Novel microsatellites for multiplex PCRs in the Humpback grouper, Cromileptes altivelis (Valenciennes, 1828), and applications for broodstock management

The Humpback grouper, Cromileptes altivelis (Valenciennes, 1828), is one of the most valuable reef fishes, especially in Southeast Asia. It was recently listed by the IUCN as vulnerable due to its rapid decline in abundance. In an attempt to reduce exploitation of wild fingerlings, captive breeding has been performed either with or without hormone injection, and the brooders allowed to communally spawn. These actions have raised concerns regarding the reduction of effective population size by various factors related to communal spawning. In the present study, the first sets of microsatellite primers for two panels of multiplex PCRs (triplex and pentaplex) were developed from the DNA of C. altivelis. These primers were employed for parentage analysis and evaluation of genetic variation of three stocks: a parental stock (P, n = 20); the first generation offspring (G1, n = 120, age = 4 months); and an unrelated potential broodstock (PP, n = 118). The parentage analysis showed that among 20 brooders (P), two females and five males contributed to the genetic composition of G1. We observed multiple paternity, demonstrating that each female mated with two or three males. Moreover, the family contribution was skewed, and G1 was dominated by a single full-sib (87.50%). These factors likely resulted in low effective population size and may enhance inbreeding in successive generations. Genetic variation was studied based on seven loci (a locus was removed from the analysis due to physical linkage) and showed that the potential broodstock had slightly lower genetic variation relative to the parental stock. However, genetic distance and genetic relatedness estimator (rxy) indicated that the stock was sufficiently diverse from the parental stock; therefore, this stock may be used as female parents for P, which will be dominated by males as the stock grows older. Due to high rxy between some pairs of P and PP, the breeding pairs should be carefully selected based on low genetic relatedness to avoid inbreeding.