Fine-mapping Of The Resistance Genes Against Soybean Mosaic Disease And Antiviral Validation Of The 3gG2 And 5gG3 Transgenic Soybean Plants

Induced by Soybean mosaic virus(SMV),the soybean mosaic disease is a serious threat to the soybean production in all the soybean-growing regions of the world.However,one of our recent studies revealed that Bean common mosaic virus(BCMV),which is closely related to SMV,often induced the mosaic symptoms in many soybeangrowing fields of south China.After inoculating with four soybean-infecting BCMV isolates,some soybean germplasms(such as PI 96983,Marshall,Ogden and York)which have been reported to harbor Rsv1 alleles resistant to multiple SMV strains,showed susceptible phenotypes.Whereas the cultivars Suweon 97 and Raiden(known to harbor SMV-resistance genes Rsv1-h and Rsv1-r,respectively)were able to resist all the four tested BCMV isolates.In order to investigate the inheritance pattern of the BCMV resistance and finemap the resistance gene in soybean,a BCMV-resistance cultivar Suweon 97 was firstly crossed with the susceptible cultivar Williams 82 to prepare the mapping population in this study.After inoculating 220 F₂ individuals with the BCMV isolate HZZB011,a 3:1(3 Resistant /1 Susceptible)segregation ratio was observed,suggesting that Suweon 97 possesses a single dominant gene against BCMV.A total of 951 simple sequence repeat(SSR)markers across the whole genome were then screened and 186 markers showed polymorphism between the two parents.By performing bulked segregation analysis(BSA)with polymorphic markers,the resistance gene was determined to be linked with the marker BARCSOYSSR?13?1109 on chromosome 13.By further determining genotypes of 220 F₂ individuals for nearby SSR markers,the BCMV-resistance gene was then defined between markers BARCSOYSSR?13?1109 and BARCSOYSSR?13?1122.Furthermore,14 F_2:3 lines(derived from a larger population established earlier,consisting of 1150 F₂ individuals)showing recombination between the two markers were assayed for their phenotypes upon HZZB011 inoculation.With new single nucleotide polymorphism(SNP)markers further developed,the resistance gene was finally mapped to a 58.1 kb interval flanked by BARCSOYSSR?13?1114 and SNP-49.According to the reference genome sequence of Williams 82,a CC-NBS-LRR(CNL)-type resistant gene Glyma.13g184800 was annotated in this interval,representing the most likely candidate conferring the BCMV resistance.Remarkably,the SMV resistance gene Rsv1-h was previously mapped(by inoculating with SMV strains SC6-N and SC7-N)to almost the same region,thereby raising a possibility that Suweon 97 likely relies on the same gene to resist against both SMV and BCMV.To verify the above hypothesis and further determine the relationship between the BCMV-and SMV-resistance genes in soybean,the F₂ and F_2:3 populations derived from a cross between Raiden and Williams 82 were used to fine map the SMV-and BCMVresistance genes.The 3R:1S segregation ratios from the 140 F₂ individuals inoculated with BCMV-HZZB011 as well as 51 F₂ plants inoculated with SMV-SC6-N supported that either the BCMV resistance or the SMV resistance in Raiden is controlled by a single dominant gene,respectively.By performing BSA with 197 polymorphic SSR markers across the whole genome,the BCMV-resistance gene was identified to be linked with SSR markers BARCSOYSSR?13?1103 and BARCSOYSSR?13?1169,near the Rsv1 locus on chromosome 13.Later,12 nearby markers were used to examine the genotypes of 140 F₂ individuals,and the HZZB011 resistance gene was mapped to the region between markers BARCSOYSSR?13?1084 and BARCSOYSSR?13?1115.Also,the same 12 SSR markers(across the Rsv1 complex locus)were used to identify the genotypes of 51 F₂ individuals inoculated with SMV-SC6-N,and the SMVresistance gene in Raiden(an Rsv1-r name was assigned in previous studies)was then delimited between markers BARCSOYSSR?13?1075 and BARCSOYSSR?13?1161.Subsequently,the markers BARCSOYSSR?13?1075 and BARCSOYSSR?13?1161 were used to screen a larger population(consisting of 1009 F₂ individuals).A total of 70 individuals,showing recombination between the two markers,were obtained and then classified into 16 genotype patterns.For each genotype,one F_2:3 line was randomly selected and inoculated by either HZZB011 or SC6-N.With the help of new SNP markers,both the BCMV-resistance gene and Rsv1-r were mapped to the same 154.5 kb region between markers SNP-38 and SNP-50.To test the antiviral function for gene(s)in this interval,we further used BCMV-DXH015 and SMV-SC7-N strains to inoculate six recombinant F_2:3 lines,and the results were consistent with previous data(inoculated with BCMV-HZZB011 and SMV-SC6-N),suggesting that one or more functional resistance genes in the mapped interval(between SNP-38 and SNP-50)are able to confer resistances to multiple SMV and BCMV strains.According to the reference genome sequence,two CNL-type genes Glyma.13g184800 and Glyma.13g184900 are located in this region.After amplifying these two genes from Raiden and comparing to their alleles in Williams 82,we found two CNL genes exhibited significant divergences between Raiden and Williams 82,and their evolution has been affected by positive selections(with higher nonsynonymous substitution rate than synonymous substitution rate detected,Dn > Ds).Previously in Suweon 97,Glyma.13g184900 was excluded from the mapping regions resistant to SMV and BCMV.After sequence analysis of Glyma.13g184800 and Glyma.13g184900 genes cloned from 12 soybean germplasms,it was further found that the Raiden gene Glyma.13g184800 shares a 100% identity with its allele only in Suweon 97.While,Glyma.13g184900 has the same gene sequence with the alleles in Suweon 97,PI 96983 and York.Since the SMV-resistance genes in PI 96983 and York(Rsv1and Rsv1-y)have been mapped to other regions in the Rsv1 complex locus and two cultivars also showed no resistances to any tested BCMV isolates including the HZZB011 and DXH015,it can be inferred that Raiden gene Glyma.13g184900 is unlikely to be a resistant candidate.Therefore,gene Glyma.13g184800 represents a strong candidate gene conferring resistances to both BCMV and SMV.Considering the previous mapping results in Suweon 97,we found that the resistant genes in Suweon 97 and Raiden,which both confer a broad-spectrum resistance to SMV and BCMV,were mapped to an overlapped region,with Glyma.13g184800 predicted as the most promising candidate(functional validation experiments are required next).Transgenic validation experiment is a critical step in the identification of the functional genes.Within the Rsv1 complex locus,several functional SMV-resistance genes have been identified and some candidate genes were proposed,according to previous and our studies.However,functional validations of these genes via transgenic experiments have not been reported yet in the research of soybean resistance to mosaic disease.In this study,two genes 3gG2 and 5gG3 were amplified from the SMVresistance cultivar PI 96983 and further cloned into an overexpression vector p FGC5941.We then used the two recombinant plasmids to transform the susceptible cultivar Williams 82.Multiple 3gG2 and 5gG3 transgenic T0 plants were obtained successfully,and their T1 seeds were further harvested.For each gene,three T1 lines were planted and inoculated by SMV-SC6-N.When Williams 82 exhibited mosaic symptoms,all the 3gG2 and 5gG3 transgenic plants showed normal phenotypes and no viral RNAs could be detected from the leaves via RT-PCR.This represents the first transgenic work to verify the SMV-resistance function for candidate genes mapped to the Rsv1 complex locus.When inoculated with BCMV-HZZB011,mosaic leaves were detected from all the transgenic plants,indicating that neither 3gG2 nor 5gG3 can confer resistance to BCMV.To sum up,the BCMV-resistance gene was firstly identified and mapped in soybean in this study,and the target genes,mapped in two crosses of Suweon 97 × Williams 82 and Raiden × Williams 82,were located in the same candidate region as the SMV-resistance genes Rsv1-h and Rsv1-r,with gene Glyma.13g184800 regarded as functional gene conferring resistance to both BCMV and SMV.Besides,3gG2 and 5gG3 genes were introduced into Williams 82 by soybean transformation experiments.The transgenic lines were demonstrated to be resistant to SMV-SC6-N,but susceptible to BCMV-HZZB011,representing the first solid work of verifying the antiviral function of SMV-resistance genes on Rsv1 locus.This work makes an important foundation for further validating the antiviral function of other candidate genes and gradually exploring the evolution mechanism of the resistant genes within this complex locus.