2022 ADF Project # 20190101 report: Cloning clubroot resistance genes from B. nigra and transferring the genes into canola through a CRISPR/Cas9 based technology
Citation
Yu F. (2022) ADF Project # 20190101 report: Cloning clubroot resistance genes from B. nigra and transferring the genes into canola through a CRISPR/Cas9 based technology.
Résumé en langage clair
Clubroot resistance gene Rcr6 was previously identified in three Brassica nigra resistant lines PI 219576, BRA and CR2716 in our group based on the B. nigra old reference genomes. In the first year of the project, the Rcr6 target region was searched in a recently published B. nigra reference genome (Bnigra_ONT_02) to identify corresponding region in the new genome, which was located into 1.2 Mb region of chromosome B3 including 7 disease resistance genes for the possible candidates of Rcr6. Short reads from bulked segregant RNA or DNA sequencing of BC1 populations derived from the three resistant lines were aligned to the new genome for developing SNP markers in the target region. As a result, ten polymorphic and robust SNP markers were developed for KASP analysis. To further fine mapping of Rcr6, a BC1S1 populations from BC1 resistant plants derived from CR2748 x (CR2716 x CR2748) was developed. A total of 2880 plants in the population were tested with clubroot pathotypes 3A and 5X respectively; and 746 susceptible plants were identified. A total of 10 recombinants were obtained through KASP analysis of the susceptible plants with the 10 SNP markers. Rcr6 was further defined into a 0.23 Mb region of chromosome B3 including only two predicted disease resistance genes, BniB03g015510.1C2 and BniB03g015520.1C2. These two genes will be used for cloning of Rcr6 and functional verification. Meanwhile, a CRISPR/Cas9-based vector system and streamlined workflow for cisgenic/intragenic plant breeding has been established and is ready to deliver functionally-verified Rcr6 into canola.
Résumé
Clubroot resistance gene Rcr6 was previously identified in three Brassica nigra resistant lines PI 219576, BRA and CR2716 in our group based on the B. nigra old reference genomes. In the first year of the project, the Rcr6 target region was searched in a recently published B. nigra reference genome (Bnigra_ONT_02) to identify corresponding region in the new genome, which was located into 1.2 Mb region of chromosome B3 including 7 disease resistance genes for the possible candidates of Rcr6. Short reads from bulked segregant RNA or DNA sequencing of BC1 populations derived from the three resistant lines were aligned to the new genome for developing SNP markers in the target region. As a result, ten polymorphic and robust SNP markers were developed for KASP analysis. To further fine mapping of Rcr6, a BC1S1 populations from BC1 resistant plants derived from CR2748 x (CR2716 x CR2748) was developed. A total of 2880 plants in the population were tested with clubroot pathotypes 3A and 5X respectively; and 746 susceptible plants were identified. A total of 10 recombinants were obtained through KASP analysis of the susceptible plants with the 10 SNP markers. Rcr6 was further defined into a 0.23 Mb region of chromosome B3 including only two predicted disease resistance genes, BniB03g015510.1C2 and BniB03g015520.1C2. These two genes will be used for cloning of Rcr6 and functional verification. Meanwhile, a CRISPR/Cas9-based vector system and streamlined workflow for cisgenic/intragenic plant breeding has been established and is ready to deliver functionally-verified Rcr6 into canola.