Genetic Mapping And Candidate Gene Analysis Of Resistance Genes To Powdery Mildew And Stripe Rust In Wheat

Powdery mildew and stripe rust caused by Blumeria graminis f.sp.tritici（Bgt）and Puccinia striiformis f.sp.tritici（Pst）,respectively,are devastating wheat diseases worldwide,which seriously affect wheat production.Development of resistant cultivars is the most economical and environment-friendly way for controlling these diseases.In our previous study,a stable quantitative trait locus（QTL）QPm.caas-3BS for adult-plant resistance to powdery mildew was mapped in a recombinant inbred line（RIL）population derived from the Zhou8425B/Chinese Spring cross.Consequently,the RIL population and their derived secondary mapping populations were used for fine mapping of QPm.caas-3BS in the present study;the re-sequenced data of Zhou8425B,RNA-Seq,bioinformatics and association analysis were used for fine mapping of QPm.caas-3BS and prediction of candidate genes.In addition,a genetic population for mapping the stripe rust resistance gene in Shaannong69 was constructed by crossing with susceptible parents Huixianhong and Avocet S.BSR-Seq（Bulked segregant RNA sequencing）,BSE-Seq（Bulked segregant exome capture sequencing）and molecular markers were used to map the Yr gene in Shaannong69.The main results are summarized below:（1）Based on the wheat 90K SNP chip mapping results,16 lines from the Zhou8425B/Chinese Spring population were selected for making 12 crosses.Based on the genotypes and phenotypes of parents and progenies of 12 crosses,two crosses ZC3B1 and ZC3B8 were screened and used to construct secondary mapping populations.One hundred and seven recombinant individuals were screened from 2027 BC₁F₂plants using the flanking markers of QPm.caas-3BS,and were divided into 17 kinds of recombinant types based on eight molecular markers.（2）Eleven groups of heterozygous recombinant individuals were used to develop BC₁F_2:3and BC₁F_2:4populations.According to the genotypes and phenotypes of BC₁F_2:3and BC₁F_2:4populations,QPm.caas-3BS was mapped to a 3.91 Mb physical interval flanked by markers 3B4625 and 3B5000,including 98 high-confidence gene.According to the re-sequenced data of Zhou8425B,48 genes showed missense mutations between Zhou8425B and Chinese Spring.Among them,six genes had a higher FPKM（Fragments per kilobase per million reads）in 3B+（Zhou8425B allele）NILs（Near-isogenic lines）than 3B-（Chinese Spring allele）NILs based on RNA-Seq data.The results of q RT-PCR（Real-time quantitative PCR）and association analysis indicated that three genes Traes CS3B02G014800,Traes CS3B02G016800 and Traes CS3B02G019900 were associated with powdery mildew resistance.（3）A partial genetic map of 3BS was re-constructed using three KASP（Kompetitive allele specific PCR）markers that were developed based on the sequences of Traes CS3B02G014800,Traes CS3B02G016800 and Traes CS3B02G019900,and 90K SNP chip and SSR markers linked with Pmx/Yr30/Lr27Sr2 and Sb3.QPm.caas-3BS was then mapped to a 0.3 c M interval,corresponding to a431 kb region according to IWGSC（International wheat genome sequencing consortium）v1.1.This interval included 16 high-confidence genes,among which Traes CS3B02G014800 is the most likely candidate gene,and Traes CS3B02G016300 and Traes CS3B02G016400 are also potential candidates based on gene annotation and sequence variations between parents.（4）Traes CS3B02G014800 encodes an uncharacterized protein and has numbers of predicted trans-membrane regions.Analysis of allelic variation showed that Traes CS3B02G014800 mainly had three haplotypes in 285 wheat cultivars from three wheat grown regions in China.The frequency of the susceptibility allele was higher than the resistance allele in these wheat cultivars.（5）Using the Pst race CYR31 to inoculate the F₁,F₂and F_2:3populations of theShaanong69/Huixianhong cross,we found that the resistance to stripe rust in Shaannong69 was conferred by a single dominant gene,designated as Yr SN69.Based on BSR-Seq and BSE-Seq of resistant and susceptible bulks from the F_2:3population as well as molecular marker analysis,Yr SN69was mapped on chromosome arm 2BL in a 3.6 c M genetic interval,with genetic distances of 2.3 c M and 1.3 c M to markers 2BC-7 and 2BA-7,respectively.