Variability in CpNpG methylation in higher plant genomes.

The methylation status of ribosomal gene (rRNA) clusters have been investigated in a large variety of angiosperm species. Here we have analysed methylation in ribosomal gene (rRNA) clusters using MspI, HpaII, BstNI, EcoRII and CfoI restriction enzymes in combination with Southern hybridization to the 25S rDNA probe. It was shown that cytosine methylation at CpG dinucleotides and CpNpG trinucleotides occurred in all plant genomes examined. Methylation of rDNA units at CpG dinucleotides (studied with CfoI) was high in all species tested with approx. 40-70% of units being completely or nearly completely methylated. In contrast, the extent of the CpNpG methylation (studied with MspI and EcoRII) varied significantly between species; the percentage of the rDNA fraction entirely methylated at CpNpG trinucleotides ranged from less than 1% to almost 90% depending on the genome studied. Larger interspecies than within species variation was also observed among several non-transcribing repetitive sequences. In a small genome of A. thaliana, the CpNpG methylation appeared to be highly compartmentalized into the repetitive fraction. The methylation of trinucleotides was abundant in large A+T-rich genomes and it is proposed that the CpA(T)pG trinucleotides may help to maintain a high density of methylatable targets in plant repeated sequences.