基于Cyt b、ND1及ND2的DNA条形码在鰤属鱼类物种鉴定中的应用

Application of DNA barcoding based on Cyt b, ND1 and ND2 in Seriola species identification

中文关键词: 鰤属鱼类  DNA条形码  物种鉴定  系统进化分析

英文关键词: Seriola species  DNA barcode  Species identification  Phylogenetic analysis

中文摘要:

      为建立我国鰤属(Seriola)鱼类快速有效的分子鉴别技术，从DNA条形码角度出发，分析了线粒体细胞色素b (Cyt b)、NADH脱氢酶(ND1和ND2)基因在黄条鰤(Seriola lalandi)、高体鰤(Seriola dumerili)和五条鰤(Seriola quinqueradiata)等鰤属鱼类物种鉴定和系统进化以及地理区域鉴别中的适用性。结果显示，Cyt b基因表现出明显的A+T偏倚性，ND2基因序列突变速率较高，变异率为20.52%，ND2基因(Hd=0.900, Pi=0.082)的遗传多样性高于ND1 (Hd=0.874, Pi=0.077)和Cyt b (Hd=0.814，Pi=0.061)。比较了鰤属鱼类3种基因序列的结构特征，基于ND1和ND2基因计算的鰤属鱼类种间遗传距离都为种内遗传距离的10倍以上，但Cyt b基因对高体鰤和几内亚鰤(Seriola carpenteri)辨识力不足。系统进化分析显示，每个物种都形成单系分支，3个基因均能对我国3种鰤属鱼类进行鉴别，且都可有效区别来自全球3个不同水域的黄条鰤种群。因此，Cyt b、ND1和ND2基因不仅可作为鰤属鱼类物种鉴定的有效DNA条形码，还可作为不同地理种群划分和种质资源科学保护的依据，为我国鰤鱼养殖产业的持续健康发展和种质资源的可持续利用提供技术依据。

英文摘要:

      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.