High throughput identification of tuna (Thunnus spp.) larvae in the Gulf of Mexico using unlabeled-probe high-resolution melting analysis (UP-HRMA).

Abstract

The genus Thunnus (family Scombridae) comprises eight species of tunas of which all but one are targeted by industrialized fisheries. While intact individuals of these species can be distinguished by morphological characteristics, researchers and managers often rely on dressed, frozen, juvenile, or larval fish samples which often necessitates molecular species identification. Here we investigate short amplicon (SA) and unlabeled probe (UP) high-resolution melting analysis (HRMA) as a low-cost, high-throughput molecular genotyping assay capable of distinguishing between albacore tuna (T. alalunga), blackfin tuna (T. atlanticus), bigeye tuna (T. obesus), Atlantic bluefin tuna (T. thynnus), and yellowfin tuna (T. albacares) in the Gulf of Mexico. While SA-HRMA of variable regions in the NADH dehydrogenase subunit 4 (ND4) and subunit 5 (ND5), and subunit 6 (ND6) of the mitochondrial DNA (mtDNA) genome did yield some species-specific diagnostic melting curves (ex. ND4 assay can reliably distinguish Atlantic bluefin tuna) genotype masking produced excessive variation in melting curves for reliable multi-species identification. To minimize the genotyping masking of SA-HRMA a 26 base pair long UP containing four SNPs was developed within a 133 bp segment of ND4. The UP-HRMA is able to reliably distinguish Gulf of Mexico species T. thynnus, T. obesus, T. albacares, and T. atlanticus by UP melting temperature at 67°C, 62°C, 59°C, and 57°C respectively. The developed UP-HRMA assay is a lower-cost, higher-throughput, alternative to previously published molecular assays for tuna identification that can be easily automated for large data sets including ichthyological larval surveys, fisheries specimens lacking distinguishing morphological characteristics, or detection of fraudulent trading of tuna species.