Inheritance,Fine Mapping And Candidate Gene Analysis Of Resistance To Soybean Mosaic Virus Strains SC5 And SC20 In Soybean(Glycine Max(L.)Merr.)

Soybean[Glycine max?L.?Merr.],grown for its edible seed protein and oil,is often called the miracle crop because of its many uses.However,one of the major constrains in soybean production is viral pathogens.Among the various viral diseases,Soybean mosaic disease is reported to be the most destructive viral disease caused by Soybean mosaic virus?SMV?and is found in all over the world including China.Early infection by the virus gives the highest reduction in yield?50-80%?.Insecticidal sprays and cultural methods are not always effective.Although,the control of the aphid,the vector has not proved practical or reliable?a means of virus management?.Exploitation of host plant resistance by means of growing resistant varieties is the best method of controlling SMV.Soybean is a vital crop in China and represents the major economic crop to small hold farmers.Three major soybean producing regions in China are Northeast China,Huang-Huai Valleys,and Southern China.One of the major constraints hindered in these areas in soybean production is SMV.National centre for soybean improvement?NCSI?,China,has been actively working on SMV research for the past few decades.Totally,22 strains,?SC1-SC22?were identified from three major soybean producing regions,by different researchers,during the period from 1999-2013.NCSI collected soybean cultivars and landraces from China and other countries and they screened against the Chinese SMV strains as well as identified as resistant sources.Meanwhile,identification and analysis of resistant genes for Chinese SMV strains are currently in progress.Recently,China has faced considerable reductions in soybean seed yields due to moderate virulent SMV strains SC5 and SC20.Kefeng.No.l and Qihuang.No.1 cultivars are showing resistance to SC5 and SC20,respectively.However,the genetic and molecular basis of resistance is unknown.Therefore,it is necessary to explore a resistance study for SC5 and SC20.That could be utilized in SMV breeding programs for the production and deployment of cultivars containing enhanced SMV resistance.With this background knowledge,the objectives of this study were proposed as;i)Inheritance analysis and identification of the location of the gene conferring resistance to SC5 and SC20,ii)Identification of the candidate genes to the resistance genes of SC5 and SC20.1.INHERITANCE,FINE-MAPPING AND CANDIDATE GENE ANALYSIS OF THE GENE RESISTANTTO SMV STRAIN SC5To examine the inheritance of resistance in SC5,P₁,P₂,F₁,F₂ and RIL populations of the cross Kefeng.No.1?resistant,R?between Nannong1138-2?susceptible,S?were used.F₁ was the same to Kefeng.No.1,F₂ and RILs segregated in phenotypic ratios of 3R:1S and 1R:1S respectively.These results confirmed that a single dominant gene confers the Kefeng No.l resistance to SC5.Subsequently,we tracked the genomic region conferring the SC5 resistance.It was found to be Bin 352-Bin353 of 500 Kb and is present on the Chromosome 2?LG Dlb?.The location was found out using 427 RILs and high density bin map composed of 4703 bin markers.The 500 Kb genomic regions contained thirty eight putative genes.To find out the'possible candidate genes' we utilized the whole genome resequencing?WGRS?data of 427 RILs SNPs information.Totally,271 SNPs are present in the 500kb genomic region.Of these 271 SNPs,107 SNPs were found on the twenty nine genes.Each gene contained one to sixteen SNPs.The remaining nine genes did not contain any SNPs.To know the association between 107 SNPs and the phenotype data of the 427 RILs,they were used to testing at cut off p<0.01 and p<0.001 significance level by chi square analysis.This showed that 60 SNPs were associated with SC5 resistance.These 60 SNPs were found on twenty one genes.Further,we used the concept that all the given SNPs and their corresponding genes are used to associate with the concerned trait.With this concept in mind;we have selected the eleven candidate genes.The functions of these genes are verified by qRT-PCR analysis.Out of the eleven genes,seven genes have functional implications after infection with SC5.Further,by combining the results of association and qRT-PCR analyses,we found that Glyma02g13495 is the 'most possible candidate gene'to SC5 resistance.2.INHERITANCE,FINE-MAPPING AND CANDIDATE GENE ANALYSIS OF THE GENE RESISTANT TO SMV STRAIN SC20The inheritance of SMV resistance in Qihuang.No.1 was determined by evaluating the resistance or susceptibility to SC20 in 20 Qihuang.No.1,Nannong 1138-2 plants,10 F₁,120 F₂ plants and 178 RILs resulting from a cross of Qihuang.No.1 and Nannong1138-2.Qihuang.No.1 and all F₁ plants were resistant to SC20 and Nannong 1138-2 was susceptible.Segregation of the F₂ and RIL population fit a 3R:1S and 1R:1S expected ratio.These results advocate that the SC20 resistance in Qihuang.No.l was controlled by a single dominant gene.Following this,a limited mapping strategy?with the support of SSR marker reported to be associated with SMV resistance?was used to effectively identify a gene associated with the SC20 resistance.The results indicated that resistance gene was positioned on chromosome 13?LG F?in an interval between markers BARCSOYSSR₁3₁099 and BARCSOYSSR₁3₁085.To narrow the region including the SC20 resistance gene,a larger segregating population,including 346 plants was adopted to further delimit the targeted gene.Totally 86 molecular markers?Genomic region between BARCSOYSSR₁3₁099 and BARCSOYSSR₁3₁185,where the SC20 resistance gene was probably located?were used to identify the exact location of SC20 resistance gene.The results indicated that resistance gene was delimited between two markers namely gm-ssr₁3-14 and gm-indel₁3-3 with genetic distances of 0.1 and 0.3 cM,respectively,which are at nucleotide positions,30795177and 30875000 respectively;the physical distance is 79 kb.This targeted region contained seven genes.Further,the expression profiling identified the two 'most likely candidate genes' Glyma.13G194700 and Glyma.13G195100 for SC20 resistance.Sequencing analysis of these two genes showed that two parental lines have different alleles and the allelic variations between Qihuang.No.1 and Nannongl 138-2 may account for the resistance to SC20 in Qihuang.No.l or for susceptibility to SC20 in Nannong 1138-2.In summary,the present study answers the question of understanding the response interms of genetic and molecular basis of resistance in Kefeng.No.1and Qihuang.No.1,to SC5 and SC20.Molecular markers linked to SC5 and SC20 resistance will facilitate marker assisted selection in breeding programs for resistance to SC5 and SC20.Besides,the identified candidate genes will be validated in the future by functional genomics approaches and might be transferred into soybean genotypes by marker assisted breeding or genetic transformation.These genes will help the development of superior genotypes/lines with enhanced resistance to SC5/SC20 that will eventually enhance crop productivity of soybean.