Plant & Animal Genome VI Conference Plant & Animal Genome VI Conference
P34
Department of Plant Science, Montana State University, Bozeman, MT 59717
A major portion of the plant genome is represented by repetitive sequences. As a consequence, significant proportion of clones in the libraries constructed from the total genomic DNA contains repetitive DNA, which hinders isolation of the single-copy DNA fragments. Since repetitive sequences are often highly methylated, and active genes, as a rule, are undermethylated, such differences in the methylation pattern could be utilized to differentiate between them. We utilized restriction enzyme McrBC (New England Biolabs), which recognizes two methylated cytosine residues situated up to 2,000 bp apart and cleaves DNA strands between them. Digestion of barley genomic DNA with McrBC results in most of the genomic DNA being degraded down to fragments less than 1 kb in size. However, a small proportion (less than 5% of the original DNA amount) remains high molecular weight, indicating the presence of relatively long continuous stretches of unmethylated sequences in the barley genome. These fragments are likely to represent gene-rich portions of the genome, and they do not contain highly repetitive sequences. This DNA fraction was used to construct a barley genomic library with inserts 10-25 kb in size, highly enriched in low-copy number sequences which are likely to represent active genes. The distribution of a few of these sequences on the barley linkage map was analyzed. A library consisting of undermethylated long DNA fragments might serve as an useful source of single-copy sequences for gene isolation, mapping and in situ hybridization experiments.