Discovery And Cloning Of Rice Black Streaked Dwarf Disease Resistance Genes

Rice black-streaked dwarf virus disease（RBSDVD）caused by rice black-streaked dwarf virus（RBSDV）and southern rice black-streaked dwarf virus disease（SRBSDVD）caused by southern rice black-streaked dwarf virus（SRBSDV）have caused great losses to rice production.It is the most economical and effective method to control RBSDVD by breeding disease resistant varieties.Therefore,exploring germplasm resources,identifying and cloning disease resistance genes have become the key to breeding disease resistant varieties.In this study,based on 3000 rice germplasm resources with genome resequencing,the quantitative trait locus（QTLs）associated with RBSDV resistance were identified by using genome-wide association study（GWAS）.In addition,a rice variety ZH5 with broad-spectrum resistance to RBSDVD and SRBSDVD was identified,and an F₂population was constructed using ZH5 and the susceptible variety Zhenshan97（ZS97）.The SRBSDV-related resistance genes were identified by bulked segregate analysis-sequencing（BSA-seq）.The candidate genes for resistance to rice black-streaked dwarf disease were cloned and their functions were preliminarily analyzed.The main results are as follows:1.Total of 608 rice germplasm resources collected from 71 different countries were identified for RBSDVD resistance.Twenty-nine RBSDVD-resistant germplasms were identified with the disease incidence of less than 15%evaluated as the standard.Combined with the comprehensive analysis of agronomic traits,12 rice varieties with strong RBSDVD resistance and better agronomic traits were identified.The results of phenotypic evaluation provided an excellent source of resistance for breeding RBSDVD resistant varieties.2.RBSDV resistance of a diverse global collection comprising 1956 rice accessions（including above 608 accessions）were evaluated under natural conditions.The average disease incidences of the Xian/indica（XI）subgroup were significantly lower than those of the Geng/japonica（GJ）subgroup.Among the five subgroups of indica rice,most of the RBSDVD-resistant accessions came from XI-1B subgroup.The rice variety ZH5 with high resistance to RBSDVD and the rice variety ZS97 with susceptibility to RBSDVD were identified.Using 44000 high-quality SNPs evenly distributed on chromosomes obtained by resequencing,five QTLs（q RBSDV-1.1、q RBSDV-6.1、q RBSDV-6.3、q RBSDV-7.1 and q RBSDV-9.1）were consistently identified by a single-locus GWAS and a multilocus GWAS.Six candidate genes were identified according to the intensity of their association signals and haplotype analysis in a single-locus GWAS.The haplotype analysis of these candidate genes suggested that LOC＿Os01g46970,LOC＿Os06g31190,LOC＿Os06g03120,LOC＿Os07g38250 and LOC＿Os09g21620 partially explain the variation in RBSDV resistance between the Xian and Geng subgroups,but LOC＿Os06g03150 partially explains the differences in RBSDV resistance within the Xian subgroup,especially between the XI-1A and XI-1B subgroups.3.Total of 2044 plants of F₂ populations of ZH5 and ZS97 were artificially inoculated with SRBSDVD,and DNA pools of resistance and susceptibility were constructed.A major QTL（q SRBSDV-6）of SRBSDV resistance on chromosome 6 was identified by BSA-seq.And two main candidate genes（LOC＿Os06g33360 and LOC＿Os06g31280）were dentified based on the results of GWAS analysis of RBSDV resistance and BSA-seq analysis of SRBSDV resistance.4.OsASP6（LOC＿Os06g03120）,a candidate gene related to RBSDV resistance on chromosome 6identified by GWAS,encoding an aspartic protease precursor,was functionally analyzed.OsASP6 was located on the cell membrane and cytoplasm,and was induced by brassinosteroid and jasmonic acid.Knockout of OsASP6 in DDQ,ZH11 and ZS97 significantly increased the resistance to RBSDV and SRBSDV;over-expression of OsASP6 could significantly reduce the resistance to RBSDV and SRBSDV,indicating that OsASP6 has a negative regulatory role in the resistance of RBSDV and SRBSDV.5.OsNB6（LOC＿Os06g33360）and OsTN6（LOC＿Os06g31280）,two candidate genes of SRBSDV resistance on chromosome 6 identified by BSA-seq,were functionally analyzed.It was found that knockout materials of the two genes with ZH5 as the receptor significantly reduced resistance to RBSDV and SRBSDV,indicating that OsNB6 and OsTN6 have positive regulatory roles in the resistance of RBSDV and SRBSDV.These results provided resistant germplasm resources and new resistance-related genes and molecular marker information for improving the resistance to rice black-streaked dwarf disease.