Multiplex PCR Assay for Simultaneous Identification of Five Types of Tuna (Katsuwonus pelamis, Thunnus alalonga, T. albacares, T. obesus and T. thynnus).

There is a need to identify the species of similar types of fish, especially those that are commercially sold. Particularly, the price of tuna varies depending on its type, which is difficult to determine as they are sold in cut or processed forms. This study developed a multiplex polymerase chain reaction (PCR) assay to identify the five most common tuna species: bigeye, skipjack, Atlantic bluefin, albacore, and yellowfin tunas. Newly designed species-specific primer sets for these five tuna species were created. Subsequently, the amplicon sizes obtained were 270, 238, 200, 178, and 127 base pairs for bigeye, skipjack, Atlantic bluefin, albacore, and yellowfin tunas, respectively. Each primer's specificity was further tested using 15 other fish species, and no cross-reactivity was observed. To identify multiple targets in a single reaction, multiplex PCR was optimized to increase its resolution and accuracy. The detection levels of the multiplex PCR assay were confirmed to be 1 pg for all the five tunas. Additionally, it was successfully applied to 32 types of commercial tuna products. Therefore, this multiplex PCR assay could be an efficient identification method for various tuna species.

Variation in the Karyotype, Cytochrome b Gene, and 5S rDNA of Four Thunnus (Perciformes, Scombridae) Tunas.

Yan-Horn Lee, Tsair-Bor Yen, Chiu-Fen Chen, and Mei-Chen Tseng (2018) Thunnus tunas in Scombridae are divided into the temperate subgenus Thunnus (bluefin group) and tropical subgenus Neothunnus (yellowfin group) species based on anatomic traits and distributions. The main purpose of this study was to examine the systematic status of T. obesus based on karyotype, cytochrome (Cyt) b gene, and 5S ribosomal DNA sequences. All T. obesus, T. albacares, T. alalunga, and T. orientalis specimens were caught in southeastern coastal waters off the main island of Taiwan. The karyotypical formula of T. obesus was 2 m + 2 st + 44 t, that of T. albacares was 2 m + 2 sm + 2 st + 42 t, that of T. alalunga was 2 m + 2 sm + 2 st + 42 t, and that of T. orientalis was 2 m + 2 sm + 44 t (m: metacentric; sm: submetacentric; st: subtelocentric; t: telocentric chromosome). According to a molecular genetics analysis for these species using Cyt b gene sequences (1141 bp), interspecific genetic distances ranged from 0.004 (T. orientalis vs. T. alalunga) to 0.038 (T. alalunga vs. T. obesus). The genealogy tree exhibited these 4 species as being categorized into 4 monophyletic groups with high bootstrapping values; T. alalunga and T. orientalis are sister species. This result suggests that the species currently allocated in Thunnus and Neothunnus might need new taxonomic characters to redefine the monophyly of the two subgenera. The sequence lengths of all cloned 5S genes from the 4 species ranged from 327-342 bp. Interspecific genetic distances ranged from 0.016 (T. orientalis vs. T. alalunga) to 0.111 (T. orientalis vs. T. albacares). The phylogenetic tree based on 5S rDNA shows T. obesus divided into 2 groups: one similar to T. albacares and the other close to T. orientalis. These results imply that Thunnus tunas have a common synapomorphic character with Scombridae fish (2n = 48) and high numbers of telocentric chromosomes (42-44). Thunnus orientalis and T. alalunga are sister based on molecular data. Thunnus obesus may have been derived from a more-complicated speciation processes.