Genetic Mapping And Cloning Of The Bacterial Wilt Resistance Genes In Tomato

Bacterial wilt is a worldwide devastating soil-borne disease caused by Ralstonia solanacearum E.F.Smith.The pathogen has a quite wide range of hosts and evolved into different races based on the different host ranges and geographical distributions.Therefore,it is very difficult to study the pathogenic and its resistant mechanism by traditional methods,since R.solanacearum has complex morphology,subspecies differentiation,pathogenicity and pathogenesis.With the development of genomic and transcriptome technologies,it has become possible to identify disease-resistance genes and study disease-resistance mechanisms at the genome-wide level.First,we established an accurate method for identification of bacterial wilt resistance tomato.Then,we hybridized the resistant inbred line’ZRS₇’and the susceptible inbred line’HTY₉’to construct F₂ isolation population;using BSA-Seq for genetic locating of bacterial wilt resistance genes and screening of candidate genes.The resistant genes were further identified by transcriptome.At last we constructed viral silencing vectors for preliminary functional validation of candidate genes.The main results are as follows:1.Different tomato lines and hybrid combinations were inoculated and identified by wounded root steeping method,and a pair of stable inbred lines with well-defined resistance/susceptiblity to bacterial wilt were screened,including resistant line’ZRS₇’and susceptible line’HTY₉’.Four different inoculation methods were compared using tomato susceptible line’HTY-9’.The results showed that all the four inoculation methods could cause disease in susceptible tomato,but pathogenicity rate of different inoculation methods were different.Stem injection inoculation>wounded root steeping>wounded root perfusion inoculation>non-wounded root inoculation.Among them,wounded root steeping method is the best identification method because of its simple operation and stable identification effect.2.The wounded root steeping method was used to identify the disease resistance of individual strains in the F₂ generation isolated population,and the selected extreme resistance pool and extreme susceptiblity pool were resequenced for SNP analysis together with the parents’ZRS₇’and’HTY₉’.A total of 136 Gbp clean reads were obtained.The average sequencing depth of the two parents was 20.1x,and the sequencing depth of the two mixed pools was 56.24x.A total of 1672851 SNP sites and 247412 InDel sites were detected.UsingΔSNPS-index method to calculate and three important intervals related to bacterial wilt resistance were detected,which located in No.2,7 and 12 chromosome.Physical distance of these intervals are 43 to52 Mb,59-60 Mb and 0-62 Mb.A total of 19 candidate genes were screened from the SNP analysis results.3.The transcriptome analysis of stems of’ZRS-7’and’HTY-9’which infected with R.solanacearum at 3 dpi（day（s）post infection,dpi）revealed that the disease resistance process of’ZRS-7’was extremely complex.GO analysis revealed that the pathways associated with disease resistance traits were mainly enriched in the molecular function category,which mainly involved proteins such as calmodulin,receptors and the binding function of cation and metal ion plasmas.KEGG analysis revealed that DEGs in resistant and susceptible plants were mainly enriched in the synthesis of secondary metabolites,phytopathogenic interactions,lignin synthesis and phenylalanine metabolism.Twelve of the 19 candidate genes screened by BSA-Seq analysis were differentially expressed in the transcriptome analysis,and four were significantly different.Using qRT-PCR further confirmed.The results showed that Solyc12g005080 and Solyc12g006380 were significantly upregulated 3 days after inoculation,while Solyc12g006990 was only significantly altered in susceptible parents.4.Two genes,Solyc12g005080 and Solyc12g006990,were silenced in the resistant plant’ZRS-7’using the transgenic virus silencing system.The Solyc12g005080 silenced plants showed obvious symptoms of infection.The results of GO annotation showed that Solyc12g005080 was involved in the synthesis of secondary metabolites which could slow down the propagation rate of the pathogen,and PAL was the key enzyme in the process of secondary metabolite synthesis.The results of physiological indexes showed that the PAL activity of Solyc12g005080silenced plants after inoculation was significantly lower than that of the control.In conclusion,it can be speculated that Solyc12g005080 may be involved in the synthesis of secondary metabolites related to disease resistance to limit the proliferation of R.solanacearum in plants and then help tomatoes to resist bacterial wilt.This gene plays a crucial role in plant disease resistance.Although the Solyc12g006990 gene-silenced plants did not show symptoms of disease susceptibility,the number of invading bacteria was increased compared with the control.This indicating that this gene also plays a role in the process of bacterial wilt resistance in tomato.