背景:DNA甲基化是影响植物生长的关键因素,适应性,和表型可塑性。虽然在模型和作物物种中进行了广泛的研究,在霍姆橡树和其他非驯化的林木中,它仍然相对未被开发。这项研究对holmoak基因组中的DNA甲基转移酶和去甲基酶基因进行了全面的计算机内挖掘,以增强我们对这些未被研究的物种中这一基本过程的理解。这些基因在成虫和幼苗叶片中的表达水平,以及胚胎,使用定量实时PCR(qRT-PCR)进行分析。通过甲基化敏感的扩增多态性(MSAP)技术评估了全球DNA甲基化模式。此外,通过MSAP测序(MSAP-Seq)鉴定特异性甲基化基因组序列.
结果:在holmoak基因组中发现了13种DNA甲基转移酶和3种脱甲基酶基因。这些基因的表达水平在器官和发育阶段之间显着变化。MSAP分析显示,表观遗传在器官和发育阶段的遗传变异中占主导地位,在成年叶片中观察到明显更高的全局DNA甲基化水平。胚胎表现出频繁的去甲基化事件,而幼苗叶片中普遍存在从头甲基化。MSAP-Seq鉴定的基因组序列中约有35%被甲基化,主要影响核基因和基因间区域,与重复序列和叶绿体基因相反。发现与它们的启动子和内含子区域相比,在核基因的外显子区域中甲基化更为明显。甲基化基因主要与关键的生物过程相关,如光合作用,ATP合成耦合电子传输,防御反应。
结论:这项研究通过评估基于DNA甲基化的表观遗传事件和机制,为分析橡树的变异性开辟了一个新的研究方向。它揭示了控制DNA(去)甲基化的酶机制,以及发育阶段不同器官中甲基化酶和去甲基化酶表达水平的变化。表达水平与观察到的DNA甲基化模式相关,显示幼苗和胚胎中从头甲基化和去甲基化事件的发生率,分别。参与调控转座因子沉默的几个甲基化基因,脂质生物合成,成长和发展,并强调了对生物和非生物胁迫的反应。MSAP-seq整合了全基因组亚硫酸氢盐测序和先进的测序技术,如PacBio或纳米孔,将阐明调节特定基因表达的表观遗传机制及其与表型变异性和对环境线索响应差异的相关性,尤其是与气候变化有关的。
BACKGROUND: DNA methylation is a critical factor influencing plant growth, adaptability, and phenotypic plasticity. While extensively studied in model and crop species, it remains relatively unexplored in holm oak and other non-domesticated forest trees. This study conducts a comprehensive in-silico mining of DNA methyltransferase and demethylase genes within the holm oak genome to enhance our understanding of this essential process in these understudied species. The expression levels of these genes in adult and seedling leaves, as well as embryos, were analysed using quantitative real-time PCR (qRT-PCR). Global DNA methylation patterns were assessed through methylation-sensitive amplified polymorphism (MSAP) techniques. Furthermore, specific methylated genomic sequences were identified via MSAP sequencing (MSAP-Seq).
RESULTS: A total of 13 DNA methyltransferase and three demethylase genes were revealed in the holm oak genome. Expression levels of these genes varied significantly between organs and developmental stages. MSAP analyses revealed a predominance of epigenetic over genetic variation among organs and developmental stages, with significantly higher global DNA methylation levels observed in adult leaves. Embryos exhibited frequent demethylation events, while de novo methylation was prevalent in seedling leaves. Approximately 35% of the genomic sequences identified by MSAP-Seq were methylated, predominantly affecting nuclear genes and intergenic regions, as opposed to repetitive sequences and chloroplast genes. Methylation was found to be more pronounced in the exonic regions of nuclear genes compared to their promoter and intronic regions. The methylated genes were predominantly associated with crucial biological processes such as photosynthesis, ATP synthesis-coupled electron transport, and defence response.
CONCLUSIONS: This study opens a new research direction in analysing variability in holm oak by evaluating the epigenetic events and mechanisms based on DNA methylation. It sheds light on the enzymatic machinery governing DNA (de)methylation, and the changes in the expression levels of methylases and demethylases in different organs along the developmental stages. The expression level was correlated with the DNA methylation pattern observed, showing the prevalence of de novo methylation and demethylation events in seedlings and embryos, respectively. Several methylated genes involved in the regulation of transposable element silencing, lipid biosynthesis, growth and development, and response to biotic and abiotic stresses are highlighted. MSAP-seq integrated with whole genome bisulphite sequencing and advanced sequencing technologies, such as PacBio or Nanopore, will bring light on epigenetic mechanisms regulating the expression of specific genes and its correlation with the phenotypic variability and the differences in the response to environmental cues, especially those related to climate change.