Development of a DNA barcode for species identification of tuna

Abstract: Today, DNA-barcoding with the gene COI is regularly used in the identification of fish. However, this is not an adequate way of identifying species of tuna due to COI lacking sufficient interspecies divergence. This is problematic since fraud and mislabeling are a major concern within the fish and tuna industries. Thus, there is a need for a new genetic barcode region when identifying the 15 tuna species within the tribe Thunnini. This study has considered six mitochondrial genetic regions (16S, ATP8, COII, CR, CytB, and ND2) and their potential as barcodes in comparison to COI. To be of practical use, the barcode has to be able to differentiate between all 15 tuna species, as well as contain conserved primer binding sites and be approximately 400 bp, or shorter. Analyses of the regions were made through Multiple Sequence Alignments built using ClustalW in Mega 11.0. The candidates were first evaluated through neighbor-joining trees and plots of inter- and intraspecies variation, and then analyzed further in search of conserved regions for primer binding, flanking a segment of approximately 400 bp (or shorter). This resulted in two possible barcode candidates with corresponding primers from the CR and ND2 genes. As a final step, these two were analyzed for specificity using BLAST, to evaluate their actual utility in differentiating the tuna species. The results show that they both can identify the different tuna species, but that ND2 is superior with 100% identification accuracy. In addition to the theoretical analysis, the ability of the primers was measured through a real PCR amplification. Unfortunately, only the CR barcode could be evaluated, but the results show it to be practically useful. Even though the utility of ND2 in PCR could not be analyzed, it is highly recommended as a region for further investigations. Given the strong theoretical support, it definitely shows promise as a new barcode for species identification of tuna.