@article {Pisupati2022.12.04.519028, author = {Rahul Pisupati and Viktoria Nizhynska and Almudena Moll{\'a} Morales and Magnus Nordborg}, title = {On the Causes of Gene-Body Methylation Variation in Arabidopsis thaliana}, elocation-id = {2022.12.04.519028}, year = {2022}, doi = {10.1101/2022.12.04.519028}, publisher = {Cold Spring Harbor Laboratory}, abstract = {Gene-body methylation (gbM) refers to sparse CG methylation of coding regions, in particular of evolutionarily conserved house-keeping genes. It is found in both plants and animals, but is directly and stably (epigenetically) inherited over multiple generations in the former. Studies in Arabidopsis thaliana have demonstrated that plants originating from different parts of the world exhibit large differences in gbM, which presumably reflects an epigenetic memory of ancestral genetic and/or environmental factors.Here we look for evidence of such factors in F2 plants resulting from a cross between a southern Swedish line with low gbM and a northern Swedish line with high gbM, grown at two different temperatures. Using bisulfite-sequencing data with nucleotide-level resolution on hundreds of individuals, we confirm that CG sites are either methylated (nearly 100\% methylation across sampled cells) or unmethylated (approximately 0\% methylation across sampled cells), and show that the higher level of gbM in the northern line is due to more sites being methylated. Furthermore, methylation variants almost always show Mendelian segregation, consistent with their being directly and stably inherited through meiosis.To explore how the differences between the parental lines could have arisen, we focused on somatic deviations from the inherited state, distinguishing between gains (relative to the inherited 0\% methylation) and losses (relative to the inherited 100\% methylation) at each site in the F2 generation. We demonstrate that deviations predominantly affect sites that differ between the parental lines, consistent with these sites being more mutable. Gains and losses behave very differently in terms of the genomic distribution, and are influenced by the local chromatin state. We find clear evidence for different trans-acting genetic polymorphism affecting both gains and losses, with those affecting gains showing strong environmental interactions (G{\texttimes}E). Direct effects of the environment were minimal.In conclusion, we show that genetic and environmental factors can change gbM at a cellular level, and hypothesize that these factors can also lead to trans-generational differences between individuals via the inclusion of such changes in the zygote.Author summary Gene-body methylation, the sparse CG methylation of house-keeping genes, is found in both plants and animals, but can be directly inherited in the former. Recently, we discovered that Arabidopsis thaliana originating from different geographic regions exhibit different patterns of gbM, presumably reflecting a trans-generational memory of genetic or environmental factors. Here we look for evidence of such factors using a genetic cross between two natural inbred lines: one with high, and one with low gbM. We confirm that methylation states are stably inherited, but also see large somatic deviations from the inherited state, in particular at sites that differ between the parental lines. We demonstrate that these deviations are affected by genetic variants in interaction with the environment, and hypothesize that geographic differences in gbM arise through the inclusion of such deviations in the zygote.Competing Interest StatementThe authors have declared no competing interest.}, URL = {https://www.biorxiv.org/content/early/2022/12/04/2022.12.04.519028}, eprint = {https://www.biorxiv.org/content/early/2022/12/04/2022.12.04.519028.full.pdf}, journal = {bioRxiv} }