Localization And Candidate Gene Prediction Of The Locus R_SC3Resistant To Soybean Mosaic Virus In Soybean

Soybean mosaic virus（SMV）disease is one of the most important factors that endanger the yield and quality of global soybean[Glycine max（L.）Merr].Soybean mosaic virus has the characteristics of many modes of transmission,rapid transmission speed,and wide host of viruses.It is difficult to prevent and control by chemical methods.Therefore,eliminating the source of infection and producing non-virus seeds is an effective way to control soybean mosaic virus.The virus transmission in the field is difficult to control.Cultivating and promoting disease-resistant varieties is an economic,effective and green method to prevent and control SMV from the source.The most important thing in cultivating disease-resistant varieties is to find resistance sites,and then select germplasm materials with disease resistance genes through molecular marker-assisted breeding and apply them to breeding work.Soybean mosaic virus SC3 strain is the dominant strain in the soybean producing areas of Huanghuaihai and Yangtze River Watershed in China.Kefeng No.1 is one of the few excellent disease-resistant varieties currently screened for differen soybean mosaic virus strains with broad-spectrum resistance.The genetic pattern of resistance of Kefeng No.1 to SC3 strain and its resistance site were studied to analyze the disease resistance of SMV.Mechanism and lay the foundation for cultivating new varieties of soybean disease resistance.In this study,427 recombinant inbred lines of Kefeng 1 and Nannong 1138-2 were initially located based on BSA-seq,and F1 and F2 were used to judge the genetic pattern of Kefeng 1 resistance to SC3.Linkage mapping was performed using 427 recombinant inbred lines.The candidate gene was screened based on the two localization results of BSA-seq,and the candidate gene was analyzed by SMV induction.The aim was to find the resistance gene of Kefeng No.1 to SC3 strain.The main results are as follows:1.Initial localization of RSC3 by Kefeng No.1 Based on BSA-seq 427 RILs of Kefeng 1 and Nannong 1138-2 were inoculated with SC3,and 20 lines with extreme resistance and extreme susceptible were selected.resistance-pools and susceptible-pools were constructed by equal amounts of mixed DNA,and whole genome resequencing was performed with the two parents pools.SNP detection and annotation were performed after the library sequencing quality and the reference genome coverage were qualified.By analyzing the SNP frequency（SNP-index）,the progeny-pool was screened and counted,and a total of 1,697,099 polymorphic marker sites were obtained.A total of 71,031 SNPs were obtained by screening two progeny SNP-index sites with significant differences across the genome.Further screening of the obtained SNPs,using a 95%confidence interval of the △（SNP-index）value as a criterion,selecting the genes with a stop loss,a stop gain,a non-synonymous mutation or an alternative splicing site in the exon region as candidate genes.Finally,1138 SNPs were obtained,which were unevenly distributed on 19 chromosomes,and the candidate genes are 505 genes in which these SNPs are located2.Genetic analysis and further localization of RSC3 to Kefeng No.1The F1,F2 and RILs with Kefeng No.1 and Nannong 1138-2 as parents were inoculated with SC3 for resistance identification.F1 all showed resistance to disease,and the chi-square test showed that the F2 separation ratio was in accordance with the 3:1 separation ratio,while in the 427 RILs family,the separation ratio was 1:1.It is proved that the resistance of Kefeng No.1 to SC3 is controlled by one dominant genes.The genetic map was constructed using 3683 SNPLDB markers in the RIL population,and the phenotypic data was used to genetically map of RSC3.Finally,Rsc3 was mapped to chromosome 2（LG D1b）,which was closely linked to Gm02_BLOCK₈658796₈849164 and Gm02_BLOCK₈465794₈561522.The physical distance between the two markers was 193 kb,and the physical position was 8465794 bp to 8658796 bp on chromosome 2.There are 19 genes in this interval.3.RSC3 candidate gene analysisThe above positioning results were integrated and combined with the gene annotation of the reference genome to identify 12 disease resistance candidate genes for verification.The expression of each gene after induction by SMV was detected by qRT-PCR,and only 5 of them were expressed normally in leaves.Glyma.02G123100 has an upward trend in both varieties after inoculation,which does not indicate that it is related to disease resistance.Both Glyma.02G100000 and Glyma.02G105900 encode a serine/threonine protein kinase gene and are up-regulated in resistant variety after infection by virus,which may be a resistance gene.he initial expression level of Glyma.02g097400 in Kefeng No.1 was lower than that of Nannong 1138-2,and it increased significantly after be induced in Kefeng No.1.Moreover,it encodes the LRR and S/TK structures,which belong to the conserved structure of the plant disease resistance gene（R gene）,which may be a disease resistance gene.Glyma.02G122700,although up-regulated in Kefeng No.1 after 24 hours of inoculation,does not encode any disease-resistant domain,and its relationship with disease resistance needs further study.