Isolation and characterization of dinucleotide-microsatellite DNA in Penaeus monodon fabricius

Abstract

Microsatellites have emerged as an important source of genetic markers for eukaryotic genomes. In this study, dinucleotide microsatellite sequences were isolated and characterized from the genome of the black tiger prawn, Penaeus monodon. Two partial genomic libraries; Library A from digestion of P. monodon genomic DNA with restriction enzmes, Alu I, Rsa I, Hae III and Hinc II and Library B from digestion with restriction enzymes, Ala I and Rsa I were constructed. For the Library A, 60 and 225 positive recombinants form 5,250 and 6,390 clones were found after screening with (CT)15 and (GT)15 probes, respectively, but no positive recombinants clones were found when (AT)15 probe was used. Nucleotide sequencing of 16 (CT)n revealed 2 perfect, 12 imperfect and 2 compound microsatellites and of 76 (GT)n, 20 perfect, 30 imperfect and 26 compound microsatellites were found. Library B, 121 out of 2,400 colonies were positive when hybridized with (GT)15 probes and 40 clones contained microsatellites. From these clones, 11 perfect, 17 imperfect and 12 compound microsatellites were found. On average, (CT)n and (GT)n microsatellites occur every 164 and 42 kb. The predominant category of P. monodon (CT)n and (GT)n was imperfect repeats; 75% and 39.5%, respectively. The most common size class was an array of 12-17 repeats for (CT)n and that of 36-46 repeats for (GT)n. Although, positive recombinant clones were not found using (AT)15 probes but the repeats of (AT)n were found with (CT)n and (GT)n microsatellites. Among 6 pairs of microsatellite primers surveyed, two produced expected PCR product but alleles were ambiguously scored.