Identification and functional analysis of a gene regulating tolerance to freezing in Brassica Napus L. seedlings using BSA-seq and RNA-seq

Rape (Brassica napus L.) is a valuable oil crop cultivated widely in the Yangtze River basin. However, the extremely low temperatures encountered in the area adversely affect Brassica napus overwintering, resulting in decreased yield. Therefore, this study examined the frost-resistant (FR) 7P76 line and the frost-susceptible (FS) Zhongshuang 11 line, as well as F2 generation crosses of the two varieties. Through bulk-sequencing analysis (BSA-seq), candidate genes associated with frost resistance in Brassica napus were identified within three intervals: the 0.02-Mb interval on chromosome C01_random, the 0.64-Mb interval on chromosome C07_random, and the 10.76-Mb interval on chromosome C07. These intervals encompass a total of 716 genes. Combined RNA-seq and BSA-seq analyses revealed that 43 genes to be associated, and BnaC07g47890D was identified as a candidate cold-resistance-associated gene in Brassica napus, based on changes in expression and the results of KEGG, KOG, and GO analyses. The function of BnaC07g47890D was investigated by the construction of an overexpression vector and transformation into Arabidopsis. Compared to wild-type plants, we found that transgenic plants exhibited less damage and a stronger recovery capacity under - 4 ℃ freezing stress. These transgenic plants had higher activities of CAT, POD, and APX, as well as increased proline content; with the activity of CAT showed the most significant difference, and relative electrical conductivity and MDA content were lower. Fluorescence quantitative analysis confirmed that BnaC07g47890D enhances plant cold tolerance by promoting catalase gene expression.