Fine Mapping And Molecular Characterization Of Resistance Genes Against Soybean Mosaic Virus In Soybean

Soybean mosaic virus(SMV)is a well-known devastating pathogen of soybean(Glycine max(L.)Merrill.)in the world,causing significant yield losses and seed quality deterioration.To prevent SMVs from causing disease,a widely adopted strategy is to cultivate and plant SMV-resistant cultivars.It therefore requires us to explore the inheritance patterns of SMV-resistance and further to uncover the functional genes that confer resistances to SMV in soybean cultivars.1.Fine mapping for the SMV resistance genes in PI 96983A cross between PI 96983 and Williams 82 was made to obtain a F2 population(a total number of 1020 individuals).Sixty-nine recombinant individuals were screened by using two polymorphic SSR markers(BARCSOYSSR₁ 3₁103 and BARCSOYSSR₁3₁187)that flank Rsv1 locus.31 groups(R1-R31)were classified based on the recombination site together with genotypes of SSR or SNP markers.According to the phenotypes of F2:3 individuals inoculated by SC7-N,RSC7-N gene was restrained to a 137.4 kb-region between SSR marker BARCSOYSSR₁3₁133 and SNP marker M23.This result was also validated by observed phenotypes of eleven F3:4 lines upon SMV inoculation.2.Fine mapping for the SMV resistance genes in Suweon 97Phenotypes of F2 populations from a cross of Suweon 97 and Williams 82 inoculated by two Chinese SMV strains(SC6-N and SC7-N infected 121 and 146 F2 individuals,respectively)demonstrated that SMV resistance in Suweon 97 was controlled by one single dominant gene locus(3R:1S,P>0.05).1150 F2 individuals were then screened for two SSR markers(BARCSOYSSR₁3₁103 and BARCSOYSSR₁3₁187)that flank the Rsv1 locus.Seventy-four recombinants were identified and 20 additional polymorphic SSR markers within the Rsvl region were then employed in genotyping these recombinants.Then,these recombinants were classified into three group including ?1103-1124,?1124-1140 and ?1140-1187.Next,F2:3 recombinant lines of three representative individuals in each group were inoculated with SC6-N and SC7-N to determine their phenotypes.The result indicateded that gene(s)conferred resistance to SC6-N and SC7-N were located between BARCSOYSSR₁3₁103 and BARCSOYSSR₁3₁124.To further narrow the above region,other F2:3 recombinant lines in group ?1103-1124 were also inoculated with SC6-N and SC7-N.Finally,Rsv1-h gene(s)in Suweon 97 that conferred resistance to SC6-N and SC7-N were constrained in an interval of 97.5 kb flanked by BARCSOYSSR₁3₁114 and BARCSOYSSR₁3₁115.The conclusion was also verified by F3:4 recombinant lines.Based on Williams 82 reference genome,eight genes were present in such region,of which two,Glyma13g184800 and Glyma13g184900,encoded CC-NBS-LRR type of resistance genes were considered potential candidates for Rsvl-h.3.Molecular characterization of NBS-LRR genes in the soybean Rsv3 locusBased on Williams 82 genome sequence,there were five NBS-LRR genes(named after NBS_A-E)on Rsv3 locus,being considered as the strongest candidates that confer resistance to SMV.However,sequences of the five genes on Rsv3 locus in Rsv3-possessing cultivars were not previously reported.Therefore,we constructed a BAC library for an Rsv3-possessing cultivar,Zaoshu 18,and screened out two positive BAC clones by utilizing SSR markers BARCSOYSSR₁4₁411-BARCSOYSSR₁4₁420.Then,the two clones were sequenced by PacBio RS ?sequencer.Compared all of annotated Zaoshu 18 genes with their Williams 82 counterparts,genes content and genes order of Zaoshu 18 were similar to these of Williams 82 on the Rsv3 locus,but there were extraordinary differences of NBS C and NBS D between Zaoshu 18 and Williams 82.Amplifying NBS_A-E genes from eight additional cultivars revealed that the NBS_A-D genes diverged into two different alleles of which nbs A-D alleles were associated with the rsv3-possessing cultivars,whereas the NBS_A-D alleles were associated with the Rsv3-possessing cultivars.For the NBS_E gene,the cultivar Columbia possessed an allele(NBS_E)that differed from that in Zaoshu 18 and rsv3-possessing cultivars(nbs_E).Furtherly,recombination analysis on alleles of the NBS_C-E genes demonstrated that recombination was a major force responsible for allele divergence.Meanwhile,the LRR domains of the NBS_C-E genes experienced strong positive selection pressure.In total,our results elucidated that not only NBS C but also NBS_D and Columbia NBS_E are most likely functional alleles confering resistance to SMV.