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.
Highlights
In the last twenty years, many studies have focused on epigenetic modifications in plants, on DNA methylation status, and on the mechanisms driven by external stimuli that are exploited by plants to counteract mid- and/or long-term changes, with or without the involvement of signalling molecules
The natural populations inhabited environments that were characterised by different pedoclimatic conditions, forming large monoclonal stands, but at the same time, their DNA methylation profiles revealed several differences specific to their geographical location, suggesting that epigenetic diversity should be considered in the diversity analyses that are commonly employed in the framework of the Convention on Biological Diversity
This study on clonal white poplar populations/stands of the Maltese archipelago focused on the integration of two reliable and powerful analytical tools—namely, the cost-effective Methylation-sensitive amplification polymorphism (MSAP) analysis and next-generation sequencing (NGS)—to identify genes and pathways affected by changes in DNA methylation
Summary
In the last twenty years, many studies have focused on epigenetic modifications in plants, on DNA methylation status, and on the mechanisms driven by external stimuli (abiotic stresses, pedoclimatic conditions, climate changes etc.) that are exploited by plants to counteract mid- and/or long-term changes, with or without the involvement of signalling molecules (e.g., jasmonic acid, salicylic acid and reactive oxygen species).In plants, DNA methylation commonly occurs within three main sequences: CG, CHG and CHH (where H is A, C or T); the symmetrical CpG and CpA(T)pG sites are reported to be the most frequent targets of cytosine modification. Several studies have investigated the possibility that DNA methylation status might better reveal the genetic biodiversity within or among populations [7,8,9], especially in species that spread through natural vegetative propagation In this case, epigenetic modifications may compensate for reduced genetic biodiversity, counteract pedoclimatic variations and present an additional source of phenotypic variation. This aspect has been investigated recently in two natural plant species that are widely distributed—namely, the white poplar (Populus alba L., a dicot species) [8] and the giant reed (Arundo donax L., a monocot species) [9] In both cases, the natural populations inhabited environments that were characterised by different pedoclimatic conditions, forming large monoclonal stands, but at the same time, their DNA methylation profiles revealed several differences specific to their geographical location, suggesting that epigenetic diversity should be considered in the diversity analyses that are commonly employed in the framework of the Convention on Biological Diversity. It has been demonstrated that epigenetic modifications are hereditable [10], and this supports the result obtained by Raj et al (2011) [11], who found that the clones with the most divergent transcriptomes and clone history had the most marked differences in the extent of total DNA methylation, suggesting an epigenomic basis for the clone-history-dependent transcriptome divergence
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