes di cult to identify genotypes
carrying combinations of more than one gene. Pyramid-
ing of resistance genes could be facilitated by marker-
assisted selection.
Nyquist (1957) used monosomic analysis to locate
Sr36 on chromosome 2B; it was later mapped on the short
arm of chromosome 2B (Gyarfas, 1978; McIntosh and
Luig, 1973). Bariana et al. (2001) identi ed 10 molecular
markers linked to the Sr36 locus. Eight were restriction
fra g ment leng t h polymorphism ( R FLP) or ampli ed frag-
ment length polymorphism (AFLP) markers, and two were
microsatellites (STM773 and GWM271). These authors
reported that STM773 gave a better ampli cation than
GWM271. However, even though the STM773 marker
could be directly used to identify homozygous genotypes
for Sr36, this marker requires careful scoring because the
primers also amplify other fragments, making it di cult
to distinguish heterozygous from homozygous genotypes.
Therefore, more robust, codominant, and easy-to-detect
microsatellite markers are needed for Sr36.
The objectives of this study were (i) to identify
codominant microsatellite markers closely linked to Sr36;
and (ii) to validate their potential use in marker-assisted
selection of Sr36 using a set of diverse wheat germplasm.
MATERIALS AND METHODS
Plant Materials
Genetic analysis of Sr36 was performed with t wo F2 mapping
populations. The 122 F2 individuals were der ived from a cross
between a susceptible wheat line, LMPG, and its near-isogenic
line Sr36/9*LMPG carrying Sr36. The genetic stock Sr36/
9*LMPG was developed by Dr. D. Knott at the University of
Saskatchewan, Saskatoon, Canada (Knott, 1990). An additional
112 F2 individuals were derived from a cross between a sus-
ceptible wheat cultivar Chinese Spring (CS) and the resistant
line W2691Sr36-1, carrying Sr36 in the genetic background
of W2691. The F2 populations and their subsequent F3 families
were grown in the greenhouse at the University of Minnesota,
St. Paul, during spring 2005 and fall 2005, respectively.
In addition to the four wheat lines used for genetic analysis
of Sr36, a diverse set of 76 wheat cultivars and breeding lines
were obtained from the USDA-ARS National Small Grains
Collection, Aberdeen, ID. These accessions and breeding
lines were selected on the basis of previously published reports
that indicated whether they possess Sr36 (accessions with a nd
without Sr36) (Table 1). The information on the pedigree and
the presence of Sr36 was obtained from two USDA Websites
(ht tp://www.ars-grin.g ov/n pgs/a c c/ac c _ quer ie s.ht m l, http://
wheat.pw.usda.gov) and McIntosh et al. (1995). It was supple-
mented with information from previous surveys of seedling
resistance conducted at the USDA-ARS Cereal Disease Lab-
oratory. The Chinese Spring nullisomic-tetrasomic (N2B-
T2D) line (Sears, 1966) was used to verify the location of the
ampli ed bands of microsatellite markers.
Stem Rust Inoculation and Evaluation
Stem rust screenings were performed on seedlings of paren-
tal lines (Sr36/9*LMPG, LMPG, W2691Sr36-1, CS), 122 F2
(LMPG × Sr36/9*LMPG) lines, and 112 F2 (CS × W2691Sr36-
1) lines. To determine the F2 genotypes and also to distinguish
heterozygous from homozygous resistant F2 line s, 16 t o 3 0 pla nt s
of each F2:3 family (seeds derived from bagged F2 spikes) were
tested for segregation at the Sr36 locus using the race QFCS
(isolate 03ND76C), which is avirulent on Sr6, Sr7b, Sr9b, Sr9e,
Sr11, Sr30, Sr36, and SrTmp. For inoculation, urediniospores
of QFCS stored at −80°C were heat shocked and suspended
in a lightweight mineral oil (soltrol 170) and sprayed on two-
leaf stage seedlings (~7 d after planting, when the primary
leaves were fully expanded) following protocols described by
Jin (2005). Inoculated seedlings were kept overnight in a dew
chamber for 16 h with no light and then exposed to 2 to 4 h of
light to complete infection. After infection, plants were placed
either in a growth chamber with 16 h of light at 20 to 22°C and
8 h of dark at 18 to 20°C or in a greenhouse set at 18 to 21°C
under 160-W very high output (VHO) uorescent tubes with a
16-h photoperiod. Infection types (ITs) were scored from pri-
mary leaves approximately 14 d after inoculation based on the
scale of 0 to 4 as stipulated by Stakman et al. (1962) and modi-
ed by Roelfs (1988b).
The presence and absence of Sr36 in 76 wheat cultivars
and breeding lines was veri ed on the basis of low infection
response (0 = immunity) to QFCS and low infection to MCCF
(Table 1). All races used for inoculation were veri ed on the
basis of their avirulence/virulence formula using 16 Sr di er-
ential lines (Roelfs and Martens, 1988; Roelfs et al., 1993) as
checks to verify the standard race designations of all the races.
Molecular Analysis
For molecular mapping of Sr36, 2 to 3 cm of leaf tissues were
collected from seedlings of parental lines, 122 F2 (LMPG ×
Sr36/9*LMPG) lines, 112 F2 (CS × W2691Sr36-1) lines, and
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CROP SCIENCE, VOL. 48, JANUARY–FEBRUARY 2008 WWW.CROPS.ORG 255
Table 1. Validation of Sr36-linked microsatellite markers using conventional screening methods and polymerase chain reac-
tion–based simple sequence repeat (SSR) markers in wheat cultivars and breeding lines derived from diverse genetic origin.
Cultivar/breeding line†Accession number Origin Puccinia graminis races Sr36‡SSR marker§
QFCS TPMK MCCF GWM 319 STM 773-2 WMC 477
1Sr36/9*LMPG Canada 0
¶40++++
2LMPG Canada3+44––––
3W2691Sr36-1 Australia031;++++
4 ‘Chinese Spring’ CItr 14108 China 4 3+ 3+ – – – –
5 ‘GA-Dozier’ PI 591000 USA 2 2 2 – – – –
6‘Fleming’ PI 599615USA ;122––––
7‘GA-Stuckey’ PI 591001USA 00;0++++
8‘Goodstreak’ PI 632434USA ;10;;––––
9‘Harry’ PI 632435USA ;0;– – – –
10‘Jaypee’ PI 592760USA 03+0++++
11‘Sisson’ PI 617053USA 0;10++++
12 ‘Morey’ PI 591428 USA 2+ 2 2 – – – –
13‘NC-Neuse’ PI 633037USA 0;20;++++
14 ‘Patterson’ PI 583825 USA 3 2, 3- 2+ – – – –
15W1656 CItr 12632USA 040+ + + +
16‘Mengavi’ PI 290912Australia040+ + + +
17‘Arthur’ CItr 14425USA 00, 40 + + + +
18 ‘Arthur 71’ CItr 15282 USA 0 0 0 + + + +
19 ‘Gouritz’ PI 479672 South Africa 0 1 0 + + + +
20W1657 CItr 12633USA 04;++++
21‘Maris Fundin’ PI 410869UK 43+4 – – – –
22‘Zaragoza 75’ PI 519305Mexico 43+4 – – – –
23 W 3496 PI 520133 Australia 0 2+ 0 + – – –
24 ‘Timson’ PI 404115 Australia 0 0; 0 + + + +
25 NE 73843 PI 519136 USA 0 0 0; + + + +
26 CI 1405 0 CI tr 14 05 0 U S A u u u + + + +
27TA 1600 PI 603223Iran uuu– – – –
28RL 6044 CItr 17752Canada021+––––
29RL 5045 PI 520492Canada0;2, 41+––––
30 ‘Tosca’ PI 479680 South Africa 4 4 4 – – – –
31 Sr 6 CItr 15082 Canada 0; 2, 4 1 – – – –
32‘Eureka’ CItr 17738USA 0;21––––
33 ‘Red Egyptian’ CItr 12345 Egypt u u u – – – –
34 ‘Excel’ PI 555465 USA 0; 2 2 +/? ± ± ±
35CK 9803 USA 00, 10++++
36‘Ernie’ PI 599615USA 0;0+ + + +
37‘Halt’ USA 1;3+1––––
38‘Intrada’ PI 631402USA ;11+––––
39 ‘Roughrider’ CItr 17439 USA 0 2+ 0 + + + +
40 ‘TAM 200’ PI 578255 USA 1; 1+ 1+ – – – –
41 ‘Vista’ PI 562653 USA 0 2 0 + + + +
42‘Brundage 96’ USA 444– – – –
43‘Chukar’ PI 628641USA 444– – – –
44 CK 9877 USA 2+, 4 2+ 0, 3+ - ± ± –
45‘Roane’ USA 42+3+––––
46‘Rosen’ CItr 17607USA 000;++++
47‘Truman’ PI 634824USA 444 – – – –
48‘TAM 105’ CItr 17826USA 2-44––––
49 Idead 59 CItr 13631 USA 0, 4 4 4 ± ± ± ±
50 ‘Timvera’ PI 351987 Australia 0 4 0 + + + +
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256 WWW.CROPS.ORG CROP SCIENCE, VOL. 48, JANUARY–FEBRUARY 2008
76 wheat cultivars and breeding lines. Total genomic DNA was
extracted from the ground tissues following protocols described
by Riede and Anderson (1996) and modi ed by Liu et al.
(2006). Since Sr36 was mapped on the short arm of chromo-
some 2BS (Gyarfas, 1978; Bariana et al., 2001), microsatellite
markers used in this study were based on previously published
wheat genetic maps of chromosome 2BS (Röder et al., 1998;
Somers et al., 2004; Song et al., 2005). A total of 51 microsat-
ellite primer pairs were screened for polymorphisms between
near-isogenic lines LMPG and Sr36/9*LMPG, and between
two diverse lines, Chinese Spring and W2691Sr36-1. Two mic-
rosatel lite markers (STM773 and GWM271) were previously
reported to be linked to Sr36 in a double haploid population of
‘Sunco’ × ‘Tasman’ (Bariana et al., 2001). The marker STM773
has since been converted into two SSR markers, STM773-1 and
STM773-2, and were included in the analysis. The sequences of
STM773-1 and STM773-2 were kindly provided by Dr. Mat-
thew Hayden, University of Adelaide, South Australia.
Polymerase Chain Reaction
and Electrophoresis
Polymerase chain reaction (PCR) was performed in a 96-well
plate with 10 L of nal reaction mixture containing 2.75 L
ddH2O, 1 L 10X PCR bu er, 0.6 L of 25 m M MgCl2, 1.6 L
of 1.25 mM dNTPs, 1 L of each 1 µM primer, 0.05 L of 5U
L−1 Taq DNA polymerase (Applied Biosystems, Branchburg,
NJ), and 3 L of 15 ng L−1 genomic DNA. For al l the SSR
markers, except STM773-1 and STM773-2, the PCR reaction
mixture was initially denatured at 94°C for 10 min, followed
by 35 cycles of 94°C for 1 min, 48 to 61°C (depending on
annealing temperature speci c to individual primer pairs) for 1
min, and 72°C for 2 min, with a nal extension step of 72°C
Table 1. Continued.
Cultivar/breeding line†Accession number Origin Puccinia graminis races Sr36‡SSR marker§
QFCS TPMK MCCF GWM 319 STM 773-2 WMC 477
51 ‘Timgalen’ Australia 0, 1 4 0 ± + + +
52‘Hand’ CItr 17288USA 000+ + + +
53‘Kenosha’ CItr 14025USA 0;0;++++
54 ‘Purdue’ USA 2, 4 1 2, 4 – – – –
55‘Centurk’ CItr 15075USA 11;––––
56II-53-764 CItr 15711USA ;21+;––––
57‘Gamut’ PI 329230Australia0;00;––––
58 ‘Songlen’ PI 404114 Australia 0 0 0 + + + +
59 ‘Timvera’ PI 237648 Australia 0 2, 4 0 + + + +
60‘Oxley’ PI 386167Australia;40––––
61‘Gatcher’ PI 377884Australia ;11;1– – – –
62‘Tarsa’ PI 422408Australia;uu––––
63‘Shortim’ PI 422407Australia0;0;++++
64 ‘Kenya Plume’ CItr 14335 Kenya 0; 1+ 0 – – – –
65Zaragoza 75 PI 428428Mexico 444 – – – –
66Zaragoza 75 PI 433770Mexico0;10;++++
67Zaragoza 75 PI 479665Mexico0;;0;?–––
68 ‘Lerma Rojo 64’ CItr 13929 Mexico ; ;1 ; – – – –
69 Red Egyptian PI 45374 South Africa 0; 2, 2+ 0 – – – –
70 Red Egyptian PI 45403 South Africa 2 2+ 4 – – – –
71 Red Egyptian PI 45415 South Africa ; 2+ ; – – – –
72 Red Egyptian PI 192020 Ethiopia ; 2 ; – – – –
73 Idaho 1877 NR AE PI 234233 Zambia 0; 0; 1 ++++
74 ‘Kenya 58’ CItr 12471 Kenya 0 ; 2- – – – –
75 ‘Marquis’/9*RE Canada 1+ u 2+ – – – –
76‘McMurachy’ PI 122985Canada;20;––––
77 ISr9a-Ra CItr 14177 USA 3 2 2- – – – –
78W2691Sr9b CItr 17386Australia12+2+––––
79 ISr9d-Ra CItr 14177 USA u 4 1; – – – –
80CnsSr9g 441+––––
Marker alleles 566
†Represents the orde r of wheat cultivars and breeding lines as i t appear s in Supple mentar y Fig. 1 (lane 1–43).
‡The prese nce of Sr36 in thes e cultivar s was based o n previous ly published reports. The status of Sr36 was veri fi ed based on re sults of QFC S. + or – repres ents presence
or absen ce, ± indicates heteroz ygosit y at the Sr36 locus, ? i ndicates u ncert ainty.
§For SSR mar kers, + or – ind icates pre sence or absence of the Sr36-linked ma rker alle le, ± indic ates heteroz ygosit y.
¶Infectio n types a s described by Stakman et al. (1962) and modi fi ed by Roelfs (1988b); u = missing data.
Reproduced from Crop Science. Published by Crop Science Society of America. All copyrights reserved.
CROP SCIENCE, VOL. 48, JANUARY–FEBRUARY 2008 WWW.CROPS.ORG 257
for 10 min and 4°C inde nitely. The PCR reaction protocol for
STM773-1 and STM773-2 primers was provided by Dr. Mat-
thew Hayden as a touchdown PCR program with 94°C for 10
min, followed by touchdown PCR program with 7 cycles of 60 s
at 92°C, 60 s at 64°C, 60 s at 72°C, and then ve cycles with 60
s at 92°C, 60 s at 57°C, and 60 s at 72°C (conditions identical to
the previous cycles, but with an annealing temperature of 57°C).
The program also included an additional 10 to 25 cycles each
of 30 s at 92°C, 60 s at 55°C, and 60 s at 72°C. The PCR was
ended by an extra incubation for 10 min at 72°C and 4°C inde -
nitely. Polymerase chain reaction thermal cycling was performed
in PerkinElmer/Applied Biosystems (Foster City, CA) thermo
cyclers. About 5L of 3X loading dye (0.02 g bromophenol blue,
0.02 g xylene cyanol, 1.6 mL 0.5 M EDTA, 38.4 mL formamide)
was added to the PCR products to make a nal volume of 15
L. A ll samples were denatured for 5 min at 95°C. The PCR
products were subjected to electrophoresis in a polyacrylamide
gel (6% [w/v] acrylamide/bisacrylamide, 20:1, 8 M urea in TBE,
pH 8.3) in 1X TBE bu er (90 mM Tris-borate [pH 8.3], 2 mM
EDTA) at a constant power of 110 W for 90 min. Gels were silver
stained (Bassam et al., 1991) and photographed.
Genetic Linkage Analysis
For the genetics analysis of Sr36, F2 genotypes inferred from
seedling reactions of F2:3 and F3:4 families were classi ed as
homozygous resistant (HR), segregating (Seg) and homozy-
gous susceptible (HS). Chi-squared (χ2) distribution analyses
were used to test if the observed segregation ratios for Sr36 and
marker loci t the Mendelian ratio of 1:2:1. Genetic linkage
analysis was performed between polymorphic microsatellite
markers and the Sr36 segregation data using Mapmaker com-
puter program version 3.0b (Lander et al., 1987).
RESULTS
Segregation Analysis of Sr36
in the Two F2 Populations
The wheat lines Sr36/9*LMPG and
W2691Sr36-1 were highly resistant to race
QFCS (infection type 0), and lines LMPG
and CS were susceptible (infection types
3+ and 4). The F2 genotypes were inferred
from F2:3 plants that were tested and grouped
based on their rust reaction when inoculated
with QFCS. The LMPG × Sr36/9*LMPG
and the CS × W2691Sr36 -1–der ived popu-
lations segregated 43HR:54Seg:24HS and
54HR:35Seg:10HS, respectively (Table 2).
The segregation patterns in both populations
were signi cantly di erent than the expected
segregat ion ratio of 1HR:2Seg:1HS (χ2 = 7.36,
P = 0.025; and χ2 = 47.6, P < 0.001).
Genetic Mapping of the Sr36 Gene
Of 53 microsatellite markers which were
previously shown to be located on chro-
mosome 2BS, the same 21 markers showed
polymorphism between LMPG and Sr36/9*LMPG, and
between Chinese Spring and W2691Sr36-1 lines. Because
of the reduced informativeness of dominant markers that
cannot distinguish heterozygous and homozygous allele
states, only codominant markers were used for further
analysis. Four markers, GWM429, GWM319, WMC477,
and STM773-2, were codominant and gave clear, readable
fragments of 220, 170, 190, and 155 bp in the resistant F2
plants and 210, 180, 160, and 190 bp in the susceptible
F2 plants, respectively (Fig. 1). However, the primers for
WMC477 also ampli ed a n add ition a l frag ment of 158 bp,
which is visible in homozygous resistant but not heterozy-
gous plants (Fig. 1C). The marker GWM429 was codomi-
nant only in the LMPG × Sr36/9*LMPG population and
was dominant in the CS × W2961Sr36-1 population. The
SSR marker data, together with rust screening data, dis-
played a similar distortion trend that favored the Sr36-
containing segment over the non-Sr36 segment (Table 2).
This implies that both populations segregated for a single
gene conferring resistance to QFCS.
A linkage map was generated for each population (Fig.
2). In the LMPG × Sr36/9*LMPG population, the three
markers, Xstm773-2, Xgwm319, and Xwmc477, showed
complete linkage to the Sr36 gene (Fig. 2). Also in the CS
population, the two markers, Xstm4773-2 and Xwmc477,
showed complete linkage to Sr36, while Xgwm319 was 0.9
cM from Sr36 (Fig. 2).
Validation of Microsatellite
Markers Tightly Linked to Sr36
To determine the diagnostic value of the microsatellite
markers identi ed in this study, a set of 76 wheat cultivars
and breeding lines with diverse origins were genotyped
with three microsatellite markers that were tightly linked
Table 2. Segregation ratios of Sr36 and linked simple sequence repeat
marker alleles in F2 populations derived from crosses between susceptible
and resistant parents.
Population Gene/
marker Tot a l†Observed‡Expected
ratio X2P
value§
X1X1X1X2X2X2
LMPG × Sr36/
9*LMPG
Sr36 121 43 54 24 1:2:1 7.36 0.025
Xgwm319 122 43 55 24 1:2:1 7.10 0.029
Xwmc477 122 43 54 25 1:2:1 6.92 0.031
Xstm773-2 122 44 54 24 1:2:1 8.16 0.017
Xgwm429 122 43 56 23 1:2:1 7.38 0.025
Chinese Spring
× W2691Sr36-1
Sr36 99 54 35 10 1:2:1 47.6 <0.001
Xgwm319 112 58 44 10 1:2:1 46.3 <0.001
Xwmc477 112 58 43 11 1:2:1 45.5 <0.001
Xstm773-2 112 58 43 11 1:2:1 45.5 <0.001
Xgwm429 112 58 54 1:3 42.9 <0.001
†Due to seed ling letha lity, the F2 plants not evaluate d at the F3 generation were re corded as missing d ata.
‡X1X1 = homozygous for re sistant parent’s allele; X1X2 = heterozygous; X 2X2 = homozygous for susceptibl e
parent’s all ele. F2 genotypes were inferred from infe ction types of F2:3 or F3:4 families to distinguish X1X1,
X1X2, and X2X2 F2s.
§P value less than 0.05 was u sed to accept a distorted segregation from exp ected ratio of 1:2:1.
Reproduced from Crop Science. Published by Crop Science Society of America. All copyrights reserved.
258 WWW.CROPS.ORG CROP SCIENCE, VOL. 48, JANUARY–FEBRUARY 2008
to Sr36. All accessions that were known to
carry Sr36 were validated based on their reac-
tion as seedlings to races QFCS, TPMK, and
MCCF (Table 1). The race QFCS is avirulent
on Sr6, Sr7b, Sr9b, Sr9e, Sr11, Sr30, Sr36, and
SrTmp; however, cultivars and lines carrying
Sr36 were clearly distinguishable with zero
infection type (immunity) compared with
the low infection type (small ecks or IT0;)
produced by lines carrying Sr6 (temperature-
sensitive stem rust resistance gene). The DNA
fragments of 170, 190, and 155 bp were ampli-
ed in cultivars and breeding lines carrying
the Sr36-containing chromosome segment for
the three microsatellite markers, Xgwm319,
Xwmc477, and Xstm773-2, respectively (Table
1; Supplementary Fig. 1, online). These frag-
ment sizes were ampli ed in 30 wheat culti-
vars and breeding lines known to carry Sr36
(Table 1). The results indicate that three mark-
ers identi ed Sr36 correctly in these Sr36-car-
rying cultivars.
Many cultivars and breeding lines that did
not carry Sr36 displayed ITs other than immu-
nity against race QFCS (Table 1). In ‘Excel’,
the marker data reveals Sr36-associated alleles
in a heterozygous state. In 2 out of 80 wheat
cultivars and breeding lines, ‘W 3496’ and
‘CK 9877’, we found that the three markers,
Xgwm319, Xmwc477, and Xstm773-2, were not
in agreement with the stem rust screening
results. The W3496 line was developed in Aus-
tralia and traces back to Verntein/CItr 12632
as ultimate parents in its pedigree. This line
showed zero IT to QFCS, meaning that it could
be carrying Sr36 from CItr 12632, a known
carrier of this gene. CK 9877 showed 2+/3+
against QFCS, indicating that this line did not
have Sr36, and only marker Xwmc477 showed
the absence of the Sr36-speci c marker allele,
whereas Xgwm319 and Xstm773-2 showed the
presence of Sr36-speci c marker alleles, mean-
ing that there could have been recombination
between Sr36 and the two markers Xgwm319
and Xstm773-2.
DISCUSSION
Mapping Sr36 in Wheat
McIntosh and Luig (1973) reported a recom-
bination frequency of 20% between Sr36 and
Sr9. The two genes were located on di erent
chromosome arms; the Sr9 locus was mapped
on 2BL (Sears and Loegering, 1968; Tsilo et
al., 2007). In a recent study, Bariana et al.
Figure 1. Gel electrophoresis showing segregation pattern of the four SSR
markers, (A) Xgwm429, (B) Xgwm319, (C) Xwmc477, and (D) Xstm773-2, in a
subset of the F2 progenies from a cross between near-isogenic lines (Sr36/
9*LMPG and LMPG); P1, resistant parent; P2, susceptible parent; R, resistant F2;
S, susceptible F2; H, heterozygous F2 progenies. The arrow points indicate the
size of the band associated with Sr36.
Figure 2. Partial genetic linkage maps of chromosome 2BS depicting the location
of Sr36 with linked codominant simple sequence repeat loci in the LMPG × Sr36/
9*LMPG population and the CS × W2691Sr36-1 population. The linkage maps
were constructed using map distances (cM) from Kosambi.
Reproduced from Crop Science. Published by Crop Science Society of America. All copyrights reserved.
CROP SCIENCE, VOL. 48, JANUARY–FEBRUARY 2008 WWW.CROPS.ORG 259
(2001) reported two microsatellite markers, Xstm773
and Xgwm271A, together with other RFLP mark-
ers that showed complete linkage to the Sr36 locus in a
double haploid (DH) population of 168 lines; the authors
reported, however, that the Xstm773 marker showed bet-
ter ampli cation than Xgwm271. In their DH population,
STM773 was able to identif y HR and HS lines. However,
the primers for Xstm773, together with primers for sev-
eral other microsatellite markers identi ed in our study,
also ampli ed other bands that can make it di cult to
distinguish homozygous from heterozygous genotypes.
This would complicate its usage in MAS. In the early gen-
erations of breeding populations (i.e., F2, BC1, BC2), for
example, the majority of individuals are heterozygous and
need to be distinguished from homozygous individuals.
To d a t e, STM773 has been converted into two sequence
tagged microsatellite markers, STM773-1 and STM773-2
(M. Hayden, personal communication, 2005).
In this study, we found that GWM319, STM773-2,
and WMC477 were diagnostic for Sr36. Two marker
loci, Xstm773-2 and Xwmc477, were i n com p lete li n k a ge
with Sr36 in both populations. Alleles at the Xgwm319
locus cosegregated with Sr36 in one population and
were tightly linked (0.9 cM) with Sr36 in another pop-
ulation. According to the genetic map of Somers et al.
(2004), Xgwm319 and Xwmc477 were mapped near the
centromere and showed no recombination, con rming
that these markers are closely linked. Therefore, these
three markers would serve as a rst step toward the
detection of Sr36 in breeding populations. Preferential
transmission of Sr36-carrying T. timo p h e e v i segment
was observed (Table 2). The exact mechanism caus-
ing preferential transmission of T. timoph e e v i chromo-
some segment is unknown. However, Nyquist (1962)
hypothesized several possible causes. In our laboratory,
studies are in progress to determine the exact cause.
Validation of Sr36-Linked
Microsatellite Markers
Based on the previous studies, all seven reference stocks
that were widely used as sources of Sr36 in wheat
breedi ng programs (McIntosh et a l., 1995) were used in
this study: two breeding lines, Sr36/9*LMPG (Knott,
1990) and W2691Sr36-1, and six cultivars, CItr 12632
(= W1656) and CItr 12633 (= W1657) (Allard and
Shands, 1954), Idaed 59, Mengavi, Timvera (PI 351987
and PI 237648) (Pridham, 1939), and CItr 14050. In
addition to these reference stocks, we examined a range
of international germplasm carrying Sr36 as listed by
Roelfs (1988a) and McIntosh et al. (1995), including
cultivars developed in Australia, Canada, Mexico,
South Africa, and the United States (Table 1). The list
included cultivars Songlen, Timgalen, Zaragosa 75
(PI 433770), Gouritz, Hand, Kenosha, Roughrider,
Shortim, Timson, Arthur, and Arthur 71. Some of the
newly developed Sr36-carrying cultivars from the U.S.
germplasm were also included—GA-Stuckey, Jaypee,
Sisson, NC-Neuse, NE 73843, Vista, Ernie, CK 9803,
and Rosen (Table 1). All these cultivars and reference
stocks were immune to QFCS and were characterized
using Sr36-linked marker alleles of Xgwm319, Xwmc477,
and Xstm773-2 (Table 1; Jin, unpublished data). Other
cultivars and breeding lines that were known to carry
Sr36 were developed in Zambia and the United King-
dom, including Idaho 1877 NR AE and Maris Fun-
din. Both the marker analysis and stem rust screening
indicate that the accession of Maris Fundin (PI 410869)
obtained from the National Small Grains Collection
was incorrect (Table 1). However, another possibility is
that Maris Fundin does not carry Sr36 and that incor-
rect information about this germplasm exists at the
GrainGenes database (http://wheat.pw.usda.gov). Idaed
59 was heterogeneous for both the Sr36 resistance and
Sr36-linked marker alleles. The heterogeneity could be
the result of seed contamination. The W3496 line, com-
monly known as Combination III, did not carry any of
the Sr36-a ssociated marker a l leles. This is in agreement
wit h an Au stralian st udy ba sed on a r ecombinant inbred
line population derived from Yarralinka/Schomburgk
(H.S. Bariana and coworkers, personal communication,
2007). A rare recombinant combining T. timopheev i seg-
ment with stem rust resistance gene Sr9e was present in
W3496 and Yarralinka (H.S. Bariana, personal commu-
nication, 2007). The CK9877 line does not have Sr36,
and only marker Xwmc477 was in agreement; however,
loci Xgwm319 and Xstm773-2 showed the presence of
Sr36-associated marker alleles. Therefore, based on
these data, Xwmc477 appears to be the most diagnostic
compared with Xgwm319 and Xstm773-2. However, it is
important to mention that although WMC477 ampli-
es 158- and 190-bp fragments in materials contain-
ing Sr36 (Fig. 1C), we consistently observed the 190-bp
fragment. We suspect that the quantity of individual
PCR products will be lower for one of the fragments;
hence, the 158-bp fragment was faint, and only the
190-bp fragment was consistently visible in all acces-
sions that carried Sr36 (Supplementary F ig. 1). Di erent
PCR-reaction protocols might lead to preferential ampli -
cation of one of the two bands (158 and/or 190 bp). A simi-
lar PCR discrepancy involving ampli cations of multiple
fragments was described by Bercovich et al. (1999).
Many accessions with similar names may be confus-
ing, especially when the name is widely used instead of
the accession number. In this study, we analyzed four
accessions of Zaragoza 75, and only PI43370 carried Sr36.
The other three accessions could be carrying di erent
stem rust resistance genes. According to Roelfs (1988a)
and McIntosh et al. (1995), Purdue carries Sr36; however,
Reproduced from Crop Science. Published by Crop Science Society of America. All copyrights reserved.
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there were many accessions of Purdue at the National
Small Grains Collection, and the one used in this study
did not carry Sr36 according to the results of reaction to
QFCS and Sr36-linked markers.
In this study, the information obtained from using
races of P. graminis f. sp. tritici alone was not diagnostic of
Sr36 in the presence of other Sr genes. This situation was
observed in two cultivars, RL 6044 and Kenya 58, which
showed immunity to QFCS, and low IT to TPMK and
MCCF (Table 1). However, immunity in these cultivars
was due to genes or combination of genes other than Sr36.
RL 6044 is known to carry Sr33 from Tetra Canthatch//
Aegilops squarrosa, whereas Kenya 58 carries Sr6 and other
genes from Red Egyptian and Kenyan cultivars (McIn-
tosh et al., 1995). With conventional screening tests, it
would require extensive seedling tests and testcrosses to
perform gene postulation in these cultivars by using the
appropriate stem rust races—a potentially time-consum-
ing process if two or more genes confer resistance to a
particular race. However, both the rust screening results
and previously published information were successful in
validating the cultivars that carried Sr36, and the results
were in agreement with the Sr36-speci c marker alleles.
From these results, it is clear that the Sr36-linked markers
are diagnostic for this gene and can be used to detect its
presence during cultivar development.
Even though Sr36 does not provide a high level of
resistance against a wide range of stem rust races, it is still
a valuable gene because it is the best available source of
resistance to the new race of stem rust, Ug99. Therefore,
tightly linked markers identi ed in this study should be
useful in marker-assisted selection of Sr36 and can be used
in selecting for genotypes possessing Sr36 during culti-
var development. These markers will accelerate the use of
Sr36 in commercial cultivars by allowing pyramiding of
Sr36 with other e ective genes to confer a more durable
resistance. Our results show that these markers are appli-
cable across di erent genetic backgrounds.
Acknowledgments
We are thankful to Dr. Matthew Hayden for providing the
information on the STM773-1 and STM773-2 markers, and
Dr. Harbans Bariana for his useful comments on the manuscript.
This research was supported in part by the Minnesota Annual
Conference of the United Methodist Church through the Project
AgGrad fellowship awarded to T.J. Tsilo, the Agricultural Research
Council of South Africa, and the USDA Cooperative Research,
Education and Extension Service, Coordinated Agricultural
Project grant number 2006-55606-16629.
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