Gene enrichment in maize with hypomethylated partial restriction (HMPR) libraries

A new technology was developed to assist gene-enrichment sequencing of any complex plant genome, employing maize as the test organism. Hypomethylated partial restriction (HMPR) libraries were constructed by using independent partial digestions with methylation-sensitive restriction enzymes HpaII (5′-CCGG-3′) and HpyCH4IV (5′-ACGT-3′). Fragments of 1-4 kb were purified and cloned, followed by sequence analysis of >2000 clones from 10 separate libraries. Organellar clones comprised ∼10% of each library but were useful in showing that no chimeric clones were generated and that digestion efficiencies were 10%-25% in different libraries. Four separate HMPR libraries, analyzed in detail, exhibited very similar degrees of gene enrichment and repeat depletion. Known gene homologies were found in ∼25% of the HMPR clones, compared with 70% for unfiltered maize sequences) and in providing an unmatched enrichment for the “unknown” sequences that contain promoters, introns, and other gene-adjacent regions.