Identification and analysis of adenine N⁶-methylation sites in the rice genome

DNA N⁶-methyladenine (6mA) is a non-canonical DNA modification that is present at low levels in different eukaryotes^1-8, but its prevalence and genomic function in higher plants are unclear. Using mass spectrometry, immunoprecipitation and validation with analysis of single-molecule real-time sequencing, we observed that about 0.2% of all adenines are 6mA methylated in the rice genome. 6mA occurs most frequently at GAGG motifs and is mapped to about 20% of genes and 14% of transposable elements. In promoters, 6mA marks silent genes, but in bodies correlates with gene activity. 6mA overlaps with 5-methylcytosine (5mC) at CG sites in gene bodies and is complementary to 5mC at CHH sites in transposable elements. We show that OsALKBH1 may be potentially involved in 6mA demethylation in rice. The results suggest that 6mA is complementary to 5mC as an epigenomic mark in rice and reinforce a distinct role for 6mA as a gene expression-associated epigenomic mark in eukaryotes.