Validation Of Clubroot Resistance Gene Within PbBa8.1 Region And Application Of This Locus For Improving Disease Resistance In Brassica Napus

Rapeseed is the most important oil crop in China,with an annual planting area of more than 100 million mus.In recent years,the production of rapeseed in China has been seriously threatened by clubroot disease,and the area of this disease has been expanding rapidly.Currently,genetic resistance is considered as the most economical and effective approach for the control of clubroot.Therefore,screening novel disease-resistant resources,genetic mapping disease-resistant genes,and establishing of molecular marker-assisted selection systems are most urgently required.Based on the development of the first set of clubroot resistance cultivar,Huashuang 5R（including Pb Ba8.1 locus）in China,and the completion of the genome sequencing of the clubroot resistant turnip ECD04,this study was continued to fine map Pb Ba8.1 and to integrate all the resistance loci from different Brassica A genome resistance materials into the genome of ECD04,to clone the candidate resistance genes and verify their functions.The detailed results obtained are as follows:1.In this study Huashuang 5R clubroot resistance locus Pb Ba8.1 was mapped in the region of 1428kb on the A8 chromosome.First,Pb Ba8.1 was mapped in the 3Mb region of chromosome A8 through BSA sequencing,and then polymorphic markers were developed in this region.The recombinant plants were screened by genetic linkage analysis of BC₃F₃ segregation population,and then phenotype of recombinants was confirmed by inoculating the selfing progenies of them.Finally,Pb Ba8.1 was narrowed between marker 4346 and marker A08-52,corresponding to the region of 1428kb on chromosome A08 of ECD04 genome.2.The 28 clubroot resistance loci mapped in different materials of the Brassica A genome around the world were compared to the ECD04 genome and integrated into 15different loci.These integrated 15 loci are located on chromosomes A01,A02,A03,A06and A08,respectively.After sequence comparison and analysis,a total of 62 R candidate genes were predicted in the above regions,including 30 NLRs,24 RLKs and 8 RLPs.Then,the Pb Ba8.1 region was divided into three different loci and named as CRA8.1,CRA8.2 and CRA8.3,respectively.In the 1428kb mapped region,there were three TIR-NBS-LLR genes namely CRA8.1.4（Br A08039211E）,CRA8.1.5（Br A08039212E）and CRA8.2.4（Br A08039305E）.3.CRA8.2.4（Br A08039305E）was identified as the candidate resistance gene in the region by comparing the gene expression patterns and protein domains of three NLRs genes with susceptible homologous genes.An overexpression vector of this gene was constructed,the then transformated into susceptible rapeseed by Agrobacterium rhizogenes mediation.After inoculation,disease resistance investigation and foreign gene expressional level analysis,the results showed that there was a significant positive correlation between the expression level of this transgene and resistance capability,indicating that this gene has clubroot resistant function.4.The gene structures of homologous to CRA8.2.4 in several plants in the genus Brassica whose genomes have been sequenced（susceptible to clubroot）have been interrupted due to the insertion of transposons.The comparative analysis of the genomic sequences showed that genes homologous to CRA8.2.4 in the Chinese cabbage Chiffu（AA,2n=20）,Indian yellow-seed Sassoon（Z1;AA,2n=20）,and European Brassica napus Darmor（AACC,2n=38）and Brassica napus Zhongshuang 11 from China（ZS11;AACC,2n=38）have undergone structural mutations due to different transposon insertion.It is speculated that these mutations may be responsible for the loss function of resistance of the original disease resistance gene in these materials.5.The CR locus Pb Ba8.1 combined with molecular marker-assisted selection was successfully employed in improving of clubroot resistance in a recessive genic male sterile line（RGMS）of Brassica napus.Huashuang 5R was used as the donor plant to cross the excellent RGMS of Brassica napus,and then backcrossed 4 generations with restore line of RGMS and self-crossed 2 generations.During the whole process,the molecular markers closely linked to Pb Ba8.1,MS/ms and Rf/rf loci were used for genotyping,and finally clubroot resistant RGMS lines were successfully developed.This laid an important foundation for breeding of novel varieties of clubroot resistance.