Abstract
Accurate analysis of DNA methylation by bisulphite sequencing depends on the complete conversion of all cytosines into uracil. Until now there has been no standard or universal gene identified as an endogenous control to monitor the conversion frequency in plants. Here, we report the development of PCR based assays for one nuclear gene IND (INDEHISCENT) and two mitochondrial genes, NAD (NICOTINAMIDE ADENINE DINUCLEOTIDE) and ATP1 (ATPase SUBUNIT 1). We demonstrated their efficacy as bisulphite conversion controls in Brassica and other plant taxa. The target regions amplified by four primer pairs were found to be consistently free from DNA methylation. Primer pairs for IND.a and NAD were effective within Brassica species, whereas two primer pairs for ATP1 provided reliable controls across a representative range of dicot and monocot angiosperm species. These primer sets may therefore be adopted as controls in plant methylation analysis for a wide range of studies.
Highlights
Methylation of cytosine plays an important role in epigenetic gene regulation in vertebrates and higher plants [1]
PCR products from bisulphite treated DNA were cloned and for eight clones selected at random, the sequences demonstrated complete conversion of all cytosine residues to uracil
As such they represent a suitable target for use as a control in DNA methylation analysis for those Brassica species possessing the A genome (i.e. B. rapa (A), B. napus (AC), B. juncea (AB))
Summary
Methylation of cytosine plays an important role in epigenetic gene regulation in vertebrates and higher plants [1]. Over the past few decades, four major approaches have been used for distinguishing the epigenetic mark 5-methylcytosine (5mC) from unmethylated cytosine These include methods based on isochizimer restriction endonucleases, bisulphite conversion of DNA, immunoprecipitation and mass spectrometry [3,4]. False-positive 5-methylcytosines (cytosine read as 5-methylcytosine) are common, since it is often difficult to determine whether an unconverted cytosine represents true methylation or incomplete treatment Both incomplete DNA denaturation prior to bisulphite treatment and reannealing during treatment can lead to incomplete bisulphite conversion, since bisulphite converts single-stranded but not doublestranded DNA [5]. The completion of bisulphite conversion can be tested by monitoring exogenously spiked DNA controls, or retention of endogenous non-target sequence cytosine dinucleotides [4]. To date the full sequence of mitochondrial genomes has only been established for a small number of plant species
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