Using mini-barcodes coupled with high resolution melting (minibar-HRM) method for species discrimination across Pangasianodon gigas, Pangasianodon hypophthalmus and Pangasius larnaudii

Abstract

Pangasianodon gigas (giant catfish) is well-recognized as a critically endangered species due to overhunting and habitat degradation. Giant catfish aquaculture has expanded to meet the ever-increasing demands with augmented prices. Contamination or substitution of P. gigas products with other related species e.g. Pangasianodon hypophthalmus and Pangasius larnaudii because of their similar morphology but lower cost requires investigation. A method was established to differentiate species by mini-barcodes or microsatellites conjugated with high resolution melting analysis (minibar-HRM or microsatellite-HRM) using mitochondrial genes cytochrome c oxidase subunit I (COI), cytochrome b (Cytb), the nuclear recombination activator gene 1 (Rag1) and two microsatellite loci (Pg-9 and Pg-13). Results revealed that primer sets for the mitochondrial genes PanCytb1 and PanCytb2 showed a significant difference in melting shapes and melting temperature (Tm) between the three species. For nuclear genes, a single locus was not sufficient to discriminate between the three species, and at least two loci such as PanRag1 + Pg9 or PanRag1 + Pg13 were required. The sufficient amount of DNA for species discrimination using minibar-HRM with PanCytb1 and PanCytb2 was suggested as at least 5 pg. This method can also be used for adulteration determination of the P. gigas substitution with P. hypophthalmus and P. larnaudii at greater than 5% adulteration. Findings demonstrated that mini-barcode or microsatellite-HRM offered optimal performance for species discrimination of P. gigas, P. hypophthalmus and P. larnaudii. This approach can be adopted for hybrid detection using PanRag1 + Pg9 or PanRag1 + Pg13-HRM and PanCytb1 or PanCytb2 for matrilineal detection to benefit aquaculture research and quality control on a commercial scale.