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 dierences 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 eective 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 aect all organisms during their lifetime. In our study, five white poplar (Populus alba L.) specimens were collected from dierent 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 dierentially methylated among the dierent specimens and link them to specific biochemical pathways. Many dierentially 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 dierent and changing pedoclimatic conditions, to which the poplar stands have been exposed. Keywords: MSAP; NGS; poplar; epigenetic; methylation
