Genetics Dissection Of Stripe Rust Resistance In Four Wheat Cultivars(Lines) And Map-Based Cloning Of QYrnap.nwafu-2BS(YrNP63)

Stripe rust is one of the most serious fungal diseases threatening wheat production.Breeding and planting cultivars with excellent resistance is the most economical,effective and eco-friendly measure to control this disease.It is an important theoretical basis for rapid and efficient development of new resistance cultivars using molecular breeding techniques to systematically dissect the genetic basis of resistance,identify,fine-map and clone the resistance genes/QTL to stripe rust in wheat.Our laboratory has been committed to screening wheat stripe rust resistance resources and mapping resistance genes/QTL for a long time.In our previous studies,we have collected an amount of wheat germplasm with excellent resistance and mapped a series of major and minor genes/QTL including Qyrnap.nwafu-2BS（YrNP63）from them.Morever,we found additive and epistatic interaction effect were usually exsited in different genes/QTL.Based on these,this study focuses on the mapping of QTL and dissection of interaction effect between different locus in four wheat cultivars（lines）Ruihuamai 520（RH520）,Fengdecunmai 12（FDC12）,P10090 and Yaco"S"with adult-plant resistance,and map-based cloning of YrNP63 to provide new resistance genes and information of ideal combination of genes/QTL for molecular breeding of wheat cultivars with durable resistance to stripe rust.The main results were summarized as follows:1.Identification of resistance genes/QTL and favorable gene combination in wheat cultivars（lines）RH520,FDC12,P10090 and Yaco"S"RH520,FDC12,P10090 and Yaco"S"all maintains excellent adult plant resistance（APR）to stripe rust at field.To map the genes/QTL in these wheats,we have developed multiple biparental genetic populations by cross with susceptible cultivar Miangxian169（MX169）,including RH520/FDC12(F_5:7,n=287),MX169/P10090(F_6:7,n=182),MX169/P10090(F_6:7,n=122)and MX169/Yaco"S"(F_5:6,n=141).All lines were planted in multiple environments for collecting resistance phenotype data and genotyped by Wheat 16K,55K and 660K SNP array as well as KASP markers for high-resolution genetic map.Using ICIM-ADD model,a total of five major QTL related with infection type（IT）and disease severity（DS）were located on chromosome 2BS,2BL,3BS,4BL and 6A.Moreover,KASP makers linked with each locus were developed for maker-assisted selection in wheat resistance breeding.QYryac.nwafu-2BS（Yr Ac）and QYryac.nwafu-2BL were identified in cultivar Yaco"S",explained 13.3-27.5%and 14.7-25.1%phenotypic variation explained（PVE）,respectively.Yr Ac was limited in a 0.66 c M interval between marker SK-29 and YM-23,corresponding to approximate 2.56 Mb（155.79-158.35 Mb）region on chromosome 2BS according to the IWGSC Ref Seq v.1.1.It may be the same as Qyrnap.nwafu-2BS（YrNP63）.QYryac.nwafu-2BL could be a novel resistance QTL to stripe rust.Yr Ac was away 10.6 c M from QYryac.nwafu-2BL,which provides a chance to select them simultaneously for pyramiding resistance in an easier way.QYr.nwafu-3BS from RH520 corresponded the pleiotropic gene Yr30/Sr2/Lr27/Sb3/Pbc1,explained 23.0%-38.0%PVE.QYrfdc.nwafu-4BL（designated as Yr FDC12）from FDC12 was located at a resistance gene-rich region,explained 11.2%-17.5%PVE.Yr30 and Yr FDC12were respectively co-segregated with Pbc1 and Pbc FDC12,which control expression of the pseudo-black chaff（PBC）through significant additive and epistatic interaction effects.Lines harboring both Yr30（Pbc1）and Yr FDC12（Pbc FDC12）displayed high resistance to stripe rust and PBC.Therefore,Yr30 and Yr FDC12 were a fine gene combination for acquisition of highly effective,durable stripe rust resistance by pyramiding strategy.QYr.nwafu-6A（Yr P10090）in P10090,explained 26.4-32.1%PVE,was located in a 3.5c M interval between KASP markers AX-94460938 and AX-110585473 where recombination has been inhibited,corresponding to a large physical region（107.1-445.6 Mb according to IWGSC Ref Seq v.1.1）strongly selected by modern breeding.The interval of Yr P10090 also harbored the favorable alleles of GPC-A1,Ta GW2-6A and Rht24.Thus,the genomic region of Yr P10090 can be used as a multifunction genetic unit for cooperative improvement of multiple traits including high yield,high quality and disease resistance.2.Fine mapping and map-based coloning of YrNP63Qyrnap.nwafu-2BS（YrNP63）derived from wheat Napo63,may be the same as Yr Ac.To narrow the interval of YrNP63,three segregating populations developed from crossing between RILs of the Napo63/Avs population and 20 KASP markers were used to construct the high-density genetic map of YrNP63.The entire map covered 6.33 c M,and YrNP63 was limited in a 0.11 c M interval between marker YM-42 and YM-22,corresponding to about 450Kb（157.3-158.7 Mb）genomic region on chromosome 2BS according to the IWGSC Ref Seq v.1.1.A total of six high confidence annotated genes existed in the 450 kb genomic region of YrNP63 based on the IWGSC Ref Seq v.1.1 and v.2.0.According to predicted function,sequence polymorphism and expression quantity of these genes in parent lines Napo63 and Avs,only Traes CS2B02G182800（Ta2800）was considered as candidate gene of YrNP63.Ta2800 contains two exons and encodes a CC-NBS-LRR protein.The 29 out of 43 SNP variations between Napo63 and Avs can cause amino acid changes.After silencing Ta2800 by barley stripe mosaic virus-mediated gene silencing,wheat with YrNP63 would lose resistance to stripe rust,indicating that Ta2800 is the coding gene of YrNP63.Ta2800 also encodes the leaf rust resistance gene Lr13.However,Lr13 and YrNP63 are only effective for leaf rust and stripe rust,respectively.Multiple sequence alignment analysis of the CDS sequences between YrNP63 and Ta2800 in 42 other wheat cultivars（lines）revealed that YrNP63 was a new allele and contained a unique SNP variant G2683A.Based on G2683A,we have developed the YrNP63-specific molecular marker KASP2683.Moreover,to explore novel alleles of Ta2800 for resistance to stripe rust in wheat,we performed haplotype analysis of Ta2800 in 1473 wheat cultivars（lines）based on 660K SNP array genotypes.Although a total of 15 different haplotypes including eight haplotypes with more than 30 varieties（lines）were found,only YrNP63 haplotype showed resistance to wheat stripe rust.YrNP63 haplotype mainly existed in CIMMYT and ICARDA germplasm,but not in Chinese wheat,indicating that YrNP63 has not be used in China yet.