Isolation and validation of microsatellite markers from a depleted South African sciaenid species, the dusky kob (Argyrosomus japonicus), by means of the FIASCO/454 approach

Abstract

The dusky kob (Argyrosomus japonicus) is a large, estuarine-dependent sciaenid fish that has been severely depleted in South African waters and that, in recent years, has received considerable attention from the local fish farming industry. Discovery and application of appropriate molecular markers is necessary to improve the understanding of wild population structure, assist the effectiveness of broodstock and breeding programmes, and ensure monitoring of potential interactions between wild and farmed fish. The present study uses a recently tested approach that combines the FIASCO enrichment protocol with 454 GS-FLX Next Generation Sequencing, to identify large numbers of microsatellite-containing sequences at a low cost and high discovery rate from the dusky kob genome. Following the FIASCO enrichment (targeting specifically tetranucleotide repeats), 2355 potential tetranucleotide microsatellites (perfect repeat motifs including eight or more repeat units flanked by regions for primer design) were identified from 1/5th of a single 454 lane. From these sequences, a test panel of 60 potential markers was selected for validation. A total of nine (15%) markers were successfully amplified from a test sample of wild dusky kob individuals and showed high levels of polymorphism (observed heterozygosity per locus ranging between 0.375 and 0.905). Cross-species amplification of eight of these markers was also successfully carried out in another closely related and commercially important South African sciaenid species, the silver kob (A. inodorus). The microsatellite markers developed in the present study are readily available tools suitable to address genetic variability of Argyrosomus species of Southern Africa.