Diversity of Apis mellifera Subspecies from Turkey Revealed by Sequence Analysis of Mitochondrial 16s rDNA Region

Mitochondrial DNA sequence variation can be used to infer honeybee evolutionary relationships. In this study, DNA sequence diversity of the mitochondrial 16s rDNA region was investigated in 112 honeybees from 15 populations in Turkey, which is mainly populated with Apis mellifera anatoliaca, A. m. caucasica, and A. m. meda. The study revealed 11 haplotypes for this segment, with 13 variable sites and nine parsimony informative sites. The haplotypes were not discriminated according to their geographical locations in a neighbor-joining dendrogram based on 16s rDNA sequences available in Genbank, but all the haplotypes obtained in this study are clustered with published haplotypes such as A. mellifera TAS (AF214666) and A. m. ligustica (EF116868) and with some unpublished Genbank records (HQ318928, HQ318934, and HQ318938). This study expands the knowledge of the mitochondrial 16s rDNA region, and it presents the first comprehensive sequence analysis of this region in Turkish honeybees.     

Genetic Structure of Marchalina hellenica (Hemiptera: Margarodidae) Populations from Turkey: Preliminary mtDNA Sequencing Data

The scale insect Marchalina hellenica (Gennadius) (Hemiptera: Margarodidae) contributes to the production of pine honey in Turkey and Greece via the honeydew excreted when it feeds on pine trees. Although it is an insect of prime economic importance, there is no information on its genetic structure. Preliminary data were obtained based on sequencing analysis of 12s rDNA and COI mtDNA gene segments from samples from four areas of Turkey. Sequences of the 12s rDNA gene segment from Greek samples available in GenBank were also included. No variability was detected concerning the COI mtDNA gene segment analysis, although 13 haplotypes were revealed based on the 12s rDNA gene segment. The most distant population was from Mudanya-Bursa Province (Turkey). Further research is necessary on the genetic structure and variability of M. hellenica populations from the two neighboring countries.     

Characterization of the native honey bee (Apis mellifera jemenitica) in the south western region of Saudi Arabia using morphometric and genetic (mtDNA COI) characteristics

Apis mellifera jemenitica incorporates a few perceived subspecies that vary in their natural properties and farming qualities. Mitochondrial COI gene sequence (mtCOI) has not been used before for bee identification in the southwestern region of Saudi Arabia. The aim of this work was to study the morphometry and analyzing the mtCOI of all collected bees. The nucleotide sequence of the mtCOI gene was analyzed. Similarity searches and distances between each obtained DNA and sequences available in GenBank were made. Morphometric analysis revealed close similarities among the studied bees, but these similarities are different from those previously indicated in earlier studies of the same region. Molecular studies revealed that the collected bees are similar to each other and some other sequences found in GenBank, but these bees are a new hybrid or subspecies that are different from those previously reported in the same region, indicating the emergence of a new hybrid.     

Cytochrome c oxidase subunit I barcoding of the green bee-eater (Merops orientalis)

DNA barcoding using mitochondrial cytochrome c oxidase subunit I (COI) is regarded as a standard method for species identification. Recent reports have also shown extended applications of COI gene analysis in phylogeny and molecular diversity studies. The bee-eaters are a group of near passerine birds in the family Meropidae. There are 26 species worldwide; five of them are found in Saudi Arabia. Until now, GenBank included a COI barcode for only one species of bee-eater, the European bee-eater (Merops apiaster). We sequenced the 694-bp segment of the COI gene of the green bee-eater M. orientalis and compared the sequences with those of M. apiaster. Pairwise sequence comparison showed 66 variable sites across all the eight sequences from both species, with an interspecific genetic distance of 0.0362. Two and one within-species variable sites were found, with genetic distances of 0.0005 and 0.0003 for M. apiaster and M. orientalis, respectively. This is the first study reporting barcodes for M. orientalis.     

Molecular phylogeny of a red-snow-crab species complex using mitochondrial and nuclear DNA markers

Nucleotide sequence variation of mitochondrial DNA COI and nuclear rRNA gene regions was used to reconstruct phylogenetic relationships for the red-snow-crab species complex, including the red snow crab, Chionoecetes japonicus, its nominal subspecies, C. japonicus pacificus, and the triangle tanner crab, C. angulatus. The topologies of the Bayesian and neighbor-joining (NJ) trees of the COI and of NJ trees of rRNA sequences placed C. japonicus and C. angulatus in a single clade. The net sequence divergence between these taxa was d(net) = 0.000 in COI, and strongly suggests that these taxa represent a single species. In contrast, haplotypes in C. j. pacificus clustered separately from the C. japonicus - C. angulatus clade. Net sequence divergence from C. japonicus - C. angulatus to C. j. pacificus was d(net) = 0.026 in COI, indicating that C. j. pacificus should be elevated to a separate species, C. pacificus. A 165 bp insert appeared in the rRNA gene of C. j. pacificus, but was absent in the remaining species of Chionoecetes. This autapomorphic condition in C. j. pacificus adds support for an independent evolution of this taxon. Evolutionary divergences between these taxa may reflect contrasting evolutionary process influenced by ocean bathymetry.     

山西翅果油树上三种重要鳞翅目害虫的形态和分子鉴定及生活史观察

[目的]明确山西翅果油树Elaeagnus mollis上发生危害的3种鳞翅目害虫形态鉴定特征及生活史特性,并基于mtDNA COI基因DNA条形码对这3个种进行快速物种识别鉴定.[方法]通过观察山西翅果油树上3种鳞翅目害虫成虫外部形态和解剖拍照雌、雄性外生殖器特征,利用PCR扩增对待测样本COI基因DNA条形码序列进...     

青海省6种裂腹鱼COI基因作为DNA条形码的适用性初探

青海省裂腹鱼鱼类至少有20种,占土著鱼类的40%以上,具有重要的生态价值。由于生态环境的恶化和人为因素的干扰,很多物种群已濒临灭绝。快速和准确的物种鉴定对于这些物种的保护至关重要,而基于形态学的传统分类法很难满足这一需求。因此,本研究通过DNA条形码技术初步探讨了在青海省裂腹鱼物种鉴定中的适用性。本研究中,测序获得了青海26尾裂腹鱼(6个物种)的细胞色素c氧化酶亚基I (COI)基因,并经GenBank数据库进行比对;基于K2P模型分析COI序列变异;运用贝叶斯法(BI)和最大似然法(ML)构建系统发育树。结果显示,6种裂腹鱼COI序列检测得到15个单倍型,且各物种间无共享的单倍型。基于K2P模型,最大种内遗传距离和最小种间遗传距离分别为(0.621±0.297)%和(2.792±0.644)%。种间平均遗传距离为(12.205±1.307)%,约为种内平均遗传距离((0.327±0.162)%)的37倍,表明各物种的COI序列间已经形成明显"条形码间隙"。AMOVA分析显示,遗传变异主要来自种间,约占97.05%;FST=0.970 54,p<0.01,说明各物种间分化程度极高。此外,基于BI和ML方法构建的系统树具有一致的拓扑结构,分辨率较高;各物种均单独聚为一个发育枝,拓扑结构合理。以上结果表明,COI基因作为DNA条形码在青海裂腹鱼物种鉴定中具有较高的适用性。     

不同地理种群银杏大蚕蛾COI基因序列变异与遗传分化

银杏大蚕蛾Caligula japonica是亚洲东部的特有种, 既是一种重要的林业害虫, 也是一种珍贵的野生蚕类资源。为了揭示银杏大蚕蛾地理种群间的内在联系, 测定了我国分布的12个地理种群的线粒体细胞色素氧化酶C亚基I（COI）基因部分序列（GenBank登录号: FJ358506-FJ358517）, 对地理种群间的序列变异和遗传分化进行了分析。结果表明: 银杏大蚕蛾地理种群间的COI序列同源性高达99%~100%, 显示出比较小的遗传差异。序列对准后从供试COI序列中仅鉴定出9个变异位点和6个单元型, 其中3种是共享单元型。系统发育分析结果表明种群间已经按地理位置形成了一定的地理格局, AMOVA分析显示北方组和南方组之间已经具有明显的遗传分化（FST=0.478, P<0.001）。综合分析, 我们认为北方组和南方组之间的遗传分化可能与差异巨大的生态条件有关。研究结果为银杏大蚕蛾的种群遗传学和生态学研究提供了一个基本的分子生物学线索。     

罗氏沼虾不同群体线粒体COI基因的序列差异和遗传标记研究

利用PCR方法扩增获得罗氏沼虾（Macrobrachiumrosenbergii）线粒体DNA的COI基因,测定该基因片段序列。分析了罗氏沼虾缅甸原种F1代、江苏养殖群体和广西选育F2代3个群体共17只个体的序列核苷酸位点差异和遗传多态。结果表明,缅甸原种F1代遗传多样性最为丰富,江苏养殖群体和广西选育群体的遗传多样性相对贫乏。在长度为498bp的基因片段中,共检测到10个多态性核苷酸位点（占2.01%）,17只个体具有5种基因型,3群体各自的平均核苷酸位点差异分别为0.88%、0.07%和0。UPGMA分子系统聚类树显示,江苏养殖群体和广西选育群体的遗传关系最近,其单倍型混杂聚成一支,而缅甸原种F1群体相对独立为另外一支。COI基因可以作为区分两分支群体的遗传标记。     

我国沿海棱鳀属鱼类的物种鉴定与系统发育

利用DNA条形码技术对中国沿海分布的6种棱鳀属(Thryssa)鱼类样品进行了物种鉴定, 并每种取5尾用于探讨该属系统发育关系。结果显示: 棱鳀属鱼类的主要形态鉴别特征为上颌骨伸达位置和第一鳃耙的下鳃耙数量。在525 bp的目的片段上有175个变异位点, 其中简约信息位点172个, 单一信息位点3个, 无插入缺失现象, 转换数为182, 颠换数为57。A+T含量明显高于G+C含量, 并且表现出明显的反G偏倚。结合GenBank中相关的同源序列进行比较发现, 所有序列明显分为10个组群, 表明已提交的棱鳀属鱼类COI基因序列中仍存在一定的问题。从各组群间的遗传距离和氨基酸遗传差异水平可以看出, 10个组群应为不同的有效种, 但是否存在隐存种还有待于进一步确定。从NJ树上可以看出, 长颌棱鳀(T. setirostris)是最先分化出的物种, 保持着最原始的特征, 而中颌棱鳀(T. mystax)与黄吻棱鳀(T. vitrirostris)聚类到一起, 二者间存在共享单倍型。棱鳀属鱼类最早分化于中新世早期。在今后的研究中仍需要结合更多的分子标记对中颌棱鳀和黄吻棱鳀的分类地位作进一步的探讨。     

斑衣蜡蝉若虫寄生蜂地理种群遗传差异及早期快速检测

【目的】明确不同地理种群斑衣蜡蝉Lycorma delicatula若虫寄生蜂种群遗传差异,实现若虫被寄生早期的快速准确检测,以评估其对斑衣蜡蝉种群的控制作用。【方法】利用DNA条形码技术获得不同地理种群斑衣蜡蝉若虫寄生蜂COI和28S rDNA序列,利用K2P模型计算不同地理种群间的遗传距离,以邻接(neighbor-joining,NJ)法构建系统发育树;基于COI序列设计种特异性PCR(SS-PCR)引物,利用SS-PCR对斑衣蜡蝉若虫DNA进行扩增,测定其体内是否有中华螯蜂Dryinus sinicus寄生;利用目测法观察和PCR扩增测定寄生蜂对不同采样点斑衣蜡蝉若虫的寄生率。【结果】经鉴定,不同地理种群斑衣蜡蝉若虫寄生蜂为中华螯蜂,该寄生蜂COI序列共检测到16个单倍型,28S rDNA序列共检测到4个单倍型。不同地理种群间中华螯蜂遗传距离在0.00691~0.01310之间。邻接法构建的系统发育树显示不同地理种群中华螯蜂均聚于一枝。基于COI序列设计的SS-PCR引物对中华螯蜂成虫、幼虫均具有良好的扩增效果,最低检测阈值为0.000005 ng/μL DNA。利用SS-PCR测定中华螯蜂对各采样点斑衣蜡蝉若虫的寄生率为22.54%~60.00%,显著高于目测统计的结果(5.63%~36.98%)。【结论】不同中华螯蜂地理种群间遗传差异较小;SS-PCR引物可用于不同地区中华螯蜂对斑衣蜡蝉早期寄生率的快速检测。     

中国大鲵四种群的遗传结构和地理分化

为了确定栖息地的破碎化和片断化引起中国大鲵的地理分化和遗传结构变异,本文测定了来自广西、河南、陕西和湖南4个地理种群的28条大鲵的mtDNAD loop基因全序列。根据这4个地理种群的地理分布,分成珠江单元(广西种群)、黄河单元(河南种群)和长江单元(湖南和陕西种群)。通过ClustalX、MEGA2.0、DnaSP4.0、Arlequin1.1分析发现,全序列长度为771bp,其中64个多态性核苷酸变异位点,占全部碱基数的8.26%。转换和颠换分别为6和2个,插入/缺失11个。27个单倍型间的序列差异平均为1.32%。3个单元的单倍型多样性指数和核苷酸多样性指数值都偏低,而且珠江单元的这两个指数值都低于长江和黄河两个单元。珠江单元和黄河、长江单元之间分化程度显著(P<0.001),而长江和黄河单元之间差异不显著(P>0.05)。地理单元内分化程度占99.31%,而单元间只有0.69%,表明遗传差异主要发生在单元内,而且各地理单元之间的基因流较频繁。构建的NJ树和MP树显示,27个单倍型呈现一种混杂的分布格局,并未分成代表3个地理单元的聚合群。     

六种灵猫科动物的分子系统关系研究

对六种灵猫科物种线粒体12 S rRNA基因及其中四种的Cytb基因部分序列进行了测定,并从Gen-Bank获得斑林狸(Prionodon pardicolor)、熊狸(Arctictis binturong)的Cytb基因同源序列。两基因整合序列比对后长755 bp,12 S rRNA基因序列中有70个变异位点,31个简约信息位点,在Cytb基因序列中,共有120个位点呈现变异,60个简约信息位点,Cytb基因的碱基变异百分比高于12 S rRNA基因的碱基变异百分比。使用邻接法(NJ)、最大似然法(ML)重建的分子系统树显示:斑林狸从灵猫亚科中分离出来,支持灵猫亚科的多系起源,而且斑林狸可能是中国起源最早且最特化的灵猫科动物。另外,同属于灵猫亚科的大灵猫(Viverra zibe-tha)、小灵猫(Viverricula indica)聚为一支,同属于棕榈狸亚科的果子狸(Viverricula indica)、熊狸聚为姐妹群,这些与传统形态学分类观点一致。     

Molecular Characterization of a Mitochondrial DNA Segment from the Japanese Scallop (Patinopecten yessoensis): Demonstration of a Region Showing Sequence Polymorphism in the Population

A 1.3-kb mitochondrial DNA segment from the Japanese scallop Patinopecten yessoensis was cloned and sequenced. This segment contained the transfer RNA^Met gene and partial sequences of 2 ribosomal RNA genes, together with 2 separate noncoding regions (designated NcR1 and NcR2). The NcR regions derived from 78 individuals cultured in Lake Saroma or Matsu Bay, were sequenced, and we found 15 loci with sequence alterations including 13 substitutions, 1 deletion, and 1 insertion (1 locus in NcR1, 14 loci in NcR2), and 17 haplotypes. Of the 17 haplotypes, 10 were found in the Saroma population only, 3 in the Mutsu population only, and 4 in both populations. The gene diversity and nucleotide diversity values were, respectively, 0.87 and 0.0069 for the Saroma population, 0.63 and 0.0040 for the Mutsu population, and 0.83 and 0.0203 overall. Thus the NcR segment was considered to have sufficient sequence variation for population genetic studies. The 16 variants of the NcR2 sequence were separated successfully by denaturing gradient gel electrophoresis, confirming the sequence variation in NcR2. Received October 3, 2001; accepted February 19, 2001.     

云南地区恒河猴线粒体DNA控制区遗传多态性研究

目的从分子水平探讨云南地区恒河猴遗传多样性,为今后开展恒河猴遗传资源的保护及合理利用提供借鉴和背景资料。方法采用PCR直接测序法测定云南地区恒河猴96份样品的线粒体DNA控制区全序列,用Mege 4.0和DNA SP软件对变异位点数、简约信息位点数、单倍型、单倍型多样度、核苷酸多样度等遗传信息进行分析,基于邻接法（neighbor-joining,NJ）和最小进化法（minimum-evolution,ME）构建系统发生树。结果在96份样品中,共检测出了149个多态性位点,定义了46种单倍型,单倍型多样度（Hd）为0.968±0.007,核苷酸多样度（Pi）为0.020。结论云南地区恒河猴存在着较丰富的遗传多态性。     

基于线粒体COI基因分析钩手水母的群体遗传结构

钩手水母(Gonionemus vertens)为大西洋和太平洋广布种, 是我国习见的有毒水母种类之一。本文对采自黄渤海海域4个地理群体的104个钩手水母线粒体COI基因序列进行扩增, 并结合GenBank上其他182个钩手水母同源序列进行序列变异分析。在286个基因序列中共检测出52个多态位点, 定义了14种单倍型。总群体的单倍型多样性和核苷酸多样性分别为0.743 ± 0.012和1.046% ± 0.097%, 与其他几种大型水母相比, 钩手水母总群体的遗传多样性处于较高水平。AMOVA结果显示, 60.17%的分子变异源于群组间, 13.37%的分子变异源于群体内, 26.46%的分子变异源于组内群体间, 群组间、群体内和组内群体间的遗传分化均极显著。F_st值统计检验表明, 中国厦门群体与乐亭、东营、烟台、大连群体间存在显著的遗传分化, 大连与东营、烟台群体间也存在显著的遗传分化。系统分析结果显示, 钩手水母群体间存在2个明显的单倍型谱系分支。不同的钩手水母地理群体间具有复杂的遗传模式, 钩手水母复杂的生活史、扩散能力、地理隔离和海流分布可能是影响钩手水母遗传结构的重要因素。     

DNA barcoding meets molecular scatology: short mtDNA sequences for standardized species assignment of carnivore noninvasive samples

Although species assignment of scats is important to study carnivore biology, there is still no standardized assay for the identification of carnivores worldwide, which would allow large-scale routine assessments and reliable cross-comparison of results. Here, we evaluate the potential of two short mtDNA fragments [ATP6 (126 bp) and cytochrome oxidase I gene (COI) (187 bp)] to serve as standard markers for the Carnivora. Samples of 66 species were sequenced for one or both of these segments. Alignments were complemented with archival sequences and analysed with three approaches (tree-based, distance-based and character-based). Intraspecific genetic distances were generally lower than between-species distances, resulting in diagnosable clusters for 86% (ATP6) and 85% (COI) of the species. Notable exceptions were recently diverged species, most of which could still be identified using diagnostic characters and uniqueness of haplotypes or by reducing the geographic scope of the comparison. In silico analyses were also performed for a 110-bp cytochrome b (cytb) segment, whose identification success was lower (70%), possibly due to the smaller number of informative sites and/or the influence of misidentified sequences obtained from GenBank. Finally, we performed case studies with faecal samples, which supported the suitability of our two focal markers for poor-quality DNA and allowed an assessment of prey DNA co-amplification. No evidence of prey DNA contamination was found for ATP6, while some cases were observed for COI and subsequently eliminated by the design of more specific primers. Overall, our results indicate that these segments hold good potential as standard markers for accurate species-level identification in the Carnivora.     

Mitochondrial DNA sequence variation in the spruce budworm species complex (Choristoneura: Lepidoptera)

A combination of polymerase-chain-reaction amplification and automated DNA sequencing was used to survey variation in a species complex of pest insects, the spruce budworms (Choristoneura fumiferana species group), and an outgroup species, C. rosaceana. We sequenced an mtDNA region of 1,573 bp that extends from the middle of cytochrome oxidase subunit I (COI) through tRNA leucine (UUR) to the end of cytochrome oxidase subunit II. In addition, we examined levels of intraspecific variation within a 470-bp region of the COI gene. Choristoneura fumiferana clearly represented the oldest lineage within its species group, with 2.7%-2.9% sequence divergence from the other species. In contrast, the four remaining species (C. pinus, C. biennis, C. occidentalis, and C. orae) had closely related or identical mtDNA, with < 1% divergence among most of their haplotypes. Despite its older lineage and widespread geographic distribution, C. fumiferana showed significantly lower intraspecific genetic diversity than did C. occidentalis. Choristoneura orae shared haplotypes with C. occidentalis and C. biennis, and species-level separation of these three species was not supported. Two divergent, uncommon haplotypes were also found in C. occidentalis and C. biennis. The divergent haplotype in C. biennis had an unusually high number of inferred amino acid replacements, suggesting selective differences between mitochondrial DNA haplotypes. Transition:transversion ratios in Choristoneura paralleled those found in Drosophila; transition:transversion ratios were highest in closely related sequences but decreased with increasing sequence divergence. Nucleotide composition showed an A+T bias that was near the high end of the range known for insects. This work illustrates the potential utility of direct DNA sequencing in assessing population structures, species limits, and phylogenetic relationships among organisms that have not previously been subjected to DNA analysis.      

Genetic diversity and phylogenetic relationship among the western and the Asian honey bees based on two mitochondrial gene segments (COI and ND5)

The Asian honey bee species i.e., Apis cerana (the eastern honey bee), A. dorsata (the giant honey bee), and the western or European honey bee (A. mellifera) collected from Pakistan were studied using partial sequences from two mitochondrial genes (i) the Cytochrome c oxidase I (COI) and (ii) the mitochondrially encoded NADH dehydrogenase 5 (ND5) and then compared with other honey bees sequences (already submitted from different countries around the globe) obtained after the national center for biotechnology information (NCBI). DNA sequences were analyzed employing molecular evolutionary genetics analysis and Kimura 2-parameter model, neighbor-joining method was applied to investigate phylogenetic relationships, and DNA sequence polymorphism was applied to measure the genetic diversity within the genus Apis. The phylogenetic analyses yielded consistent results. Based on COI gene fragment in two Asian and European honey bee species from Pakistan and from other countries showed considerable genetic diversity levels and deviation among the species. While in contrast the phylogenetic analyses based on ND5 gene fragment in Asian and European honey bee species from Pakistan and other countries showed comparatively higher genetic diversity indices and variations than the COI gene. So, in the genus Apis, the mitochondrial ND5 region has shown the possibility to answer the interactions among species. A further detailed work (by linking the analysis of other genomic and mitochondrial genes) is required for good quality solution to establish the concise genetic diversity and interaction among the Apis species. The objective of this study was to explore the extent of genetic differences and phylogenetic links among the three kinds of honey bee species from Pakistan and comparing them with other bee species around the globe.     

Sequence diversity of MHC class-II <Emphasis Type="BoldItalic">DRB</Emphasis> gene in gazelles (<Emphasis Type="BoldItalic">Gazella subgutturosa</Emphasis>) raised in Sanliurfa of Turkey

In this study, we aimed to assess the sequence diversity of major histocompatibility complex (MHC) class-II DRB gene at exon 2 in gazelles raised in Sanliurfa Province of Turkey. Twenty DNA samples isolated from gazelles (Gazella subgutturosa) were used for sequencing exon 2 of MHC class-II DRB gene. Target region was amplified by polymerase chain reaction (PCR) and their products were directly sequenced. Nine of these 20 samples yielded unambiguously readable sequences. Three of the nine samples were homozygotes and each showed different sequences. A 262-bp sequence obtained from the three homozygote samples were submitted to GenBank (accession numbers: KC309405, KC309406 and KC309407). Using an allele specific PCR, we detected 10 additional haplotypes. Among 13 haplotypes, 45 nucleotide positions were polymorphic and most of the polymorphic nucleotide positions localized at peptide-binding region (PBR). Rates of nonsynonymous substitutions were significantly higher than synonymous substitutions at PBR. Phylogenetic analysis of the haplotypes showed that 10 haplotypes of the gazelles were clustered together while three were clustered with ovine and bovine haplotypes. The results indicated that at least 13 haplotypes at exon 2 of MHC class-II DRB gene were showing high degree of nucleotide and amino acid diversity, and certain haplotypes of G. subgutturosa were more similar to haplotypes from sheep or cattle than to each other. Rates of synonymous and nonsynonymous substitutions suggested that positive selection was a driving force for diversity at this locus in G. subgutturosa.