Genome-wide Association Study Reavels Novel Loci For Soybean Mosaic Virus SC7 Resistance And Functional Study Of Rsc7-1

Soybean(Glycine max(L.)Merr.)is an important oil crop which contains abundant protein and oil.The soybean mosaic virus(SMV)affects the growth of soybean and sharply reduces the yield.All main soybean production areas in China are affected by different SMV strains.SC7 is a prevalent strain in Huang-Huai and Yangtze River basin.Through screening resistant germplasm resources,using genetic methods to investigate resistant loci,studying the function of resistant genes and exploring resistant mechanisms that will provide a theoretical basis for breeding resistant varieties.SMV resistance can be divided into two types.Resistance to SMV infection is a qualitative trait that is controlled by a pair of single dominant genes and confer complete resistance to some SMV strains but susceptibility to others.The damage of the prevalent SMV strains in soybean production areas can be effectively reduced by using this resistant type,but the resistance will be lost due to the variation of the strains.The other types,resistance in development to SMV is a quantitative trait controlled by one additive maj or gene plus multiple genes in soybean.This is a broad spectrum and durable resistance and can achieve satisfactory yield under outbreak of disease.In order to map quantitative trait loci(QTLs)for SMV resistance,a Genome-Wide Association Study(GWAS)was performed to a diverse panel of 165 soybean mutants and 219 soybean landraces genotyped with the 355 K SoySNP array and phenotyped with disease rates(DR)to SMV strain of SC7 in multiple environments.The aim of this study is to identify candidate genes and haplotypes significantly associated with resistance in development to SC7.The main results are as follows:1.A soybean mutant panel through compound chemical(ethyl methanesulfonate,EMS)and physical(60Coy)mutagenesis was used in this study.A genome-wide association analysis of disease rates to SC7 in four environments were conducted.The analysis revealed abundant phenotypic diversity in this mutant panel.It can be clustered into two major subpopulations,Subpopulation I contains 93 accessions including the parent Nannong 86-4.And Subpopulation ? contains 72 accessions including the parent Nannong 94-16.The linkage disequilibrium decay was approximately 1 Mb.Using a general linear model,104 SNPs significantly associated with DR to SMV were identified.Among them,52 SNPs were repetitively detected in both E3 and BLUP on chromosome 12,the rest SNPs were only detected in a single environment.These novel SNPs located in or near biotic stress QTLs that were previously reported.Our results will provide insights for improving marker-assisted selection and molecular designed breeding strategies for new accession with resistance to SMV.Here,we selected two resistance and two susceptible mutants for SMV strain of SC7 from a soybean mutant population compound induced by EMS and 60Coy.These four soybean mutants and their wild type(WT)parent Nannong 86-4 were re-sequenced to identify mutations by using the Illumina HiSeq 4000 sequencer.With an average 95.95%genome coverage and an average 27.89×sequencing depth for each mutant.Through intergenomic comparison analysis,we identified abundant single nucleotide polymorphisms(SNPs),small insertions or deletions(Indels),copy number variations(CNVs)and structural variations(SVs)between mutants and their WT parent.The average SNP,indel,CNV and SV mutation density was 3.3 kb,51.4 kb,185.2 kb and 1.8 Mb respectively.Based on Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis,some putative genes were significantly enriched in defense relative categories or pathways.Our work will be helpful for understanding of genomic variation induced by EMS and 60Coy.Moreover,different variable genes between resistance and susceptible mutants were valuable resource to facilitate functional study of resistance genes to SC7.2.We evaluated a group of 219 soybean landraces in four environments for detecting the SNPs significantly associated with resistance to SC7 via GWAS.The results showed there was abundant phenotypic diversity in this population.The variable coefficients were ranged from 31.43%to 56.25%and the broad-sense heritability was 88.73%indicating this trait can stable deliver to the progeny.Forty-three SNPs exceeding a significant threshold using a mix linear model in GWAS results.Among these 43 SNPs,there were 29 SNPs,9 SNPs and 5 SNPs were detected on chromosome 2,13 and 19,respectively.On chromosome 13,two SNPs were repetitively detected in 2 environments,the other 5 SNPs were only detected in a single environment.Five SNPs on chromosome 19 were also detected only once.In addition,four SNPs on chromosome 2 were repetitively detected in all four environments and BLUP.The phenotypic variation explained by these 4 SNPs were ranged from 23.34%to 40.24%,indicating these loci were major QTLs for controlling disease rates.Two of four SNPs were located in the 5'-untranslated region(5'-UTR)and coding exonic region of gene Rsc7-1,respectively.The 219 soybean accessions were divided into haplotype A and haplotype B based on these two polymorphisms.Haplotype A contained 160 accessions with an average disease rate 0.79,while haplotype B contained 59 accessions had an average disease rate 0.45.An ANOVA analysis showed that the difference of disease rates between haplotype A and B reached an extreme significant level.Therefore,Rsc7-1 is a candidate gene for subsequent functional study.3.To further explore the function of Rsc7-1,a Rsc7-1 overexpression or silence vector was constructed and transformed into soybean root hairy system.Then we analyzed the expression levels of defense-related marker gene GmPR1 in transgenic hairy roots.In contrast with the vector control lines,the transcripts of GmPR1 were significant up-regulated in OE-Rsc7-1 lines,while significant decreased in RNAi-Rsc7-1 lines,suggested Rsc7-1 was involving in positively regulate defense response.The results of ROS level measurements indicated that the OE-Rsc7-1 transgenic hairy roots were stained deeper color than vector controls.Bean pod mottle virus(BPMV)based gene silenced Rsc7-1 showed more SMV accumulation levels than control plants at 14,21 days past inoculation.Meanwhile,the expression of defense-related marker gene Pathogenesis-related 1(GmPR1)was significant lower than control plants.The ROS levels in Rsc7-1-silenced plants was also lower than that in control plants.These results suggested Rsc7-1 playing a positive regulated role in immunity and ROS burst.Furthermore,a total of 11 SNPs and an indel were identified with minor allele frequency? 5%by sequencing Rsc7-1 full lengthes in 38 soybean accessions.We observed 4 haplotypes based on these 12 polymorphisms,haplotype 1 was the best haplotype with significant lower disease rates than other three haplotypes.The identification of Rsc7-1 resistant haplotype will facilitates SMV resistant breeding program.