MiRNA-mediated Changes in DNA Methylation Affect the Expression of Genes Involved in the Thickness of Pod Canopy Trait in Brassica Napus

Abstract

Background: Methylation plays an important role in regulating crop development, but little is known about how methylation regulates plant architecture in rapeseed (Brassica napus). Here, we examined how methylation affects the TPC (thickness of pod canopy) trait in rapeseed by performing genome-wide methylation analysis of two extreme TPC lines. Results: We detected significant differences in overall methylation levels between the high- and low-TPC lines in the CG, CHG, and CHH contexts in the promoters of genes in the stem apex and flower bud. In flower buds, 26 genes had significantly higher methylation levels in the high-TPC samples compared to the low-TPC samples, resulting in significantly reduced gene expression. By contrast, in the stem apex samples, the promoter regions of 22 genes were hypermethylated in the high- vs. low-TPC samples. The promoters of 19 and 21 genes had significantly reduced methylation levels in the flower bud and stem apex, respectively, of the high- vs. low-TPC samples, resulting in significantly higher expression levels. Some of these differentially expressed genes are associated with TPC-related traits, such as BnaC03g53050D (UBC32), BnaA05g26660D (CYSB), BnaA10g07880D (TCP 1), BnaAnng09670D (SMP1), BnaA09g02000D (SDH2-2), BnaC01g12960D (NRT1.8), and BnaC09g30490D (TAF15b). In addition, 14 important genes related to growth and development were differentially regulated between the two groups due to miRNA-mediated differences in methylation levels in their promoters. For example, hypermethylation in the promoter region of BnaCnng64040D (Lipase family protein) mediated by miR159a led to significantly reduced gene expression in flower buds of high-TPC vs. low-TPC lines. Conclusions: These results, together with our previously generated RNA-seq and miRNA profiling data, indicate that both methylation and miRNAs are involved in regulating the expression of genes in nitrogen-related metabolic pathways, thereby affecting the TPC trait in B. napus, providing a reference for uncovering the molecular mechanism regulating this crucial trait.