Fine Mapping And Identification Of Genes Underlying Soybean Resistance To Soybean Mosaic Virus

Soybean mosaic virus(SMV)seriously impairs the yield and seed quality on soybean[Glycine max(L.)Merr.].Totally,22 SMV strains(SC1-SC22)have been identified in China,and SC 15 and SC 18 are popular SMV strains in major soybean producing areas of southern and northeast China,and SC 15 can break the resistance of all 10 differential hosts,and cause serious threat to soybean production of China.Breeding of resistant cultivars is the most cost-effective and eco-friendly responsible approach for SMV management in soybean production,and genetic analysis of resistance genes is the theoretical basis of resistant cultivars.The availability of soybean reference whole genome sequence(WGS)has laid the basis for the development of new markers and identification of candidate genes.Thus,the objectives of the present study were to:(1)explore the inheritance patterns of soybean resistance to SMV strains and the allelic relationships among the resistant genes by making crosses between different soybean genotypes;(2)fine map the genes underlying the resistance to SC 15 and SC 18 using newly developed SSR markers;(3)identify the most likely disease-resistant candidate genes through prediction,sequence and expression analyses;(4)explore the likely involvement of H2O2,antioxidant enzymes(POD and CAT)and plant hormones in resistance mediated by Rsc15.The main results were as follows:1.Complete genome sequence analysis of different SMV strainsThe complete genome sequences of the four SMV strains(namely SC 10,SC 14,SC 15 and SC 18)were obtained,and the complete genomes of all the four strains were all 9529 nt in length.Each of them contains a single open reading frame(ORF)and the nucleotide sequence identities of the four SMV strains were very high.Alignment and phylogenetic analysis of the complete sequences of the four strains with 47 relevant Potyvirus strains and isolates revealed that the nucleotide sequence identities of the four SMV strains with Chinese strains(i.e.,SC3 and SC6)is higher than that of with SMV strains abroad.Meanwhile,the N-untranslated regions(N-UTR)and the first protein(P1)gene showed higher variability in the whole genome.2.Genetic analysis of resistancePopulations derived from Nannong 1138-2 × Kefeng No.l,Nannong 1138-2 x RN-9 and Qihuang No.1 × Nannong 1138-2 were inoculated with SC18.Results showed that all F1 plants were extremely resistance to SC18,indicating that resistance is dominantly inherited.Segregation of resistant(R)and susceptible(S)individuals fitted a 3:1 ratio in the F2 populations,and it fitted a 1:2:1 ratio in the segregation of R,Seg and S in the F2:3 lines.All these results suggested that Kefeng No.1,RN-9 and Qihuang No.l all possess one dominant SC 18 resistance gene.Populations derived from RN-9 × Kefeng No.1,Qihuang No.1 × Kefeng No.l and Qihuang No.1 × RN-9 were inoculated with SC 18.All F1 plants were extremely resistance to SC18,and segregation of R and S individuals fitted a 15:1 ratio in F2 populations,supporting the hypothesis that three independent dominant genes confer SC18 resistance in Kefeng No.1,RN-9 and Qihuang No.1.For investigating the inheritance of resistance to SMV strains with different virulence in RN-9,the F2 and recombinant inbred line(RIL)populations derived from RN-9 × 7605 were inoculated with the four strains(SC 10,SC 14,SC 15 and SC 18).It showed that all F1 plants were symptomless,while the susceptible parent 7605 plants developed a typical mosaic symptom.The segregation of all F2 populations fitted a good 3R:1S ratio(P>0.05),and that of all RILs(216 lines)showed a satisfactory fit of 1R:1S(P>0.05).All these results suggested that the resistance to SC10,SC14,SC15 and SC18 in RN-9 were all controlled by single dominant genes.The segregation ratios revealed that gene(s)confer resistance to the four strains in RN-9 were all single dominantly inherited.Totally,53 lines with phenotypic recombination events were screened out and the recombination value between each four resistance genes ranged from 6.48%to 12.50%,indicating that resistance genes for the four strains might be different.3.Fine mapping and identification of gene underlying resistance to SC15 in RN-9In this study,Rsc15 was located between Sat₂46 and Satt286 using the F2 and the RIL(216 lines)populations,which were consistent with previous results.In our study,resistance genes for SC 10 and SC 18 were both positioned between the two SSR markers Satt286 and Satt277,and the resistance genes for SC 14 and SC 15 were both located between Satt246 and Satt286.It suggested that gene(s)conferred the resistance to the four SMV strains might be clustered on chromosome 6.Using the RIL population and newly developed SSR markers,Rsc15 was fine mapped within the interval flanked by the two markers SSR₀6₁7 and BARCSOYSSR₀6₀835,at genetic distances of 2.38 cM and 1.66 cM,respectively.According to the WGS,five gene models were predicted in this 95-kb interval.None of 136 genes identified between Sat₂46 and Satt286 were found to belong to NBS-LRR family,while two genes encoded RLK and five genes encoded STK.For the five genes predicted in the 95-kb region,gene Glyma.06g182500 encodes a protein of unknown function;Glyma.06g182600(designated as GmPEX14)encodes a peroxisomal membrane anchor protein;Glyma.06g182700 encodes a predicted carbonic anhydrase involved in protection against oxidative damage;Glyma.06g182800 encodes a tetratricopeptide repeat(TPR)-like superfamily protein;and·Glyma.06g182900 encodes an STK.Allelic sequence comparison was conducted using the DNA/cDNA sequences from the two parents.The results showed that the coding sequences(CDS)of all the five candidate genes and the promoter sequences of three genes(Glyma.06g182700,Glyma.06g182800 and Glyma.06g182900)were conserved between the two parents,while a nucleotide insertion/deletion mutation(InDel;8 bp)and a single nucleotide polymorphism(SNP)site were detected in the promoter sequence of Glyma.06g182600.Moreover,the CDS of all three STK-encoding genes(Glyma.06gl 76800,Glyma.06g177700 and Glyma.06g183300)were conserved.In contrast,3 and 6 SNPs were identified in the CDS or promoter of Glyma.06g175100 and Glyma.06g183500,respectively.For another RLK-encoding gene,Glyma.06g184400,a single base pair deletion close to the initiation codon delayed transcription in 7605.Based on architectural features,these five genes,along with twelve other genes near Rsc15(encoding RLK,STK or disease resistance-related proteins),were further selected for expressio analysis using qRT-PCR when inoculated with SC 15.For the five genes located in the Rsc15 flanking region,only GmPEX14 was up-regulated in cultivar RN-9 from 1 to 12 hpi,but its expression was significantly suppressed in 7605.Other plant immunity-related genes near Rsc15 could,to some extent,be induced by SMV infection.The expression of candidate genes,including RLK-and STK-encoding genes and Glyma.06g182600,was further examined under various stress treatments,and all eight genes showed significant differences on average.And the expression of these genes in the leaves was generally higher than that of in other tissues(root,stem,flower and immature pod).All these results inferred that the gene GmPEX14 was the best candidate gene contributing to the resistance of Rsc15 and that genes encoding RLK/STK)(i.e.,Glyma.06g175100,Glyma.06g182900,Glyma.06g183500 and Glyma.06g184400)were also potential candidates.The detection of H2O2 and CAT and POD assays were performed after SC 15 inoculation,and results revealed that the levels of endogenous H2O2 in RN-9 were higher than those of in Nannong 1138-2,and they were significantly increased from 2 to 12 hpi in RN-9;The activity of CAT in Nannong 1138-2 was higher than that of in RN-9,and two peaks were observed at 2 and 24 hpi;However,no significant difference in POD activity was detected between Nannong 1138-2 and RN-9.Moreover,high correlations were established between the relative expression level of GmPEX14 and the H2O2 concentration,as well as the activities of CAT and POD,during the early stages(0-12 hpi)of SMV infection in RN-9.4.Fine-mapping and identification of SC18 resistance gene in Kefeng No.1Linkage analysis of 180 F2 individuals and 5 markers(viz.Satt558,Satt634,Satt266,Satt698 and Satt537)genotype data showed that Satt634 and Satt266 on chromosome 2(linkage group D1b)were genetically linked to the resistance locus in Kefeng No.1 with genetic distance of 5.5 cM and 15.3 cM,respectively.Two RIL populations and 35 polymorphic markers were screened out of 101 SSR markers at the target region were used for fine mapping.Finally,the resistant gene was delimited within the interval between the two markers BARCSOYSSR₀2₀667 and BARCSOYSSR₀2₀670.Based on the WGS(Glyma 2.0),the target region was approximately 80 kb in physical distance,containing 6 putative genes.Sequence analysis of resistant candidate genes between the two parents showed that gene Glyma.02G127800 was grouped with co-regulators of receptor kinase-mediated immunity and there are 13 SNP sites in the CDS resulting in four AA substitutions;there's no annotation for Glyma.02G127900;Glyma.02G128000(GmSAMDC1)encodes an S-adenosylmethionine decarboxylase(SAMDC)active protein,and is implicated in response to multiple-stress conditions(salt,drought and cold)in soybean,are all conserved in CDS sequences between the two parents..Glyma.02G128200 encodes a putative S-adenosyl-L-methionine-dependent methyltransferase(SAM-Mtase)protein,which kind of protein is the key enzymes in important biotechnological pathways(e.g.phenylpropanoid and flavonoid et al.),and the two parent shared 98.8%identity in CDS,and there were 10 AA substitutions located in the Mtase domain.Glyma.02G128300 encodes one CDP-alcohol phosphatidyltransferase active protein conferring plasma membrane biosynthesis,and there were 4 SNP resulting in one AA substitution in the CDP-APT domain.Expression analysis of SC18 resistance candidate genes were carried out by qRT-PCR.The gene Glyma.02G127800 was significantly up-regulated between 2 hpi and 9 hpi in Kefeng No.1;Glyma.02G128000(including Glyma.02G128100)was significantly up-regulated only at 2hpi in Nanong1138-2;Glyma.02G128200 showed an earlier response in the resistant parent than that of in the susceptible parent.Gene Glyma.02G128300 was significantly up-regulated at 1 hpi in both two parental plants,but the magnitude of the relative expression level in Nannong1138-2 was about 3 times lower than that of in Kefeng No.1.Sequence and expression analyses of these genes revealed that SMV resistance in Kefeng No.1 was probably attributable to three of the candidate genes(i.e.Glyma.02G127800,Glyma.02G128200 and Glyma.02G128300).The subcellular localization results showed that the fusion protein Glyma.02G127800-GFP was only located on the cell membrane;Glyma.02G128000-GFP was located on the cell membrane,cytoplasm and nucleus;Glyma.02G128300-GFP is localized on the cell membrane and cytoplasm,and all these results were consistent with its protein structure prediction.Collectively,the results of this study will greatly facilitate the cloning of SC 18 resistance genes and marker-assisted breeding of SMV-resistant soybean cultivars.