Identification of southern hemisphere abalone (Haliotis) species by PCR-RFLP analysis of mitochondrial DNA

Illegal fishing and species-substitution of abalone (genus Haliotis), a highly valuable marine gastropod, are of worldwide concern. A mitochondrial DNA PCR-RFLP analysis of fragments of the cytochrome oxidase I (mtCOI) and II (mtCOII) genes was developed for the identification of II Southern Hemisphere species of abalone. These included five temperate and one tropical species from Australian waters, three temperate species from New Zealand and two temperate species from South Africa. All species, with the exception of the Haliotis rubralH. conicopora complex, can be unequivocally identified using the combined profiles from four individual restriction enzyme digests (Ddel, Hhal, HinFl and Hpall) on a 193 base pair fragment of mtCOI. Six species each displayed a unique profile for a single restriction enzyme. A one hundred and fifty-nine base pair fragment of mtCOII allowed individual identification of six of the species using the combined profiles from five individual restriction enzyme digests (DdeI. EcoRV, Hhal, Hpall, and RsaI). These primers failed to amplify in H. iris. Again H. rubra and H. conicopora could not be separated, and neither could H. australis and H. spadicea. No DNA sequence variation in either fragment was observed between H. rubra and H. conicopora the latter may be a Subspecies of H. rubra. The use of both fragments and a minimum of two restriction enzymes is recommended for species differentiation. DNA was successfully extracted, PCR amplified and identified front canned tissue and mucous samples of H. rubra. A conformational mutation in the mtCOI fragment was observed in H. midae. but in no other species nor in the mtCOII fragment.