Data from: BsRADseq: screening DNA methylation in natural populations of non-model species

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.

表观遗传修饰的发生速率预计远快于基因突变，这可能使隔离种群发生随机漂变分化；若种群面临不同的选择机制，则会出现定向分化。因此，我们可以预测，栖息于不同生境的个体之间，全基因组范围的表观遗传分化程度会较高。在此，我们介绍亚硫酸氢盐转化限制性酶切位点相关DNA测序（bisulfite-converted restriction site associated DNA sequencing，bsRADseq），一种可量化多个个体间DNA甲基化分化水平的方法。该简化基因组测序方法具有良好的灵活性，可按需调整检测的DNA序列范围。我们通过三类自然研究体系展示了该方法的适用性，每类体系均包含适应不同环境的种群：二倍体植物（麦瓶草属 *Heliosperma*，石竹科 Caryophyllaceae）、四倍体植物（红门兰属 *Dactylorhiza*，兰科 Orchidaceae）以及动物（三刺鱼 *Gasterosteus aculeatus*，刺鱼科 Gasterosteidae）。我们开发了一套稳健的生物信息学分析流程，整合了RAD位点组装、单核苷酸多态性（single nucleotide polymorphism，SNP）调用、亚硫酸氢盐转化读段比对以及DNA甲基化调用等工具，可在有无参考基因组的情况下分析bsRADseq测序数据。尤为关键的是，该方法可精准区分单核苷酸多态性与甲基化多态性（methylation polymorphism，SMPs）。尽管基因组不同位点的DNA甲基化频率差异悬殊，但我们发现，栖息于不同生态环境的个体之间，其甲基化谱却具有惊人的高度一致性，在麦瓶草属物种中表现尤为突出。这种组成型稳定性表明，DNA甲基化变异受到了显著的分子或发育约束作用。总而言之，通过将RADseq的灵活性与亚硫酸氢盐测序在DNA甲基化定量方面的精准性相结合，bsRADseq方法与我们的生物信息学流程为非模式生物的进化与生态相关全基因组表观遗传研究开辟了新的可能，且不受其基因组特征的限制。