Development of microsatellite markers for commonbean (Phaseolus vulgaris L.) based on screening ofnon-enriched, small-insert genomic libraries

摘要

Microsatellite markers are useful genetic tools for a wide array of genomic analyses although their developmentis time-consuming and requires the identification of simple sequence repeats (SSRs) from genomic sequences. Screeningof non-enriched, small-insert libraries is an effective method of SSR isolation that can give an unbiased picture of motiffrequency. Here we adapt high-throughput protocols for the screening of plasmid-based libraries using robotic colony pick-ing and filter preparation. Seven non-enriched genomic libraries from common bean genomic DNA were made by diges-tion with four frequently cutting restriction enzymes, double digestion with a frequently cutting restriction enzyme and aless frequently cutting restriction enzyme, or sonication. Library quality was compared and three of the small-insert libra-ries were selected for further analysis. Each library was plated and picked into 384-well plates that were used to createhigh-density filter arrays of over 18 000 clones each, which were screened with oligonucleotide probes for various SSRmotifs. Positive clones were found to have low redundancy. One hundred SSR markers were developed and 80 were testedfor polymorphism in a standard parental survey. These microsatellite markers derived from non-SSR-enriched librariesshould be useful additions to previous markers developed from enriched libraries.