印度-西太平洋胡椒鲷亚科鱼类DNA条形码及分子系统进化研究

为探讨COⅠ基因作为条形码在胡椒鲷亚科鱼类物种鉴定的可行性, 研究测定了胡椒鲷亚科8种鱼类51个个体线粒体COⅠ基因长度为651 bp的序列, 利用MEGA 5.0计算胡椒鲷亚科种内与种间的遗传距离, 基于最大似然法与贝叶斯法构建了系统进化树。结果显示: 胡椒鲷亚科鱼类种间平均遗传距离(0.142)显著大于种内平均遗传距离(0.003), 物种间遗传距离均大于Hebert推荐的物种鉴定最小种间遗传距离0.020(2%)。系统进化树上, 同一物种不同个体间均能形成独立的单系分支, 表明COⅠ基因可作为胡椒鲷亚科物种鉴定的有效条形码基因。研究同时揭示, 在形态分类上被认为是同种异名的两种胡椒鲷(条纹胡椒鲷与东方胡椒鲷)的COⅠ基因序列差异达到0.070, 有可能是两个独立的物种。此外, 在属级水平, 少棘胡椒鲷属与胡椒鲷属种类之间的平均遗传距离小于胡椒鲷属内部种间的遗传距离, 支持少棘胡椒鲷归属于胡椒鲷属的观点。       

基于线粒体COⅠ基因部分序列的长江口舌鳎科鱼类系统分类研究

为了确定线粒体COⅠ基因在长江口舌鳎科鱼类系统分类及物种鉴定中的作用,本实验采用线粒体COⅠ基因特异扩增测序及GenBank已有序列联合配对分析的方法,对长江口舌鳎科2属9种鱼类39个COⅠ基因片段的序列进行比较和系统进化研究。采用MEGA 5.0软件进行统计分析,舌鳎科鱼类该片段的平均AT含量高于GC含量,第1密码子位点含量最高(51.8%～57.3%,平均53.8%),第2密码子的含量稳定,均为42.0%,第3密码子变化范围最大(28.1%～37.8%,平均32.4%)。依据Kimura-2-parameter模型,9种舌鳎科鱼类种间遗传距离平均值为0.191,种内为0.003,种间遗传距离是种内的63.7倍。采用最大简约法(MP) 和邻接法(NJ)构建系统发育树,显示长江口舌鳎科鱼类为明显的单系群,但舌鳎科鱼类内部的系统发育关系与形态分类划分的亚属并不完全一致,其中须鳎属的日本须鳎(Paraplagusis japonica)与拟舌鳎亚属的宽体舌鳎(Cynoglossus robustus)聚为一支。虽然三线舌鳎亚属的种类均可聚为独立的分支,但短吻三线舌鳎(C. abbreviatus)与紫斑舌鳎(C. purpureomaculatus)、长吻红舌鳎(C. lighti)与短吻红舌鳎(C. joyneri),两组间种间遗传距离分别为0.002和0.007,两组物种间均存在同种异名现象。本研究表明,线粒体COⅠ基因作为分子标记除了能筛选出同种异名种类外,还能够对舌鳎科鱼类进行有效的物种鉴定和系统进化分析,线粒体COⅠ基因作为分类条形码是可行的。     

基于线粒体CO 1基因序列的DNA条形码在鲤科鲌属鱼类物种鉴定中的应用

本研究探讨了线粒体CO1基因作为DNA条形码对鲌属鱼类进行物种鉴定的可行性。研究中获得了鲌属4种鱼类共32个个体长度为816bp的CO1基因序列。利用MEGA软件计算鲌属鱼类种间及种内遗传距离,利用邻接法、最大简约法、最大似然法和Bayesian方法分别构建分子系统树。结果显示,鲌属鱼类的种间遗传距离显著大于种内遗传距离。在系统树中,鲌属鱼类每一物种的个体分别形成各自独立的分支。基于CO1基因的DNA条形码在识别鲌属鱼类物种方面和传统形态学基本一致,而且该基因可以探讨鲌属鱼类种间的系统发育关系。本研究表明以CO1基因作为鲌属鱼类DNA条形码进行物种鉴定具有一定的可行性。     

基于线粒体COⅠ基因的齿小蠹属昆虫DNA条形码研究

齿小蠹属（鞘翅目： 小蠹科）昆虫是植物检疫中经常截获的类群, 为探讨线粒体细胞色素C氧化酶亚基Ⅰ(COⅠ)基因的特定区段作为DNA条形码快速准确鉴定齿小蠹种类的可行性, 以齿小蠹属昆虫为研究对象, 测定分析了线粒体COⅠ基因462 bp碱基序列。序列分析结果显示： 变异位点为259个, 保守位点203个, 简约信息位点181个, 自裔位点78个。所有位点中, A, G, C和T碱基平均含量分别为30.7%, 16.5%, 17.0%和35.8%。A+T含量较高, 为66.5%, 明显高于G+C含量, 表现明显的A+T碱基偏嗜, 且A与T含量相当, 符合昆虫线粒体基因碱基组成的基本特征。转换与颠换结果显示： 该段序列未达到饱和, 可以得到准确的进化分析。利用Kimura 2-parameter模型分析遗传距离得到, 同物种间的遗传距离介于0.002~0.007之间, 不同种间的遗传距离介于0.056~0.431间, 平均遗传距离为0.199, 说明该段序列能够区分不同物种。基于COⅠ基因序列构建的邻接法系统发育树（NJ树）显示, 同一物种聚为同一小支, 且分支自展值均为100%; 近缘种能聚集在一起, 且置信度很高（≥97%）。结果表明应用基于COⅠ基因片段的DNA条形码进行齿小蠹属昆虫分类鉴定具有可行性。     

基于DNA条形码的四川老君山国家级自然保护区尺蛾科昆虫种类鉴定

为了解四川老君山国家级自然保护区鳞翅目Lepidoptera尺蛾科Geometridae资源,于2019年6—8月和2020年5—6月通过灯诱法采集2 402号标本,运用DNA条形码技术对标本进行分子鉴定。通过BOLD数据库,邻接法构建系统发育树和ABGD在线工具对获得的297条线粒体细胞色素C氧化酶亚基Ⅰ(COⅠ)基因序列进行物种界定,结合BOLD和GenBank中70条相关种序列共367条COⅠ基因序列分析遗传距离。发现297条序列隶属于191种,其中95种鉴定到种,35种鉴定到属;鉴定到属或种水平的序列隶属于92属。计算遗传距离发现,种内遗传距离为0～4.36%,平均种内遗传距离为0.63%,种间遗传距离为3.43%～19.12%,平均种间遗传距离为11.91%。结果表明,保护区尺蛾科昆虫种类丰富,基于COⅠ基因的DNA条形码技术能有效区分尺蛾科物种。     

基于线粒体COⅠ基因的沙鳅亚科鱼类DNA条形码及其分子系统发育研究

选择线粒体COⅠ基因作为分子标记,进行沙鳅亚科鱼类（Botiinae）DNA条形码及其分子系统发育研究。研究获得了沙鳅亚科7属19种共131个个体的COⅠ基因序列,利用MEGA5.0软件分析了沙鳅亚科鱼类COⅠ基因的序列特征,计算了种内及种间遗传距离。沙鳅亚科鱼类的分子系统发育关系的重建分别采用NJ法和Bayesian法。研究发现,沙鳅亚科COⅠ基因的碱基组成为: A 24.4%、T 29.5%、G 18.0%、C 28.1%。沙鳅亚科鱼类种内平均遗传距离为0.0020.000,种间平均遗传距离为0.1480.008。DNA条形码研究结果显示,所分析的19种沙鳅鱼类各自分别聚成单系分支,表明COⅠ基因在本研究中具有100%的物种鉴别率。同时,系统发育分析支持各属的单系性,并且结果显示沙鳅亚科鱼类聚为两个分支,其中一支由薄鳅属和副沙鳅属构成,另一分支则包括: （沙鳅属、色鳅属）和 中华沙鳅属、（缨须鳅属、安彦鳅属）。因此,COⅠ基因可以作为有效的分子标记对沙鳅亚科进行DNA条形码研究以及分子系统发育研究。       

我国32种鸟类DNA条形码分析

DNA条形码是一种分子分类方法,近年来在物种鉴定方面得到迅速的发展和应用.本研究分析了我国27属32种鸟类(61只)的线粒体细胞色素c氧化酶亚基Ⅰ(COⅠ)基因的条形码片段,分别用阈值法、聚类法和诊断核苷酸进行了分析,探究DNA条形码鉴定我国鸟类的准确性.结果显示,种内CO Ⅰ序列变异很小,种间存在较多的变异位点,种间的遗传距离显著大于种内的遗传距离,DNA条形码序列能够鉴定所有鸟类.     

基于CO Ⅰ基因的龟鳖目动物鉴定与分类进化分析

为探索COⅠ基因条形码技术在龟鳖目物种分类与鉴定中的适用性,本研究应用通用引物PCR扩增获得龟鳖目动物9科26属45种共205个样品的634 bp COⅠ基因序列片段进行研究分析。结果显示,COⅠ基因在龟鳖目动物中存在少量的碱基插入和缺失,共计12种20个位点,物种占比34.28%;平均碱基含量(A+T)为55.42%,显著高于(G+C)的44.58%。通过Kimura's 2-parameter计算45个物种的种内遗传距离为0～0.023 98,平均为0.003 2;种间遗传距离为0.022 3～0.334 9,平均为0.1 637,种间平均遗传距离约为种内平均遗传距离的51.16倍;有3个物种的种内遗传距离大于Hebert推荐区分物种的最小种间遗传距离0. 02,其中1个物种的种内遗传距离大于45个物种的种间最小遗传距离0.022 3。同科内不同属间及同目内不同科间的遗传距离分别为0.146 7、0.212 7,显示物种间遗传距离随着分类阶元的上升而逐步扩大。系统进化树聚类结果显示,不同个体依据亲缘关系程度不同分别聚为独立的一支,没有出现物种交叉分布,能准确反映个体间的亲缘关系。结果表明利用COⅠ基因鉴定龟鳖目物种具有可行性,但科内属间及以上分类阶元进化节点自展率不高,无法通过COⅠ基因明确其进化关系,须结合其他DNA指标加以确认。     

线粒体COI基因在少鳞鳜属(Coreoperca)鱼类条形码区段的变异分析

为了研究线粒体COI基因作为DNA条形码对少鳞鳜属(Coreoperca)鱼类进行物种鉴定的可行性.采用PCR扩增与测序技术与GenBank已有序列联合分析的方法,对少鳞鳜属3种84个个体的条形码区段进行比较分析.结果表明,碱基T(U)的平均含量为30.3％,C的平均含量为27.8％,A的含量为23.1％,G的含量为18.8％.在4种碱基中,G的含量最低,其在第三密码子的使用率上也低至9.3％.少鳞鳜属3种鱼类种内平均遗传距离值为0.003 8,种间为0.146 3,种间遗传距离是种内的38倍;通过构建的系统发育树,少鳞鳜属中的分支明确,种间无交叉情况,说明种间具有明显差异,种内没有亚种的分化.研究证明,线粒体COI基因可作为条形码序列对少鳞鳜属鱼类物种鉴别的有效片段,并且还为其物种的鉴定与分类提供辅助方法.     

猫科动物DNA条形码及线粒体假基因对物种鉴定的影响

在多种动物类群中,基于线粒体细胞色素c氧化酶亚基Ⅰ(COⅠ)基因的DNA条形码是一种高效的物种鉴别手段,然而猫科Felidae动物中广泛存在的线粒体假基因可能影响DNA条形码的有效性。本研究共涉及猫科动物12属25种119个样本。采用3对条形码通用引物对6属11种29个猫科动物样本进行了扩增及测序。结果3个样本扩增失败,8个样本得到假基因,18个样本获得了条形码序列。结合另外93条猫科动物条形码序列(源自BOLD Systems),采用Kimura 2-parameter模型计算遗传距离,构建Neighbor-Joining(NJ)树。结果显示,遗传距离种内为0%~8.1%,平均0.8%;种间为1.4%~13.1%,平均8.7%;属间为8.2%~21.8%,平均15.1%。NJ树显示,除3个种外,其余物种均以极高的置信度(99%)形成单系分支。而假基因序列有些可以单独形成分支,有些夹杂在COⅠ序列形成的分支中,对物种鉴定产生干扰。     

基于16S rDNA部分序列探讨中国近海30种石斑鱼类的分子系统进化关系

石斑鱼因其种类繁多、分布广泛及缺乏显著的形体特征,使其系统分类的研究颇为困难。为探讨中国近海石斑鱼类的系统进化关系,通过PCR扩增获得了石斑鱼亚科(Epinephelinae)6属30个种类的线粒体16SrDNA基因片段序列。采用多个生物软件对序列变异和碱基组成进行分析,计算了Kimura2parameter遗传距离、转换/颠换比等遗传信息指数,并结合GenBank石斑鱼属的同源序列,以多纹长尾(Pronotogrammusmultifasciatus)和皮氏叫姑鱼(Johniusbelengerii)为外群构建NJ、MP和ML系统树。根据所得分子依据并结合形态学特征,推论如下:1)在本研究的30种石斑鱼中,鳃棘鲈属(Plectropomus)最先分化,并呈明显单系性;九棘鲈属(Cephalopholis)是一个单系群,并且较石斑鱼属(Epinephelus)原始;侧牙鲈属(Variola)的进化地位介于鳃棘鲈属与九棘鲈属之间;2)宽额鲈(Promicropslanceolatus)可以归入石斑鱼属,而驼背鲈(Cromileptesaltivelis)也与石斑鱼属有很近的亲缘关系,甚至可能是石斑鱼属内的特化类群;3)石斑鱼属内部存在两个平行进化的姐妹分支,分支内部的种类组成与地理分布无关,暗示了石斑鱼属早期的分化模式。     

从细胞色素b部分序列探讨石斑鱼属的分子系统发育关系

石斑鱼的分类在学术界尚存在很多争议。本文采用PCR技术,测定了分布于中国的点带石斑鱼(Epinepheluscoioiaes)、青石斑鱼(E.awoara)、六带石斑鱼(E.sexfasciatus)、褐石斑鱼(E.brunneus),长棘石斑鱼(E.longispinis),吻斑石斑鱼(E.spilotoceps),巨石斑鱼(E.tauvina)以及石斑鱼亚科侧牙鲈属的侧牙鲈(Variola louti)的线粒体细胞色素b基因402bp的序列,结合GenBank提供的7种中国无记录的石斑鱼:青铜石斑鱼(E.aeneus)、海丰石斑鱼(E.haifensis)、犬牙石斑鱼(E.caninus)、东大西洋石斑鱼(E.marginatus)、白斑石斑鱼(E.multinotatus)、德氏石斑鱼(E.drummondhayi)、淡点石斑鱼(E.labriformis),把这14种石斑鱼作为内群,对他们的序列组成和结构特点进行了分析;并以同为科(Serranidae)、石斑鱼亚科(Epinephelinae)的侧牙鲈属(Variola)的侧牙鲈作外群,分别用MP法、NJ法对内、外群构建了分子系统树,结果表明:(1)14种石斑鱼402bp的mtDNA Cytb部分序列的碱基组成上,A+T的含量为53.6%高于G+C含量(46.4%),序列中转换颠换比为4.78,没有突变饱和;(2)青石斑鱼与六带石斑鱼、点带石斑鱼与巨石斑鱼、长棘石斑鱼与吻斑石斑鱼分别聚在一起,与形态分类结果一致;(3)系统树中,大西洋与太平洋种类各枝交替汇合,表明石斑鱼的Cytb序列有严格的保守性,也可能是协同进化的结果;(4)揭示在良种选配或遗传管理时,避免居于同枝的石斑鱼混交,将大西洋种类与太平洋种类杂交可能是遗传改良的一种途径。       

Molecular phylogenetic relationship of Eplnephelus based on sequences of mtDNA Cty b

The mtDNA Cyt b gene was sequenced partially for Variola louti of Serranidae,Epinephelinae and seven endemic species of groupers-Epinephelus awoara,E.brunneus,E.coioides,E.longispinis,E.sexfasciatus,E.spilotoceps and E.tauvina in China.The seven endemic species and other seven foreign species of groupers--E,aeneus,E.caninus,E.drummondhayi,E,haifensis,E.labriformis,E.marginatus and E.multinotatus from the GenBank were combined and analysed as ingroup,while Variola louti was used as outgroup.We compared the 420 bp sequences of Cyt b among the 15 species and constructed two types of molecular phylogenetic trees with maximum parsimony method (MP)and neighbor-joining method (NJ) respectively.The results were as follows:(1) As to the base composition of mtDNA Cyt b sequence (402 bp) of 14 species of Epinepkelus,the content of (A + T) was 53.6%,higher than that of (G + C) (46.4%).The transition/transversion ratio was 4.78 with no mutation saturation.(2) The duster relationships between E.awoara and E.sexfasciatus,E.coioides and E.tauvina,E.longispinis and E.spilotoceps were consistent with phenotypes in taxonomy.(3) In the phylogenetic tree,the species in the Atlantic Ocean were associated closely with those in the Pacific Ocean,which suggested that the Cyt b sequences of Epinephelus were highly conserved.This may be attributed to the coordinate evolution.(4) In well-bred mating or heredity management,mating Epinephelus of the same branch should be avoided.It is likely to be an effective way to mate the species of the Atlantic Ocean with those of the Pacific Ocean to improve the inheritance species.     

Are all species of Pseudorhabdosynochus strictly host specific? — A molecular study

Species of the diplectanid monogenean genus Pseudorhabdosynochus are strictly host-specific (specialist), with the exception of P. cyanopodus, which was reported in New Caledonia, South Pacific, from two host species, Epinephelus cyanopodus and E. chlorostigma. We sequenced the COI gene of both host fish species and of their monogeneans. Morphological identification and pairwise distances showed that the two fish species were distinct (difference 6.1-6.6%), but that their monogeneans were not (difference 0-1.5%). A morphological study of sclerotised parts showed that specimens of P. cyanopodus are similar in both fish. Most species of groupers and their associated Pseudorhabdosynochus species are from warm surface waters, but the two groupers E. cyanopodus and E. chlorostigma are usually caught in deep-sea on the outer slope of the coral reef. This suggests that acquisition of a less strict host specificity is an adaptation of P. cyanopodus to deep-sea hosts.     

Comparative ultrastructural studies of the zona radiata of marine fish eggs in three genera in Perciformes

The eggs of Mugil cephalus were significantly larger than those of Epinephelus malabaricus , and E. coioides , while those of Sciaenops ocellatus were intermediate between E. coioides and M. cephalus . The distribution density of pores in the egg envelope of S. ocellatus was significantly different from that of E. malabaricus and E. coioides . The micropyle diameters were significantly different in the four species. The ultrastructure of the zona radiata surface, the distribution density of pores and the size of eggs were also useful characters for distinguishing among the four species, but the ultrastructural features of the micropyles were the most important of all for egg identification.     

Molecular phylogenetic relationships of China Seas groupers based on cytochrome b gene fragment sequences

The classification and evolutionary relationships are important issues in the study of the groupers. Cytochrome b gene fragment of twenty-eight grouper species within six genera of subfamily Epinephelinae was amplified using PCR techniques and the sequences were analyzed to derive the phylogenetic relationships of the groupers from the China Seas. Genetic information indexes, including Kimura-2 parameter genetic distance and Ts/Tv ratios, were generated by using a variety of biology softwares. With Niphon spinosus, Pagrus major and Pagrus auriga as the designated outgroups, phylogenetic trees, which invoke additional homologous sequences of other Epinephelus fishes from GenBank, were constructed based on the neighbor-joining (NJ), maximum-parsimony (MP), maximum-likelihood (ML) and minimum-evolution (ME) methods. Several conclusions were drawn from the DNA sequences analysis: (1) genus Plectropomus, which was early diverged, is the most primitive group in the subfamily Epinephelinae; (2) genus Variola is more closely related to genus Cephalopolis than the other four genera; (3) genus Cephalopolis is a monophyletic group and more primitive than genus Epinephelus; (4) Promicrops lanceolatus and Cromileptes altivelis should be included in genus Epinephelus; (5) there exist two sister groups in genus Epinephelus.     

Molecular phylogenetic relationship of Epinephelus based on sequences of mtDNA Cty b

The mtDNA Cyt b gene was sequenced partially for Variola louti of Serranidae, Epinephelinae and seven endemic species of groupers—Epinephelus awoara, E. brunneus, E. coioides, E. longispinis, E. sexfasciatus, E. spilotoceps and E. tauvina in China. The seven endemic species and other seven foreign species of groupers—E. aeneus, E. caninus, E. drummondhayi, E. haifensis, E. labriformis, E. marginatus and E. multinotatus from the GenBank were combined and analysed as ingroup, while Variola louti was used as outgroup. We compared the 420 bp sequences of Cyt b among the 15 species and constructed two types of molecular phylogenetic trees with maximum parsimony method (MP) and neighbor-joining method (NJ) respectively. The results were as follows: (1) As to the base composition of mtDNA Cyt b sequence (402 bp) of 14 species of Epinephelus, the content of (A + T) was 53.6%, higher than that of (G + C) (46.4%). The transition/transversion ratio was 4.78 with no mutation saturation. (2) The cluster relationships between E. awoara and E. sexfasciatus, E. coioides and E. tauvina, E. longispinis and E. spilotoceps were consistent with phenotypes in taxonomy. (3) In the phylogenetic tree, the species in the Atlantic Ocean were associated closely with those in the Pacific Ocean, which suggested that the Cyt b sequences of Epinephelus were highly conserved. This may be attributed to the coordinate evolution. (4) In wel1-bred mating or heredity management, mating Epinephelus of the same branch should be avoided. It is likely to be an effective way to mate the species of the Atlantic Ocean with those of the Pacific Ocean to improve the inheritance species.     

Assessment of genetic diversity and phylogenetic relationship among grouper species Epinephelus spp. from the Saudi waters of the Arabian Gulf

Understanding of fish genetic characterization plays a vital role in the conservation and utilization of fish genetic resources of grouper species. The present study was carried out to assess the genetic diversity and phylogenetic relationships in five grouper species, Epinephelus spp. from eastern Saudi Arabian coast using two molecular marker systems, inter simple sequence repeat (ISSR) and microsatellite (SSR) markers. In total, 219 individuals grouper specimens (Epinephelus tauvina, E. coioides, E. bleekeri, E. malabaricus, and E. areolatus) were genotyped with 10 ISSR and 11 SSR selected primers. The ISSR produced 94 DNA fragments, of which 44 were polymorphic with an average of 2.13 fragment per primer. While SSR primers generated 107 alleles, all of them were polymorphic with an average 9.72 per primer. ISSR and SSR techniques demonstrated a high level of gene diversity and genetic distances illustrated by UPGMA dendrograms among the grouper species. The results proved that the SSR markers were highly informative and efficient in detecting genetic variability and relationships of the Epinephelus spp.