Identification Of Genes Resistance To Gray Mold By Virus-Induced Gene Silencing In Tomato

Tomato has abundant nutrition,so more and more people like this vegetable.Then the cultivation area increasing rapidly,meanwhile,more and more disease appeared,such as gray mold.Among them Botrytis cinerea is a kind of typical necrotrophic pathogen,it can infect many plants,from seedling to adult plants,even in postharvest period,cause serious loss.So it is very important to understanding its molecular mechanism further.In this study,300 sequenced tomato materials were used to identify the phenotype after Botrytis cinerea infection.Identify the phenotypes of different tomato materials 4 days after Botrytis cinerea infection,then calculated the lesion area,further genome wide association analysis were performed by the data of average lesion area and SNP.We use virus-induced gene silencing to screen the candidate genes,use transgenic technology to further identify the function of resistance genes.The main results as follows:1.The phenotype of 300 sequenced tomato materials after Botrytis cinerea infection show that:the resistance is different for different materials we pick out the extreme resistance and susceptible materials.2.Genome wide association analysis were performed,the result showed that,some locations are the same and some of them is new compared with previous results.These sites are mainly located on the six chromosomes,nine loci that significantly associated with Botrytis cinerea.3.We identified some candidate genes using virus-induced gene silencing method.We found some genes resistance to gray mold.These resistance genes are belong to transcription factor、non-specific lipid transfer protein、protein kinase、cellulose synthase and so on.4.By detecting the expression of candidate gene in virus-induced plants,we discovered that the expression of these genes reduced about 40%-80%.5.The results of H₂O₂detection in situ,showed that a amount of H₂O₂accumulated after Botrytis cinerea infection,and H₂O₂gathered mainly on disease spots,this demonstrate that reactive oxygen species can accelerate Botrytis cinerea infection.6.Tomato materials were treated with exogenous SA、Me JA and ACC,candidate gene display different expression level.After treatment with SA,contrast with control,the expression level of Sl5110、Sl5190、Sl14760、Sl117910 elevated significantly.After treatment with Me JA,contrast with control,the expression level of Sl90340、Sl5190、Sl117910、Sl14760 these genes elevated.After treatment with ACC,contrast with control,the expression level of Sl5110、Sl90840 elevated at 24 h,and then rapid declined,other genes do not have significantly change.7.After 24 hours inoculation with Botrytis cinerea,we detecting the marker gene expression,we found that the expression of SA pathway marker gene Sl PR1display different expression level in different silenced plants,the expression level of TRV-90340 do not have significantly change contrast with control,the expression level of TRV-5450、TRV-5110 declined.But the expression level of TRV-90840 and TRV-5190 elevated.and the expression of JA pathway marker gene Sl PI-I、Sl PI-II decreased.This also demonstrated that these genes are related to disease resistance.In summary,through Genome wide association analysis,we found nine loci that significantly associated with Botrytis cinerea.We use virus-induced gene silencing to screen theses candidate genes of gray mold.Use exogenous SA、Me JA and ACC treated tomato materials,found candidate genes display different respond patterns,demonstrate that they maybe use different patterns to resistance pathogen infection.For resistance gene,after infection with Botrytis cinerea,there disease area increased,accumulated a large amount of H₂O_2,the expression level of JA marker gene declined.