PDF(Sci-hub)
Abstract：
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 (EcoRI-HpaII and EcoRI-MspI) 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 (Populus alba 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 P. alba (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.
摘要：
在过去的几十年里，一些证据表明，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通常是高度甲基化的,尽管物种之间存在显着差异。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，然而,随着下一代测序(NGS)的出现,出现了质的飞跃.在大的情况下，重复,或尚未测序的基因组以高水平的DNA甲基化为特征，亚硫酸氢盐预处理DNA的NGS分析是昂贵且耗时的，而且,在某些情况下，数据分析是一个重大挑战。甲基化敏感扩增多态性(MSAP)分析是研究DNA甲基化的高效方法。该方法基于双重DNA消化谱（EcoRI-HpaII和EcoRI-MspI）的比较，以揭示甲基化模式的变化。这些通常可归因于在其一生中影响所有生物体的动物性和应激条件。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（PopulusalbaL.）标本，他们的DNA通过一种创新的方法进行处理，其中MSAP分析之后是NGS。这使我们能够识别不同样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。发现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未测序基因组的表观遗传学研究，如白杨（在研究时）。并确定与压力相关的表观遗传变异，可能是由不同和变化的气候条件引起的，白杨树所暴露的地方。
