Isolation and identification of a new set of microsatellite loci from Ucides cordatus genome

Abstract

A new set of microsatellite loci (simple sequence repeats, SSRs) from the overexploited mangrove crab Ucides cordatus is described in this study. Microsatellite isolation used a highly simplified and inexpensive protocol based on (i) multiple enzyme digestion/ligation; (ii) mixed biotin-labeled probes and streptavidin-coated magnetic bead hybridization capture strategy, and (iii) a double-repeat-enrichment procedure. A genomic library, double-enriched for inserts containing tetranucleotide repeat motifs [(GACA)6, (GATA)7, (GGAT)5 and (GTAT)5], was constructed to increase the chance of recovering SSR-containing sequences within DNA fragments. Amplified enriched DNA was cloned and transformed into competent E. coli. Then, positive clones were identified by 'white/blue plaque selection’. One hundred and five colonies were PCR-screened for sequencing, and 72 of these were found to have unique SSR inserts. Microsatellite motifs contained more than five repeats, and most loci were found to have perfect tandem repeats (51.4%), of which 94.4% were dinucleotide and 5.5% trinucleotide. Only 20% of all loci were compound and 28.6% were imperfect repeats containing di-, tri- and/or tetranucleotides. The high frequency of perfect repeat motifs after enrichment is additional evidence of the importance of adopting this procedure for the isolation of SSR. The novel 34 SSRs described in this study are expected to be highly polymorphic and, therefore, useful in population/stocks discrimination of this valuable mangrove species throughout its range, currently subjected to excessive fishing efforts.

Keywords

• microsatellite loci
• short tandem repeats
• double enrichment
• molecular markers
• genetic diversity