Estación Biológica de Doñana, Consejo Superior de Investigaciones Científicas (CSIC), Sevilla, Spain.
Department of Terrestrial Ecology, Netherlands Institute of Ecology (NIOO-KNAW), Wageningen, The Netherlands.
PLoS One. 2023 Sep 8;18(9):e0291202. doi: 10.1371/journal.pone.0291202. eCollection 2023.
DNA cytosine methylation is an epigenetic mechanism involved in regulation of plant responses to biotic and abiotic stress and its ability to change can vary with the sequence context in which a cytosine appears (CpG, CHG, CHH, where H = Adenine, Thymine, Cytosine). Quantification of DNA methylation in model plant species is frequently addressed by Whole Genome Bisulfite Sequencing (WGBS), which requires a good-quality reference genome. Reduced Representation Bisulfite Sequencing (RRBS) is a cost-effective potential alternative for ecological research with limited genomic resources and large experimental designs. In this study, we provide for the first time a comprehensive comparison between the outputs of RRBS and WGBS to characterize DNA methylation changes in response to a given environmental factor. In particular, we used epiGBS (recently optimized RRBS) and WGBS to assess global and sequence-specific differential methylation after insect and artificial herbivory in clones of Populus nigra cv. 'italica'. We found that, after any of the two herbivory treatments, global methylation percentage increased in CHH, and the shift was detected as statistically significant only by epiGBS. As regards to loci-specific differential methylation induced by herbivory (cytosines in epiGBS and regions in WGBS), both techniques indicated the specificity of the response elicited by insect and artificial herbivory, together with higher frequency of hypo-methylation in CpG and hyper-methylation in CHH. Methylation changes were mainly found in gene bodies and intergenic regions when present at CpG and CHG and in transposable elements and intergenic regions at CHH context. Thus, epiGBS succeeded to characterize global, genome-wide methylation changes in response to herbivory in the Lombardy poplar. Our results support that epiGBS could be particularly useful in large experimental designs aimed to explore epigenetic changes of non-model plant species in response to multiple environmental factors.
DNA 胞嘧啶甲基化是一种参与调控植物对生物和非生物胁迫反应的表观遗传机制，其变化能力可能因胞嘧啶出现的序列上下文而异（CpG、CHG、CHH，其中 H=腺嘌呤、胸腺嘧啶、胞嘧啶）。模型植物物种的 DNA 甲基化定量通常通过全基因组亚硫酸氢盐测序（WGBS）来解决，这需要一个高质量的参考基因组。简化代表性亚硫酸氢盐测序（RRBS）是一种具有成本效益的替代方法，适用于具有有限基因组资源和大型实验设计的生态研究。在这项研究中，我们首次提供了 RRBS 和 WGBS 输出结果的全面比较，以表征对给定环境因素的 DNA 甲基化变化。特别是，我们使用 epiGBS（最近优化的 RRBS）和 WGBS 来评估昆虫和人工取食后黑杨无性系 Populus nigra cv. 'italica' 中全基因组和序列特异性差异甲基化。我们发现，在任何两种取食处理后，CHH 中的全甲基化百分比增加，这种变化仅通过 epiGBS 检测到具有统计学意义。关于取食诱导的特定基因座的差异甲基化（epiGBS 中的胞嘧啶和 WGBS 中的区域），两种技术都表明了昆虫和人工取食引发的反应的特异性，同时 CpG 中的低甲基化和 CHH 中的高甲基化频率更高。当存在于 CpG 和 CHG 时，甲基化变化主要发生在基因体和基因间区，而在 CHH 背景下则发生在转座元件和基因间区。因此，epiGBS 成功地描述了针对取食的隆巴第杨的全基因组甲基化变化。我们的结果支持 epiGBS 特别适用于旨在探索非模式植物物种对多种环境因素的反应的大型实验设计。
