Divergent DNA methylation contributes to duplicated gene evolution and chilling response in tea plants

Tea plant is a thermophilic cash crop and contains a highly duplicated and repeat-rich genome. It is still unclear how DNA methylation regulates the evolution of duplicated genes and chilling stress in tea plants. We therefore generated a single-base resolution DNA methylation map of tea plants under chilling stress. We found that compared with other plants, the tea plant genome is highly methylated in all three sequence contexts, including CG, CHG, and CHH (H = A, T, or C), which is further proved to be correlated with its repeat content and genome size. We show that DNA methylation in the gene body negatively regulates the gene expression of tea plants, while non-CG methylation in the flanking region enables a positive regulation of gene expression. We demonstrate that transposable element-mediated methylation dynamics significantly drives the expression divergence of duplicated genes in tea plants. DNA methylation and expression divergence of tea plant duplicated genes increase with their evolutionary ages and selective pressure. Besides, we detect thousands of differentially methylated genes, some of which are functionally associated with chilling stress. We also experimentally reveal that DNA methyltransferase genes of tea plants are significantly down-regulated, while demethylase genes are up-regulated at the initial stage of chilling stress, which is in line with the significant loss of DNA methylation of three well-known cold-responsive genes at their promoter and gene body regions. Overall, our findings underscore the importance of DNA methylation regulation and offer new insights into the duplicated gene evolution and chilling tolerance in tea plants.