Genome-Wide Association Study and Gene Specific Markers Identified 51 Genes or QTL for Resistance to Stripe Rust in U.S. Winter Wheat Cultivars and Breeding Lines.

Stripe (yellow) rust, caused by fungal pathogen Puccinia striiformis f. sp. tritici (Pst), is a serious disease of wheat in the United States and many other countries. Growing resistant cultivars has been approved to be the best approach for control of stripe rust. To determine stripe rust resistance genes in U.S. winter wheat cultivars and breeding lines, we analyzed a winter wheat panel of 857 cultivars and breeding lines in a genome-wide association study (GWAS) using genotyping by multiplexed sequencing (GMS) and by genotyping with molecular markers of 18 important stripe rust resistance genes or quantitative trait loci (QTL). The accessions were phenotyped for stripe rust response at adult-plant stage under natural infection in Pullman and Mount Vernon, Washington in 2018 and 2019, and in the seedling stage with six predominant or most virulent races of Pst. A total of 51 loci were identified to be related to stripe rust resistance, and at least 10 of them (QYrww.wgp.1D-3, QYrww.wgp.2B-2, QYrww.wgp.2B-3, QYrww.wgp.2B-4, QYrww.wgp.3A, QYrww.wgp.5A, QYrww.wgp.5B, QYrww.wgp.5D, QYrww.wgp.6A-2 and QYrww.wgp.7B-3) were previously reported. These genes or QTL were found to be present at different frequencies in breeding lines and cultivars developed by breeding programs in various winter wheat growing regions. Both Yr5 and Yr15, which are highly resistant to all races identified thus far in the U.S., as well as Yr46 providing resistance to many races, were found absent in the breeding lines and commercially grown cultivars. The identified genes or QTL and their markers are useful in breeding programs to improve the level and durability of resistance to stripe rust.

Genome-Wide Linkage Mapping Reveals Stripe Rust Resistance in Common Wheat (Triticum aestivum) Xinong1376.

Stripe rust, also known as yellow rust, is a significant threat to wheat yield worldwide. Adult plant resistance (APR) is the preferred way to obtain durable protection. Chinese winter wheat cultivar Xinong1376 has maintained acceptable APR to stripe rust in field environments. To characterize APR in this cultivar, 190 F10 recombinant inbred lines (RILs) developed from Xiaoyan81 × Xinong1376 were evaluated for infection type and disease severity in fields either artificially or naturally inoculated. The population along with parents were genotyped using the Illumina 90K single-nucleotide polymorphism arrays. Six quantitative trait loci (QTL) were detected using the inclusive composite interval mapping method. QYr.nwafu-4AL and QYr.nwafu-6BL.3 conferred stable resistance in all environments, and likely corresponded to a gene-rich region on the long arm of chromosomes 4A and 6B. QYr.nwafu-5AL, QYr.nwafu-5BL, QYr.nwafu-3BL.1, and QYr.nwafu-3BL.2 were detected only in some environments but enhanced the level of resistance conferred by QYr.nwafu-4AL and QYr.nwafu-6BL.3. Kompetitive allele-specific PCR (KASP) markers developed for QYr.nwafu-4AL and QYr.nwafu-6BL.3 were confirmed in a subset of RILs and 133 wheat genotypes. The QTL on 4AL and 6BL with their linked KASP markers would be useful for marker-assisted selection to improve stripe rust resistance in breeding programs.

A major QTL co-localized on chromosome 6BL and its epistatic interaction for enhanced wheat stripe rust resistance.

KEY MESSAGE: Co-localization of a major QTL for wheat stripe rust resistance to a 3.9-cM interval on chromosome 6BL across both populations and another QTL on chromosome 2B with epistatic interaction. Cultivars with diverse resistance are the optimal strategy to minimize yield losses caused by wheat stripe rust (Puccinia striiformis f. sp. tritici). Two wheat populations involving resistant wheat lines P10078 and Snb"S" from CIMMYT were evaluated for stripe rust response in multiple environments. Pool analysis by Wheat660K SNP array showed that the overlapping interval on chromosome 6B likely harbored a major QTL between two populations. Then, linkage maps were constructed using KASP markers, and a co-localized locus with large effect on chromosome 6BL was detected using QTL analysis in both populations. The coincident QTL, named QYr.nwafu-6BL.2, explained 59.7% of the phenotypic maximum variation in the Mingxian 169 × P10078 and 52.5% in the Zhengmai 9023 × Snb"S" populations, respectively. This co-localization interval spanning 3.9 cM corresponds to ~ 30.5-Mb genomic region of the newest common wheat reference genome (IWGSC RefSeq v.1.0). In addition, another QTL was also detected on chromosome 2B in Zhengmai 9023 × Snb"S" population and it can accelerate expression of QYr.nwafu-6BL.2 to enhance resistance with epistatic interaction. Allowing for Pst response, marker genotypes, pedigree analysis and relative genetic distance, QYr.nwafu-6BL.2 is likely to be a distinct adult plant resistance QTL. Haplotype analysis of QYr.nwafu-6BL.2 revealed specific SNPs or alleles in the target region from a diversity panel of 176 unrelated wheat accessions. This QTL region provides opportunity for further map-based cloning, and haplotypes analysis enables pyramiding favorable alleles into commercial cultivars by marker-assisted selection.

Utilization of the Genomewide Wheat 55K SNP Array for Genetic Analysis of Stripe Rust Resistance in Common Wheat Line P9936.

Breeding for resistance to stripe rust (caused by Puccinia striiformis f. tritici) is essential for reducing losses in yield and quality in wheat. To identify genes for use in breeding, a biparental population of 186 recombinant inbred lines (RILs) from a cross of the Chinese landrace Mingxian 169 and CIMMYT-derived line P9936 was evaluated in field nurseries either artificially or naturally inoculated in two crop seasons. Each of the RILs and parents was genotyped with the wheat 55K single-nucleotide polymorphism (SNP) 'Breeders' array and a genetic linkage map with 8,225 polymorphic SNP markers spanning 3,593.37 centimorgans was constructed. Two major quantitative trait loci (QTL) and two minor QTL were identified. The major QTL QYr.nwafu-3BS.2 and QYr.nwafu-7BL on chromosomes arms 3BS and 7BL were detected in all field locations and explained an average 20.4 and 38.9% of phenotypic variation stripe rust severity, respectively. QYr.nwafu-3BS.2 likely corresponds to the locus Yr30/Sr2 and QYr.nwafu-7BL may be a resistance allele identified previously in CIMMYT germplasm. The other minor QTL had limited individual effects but increased resistance when in combinations with other QTL. Markers linked to QYr.nwafu-7BL were converted to kompetitive allele-specific polymerase chain reaction markers and validated in a panel of wheat accessions. Wheat accessions carrying the same haplotype as P9936 at the identified SNP loci had lower average stripe rust severity than the average severity of all other haplotypes.

Genome-wide Mapping for Stripe Rust Resistance Loci in Common Wheat Cultivar Qinnong 142.

Stripe rust caused by Puccinia striiformis f. sp. tritici threatens worldwide wheat production. Growing resistant cultivars is the best way to control this disease. Chinese wheat cultivar Qinnong 142 (QN142) has a high level of adult-plant resistance to stripe rust. To identify quantitative trait loci (QTLs) related to stripe rust resistance, we developed a recombinant inbred line (RIL) population from a cross between QN142 and susceptible cultivar Avocet S. The parents and 165 F6 RILs were evaluated in terms of their stripe rust infection type and disease severity in replicated field tests with six site-year environments. The parents and RILs were genotyped with single-nucleotide polymorphism (SNP) markers. Four stable QTLs were identified in QN142 and mapped to chromosome arms 1BL, 2AL, 2BL, and 6BS. The 1BL QTL was probably the known resistance gene Yr29, the 2BL QTL was in a resistance gene-rich region, and the 2AL and 6BS QTLs might be new. Kompetitive allele specific polymerase chain reaction markers developed from the SNP markers flanking these QTLs were highly polymorphic in a panel of 150 wheat cultivars and breeding lines. These markers could be used in marker-assisted selection for incorporating the stripe rust resistance QTL into new wheat cultivars.

Genetic architecture of wheat stripe rust resistance revealed by combining QTL mapping using SNP-based genetic maps and bulked segregant analysis.

KEY MESSAGE: A major stripe rust resistance QTL was mapped to a 0.4 centimorgan (cM) genetic region on the long arm of chromosome 7B, using combined genome-wide linkage mapping and bulk segregant analysis. The German winter wheat cv. Centrum has displayed high levels of adult plant stripe rust resistance (APR) in field environments for many years. Here, we used the combined genome-wide linkage mapping and pool-extreme genotyping to characterize the APR resistance. One hundred and fifty-one F2:7 recombinant inbred lines derived from a cross between susceptible landrace Mingxian 169 and Centrum were evaluated for stripe rust resistance in multiple environments and genotyped by the wheat 35K single nucleotide polymorphism (SNP) array. Three stable quantitative trait loci (QTL) were identified using QTL analysis across five field environments. To saturate the major QTL, the wheat 660K SNP array was also used to genotype bulked extremes. A major QTL named QYrcen.nwafu-7BL from Centrum was mapped in a 0.4 cM genetic interval flanking by AX-94556751 and AX-110366788 across a 2 Mb physical genomic region, explaining 19.39-42.81% of the total phenotypic variation. It is likely a previously uncharacterized QTL based on pedigree analysis, reaction response, genotyping data and map comparison. The SNP markers closely linked with QYrcen.nwafu-7BL were converted to KASP markers and validated in a subset of 120 wheat lines. A 211 F2 breeding population from a cross of an elite cultivar Xinong 979 with Centrum were developed for marker-based selection. Three selected lines with desirable agronomic traits and the positive alleles of both KASP markers showed acceptable resistance which should be used as resistance donors in wheat breeding programs. The other QTL QYrcen.nwafu-1AL and QYrcen.nwafu-4AL with additive effects could enhance the level of resistance conferred by QYrcen.nwafu-7BL.

Comparative genome-wide mapping versus extreme pool-genotyping and development of diagnostic SNP markers linked to QTL for adult plant resistance to stripe rust in common wheat.

KEY MESSAGE: A major stripe rust resistance QTL on chromosome 4BL was localized to a 4.5-Mb interval using comparative QTL mapping methods and validated in 276 wheat genotypes by haplotype analysis. CYMMIT-derived wheat line P10103 was previously identified to have adult plant resistance (APR) to stripe rust in the greenhouse and field. The conventional approach for QTL mapping in common wheat is laborious. Here, we performed QTL detection of APR using a combination of genome-wide scanning and extreme pool-genotyping. SNP-based genetic maps were constructed using the Wheat55 K SNP array to genotype a recombinant inbred line (RIL) population derived from the cross Mingxian 169 × P10103. Five stable QTL were detected across multiple environments. A fter comparing SNP profiles from contrasting, extreme DNA pools of RILs six putative QTL were located to approximate chromosome positions. A major QTL on chromosome 4B was identified in F2:4 contrasting pools from cross Zhengmai 9023 × P10103. A consensus QTL (LOD = 26-40, PVE = 42-55%), named QYr.nwafu-4BL, was defined and localized to a 4.5-Mb interval flanked by SNP markers AX-110963704 and AX-110519862 in chromosome arm 4BL. Based on stripe rust response, marker genotypes, pedigree analysis and mapping data, QYr.nwafu-4BL is likely to be a new APR QTL. The applicability of the SNP-based markers flanking QYr.nwafu-4BL was validated on a diversity panel of 276 wheat lines. The additional minor QTL on chromosomes 4A, 5A, 5B and 6A enhanced the level of resistance conferred by QYr.nwafu-4BL. Marker-assisted pyramiding of QYr.nwafu-4BL and other favorable minor QTL in new wheat cultivars should improve the level of APR to stripe rust.

SNP-based pool genotyping and haplotype analysis accelerate fine-mapping of the wheat genomic region containing stripe rust resistance gene Yr26.

KEY MESSAGE: NGS-assisted super pooling emerging as powerful tool to accelerate gene mapping and haplotype association analysis within target region uncovering specific linkage SNPs or alleles for marker-assisted gene pyramiding. Conventional gene mapping methods to identify genes associated with important agronomic traits require significant amounts of financial support and time. Here, a single nucleotide polymorphism (SNP)-based mapping approach, RNA-Seq and SNP array assisted super pooling analysis, was used for rapid mining of a candidate genomic region for stripe rust resistance gene Yr26 that has been widely used in wheat breeding programs in China. Large DNA and RNA super-pools were genotyped by Wheat SNP Array and sequenced by Illumina HiSeq, respectively. Hundreds of thousands of SNPs were identified and then filtered by multiple filtering criteria. Among selected SNPs, over 900 were found within an overlapping interval of less than 30 Mb as the Yr26 candidate genomic region in the centromeric region of chromosome arm 1BL. The 235 chromosome-specific SNPs were converted into KASP assays to validate the Yr26 interval in different genetic populations. Using a high-resolution mapping population (> 30,000 gametes), we confined Yr26 to a 0.003-cM interval. The Yr26 target region was anchored to the common wheat IWGSC RefSeq v1.0 and wild emmer WEWSeq v.1.0 sequences, from which 488 and 454 kb fragments were obtained. Several candidate genes were identified in the target genomic region, but there was no typical resistance gene in either genome region. Haplotype analysis identified specific SNPs linked to Yr26 and developed robust and breeder-friendly KASP markers. This integration strategy can be applied to accelerate generating many markers closely linked to target genes/QTL for a trait of interest in wheat and other polyploid species.

Combining Single Nucleotide Polymorphism Genotyping Array with Bulked Segregant Analysis to Map a Gene Controlling Adult Plant Resistance to Stripe Rust in Wheat Line 03031-1-5 H62.

Stripe rust, caused by Puccinia striiformis f. sp. tritici, is one of the most devastating diseases of wheat worldwide. Growing resistant cultivars is considered the best approach to manage this disease. In order to identify the resistance gene(s) in wheat line 03031-1-5 H62, which displayed high resistance to stripe rust at adult plant stage, a cross was made between 03031-1-5 H62 and susceptible cultivar Avocet S. The mapping population was tested with Chinese P. striiformis f. sp. tritici race CYR32 through artificial inoculation in a field in Yangling, Shaanxi Province and under natural infection in Tianshui, Gansu Province. The segregation ratios indicated that the resistance was conferred by a single dominant gene, temporarily designated as YrH62. A combination of bulked segregant analysis (BSA) with wheat 90K single nucleotide polymorphism (SNP) array was used to identify molecular markers linked to YrH62. A total of 376 polymorphic SNP loci identified from the BSA analysis were located on chromosome 1B, from which 35 kompetitive allele-specific PCR (KASP) markers selected together with 84 simple sequence repeat (SSR) markers on 1B were used to screen polymorphism and a chromosome region associated with rust resistance was identified. To saturate the chromosomal region covering the YrH62 locus, a 660K SNP array was used to identify more SNP markers. To develop tightly linked markers for marker-assisted selection of YrH62 in wheat breeding, 18 SNPs were converted into KASP markers. A final linkage map consisting of 15 KASP and 3 SSR markers was constructed with KASP markers AX-109352427 and AX-109862469 flanking the YrH62 locus in a 1.0 cM interval. YrH62 explained 63.8 and 69.3% of the phenotypic variation for disease severity and infection type, respectively. YrH62 was located near the centromeric region of chromosome 1BS based on the positions of the SSR markers in 1B deletion bins. Based on the origin, responses to P. striiformis f. sp. tritici races, and marker distances, YrH62 is likely different from the other reported stripe rust resistance genes/quantitative trait loci on 1B. The gene and tightly linked KASP markers will be useful for breeding wheat cultivars with resistance to stripe rust.

Rapid identification of an adult plant stripe rust resistance gene in hexaploid wheat by high-throughput SNP array genotyping of pooled extremes.

KEY MESSAGE: High-throughput SNP array analysis of pooled extreme phenotypes in a segregating population by KASP marker genotyping permitted rapid, cost-effective location of a stripe rust resistance QTL in wheat. German wheat cultivar "Friedrichswerther" has exhibited high levels of adult plant resistance (APR) to stripe rust in field environments for many years. F2:3 lines and F6 recombinant inbred line (RILs) populations derived from a cross between Friedrichswerther and susceptible landrace Mingxian 169 were evaluated in the field in 2013, 2016 and 2017. Illumina 90K iSelect SNP arrays were used to genotype bulked extreme pools and parents; 286 of 1135 polymorphic SNPs were identified on chromosome 6B. Kompetitive Allele-Specific PCR (KASP) markers were used to verify the chromosome region associated with the resistance locus. A linkage map was constructed with 18 KASP-SNP markers, and a major effect QTL was identified within a 1.4 cM interval flanked by KASP markers IWB71602 and IWB55937 in the region 6BL3-0-0.36. The QTL, named QYr.nwafu-6BL, was stable across environments, and explained average 54.4 and 47.8% of the total phenotypic variation in F2:3 lines and F6 RILs, respectively. On the basis of marker genotypes, pedigree analysis and relative genetic distance QYr.nwafu-6BL is likely to be a new APR QTL. Combined high-throughput SNP array genotyping of pooled extremes and validation by KASP assays lowers sequencing costs compared to genome-wide association studies with SNP arrays, and more importantly, permits rapid isolation of major effect QTL in hexaploid wheat as well as improving accuracy of mapping in the QTL region. QYr.nwafu-6BL with flanking KASP markers developed and verified in a subset of 236 diverse lines can be used in marker-assisted selection to improve stripe rust resistance in breeding programs.

Saturation Mapping of a Major Effect QTL for Stripe Rust Resistance on Wheat Chromosome 2B in Cultivar Napo 63 Using SNP Genotyping Arrays.

Stripe rust or yellow rust (YR), caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most important diseases of wheat (Triticum aestivum L.). Widespread deployment of resistant cultivars is the best means of achieving durable disease control. The red grain, spring wheat cultivar Napo 63 produced by CIMMYT in the 1960s shows a high level of adult-plant resistance to stripe rust in the field. To elucidate the genetic basis of resistance in this cultivar we evaluated 224 F2:3 lines and 175 F2:6 recombinant inbred lines (RILs) derived from a cross between Napo 63 and the Pst-susceptible line Avocet S. The maximum disease severity (MDS) data of F2:3 lines and the relative area under the disease progress curve (rAUDPC) data of RILs were collected during the 2014-2015 and 2015-2016 wheat growing seasons, respectively. Combined bulked segregant analysis and 90K single nucleotide polymorphism (SNP) arrays placed 275 of 511 polymorphic SNPs on chromosome 2B. Sixty four KASP markers selected from the 275 SNPs and 76 SSR markers on 2B were used to identify a chromosome region associated with rust response. A major effect QTL, named Qyrnap.nwafu-2BS, was identified by inclusive composite interval mapping and was preliminarily mapped to a 5.46 cM interval flanked by KASP markers 90K-AN34 and 90K-AN36 in chromosome 2BS. Fourteen KASP markers more closely linked to the locus were developed following a 660K SNP array analysis. The QTL region was finally narrowed to a 0.9 cM interval flanked by KASP markers 660K-AN21 and 660K-AN57 in bin region 2BS-1-0.53. The resistance of Napo 63 was stable across all environments, and as a QTL, explained an average 66.1% of the phenotypic variance in MDS of F2:3 lines and 55.7% of the phenotypic variance in rAUDPC of F5:6 RILs. The short genetic interval and flanking KASP markers developed in the study will facilitate marker-assisted selection, gene pyramiding, and eventual positional cloning of Qyrnap.nwafu-2BS.

Development and Validation of KASP-SNP Markers for QTL Underlying Resistance to Stripe Rust in Common Wheat Cultivar P10057.

Stripe rust (Puccinia striiformis f. sp. tritici) is among the most important diseases of wheat (Triticum aestivum L.) globally. Utilization of adult plant resistance (APR) constitutes a key tool for maintaining protection against this disease. The CIMMYT wheat cultivar P10057 displayed a high level of APR to stripe rust in germplasm evaluation in field environments. To clarify the genetic basis and identify quantitative trait loci (QTLs) involved in stripe rust resistance in P10057, three wheat populations were used: 150 F5:6 recombinant inbred lines (RILs) derived from the cross Mingxian 169 × P10057, and 161 and 140 F2:3 lines from Avocet S × P10057 and Zhengmai 9023 × P10057, respectively. These three populations were evaluated for infection type (IT) and disease severity (DS) in Shaanxi, Gansu, and Sichuan during the 2014-15 and 2015-16 cropping seasons. Genotyping was performed with Kompetitive Allelic Specific PCR (KASP) and simple sequence repeat (SSR) markers linked to the resistance loci. Using QTL analysis, two genomic regions associated with resistance were found on chromosome arms 2BS and 3BS, respectively. These two stable QTLs, designated Qyrlov.nwafu-2BS and Qyrlov.nwafu-3BS, were detected across all environments and explained average 22.6 to 31.6% and 21.3 to 32.3% of stripe rust severity phenotypic variation, respectively. Qyrlov.nwafu-2BS may be the resistance allele derived from CIMMYT germplasm and Qyrlov.nwafu-3BS likely corresponds to the locus Sr2/Lr27/Yr30/Pbc. The KASP markers IWA5377, IWA2674, and IWA5830 linked to QYrlov.nwafu-2BS and IWB57990 and IWB6491 linked to Qyrlov.nwafu-3BS were reliable for marker-assisted selection (MAS) in the Zhengmai 9023 × P10057 population. These QTLs with KASP markers are expected to contribute in developing wheat cultivars with improved stripe rust resistance.