MSAP markers and global cytosine methylation in plants

17 Methylation of DNA cytosines affects whether transposons are silenced and genes are 18 expressed, and is a major epigenetic mechanism whereby plants respond to 19 environmental change. Analyses of Methylation-Sensitive Amplification Polymorphism 20 (MS-AFLP or MSAP) have been often used to assess methyl-cytosine changes in 21 response to stress treatments and, more recently, in ecological studies of wild plant 22 populations. MSAP technique does not require a sequenced reference genome and 23 provides many anonymous loci randomly distributed over the genome for which the 24 methylation status can be ascertained. Scoring of MSAP data, however, is not 25 straightforward and efforts are still required to standardize this step to make use of 26 the potential to distinguish between methylation at different nucleotide contexts. 27 Furthermore, it is not known how accurately MSAP infers genome-wide cytosine 28 methylation levels in plants. Here we analyze the relationship between MSAP results 29 and the percentage of global cytosine methylation in genomic DNA obtained by HPLC 30 analysis. A screening of literature revealed that methylation of cytosines at cleavage 31 sites assayed by MSAP was greater than genome-wide estimates obtained by HPLC, 32 and percentages of methylation at different nucleotide contexts varied within and 33 across species. Concurrent HPLC and MSAP analyses of DNA from 200 individuals of 34 the perennial herb Helleborus foetidus confirmed that methyl-cytosine was more 35 frequent in CCGG contexts than in the genome as a whole. In this species, global 36 methylation was unrelated to methylation at the inner CG site. We suggest that global 37 HPLC and context-specific MSAP methylation estimates provide complementary 38 information whose combination can improve our current understanding of 39 methylation-based epigenetic processes in non-model plants. 40