Identifation Of Resistance Sources To Stripe Rust Pathogen Based On Linkage Mapping And Association Analysis And Development Of Wheat Lines With Pyramided Genes Yr64 And Yr65

Stripe rust or yellow rust?Yr?,caused by the fungus Puccinia striiformis Westend.f.sp.tritici Erikss.?Pst?,is one of the most destructive diseases and has long been considered the major constraint to wheat production.Identification of new sources of resistance and breeding resistant cultivars were the most economic,eco-friendly and effective method to control this disease.The sixty accessions and fifteen Yr single-gene lines were tested at the seedlings in the greenhouse with ten Pst races and at the adult-plant stage in the fields with mixed Pst races.Molecular markers detection of known genes was also used.The results showed the resistance characteristics of sixty accessions and their values on breeding program.In addition,The German wheat cultivar‘Centrum'and Chinese common wheat cultivar‘xinong 1376'showed high level of APR against stripe rust despite the several changes in predominant Pst races.Thus,dissecting genetic basis of resistance in Centrum and Xinong1376 is important for developing new resistant cultivars with higher yields and adaptation.In addition,genome-wide association study?GWAS?was implemented in 857 wheat cultivars/breeding lines from National Small Grains Collection?NSGC?winter wheat germplasm collection to identify new resistance gene loci.To develop durable resistance breeding line to stripe rust,two Yr genes including Yr64 and Yr65 in the same chromosome arm were pyramided using traditional intercross and backcross method,the line carrying Yr64 and Yr65 would be useful in the wheat breeding program.The 60 winter wheat accessions collected around the world and fifteen Yr single-gene lines were tested in seedlings with ten Chinese Pst races including CYR23,CYR29,CYR31,CYR32,CYR33,Sull-4,Sull-5,Sull-7,V26/CH42 and V26/Gui22.Gene postulation was implemented based on resistance spectra of single gene.Marker test of known genes were also implemented to validate gene postulation.In addition,adult plant resistance tests were implemented in Yangling,Shaanxi and Tianshui,Gansu,respectively.The results showed a total of fifty accessions performed stable adult-plant resistance to stripe rust.Among of these,13 were postulated to have Yr9,Yr10,Yr17 and Yr26.The remaining accessions were not tested any resistant genes,and possibly have unknown new resistant genes.4 accessions were postulated to have unknown all-stage resistant genes.The 4 accessions were not suggested to apply in the breeding program.But the fifty accessions can be used in current breeding program.The German wheat cultivar‘Centrum'has shown a high level of APR against stripe rust despite the several changes in predominant Pst races.A recombinant inbred line?RIL?population was developed from a cross of Centrum with the susceptible Chinese landrace Mingxian 169?MX169?.Meanwhile,a F₂ breeding population was developed from Xinong979×Centrum,as Xinong 979 has many desirable agronomical traits and has been a major cultivar grown in several provinces of China.Seedling test was conducted under greenhouse conditions to characterize the stripe rust reaction of Centrum.stripe rust test in fields were implemented in Yangling of Shaanxi province and Tianshui of Gansu province in 2016 and2017 and in Jiangyou of Sichuan province in 2017.All 151 lines and their parents were genotyped with 35K array by Capital Bio Corporation.Stable QTL were identified on chromosomes 1AL,4AL and 7BL and designated as QYrcen.nwafu-1AL,QYrcen.nwafu-4AL and QYrcen.nwafu-7BL,respectively.All detected QTL were derived from the resistant parent Centrum using ICIM analysis.QYrcen.nwafu-7BL explained 19.3–42.8%on phenotypic variation.To saturate marker density in the major QTL QYrcen.nwafu-7BL,we combined BSA with 660K SNP arrays and narrowed down the genetic distance to 0.4Cm flanking markers AX-94556751 and AX-110366788.Comparing with previous QTL on chromosome 7BL,QYrcen.nwafu-7BL can be a new QTL to stripe rust.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?IT?and disease severity?DS?in fields either artificially or naturally inoculated.The population along with parents were genotyped using the Illumina 90K single nucleotide polymorphism?SNP?arrays.Six quantitative trait loci?QTL?were detected using Inclusive composite interval mapping?ICIM?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.1and 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.Germplasm core collections that reflect the range of genetic and phenotypic diversity of crop species are ideal platforms for examining the genetic architecture of complex traits such as resistance to stripe rust.We reported the results of genetic characterization and genome-wide association analysis?GWAS?for resistance to stripe rust in a core subset of 857accessions in the National Small Grains Collection?NSGC?winter wheat germplasm collection,based on genotyping with the wheat 3K single nucleotide polymorphism?SNP?iSelect assay and phenotyping of seedling and adult plants under natural disease epidemics in east Washington state Pullman and West Washington state Mount Vernon in 2018 and2019 crop seasons.High correlations among the field data were translated into high heritability values within and across locations.Population structure was evident when accessions were grouped by stripe rust reaction.GWAS identified 34 resistance loci that were effective across at least two environments.Based on the relative positions of the integrated linkage map,one of the QTL with significant genome-wide adjusted P-values in this study represent potentially new locus.This study provides an overview of the diversity of Pst resistance in the NSGC winter wheat germplasm core collection,which can be exploited for diversification of stripe rust resistance in breeding programs.Yr64 and Yr65 are effective against all Pst races identified in the United States,however,both genes with all-stage resistance may not have durable resistance.Based on the common SSR markers in the linkage maps of Yr64 and Yr65,the two genes were estimated to be linked in repulsion and separated by 7.8 cM.To achieve high-level and long-lasting resistance,we developed a wheat line,F₃-Yr64/Yr65,by pyramiding Yr64 and Yr65,both on the chromosome 1BS and providing high resistance to all tested Pst races.To validate F₃-Yr64/Yr65 possessing both genes,we crossed it to Avocet S?AvS?.The F₂populations from this cross were phenotyped with Pst races under controlled greenhouse conditions and under natural Pst infection in the field.The population was genotyped with SSR markers previously reported to be linked to the resistance gene loci and with additional SSR and SNP-KASP markers along chromosome 1B.Both phenotype and genotype data confirmed the combination of Yr64 and Yr65 in F₃-Yr64/Yr65,and the high-resolution linkage map dissected the chromosomal regions and traced their origins.