Development of type I microsatellite markers and application in genome mapping of the black tiger shrimp Penaeus monodon

Abstract

Microsatellites are useful markers for numerous applications in aquaculture and fisheries research. In this study, type I microsatellite markers were developed for use in constructing a genetic linkage of Penaeus monodon. A software tool was applied for the identification of microsatellite repeats in the black tiger shrimp (P. monodon) ESTs database (http://pmonodon.biotec.or.th). Abioinformatics analysis of 10,100 ESTs identified 1,381 ESTs containing microsatellites. Clustering analysis indicated that 513 of these ESTs fell into 129 contigs, and the remaining 868 ESTs were singletons. A total of 2,165 microsatellite were identified. Perfect microsatellites were predominant in this study whereas imperfect and compound microsatellites were found at a much lesser extent (16.7% and 5.2%, respectively). Trinucleotide AAT repeat type appeared to be the most abundant type distributed in P. monodon database, followed by dinucleotide AT repeat type. Homologysearching by the BLASTX program revealed that the microsatellite containing clones represent 11.6% know gene products, 37.3% hypothetical protein and 51.1% unknown gene. Microsatellite in ESTs were mainly located in coding region, followed by 3{7f2019}-UTR and 5{7f2019}-UTR region, respectively. One hundred and fifty-four primer pairs flanking microsatellite loci have been used for screening polymorphism. As results, 126 primer pairs produced PCR products and 50 pairs were polymorphic. Characterization of the 50 new microsatellite markers on a panel of 35-48 unrelated shrimps showed high levels of genetic polymorphism with the average of 12.6 alleles per locus and the average polymorphic information content (PIC) of 0.723. The average observed and expected heterozygosities were 0.698 and 0.759, respectively. These 50 microsatellite loci were used to genotype the reference family for international genetic mapping of P. monodon. The genotyping data was analyzed with AFLP primer combination, microsatellites, and other markers with LOD score of 3.5 and maximum [theta] of 0.30. Thirty-six microsatellite markers were integrated into the previously shrimp genetic linkage map. The total lengths of linkage groups covering the male and female maps were 1,101 and 891.4 cM, respectively. The average spacing between 2 markers of male and female maps were 7 and 8 cM, respectively.