EXPLORING RELATIONSHIPS BETWEEN METHYL CYTOSINE DISTRIBUTION AND GENOME STRUCTURE IN BARLEY (Hordeum vulgare)

Department of Plant Sciences, Montana State University, Bozeman, MT 59715 USA

Cytosine methylation appears to be a useful physical tag which discriminates among functionally active and inactive genomic regions. Restriction enzyme McrBC (New England Biolabs) which degrades methylated DNA, was utilized to isolate undermethylated portion of the barley genome. Significant part of the undermethylated genome fraction (approximately 10% of the total nuclear DNA) could be recovered as fragments over 10,000 bp in length. Transformation of cloned fragments into Mcr⁺ and Mcr^- strains of E.coli confirmed that this fraction contains reduced levels of methyl cytosine. Analysis of DNA renaturation kinetics demonstrated that the amount of the repetitive DNA in the McrBC-resistant genome portion is reduced, while complexity of this fraction remains high. This suggests that it is enriched for low copy, undermethylated sequences. AFLP-based mapping analysis using DNA templates digested with isoschisomers possessing different methylation sensitivity (HpaII/MspI) demonstrated that centromeric regions of barley chromosomes are recombinationally inactive and heavily methylated. These results confirm observations that recombinationally active and presumably gene-rich chromosomal regions are more likely to contain low levels of methyl cytosine. Utilization of methylation differences between active portions of the genome and heavily methylated heterochromatic portions might be useful for better characterization of the functionally important portions of the plant genomes. Ability to separate these fractions utilizing different methylation sensitivity of restriction enzymes suggests that this approach might be useful in facilitating selective cloning of the gene-rich regions of cereal genomes.