Development of microsatellite markers for common bean (Phaseolus vulgaris L.) based on screening of non-enriched, small-insert genomic libraries.
Microsatellite markers are useful genetic tools for a wide array of genomic analyses although their development is time-consuming and requires the identification of simple sequence repeats (SSRs) from genomic sequences. Screening of non-enriched, small-insert libraries is an effective method of SSR isolation that can give an unbiased picture of motif frequency. Here we adapt high-throughput protocols for the screening of plasmid-based libraries using robotic colony picking and filter preparation. Seven non-enriched genomic libraries from common bean genomic DNA were made by digestion with four frequently cutting restriction enzymes, double digestion with a frequently cutting restriction enzyme and a less frequently cutting restriction enzyme, or sonication. Library quality was compared and three of the small-insert libraries were selected for further analysis. Each library was plated and picked into 384-well plates that were used to create high-density filter arrays of over 18 000 clones each, which were screened with oligonucleotide probes for various SSR motifs. Positive clones were found to have low redundancy. One hundred SSR markers were developed and 80 were tested for polymorphism in a standard parental survey. These microsatellite markers derived from non-SSR-enriched libraries should be useful additions to previous markers developed from enriched libraries.
Blair
Matthew W
MW
International Center for Tropical Agriculture (CIAT), A.A. 6713, Cali, Colombia. m.blair@cgiar.org
Torres
Monica Muñoz
MM
Pedraza
Fabio
F
Giraldo
Martha C
MC
Buendía
Hector F
HF
Hurtado
Natalia
N
D003001:Cloning, Molecular; D018744:DNA, Plant / Q000737:chemistry / Q000235:genetics*; D015698:Genomic Library; D018895:Microsatellite Repeats / Q000235:genetics*; D027805:Phaseolus / Q000235:genetics*; D017422:Sequence Analysis, DNA
D018744:DNA, Plant