Genome-Wide Association Study And Fine Mapping Of QRBSDV6 On Resistance To Rice Black-Streaked Dwarf Virus Disease In Rice

Rice black-streaked dwarf virus disease(RBSDVD)is a serious rice disease,which pathogen is rice black-streaked dwarf virus(RBSDV),transmitted by small brown planthopper(Laodelphax striatellus Fallen,SBPH),an insect belong to Delphacidae of Homoptera.Once infected with this disease,rice plant would become dwarf,produce darkgreen leaves and extremely low seed fertility,leading to a sharp decline in production.At present,the main means of dealing with RBSDVD is to apply pesticides to control the SBPHs,which consumes a lot of manpower and resources,and threatens the environment and ecology.Discovering new resistance materials,searching for resistance genes,and breeding resistant varieties can not only significantly reduce disease epidemics,but also avoid negative impacts on the environment and ecology.It is considered to be an effective strategy to fundamentally control RBSDVD.However,since the RBSDV is transmitted by the insect vector,which are easily affected by the environment,makes the phenotypic identification more difficult.Therefore,few QTLs for disease resistance were currently identified,and only qRBSDV6MH was fine mapped.The lag of research on RBSDVD has become barriers of breeding advance,so it is urgent to seek new resistance resources and QTLs,map and clone resistance gene,and then lay a foundation for elucidating the molecular mechanism of rice resistance to RBSDVD and establishing a molecular breeding platform against RBSDVD.To this end,the study completed the identification on resistance to RBSDVD of 1070 rice resources from 72 countries and regions,and obtained a number of new resistance resources.Then we validated the differences on resistance between different rice subspecies,and detected anti-RBSDVD locus by genome-wide association study(GWAS).In addition,based on the predecessor's mapping of the main QTL qRBSD V6 against RBSDVD,this study completed the fine mapping of the QTL.The main research contents and related conclusions are as follows:?.Genome-wide association study on resistance to RBSDVD1.We carried out research on RBSDVD resistance selecting 1070 rice from all over the world,these rice resources are rich in genetic background and contain various indica and japonica,as well as aus rice and aromatic rice.There are significant differences on the RBSDVD resistance among different backgrounds of rice germplasm resources.we found that the average incidence of all samples was 35.2%,and the average incidence of indica was only 29.5%,which was significantly lower than the average incidence of whole panel and japonica with 46.5%.The mean incidence of aus rice was 42.1%,and this value of aromatic rice was 34.2%.2.Combined the SNP data of the 1070 rice samples sequenced,we used the mixed linear model(MLM)to perform GWAS on RBSDVD resistance.Significant association signal was detected on chromosome 6.At the same time,GWAS was also carried out using indica and japonica panel respectively,this locus was confirmed by indica panel.3.Based on the annotation of the reference genome in the interval and the annotation of the SNPs,the gene LOC_Os06g03120 encoding an aspartic protease family protein was selected as a candidate gene.4.Haplotype analysis demonstrated that there are five major haplotypes on this gene,and the germplasm resources with haplotype 1 are more resistant to RBSDVD than the other four.Statistical analysis showed haplotype 1 mainly exist in indica.?.Fine mapping of a major QTL qRBSDV6 conferring RBSDVD resistance1.Through previous studies,a major RBSDVD resistance locus qRBSDV6 was detected on chromosome 6.Another linkage analysis was conducted using F2 population derived from a crossing of 9194 and Suyunuo in order to validate previous studies,and qRBSD V6 was still detected with a LOD score of 4.48 and PVE of 16.7%,which confirmed this locus is stable.2.In order to exclude the interference of QTLs at other locations when fine mapping,Suyunuo was selected as recurrent parent,through multiple generations of backcrossing,we constructed the secondary separation population BC3F2 of qRBSDV6.After linkage analysis using this population,the presence of resistant QTL on this locus was further confirmed,with LOD score of 13.7 and PVE of 23.4%.3.The individuals which recombined within the qRBSDV6 interval were screened from the backcrossed population.The offspring lines of each individual were evaluated for resistance to RBSDVD.By continuously narrowing the mapping segment,the gene was finally mapped to a physical interval with 201.7 kb on chromosome 6.4.Based on the annotation of genes in the reference variety in public database,combined with previous reports of genes on biotic tolerance,we focused on genes LOC_Os06g34070 and LOC_Os06g34120,which two encode GDSL family lipases.After sequencing the genes of two parents,multiple mutations were found in the upstream of LOC_Os06g34070,then it was tentatively set as a candidate gene and related genetic transformation has been conducted.Screening of rice germplasm resources with RBSDVD resistance provides abundant original materials for breeding and research.The resistance locus obtained by GWAS is valuable and worthy of further study,at the same time,excellent haplotype of this genes could apply to breeding.The fine mapping of qRBSDV6 not only laid a foundation for further cloning of this gene,but also help to break the linkage drag,introduce it into new germplasm through molecular marker-assisted selection,and breed a new variety with RBSDVD resistance.