Published online 7 August 2009
Published in Crop Sci 49:1786-1790 (2009)
© 2009 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
Open Access Article
Linkage Maps of Wheat Stripe Rust Resistance Genes Yr5 and Yr15 for Use in Marker-Assisted Selection
Lesley R. Murphya,
Dipak Santrab,
Kimberlee Kidwellb,
Guiping Yanc,
Xianming Chena and
Kimberly Garland Campbella,*
a U.S. Department of Agriculture, Agricultural Research Service, Wheat Genetics, Quality, Physiology, and Disease Research Unit, Pullman, WA, 99164-6430
b Dep. of Crop and Soil Sciences, Washington State Univ., Pullman, WA 99164-6430
c Dep. of Plant Pathology, Washington State Univ., Pullman, WA 99164-6430, Current address: Oregon State Univ., Columbia Basin Agricultural Research Center, Pendleton, OR 97801
* Corresponding author (kgcamp{at}wsu.edu).
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ABSTRACT
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Stripe rust (caused by Puccinia striiformis Westend. f. sp. tritici Eriks.) is a serious disease of wheat (Triticum aestivum L.). Resistance genes Yr5 and Yr15 are the only known all-stage resistance genes that defeat all stripe rust races currently found in the United States. Previously mapped markers for these genes, however, show limited polymorphism across diverse genotypes and/or map at a distance from the genes, reducing the effectiveness of marker-assisted selection. Our objective was to create new linkage maps for both genes using sequence tagged site (STS) and simple sequence repeat (SSR) loci and to evaluate closely linked markers across a diverse panel of wheat genotypes. Two recombinant inbred populations created using Avocet-Susceptible as a susceptible parent and Triticum aestivum L. ssp. spelta (L.) Thell. ‘Album’ and Triticum dicoccoides Koern. as the Yr5 and Yr15 donors, respectively, were evaluated for resistance to multiple races of stripe rust. Molecular markers that had been previously mapped to wheat chromosome 1B (Yr5) or 2B (Yr15) were mapped on the appropriate population. Markers most closely linked to each gene were evaluated against a panel of genotypes collected from active introgression programs in the United States. The Yr5 gene is flanked on the distal side by STS7/8 marker and by Xbarc349 and Xbarc167 on the proximal side, although none of these markers were diagnostic in all backgrounds. For Yr15, Xbarc8 and Xgwm413 appear to be completely linked with the gene in our population, along with resistance gene analog polymorphism marker Xwgp34. These two SSR markers also appear to be diagnostic in all backgrounds tested with one exception (Zak). We have developed linkage maps for both genes and identified several useful SSR and STS markers for introgression of Yr5 and Yr15.
Abbreviations: AVS, Avocet-SusceptibleM • HTAP, high-temperature adult-plant • MAS, marker-assisted selection • NIL, near-isogenic line • PCR, polymerase chain reaction • RAPD, random amplified polymorphic DNA • RFLP, restriction fragment length polymorphism • RGAP, resistance gene analog polymorphism • SSR, simple sequence repeat • STS, sequence tagged site
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INTRODUCTION
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THE FUNGUS Puccinia striiformis Westend. f. sp. tritici Eriks. causes the wheat disease stripe (also known as yellow) rust. This disease is highly destructive and can cause up to 100% crop loss, though commonly in the range of 10 to 70% (Chen, 2005). In the United States, this disease is especially problematic in the West; however, prevalence of the disease in the eastern and midwestern United States has increased since 2000 (Chen et al., 2002; Chen, 2007). Many cultivars of wheat have been developed to reduce crop loss by breeding in race-specific all-stage (also referred to as seedling) resistance and non–race-specific high-temperature adult-plant (HTAP) resistance genes (Chen, 2007). In the 1990s, in Washington and the rest of the Pacific Northwest, losses were reduced primarily through incorporation of HTAP resistance genes (Chen, 2007) but these genes are not always effective when used alone, especially when infection occurs early in plant development and inoculum levels are high. Fungicide usage has also dramatically reduced crop losses, but the cost, potential environmental problems, and increased fungicide tolerance through selective pressure on P. striiformis make breeding for resistance a better solution (Chen, 2007).
Currently, the all-stage resistance genes Yr5 and Yr15 provide resistance to all known races of stripe rust (Chen et al., 2003; http://maswheat.ucdavis.edu/protocols/Yr15/index.htm). It has been hypothesized that pyramiding various stripe rust genes into barley (Hordeum vulgare L.), together and in combination with HTAP resistance, would provide a more durable and effective means of controlling stripe rust than using either gene alone or using HTAP resistance alone, and it would have the potential to eliminate the need for fungicide application (Castro et al., 2003). It is reasonable to believe this would also apply to wheat. Methods for evaluating stripe rust resistance during the breeding process include artificially inoculated greenhouse experiments and both artificially and naturally infected field experiments. These methods can be time consuming, are limited to the growing season, and require maintenance of the various stripe rust races to detect specific genes for resistance (Chen et al., 2003; Singh et al., 2000). In addition, races of P. striiformis that differentiate Yr5 and Yr15 have not been identified so it is currently not possible to use greenhouse or field resistance phenotyping to identify genotypes possessing both resistance genes, unless they are identified using progeny testing and test crosses. Alternatively, as for many traits, marker-assisted selection (MAS) is an efficient tool to select for and predict phenotype. The challenge in the use of MAS in large breeding programs is the need for tightly linked markers to the gene of interest that are polymorphic across a range of genetic backgrounds and, preferably, easy to use (reliable, repeatable, with fragments that are easily detected).
The chromosomal locations of these genes are already known. Yr5 was mapped cytogenetically by Macer (1966) and further by Law (1976); while Yr15 was initially mapped cytogenetically and then with restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) markers (McIntosh et al., 1996, Sun et al., 1997). Chen et al. (2003) and Yan et al. (2003) subsequently used a map of resistance gene analog polymorphism (RGAP) markers to identify the location of the Yr5 gene and develop the Yr5STS-7/8 marker, but this map was not integrated with other wheat cytogenetic or molecular linkage maps. The Yr5STS-7/8 marker shows limited polymorphism across several wheat genotypes (Chen et al., 2003; unpublished data). Therefore, Yr5STS-7/8 was converted to a cleaved amplified polymorphic marker to improve polymorphism frequency (Chen et al., 2003), but its use is still limited by low rates of polymorphism in North American germplasm. For Yr15, several molecular markers have been identified and used for MAS, both RFLPs and microsatellites, but again, many are not polymorphic across genotypes and populations and those that are have been mapped at some distance from the putative gene location. Peng et al. (2000) created a map of the Yr15 region but the SSR marker distances from the gene, both proximal and distal, were found to be greater than 4 cM (Xgwm413 at 4.3 cM and Xgwm911 at 19.9 cM, respectively). Chagué et al. (1999) found Xgwm33 to be 5.7 cM from the Yr15 gene and the BARC map places Xbarc8 at 9 cM distal from the Yr15 gene (http://maswheat.ucdavis.edu/protocols/Yr15/index.htm). It is the goal of this study, in regards to Yr15, to saturate the area around the putative gene location with SSR markers to determine whether these previously published markers are the closest.
In this paper, our objectives were to develop linkage maps of the chromosome regions containing Yr5 and Yr15 using both sequence tagged site (STS) and SSR markers and to identify easy-to-use SSR markers that are tightly linked to these two important stripe rust genes with a high degree of polymorphism among genotypes. The SSR markers tested were chosen from maps of the chromosomes in question (2B for Yr5 and 1B for Yr15) (Röder et al., 1998, Somers et al., 2004).
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MATERIALS AND METHODS
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Plant Materials and Evaluation for Stripe Rust Resistance
Two mapping populations were created by backcrossing Avocet-Susceptible (AVS) to AVS near-isogenic lines (NILs) possessing either Yr5 (Yr5/6*AVS), from Triticum aestivum L. ssp. spelta (L.) Thell. ‘Album’, or Yr15 (Yr15/6*AVS), from T. dicoccoides Koern. derivative V763-251-wb (Wellings and McIntosh, 1998) as described in more detail in Yan et al. (2003). After the seventh backcross, each population was advanced by single seed descent. A BC7F3 population of 93 individuals was created for Yr5 and a BC7F4 population of 136 individuals was created for Yr15. Thus each population is expected to segregate mainly in the region of the rust resistance gene.
Fifteen to 20 plants of each line were evaluated in the seedling stage with multiple races of stripe rust. The Avocet-Yr5 F3 population and its evaluation are described in detail in Yan et al. (2003). Evaluation of rust resistance was also performed as in Yan et al. (2003) for the Avocet-Yr15 F4 population except that the population was only screened with P. striiformis races PST-29, PST-37, and PST-43.
DNA Extraction, PCR Amplification, Electrophoresis, and Data Analysis
Extraction of genomic DNA of parents and progeny of the Avocet-Yr5 F3 lines is described in Yan et al. (2003). DNA of the Avocet-Yr15 F4 lines was extracted in the same manner. Polymerase chain reaction (PCR) amplifications were run on several platforms with different conditions for each. For fragment separation in the ABI 3130xL (Applied Biosystems, Foster City, CA), PCR amplifications were performed with final concentrations of 1x PCR buffer and 2.5 mM MgCl2, 0.2 mM dNTPs, 0.5 pmol of M-13–tagged forward primer, 3.75 pmol of reverse primer, and 2.0 pmol of M-13–tagged dye (6'-FAM [MWG], VIC, NED, or PET) (ABI), 0.75 U of New England Biolabs (Ipswich, MA) Taq DNA polymerase, and 80 ng of genomic DNA in a 15-µL reaction. For fragment detection using the Li-Cor IR2 DNA Analyzer (Li-Cor Biosciences, Lincoln, NE), PCR conditions were the same as above with the exception of a final concentration of 1.2x PCR buffer, 0.25 mM dNTPs, 0.1 pmol M-13–tagged forward primer, 0.125 pmol reverse primer, and 0.05 pmol M-13–labeled Li-Cor fluorophore (IR700). Polymerase chain reaction products that were detected on 6.6% non-denaturing polyacrylamide gels with ethidium bromide staining were done using final concentrations of 1x PCR buffer and 1.5 mM MgCl2, 0.2 mM dNTPs, 3.75 pmol of forward and reverse primer, 0.75 U of New England Biolabs Taq DNA polymerase, and 50 ng genomic DNA in a 15-µL reaction. Eppendorf Mastercycler (Eppendorf-Netheler-Hinz GmbH, Hamburg, Germany), Applied Biosystem GeneAmp 9700, and PerkinElmer GeneAmp 9600 (PerkinElmer, Boston, MA) thermocyclers were used. The conditions used were 3 min of initial denaturation at 94°C, 1 min at 94°C, 1 min at annealing temperature, and 1 min at 72°C for 42 cycles, then a final extension step of 10 min at 72°C for all primer combinations except Xgwm934, which had 45 s, 30 s, and 45 s for the 42 repeated cycles; Xbarc8 and Xgwm582 had 2-min extensions; and for Xgwm273 a touchdown protocol was used with decreasing temperatures from 62 to 57°C, then 45 cycles at 56°C with annealing time reduced to 30 s. Annealing temperatures used were those published on GrainGenes (http://wheat.pw.usda.gov/GG2/index.shtml) for each primer combination. Polymerase chain reaction products separated using the ABI 3130xL were scored using Gene Marker V.1.5 (Softgenetics, College Park, PA), on the 6.25% polyacrylamide gels on a Global IR2 Analysis System, scoring was done with GeneImagIR software (Li-Cor Biosciences). Polymerase chain reaction products run on 6.6% polyacrylamide (non-denaturing) gels were stained with ethidium bromide and scored visually. STS markers were run as described at http://maswheat.ucdavis.edu/protocols/Yr5/index.htm except that 100 ng of genomic DNA was used; ethidium bromide, not silver, was used to stain; and PCR products were separated in 2.8% agarose gels at 80 V for 2 h. Yan and Chen (personal communication, 2003) developed an STS marker for Yr15, derived from RGAP markers. The marker, Xwgp34 was amplified with RGA primers Xa1LRF (5'-CTCACTCTCCTGAGAAAATTAC-3') and PtoFen-S (5'- ATGGGAAGCAAGTATTCAAGGC-3'). The RGAP markers were screened as described in Yan et al. (2003).
Linkage mapping was performed with JoinMap 3.0 (Van Ooijen, 2006) utilizing Haldane's mapping function. The phenotype data from stripe rust resistance screening of each population was included as single locus as described for Yr5 from Yan et al. (2003).
Based on the results of the linkage mapping, we then tested the primers most closely flanking either Yr5 or Yr15 on an array of wheat breeding lines from several geographically distinct breeding programs with active Yr5 and Yr15 introgression efforts (genotypes were generously donated by J. Dubcovsky, K. Gill, J. Johnson) to determine whether the primers would be polymorphic and diagnostic. DNA was extracted from the donated samples using the protocol of Dellaporta et al. (1983).
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RESULTS AND DISCUSSION
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Resistant NILs in both populations had infection type 1 (tiny necrotic flecks without sporulation) to races PST-17, PST-37, PST-43, and PST-45 in the Yr5 population and to races PST-29, PST-37, and PST-43 for the Yr15 population in the seedling stage under controlled greenhouse conditions and infection type 0 in fields under natural infection. In the Yr15 population, of the 136 individuals used for mapping, 73 were highly resistant and the remaining 63, highly susceptible.
Based on the maps created with the population data (Fig. 1
), the previously developed Yr5STS-7/8 marker was closest proximally to the Yr5 gene, located only 0.3 cM away, with SSR marker Xwmc175 located 1.4 cM away from the gene. Dominant SSR marker Xbarc349 is located 0.4 cM distally, and codominant Xbarc167 is 2.6 cM distal from the gene. For Yr15, Xbarc8 and Xgwm413 appear to be completely linked with the gene in our population, along with RGAP marker Xwgp34. This complete linkage may be due to the fact that Yr15 is an introgression from a wild wheat species, T. dicoccoides, and recombination can be expected to be reduced in the region surrounding Yr15 (Riley and Chapman, 1958). In comparing the performance of these markers in diverse backgrounds, it was determined that Xbarc8 and Xgwm413 are diagnostic of the Yr15 gene across all backgrounds tested with the exception of the soft white spring wheat cultivar Zak (Table 1
). This may indicate a recombination in the T. dicoccoides introgression in this line. Conversely, the SSR primers that mapped the closest to the Yr5 gene in the AVS mapping population were not diagnostic across the backgrounds tested (Table 2
). Xbarc349, which is a dominant marker for the presence of the Yr5 gene in the AVS background, was present in all backgrounds, thus rendering it useless in the backgrounds tested here. Two other SSR markers, Xwmc175 and Xbarc167, had mixed results. To further examine this problem, the published markers (http://maswheat.ucdavis.edu/protocols/Yr5/index.htm) for Yr5, which had proven to be nonpolymorphic or nondiagnostic in other germplasm that we evaluated previously, were then tested on the germplasm included in this paper using all possible combinations (STS-7/8, STS-9/10, alone and as a cleaved amplified polymorphic sequence marker, digested with DpnII, as well as STS-7/10). The STS-7/8 primer combination proved to be the most reliable, and with DpnII digestion, the most polymorphic of the STS markers. However, it was not diagnostic in two of the backgrounds tested, Alpowa and Zak. We are currently conducting test crosses of the Zak Yr5 and Yr15 lines to the Avocet-Yr5 and Avocet-Yr15 donors because these genotypes were developed through phenotypic selection of rust resistant progeny, without the use of any markers.
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Figure 1. Simple sequence repeat (SSR) maps developed from (A) BC7F3 populations of Yr5/6*AVS and (B) BC7F4 populations of Yr15/6*AVS. AVS, Avocet-Susceptible.
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Table 1. Results of marker screening over multiple wheat backgrounds with simple sequence repeat (SSR) markers for Yr15. Yes denotes that the marker is diagnostic, while No indicates that it is not.
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Table 2. Results of marker screening over multiple wheat backgrounds with published and proposed STS and simple sequence repeat (SSR) markers for Yr5. Yes denotes that the marker is diagnostic, while No indicates that it is not.
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Previously, Peng et al. (2000) located Xgwm413 4.3 cM proximal to Yr15. However, the map generated in this study shows Xgwm413 to be more tightly linked to the Yr15 gene. The mapping population used by Peng et al. (2000) was an F2:3 population consisting of 121 lines of a T. dicoccoides x T. durum Desf. cross. The population used in this study was 136 lines of a BC7F4 T. aestivum recombinant inbred line. These differences and possible suppression of recombination in the hexaploid derivative, may have contributed to the linkage map differences. Additionally, for Yr15, two SSR markers are located within 2 cM of the gene, although only the Yr15 marker Xgwm413 produced a positive result in all of the lines tested. The other published markers mentioned previously, Xgwm33 and Xgwm911, were not polymorphic in the mapping population used in this study.
We have identified microsatellite markers that can be used in MAS for breeding of stripe rust resistance in wheat that are both easy to use and closely associated to these two important stripe rust resistance genes across a variety of diverse backgrounds. This result will give breeders alternative markers to evaluate if the previously published markers are not polymorphic in certain genotypes. This result also locates both Yr5 and Yr15 in linkage maps comprised of RGAP, STS, and SSR markers, facilitating additional genetic research and comparison to previously published linkage maps.
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ACKNOWLEDGMENTS
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Our thanks to J. Dubcovsky, K. Gill, and J. Johnson for generously donating their seeds for us to test. This research was funded by USDA-ARS in-house projects: 5348-21000-023-00D (KGC) and 5348-22000-014-00D (XMC) and by the USDA-CSREES-NRI-Wheat Coordinated Agriculture Project Grant: CA-D*-PLS-7489-CG.
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NOTES
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All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permission for printing and for reprinting the material contained herein has been obtained by the publisher.
Received for publication October 24, 2008.
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ABSTRACT
INTRODUCTION
