Source: APPLIED FOOD TECHNOLOGIES LLC submitted to
DEFINITIVE MOLECULAR MARKERS FOR SPECIATION OF TUNA: GENUS THUNNUS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
SMALL BUSINESS GRANT
Reporting Frequency
Annual
Accession No.
0225224
Grant No.
2011-33610-30426
Project No.
FLAW-2011-00234
Proposal No.
2011-00234
Multistate No.
(N/A)
Program Code
8.5
Project Start Date
Jun 15, 2011
Project End Date
Feb 14, 2012
Grant Year
2011
Project Director
Applewhite, L.
Recipient Organization
APPLIED FOOD TECHNOLOGIES LLC
3610 NW 42ND TERRACE
GAINESVILLE,FL 32606-8110
Performing Department
(N/A)
Non Technical Summary
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.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20150101000100%
Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
5010 - Food;

Field Of Science
1000 - Biochemistry and biophysics;
Goals / Objectives
The long-term goal of this project is to develop a reproducible, cost-effective molecular diagnostic that will unambiguously identify and distinguish the major commercially traded tuna species. While ongoing programs at AFT as well as efforts in several academic and regulatory groups continue to extend our capabilities to speciate many commercially important fish, the genetic complexity and relatedness of the major tuna species has presented significant challenges in developing a definitive diagnostic for fish of the Thunnus genus. The main technical objectives of this project are to procure and authenticate reference specimens representing six of the Thunnus species; screen for gene marker(s) and design primer/probe for differentiating highly genetic- related tuna specie; and test tuna species specific primers and primer/probes on PCR platforms. Successful development and implementation of this SBIR deliverable will provide a species authentication tool of significant value to seafood distributors, processors, and federal and state regulatory agencies that is currently unavailable.
Project Methods
Taxonomical verification of tuna species is one of the most important aspects of developing a fully validated diagnostic in order for the test to stand up to regulatory and legal scrutiny. FDA has established protocols for fish authentication and these include a thorough taxonomic verification of the reference species. We have previously collected and established a taxonomically validated reference library for numerous fish species and will add the validated tuna specimens to this database. Once validated and cataloged a series of primers targeting mitochondrial and/or nuclear genes for discriminating between the six Thunnus species will be designed by data-mining both information of genetic targets in previously published phylogenetic studies and in-house fish genetic markers. A strategy incorporating sequencing, alignments, phylogenetic analyses, minibarcode method and primer/probe approach for the most promising gene target(s) will be taken in developing these primers. DNA extracted from tuna tissue plugs in accordance with our previously established methods will be assessed for quality by agarose gel and quantitated by UV absorbance using a NanoDrop or by a fluorescent dsDNA-selective dye binding assay. All reference tuna species DNA will be amplified with degenerative primers corresponding to each of the identified target genes. All amplicons generated will be bi-directionally sequenced, edited and assembled (using Sequencher 4.1) then trace files meeting a defined minimum base length cut off for each target gene will be uploaded for further analysis. For each target gene, sequences representing all tuna species in this study will be analyzed using a series of alignment and phylogentic analysis tools to distinguish base changes associated with divergence within species verses between species and nearest neighbor values will be calculated. Identified differential bases and base stretches between species will be the target of subsequent primer design. Both a conventional PCR and real time PCR platform will be used in testing tuna species specific primers and 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.

Progress 06/15/11 to 02/14/12

Outputs
OUTPUTS: Tuna is one of the world's most valuable commercial seafoods. The six species in the Thunnus genus (Atlantic bluefin, Southern bluefin, Pacific bluefin, albacore, yellowfin and bigeye) are considered the most popular tuna species and are the focus of this SBIR project. Increasing international demand for these high-value tuna species highlights a growing regulatory concern for proper species identification of tuna in commerce as well as stock assessment, management and conservation of these fish. Currently there are no validated diagnostics available for distinguishing all of these commercially important species of tuna. The objective of this Phase I proposal was to show proof-of-concept for a reliable and species discriminating DNA-based diagnostic that could be developed to definitely distinguish the various high value tuna species in commerce. The Outputs for the project include the following: Activities: procurement and taxonomical validation of the six most popular tuna species; conducting and analyzing experiments extracting genomic DNA, identifying and amplifying species specific gene targets, and designing primers and probes to discriminate the tuna species. Services: After this diagnostic is optimized and validated in Phase II we will utilize the diagnostic in our in-house species identification testing service. We will also train others on the use of this diagnostic. Products: The six most valued tuna species have been added to our database of taxonomically validated reference fish. Methods were developed to utilize DNA PCR and sequencing to identify these tuna species in commerce. The long term goal of this proposal is to develop a DNA diagnostic that will provide an accurate evaluation tool for tuna distributors, processors, wholesalers, retailers and government regulators for properly labeling fresh, frozen and canned tuna products. This molecular diagnostic will also be beneficial to tuna fisheries management practices and goals. Dissemination: Once Phase II is completed we will advertise that this new tuna species identification diagnostic is available. The DNA-based method will be streamlined into kit form. This kit will contain detailed sampling protocols, extraction method and materials, all test reagents and proprietary primer/probe sets. The primary end-users will be Federal and state regulatory agencies including FDA, NMFS, US Customs, State Departments of Agriculture, Environmental Protection and Natural Resource Departments. The kits will also be available to public and private laboratories and inspection services. PARTICIPANTS: Project Director, LeeAnn Applewhite, organized and directed all the key activities of the project. Applewhite established time lines for the objectives, administered the grant funds and assisted in preparing all reports. Research Scientist, Barbara Carter, designed and implemented all the research protocols for the project, thoroughly reviewed all data obtained, and closely supervised all the laboratory work. Dr. Maureen Dolan, ASU subcontract, assisted with DNA extraction protocols and development of tuna species specific primers and probes. TARGET AUDIENCES: Once the DNA diagnostic is further validated, members of the seafood industry, commercial laboratories, and Federal and State laboratories will have the first DNA based diagnostic to definitively distinguish the top value tuna species from other commercially important fish species. AFT is a leader in seafood species identification diagnostic development. All the diagnostics developed have been commercialized in testing programs for the food industry. The development of DNA based species identification diagnostics and their implementation in monitoring seafood in commerce has dramatically reduced intentional mislabeling of seafood. This tuna diagnostic will also be utilized in fishery management programs worldwide. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
We succeeded in meeting the goals set forth in our Phase I Research Proposal technical objectives. Success achieved in the 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 including skipjack, (Katsuwans pelamis), blackfin (Thunnus atlanticus) and Atlantic bonito (Sarda sarda) 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 pouches). Our findings in this Phase I feasibility study establishes that AFT has a scientifically sound and effective diagnostic design strategy for delivering a species discriminating DNA I.D. test for tuna. New methods and techniques were utilized that led to the success of this project. These improved technologies will be adopted and used in our testing service as well as in updated reference methods such as FDA's DNA Barcoding for Fish Species Identification. This diagnostic will address the immediate needs of the commercial seafood industry in eliminating tuna mislabeling in commerce as well as in providing a valuable tool for conservation management of one of the most endangered trade fish in the world. This project has commercialization potential for the development of user friendly test methods that can be run with minimal laboratory equipment and minimal training.

Publications

  • No publications reported this period