Development of a multiplex microsatellite assay for parentage assignment in orange-spotted groupers (Epinephelus coioides)

Establishment of reliable pedigree information of different lineages is crucial for selective breeding and stock enhancement programs. However, owing to the absence of a reliable assay for parentage assignment, such information is lacking in orange-spotted grouper (Epinephelus coioides), which is an economically important coral-reef fish. In this study, we developed two multiplex microsatellite polymerase chain reaction systems that consisted of 12 microsatellite markers (seven in system A and five in system B) for parentage assignment of orange-spotted groupers. The theoretical exclusion probability per locus ranged from 0.079 to 0.436 when both parents were unknown (E-1P) and from 0.191 to 0.614 when one parent was known (E-2P). The combined exclusion probability values of 12 loci were 98.8% (E-1P) and 100% (E-2P), respectively. The accuracy of the parentage test for a mixed family (15 dams and 12 sires, 226 offspring) was 100%. In addition, two wild populations (79 individuals) were added as interferential offspring, only four of which were misassigned to the stock. The success rate of assignment was 98.69%. We provide a reliable microsatellite-based assay for parentage assignment in orange-spotted groupers, which will be useful for selective breeding and stock enhancement programs for this species and maybe other groupers.