Genome-wide Association Study, Fine Mapping And Candidate Gene Analysis Of Rice Seedling Resistance To Srbsdv

Rice(Oryza sativa L.)is one of the most important crops in the world that feeds more than half population of the world.The outbreak of southern rice black-streaked dwarf virus(SRBSDV)spread by whitebacked planthopper caused great losses to rice production.The practice of rice production proves that it is the most cost-effective and environmental protection measures to control rice virus by exploring and utilizing new sources of resistance and cultivating resistant varieties.However,there are no genes or QTL have been reported related to the resistance to SRBSDV until now.In this study,we extensively examined the resistance in genome-wide association study(GWAS)based on genotyping SNPs variants across SRBSDV of Guangxi core germplasms rice landraces.At the same time,the new genes of resistance to SRBSDV from common wild rice were finely mapped,and the candidate genes were screened by gene expression analysis,which provided a theoretical basis for the further study of virus resistance genes and provided materials and tightly linked molecular markers for disease-resistant breeding.1.Genome-wide association study of resistance to SRBSDV in core germplasm of Guangxi rice landraces.Using artificial inoculation method,the resistance of the core germplasm of rice landraces in Guangxi was identified,and the results showed that there were significant differences in the resistance of SRBSDV of 419 Guangxi core germplasms rice landraces.The incidence variation of SRBSDV ranged from 6.11%to 100%,with an average of 84.80%.GWAS detected 5 QTLs related to SRBSDV resistance by using 211,818 SNPs,which can explain 7.84-17.80%of the phenotypic variation.Among all associated SNPs,a total of 221 candidate genes were detected,46 candidate genes were detected both in indica and full population.2.Resistance characteristics and genetic analysis of D4 to SRBSDV.D4 is a highly resistant to SRBSDV material with wild rice lineage.In this study,the resistance level of D4 to whitebacked planthopper were analyzed by displacement test and resistance test.The results showed that D4 had no displacement and no resistance to the virus vector whitebacked planthopper,indicating that the resistance of D4 to SRBSDV is antiviral rather than insect-resistant.At the same time,genetic analysis of SRBSDV resistance in the 998/D4 F2 generation was conducted using the artificial inoculation identification.The skewed normal distribution of the incidence of disease for SRBSDV indicated that the resistance of SRBSDV was controlled by major gene and minor genes.3.Use QTL-seq technology based on high-throughput sequencing to locate major QTL related to resistance of SRBSDV.According to the SRSBSDV disease as determined by artifcial inoculation identifcation of 357 F2:3 families,derived from the cross between Guanghui 998 and D4,50 individual strains of extreme resistance and susceptibility of F2 were selected to be mixed equal amounts of DNA to construct an anti-sensing pool,and re-sequencing with both parents.Using the SNP-index correlation analysis technique,major QTLs associated with resistance of SRBSDV were located within 1.40M on chromosome 9,with a physical distance of 16,300,001-17,700,OOlbp,the interval contains 218 genes,named qSRBSDV9.4.Identification and fine mapping of qSRBSDV9 for rice seedling resistance to SRBSDV.InDel markers were used for linkage genetic analysis of resistance site qSRBSDV9.Using interval mapping method,the QTL associated with southern rice black-streak dwarf virus resistance was detected on chromosome 9 between Indel7 and Indel40,which coincided with the location of qSRBSDV9 detected by QTL-seq.The LOD value of this region is 4.8,which can explain 34.6%of the phenotypic variation.According to the location interval of QTL-seq,InDel primers were designed to amplify the parents and isolate population individual plants.The InDel markers with polymorphism between parents were screened first,and then the allele lines were selected by using InDel markers with polymorphism between parents,twelve recombinant individuals were finally found.The marker differences between the recombined plants and the resistant and susceptible parents were compared.Finally,the major QTLs were located between ID32 and ID35(physical distance was 17,393,019-17,495,354bp)with 102.3 kb base on substitution mapping method,the interval only contains 21 candidate genes.5.Analysis of candidate genes for resistance of SRBSDVBy referring to the annotation information of the rice genome,the function of the genes in the fine mapping interval were annotated.By artificial inoculation of SRBSDV,21 candidate genes in the resistance site qSRBSDV9 were analyzed for expression levels,and the optimal candidate genes(LOC_Os09g28690 and LOC_Os09g28730)which were involved in the regulation of SRBSDV resistance were finally obtained.6.Development the linked molecular marker and breeding evaluation of qSRBSDV9The seedling resistance performance of SRBSDV in the BC3F3:4 population constructed by Guiyu 7/D4 was evaluated through natural inoculation?InDel markers ID32 and ID35 closely linked to qSRBSDV9 were used to test the antagonistic and sensory lines.The results show,both labeled bands are clear and stable,the genotypic and phenotypic apposition rate reached more than 90%.Using these two markers to screen near-isogenic lines containing qSRBSDV9,the disease resistance was significantly improved over the susceptible parents.Our research indicates the main QTL qSDBSDV9 and its linkage markers ID32 and ID35 of rice seedling resistance to SRBSDV can be used for molecular marker-assisted selection breeding.