Scoring and analysis of methylation‐sensitive amplification polymorphisms for epigenetic population studies

Abstract

DNA methylation is an important, heritable epigenetic modification in most eukaryotic organisms that is connected with numerous biological processes. To study the impact of natural epigenetic variation in an ecological or evolutionary context, epigenetic studies are increasingly using methylation-sensitive amplification polymorphism (MSAP) for surveys at the population or species level. However, no consensus exists on how to interpret and score the multistate information obtained from the MSAP banding patterns. Here, we review the previously used scoring approaches for population epigenetic studies and develop new alternatives. To assess effects of the different approaches on parameters of epigenetic diversity and differentiation, we applied eight scoring schemes to a case study of three populations of the plant species Viola elatior. For a total number of 168 detected polymorphic MSAP fragments, the number of ultimately scored polymorphic epiloci ranged between 78 and 286 depending on the particular scoring scheme. Both, estimates of epigenetic diversity and differentiation varied strongly between scoring approaches. However, linear regression and PCoA revealed qualitatively similar patterns, suggesting that the scoring approaches are largely consistent. For single-locus analyses of MSAP data, for example the search for loci under selection, we advocate a new scoring approach that separately takes into account different methylation types and thus seems appropriate for drawing more detailed conclusions in ecological or evolutionary contexts. An R script (MSAP_score.r) for scoring and basic data analysis is provided.

Genetic and epigenetic divergence between disturbed and undisturbed subpopulations of a Mediterranean shrub: a 20‐year field experiment

“…2013; Fulneček and Kovařik 2014) techniques. The MSAP technique is useful to identify genome‐wide methylation patterns in ecological epigenetics studies of species without detailed genomic information (Schrey et al.…”

“…Differences in the products obtained with Hpa II and Msp I thus reflect different methylation states at the cytosines of the CCGG sites recognized by Hpa II or Msp I cleavage sites (Schulz et al. 2013; Fulneček and Kovařik 2014). MSAP assays for this study were conducted using four Hpa II‐ Msp I + 2/ Mse I + 3 primer combinations (Table S1).…”

“…Different MSAP ‘scoring’ methods can be used to obtain from these data the sample x marker matrix containing information on the methylation status of cytosines at the CCGG end of fragments (MSAP ‘fingerprint’ matrix) (Herrera and Bazaga 2010; Schulz et al. 2013; Fulneček and Kovařik 2014). We used the ‘Mixed Scoring 2’ scheme of Schulz et al.…”

“…We used the ‘Mixed Scoring 2’ scheme of Schulz et al. (2013). The 173 MSAP fragments obtained (Table S1) were transformed into three distinct sets of markers corresponding to unmethylated ( u ‐type in Schulz et al.…”

“…The 173 MSAP fragments obtained (Table S1) were transformed into three distinct sets of markers corresponding to unmethylated ( u ‐type in Schulz et al. 2013 terminology), HMe CG +  Me CG methylation (internal methylation plus hemimethylation, m ‐type) and HMe CCG methylation (external hemimethylation, h ‐type) markers. Plants sampled were characterized epigenetically by means of the presence – absence scores for u ‐type, h ‐type and m ‐type MSAP markers, using the Extract_MSAP_epigenotypes function from Schulz et al.…”