Market transparency is in strong demand by consumers, and the authentication of species is an important step for seafood traceability. In this study, a simple molecular strategy, COIBar–RFLP (cytochrome oxidase I barcode–restriction fragment length polymorphism), is proposed to unveil commercial fraud based on the practice of species substitution in the swordfish trade. In particular, COI barcoding allowed the identification of the species Prionace glauca, Mustelus mustelus, and Oxynotus centrina in slices labeled as Xiphias gladius. Furthermore, the enzymatic digestion of COI amplicons using the MboI restriction endonuclease allowed the simultaneous discrimination of the four species. Interestingly, an intraspecific differential MboI pattern was obtained for the swordfish samples. This pattern was useful to differentiate the two different clades revealed in this species by phylogenetic analyses using several molecular markers. These results indicate the need to strengthen regulations and define molecular tools for combating the occurrence of fraud along the seafood supply chain and show that COIBar–RFLP could become a standardized molecular tool to assess seafood authenticity.
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