基于线粒体COI和ND1基因分析 三尾凤蝶遗传多样性

以云南和四川分布的3个种群(云南丽江种群、云南东川种群、四川种群)共36头三尾凤蝶为研究对象,选取线粒体COI和ND1基因作为分子标记,对三尾凤蝶的遗传多样性进行了研究.结果表明,PCR扩增测得COI和ND1基因联合序列长度为1913 bp,A+T含量为74.3%,表现出明显的A/T碱基偏向性;共检测到4个变异位点,定义了5个单倍型,其中,Ha1出现的频率较高,为共享单倍型.核苷酸和单倍型多样性指数分别为0.0002和0.2180;遗传分化系数(Gst)、固定系数(Fst)、基因流(Nm)和遗传距离分别为0.05998、0.05995、3.92000和0.分析显示,三尾凤蝶遗传多样性低,各地理种群基因交流频繁,没有形成明显分化.     

Phylogenetic relationships between purple and tea-green individuals of the blue swimming crab Portunus trituberculatus by PCR-SSCP analysis

Given the morphological characters of purple crabs identical with that of the tea-green ones except the body color, it is expected that the purple crabs and the tea-green ones were from the same species. In order to identify the phylogenetic relationships between them at the molecular level, we amplified and compared two conserved mitochondrial gene fragments of purple and tea-green crabs through PCR-SSCP and DNA sequencing analyses. The results showed that there was no distinct variation of DNA bands of both the CO1 and 16srRNA genes detected in polyacrylamide gels through SSCP analysis between purple and tea-green crab samples, and the following sequence analysis of color-different crab individuals presented 99.81% and 99.91% nucleotide sequence identity of the CO1 and 16srRNA genes respectively. These indicated that there was no species or subspecies differentiation between purple and tea-green crabs, meaning that they belonged to the same species, Portunus trituberculatus.     

驯鹿狂蝇(Cephenemyia trompe)线粒体COI基因序列研究

利用分子生物学技术对驯鹿狂蝇蛆及其成蝇（Ccphenemyia trompe）的线粒体COI基因种属特异性序列进行了研究。DNA核苷酸测序结果证实：中国内蒙古株驯鹿狂蝇蛆（C.trompe CIML）及其成蝇（C．trompe CIMA）线粒体COI种属特异性基因片段长度约为672bp～676bp。种系发生进化树分析显示：第三期幼虫与成蝇两者在核苷酸碱基序列上存在一定差异；它们与同种不同挪威株（C．trompel同源性较为接近：与同属不同种（C．stimulator和C．ulrichii）同源性稍差；与不同属种的羊狂蝇（Oestrusovis）同源性较远，狂蝇科不同属间序列差异明显，差异性在18．6％～23％之间；鹿蝇属不同种间差异性在1．9％～9．9％之间：驯鹿狂蝇不同株间差异性为0．9％。说明生物线粒体COI基因核苷酸序列在一定程度上．可反映出种属及株间在进化上的差异性。     

4种常见鲤科鱼类DNA条形码的研究

测定了天津地区养殖的彭泽鲫、黄金鲫、乌克兰鳞鲤、鲤鱼4种共13尾鱼长度为814 bp的COI部分基因序列,以白鲢、罗非鱼作为外群.利用Mega4.1软件进行序列组成统计分析、种内及种间遗传距离分析,并用邻接法构建系统发育树（NJ树）,在NJ树上共发现由不同物种组成的5个分支,这与传统的分类学结果一致.该研究结果显示,COI基因部分序列不但可以作为物种辨识的良好DNA条码,而且在鲤科鱼类的种间系统发生关系分析方面也具有一定的适用性.     

高通量测序分析5例进口鱼粉鱼源性成分构成

为揭示鱼粉原料构成,保证鱼粉质量,本研究应用高通量测序技术对来源于不同国家的5例进口鱼粉样品进行测序,并基于COI基因及其丰度对鱼粉鱼源性成分构成进行分析。提取鱼粉样品DNA后应用鱼类通用COI基因扩增引物进行PCR扩增,得到鱼类混合COI基因序列产物,将扩增产物送测序公司进行高通量测序,将COI基因测序数据与鱼类相关数据库进行比对,通过比对结果进行鱼粉的物种源性成分分析。结果显示,成功获得了5例鱼粉样品中原料鱼成分,其中白鱼粉原料鱼主要成分为鳕鱼;而红鱼粉原料鱼成分较为多样,涉及鲱鱼、竹刀鱼等。通过Python对数据进行统计分析,绘制PCR扩增后鱼类COI基因丰度图,进一步直观提示鱼粉样品中原料的成分构成。本研究结果表明,高通量测序技术可应用于鱼粉质量评估,有助于快速了解鱼粉构成,对其质量及用途进行指导,同时,提示该技术可进一步应用于鱼粉掺杂掺假及致病微生物的监测工作中,对保障鱼粉产品质量、维护正常市场秩序具有重要意义。     

太平洋牡蛎核糖体DNA转录间隔子和线粒体基因片段序列测定

以相应引物经PCR扩增了太平洋牡蛎 (Crassostreagigas)的核糖体转录间区域 (ITS 1和ITS 2 )及线粒体 16SrDNA和COI基因片段。PCR产物经T 载体连接后进行克隆和测序 ,分别得到长度为 5 4 3、791、5 30和 70 0bp的核苷酸序列。 4个DNA片段的A、T、G和C碱基含量分别为 2 3.5 7%、2 0 .0 7%、2 9.4 7%和 2 6 .89% (ITS 1) ,2 7.4 3%、19.2 2 %、2 7.0 5 %和2 6 .30 % (ITS 2 ) ,2 9.2 5 %、2 9.2 5 %、2 3.0 2 %和 18.4 9% (16SrDNA) ,2 2 .71%、39.4 3%、2 0 .4 3%和 17.4 3% (COI)。实验证明ITS 1和ITS 2引物在贝类中通用性良好。文中同时讨论了 4个序列在我国几种牡蛎的种类鉴别及相关研究的应用潜力     

Identifying earthworms through DNA barcodes

With almost 3000 species, earthworms provide important model systems for studying soil fauna. However, species identification of earthworms is difficult and therefore limiting. The use of DNA barcodes, which are short sequences from standardized regions of the genome, has been regarded as a promising approach to resolve this taxonomic dilemma. We evaluated sequence diversity in the mitochondrial cytochrome-c oxidase I (COI) gene as a tool for resolving differences among species of Chinese earthworms. Members of six genera and 28 species were examined, and species were successfully discriminated in all cases. Sequence divergence within species was generally less than 1%, whereas divergence between species was greater than 15% in all cases. Divergence among individuals of Eisenia fetida were much higher (up to 7.8%); however, this may represent the presence of unrecognized sibling species or subspecies. We conclude that although it cannot completely replace taxonomy, the DNA barcode is a powerful tool for identifying species of earthworms and provides a useful complement to traditional morphological taxonomy.     

Molecular epidemiological characterization of poultry red mite, Dermanyssus gallinae,in Japan

Dermanyssus gallinae, the poultry red mite, is an obligatory blood-sucking ectoparasite. The genetic diversity of D. gallinae has been examined in some countries, but so far not in Asian countries. Here, we sequenced a part of the mitochondrial cytochrome oxidase subunit I (COI) and16S rRNA genes and nuclear internal transcribed spacers (ITS) region in 239 mite samples collected from 40 prefectures throughout Japan. The COI and 16S rRNA nucleotide sequences were classified into 28 and 26 haplotypes, respectively. In phylogenetic trees, the haplotypes clustered into 2 haplogroups corresponding to haplogroups A and B, which were previously reported. Haplogroups A and B were further subdivided into sub-haplogroups AJ1 and AJ2, and BJ1 and BJ2, respectively. In both trees, the sequences of haplotypes in AJ1 and BJ2 were relatively distant from those reported in other countries, while some sequences in AJ2 and BJ1 were identical to those in Europe. In addition, the ITS sequences were classified into two sequences, and both sequences were closely related to the sequences found in European countries. These findings indicate a possibility of international oversea transmission of D. gallinae.     

高榕榕果内Eupristina属两种榕小蜂的遗传进化关系

对生长在福州地区的高榕进行长期追踪观察,发现高榕榕果内仅生活着Eupristina altissima和Eupristina sp.榕小蜂,前者为高榕的传粉小蜂,后者无传粉行为,两者雌蜂之间在体色、触角、花粉袋和花粉刷等部位存在细微的差异,而两者雄蜂之间无形态差异。通过克隆福建地区5个样地的高榕榕果内收集到的E. altissima和Eupristina sp.榕小蜂,以及细叶榕的传粉小蜂Eupristina verticillata(外群)的Cytb及COI基因,并进行碱基组成及遗传距离分析,用邻接法构建系统发育树,分析两榕小蜂群体之间的遗传进化关系,结果显示:(1)榕小蜂COI及Cytb序列碱基组成中A+T的含量(Cytb序列中A+T=75.3%,COI序列中A+T=75.5%)显著高于G+C,符合膜翅目昆虫线粒体基因碱基组成特征。(2)对两群体小蜂进行遗传距离分析显示,Cytb序列中E. altissima和Eupristina sp. 群体内各样本之间的平均遗传距离分别为0.0092和0.0030,而E. altissima与Eupristina sp. 群体间的平均距离为0.1588;COI序列中E. altissima 与Eupristina sp. 群体内各样本之间的平均遗传距离分别为0.0065和0.0205,而二者群体间的平均遗传距离为0.1043,表明两者群体间的遗传距离明显大于各自群体内各样本间的遗传距离。统计GenBank中下载的6个属34种榕小蜂Cytb序列的种间遗传距离为0.0811-0.1723,6个属28种榕小蜂COI序列的种间遗传距离为0.0939-0.1986。由此认为E. altissima和Eupristina sp.之间的遗传距离差异已经达到了种间水平,即E. altissima与Eupristina sp.为两个不同的种。(3)在形态上,两种小蜂的雌蜂之间有微小差异,而二者雄蜂之间无差异,但Cytb与COI序列分析结果一致表明:E. altissima与Eupristina sp.雄蜂之间,以及二者雌蜂之间的遗传距离均差异显著,表明形态变异滞后于基因变异。雌蜂在表型上进化快于雄蜂,可能是由于雌蜂羽化后从榕果出飞,受到外界环境因素的影响较大,且两种雌蜂在传粉功能上存在差异,故二者之间的形态差异较大,而雄蜂寿命短,又终生生活在黑暗封闭、环境变化相对恒定的榕果内,两种雄蜂在行为上不存在差异,故二者表型变异较为缓慢。E. altissima和Eupristina sp.小蜂对宿主的专一性不强,在榕-蜂协同进化过程中,可能发生过宿主转移事件。     

甘肃河西走廊苹果蠹蛾种群遗传分化研究:基于COI基因序列分析

【目的】揭示河西走廊苹果蠹蛾不同地理种群间的联系,分析地理种群间的序列变异、遗传多样性和遗传分化。【方法】采用PCR和基因测序技术,扩增并分析了河西走廊8个地理种群132个苹果蠹蛾个体的线粒体细胞色素氧化酶Ⅰ亚基(COI)基因片段,应用DnaSP 5.10计算单倍型多样性指数(Hd)、核甘酸多样性指数(Pi)和Tajima’s D值,采用ZT软件包进行Mantel检验,分析种群间遗传距离与地理距离的相关性,利用软件Arlequin 3.11计算成对种群间的固定系数(Fst)。【结果】在获得的132条序列中共发现了8个变异位点和5个单倍型,其中3个单倍型为种群共享单倍型。总体单倍型多样性指数为0.578,种群内单倍型多样性为0.000~0.700。各种群的Tajima’s D值中性检验符合中性突变,说明河西走廊苹果蠹蛾在历史上没有出现群体扩张,群体大小稳定。Fst值表明,河西走廊苹果蠹蛾种群间具有一定程度的遗传分化,而各地理种群的遗传距离与地理距离无显著的相关性。【结论】河西走廊苹果蠹蛾遗传多样性较低,且地理种群间有一定程度的遗传分化。