Mapping genetic modifiers of epimutation rates reveals a punctuated-equilibrium model of CG methylome evolution

Spontaneous epimutations—stochastic changes in cytosine methylation—can persist across generations in plants and are thought to contribute to phenotypic variation. Although epimutations are increasingly studied for their potential long-term effects, it remains unclear why their accumulation varies across genotypes. Here, we tracked DNA methylation across ten generations in ∼400 mutation accumulation lineages derived from ∼70 Arabidopsis Ler x Cvi recombinant inbred lines. Treating epimutation rates as quantitative molecular traits, we mapped a major QTL to a Cvi-derived deletion near VIM2 and VIM4 , two genes involved in CG methylation (mCG) maintenance. We show that this deletion rapidly reduces genome-wide methylation to a lower steady-state and compromises mCG maintenance fidelity across generations, resulting in a ∼1.5-fold increase in epimutation rates. Genotypes with elevated rates exhibited accelerated epigenetic drift and phenotypic divergence. Our findings support a punctuated-equilibrium model of mCG evolution, in which sudden disruptions to methylation homeostasis can destabilize epigenetic inheritance over longer time-scales.