Combined Bulked Segregant Analysis-Sequencing and Transcriptome Analysis to Identify Candidate Genes Associated with...

Cold tolerance in rapeseed is closely related to its growth, yield, and geographical distribution. However, the mechanisms underlying cold resistance in rapeseed remain unclear. This study aimed to explore cold resistance genes and provide new insights into the molecular mechanisms of cold resistance in rapeseed. Rapeseed M98 (cold-sensitive line) and D1 (cold-tolerant line) were used as parental lines. In their F₂ population, 30 seedlings with the lowest cold damage levels and 30 with the highest cold damage levels were selected to construct cold-tolerant and cold-sensitive pools, respectively. The two pools and parental lines were analyzed using bulk segregant sequencing (BSA-seq). The G'-value analysis indicated a single peak on Chromosome C09 as the candidate interval, which had a 2.59 Mb segment with 69 candidate genes. Combined time-course and weighted gene co-expression network analyses were performed at seven time points to reveal the genetic basis of the two-parent response to low temperatures. Twelve differentially expressed genes primarily involved in plant cold resistance were identified. Combined BSA-seq and transcriptome analysis revealed BnaC09G0354200ZS, BnaC09G0353200ZS, and BnaC09G0356600ZS as the candidate genes. Quantitative real-time PCR validation of the candidate genes was consistent with RNA-seq. This study facilitates the exploration of cold tolerance mechanisms in rapeseed.