Abstract:Seriola is a genus of Carangidae in Perciforms; they are long-distance migratory oceanic species with global distribution and inhabit temperate and subtropical marine waters worldwide. There are nine species in Genus Seriola. Three species, including S. lalandi, S. dumerili, and S. quinqueradiata, are found in China's coastal waters. Seriola fish is large, fast-growing, and highly favored by the international consumption market due to its excellent taste, nutritional quality, and economic value; furthermore, they are promising for open ocean aquaculture. In 2017, a significant breakthrough was achieved in seedlings production of S. lalandi by Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences in China, and the scaled juveniles' production technology was established in 2019. In 2020, another breakthrough was achieved in seedlings production of S. quinqueradiata in China. Since 2017, the farming industry of Seriola fish in China has entered the fast development era. Nowadays, the Seriola species are farmed in Liaoning, Fujian, Shandong provinces of China, etc., and the annual farming yield was about 20 thousands of tons in China since 2018. These three Seriola species, natively distributed in China, have similar exterior morphology and are hard to differentiate with naked eye and traditional differentiation methods, especially in case of the juveniles. In addition, an allotype phenomenon exists in Seriola fish in global oceanic waters. Therefore, it is necessary to establish a simple and efficient molecular species identification method to facilitate the species and population determination of Seriola fishes in international oceanic waters. Moreover, it also could be beneficial for discrimination, conservation, and sustainable utilization of natural fishery resources in international public waters. Many scientists have studied the germplasm and population genetic characteristics of S. lalandi, S. dumerili, and S. quinqueradiata from international waters using nuclear genes and mitochondrial markers. Still, there is no information on the genetic background of Seriola fishes in China. DNA barcodes are widely applied to species identification, systematic analysis, and population genetics of fish because of their sensitivity, accuracy, and reliability, especially for CO I, cytochrome oxidase b (Cyt b) genes, etc. Furthermore, when DNA barcodes are applied to species identification or hidden species analysis, the combination of several DNA barcodes could be more high-efficient and accurate. Thus, in the present study, to explore the applicability of Cyt b and NADH dehydrogenase subunit 1 and 2 (ND1 and ND2) to species identification and evolutionary analysis of three Seriola species, including S. lalandi, S. dumerili, and S. quinqueradiata were investigated and determined. The finclips samples were collected from three native distributed Seriola species. S. lalandi samples were also collected from Australian and Japanese populations. Genomic DNA was extracted from finclips, specific primers for Cyt b, ND1, and ND2 genes were designed. The PCR reaction system of the three genes totalled 50 μL each, Including rTaq enzyme 25 μL, template 2 μL, upstream and downstream primer 1 μL each, and ddH2O 21 μL. The results showed that the target fragments of three genes could be amplified from the genomes of three Seriola species, and 68 gene sequences were obtained. The sizes of Cyt b, ND1, and ND2 fragments were 538 bp, 673 bp, and 907 bp in length, respectively, which were consistent with the predicted results, wherein Cyt b gene sequence showed obvious A+T bias characteristics. The mutation rate of the ND2 gene was 20.52%. The genetic diversity of ND2 gene (Hd = 0.900, Pi = 0.082) was higher than that of ND1 (Hd = 0.874, Pi = 0.077) and Cyt b (Hd= 0.814, Pi = 0.061). The results indicated that Cyt b, ND1, and ND2 could be used to identify the three Seriola species natively distributed in the China oceans. In Seriola species, the interspecific genetic distances of the three genes were more than ten times the intraspecific genetic distances. Furthermore, the evolutionary tree based on Cyt b, ND1, and ND2 shows that Chinese and Japanese S. lalandi are clustered into independent branches, effectively distinguishing Chinese, Japanese, and Australian S. lalandi populations. In addition, S. lalandi and S. quinqueradiata were clustered together, while S. dumerili and S. rivoliana were clustered together, showing similar genetic relationships with each other. Based on the Cytochrome b gene, S. carpenteri and S. dumerili showed high sequence homology, were clustered on the same node, and could not be effectively distinguished. It can be seen that except for Cyt b, the maximum-likelihood tree constructed based on ND1 and ND2 genes has accurate identification ability for different fish species from the Seriola genus. In summary, Cyt b, ND1, and ND2 could be used as DNA barcodes for species identification and geographical differentiation of Seriola fishes. A variety of DNA barcodes could be combined to achieve more precise identification results. These DNA barcodes could also be applied to evaluate and conserve the genetic diversity of farmed Seriola species, which could be beneficial for sustainable utilization of the germplasm resources of Seriola species.
