Development of two microsatellite multiplex systems for black tiger shrimp Penaeus monodon and its application in genetic diversity study for two populations

Abstract

Despite large numbers of putative microsatellites currently listed in databases for Penaeus monodon, there are no publications on assessing these markers for multiplexed high throughput systems either for fingerprinting or population genetics study purposes in P. monodon. Accordingly, we started our investigation on the development of high throughput systems for P. monodon. Ninety publicly-available P. monodon microsatellite sequences were initially screened for suitability. They were assessed for the presence of tri- or tetra-nucleotide repeats, repeat number and type, suitability of flanking sequences for primer design and estimated size of product (100 to 350 bp). Nineteen sequences were chosen for preliminary assessment on a panel of 15 animals. Of the 19 tested, only 12 were suitable for further investigation. Therefore a 2-step enrichment library approach was adopted to develop additional microsatellites. Of 42 new unique microsatellite sequences obtained, eight sequences were assessed and seven showed polymorphism. Together, these 19 markers were examined further for their ease of amplification and reliability of allele calling for inclusion in high throughput systems. Thirteen polymorphic markers were incorporated into two multiplex systems (six and seven markers, respectively). These multiplexed systems were then used to evaluate the genetic diversity between two populations of P. monodon, one from the East Coast of Australia and a single pond containing farmed animals from Thailand. There were significant differences between the two populations. Three markers in system 1 showed Hardy-Weinberg disequilibrium in both populations, indicating their unsuitability as high throughput system markers. Using two systems and the UPGMA clustering methods revealed the existence of sub-populations within the Australian wild population. The results indicate the usefulness of the two multiplexed microsatellite systems in genetic diversity studies. Crown Copyright © 2007.