RAPID ISOLATION AND CHARACTERIZATION OF MICROSATELLITES IN ASIAN SEABASS (Lates calcarifer)

¹ Laboratory of Fish Biotechnolgy Institute of Molecular Agrobiology 1 Research Link National University of Singapore 117604 Singapore

² Primary Production Department Ministry of Natinal Development Singapore

Microsatellites are widely used as genetic markers because they are co-dominant, multi-allelic, highly polymorphic and easy to score with PCR. A main drawback of microsatellites is the time and cost required to isolate them from genomic library. We have adapted a rapid and non-radioactive method described by Fischer et al. (BioTechniques. 1998, 24: 796-802) with some modifications for isolation of di-, tri- and tetranucleotide microsatellites in Asian seabass. The putative microsatellite elements were captured from digested genomic DNA by using biotin-labeled microsatellite oligonucleotides (CA)10, (AAC)9, (GACA)5 and (GATA)6 attached to the streptavidin-coated magenetic beads. The captured fragments were amplified by adapter PCR and the PCR products enriched for microsatellites were cloned into pPCR-Script Cam SK(+) plasmid vector for sequencing. In the library enriched for CA-microsatellites 5,300 clones were obtained, while in the one enriched for AAC-, GACA- and GATA-microsatellites more than 10,000. The lengths of inserts were determined using colony PCR. Eighty-five percent (244/288) of the clones contained inserts with length between 250-1000 bp. One hundred and twenty clones (60 clones for each library) with insert lengths between 250 and 1000 bp were sequenced. Forty-eight (40%) sequenced clones contained microsatellites, proving that this approach is highly effective for isolating microsatellites. Thirty-five microsatellites were characterised in unrelated individuals of Asian seabass using an automated DNA sequencer (ABI 377). The allele number at loci ranged from 2 to 21 and observed heterozygosity was between 0.16 and 0.92. Nine of microsatellites were selected for studying the genetic structure of one broodstock of Asian seabass in Singapore, the results of this study will be presented.