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Published in Crop Sci. 44:1403-1411 (2004).
© 2004 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA

GENOMICS, MOLECULAR GENETICS & BIOTECHNOLOGY

Microsatellite Markers Linked to the Stb2 and Stb3 Genes for Resistance to Septoria Tritici Blotch in Wheat

Tika B. Adhikaria, Hugh Wallworkc and Stephen B. Goodwinb,*

a Department of Plant Pathology, 306 Walster Hall, North Dakota State University, Fargo, ND 58105-5012
b USDA-ARS, Crop Production and Pest Control Research, Department of Botany and Plant Pathology, 915 West State Street, Purdue University, West Lafayette, IN 47907-2054
c Cooperative Research Centre for Molecular Plant Breeding, South Australian Research and Development Institute (SARDI), GPO Box 397, Adelaide, SA 5001, Australia

* Corresponding author (sgoodwin{at}purdue.edu).


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Septoria tritici blotch (STB) of wheat (Triticum aestivum L.), caused by the fungal pathogen Mycosphaerella graminicola (Fuckel) J. Schröt. in Cohn (anamorph: Septoria tritici Roberge in Desmaz.), occurs naturally in all wheat production areas around the world. Two genes for resistance to this disease, Stb2 and Stb3, have been identified in wheat germplasm and together confer resistance to the most prevalent strains of M. graminicola in Australia and the USA. However, so far neither gene has been mapped in the wheat genome and their linkage relationships to other markers are not known. The objectives of this study were to identify molecular markers linked to the STB resistance genes Stb2 and Stb3 and to map these genes in the wheat genome. Genetic families of doubled-haploid populations segregating for Stb2 and Stb3 were evaluated in the greenhouse for STB reaction during the spring and fall seasons of 2002 and 2003. Genomic DNA isolated from each segregating population was analyzed with microsatellite or simple-sequence repeat (SSR) markers in bulked-segregant analysis to identify those that cosegregated with the STB phenotypes. Linkage analysis identified five SSR markers near the Stb2 gene on the distal region of the short arm of chromosome 3B. Loci Xgwm389 and Xgwm533.1 were approximately 1 cM distal to Stb2, which itself was 3.7 cM distal to Xgwm493. In addition to Stb2, this genomic region contains multiple genes conferring resistance to taxonomically diverse fungal pathogens of wheat, including a major quantitative trait locus for resistance to Fusarium head blight (caused by Fusarium graminearum Schwabe). The SSR marker Xgdm132 was linked to the Stb3 gene at a distance of approximately 3 cM on the short arm of chromosome 6D. The microsatellite markers identified in this study should facilitate marker-assisted selection and pyramiding of Stb2 and Stb3 with other STB resistance genes for more durably resistant wheat.

Abbreviations: BSA, bulked-segregant analysis • DH, doubled haploid • PCR, polymerase chain reaction • RFLP, restriction fragment length polymorphism • SSR, simple-sequence repeat • STB, Septoria tritici blotch


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
SEPTORIA TRITICI BLOTCH, caused by the ascomycete fungus M. graminicola, is an economically important disease of wheat worldwide (Eyal et al., 1985). Yield losses as high as 60% have been reported for susceptible wheat cultivars under severe epidemics (King et al., 1983). In both Australia and the USA, fungicide sprays against STB are not economical. Therefore, genetic resistance is the most effective and environmentally safe approach to combat this disease (Eyal, 1981).

Up to now, eight loci for resistance to STB have been identified, which have been designated in a series from Stb1 to Stb8 (Adhikari et al., 2003; Adhikari et al., 2004; Arraiano et al., 2001; Brading et al., 2002; McCartney et al., 2003; Rillo and Caldwell, 1966; Somasco et al., 1996; Wilson, 1985). None of these genes had been mapped until very recently, but progress has been made during the past 2 yr mostly by determination of linkages with previously mapped microsatellite or SSR (Pestsova et al., 2000; Röder et al., 1998) and RFLP (Nelson et al., 1995a, 1995b, 1995c) markers. Map locations and linked molecular markers now have been published for genes Stb5 through Stb8 (Adhikari et al., 2003; Arraiano et al., 2001; Brading et al., 2002; McCartney et al., 2003), but the locations for the other genes are unknown.

Two potentially important resistance genes, Stb2 and Stb3, were identified in the wheat cultivars Veranopolis and Israel 493, respectively (Wilson, 1985). STB resistance at both loci was conditioned independently and the resistance at each locus appeared to be dominant (Wilson, 1979). However, because neither Stb2 nor Stb3 has been mapped, a lack of linked molecular markers has limited the utility of these genes for cultivar development and germplasm enhancement. Thus, the objectives of this study were (i) to identify SSR markers linked to Stb2 and Stb3 to facilitate marker-assisted selection and (ii) to map Stb2 and Stb3 in the wheat genome.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mapping Populations
The plant populations used in this study were generated at the South Australian Research and Development Institute (SARDI), Adelaide, Australia. One hundred six doubled-haploid (DH) lines (hereafter, Stb2 population) were derived from a cross between Veranopolis, the STB-resistant wheat cultivar carrying the Stb2 gene (Wilson, 1985), and the susceptible line RAC875-2. RAC875-2 is a hard white spring wheat in the breeding program at the University of Adelaide. Similarly, a second population of 97 DH individuals (hereafter, Stb3 population) from a cross between the resistant cultivar Israel 493, possessing the Stb3 gene (Wilson, 1985), and the susceptible line RAC875-2 was developed and tested for phenotypic and marker analysis.

Plant Inoculations and Disease Scoring
Both the Stb2 and Stb3 mapping populations were evaluated for resistance to M. graminicola in a greenhouse of the Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN, USA, during the fall and spring seasons of 2002 and 2003. Wheat seeds were vernalized for 7 d at 4°C then transplanted individually into 10-cm diameter pots filled with a 1:1 peat moss: perlite mix (Sun Grow Horticulture Inc., Bellevue, WA, USA) 10 d after seeding. Plants were fertilized at the four-leaf stage with MiracleGro water-soluble fertilizer (15 N:30 P:15 K). The STB reaction of each DH line from both mapping populations was determined by inoculating with an isolate of M. graminicola collected at Paskeville, South Australia. This isolate was chosen following initial pathogenicity testing (see Results). Inoculum was prepared by placing a 1-cm2 piece of agar culture into 250-mL Erlenmeyer flasks containing 100 mL of yeast-sucrose liquid medium (10 g of sucrose and 10 g of yeast extract in 1 L of distilled water) and 100 µL of kanamycin sulfate (25 mg/mL). Flasks were plugged with cotton and placed on an orbital shaker (Barnstead/Thermolyne, Dubuque, IA, USA) at 150 rpm at room temperature for 3 to 5 d. The inoculum suspension was adjusted to approximately 4 x 106 spores/mL by hemacytometer before inoculation. One drop of Tween 20 (polyoxyethylene-sorbitan monolaurate, Sigma Chemical Co., St. Louis, MO, USA) was added per 100 mL of spore suspension. Plants were inoculated with a hand sprayer approximately 7 wk after transplanting once the flag leaf of each plant was fully emerged from the boot. Mist chambers were built around each inoculated greenhouse bench and the plants were misted to maintain high humidity for 72 h, after which the plastic was removed and the plants remained at ambient greenhouse conditions.

Plants were assessed for STB reaction approximately 3 wk after inoculation. Reactions were recorded by two disease-scoring methods (Gaunt et al., 1986; Rosielle, 1972): (i) disease severity (DS) was based on the visually estimated percentage leaf area of necrotic lesions with or without pycnidia; and (ii) pycnidial density was rated within necrotic lesions with a score ranging from 0 to 5, where 0 = no sporulation and 5 = maximum pycnidial production. The inoculated plants that showed near-immune responses with DS of less than 5% and pycnidial density of 0 to 1 were considered resistant (R). Diseased plants with DS of more than 5% and pycnidial density higher than 1 were considered susceptible (S). These criteria gave good separation between resistant and susceptible individuals in previous analyses (Adhikari et al., 2003). Each pot contained a single plant of a DH or parental line and each inoculated plant was treated as an experimental unit. From six to eight plants of each line were tested each season. Scores for DS and pycnidial density from all seasons were combined and averaged. Chi-square statistics were used to test for goodness of fit of the observed data to expected segregation ratios.

DNA Extraction
One-week-old expanding leaves were harvested from the parents and the 203 lines of both DH populations and placed in 1.5-mL microcentrifuge tubes. The leaves were lyophilized for 72 h and stored at –80°C. Wheat leaves were ground in liquid nitrogen to a fine powder with a mortar and pestle. Genomic DNA was prepared from frozen leaves of the parents and all lines comprising the Stb2 and Stb3 populations with the DNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA) according to the specifications of the manufacturer. The concentration of each DNA sample for microsatellite analysis was adjusted to 6 ng/µL with a fluorometer (Hoefer Scientific Instruments, San Francisco, CA, USA).

Microsatellite Analysis
A total of 140 wheat SSR markers designated as either Xgwm for Gatersleben (Germany) wheat microsatellite (Röder et al., 1998) or Xgdm for Gatersleben D-genome microsatellite (Pestsova et al., 2000), selected to cover each chromosome arm of all three wheat genomes, was tested for useful polymorphisms in both DH mapping populations. Once the approximate chromosomal locations of the Stb2 and Stb3 genes were confirmed by amplifying genomic DNA of nullisomic-tetrasomic lines (Sears, 1966) with specific SSR markers, 14 and 12 Beltsville Agriculture Research Center (BARC) primers from wheat chromosomes 3B and 6D, respectively, also were evaluated for polymorphism on the Stb2 and Stb3 populations. Additional markers on chromosome 3BS also were tested on the Stb2 DH population. These included 20 expressed sequence tag (EST) primers kindly provided by J.A. Anderson, University of Minnesota, St. Paul, MN, USA. These EST primers were developed through the NSF-supported U.S. EST project (http://wheat.pw.usda.gov/NSF; verified 9 March 2004) and were requested because they are associated with a major quantitative trait locus (QTL) for resistance to Fusarium head blight (FHB) on wheat chromosome 3BS (Liu and Anderson, 2003). Another marker in the same chromosomal region was an amplified fragment length polymorphism (AFLP) primer combination that was converted into a sequence-tagged site (STS) marker because it also was associated with the major QTL for resistance to FHB on chromosome 3BS (Guo et al., 2003). All SSR, BARC, STS, and EST primers were synthesized by MWG Biotech Inc. (Charlotte, NC, USA). The letter "X" in front of each primer refers to the basic symbol for molecular marker locus with unknown function in wheat.

Polymerase chain reaction (PCR) for each micosatellite and BARC primer was performed as described previously by Adhikari et al. (2003). Each PCR reaction (25 µL) contained 5.8 µL of sterile deionized water, 4 µL of 10x Q-solution, 2 µL of 10x PCR buffer, 1 µL of 25 mM MgCl2, 2 µL of 10 mM dNTPs, 4 µL of primer solution (4 µM each), 0.2 µL of Hot Star Taq DNA polymerase (5 units/µL), and 6 µL of 6 ng/µL template DNA. PCR was performed in a PTC-100 Thermal Cycler (MJ Research, Watertown, MA, USA) at standard amplifications of 94°C for 3 min, followed by 44 cycles of 94°C for 1 min, 50, 55, or 60°C (based on primer annealing temperature) for 1 min, and 72°C for 2 min, then with a final extension at 72°C for 7 min before cooling to 4°C. For STS and EST primers, PCR reactions and conditions were according to Guo et al. (2003) and Liu and Anderson (2003), respectively. Aliquots (20 µL) of PCR products were separated by electrophoresis in 3% (w/v) agarose gels in 0.5x TAE buffer (0.02 M Tris, 0.0095 M glacial acetic acid and 0.005 M EDTA). Gels were stained with ethidium bromide and visualized by means of an ultraviolet transilluminator. PCR products also were separated on 12% (29:1 acrylamide: Bis) nondenaturing polyacrylamide gels and were detected by silver staining as described by Adhikari et al. (2003).

On the basis of the phenotypic data, molecular markers linked to each resistance gene were identified by bulked-segregant analysis (BSA) (Michelmore et al., 1991). For mapping each population, resistant and susceptible bulks consisted of equal amounts of DNA from 10 homozygous resistant and 10 homozygous susceptible individuals. After the identification of a specific polymorphism between a resistant and susceptible bulk by BSA, cosegregation analysis and mapping in each population was performed to confirm and determine the genetic linkage between the resistance genes and the molecular markers.

Chromosomal Location of Stb2 and Stb3
Genomic DNA from wheat cultivar Chinese Spring (CS) and 42 nullisomic-tetrasomic (NT) lines of CS and the resistant and susceptible parents was amplified by PCR with the specific microsatellite primers Xgwm389, Xgwm533 and Xgwm493 for Stb2, and Xgdm132 and Xgdm141 for Stb3. All PCR conditions were similar to those described above.

Linkage Analysis
Segregation ratios for STB disease reaction and molecular markers from both the Stb2 and Stb3 DH mapping populations were tested for goodness of fit to the 1:1 ratio expected for a single gene. Linkage analyses between the resistance genes and molecular markers were performed with the program MAPMAKER version 2.0 (Lander et al., 1987). The marker order was obtained by a multipoint analysis. Centimorgan (cM) units were calculated using the Kosambi mapping function (Kosambi, 1944). A log of the odds (LOD) score of 3.0 and maximum recombination fraction of 0.4 were used to determine the order of the loci.


   RESULTS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Selection of a Tester Isolate
Initial analyses were done with our standard Indiana tester isolate IN95-Lafayette-1196-WW-1-4 (Adhikari et al., 2003, 2004). However, all three parental lines, including the "susceptible" parent RAC875-2, were resistant to this isolate. Each DH progeny population segregated in an approximately 3:1 ratio of resistant:susceptible individuals when inoculated with our Indiana tester isolate, consistent with a single, independent resistance gene in each parent, including RAC875-2 (data not shown). If this hypothesis is correct, then the resistant progeny lines could have one or two resistance genes. Because our Indiana tester isolate could not differentiate lines containing only Stb2 or Stb3, it was not used for further testing.

To identify a tester isolate that would overcome the resistance gene in the Australian parent RAC875-2 but not Stb2 or Stb3, four isolates from Australia were tested for pathogenicity to Veranopolis (Stb2), Israel 493 (Stb3), Tadinia (Stb4), and Chinese Spring (no known resistance genes) in addition to RAC875-2. The test was conducted during April of 2002 when greenhouse temperatures were higher than desired. Nevertheless, some useful differences were identified (Table 1).


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  		Table 1. Tests of four isolates of M. graminicola from South Australia on five lines of wheat with or without resistance genes.

 
All isolates infected the susceptible cultivars Chinese Spring and RAC875-2, although the levels of disease were low because of the suboptimal testing conditions. The Hardewick Bay isolate gave the best infection on RAC875-2 but also infected Israel 493 (Table 1). Surprisingly, all isolates infected the Stb4-containing cultivar Tadinia to a higher level than the susceptible cultivar Chinese Spring. Veranopolis was the most resistant cultivar, showing little damage from any isolate (Table 1). From these results, the Paskeville isolate was chosen for all further analyses as it infected RAC875-2 but did not cause significant disease on Veranopolis (Stb2) or Israel 493 (Stb3).

Phenotypic Analysis of the Stb2 Population
The distribution of STB severity (Fig. 1A) and pycnidial density (Fig. 1B) in the Stb2 population showed a slightly higher number of progeny in the resistant class. Testing of this population was complicated by segregation of genes for maturity and plant height from the Veranopolis parent, which is a very tall, late cultivar. The DH lines were grouped and tested by maturity so that the plants, as nearly as possible, were at the same developmental stage when inoculated. This required numerous inoculation dates during the testing period. The observed segregation of 57 resistant and 49 susceptible lines in a DH population derived from the cross between Veranopolis and RAC875-2 was consistent with a 1:1 ratio ({chi}2 = 0.60; 0.25 < P < 0.50).



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  		Fig. 1. Segregation of specific resistance to an Australian isolate of M. graminicola in the DH progeny developed from a cross between Veranopolis (containing the Stb2 gene for resistance) and the susceptible parent RAC875-2. Histograms showing frequency distribution of (A) percent leaf area covered with lesions of Septoria tritici blotch and (B) pycnidial density among 106 DH lines. Susceptible classes are indicated with horizontal lines. Phenotypic values of the parents are shown by arrows.

 
Phenotypic Analysis of the Stb3 Population
The Stb3 population was much more uniform for plant height and maturity, which greatly simplified the phenotypic analysis. The distribution of STB severity and pycnidial density among 97 doubled-haploid lines derived from a cross between Israel 493 and RAC875-2 was clearly bimodal with only a few intermediate responses (Fig. 2) . The overall 49:48 segregation of resistant to susceptible plants was in agreement with a 1:1 ratio ({chi}2 = 0.01; 0.90 < P < 0.95), confirming that a single gene is responsible for the resistance to M. graminicola in Israel 493.



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  		Fig. 2. Segregation of specific resistance to an Australian isolate of M. graminicola in the DH progeny developed from a cross between Israel 493 (with the Stb3 gene for resistance) and the susceptible parent RAC875-2. Histograms showing frequency distribution of (A) percent leaf area covered with lesions of Septoria tritici blotch and (B) pycnidial density among 97 DH lines. Susceptible classes are indicated with horizontal lines. Phenotypic values of the parents are shown by arrows.

 
Molecular Mapping of the Stb2 Population
Among the 140 Gatersleben microsatellites and 14 BARC markers assayed, the three SSR loci Xgwm389, Xgwm533.1, and Xgwm493, and four BARC primers Xbarc75, Xbarc77, Xbarc133, and Xbarc147, were polymorphic in the BSA and segregated normally in the Stb2 population. Linkage analysis indicated that the two BARC primers Xbarc77 and Xbarc147 were not linked to the Stb2 gene. The five markers Xbarc75, Xbarc133, Xgwm389, Xgwm533.1, and Xgwm493 segregated in ratios of 56:50 ({chi}2 = 0.34; 0.50 < P < 0.75) or 57:49 ({chi}2 = 0.60; 0.25 < P < 0.50), which did not differ significantly from 1:1. Primer pair Xgwm389 (Fig. 3) amplified a band of 125 base pairs (bp) from the resistant parent Veranopolis and 56 of the 57 resistant progeny. Likewise, primers Xbarc75, Xbarc133, Xgwm533, and Xgwm493 amplified bands of 125, 115, 150, and 125 bp, respectively, from the resistant parent and from 54 to 56 of the resistant progeny. Based on these results, the 125-bp allele amplified by the primer pair Xgwm493 and the 150-bp band produced with the primers for locus Xgwm533.1, when used together, were diagnostic for the presence of Stb2 in the DH lines tested; all lines with both markers also were resistant.



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  		Fig. 3. DNA bands amplified from parents and nine DH progeny derived from a cross between the resistant wheat cultivar Veranopolis (containing the Stb2 gene for resistance to M. graminicola) and the susceptible parent RAC875-2 with microsatellite primer pair Xgwm389 shown in a 3% agarose gel. A 25-bp DNA ladder was used as a standard size marker. Resistant and susceptible progeny are indicated by R and S, respectively. The 125-bp DNA fragment amplified from the resistant parent Veranopolis and resistant lines is indicated by the arrow on the left.

 
Mapmaker analysis placed loci Xgwm533.1 and Xgwm493 in flanking positions relative to Stb2 at distances of 0.9 and 3.7 cM, respectively (Fig. 4) . No recombination was observed between Xgwm533.1 and Xgwm389, so these loci mapped to the same position. Markers Xbarc75 and Xbarc133 also were close (from 2.8–4.7 cM), but distal, to Stb2. Unfortunately, none of the EST and STS markers showed polymorphism in the BSA. Analysis of genomic DNA of nullisomic–tetrasomic lines with the SSR primers for loci Xgwm389, Xgwm533.1, and Xgwm493 confirmed that all of these markers plus the Stb2 gene are located on chromosome 3B (data not shown).



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  		Fig. 4. Molecular map of the short arm of wheat chromosome 3B showing the genetic location of the Septoria tritici blotch resistance gene Stb2. Markers were mapped in the 106 DH lines developed from a cross between the resistant cultivar Veranopolis (which carries the Stb2 gene for resistance to M. graminicola) and the susceptible parent RAC875-2. Approximate distances in centimorgans (cM) are indicated on the left; molecular markers and the resistance locus are on the right. The letter X in front of each SSR locus name indicates the basic symbol for a molecular marker with unknown function in wheat. Map distances are proportional to the distances in cM.

 
Molecular Mapping of the Stb3 Population
The level of polymorphism in the Stb3 population was low for the 140 SSR and 12 BARC primers assayed in the BSA. The SSR marker Xgdm132 amplified a three-banded pattern with amplicons of 150, 168, and 180 bp from the resistant parent Israel 493 and most of the resistant progeny (Fig. 5) . This locus was linked to the Stb3 gene at a genetic distance of approximately 3.0 cM. Among the 12 BARC primers tested from the genomic region near Xgdm132, none showed polymorphism in the BSA. Nullisomic–tetrasomic analysis confirmed that Xgdm132 and, presumably, the closely linked Stb3 gene were located on chromosome 6D (data not shown).



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  		Fig. 5. Segregation of amplification products of microsatellite locus Xgdm132 in a silver-stained polyacrylamide gel (12%) after polymerase chain reaction. DNA of 97 DH lines from a cross between the resistant wheat cultivar Israel 493 (P1) and the susceptible line RAC875-2 (P2) was evaluated; R = resistant, and S = susceptible to an Australian isolate of the Septoria tritici blotch pathogen, M. graminicola. A 25-bp DNA ladder was used as a standard size marker. The 150-bp DNA fragment amplified from the resistant parent Israel 493 and resistant lines is indicated by the arrow on the right. Two larger bands also amplified in each progeny line but gave the same pattern as the 150-bp band, so were considered to represent the same allele.

 

   DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The DH populations analyzed in this study were highly efficient for the characterization of resistance to M. graminicola in wheat and permitted us to identify the map locations of two specific resistance genes. The 1:1 segregation of resistance to susceptibility in both DH populations confirmed that the resistances to our Australian tester isolate in Veranopolis and Israel 493 were each controlled by a single major gene. These results support those of Wilson (1979)(1985) who named the resistance genes in Veranopolis and Israel 493 as Stb2 and Stb3, respectively.

Bulked segregant analysis with the primer pairs for more than 130 SSR loci identified the markers Xgwm533.1 and Xgwm493 that flanked the Stb2 gene at distances of 0.9 and 3.7 cM, respectively. In our analysis, locus Xgwm389 mapped to the same location as Xgwm533.1, even though they were separated by more than 3 cM in two other studies (Buerstmayr et al., 2003; Spielmeyer et al., 2003). All three of these markers have been located previously to the distal region of the short arm of chromosome 3B (Röder et al., 1998), along with Xbarc75 and Xbarc133, which also were polymorphic in the DH population and mapped within 5 cM of Stb2. These SSR markers are not only highly polymorphic, but most of them also are specific and amplify only a single locus from one of the three wheat genomes (Pestsova et al., 2000; Röder et al., 1998). These previously mapped SSR markers are an invaluable resource that permitted rapid localization of the Stb2 gene to the short arm of wheat chromosome 3B.

The genomic region that contains Stb2 is particularly rich in genes for resistance against a wide variety of fungal pathogens. For example, the Sr2 locus for resistance to wheat stem rust (incited by Puccinia graminis Pers.:Pers.) also was mapped recently with the three SSR markers Xgwm493, Xgwm533, and Xgwm389, and RFLP marker glk683, to the short arm of chromosome 3B (Spielmeyer et al., 2003). Marker Xgwm533 was tightly linked (within 1.6 cM) and was considered diagnostic for the Sr2 gene among a worldwide collection of 51 germplasm accessions tested (Spielmeyer et al., 2003). The SSR markers Xgwm389 and Xgwm493 also were linked to Sr2 at distances of 2.7 and 6.6 cM, respectively. However, compared with Xgwm533, the level of polymorphism for Xgwm389 was too low for this locus to be diagnostic for Sr2 in a range of Australian germplasm (Spielmeyer et al., 2003). The location of the SSR markers Xgwm389, Xgwm533, and Xgwm493 in the current study was the same as that reported by Spielmeyer et al. (2003) and those of previously published genetic maps (Buerstmayr et al., 2003; Pestsova et al., 2000; Röder et al., 1998). However, locus Xbarc133 was much more proximal in three other maps that were published recently (Bourdoncle, 2003; Buerstmayr et al., 2003; Liu and Anderson, 2003). The reasons for the discrepancies among these maps are not known, but could be because of one or more undetected errors in the data set, possibly in the phenotypic scoring of resistance responses.

Other resistance genes in the same chromosomal region as Stb2 include a major QTL designated Qfhs.ndsu-3BS that confers resistance to F. graminearum (causal agent of head blight or scab), which was mapped with SSR, EST and STS markers (Bourdoncle, 2003; Buerstmayr et al., 2003; Guo et al., 2003; Liu and Anderson, 2003). In addition, the Lr27 gene that confers seedling resistance to leaf rust, caused by Puccinia triticina Eriks., and a gene coding for phenylalanine ammonia lyase, which is believed to be involved in defense responses (Faris et al., 1999), have been mapped to the same genomic region as Stb2. A major QTL for resistance to Stagonospora nodorum glume blotch [Sng; caused by Stagonospora nodorum (Berk.) Castellani & E.G.], QSng.sfr-3BS, was not mapped precisely but probably is slightly distal to SSR locus Xgwm389 (Schnurbusch et al., 2003). This location would place QSng.sfr-3BS close to Stb2. Genes for resistance to yellow or stripe rust, caused by Puccinia striiformis Westend. (Manilal et al., 2003), also are located on chromosome 3BS and may be part of the same cluster. A QTL for resistance to Karnal bunt, caused by Tilletia indica Mitra, is on chromosome 3BS (Nelson et al., 1998) but is much closer to the centromere than the other genes. The presence of multiple disease resistance genes or QTL and the identification of closely linked markers make this genomic region attractive for future analyses and possible map-based cloning of resistance loci.

Resistance gene clustering has been observed at a larger genomic scale than that of specific gene families (Hulbert et al., 2001). Specifically, some clusters have been viewed as chromosomal regions where numerous resistance genes grouped within a span of a few to 20 cM (Pryor, 1987). Some examples of clusters of genes conferring resistance to single or to taxonomically diverse plant pathogens were reported in soybean [Glycine max (L.) Merr., Ashfield et al., 1998; Bachman et al., 2001; Polzin et al., 1994], maize (Zea mays L., Hulbert et al., 2001), tomato (Lycopersicon esculentum Mill., Dickinson et al., 1993), and rice (Oryza sativa L., Mackill and Bonman, 1992; Yoshimura et al., 1983). Furthermore, related structure and function among resistance genes in these clusters have provided evidence that they occur in multigene families and have a common origin (Ronald, 1998). The region of wheat chromosome 3BS that contains Stb2 appears to represent another example of a superfamily of genes that encode resistance against a wide variety of fungal pathogens. A similar supercluster of resistance genes with specificities to diverse pests and pathogens, including the Stb4 and Stb5 genes for resistance to M. graminicola, was noted previously near the centromere of wheat chromosome 7D (Adhikari et al., 2004).

With genes for resistance against stem rust, leaf rust, Karnal bunt, Fusarium head blight and Stagonospora nodorum glume blotch in addition to STB, this region of chromosome 3BS holds great promise for increasing disease resistance in wheat. Although mapped in different populations, many of the same markers were analyzed in multiple studies so it is possible to deduce a likely arrangement of these genes in relation to the molecular markers in the distal region of chromosome 3BS (Fig. 6) . From this analysis, it appears that a likely order of the resistance genes and representative molecular markers starting from the telomere of chromosome 3BS may be Xbarc75-QSng-Xgwm389-Sr2-Xgwm533.1-Stb2-Lr27-Qfhs-Xgwm493. More work is needed to identify the location of Stb2 within this cluster more precisely. The gene for Karnal bunt resistance is much more proximal compared with the others. These genes do not appear to be allelic so possibly could be combined in cis into a single large linkage block that could be used in breeding programs for simultaneous transfer of multiple resistances into improved cultivars.



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  		Fig. 6. Locations of resistance genes and common microsatellite markers redrawn from different genetic maps of the distal region of wheat chromosome 3BS. The stem rust resistance gene Sr2 (A) was mapped by Spielmeyer et al. (2003). A major QTL for resistance to Fusarium head scab (Qfhs) was mapped by (B) Liu and Anderson (2003) and (C) Buerstmayr et al. (2003). The Septoria tritici blotch resistance gene Stb2 (D) was mapped in the present study. Genetic distances in cM are indicated to the left of the respective maps for those studies in which they were reported. Approximate locations of the Lr27 gene for resistance to leaf rust and a gene for phenylalanine ammonia lyase (PAL) from Faris et al. (1999), plus a major QTL for resistance to Stagonospora nodorum glume blotch (QSng) from Schnurbusch et al. (2003)(personal communication), are indicated on the far right. Common markers in different maps are joined by dashed lines.

 
In contrast to Stb2, only one marker, microsatellite locus Xgdm132, was linked to the Stb3 gene at a distance of approximately 3 cM. This marker, and presumably also the Stb3 gene, is near the telomere of chromosome 6DS (Pestsova et al., 2000). This chromosome has a much lower density of known resistance genes compared to chromosome 3BS. However, a few genes effective against insect pests and pathogens have been mapped previously to wheat chromosome 6D. The Cmc4 gene for resistance to wheat curl mite was mapped to chromosome 6DS (Malik et al., 2003) but, on the basis of published map distances of common markers, it probably is 25 to 30 cM proximal to Stb3 so is not part of the same cluster. The Pm24 gene for resistance to powdery mildew [causal agent: Blumeria graminis (DC.) E.O. Speer] (Huang et al., 1997), and the Yr20 and Yr23 genes for resistance to stripe rust (Chen et al., 1995), also are located on chromosome 6D. However, whether these latter three genes are on the short or the long arm and how they are located relative to mapped molecular markers are not known.

The level of polymorphism for SSR loci in the Stb3 population was much lower, which greatly limited the identification of useful markers compared with Stb2. This occurred even though virtually every marker near locus Xgdm132 was tested. The resistant cultivar Israel 493 (which carries the Stb3 gene) and the susceptible line RAC875-2 were the parents of the Stb3 DH population. Both parents are semidwarf wheats but otherwise appear to be quite unrelated. Although the 150-bp allele at the Xgdm132 locus was tightly linked to the Stb3 gene and could be useful in transferring this resistance into improved wheat cultivars, identification of additional molecular markers with tighter linkages and that flank the Stb3 gene would be desirable to improve selection efficiency, particularly considering the low level of polymorphism.

On the basis of our limited inoculation tests, Stb2 may be more likely to provide effective resistance in the field when deployed singly. None of the four Australian isolates tested infected Veranopolis, but one isolate did infect Israel 493, which suggests that the Stb3 resistance gene may break down rapidly if deployed widely. Veranopolis was one of the most uniformly resistant cultivars in a recent worldwide virulence survey (Kema et al., 1996). However, virulence on Veranopolis occurs in New South Wales, Australia (Peter Martin, personal communication), so testing within each geographical region may be advisable before deployment. Unfortunately, Israel 493 was not tested in the survey by Kema et al. (1996), so nothing is known about worldwide frequencies of virulence to Stb3. All of the Australian isolates tested infected Tadinia better than the susceptible cultivar Chinese Spring, so the Stb4 resistance gene is not likely to be effective at all in Australia. This is consistent with past experience, as Stb4 broke down recently in California (Jackson et al., 2000) after being effective for more than 15 yr (Somasco et al., 1996) following its release in 1984 (Gilchrist et al., 1990). Isolates of M. graminicola from bread wheat generally do not infect durum wheat (T. durum Desf.) and vice versa (Kema et al., 1996). Therefore, it is possible that resistance genes on the A and B genomes may be more effective than those on the D genome against bread-wheat-adapted isolates of M. graminicola. This hypothesis must be tested by additional experimentation.

In addition to Stb2 and Stb3, previously mapped genes for resistance to STB include Stb1 on chromosome 5BL (Yang, 2000), Stb4 and Stb5 near the centromere of chromosome 7DS (Adhikari et al., 2004; Arraiano et al., 2001), Stb6 on chromosome 3AS (Brading et al., 2002), Stb7 on chromosome 4AL (McCartney et al., 2003) and Stb8 on chromosome 7BL (Adhikari et al., 2003). It is interesting that all of these genes except for Stb4 and Stb5 are on different wheat chromosomes, which should make it possible to pyramid them into a single cultivar. These resistance genes also span all three wheat genomes. Therefore, each of the original diploid parents of hexaploid wheat may have contributed independent genes for resistance to STB, although those identified so far came mostly from the B and D genome donors.

One major challenge to wheat breeders and plant pathologists is the selection and development of cultivars with durable resistance to M. graminicola. To achieve this goal, a simple but efficient method to identify STB resistance genes is necessary for breeding programs where many cultivars with different resistance genes are used for crossing. The results of this study are of practical significance to STB resistance breeding. The Stb2 and Stb3 genes together confer resistance to the most virulent strains of M. graminicola in Australia and the USA. The PCR-based SSR markers linked to the Stb2 and Stb3 genes identified in this study not only can assist plant breeders in making parental selection but also will facilitate stacking the resistance genes into advanced breeding lines. The SSR loci Xgwm389, Xgwm533.1 and Xgwm493 for Stb2 and the single locus Xgdm132 for Stb3 can be used to mark these resistance genes. Thus, incorporation of marker-assisted selection into breeding programs will speed pyramiding of the Stb2 and Stb3 genes with the other recently mapped STB resistance genes into a single wheat cultivar to develop broad-spectrum and durable resistance to M. graminicola.


   ACKNOWLEDGMENTS
 
This work was supported by USDA-ARS CRIS project 3602-22000-013-00D. We thank Jill Breeden, Jessica Cavaletto and Kristi Brikmanis for their excellent technical assistance, Drs. P. Cregan and Q. J. Song for providing the sequences of the BARC microsatellites, Dr. J. A. Anderson for sequences of the EST primers, and Dr. Herb Ohm for enlightening discussions about the 3BS resistance cluster. Dr. Jorge Dubcovsky kindly provided helpful comments on the mapping analysis of the Stb2 gene. Published as paper 17249, Purdue University Agricultural Experiment Station.


   NOTES
TOP
 NOTES
ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Names are necessary to report factually on available data. However, the USDA neither guarantees nor warrants the standard of the product, and the use of the name implies no approval of the product to the exclusion of others that also may be suitable.

Received for publication July 11, 2003.


   REFERENCES
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 


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