Differentiation of Epinephelus moara from E. bruneus by improved nest-tetra-primer-specific PCR

Epinephelus moara and E. bruneus are closely related species in the genus Epinephelus (Perciformes, Serranidae). Their morphological similarity, changing color pattern at different stages and living conditions make them difficult to be differentiated. To identify these two species, an improved nest-tetra-primer-specific PCR assay was developed. Three specific molecular markers, the control region NC1 (394 bp), species-specific internal region ND2-M (268 bp) and ND2-B (122 bp), were identified in the mitochondrial ND2 gene from these two grouper species. Five markers were also discovered in the ITS1 regions of their nuclear ribosomal DNA, which were the control regions NC2 (588 bp) and NC3 (563 bp), and species-specific internal regions rDNA-M (426 bp), ITS1-M (488 bp) and ITS1-B (304 bp). This method provided a highly specific, precise, reliable and rapid molecular marker technique to discriminate between the two grouper species, as well as a new way of DNA identification to differentiate closely related species in fishes.