Definitive Molecular Markers for Speciation of Tuna: Genus Thunnus

Ranked as one of the world's most valuable commercial seafood, tuna species belonging to the Thunnus genus are considered the most important and endangered fish in worldwide trade. Extensive research to develop methods to distinguish Thunnus at the species level has significantly contributed to the enormous databanks of genetic information on these fish. However to date, no single approach has been successful in completely discriminating the Thunnus species. Applied Food Technologies (AFT) has been successful in the development of molecular diagnostics for fish species identification. Our two most common approaches include PCR multiplexing utilizing species specific primers and DNA barcoding based on the COI gene sequence alignments. However as others, we have shown that neither approach is specific enough to distinguish these closely related species. A number of other methods based on RFLPs, RAPDs, SSCPs or AFLPs as well as sequencing with numerous different gene targets have not been successful in distinguishing species in the Thunnus genus. To develop a reproducible, cost-effective molecular diagnostic that unambiguously identifies and distinguishes the major commercially traded tuna species our Phase I proposal will initially target the six most valued Thunnus species. Data mining both the information of genetic targets in previously published phylogenetic studies and in-house fish genetic markers, a series of primers targeting mitochondrial and/or nuclear genes for discriminating these tuna will be designed. A multipronged strategy incorporating sequencing, alignments, phylogenetic analyses, minibarcode method and primer/probe approach will be taken in developing primers to the most promising gene target(s) identified in our search. A conventional PCR and real time PCR platform will be used in testing tuna species specific primers or primer/probes. This work will enable us to determine the best strategy and target gene(s) to use in follow-on development of a validated species ID diagnostic for all major tuna in trade. To date, regulators monitoring product labels in commerce and fisheries management groups do not have an available tool to distinguish Thunnus species. The diagnostic developed as part of this USDA SBIR will be commercially available for use in Federal, State, public and private laboratories. This work also has potential to serve as platform technology in developing DNA-based methods for distinguishing other genetically close related species.