Genome-wide CG hypomethylation of the <i>Arabidopsis</i> ecotype Cvi linked to structural variation and RNAi at the <i>VIM4</i>-<i>VIM2</i> locus.

Abstract

The Cape Verde Island (Cvi)-0 ecotype, a relict lineage of <i>Arabidopsis thaliana</i>, is characterized by exceptionally low CG methylation levels genome-wide. Numerous population-level and molecular studies have highlighted links between this epigenomic feature, phenotypic traits and environmental factors; however, the molecular mechanism underlying Cvi-0's distinctive methylation profile remains unknown. Using multiomics comparisons between Cvi-0 and Col-0, we identified Cvi-specific small-RNAs derived from an inverted repeat (IR) formed by two inverted-orientation DNA methylation pathway genes, <i>VARIANT IN METHYLATION 2</i> and <i>4</i> (<i>VIM2</i> and <i>VIM4</i>). An intergenic 2.8-kb deletion brings these genes closer in Cvi-0, enabling read-through transcription and formation of chimeric transcripts. Targeted 5' RACE and WGBS showed that the resulting siRNAs mediate both posttranscriptional gene silencing and local DNA hypermethylation <i>in cis</i> and <i>trans,</i> reducing the expression of the <i>VIM1</i> gene, which is required for methyltransferase activity in <i>A.thaliana</i>. Comparative analysis across <i>A. thaliana</i> ecotypes and related species revealed substantial structural polymorphism at the <i>VIM4</i>-<i>VIM2</i> IR locus, likely shaped by nonallelic homologous recombination-facilitated by local palindromic structures, intronic repeats, and transposable element activity. Notably, the Cvi-0 <i>VIM4</i>-<i>VIM2</i> IR configuration was unique, suggesting it is a rare, lineage-specific natural variant. Taken together, natural variation at the <i>VIM4</i>-<i>VIM2</i> palindrome locus, uniquely found in Cvi-0, appears to be the causal variant driving siRNA biogenesis and down-regulation of the methyltransferase pathway. This distinctive regulatory mechanism is likely to have contributed to long-term reduction of CG methylation in Cvi-0, potentially maintained over millennia due to its geographical isolation from the mainland.