Mitochondrial DNA and the largest nuclear-mitochondrial DNA in Arabidopsis can be separated by their methylation levels

Methylation of cytosines in plant mitochondrial DNA (mtDNA) has been a controversial issue. Results supporting mtDNA methylation may have been subject to contamination due to the presence of nuclear sequences originating from the mitochondrial genome called nuclear mitochondrial insertions (NUMT). In Arabidopsis (Arabidopsis thaliana) Columbia 0 (Col-0), the largest NUMT, located on Chromosome 2, is nearly twice the size of the entire mitochondrial genome and exhibits a sequence almost identical to the mitochondrial genome, albeit with shuffling and repeats. In the presence of such high similarity, it is challenging to eliminate interference when determining mtDNA methylation levels. Here, we applied a methyl-CpG-binding domain (MBD) protein-based affinity assay to separate total DNA, applied next-generation sequencing to the pre- and postseparation DNA samples, and examined the single nucleotide polymorphism (SNP) sites between NUMT and mtDNA. The results revealed successful separation of methylated and non-methylated DNA within the total DNA, with simultaneous separation achieved between NUMT DNA and mtDNA. These results suggest that our method can achieve separation based on the differential methylation levels of the whole lengths of NUMT and mtDNAs. The bisulfite sequencing results for the postseparation DNA samples suggest that mtDNA exhibits not only a lack of CpG methylation but also an absence of CHH and CHG methylation. In contrast, the NUMT shows high levels of methylation across all 3 contexts, at least in the Col-0 accession.