Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq)—A Method for High-Throughput Analysis of Differentially Methylated CCGG Sites in Plants with Large Genomes

Abstract

Epigenetic mechanisms, including histone modifications and DNA methylation, mutually regulate chromatin structure, maintain genome integrity, and affect gene expression and transposon mobility. Variations in DNA methylation within plant populations, as well as methylation in response to internal and external factors, are of increasing interest, especially in the crop research field. Methylation Sensitive Amplification Polymorphism (MSAP) is one of the most commonly used methods for assessing DNA methylation changes in plants. This method involves gel-based visualization of PCR fragments from selectively amplified DNA that are cleaved using methylation-sensitive restriction enzymes. In this study, we developed and validated a new method based on the conventional MSAP approach called Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq). We improved the MSAP-based approach by replacing the conventional separation of amplicons on polyacrylamide gels with direct, high-throughput sequencing using Next Generation Sequencing (NGS) and automated data analysis. MSAP-Seq allows for global sequence-based identification of changes in DNA methylation. This technique was validated in Hordeum vulgare. However, MSAP-Seq can be straightforwardly implemented in different plant species, including crops with large, complex and highly repetitive genomes. The incorporation of high-throughput sequencing into MSAP-Seq enables parallel and direct analysis of DNA methylation in hundreds of thousands of sites across the genome. MSAP-Seq provides direct genomic localization of changes and enables quantitative evaluation. We have shown that the MSAP-Seq method specifically targets gene-containing regions and that a single analysis can cover three-quarters of all genes in large genomes. Moreover, MSAP-Seq's simplicity, cost effectiveness, and high-multiplexing capability make this method highly affordable. Therefore, MSAP-Seq can be used for DNA methylation analysis in crop plants with large and complex genomes.

Prospects for incorporation of epigenetic biomarkers in human health and environmental risk assessment of chemicals

“…Finally, there are several variations of methylation‐sensitive amplified polymorphism (MSAP) that allow the parallel and direct analysis of DNAm for a high number of sites across the genome (Yaish, Peng & Rothstein, ). A variation of the MSAP approach that involves next generation sequencing and automated data analysis is a suitable option for plant species (Chwialkowska et al ., ). Methylation ‐sensitive amplified fragment length polymorphism (MS‐ AFLP) is a modification of amplification fragment length polymorphism (AFLP) that is based on DNA restrictive digestion with enzymes; this technique is also used to profile methylation patterns without requiring detailed knowledge of the genome sequence (Fulneček & Kovařík, ; Kurdyukov & Bullock, ).…”

The assessment of epigenetic diversity, differentiation, and structure in the ‘Fuji’ mutation line implicates roles of epigenetic modification in the occurrence of different mutant groups as well as spontaneous mutants

“…Many techniques have been developed to analyze global DNA methylation and its alterations [12]. The method of methylation-sensitive amplified polymorphism (MSAP) is one of the most efficient, economical, and widely one used for the detection of DNA methylation events [13][14]. This approach is a modified version of the amplified fragment length polymorphism (AFLP) method based on the differential sensitivity of isoschizomeric restriction endonucleases to site-specific cytosine methylation [15].…”

“…So, on the basis of variant band patterns resulting from differential digestion of the genome by HpaII/MspI isozymes, variation in the DNA methylation level and pattern in the whole genome is detected [15,17]. MSAP has been successfully applied for the analysis of the variation in methylation levels and patterns in a variety of plant species [13].…”

Epigenetic Analysis through MSAP-NGS Coupled Technology: The Case Study of White Poplar Monoclonal Populations/Stands

“…Analysing two wheat genotypes in combination with their somaclones, characterized by a changed heritable phenotype, the authors were able to study epigenetically induced changes and, at the same time, identify over 100 amplicons that were differentially methylated among the samples. Another MSAP-based approach combined with NGS is MSAP-Seq [ 14 ], which is also integrated with an automatic pipeline MSEQER. This method used conventional MSAP analysis (digestion of DNA with Eco RI and Hpa II enzymes) and replaced the gel separation of amplicons (from selective PCR) with NGS.…”

“…At the present, only two studies have reported the combined use of MSAP analysis and NGS in plants, but their results were limited to the identification of polymorphic amplicons, without the determination of the DNA methylation status of the sequenced fragments or the identification of the pathways that were enriched for differentially methylated sequences. In other studies, some authors applied bisulphite NGS sequencing after MSAP [ 13 ], particularly when the genome sequence was known, as in the case of rice as an alternative, they proceeded with amplicon electrophoresis on polyacrylamide gels and the isolation of polymorphic bands, then sequenced through Sanger technology [ 14 ]. Our study promises to make a step forward in this kind of molecular analysis, providing a new protocol of coupled MSAP-NGS and a new flowchart for data mining, useful for obtaining information about the methylation status of the sequenced fragments and their involvement in specific genetic pathways and biological processes.…”