Analysis of DNA methylation in Arabidopsis thaliana based on methylation-sensitive AFLP markers

Abstract

AFLP analysis using restriction enzyme isoschizomers that differ in their sensitivity to methylation of their recognition sites has been used to analyse the methylation state of anonymous CCGG sequences in Arabidopsis thaliana. The technique was modified to improve the quality of fingerprints and to visualise larger numbers of scorable fragments. Sequencing of amplified fragments indicated that detection was generally associated with non-methylation of the cytosine to which the isoschizomer is sensitive. Comparison of EcoRI/ HpaII and EcoRI/ MspI patterns in different ecotypes revealed that 35-43% of CCGG sites were differentially digested by the isoschizomers. Interestingly, the pattern of digestion among different plants belonging to the same ecotype is highly conserved, with the rate of intra-ecotype methylation-sensitive polymorphisms being less than 1%. However, pairwise comparisons of methylation patterns between samples belonging to different ecotypes revealed differences in up to 34% of the methylation-sensitive polymorphisms. The lack of correlation between inter-ecotype similarity matrices based on methylation-insensitive or methylation-sensitive polymorphisms suggests that whatever the mechanisms regulating methylation may be, they are not related to nucleotide sequence variation.

Differential Methylation during Maize Leaf Growth Targets Developmentally Regulated Genes

“…Similar results were found in MSAP experiments conducted in Arabidopsis (Cervera et al, 2002), pepper (Capsicum annuum; Portis et al, 2004), and maize (Lu et al, 2008). Also, the occurrence of two methylation changes between two zones was quite uncommon.…”

“…These gradual methylation changes are most likely caused by unequal changes in the methylation state of the different cell types that make up the tissues of the harvested samples (Xiong et al, 1999;Peraza-Echeverria et al, 2001;Cervera et al, 2002). For our objectives, the most interesting loci were the CCGG sites that abruptly changed their methylation from one zone to the next.…”

Epigenetic Variation, Inheritance, and Selection in Plant Populations

“…However, because such unique epialleles that cause phenotypic changes can only be distinguished from genetic variants by in-depth molecular analyses, epigenetic alterations independent of observable phenotypes are now being analyzed at the genomewide level. Several studies in nonmodel organisms have shown by methylation-sensitive amplified polymorphism (MSAP) analyses that natural populations vary more in DNA methylation than in DNA sequence, that is, their epigenetic diversity is higher than their genetic diversity (Cervera et al 2002;Keyte et al 2006;Salmon et al 2008;Herrera and Bazaga 2010;Lira-Medeiros et al 2010). In addition, DNA methylation patterns were analyzed in Arabidopsis using high-throughput sequencing of bisulfite-treated genomic DNA (BS-Seq).…”

Analysis of DNA methylation of perennial ryegrass under drought using the methylation-sensitive amplification polymorphism (MSAP) technique

“…In addition, both of the two enzymes can digest the non-methylated 5′-CCGG-3′ sequences. Thus, for a given DNA sample, full methylation of the internal cytosine or hemi-methylation of the external cytosine at the 5'-CCGG-3' sites can be unequivocally distinguished by MspI and HpaII, respectively (Cervera et al 2002). Due to its advantages, such as simple operation, the high number of available polymorphisms, and convenient primer design, the MSAP technique is widely used.…”