Genome-wide analysis of DNA methylation and transcriptional changes associated with overwintering memory in Brassica rapa L. grown in the field

Winter rapeseed, the sole overwintering oilseed crop in northern China, emphasizes winter resilience, yet epigenetic regulatory mechanisms governing overwintering memory remain poorly understood. In this study, the root collar tissues from the robust cold-resistant variety Longyou-7 were sampled during the pre-winter period (S1), overwintering periods (S2–S5), and re-greening period (S6), to analyze overall genomic DNA methylation levels using high-performance liquid chromatography (HPLC). The result showed that DNA methylation level exceeded 80% in the S1 stage. Throughout the overwintering periods, methylation levels displayed a decreasing trend in S3, followed by an increase in S5, and a pronounced decrease in S6. Consequently, S1, S3, S5, and S6 periods were chosen for whole-genome bisulfite sequencing analyses to elucidate the overwintering memory mechanisms of Longyou-7. The result revealed that DNA methylation primarily occurs in the CG context in Longyou-7. However, methylation of mC sites is most prevalent in the CHH type, gradually decreasing during overwintering periods. Analysis of methylation patterns in specific genomic regions of Longyou-7 showed that the highest methylation levels in the intergenic region. Moreover, mC sites in repeats and transposon elements are distributed differently across the three contexts. Subsequently, differentially methylated regions and promoters of Longyou-7 were identified during various periods compared to the S1 stage, followed by joint analysis with transcriptome sequencing. Functional enrichment analysis highlighted the involvement of most overlapping genes in the MAPK signaling pathway, plant hormone signal transduction, and starch and sucrose metabolism pathways. Changes in candidate gene expression within these three pathways correlated closely with DNA methylation levels. Our findings underscored the critical role of DNA methylation in regulating the expression of overwintering memory genes in winter rapeseed. These results offer a comprehensive insights into the epigenetic regulatory mechanisms governing winter rapeseed's overwintering memory, while identified overwintering memory genes served as crucial genetic resources for multifaceted breeding of winter-resistant varieties.