Abstract

BackgroundVariation in DNA methylation across distinct genetic populations, or in response to specific biotic or abiotic stimuli, has typically been studied in leaf DNA from pooled individuals using either reduced representation bisulfite sequencing, whole genome bisulfite sequencing (WGBS) or methylation sensitive amplified polymorphism (MSAP). The latter represents a useful alterative when sample size is large, or when analysing methylation changes in genomes that have yet to be sequenced. In this study we compared variation in methylation across ten individual leaf and endosperm samples from maize hybrid and inbred lines using MSAP. We also addressed the methodological implications of analysing methylation variation using pooled versus individual DNA samples, in addition to the validity of MSAP compared to WGBS. Finally, we analysed a subset of variable and non-variable fragments with respect to genomic location, vicinity to repetitive elements and expression patterns across leaf and endosperm tissues.ResultsOn average, 30% of individuals showed inter-individual methylation variation, mostly of leaf and endosperm-specific differentially methylated DNA regions. With the exception of low frequency demethylation events, the bulk of inter-individual methylation variation (84 and 80% in leaf and endosperm, respectively) was effectively captured in DNA from pooled individuals. Furthermore, available genome-wide methylation data largely confirmed MSAP leaf methylation profiles. Most variable methylation that mapped within genes was associated with CG methylation, and many of such genes showed tissue-specific expression profiles. Finally, we found that the hAT DNA transposon was the most common class II transposable element found in close proximity to variable DNA regions.ConclusionsThe relevance of our results with respect to future studies of methylation variation is the following: firstly, the finding that inter-individual methylation variation is largely restricted to tissue-specific differentially methylated DNA regions, underlines the importance of tissue-type when analysing the methylation response to a defined stimulus. Secondly, we show that pooled sample-based MSAP studies are methodologically appropriate to study methylation variation. Thirdly, we confirm that MSAP is a powerful tool when WGBS is not required or feasible, for example in plant species that have yet to be sequenced.

Highlights

  • Variation in DNA methylation across distinct genetic populations, or in response to specific biotic or abiotic stimuli, has typically been studied in leaf DNA from pooled individuals using either reduced representation bisulfite sequencing, whole genome bisulfite sequencing (WGBS) or methylation sensitive amplified polymorphism (MSAP)

  • We demonstrated that the 13% reduction in DNA methylation in maize endosperm relative to leaf and embryo tissues largely resulted from maternal hypomethylation [42], results that were subsequently confirmed by high-throughput bisulfite sequencing of arabidopsis, rice, sorghum, maize and castor bean genomes [34, 43,44,45,46,47]

  • Characterization of inter-individual methylation variation (ii-MV) in maize endosperm and leaf using MSAP MSAP was employed to characterize ii-MV. This technique is a modification of AFLP (Amplified Fragment Length Polymorphism), which is based on random amplification of restriction fragments typically generated by digestion of genomic DNA with EcoRI and MseI restriction enzymes [48]

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Summary

Introduction

Variation in DNA methylation across distinct genetic populations, or in response to specific biotic or abiotic stimuli, has typically been studied in leaf DNA from pooled individuals using either reduced representation bisulfite sequencing, whole genome bisulfite sequencing (WGBS) or methylation sensitive amplified polymorphism (MSAP). The genome-wide distribution of DNA methylation has been detailed both in arabidopsis [14, 15] and agronomically important plants such as rice, maize, soybean, cassava, soybean, common bean, wheat and cotton [16,17,18,19,20,21,22,23] These studies show that the bulk of DNA methylation is located within transposable elements (TEs), underlining its important and well-characterized function - proposed several years ago - in regulating TE activity [24, 25]. Those data uncovered the prevalence of CG methylation within the gene-body

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