Candidate Analysis Of Resistance Genes To Bean Common Mosaic Virus In Soybean Cultivar V94-5152 And Evolutionary Analysis Of Candidate Gene

Soybean is one of the most important economic and food crops in the world.It is also an important source of human fat and protein,and an important raw material for industries such as medicine and cosmetics.China is the world’s largest soybean consumer and also the largest soybean importer.Imported soybeans can reach 80% of the total demand.Therefore,to reduce soybean dependence on imports of China,it is very important to increase soybean yield in China.Soybean mosaic disease is one of the main diseases in soybean production,which has a serious impact on soybean varieties and yields in Northeast and North China.It has always been believed that Soybean mosaic virus（SMV）is the pathogen that causes soybean mosaic disease,but recent studies have found that Bean common mosaic virus（BCMV）can also cause soybean mosaic disease.Even in some soybean planting areas,the number of isolated BCMV exceeds SMV.Because its symptoms are very similar to soybean mosaic virus,it is easy to be ignored.The research on soybean BCMV-resistance candidate genes is of great significance.There are many natural anti-BCMV genes in soybean germplasm.In this study,the phenotype of soybean cultivar V94-5152 inoculated with BCMV-HZZB011 and the genotype results of 7 SSR markers were enabled us to draw a genetic linkage map,which indicated that the BCMV-resistance gene is located 0.2 c M downstream of BARCSOYSSR₀2₀617.According to the Williams 82 reference genome sequence in the positioning interval,five candidate genes upstream of the marker（Glyma.02g121300-Glyma.02g121700）and five downstream candidate genes（Glyma.02g121800-Glyma.02g122200）were amplified and sequenced.After sequence analysis,four genes Glyma.02g121400,Glyma.02g121600,Glyma.02g121900 and Glyma.02g122000 were found to have non-synonymous mutations or Indels between V94-5152 and Williams 82.Also,this location is near to the recently cloned SMV-resistance Rsv4 locus from the cultivar Peking.At the same time,Ishibashi et al.mapped and cloned the soybean Peking anti-SMV Rsv4 gene in 2019.This gene has two consecutively repeated ORFs（NM₀01249088 and NM₀01253944,Fig.1-2）in Enrei and Williams 82.There is a3.6 kb deletion in this segment in SMV resistant varieties,forming a recombinant ORF.To explore whether the BCMV-resistance gene is tightly associated with the Rsv4 gene,we further amplified the gene from multiple recombinant F2 individuals.The BCMV inoculation phenotypes of these recombinant F2 and F2:3 progenies were consistent with the genotypes of Rsv4 locus,raising a possibility that V94-5152 relies on Rsv4 gene to confer resistance against both SMV and BCMV.To further explore how and when Rsv4 resistances had evolved,the 10 newly obtained Rsv4 sequences from Kefeng No.1,Columbia and 8 wild soybean materials were analyzed together with 10 representative sequences reported by Ishibashi et al.In terms of insertion and deletions present in these sequences,five types of Rsv4 alleles could be classified.A new type of Rsv4 allele was detected in the wild soybean W58,which showed a 1150 bp-deletion covering the whole ORF1 gene.In such case,the ORF2 gene became the only gene remained at the Rsv4 locus.It seems more likely that the Enrei-or Williams 82-type allele represents an ancestral state,from which two kinds of deletion alleles were derived.Among 17 soybeans sharing the 3.6 kb deletion at the Rsv4 locus,the SMV-susceptible wild soybean PI 464925 showed an identical sequence to Williams 82_rsv4 except the 3.6 kb region.It therefore likely represents an ancestral state before resistances further evolved.To further explore how the Rsv4 resistance had evolved among soybeans,the 17 sequences sharing the 3.6 kb deletions were used to reconstruct an evolutionary tree.Since the wild soybean PI 464925（susceptible to SMV）shared an identical sequence to Williams 82 except the 3.6 kb deletion region,the PI 464925_rsv4 allele was proposed to represent an ancestral state before resistance emerged.It was evident that positive selections had been acting on this locus,with one critical amino acid change（R55P）shared by all resistance soybeans tested.Critically,one such substitution G164C（R55P）was shared by all eight resistance soybeans analyzed（Figure 4B）.Another substitution G199T（D67Y）was shared by Peking,Beeson,Columbia,Kefeng No.1 and V94-5152.The Beeson and Columbia further accumulated two substitutions C725T（P242L）and G787A（E263K）;while one substitution A574G（R192G）further occurred in V94-5152 and Kefeng No.1.A significantly higher value of Dn than Ds was revealed when comparing PI 464925_rsv4 allele with other Rsv4 alleles,indicating that positive selection had been acting and driving the divergence of these two alleles.