Stable and temporally shifted DNA methylation differences between 14-year-old Norway spruce epitypes under common garden conditions.

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conclusionDynamic DNA methylation differences between epitypes throughout the annual cycle, circannual clocks affect methylation levels, stable methylation marks in the promoters of 30 candidate genes, embryo-adult transmission of methylation marks. DNA methylation can change DNA properties, affecting chromatin accessibility, gene expression, and phenotypic variation. In clonal Norway spruce, warmer (WE) versus cooler (CE) embryogenic conditions produce phenotypically different trees. This climatic memory, induced during embryogenesis, remains stable in the resulting epitype trees, and the epigenetically altered timing of bud phenology persists between WE and CE epitypes even after decades under common garden conditions. We examined DNA methylation patterns in 14-year-old epitypes throughout the annual developmental cycle. Using targeted bisulfite sequencing, we screened for differential DNA methylation over a 3000 bp region in 2744 genes related to the epigenetic machinery, circadian clock, and phenology. Clustering DNA methylation differences in the CG context clearly separated epitype trees, confirming epigenetic mark differences. Differences in methylation of cytosines in all contexts were highly dynamic and varied markedly among annual developmental stages, suggesting the existence of circannual clocks affecting methylation levels in the studied genomic regions. Most stable methylation marks were identified in CG contexts, fewer in CHG and none in CHH contexts, consistent with differences in inheritance among methylation contexts. We identified stable CG and CHG methylation marks in the promoter regions of 30 specific genes. Two ARGONAUTE genes and 4 other genes exhibited stable marks across all time points for CE or WE, and putative embryo-adult transmission for some genes. These findings indicate that DNA methylation marks maintained in genomic regions throughout the annual cycle may contribute to an induced epigenetic memory established in embryos and later manifested as phenologically different epitype trees.