Mitochondrial DNA and the 641kb nuclear-mitochondrial DNA inArabidopsiscan be separated by their CpG methylation levels

Methylation on the cytosine in plant mitochondrial DNA has been a controversial issue. Results supporting mitochondrial DNA methylation may have been subject to contamination due to the presence of the nuclear sequence originating from the mitochondrial genome called NUMT (nuclear mitochondrial DNA insertions). Especially in Arabidopsis thaliana Columbia 0, the largest NUMT located on chromosome 2 is nearly twice the size of the entire mitochondrial genome and almost identical. In the presence of such high similarity, it is challenging to eliminate interference in the determination of mitochondrial DNA methylation levels. Here we applied MBD (methyl-CpG binding domain) protein-based affinity assay to separate total DNA, and applied next generation sequencing to the pre- and post-separation DNA samples, and checked their SNP sites. The results revealed successful separation of methylated and non-methylated DNA within the total DNA, with separation achieved between NUMT DNA and mitochondrial DNA. The result suggests that our method can achieve separation based on the differential methylation levels of the whole lengths of NUMT and mitochondrial DNAs, and that mitochondrial DNA barely exhibits CpG methylation, at least in the Columbia 0.