Department of Chemistry and Biology "A. Zambelli", University of Salerno, via Giovanni Paolo II 132, 84084 Fisciano (SA), Italy.
Department of Forest Genetics, Austrian Federal Research Centre for Forests, 1131 Vienna, Austria.
Int J Mol Sci. 2020 Oct 7;21(19):7393. doi: 10.3390/ijms21197393.
Over the last several decades, several lines of evidence have shown that epigenetic modifications modulate phenotype and mediate an organism's response to environmental stimuli. Plant DNA is normally highly methylated, although notable differences exist between species. Many biomolecular techniques based on PCR have been developed to analyse DNA methylation status, however a qualitative leap was made with the advent of next-generation sequencing (NGS). In the case of large, repetitive, or not-yet-sequenced genomes characterised by a high level of DNA methylation, the NGS analysis of bisulphite pre-treated DNA is expensive and time consuming, and moreover, in some cases data analysis is a major challenge. Methylation-sensitive amplification polymorphism (MSAP) analysis is a highly effective method to study DNA methylation. The method is based on the comparison of double DNA digestion profiles (RI-II and RI-I) to reveal methylation pattern variations. These are often attributable to pedoclimatic and stress conditions which affect all organisms during their lifetime. In our study, five white poplar ( L.) specimens were collected from different monoclonal stands in the Maltese archipelago, and their DNA was processed by means of an innovative approach where MSAP analysis was followed by NGS. This allowed us to identify genes that were differentially methylated among the different specimens and link them to specific biochemical pathways. Many differentially methylated genes were found to encode transfer RNAs (tRNAs) related to photosynthesis or light reaction pathways. Our results clearly demonstrate that this combinatorial method is suitable for epigenetic studies of unsequenced genomes like (at the time of study), and to identify epigenetic variations related to stress, probably caused by different and changing pedoclimatic conditions, to which the poplar stands have been exposed.
在过去的几十年中，有几条证据表明，表观遗传修饰调节表型，并介导生物体对环境刺激的反应。植物 DNA 通常高度甲基化，尽管在不同物种之间存在显著差异。已经开发了许多基于 PCR 的生物分子技术来分析 DNA 甲基化状态，但是随着下一代测序（NGS）的出现，出现了定性飞跃。在具有高水平 DNA 甲基化的大型、重复或尚未测序的基因组的情况下，NGS 分析亚硫酸氢盐预处理的 DNA 既昂贵又耗时，而且在某些情况下数据分析是一个主要挑战。甲基化敏感扩增多态性（MSAP）分析是一种研究 DNA 甲基化的高效方法。该方法基于比较双 DNA 消化谱（RI-II 和 RI-I）来揭示甲基化模式的变化。这些变化通常归因于影响所有生物体一生的土壤气候和压力条件。在我们的研究中，从马耳他群岛的不同单克隆林分中收集了五个白杨（L.）标本，并通过一种创新的方法处理它们的 DNA，其中 MSAP 分析后进行 NGS。这使我们能够鉴定出在不同标本中差异甲基化的基因，并将它们与特定的生化途径联系起来。许多差异甲基化基因编码与光合作用或光反应途径相关的转移 RNA（tRNA）。我们的研究结果清楚地表明，这种组合方法适用于像（在研究时）这样的未测序基因组的表观遗传学研究，并确定与压力相关的表观遗传变异，这些压力可能是由不同和不断变化的土壤气候条件引起的，白杨林分已经暴露在这些条件下。
