Mapping of unmethylated sites in rDNA repeats in barley NOR deletion line

Extensive cytosine methylation is characteristic of plant rDNA. Evidence exists, however, that the active rRNA genes are less methylated. In this work we report on the mapping of unmethylated CCGG sites in Hordeum vulgare rDNA repeats by digestion with methylation sensitive restriction enzyme HpaII and indirect end-labeling of the generated fragments. For mapping we used genomic DNA from barley deletion line with a single NOR on chromosome 5H. This NOR is more active in order to compensate for the missing NOR 6H. The enhanced NOR 5H activity in the deletion mutant is not due to higher multiplicity of the rRNA genes or, as sequencing showed, to changes in the subunit structure of the intergenic spacer. The HpaII sites in barley rDNA are heavily methylated. Nevertheless, a fraction of the rDNA repeats is hypomethylated with unmethylated CCGG sites at various positions. One unmethylated CCGG sequence is close to the transcription start site, downstream of the 135bp subrepeats. Unmethylated sites are also present in the external transcribed spacer and in the genes coding mature rRNAs. The patterns of unmethylated sites in the barley deletion line and in lines with two NORs were compared. It is hypothesized that the occurrence of unmethylated sites on a fixed subset of rDNA repeats correlates with their transcriptional activity.