Development of DNA typing kit in black tiger prawn Penaeus monodon fabricius by microsatellite technique

Abstract

Eight microsatellite loci, CSCUPmo1, CSCUPmo2, CSCUPmo3, CSCUPmo4, CSCUPmo6, CSCUPmo7, CSCUPmo9 and CSCUPmo11 were investigated to select suitable microsatellite loci for use in DNA typing of the black tiger prawn Penaeus monodon. Allelic variations were examined in 50 individuals P.monodon from Trad. All eight loci were be highly polymorphic which exhibited number of alleles at each locus of 29, 27, 27, 21, 29, 22, 33 and 10 alleles, respectively and heterozygosities between 0.21-0.90. Size ranges of alleles varied from 132-380 bp. Six microsatellite loci were chosen based on the level of polymorphism and the ease of allele scoring. The CSCUPmo3 and CSCUPmo7 loci that gave ambiguous allelic patterns and an excess of homozygous genotypes were discarded from further analysis. Approximately 10 mg and 5 microlitres of the tissue and blood/ethanol respectively were using for DNA typing of P.monodon by microsatellite technique. Alkaline extraction method was the most simple and appropriate DNA isolation when dealing with a large number of specimens. Multiplex analysis was developed to provide rapid amplification of multiple loci simultaneously. Successful analyses were multiplex PCR of loci CSCUPmo1 and CSCUPmo2 and single loading of combined amplified products of paired microsatellites (CSCUPmo4+CSCUPmo9, CSCUPmo6+CSCUPmo11). Non isotopic method were used to detect amplified alleles by separating in 8% denaturing polyacrylamide sequencing gels and visualization of amplified alleles using silver staining. Allele difference of 3 bases such as those of the locus CSCUPmo11 could also be detected by using 15% denaturing polyacrylamide minigel. This allowed simpler and more rapid detection of microsatellite alleles. Construction of allelic ladders for use as standard DNA markers was succeeded for the locus CSCUPmo11. The allelic ladders make band scoring simple and more precise.