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Abstract
This study explores the stochastic gains and losses of DNA methylation (epimutations) at CG sites in plant genomes and their innovative application in evolutionary research. Plant CG epimutations occur at rates of approximately 10^(-4) to 10^(-3) per site per generation, four to five orders of magnitude faster than traditional DNA sequence mutations (~10^(-8)). These epimutations are stably inherited through mitotic and meiotic cell divisions and can display clock-like behavior.We identified and validated clock-like regions in plant methylomes. Our mathematical models successfully described how epimutations accumulate in plant genomes. We demonstrated the feasibility of an "evolutionary epigenetic clock" in both self-fertilizing (Arabidopsis thaliana) and clonal (Zostera marina) plant species, representing two major modes of plant reproduction. Results indicate that this clock is applicable for phylogenetic studies on a scale of years to centuries. Compared to SNP-based results, our approach produces less variation, providing unprecedented high-resolution temporal markers for recently diverged plant species or populations.
We developed MethPhylo, a comprehensive pipeline designed for phylogenetic analysis using single-base resolution DNA methylation data. It integrates the complete workflow from sequencing data processing to phylogenetic tree construction, addressing technical challenges in applying DNA methylation data to evolutionary research. Analysis of A. thaliana mutation accumulation lines using MethPhylo further confirmed the clock-like evolutionary patterns on gene-body-methylated genes.
To investigate the molecular mechanisms of epimutations, we conducted long-term (>15 generations) experiments with mutants of various RNA-directed DNA methylation (RdDM) pathway components (dcl3, nrpd1a, nrpd1b, rdr2), and with CHG methylation mutant cmt3 as well as ddc triple mutant (drm1 drm2 cmt3). Results showed that CG methylation levels in transposable elements decreased in these mutants, whereas methylation levels and change rates in gene-body-methylated genes remained relatively stable. This finding confirms that gene body CG methylation maintenance is relatively independent of the RdDM pathway and the ddc pathway, revealing differentiated epigenetic regulation mechanisms in different regions of plant genomes.
This research not only reveals new mechanisms of plant epigenetic dynamics but also pioneers the evolutionary epigenetic clock as a novel method for studying recent evolutionary events, opening new avenues for high-resolution temporal studies of plant biodiversity.