Epigenetic modifications are expected to occur at a much faster rate than genetic mutations, potentially causing isolated populations to stochastically drift apart, or if they are subjected to different selective regimes, to directionally diverge. A high level of genome-wide epigenetic divergence between individuals occupying distinct habitats is therefore predicted. Here, we introduce bisulfite-converted restriction site associated DNA sequencing (bsRADseq), an approach to quantify the level of DNA methylation differentiation across multiple individuals. This reduced representation method is flexible in the extent of DNA sequence interrogated. We showcase its applicability in three natural systems, each comprising individuals adapted to divergent environments: a diploid plant (Heliosperma, Caryophyllaceae), a tetraploid plant (Dactylorhiza, Orchidaceae) and an animal (Gasterosteusaculeatus, Gasterosteidae). We present a robust bioinformatic pipeline, combining tools for RAD locus assembly, SNP calling, bisulfite-converted read mapping and DNA methylation calling to analyse bsRADseq data with or without a reference genome. Importantly, our approach accurately distinguishes between SNPs and methylation polymorphism (SMPs). Although DNA methylation frequency between different positions of a genome varies widely, we find a surprisingly high consistency in the methylation profile between individuals thriving in divergent ecological conditions, particularly in Heliosperma. This constitutive stability points to significant molecular or developmental constraints acting on DNA methylation variation. Altogether, by combining the flexibility of RADseq with the accuracy of bisulfite sequencing in quantifying DNA methylation, the bsRADseq methodology and our bioinformatic pipeline open up the opportunity for genome-wide epigenetic investigations of evolutionary and ecological relevance in non-model species, independent of their genomic features.
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