Alternative PCR–RFLP methods for mussel Mytilus species identification

Mussels are the most important bivalves in both European aquaculture and market. Together with mussels produced in EU, the European market also allows the entrance of several mytilid species from up to 16 non-EU countries. Therefore, traceability methodologies are needed for the protection of consumers’ rights and for reinforce labeling regulations. The aim of this study was to establish a methodology that allows the accurate identification of 6 different mussel species, as in fresh as in processed forms. Amplification of mussel samples with primers Myti-F/R, which anneal in a region of the adhesive foot protein gene, yielded a 190-bp fragment in M . edulis, a 178-bp in M . trossulus, a 202-bp in M . californianus / M . coruscus , and a 136-bp in M . galloprovincialis / M . chilensis . Hence, differentiation among M . edulis , M . trossulus , and the rest of Mytilus species was achieved in a first step. Further digestion with A cl I in M . coruscus / M . californianus samples and with A ci I in M . galloprovincialis / M . chilensis allowed the accurate identification of these species. A second methodology based on amplification by PCR of mitochondrial cytochrome oxidase subunit I (COI) gene and digestion with X ba I was developed to further confirmation of M . chilensis samples. The current methodology was applied in cultured and natural samples and in commercial forms. The protocol described in this study provides an accurate, reliable, and a powerful tool for mussel species identification, traceability, and for reinforcement of labeling regulations.