Abstract:Tuna is rich in nutrients and one of three major fish recommended by the International Nutrition Society. Large tuna such as Thunnus albacares, T. maccoyii, T. obesus, and T. thynnus are used as top-grade aquatic products, mainly in the form of sushi or other fresh fish products. Small tuna such as T. alalunga and Katsuwonus pelamis are mainly used in canned tuna and processed katsuobushi. The price of different species of tuna varies greatly, especially because selling tuna without the head or skin can lead to mislabeling and substandard products, which harms the interests of consumers and undermines market functions. This study was aimed at the identification T. albacares, T. maccoyii, T. obesus, T. thynnus, T. alalonga, and K. pelamis. Using tuna DNA as a template, three pairs of universal primers were used to amplify and sequence the DNA fragments of three genes (16S rRNA, Cyt b, and COⅠ) from six species of tuna. The sequencing results were manually corrected to remove the primer regions, which were compared with reference sequences in GenBank by BLAST analysis on the National Center for Biotechnology Information website. The COⅠ gene was analyzed by the Barcode of Life Data System (www.boldsystems.org). As a result, DNA sequences of 16S rRNA (576 bp), Cyt b (652 bp) and COⅠ (307 bp) were obtained from 63 samples of six species of tuna. Genetic diversity parameters such as Number of Haplotypes (h), Haplotype diversity (Hd), Average number of nucleoside difference (k), and Nucleotide diversity (π) were calculated by DnaSP 5.10 software. C-conserved sites, PI-parsimony-informative sites, and V-variable sites were calculated using Mega 7.0 software, which performed 1000 bootstrap tests with neighbor joining. The Kimura 2-parameter model was used to construct a phylogenetic evolutionary tree. The experimental results showed that 16S rRNA was relatively conservative and could not distinguish among the six tuna species. Cyt b and COⅠ could be used to identify the species. However, it is possible that COⅠ and Cyt b sequences can be used together as a DNA barcode for tuna research. A multi-gene DNA-barcode species-identification method that provides technical support for the identification of tuna and its fish products would be of great significance in promoting the accurate identification and healthy development of aquatic products.
