Source: WASHINGTON STATE UNIVERSITY submitted to
MAPPING AND CLONING HIGH TEMPERATURE ADULT PLANT STRIPE RUST RESISTANT GENES IN WHEAT
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0203894
Grant No.
2005-35301-15906
Project No.
WNP07333
Proposal No.
2005-00975
Multistate No.
(N/A)
Program Code
52.2
Project Start Date
Sep 1, 2005
Project End Date
Aug 31, 2008
Grant Year
2005
Project Director
Kidwell, K. K.
Recipient Organization
WASHINGTON STATE UNIVERSITY
(N/A)
PULLMAN,WA 99164
Performing Department
CROP & SOIL SCIENCES
Non Technical Summary
The foliar fungal disease stripe rust (also known as yellow rust) is one of the most destructive diseases of wheat throughout the U.S. and the world. Two major types of genetic resistance have been identified; seedling resistance that is race-specific and is not durable and high temperature adult plant (HTAP) resistance that is race non-specific and has provided a significant level of resistance to stripe rust for the last 27 years. HTAP resistance genes have been identified in Triticum dicoccoides and in several wheat cultivars, including `Stephens' and `Druchamp'. Full utilization of these resistance sources depends on knowledge of the location and mechanism of HTAP resistance; however, no HTAP resistance gene has been cloned to date. Our long-term goal is to understand the mechanism of HTAP resistance to stripe rust in wheat. DNA marker technology, genome mapping and positional cloning will be used to achieve this goal. Outcomes of the proposed research will include the cloning of the first HTAP resistance gene in wheat and the identification and fine mapping of major QTL for HTAP resistance. This will lead to the development of breeder friendly molecular markers to use in marker-assisted selection strategies for HTAP resistance gene integration, and the development of germplasm with durable stripe rust resistance. All of these tools eventually will assist breeders with the rapid, efficient development of wheat cultivar with durable stripe rust resistance.
Animal Health Component
20%
Research Effort Categories
Basic
80%
Applied
20%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20115491080100%
Goals / Objectives
The specific objectives of this research are: Objective-1 Clone the HTAP stripe rust resistance gene from T. dicoccoides on chromosome 6BS using a map-based cloning approach and determine if it is allelic to the QTL on chromosome 6BS in Stephens. Objective-2 Identify additional QTL with significant effects in Stephens (other than those on 6BS) and major QTL Druchamp, through genetic linkage analysis.
Project Methods
Objective-1: (a) High-density map of the HTAP stripe rust resistance gene from T. dicoccoides on chromosome 6BS (Yr36) will be developed to identify new markers and recombinants until markers flanking the gene at less that 0.2cM are found. (b) Physical map: A BAC contig will be developed by screening the RSL#65 (resistant parent of the mapping population) BAC library using markers completely linked to Yr36, as well as the closest flanking markers. Once the contig is constructed, a few selected BACs will be sequenced, annotated and candidate genes will be identified. (c) Genetic linkage map locations of Yr36 specific markers will be determined in the Stephens/Michigan Amber mapping population, and results will be compared to determine whether the major HTAP QTL in Stephens is located in the same genomic region as Yr36 based on flanking markers. In order to determine if the major QTL in Stephens is allelic to Yr36, allelism test will be conducted in an F2 population from a cross between Stephens and Yecora Rojo Yr36 (PI638740), a hexaploid wheat genotype carrying Y36 from T. dicoccoides. Objective-2: (a) In addition to the major QTL associated with HTAP on 6BS in Stephens, QTL with lesser effects will be identified using a composite interval mapping approach. (b) Major HTAP QTL in Druchamp will be identified through genetic linkage and QTL mapping using a RIL population obtained from Druchamp/Michigan Amber cross.

Progress 09/01/05 to 08/31/08

Outputs
OUTPUTS: In the previous report, the quantitative gene for HTAP resistance was Mendelized into a single locus, Yr36, and was precisely mapped within a 0.14-cM genetic interval flanked by markers Xucw113 and Xucw111. In order to clone this gene, we developed a physical map and TLLING population of Yr36. The DNA pools from the LDN BAC library were screened with B-genome specific primers for Xucw111 and Xucw113. Five BAC clones were obtained using the Xucw113 primers and six with the Xucw111 primers. Since Yr36 is closer to Xucw113 than to Xucw111 we initiated the chromosome walk from Xucw113. The presence of markers Xucw70 and Xucw112 in some of the BACs from the distal contig facilitated its orientation relative to the genetic map. We sequenced the BAC ends and developed marker Xucw125 from BAC 4. This marker was completely linked to Yr36 and 0.2 cM proximal to Xucw113, suggesting that one additional BAC might be sufficient to find a recombination event on the proximal side of Yr36. We used Xucw125 to re-screen the BAC library and selected six new BACs. We are currently sequencing these BAC ends to take the next step in the chromosome walk. A total of 3000 seeds of the isogenic line UC1041-Yr36 were mutagenized with 1% EMS. We recovered M1 seeds from 1,200 plants. One M2 seed from each M1 plant is currently being grown in the greenhouse to produce DNA pools. Previously we reported two putative QTLs for HTAP resistance in Stephens through genetic linkage analysis of 496 DNA markers and stripe rust disease data of 101 recombinant inbred lines (RILs) obtained from Stephens (resistant) x Michigan Amber (susceptible). Here we reported precise analysis of the QTLs, their physical location on the wheat genome and potential of the DNA markers flanking the QTLs in marker-assisted selection. Two QTLs (QYrst.wgp-6BS.1and QYrst.wgp-6BS.2) explained 48 to 61% of the total phenotypic variation of the HTAP resistance in Stephens. QYrst.wgp-6BS.1 was within a 3.9 cM region flanked by Xbarc101 and Xbarc136. QYrst.wgp-6BS.2 was mapped in a 17.5 cM region flanked by Xgwm132 and Xgdm113. Both QTLs were physically mapped to the short arm of chromosome 6B, but in different bins. Validation and polymorphism tests of the flanking markers in 43 wheat genotypes indicated that the molecular markers associated with these QTL should be useful in marker-assisted breeding programs to efficiently incorporate HTAP resistance into new wheat cultivars. One hundred RILs developed from Druchamp (resistant) x Michigan Amber (susceptible) were used to identify QTLs for HTAP resistance in Drucham. Stripe rust data from the field trials, seedling resistance against Pst43 and Pst45 conducted in green house and 216 DNA markers were used in the analysis. Two QTLs (QYrst.wgp-5BS and QYrst.wgp-7BS), which explained 15 to 25% of the total phenotypic variation of the HTAP resistance in Druchamp, were identified. QYrst.wgp-5BS was mapped to chromosome 5B within 14.3 cM region flanked by Xgwm335 and Xbarc004 and QYrst.wgp-7B was mapped to chromosome 7B within a 15.1 cM region flanked by Xwmc396 and Xbarc072. Resistance genes for stripe rust pathogen races Pst43 and Pst45 were mapped to chromosome 5B. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Primary audience are wheat breeders and other researchers who will utilize the outcome of this project for wheat cultivars with durable resistanc eto stripe rust. Other audience are growers, county based extension faculty for understanding how modern genomic tools have potential to improve breeding efficiency. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Cloning of Yr36 will not only generate new tools to fight stripe rust disease but might also provide valuable insights on the relationship between temperature and rust resistance. The soft white winter wheat cultivar Stephens was released in 1978 for growing in the Pacific Northwest (PNW) region of the USA. It has remained resistant to stripe rust and is still grown in major acreage of this region even though stripe rust pathogen races, which are virulent on Stephens in the seedling stage, were known for the last 30 years. The susceptibility in seedling stage suggests that over the last 30 years Stephens was protected from stripe rust damage mainly by its non-race specific HTAP resistance. Using the QTL mapping approach, we identified two QTL with major effect on stripe rust resistance. One of these two QTL was consistently identified precisely at the same chromosome region in all years and locations. However, the second QTL was not detected in one year, and when detected during other three years, it could not be mapped at the precise location of the chromosome. These two major QTL together explained 48 to 61% of the total variation of mean rAUDPC. It is easier to introgress resistance into cultivars through breeding programs when it is controlled by relatively few genes. The major effect of both of these QTL for HTAP resistance makes introgression of these QTL relatively easy in breeding programs. The level of HTAP resistance in Stephens is higher than that in many spring wheat genotypes, which have HTAP resistance. It is imperative to pyramid different HTAP QTL and effective seedling resistance genes simultaneously into future wheat cultivars in order to provide long-term genetic insurance against this devastating disease. The seedling resistance genes provide complete resistance against the avirulent races that have been periodically predominant in most wheat production regions of the PNW. When effective seedling and HTAP resistances are present together in a single genotype, seedling resistance genes mask our ability to detect characteristic phenotypes of HTAP resistance. It is impossible to differentiate the HTAP resistance phenotype from seedling resistance or HTAP resistance conferred by different QTL in a single genotype. Therefore, it is imperative to use diagnostic molecular markers for HTAP QTL of Stephens and Druchamp to combine the QTLs with effective seedling resistance genes. The molecular markers associated with the QTL for HTAP resistance to stripe rust in Stephens and Druchamp will be used in marker-assisted breeding programs to efficiently incorporate HTAP resistance into regionally adapted cultivars.

Publications

  • Santra D. K., M. Santra, X. Chen, K.G. Campbell and K.K. Kidwell. 2008. Mapping QTL for high temperature, adult plant resistance to stripe rusts in wheat (Triticum aestivum L.). Theoretical and Applied Genetics 117:793-802.


Progress 09/01/06 to 08/31/07

Outputs
Among the few known "slow rusting" genes Yr36 confers partial resistance to a broad range of stripe rust races. This gene from Triticum turgidum ssp. dicoccoides (DIC) was originally classified as a high-temperature adult-plant (HTAP) resistance gene. We show here that the resistance is expressed both in adult and young plants (fifth leaf stage). The resistance is expressed only under high temperatures (e.g. 25 C), and is not evident at low temperatures (e.g. 15 C). We previously mapped Yr36 within a 2-cM interval in the short arm of chromosome 6B. In this study, we report a high-density map of the Yr36 region based on 9,000 gametes from the cross between durum wheat cv. Langdon (LDN) and a near isogenic line which carries a 30-cM segment of chromosome arm DIC-6BS. Yr36 was precisely mapped within a 0.14-cM genetic interval flanked by markers Xucw113 and Xucw111.These flanking markers were used to initiate a chromosome walk towards Yr36. Based on the results of allelism test using 10,000 F2 plants from Stephens x Yecora Rojo Yr36, we determined that the major QTL on 6BS in Stephens is different from Yr36. One hundred and one recombinant inbred lines (RILs) developed from Stephens (resistant) x Michigan Amber (susceptible), named SMRILs and 100 RILs developed from Druchamp (resistant) x Michigan Amber (susceptible), named DMRILs were used as mapping population. F5, F6, and F7 RIL derivatives were evaluated for three years at one location, whereas F8 and F9 derivatives were evaluated at four locations in 2006 and 2007, respectively. Mean values of the infection type (IT) as well as area under disease progression curve (AUDPC) values were calculated for each RIL using disease infection type and percentage of infected leaf area recorded on three different dates for each site year. The 101 RILs were evaluated with 250 resistance gene analog polymorphism (RGAP), 245 simple sequence repeat (SSR) and 1 sequence tagged site (STS) markers for genetic linkage map construction. Two QTL were associated with HTAP resistance in Stephens; however, variation in the level of significance of associations was detected among locations and across years. QYrst.wgp-6BS (LOD = 3.89-6.83, Rsq = 0.32-0.45, P < 0.0001) and QYrst.wgp-6BL (LOD = 1.4-8.31, Rsq = 0.25-0.43, P < 0.0001) were detected on chromosome 6B within a 3.9 cM region flanked by Xbarc101 and Xbarc136 and a 17.5 cM region flanked by Xgwm132 and Xgdm113, respectively. Two hundred sixty four SSR markers out of the 1,032 tested were polymorphic between Druchamp and Michigan Amber. The RILs were genotyped with 212 SSR markers and 40 linkage groups were established. We identified two QTL associated with HTAP resistance in Druchamp; however, variation in the level of significance of associations was detected among locations and across years. Q.Dru.htap-1 (LOD=2.2-3.6, Rsq=0.10-0.15, P<0.001) was identified on chromosome 5B within a 14.3 cM distance flanked by molecular markers Xgwm335 and Xbarc004. The second QTL, Q.Dru.htap-2 (LOD=2.2-2.7, Rsq=0.10-0.12, P<0.001) was detected on chromosome 7B within a 15.1 cM region, which was flanked by Xwmc396 and Xbarc072.

Impacts
The cloning of Yr36 will not only generate new tools to fight stripe rust disease but might also provide valuable insights on the relationship between temperature and rust resistance. The soft white winter wheat cultivar Stephens was released in 1978 for growing in the Pacific Northwest (PNW) region of the USA. It has remained resistant to stripe rust and is still grown in major acreage of this region even though stripe rust pathogen races, which are virulent on Stephens in the seedling stage, were known for the last 30 years. The susceptibility in seedling stage suggests that over the last 30 years Stephens was protected from stripe rust damage mainly by its non-race specific HTAP resistance. Using the QTL mapping approach, we identified two QTL with major effect on stripe rust resistance. One of these two QTL was consistently identified precisely at the same chromosome region in all years and locations. However, the second QTL was not detected in one year and when detected during other three years, it could not be mapped at precise location of the chromosome. These two major QTL together explained 48 to 61% of the total variation of mean rAUDPC. It is easier to introgress resistance into cultivars through breeding programs when it is controlled by relatively few genes. The major effect of both these QTL for HTAP resistance makes introgression of these QTL relatively easy in breeding programs. The level of HTAP resistance in Stephens is higher than that in many spring wheat genotypes, which have HTAP resistance. It is imperative to pyramid different HTAP QTL and effective seedling resistance genes simultaneously into future wheat cultivars in order to provide long-term genetic insurance against this devastating disease. The seedling resistance genes provide complete resistance against the avirulent races that have been periodically predominant in the most wheat production region of the PNW. When effective seedling and HTAP resistances are present together in a single genotype, seedling resistance genes mask our ability to detect characteristic phenotype of HTAP resistance. It is impossible to differentiate HTAP resistance phenotype from seedling resistance or HTAP resistance conferred different QTL in a single genotype. Therefore, it is imperative to use diagnostic molecular markers for HTAP QTL of Stephens to combine it with effective seedling resistance genes or with different HTAP resistance. The molecular markers associated with the QTL for HTAP resistance to stripe rust in Stephens will be used in marker-assisted breeding programs to efficiently incorporate HTAP resistance into regionally adapted cultivars.

Publications

  • Uauy, C., J.C. Brevis, X. Chen, I.A. Khan, L. Jackson, O. Chicaiza, A. Distelfeld, T. Fahima, and J. Dubcovsky . 2005. High-temperature adult plant (HTAP) stripe rust resistance gene Yr36 from Triticum turgidum ssp. dicoccoides is closely linked to the grain protein content locus Gpc-B1. Theoretical & Applied Genetics. 112: 97-105.


Progress 09/01/05 to 08/31/06

Outputs
The objectives of this research are to: 1) Clone the HTAP stripe rust resistance gene from T. dicoccoides on chromosome 6BS using a map-based cloning approach and determine if it is allelic to the QTL on chromosome 6BS in Stephens (J. Dubcovsky et al., UC-Davis); and 2) Identify additional QTL with significant effects in Stephens (other than those on 6BS) and major QTL Druchamp, through genetic linkage analysis (K. Kidwell et al., WSU). F5, F6, and F7 RIL derivatives were evaluated for three years at one location, whereas F8 derivatives were evaluated at four locations in one year. Area under disease progression curve (AUDPC) values were calculated for each RIL using disease infection type and percentage of infected leaf area recorded on three different dates for each site year. AUDPC data from a subset of 114 RILs that were evaluated with 375 polymorphic DNA markers were used for genetic linkage map construction and QTL mapping. Three major QTL associated with HTAP resistance were identified; however, variation in the level of significance of associations was detected among locations and across years. Q.htap-1 (LOD=6.45-14.58, R2=0.23-0.44, P<0.0001) and Q.htap-2 (LOD=5.54-7.74, R2=0.20-0.26, P<0.0001) were detected on chromosome 6BS within a 23.1 cM region flanked by Xgwm88.2 and Xucw71 and a 14.9 cM region flanked by Xgwm508.2 and Xgwm132, respectively, whereas, Q.htap-3 (LOD 2.4-5.4, R2=0.09-0.20, P<0.0001) mapped within a 45.2cM region flanked by XwlSAS5 and Xbarc243 on 5B. Our ability to detect QTL associated with HTAP resistance was greatly influenced by changes in pathogen race structure over time.

Impacts
We identified a major quantitative trait locus on chromosome 6BS that is associated with high-temperature adult plant resistance to stripe rust, a major disease of wheat worldwide. Tightly linked DNA marker associated with this region on 6BS can be used to introgress the HTAP resistance from Stephens into other cultivars, and in gene pyramiding strategies to combine HTAP with effective seedling resistance genes to develop cultivars with durable resistance to stripe rust, thereby reducing or eliminating the need to control this disease with fungicides. The Dubcovsky lab has made significant progress towards cloning HTAP resistance gene Yr36, which we hypothesize as being allelic to the HTAP resistance in Stephens that we are mapping.

Publications

  • No publications reported this period