Long-read methylome profiling reveals context-specific epigenetic responses of Brassica napus to Verticillium longisporum

Canola ( Brassica napus L.) is a commercially important oilseed crop threatened by Verticillium longisporum , a soil-borne vascular fungal pathogen. While genetic resistance has been widely studied, the role of epigenetic regulation in plant responses to vascular fungal infection remains poorly understood, particularly in polyploid crop species. This study aimed to characterize genome-wide DNA methylation dynamics in B. napus following V. longisporum infection using Oxford Nanopore Technologies (ONT) sequencing. ONT sequencing enabled direct profiling of DNA methylation in infected and mock-inoculated plants. A total of 824, 267, and 19 differentially methylated windows (DMWs) were identified in CG, CHG, and CHH contexts, respectively. DMWs were distributed across both gene-associated and transposable element regions, with distinct context-specific patterns. Multiple DMWs overlapped genes associated with plant defense, including those involved in receptor-like kinase signaling, oxidative stress responses, calcium signaling, and transcriptional regulation. In addition, intergenic methylation changes proximal to defense-related genes identify candidate regulatory regions that warrant future functional validation. V. longisporum infection was associated with localized and context-dependent changes in DNA methylation in B. napus , with most variation occurring in CG and CHG contexts. The association of DMWs with defense-related genes suggests that DNA methylation may contribute to the regulation of plant responses during vascular fungal infection. These findings provide new insights into epigenetic responses in a complex polyploid crop and establish a foundation for future studies investigating the role of DNA methylation in plant disease responses.