Fine Mapping And Identification Of Soybean Resistance Genes R_SC3Q,R_SC7Qin Soybean

Soybean mosaic virus（SMV）disease is a worldwide disease in soybean[Glycine max（L.）Merr.],which results in yield loss and seed quality deficiency seriously.Utilization of resistant varieties is the most economical and environmentally safe method to ensure stable production,high-quality and high yield of soybean.In order to obtain more reliable disease resistance candidate genes for SMV-SC3 and SMV-SC7,which are popular strains in Huang-Huai and Yangtze River.On the basis of preliminary work,we carried on narrowing the regions.We take advantages of Quantitative Real-time PCR（QRT-PCR）echnology and some analysis such as transcriptome analysis,re-sequencing analysis to Screen candidate genes or resistant genes within the target regions.The main results were as follows:1、Fine mapping and identifying candidate genes of RSC3QIn this study we developed four SSR makers（S-37,S-93,S-110 and S-35）and four InDel makers（K26-2,25-57,G25-19 and G25-45）which were stable polymorphic between parents in the target region of RSC3Q.Combined screened recombinant plants with eight newly developed makers,we narrowed RSC3Qloci from the region approximately 651kbp to 180kbp flanked by maker S-110 BARCSOYSSR 13 1136 on chromosome 13.The number of predicted genes in this region was 17,and four of 17 genes were belong to NBS-LRR gene family and were positive response genes to biotic and abiotic stresses（Glyma13g25920,Glyma13g25950,Glyma13g25970 and Glyma13g26000）.Transcriptome data analysis showed that the expressions of these four genes were significant difference between resistant and susceptible families.Resequencing results showed that these four genes have more single nucleotide than other genes within the region between resistant and susceptible families.According to the above evidences,these four genes are more likely to be the genes resistant to SMV strain SC3.2、Cloning and bioinformatics analysis of soybean resistance candidate genes Rsc3QWe designed specific primers that in coding sequence of four candidate genes（Glyma13g25920、Glyma13g25950、Glyma13g25970 and Glyma13g26000）for PCR amplification.After sequence alignment and bioinformatics analysis,we found that there was no transmembrane region and no obvious hydrophobic domain in the proteins encoded by four genes.And there was no signal peptide sequence in proteins encoded by these four genes except for Glyma13g26000.Amino acid sequences analysis of the four candidate genes in Qihuang No.l indicated that four genes with higher similarity,even the similarity of Glyma13g25920 with Glyma13g26000 was 93.63%.Phylogenetic analysis based on the amino acids sequence showed that the proteins encoded by these genes had a dose relationship with Rsvl locus gene cluster which resisted to SMW,except for Glyma13g25950 had a close relationship with the reported R gene Rpg1-b（Pseudomonas syringae pv.glycinea）.3、Fine mapping and identifying candidate genes of Rsc7QCombining screened recombinant plants with four newly developed makers（S-16,S-42,K-20 and S-36）,we narrowed RSC7Q loci to the region about 250.6kbp flanked by maker S-16 and BARCSOYSSR₁3₁158 on chromosome 13.There were 17 genes predicted in this region,including five NBS-LRR genes and one PK（Protein kinase domain）gene which were positive response genes to biotic and abiotic stresses.QRT-PCR analysis was used to examine the expression pattern of six genes post SMV-SC7 inoculated.The results demonstrated that Glyma13g26310,Glyma13g26420 Glyma13g26460 and Glyma13g26530 exhibited significantly different expression pattern between resistant and susceptible plants.Resequencing results showed that these four genes have more single nucleotide than other genes within the region between resistant and susceptible families.It suggested these four genes were most likely related to disease resistance genes.At the same time,QRT-PCR analysis was used to examine the expression pattern of four key genes in plant hormone pathway post SMV-SC7 inoculated.It showed GmPR1 and GmNCED3 exhibited significantly different expression pattern between resistant and susceptible plants,and the expression peak of GmPRl and GmNCED3 in resistant plants was ten times and three times of control,respectively,but no difference in susceptible plants.Therefore we presumed that it was likely induced SA and ABA hormone pathway to resist SMV-SC7 in soybean,and SA was the major plant hormone pathway.