DNA barcoding of fishes from Zhoushan coastal waters using mitochondrial COI and 12S rRNA genes

Accurate species identification is a key component of biodiversity research.DNA barcoding is an effective molecular method used for fish species identification.We aimed to study the DNA barcoding of fish in Zhoushan coastal waters,explore the differences and applicability of two gene fragments(12S rRNA and COI)of DNA barcoding in fish species identification,and established a comprehensive fish barcoding reference database.Two hundred and eighty-seven captured fish samples from Zhoushan coastal waters were identified using morphological characteristics and DNA barcoding.A total of 26412S rRNA sequences(belonging to eight orders,31 families,55 genera,and 66 species)and 188 COI sequences(belonging to seven orders,30 families,48 genera,and 58 species)were obtained.The lengths of the 12S rRNA sequences ranged from 165 to 178 bp,and the guanine-cytosine(GC)content was 45.37%.The average 12S rRNA interspecific and intraspecific genetic distances(K2P)were 0.10%and 26.66%,respectively.The length of the COI sequence ranged 574–655 bp,and the content of GC was 45.97%.The average 12S rRNA interspecific and intraspecific genetic distances(K2P)were 0.16%and 27.45%,respectively.The minimum interspecific genetic distances of 12S rRNA and COI(1.23%and 1.86%)were both greater than their maximum intraspecific genetic distances(2.42%and 8.66%).Three molecular analyses(NJ tree,ABGD,and GMYC)were performed to accurately identify and delineate species.Clustering errors occurred when the 12S rRNA sequences were delimited using the NJ tree method,and the delimitation results of ABGD and GMYC are consistent with the final species identification results.Our results demonstrate that DNA barcoding based on 12S rRNA and COI can be used as an effective tool for fish species identification,and 12S rRNA has good application prospects in the environmental DNA(eDNA)metabarcoding of marine fish.

Hippocampal mitochondrial cytochrome C oxidase activity and gene expression in a rat model of chronic cerebral ischemia

The present study established a rat model of chronic cerebral ischemia using bilateral common carotid artery permanent ligation to analyze cytochrome C oxidase activity and mRNA expression in hippocampal mitochondria. Results showed significantly decreased cytochrome C oxidase activity and cytochrome C oxidase II mRNA expression with prolonged ischemia time. Further analysis revealed five mitochondrial cytochrome C oxidase II gene mutations, two newly generated mutations, and four absent mutational sites at 1 month after cerebral ischemia, as well as three mitochondrial cytochrome C oxidase III gene mutations, including two newly generating mutations, and one disappeared mutational site at 1 month after cerebral ischemia. Results demonstrated that decreased cytochrome C oxidase gene expression and mutations, as well as decreased cytochrome C oxidase activity, resulting in energy dysmetabolism, which has been shown to be involved in the DatholoQical Process of ischemic brain iniurv.

Genetic difference of Chinese horseshoe crab(Tachypleus tridentatus) in southeast coast of China based on mitochondrial COI gene analysis

Population genetic structure and historical demography of Chinese horseshoe crab (T.tridentatus)along southeast coast of China were inferred from the sequence data of mitochondrial cytochrome c oxidase subunit Ⅰ (COI) fragment.The sequence analysis for 964 bp COI fragment was conducted in 28 individuals collected from five localities:Ninghai in Zhejiang Province,Meizhou and Zhangpu in Fujian Province,Beihai of Guangxi Zhuang Autonomous Region and Danzhou of Hainan Province.Sequence variation was relatively low with a total of seven transitions observed.In all localities,Haplotype H3 was the dominant type observed among eight haplotypes defined previously,and was at the center of radiation in Median-Joining network.The prolonged star-like network suggests a signature of population expansions.The level of diversity was low in total,with haplotype diversity ( Hd) being equal to 0.765 and nucleotide diversity (π) being equal to 0.00118,respectively.The genetic structure analysis revealed the significant genetic difference between Ninghai and Danzhou populations.Both mismatch distribution analysis and Fu's Fs test provided consistent inference of historic population expansion.The low genetic diversity of horseshoe crab observed along China coast indicated that urgent measures should be taken to protect this rare marine animal.

Molecular Phylogenetic Analysis of the Main Lineages of Nymphalinae(Nymphalidae: Lepidoptera) Based on the Partial Mitochondrial COI Gene

The phylogenetic relationships of the subfamily Nymphalinae （sensu Chou 1994） were analyzed based on 1 488 bp of mtDNA cytochrome oxidase subunit I （COI） gene sequence data obtained from 24 individuals, along with those of eight species obtained from GenBank. The base compositions of this COI fragment varied among the individuals as follows： T 39.9%, C 14.6%, A 32.2%, and G 13.4%, with a strong AT bias （72.1%）, as usually found in insect mitochondrial genomes. The A ＋T contents of the third, second, and first codon positions of the COI fragments in this study was 92.4, 62.2, and 61.4%, respectively. The phylogenetic trees were reconstructed by neighbor-joining （NJ）, maximum likelihood （ML）, and Bayesian methods by using Byblia anvatara as outgroup. Phylogenetic analyses based on the COI gene sequence data created very similar topologies, which were producing trees with two main clades A and B, and five subclades. The data indicated that the tribes Nymphalini and Hypolimni （sensu Chou 1994） are not monophyletic groups, and the genus Junonia should be removed from Nymphalini to Hypolimni （=Junoniini）. On the basis of the data, the Symbrenthia and Araschnia had a relative distant relationship with the rest of Nymphalini. The relationships of species in the Nymphalini were confirmed via the NJ, ML, and Bayesian methods, namely （（（（Nymphalis ＋ Kaniska） ＋ Polygonia） ＋Aglais） ＋ Vanessa） ＋ （Symbrenthia ＋Araschnia）. This investigation provides a little novel information for Chinese researches of butterflies.

Cryptic species composition and genetic diversity within Bemisia tabaci complex in soybean in India revealed by mtCOI DNA sequence

Bemisia tabaci is a cryptic species complex, causing signiifcant loss on many agricultural y important crops worldwide. Knowledge on species composition and diversity within B. tabaci complex is critical for evolving sustainable pest management strategies. Here we investigate the whitelfy species complex in soybean in major soybean growing states of India. The mitochondrial cytochrome oxidase gene subunit-1 (mtCOI) based phylogenetic relationships established using Bayesian methods indicated the existence of three cryptic species namely Asia I, Asia II 1, and Asia II 7. Al the haplotypes detected in the study could be assigned to these three cryptic species fol owing the species demarcation criteria of 3.5%divergence threshold. Of these, Asia II 1 was found to be predominant with wide spread distribution across the surveyed regions from cool temperate zones to hot and humid tropical plains. On the contrary, cryptic species Asia II 7 showed localized distribu-tion. The Asia II 1 exhibited the highest haplotype diversity and Asia I showed high level of nucleotide diversity. There was a signiifcantly high genetic differentiation among these three cryptic species. The MEAM 1, a dreadful invasive species was not detected in the specimens tested in the current study. The diversity and distribution of three cryptic species is discussed in the light of current knowledge on distribution of whitelfy species in India and yel ow mosaic disease observed during sampling survey.

The complete mitochondrial genome of Triuncina daii(Lepidoptera:Bombycidae) and its phylogenetic implications

The bombycid moth, Triuncina daii Xing Wang ＆ Zolotuhin, 2015, plays an important role for analyzing the phylogenetic relationships of the family Bombycidae （Lepidoptera： Bombycoidea）. Here we first describe the complete mitochondrial genome （mitogenome） of T. daii, which includes thirteen protein-coding genes （PCGs）, twenty-two transfer RNA genes, two ribosomal RNA genes and an A＋T-rich region, and we find the mitogenome is 15,482 bp in length （GenBank no. KY091643）. The genes order and orientation in the T. daii mitogenome are similar to other sequenced lepidopteran species. Except for cox1, all of the PCGs started with ATN. Twelve PCGs stopped at TAA except for cox1 which stops at a single T. Thirteen PCGs of available species are used to demonstrate the inner phylogenetic relationships of Bombycoidea. The bombycid species form a monophyletic clade with a bootstrap value of 100% and a posterior probability of 1.00.

Genetic Variability and Reproduction Structure of <i>Corbicula japonica</i>in Major Fishing Brackish Lakes in Japan

Corbicula japonica is the best-known bivalve inhabiting widely in brackish estuaries and lakes in Japan. Although this species has been most commercially important species of inland fisheries in Japan, the gradual decline in its production over 40 years caused not only economic problems in fishery industry but also ecological disturbances in biodiversity conservation. The aim of this study was to evaluate the reproduction structure of C. japonica populations in major fishing brackish lakes based on the genetic diversity inferred by mitochondrial DNA sequence analysis of the cytochrome oxidase c subunit I gene. Of a total of 188 C. japonica individuals collected in Lakes Shinji, Jusan, Ogawara and Abashiri, 25 haplotypes were obtained, and only the haplotype HT01 was apparent with relatively high abundance in all lakes. Minimum spanning network analysis of haplotypes showed different population structures between Lake Shinji and Lakes Jusan, Ogawara and Abashiri. In addition, pairwise population genetic distance FST and ΦST values were significantly higher in Lake Shinji than Lakes Jusan, Ogawara and Abashiri. The mismatch distribution analysis showed unimodal profile for Lakes Jusan and Ogawara and bimodal profile for Lakes Shinji and Abashiri. Those results indicate a recent population expansion in all lakes, and Lakes Shinji and Abashiri and Lakes Jusan and Ogawara maintained continuous reproduction structure and suffered to rapid population growth, respectively.

Genetic diversity and population structure of the sea star Asterias amurensis in the northern coast of China

The sea star Asterias amurensis is widely viewed as a severe“marine pest”because of its broad feeding habits.Over the past few decades,A.amurensis undergoes massive and sporadic population outbreaks worldwide,causing extensive economic and ecological losses to the local aquaculture industry and marine ecosystem.Understanding the genetic diversity and population structure of A.amurensis can provide vital information for resource management.By analyzing the polymorphism of the mitochondrial cytochrome C oxidase subunit I(COI)gene and ten simple sequence repeat(SSR)microsatellites markers,the genetic diversity and population structure of A.amurensis of four populations along the northern coast of China was uncovered.A total of 36 haplotypes were identified,and a main haplotype was found in four populations.The Qingdao(QD)population displayed the highest genetic diversity among all the populations.The AMOVA and pairwise F_(st)showed that there was small but statistically significant population differentiation among the four populations,especially between QD and Weihai(WH).Moreover,the principal component analysis(PCA)and admixture analysis showed that several individuals in Yantai(YT)and Dalian(DL)had little genetic association with other individuals.Overall,this study provided useful information of the genetic diversity and population structure of A.amurensis and will contribute to the resource management of A.amurensis in China.

Availability of Myoglobin as a Molecular Marker for Phylogenetic Relationships of Fish

In order to elucidate the phylogenetic relationship of fish, DNA and deduced amino acid sequences of myoglobin （Mb） were used for the phylogenetic analyses based on different approaches, namely, maximum likelihood （ML）, neighbor joining （NJ）, unweighted pair group method with arithmetic means （UPGMA） and maximum parsimony （MP） methods in comparison with the conventional molecular markers, mitochondrial cytochrome b （cyt-b） and cytochrome c oxidase subunit I （COI）. The phylogenetic trees drawn based on Mb sequences were similar to those by the traditional classification based on the other molecular markers. The primary and secondary structures, as well as the modeled tertiary structures of Mbs were similar to each other, but were clearly distinguishable among those species. Such differences in structure would be associated with adaptation of Mb molecule to the physiological conditions of each species. These results suggest that Mb can be a molecular marker for the phylogenetic relationship of fish.

两种鲷属鱼类线粒体COI基因片段序列的比较

利用通用引物成功扩增了黄鳍鲷和黑鲷的线粒体细胞色素氧化酶Ⅰ亚基（COI）基因序列。通过序列测定，得到581bp的基因片段，碱基A、T、G、C平均含量分别为25．8％、32．6％、18．0％和23．6％，序列中的A＋T含量明显高于G＋C含量，与其他鱼类的C01基因片段研究结果相一致。通过序列分析发现黄鳍鲷和黑鲷两序列共存在14处碱基变异，其中在第19～139bp之间检测到13个变异位点，是变异频率较高的区段，可以考虑作为鲷属或者种群鉴别的分子标记。与黄鲷、真鲷、三长棘真鲷等鱼类比较，无插入和缺失位点，转换明显高于颠换。序列差异比较结果显示，真鲷与黄鳍鲷的序列差异在这5种鱼类中最大，达到了18．2％，黄鳍鲷和黑鲷的筹异最小（2．4％）。两个序列已提交到GenBank数据库中．序列臀录号为DQ185608和DQ185609.

3种鲳属鱼类线粒体COI基因序列变异及系统进化

对采自东海的银鲳（Pampus argenteus）、翎鲳（P.punctatissimus）和中国鲳（P.chinensis）3种鲳属鱼类的线粒体COI基因序列片段进行扩增和序列测定，得到603bp的基因片段，编码201个氨基酸，碱基T、C、A、G平均含量分别为34．2％、22．3％、25．9％和17．6％。所得序列共定义12个单倍型，中国鲳只有1个单倍型，银鲳3个单倍型，翎鲳8个单倍型，在这12个单倍型中共检测到109个变异位点。3种鲳密码子的使用均存在明显的偏向性，且同义密码子使用偏向性高度一致。银鲳和中国鲳的遗传距离最大（0．165～0．168），与翎鲳的遗传距离次之（0．151～0．162），翎鲳和中国鲳的遗传距离最小（0．058～0．065）。由进化树可知，翎鲳和中国鲳的进化关系最近，它们与银鲳的进化关系较远，银鲳是最早形成的种。分子水平自然选择的检验结果表明，自然选择在银鲳和翎鲳线粒体COI基因差异形成过程中起一定的作用。本研究从分子水平研究中国东海海域鲳属鱼类的分类、遗传关系和系统进化，以其了解鲳属鱼类以及与鲳科之间的亲缘关系，又为鲳属鱼类保护生物学和系统进化提供分子生物学依据。[中国水产科学，2008，15（3）：392-399]

基于线粒体COI基因部分序列的长江口虾虎鱼科鱼类系统分类

对采自长江口的7种虾虎鱼(Gobiidae)19个个体的线粒体COI基因进行测序,其他6种虾虎鱼的相应序列从Genbank中获得。序列分析结果显示7种虾虎鱼的COI基因部分序列密码子的第一位碱基的GC碱基含量最恒定,第三位碱基的GC含量最高,平均A+T的含量要高于G+C的含量,与多种鱼类的COI基因的碱基特点相似。运用MEGA5.0计算13种虾虎鱼的种间遗传距离,表明拉氏狼牙虾虎鱼(Oxyurichthy tentacularis)与孔虾虎鱼(Chaeturichthys stigmatias)归属于近盲虾虎鱼亚科,根据种间遗传距离与系统发育树的结构特征推测,触角沟虾虎鱼和矛尾虾虎鱼可能具有共同的起源。

青蟹线粒体COI假基因的分离和特征分析

线粒体DNA标记在遗传结构和系统进化研究中得到广泛应用,然而核假基因的存在对此有很大威胁。本文以中国东南沿海的青蟹(Scylla paramam osain)为研究对象,利用线粒体COI基因的通用引物和特异性引物进行扩增,分别得到34个假基因(nuc learm itochondrial pseudogenes,Num ts)和5个线粒体COI基因序列。在所获得的34个假基因中共定义了29种单倍型,根据序列的相似度,这些假基因可以分为2类,每类假基因都有各自保守的核苷酸序列。第Ⅰ类假基因存在2处插入序列和1处8 bp的缺失序列,这些位点导致了整个阅读框的移位;在第Ⅱ类假基因和5个线粒体COI序列中只有碱基替换,未发现插入和缺失序列。实验结果分析表明,这两类假基因分别代表了2次核整合事件,即核转移事件的最低值。研究结果提示了利用线粒体DNA进行青蟹遗传结构和系统进化研究时应警惕假基因的干扰。

大豆蚜捕食性天敌捕食行为的COI基因标记检测

【目的】探索大豆田捕食性天敌对大豆蚜的捕食作用。【方法】根据基因库NCBI中的一段大豆蚜细胞色素氧化酶I(COI)的基因序列(登录号为AY842503),设计了2对大豆蚜特异引物A和B,其扩增片段大小分别约为197和253 bp,应用DNA标记方法检测天敌对大豆蚜的捕食行为。【结果】种特异性检验表明,所设计引物只对大豆蚜DNA具有扩增效果,对与其同域发生的其它种类不具扩增作用;室内以引物对A与B分别检测喂食单头大豆蚜的异色瓢虫成虫和大草蛉成虫腹内靶标食物的衰变情况,结果表明,异色瓢虫成虫阳性比率的半衰期分别为喂食后3.371 h和2.814 h,大草蛉成虫分别为喂食后1.312 h和1.032 h;以A作为引物对田间捕食性天敌进行检测,结果表明,在所检测的7类群14种捕食性天敌中,5种不同种类或虫态的天敌对大豆蚜的捕食作用检出率高于50%,从高到低依次为异色瓢虫幼虫、草蛉成虫、东亚小花蝽若虫、异色瓢虫成虫、东亚小花蝽成虫;此外,田间检测表明,捕食性天敌的阳性比率与大豆蚜的种群密度呈显著正相关。【结论】DNA标记技术是探索捕食行为的有效方法之一。

三种蚊虫COII基因的克隆与序列分析

目的 测定和比较中华按蚊、白纹伊蚊和致倦库蚊细胞色素C氧化酶亚基II(COII)基因全序列 ,分析 3种蚊虫之间及与其它几种蚊虫的COII基因序列的同源性。方法 运用T A克隆技术分别获得 3种蚊虫的COII基因重组质粒 ,并进行DNA测序和结构分析。结果 3种蚊虫COII基因的核苷酸序列同源性为 84.1%～87.9% ,氨基酸序列的同源性为 85 .1%～ 89.5 % ;C +G含量为 2 3 .2 %～ 2 4.9% ,颠换频率高于转换频率。结论 基因序列同源性比较结果显示 :在COII分子结构 ,白纹伊蚊与致倦库蚊具有更近的亲缘关系。

江苏口岸小家鼠细胞色素氧化酶亚基COI基因的多态性研究

目的研究小家鼠细胞色素氧化酶亚基COI基因的多态性特征,为国境口岸鼠类的鉴定和溯源提供技术支持。方法通过对江苏口岸本底及输入性小家鼠COI基因的扩增、测序和比对,结合Gen Bank的小家鼠信息,对小家鼠进行遗传多态性分析和分子系统树研究。结果获得81个国内外小家鼠COI基因序列,测出的590个序列位点中保守位点555个,可变异位点35个,核苷酸平均差异数为7.931。建立这些小家鼠COI序列的分子系统树,该分子系统树共分为两支,其中一个分支多以江苏本底的小家鼠为主,另一分支构成复杂。结论不同地区的小家鼠之间具有一定的种内差异,小家鼠种内亚种的划分与地理位置有一定的联系。

渤海湾7种虾虎鱼科鱼类COI基因序列特征及分子分类研究

为获得渤海湾天津海域常见虾虎鱼线粒体条形码序列及系统分类信息,采集渤海湾7种虾虎鱼样本,通过形态初步鉴定物种,并提取DNA进行线粒体COI基因部分序列扩增测序,用系统进化分析及分子鉴定软件MEGA对渤海湾常见12种虾虎鱼COI基因部分片断进行序列特征分析及分子分类分析。扩增得到7种虾虎鱼COI基因片断,分析得到其平均GC含量低于AT含量,碱基在密码子位置分布中具有偏向性,且GC含量最高为第2位碱基,与多种鱼类COI基因碱基特点相似。基因密码子碱基变异率为19.4%,转换颠换比为1.3。遗传距离分析及系统进化树分析理清了渤海湾天津海域12种主要虾虎鱼的系统分类及进化关系。遗传距离显示,矛尾刺虾虎鱼(Amblychaeturichthys hexanema)和斑尾复虾虎鱼(Synechogobius ommaturus)为同种鱼。虾虎鱼COI编码基因密码子高变异率可能与其良好的环境适应性有关。

基于线粒体COI序列的中华绒螯蟹养殖群体与野生群体遗传多样性与遗传结构分析

为了解不同地区野生、养殖中华绒螯蟹Eriocheir sinensis群体的遗传多样性、遗传结构及遗传分化情况,利用线粒体细胞色素氧化酶亚基I(COI)基因序列,研究中华绒螯蟹长江流域安徽无为江段、山东东营黄河口、文献报道的辽河野生群体以及湖南大通湖、辽宁盘锦养殖群体的遗传学特征。结果显示:上述5个群体共有36个变异位点,其中简约信息位点8个。盘锦养殖群体(PJ)、东营野生群体(DY)、无为野生群体(WW)、大通湖养殖群体(DTH)以及辽河野生群体(LH)的单倍型数分别为6个、5个、9个、5个和7个,其中单倍型Hap15、16、17为LH群体的独有单倍型,单倍型Hap6、7、8、10为DY群体的独有单倍型,单倍型Hap11为PJ群体独有单倍型,单倍型Hap12、13、14为DTH群体独有单倍型,其中DTH08个体的单倍型与合浦绒螯蟹更接近。LH群体的单倍型多样性(Hd)最高,而DTH群体的核苷酸多样性(Pi)最高;WW群体的单倍型与核苷酸多样性最低。中华绒螯蟹5群体间基于COI序列的固定指数FST值为-0.034~0.089,分化程度极低,群体间的基因流为5.119~∞,显示不同种群之间基因交流较为频繁,没有明显的遗传结构。系统发育树和单倍型网络图结果显示:单倍型之间不具有明显的地理分化特征,不同地理群体之间的种质混杂情况较为严重。

叶螨线粒体COI基因中央区段的PCR扩增

根据二点叶螨线粒体COI基因序列设计1对PCR引物,对叶螨科不同种属的线粒体COI基因中央区段进行了PCR扩增。结果表明该对引物能成功扩增叶螨科7属9种叶螨的约340 bp的同源片段。由于叶螨DNA序列资料非常有限,扩大引物的使用范围成为快速获得特定基因序列的有效途径。研究结果使根据叶螨线粒体COI基因序列信息探索其系统演化成为可能。另外,还对模板DNA分离方法进行了优化。

Epiphytic zooplankton community profiles in a typical urban wetland as revealed by DNA metabarcoding

Zooplankton,a crucial component of urban wetland,are one of the effective bioindicators for monitoring the feeding stocks of organisms at higher trophic levels and assessing the ecological quality of ecosystems.However,information about the characteristics of epiphytic zooplankton community structure resulted from traditional methods is limited and hindered by the large amount of detritus and sludge attached to the macrophytes.We investigated the epiphytic zooplankton communities associated with macrophytes(Vallisneria,Nymphaea,and Thalia dealbata)in a subtropical wetland using as DNA markers of the 18 S rRNA gene and the mitochondrial cytochrome c oxidase subunit I(COI)gene.A total of 241 OTUs of zooplankton were obtained from COI amplicons,including 194 OTUs of Rotifera,22 of Cladocera,and 25 of Copepoda,while only 62 OTUs of zooplankton were obtained from 18 S rDNA amplicons including 34 OTUs of Rotifera and 28 of Copepoda.The zooplankton communities associated with the three macrophytes were similar,but they differed significantly from those in the open waters.However,there were no significant temporal differences among the zooplankton communities.Epiphytic zooplankton communities were dominated by littoral zooplankton such as Testudinella,Lecane,and Philodina.Microzooplankton,especially littoral species,utilize macrophytes as food sources and as refuges against predation.This further led to an increase inαandβdiversity of zooplankton communities in urban wetlands.Our result suggests that the joint use of multiple molecular markers could improve the taxonomic resolution and generate a comprehensive biodiversity profile of zooplankton.