Definitive Molecular Markers for Speciation of Fresh and Canned 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 all 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 also not been successful in distinguishing all species in the Thunnus genus. In Phase I of this project we targeted a different gene sequence to the COI gene that can be utilized to distinguish the six most popular Thunnus species in the fresh form and designed and tested primers that are unique to three of the six species. Success achieved in Phase I feasibility study has positioned AFT, in Phase II, to focus research and development efforts on final optimization of molecular diagnostic methods developed in Phase I and integrate additional commercially important species that are sold under the "tuna" label. Once optimized for fresh/frozen tuna, we will investigate modifications in DNA extraction protocols to enable the use of the diagnostic to identify tuna species in further processed tuna products (canned and in pouches). Our overarching goal is to deliver a commercialized multiplex diagnostic test that will readily distinguish the eight major, high-valued tuna species in commerce from one another as well as from the commonly substituted species found in commerce. The global potential of this research will serve more broadly as a template for the regulatory management of other species with close evolutionary relatedness that are also found subject to fraudulent labeling practice by providing regulators and the industry with a powerful set of tools enabling species identification and authentication. To date, regulators monitoring product labels in commerce and fisheries management groups do not have an available tool to distinguish all Thunnus species. The diagnostic developed as part of this USDA SBIR will be commercially available in our current species identification testing service as well as 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.