Cloning Of Tomato Bacterial Wilt Resistance Gene And Development Of High-throughput Molecular Markers

The bacterial wilt（BW）,which is caused by Raslstonia solanacearum is a severe crop disease worldwide.The host of R.solanacearum are extremely broad,cover various types of crop such as potato,tomato,tobacco,eggplant and pepper.The research on identification of BW resistance genes has no measurable progress,beside that resistant genes have been cloned in the model plant Arabidopsis thaliana ten years ago.In our study,we constructed a F₂ population by the BW resistant parent HG64 and susceptible parent HG70 and a Genome-wide association study（GWAS）population contained 26 resistant cultivars for cloning the BW resistance gene.Firstly,by the Bulked Segregant Analysis（BSA）on F₂ population,we found that the locus for HG64 BW resistance was same with Bwr12 which had identified before.The results of GWAS showed that there was a major loci on chromosome 12,and a LRR-RLP gene cluster was present near the lead SNP.Then we analyzed a set of published transcriptome data about BW response in resistant and susceptible lines,we found that an LRR-RLP gene located in the Bwr12 region exhibited different expression pattern between resistant and susceptible lines,and this gene is one copy of the LRR-RLP cluster near the GWAS lead SNPs.We further verified this different expression pattern by q PCR.Finally,we conducted the transgenic complementation through knock out this gene in HG64 and overexpressed it in susceptible line.The knockout HG64 lines became susceptible and the resistance of overexpression lines were improved,these results indicated that the LRR-RLP gene responsible for the resistance underlies Bwr12.We firstly identified the BW resistance gene in crops,our results laid the foundation for the research work about molecular mechanism of crop BW resistance,and also promoted the application of molecular breeding for BW resistance.Molecular marker-assisted selection（MAS）is an important part of breeding work.However,the through-out level and accuracy of molecular markers have been plaguing breeder.Next Generation Sequencing（NGS）is a popular technology in the field of high-throughput sequencing in the past decade.A mount of resequencing data and different sequencing strategies were produced based on NGS,these provided new choices for molecular marker development.Based on the NGS method and resequencing data,we developed a high-throughput molecular marker detected method and improved marker specificity.We chose the 31 tomato agronomic trait loci as genotyping targets and then test this method with 2565 samples.90%of the samples detected more than 27 loci.We also used the resequencing data of tomato natural population for Ph-3 and Bwr6 marker development,the newly developed Ph-3and Bwr6 markers exihibted high specifity.Our result exhibited the huge benefit of NGS based method and resequencing data in marker development.