16S rRNA和COI基因序列对长江口虾虎鱼科鱼类种类鉴定和系统分类的对比研究

Abstract

为了对比线粒体16S rRNA和COI基因片段在长江口虾虎鱼科鱼类种类鉴定和系统分类研究中的适用性，本文运用PCR技术，扩增了虾虎鱼科7属8种24个个体的线粒体16S rRNA和COI基因片段，并对其种间的序列差异进行比较分析。经比对后得到16S rRNA基因长度为535bp的序列，共编码178个氨基酸，24条16S rRNA序列共有12个单倍型，共检测到变异位点161个，约占总位点数的30.1%，插入/缺失位点15个，转换与颠换比值(Si/Sv)为1.51；同时，获得COI基因长度641 bp，共编码213个氨基酸，单倍型个数与16S rRNA序列相同，变异位点221个，所占百分比高于16S rRNA序列(约34.5%)，无插入/缺失位点，转换/颠换值为1.29。两种基因的转换/颠换值均小于2，因此对其做了相应的突变饱和性分析，结果显示，两种基因在虾虎鱼科鱼类中不存在突变饱和现象。遗传距离结果显示，16S rRNA基因种间序列变异程度均小于COI基因，16S rRNA基因的种内和种间平均遗传距离分别为0.002和0.169，种间约为种内遗传距离的84倍，种间遗传距离最小值存在于纹缟虾虎鱼和髭缟虾虎鱼之间为0.100；而COI序列的种内和种间平均遗传距离分别0.001和0.215，种间为种内遗传距离的215倍。因此，16S rRNA和COI序列均适用于虾虎鱼科的种类鉴定。基于16S rRNA和COI基因序列获得的UPGMA系统树显示虾虎鱼科鱼类均可形成单系群，不同之处在于16S rRNA基因序列主要表现种、属间序列差异，而COI则突出表现种、属、科间序列差异。因此，在应用这两种基因片段做系统分类研究时，应根据研究的不同系统水平选择适当的分子标记。 In order to compare the applicability of mitochondrial 16S rRNA and COI (mitochondrial cytoch-rome oxidase subunit I) in species identification and phylogeny of Gobiidae from Yangtze Estuary, this study investigated the genetic characteristics of these two gene fragments. The two gene fragments of 8 species in Gobiidae were amplified by PCR, and the sequence variation among in-terspecies was analyzed. A total of 535 bp, 12 haplotypes, 161 variation sites (about 30.1% of all), 15 insertion or deletion of sites were detected for 16S rRNA of 24 individuals, and 178 amino acids were encoded. All of the transitional pairs (si) was slightly more than that of the transversional pairs (sv), and the ratio (R = si/sv) was 1.51; while 641 bp and 221 variation sites were detected for COI, and no insertion or deletion of sites was found in the COI gene, the ratio of si and sv was 1.29. Both the ratios of the two genes from 24 individuals were less than 2, so mutation saturation of the two genes was analyzed. However, the result showed that there was no mutation saturation in the two genes of Gobiidae. The results of genetic distance showed that the degree of variation of 16S rRNA in interspecific sequence was less than that of COI sequence. Intraspecific and interspecific K2P genetic distances were respectively 0.002 and 0.169 for 16S rRNA sequences, and 0.001 and 0.215 for COI sequences. Genetic differences of 16S rRNA sequences between congeners were, on average, about 84 times higher than the differences within species, and the minimum value (0.100) was existed between Tridentiger trigonocephalus and Tridentiger barbatus; while genetic differences of COI sequences were 215 times between intraspecies and interspecies, which met Hebert’s species identification standard (more than 10 times the difference between intraspecies and interspecies). Therefore, 16S rRNA and COI sequences were both applicable for species identi-fication in Gobiidae. The UPGMA phylogenetic tree was constructed based on 16S rRNA and COI, which demonstrated that Gobiidae in the Yangtze Estuary was a monophyletic group. The major differences were that interspecies and genera were more emphasis for 16S rRNA gene, while it highlighted interspecies, genera and family more for COI gene. As a result, it is advisable to select appropriate molecular markers according to different classified levels when applying the two gene fragments for studying species identification and phylogeny.