A small-insert bovine genomic library highly enriched for microsatellite repeat sequences

Abstract

A bovine genomic phagemid library was constructed with randomly sheared DNA. Enrichment of this single-stranded DNA library with CA or GT primers resulted in 45% positive clones. The 14% of positive clones with (CA.GT) > 12, and not containing flanking repetitive elements, were sequenced, and the efficiency of marker production was compared with random M13 bacteriophage libraries. Primer sequences and genotyping information are presented for 390 informative bovine microsatellite markers. The genomic frequency for 11 tri- and tetranucleotide repeats was estimated by hybridization to a lambda genomic library. Only GCT, GGT, and GGAT were estimated to have a frequency of > 100 per genome. Enrichment of the phagemid library for these repeats failed to provide a viable source of microsatellite markers in the bovine. Comparison of map interval lengths between 100 markers from the enriched library prepared from randomly sheared DNA and M13 bacteriophage libraries prepared from Mbo1 restriction digests suggested no bias in skeletal genomic coverage based on source of small insert DNA. In conclusion, enrichment of the bovine phagemid library provides a sufficient source of microsatellites so that small repeat lengths and flanking repetitive sequences common in the bovine can be eliminated, resulting in a high percentage of informative markers.