| Soybean is one of the main grain,oil and feed crops.The demand for soybean consumption is rapidly increasing in China.However,the amounts of soybean production in China is far from meeting domestic demand and over 85%of total soybean consumption depended on import which largely threatened food security of China.Therefore,increasing soybean annual yield in China will definitely be helpful to alleviate the contradiction between soybean supplying and demanding in China.Soybean mosaic virus(SMV)is main causal pathogen of soybean mosaic disease which causing annually yield loss in soybean production and seriously affecting the appearance of soybean quality.Currently,there is no effective chemical method for controlling SMV disease,however,soybean varieties with different genotypes display significant differences in symptom responses and yield loss to SMV infection.Therefore,breeding resistant varieties to reduce yield loss caused by SMV disease is an economical and effective prevention and control strategy.In previously studies,it has been reported that two genetic mechanisms,resistance to initial infection(RI)and resistance to spread(RS)existed in soybean resistance process.Although,varieties with capacity of RI could protect soybean production from special strain causing yield loss,high selection pressure for virus survival will likely cause new dominant SMV strains occurring which able to overcome RI of current varieties.Whereas,varieties with capacity of RS can obtain a broad spectrum and persistence and subjected little or no yield loss.Unfortunately,the genetic mechanisms were mainly focuse on the RI,but few studies were focus on RS.In addition,as a newly discovered virus type in recent years,recombinant SMV is widely prevalent in many soybean producing areas in China.However,there are few studies on recombinant SMV,so it is necessary to carry out research on recombinant SMV.In order to address the inadequacy of previous studies on RI and RS,we are try to conduct following studies:(1)Fine mapping the candidate gene for RI though map-based cloning strategy by using stock genetic material with relative clearly genetic mechanism.(2)Examining the candidate genes which involving in resistance process by using qRT-PCR and VIGS et al.methods,to obtain RI gene with clearly function.(3)Constructing genetic population consist of F1?F2?F2:3 and RIL which derived from cross between RI and RS to determine the genetic mechanism of RI and RS in the population.Constructing a genomewide genetic map to determine the QTLs for RI and RS in RIL population.(4)To determine the casual symptoms relative to genes of RS by using near isogenic line(NIL).(5)Map-based cloning the candidate genes for RS.In brief,our study aims to enrich the theory of genetic mechanism for disease resistance to recombined SMV and expand the application of resistant gene resources in soybean MAS breeding and lay basics for cloning of resistant gene in soybean.The main results of our studies are as follows:1.Genetic analysis and allelic test for resistance genesSix reciprocal crosses were made to determine the genetic base and allele of resistance genes in PI 96983 and Qihuang No.1.The results showed that all the F1 plants derived from the six reciprocal crosses were resistance to HB-RS infection,while the respective segregation ratios in F2 and F2:3 plants fit the theoretical ratio of 3 resistance(R+N):1 susceptible(S)(P=0.245?0.931)and 1 resistance(R+N):2 segregation(R+N+S):1 susceptible(S).Meanwhile,none of the observed plants in F2 and F2:3 derived from the crosses of allelic test showed susceptible to SMV infection and neither mosaic nor related symptoms were observed in progenies of allelic test.All above results suggested that the resistance in PI 96983 and Qihuang No.1 were controlled by a single dominant gene or multi closely linked dominant genes,meanwhile,the resistance locus in PI 96983 and Qihuang No.1 were allelic or closely linked.In additional,some progenies containing resistance gene derived from PI 96983 showed necrosis,but not for that of Qihuang No.1,indicating that the resistance genes in PI 96983 and Qihuang No.1 were different from each other,which is hereby named resistance locus in Qihuang No.1 as Rsc-q.2.Fine mapping of the resistance gene to HB-RSRe-sequencing results of near isogenic lines(NIL-Q and NIL-N)derived from Qihuang No.1 and Nannong 1138-2 showed that 4014 genetic variation including SNP and Indel were determined between NIL-Q and NIL-N in genomic encoding region,among them,2626 of these variations which accounting for 65.4%of total variation were located on chromosome 13,furthermore,over 95%of variation on chromosome 13 were clustered in physical region of 28Mb?30Mb,suggested that the genetic background were high homozygosis.In addition,NIL-Q and NIL-N showed stable resistance and susceptible to SMV infection,respectively.Further,all sub-F1 plants showed resistance,the observation of sub-F2 and sub-F2:3 fitted for expected segregation ratios of 3 resistance:1 susceptible and 1 resistance:2 segregation:1 susceptible,suggested that Rsc-q locus was posited in physical interval of 28Mb?30Mb on chromosome 13.To fine mapping the resistance gene,Recombinant plants were screened from 4380 sub-F2 plants by using 8 SSR markers and finally 423 recombinant plants were screen out.The results of resistance identification for these recombinant plants suggested that all resistance,all susceptible or segregation were possible could be observed in F2:3 family with cross-over points between M1132 and M1136,strongly indicated that Rsc-q was located in interval of M1132-M1136 which being composed by approximately?200 kb and containing 18 candidate annotation genes;In other hand,dwarfing and shrinking symptoms possible be occurred in some recombinant plants with cross-over points between M1150 and M1155,suggested that there existed a new resistance locus(where,hereby,name as Rsc-dw)in candidate interval of M1150?M1155 which being composed by approximately?100 kb and containing only 5 candidate annotation genes.3.Analysis of biological information and expression pattern of candidate genesAccording to annotation file of whole soybean genome,the distribution of gene with resistance domain in soybean were analyzed and results revealed that there is a resistance gene cluster was located beside or in Rsc-q and Rsc-dw loci and each candidate interval of Rsc-q and Rsc-dw contained four annotated genes with disease-resistant domains.The analysis of phylogenetic tree revealed that the candidate genes of Rsc-q and Rsc-dw belonged to sub gene family of NB-ARC-LRR and TIR-NBS-LRR,respectively,suggested the differences domains of these candidate gene were closely related to the difference resistance.Meanwhile,the genetic variation analysis showed that a total of 658 SNP and 17 Indel,which accounting for 16.82%variation of whole genome between the NILs,were identified in 18 resistance gene flanked Rsc-q and Rsc-dw,and average number of variation for each gene was 37.5.Further,both transcriptome analysis revealed that Glyma13g25920,Glyma13g25950 and Glyma13g25970 were lower expressed in plants with mosaic symptoms than in that plants with symptomless.While Glyma13g26420 and Glymal3g26460 were lower expressed in dwarf plants than in that plants with symptomless.Taken together,all the results strongly suggested that the expression of Glyma13g25920,Glymal3g25950 and Glyma13g25970 have a high relation to mosaic symptom which speculated as regulators of resistance gene in Rsc-q,while Glyma13g26420 and Glymal3g26460 or one of them have a high relation to dwarf symptom which speculated as regulators of resistance gene in Rsc-dw.4.Functional confirmation of candidate genes by VIGSVIGS technology was used to determine the gene function.Eight silencing vectors were successful constructed for candidate gene of Rsc-q and Rsc-dw loci and some flanked genes.The results showed that simultaneously silence Glyma13g25920,Glyma13g25950,Glymal3g25970 and Glyma13g26000 or simultaneously silence Glyma13g25750 and Glyma13g25420 could cause typical viral symptoms related to mosaic.Whereas,simultaneously silence Glymal3g26420 and Glymal3g26460 could cause typical viral symptoms related to mosaic and dwarfing.All of above results suggested that all or some of these candidate genes were also involved in SMV resistance.As results of ELSA showed that after silenced the candidate genes,Glymal 3g26420 and Glymal 3g26460,of Rsc-dw,the virus concentration in PI 96983 and Qihuang 1 was more than 1.5 times higher than that in the positive control suggested that the resistance in PI 96983 and Qihuang No.1 were completely lost and they played important fundamental roles in disease resistance.After silenced the candidate genes,Glymal3g25920,Glyma13g25950 and Glymal3g25970,of Rsc-q,the virus concentration in PI 96983 and Qihuang 1 was more than 0.5 times higher than that in the positive control suggested that the resistance in PI 96983 and Qihuang No.1 were considerable lost and they played an important roles in SMV resistance.In addition,the resistance of PI 96983 and Qihuang No.1 were partial lost due to he virus concentration in PI 96983 and Qihuang No.1 was more than 2 times higher than that in the negative control when Glyma13g25750,Glymal3g25420,Glyma13g257800 and Glymal3g25724 were silenced,implying that these genes are also involved in resistance to SMV infection.In all,all above results strongly declare that the resistance in PI 96983 Qihuang No.1 was synergistic controlled by multiple genes.5.Genetic analysis and map cloning of RS genesTo determine the genetic mechanism and identify RS genes,F1,F2,F2:3 and RIL populations were contrasted by using two contrasting reactions genotype parents,Jidou 12(JD12)(RI)and HT(RS)and the genetic mechanism were evaluated by both RI and RS evaluation methods.After infection,all F1 plants showed resistance to SMV infection and the observation segregation ratios in F2 and F2:3 plants fit the theoretical ratio of 3 resistance:1 susceptible(P=0.12)and 1 resistance:2 segregation:1 susceptible,suggested that the resistance in JD12 was controlled by a single dominant gene.Meanwhile,according to the kurtosis(0.57)and skewness(-1.21)values calculated for three biological replicates,the distribution of the DGs for RILs varied slightly from the normal distribution,suggesting that RS to SMV was a quantitative trait.QTL analysis results revealed that three QTL were identified in RIL population.Among them,qRsmv-13 was a RI locus whereas qTsmv-2 and qTsmv-3 were RS loci.Meanwhile,the resistance loci,Rsvl and Rsv4,were located in the confidence interval of qRsmv-13 and qTsmv-2,respectively,we speculated that they may belonged to same locus,while qTsmv-3 was a newly identified resistance locus.Further,results derived from NIL,sub-F2:3 and F3:4 also indicated that that Tsmv2 could limit proliferation while Tsmv3 could relieve SMV related symptoms under same virus concentration in plants leaves.Further,qTsmv-3 was delimited to an interval of approximately 86 kb with a map-based cloning strategy.Only two of five candidate genes,Glyma.03G00550 and Glyma.03G00570,varied between the parents.Additionally,Glyma.03G00550,which is a multidrug and toxic compound extrusion transporter gene,is the likely candidate gene for qTsmv-3.Summary,in this study we determined functional gene of two RI gene loci,enriched the theory of the genetic mechanism of RS,and fine mapped and map-based cloned the candidate genes of RS.Our results provided a theoretical foundation for RS genetic breeding and the resources of resistant genes for future transgenic breeding which finally facilitate the soybean molecular breeding process of SMV resistance in China. |
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