DNA条形码技术在小型兽类鉴定中的探索:以甘肃莲花山为例

为验证DNA条形码技术在小型兽类物种鉴定中的可行性,我们使用传统的形态学方法和DNA条形码技术对甘肃莲花山国家级自然保护区采集的77号小型兽类标本进行鉴定.共鉴定出物种13种,分别属于3目7科.其中小纹背鼩鼱(Sorex bedfordiae)、藏鼩鼱(S.thibetanus)、甘肃鼹(Scapanulus oweni)黑线姬鼠(Apodemus agrarius)、社鼠(Niviventer confucianus)、斯氏鼢鼠(Eospalax smithi)、高原鼠兔(Ochotona curzoniae)等7种为莲花山首次报道.另外,采自海拔2,800 m的黑线姬鼠是该物种全球海拔最高的分布记录.通过对两种方法进行比较,我们发现形态鉴定需要研究者具有足够的分类学背景,基于DNA条形码的物种鉴定则限制于网络数据库中缺乏足够的序列信息导致成功率不高.因此我们认为现阶段形态鉴定依然是小型兽类鉴定的基石和主要手段,DNA条形码技术可以作为重要的补充手段,对形态鉴定的结果进行检验.最后本文认为DNA条形码鉴定前景非常乐观,但在现阶段的使用中应与经典分类紧密结合并尽可能保存凭证标本.     

DNA条形码技术在青海海东地区小型兽类鉴定中的应用

为弥补传统形态分类方法的不足,探究应用DNA条形码技术进行分子生物学鉴定的可行性,本研究用DNA条形码技术检测了青海省海东地区3目6科14属18种110只小型兽类的COI基因部分序列。分析所测COI基因序列可知:种内遗传距离≤3%,种间遗传距离5-10%,属间遗传距离12-19%,种间遗传距离显著大于种内遗传距离。NJ树显示同种个体聚为有很高支持度的单一分支。有6个个体(4只黄胸鼠、2只小家鼠)在现场鉴定中被误定为其他种类。研究结果表明使用条形码技术能纠正形态学鉴定中的错误,也说明动物线粒体COI基因是一个有效的DNA条形码标准基因。     

DNA条形码技术在植物中的研究现状

DNA条形码技术(DNA barcoding)是用短的DNA片段对物种进行识别和鉴定的分子生物学技术。在动物研究中该技术已经成功应用于利用线粒体细胞色素c氧化酶亚基I(COI)进行物种鉴定和发现隐种或新物种。相对于动物, COI基因在高等植物中进化速率较慢, 因此植物条形码研究以叶绿体基因组作为重点, 但目前还处于寻找合适的基因片段阶段。许多学者对此进行了积极的探索, 报道了多种植物条形码的候选片段或组合, 但还没有获得满足所有标准的特征位点片段。该文介绍了DNA条形码的标准、优点、工作流程及数据分析方法, 总结了DNA条形码在植物中的研究现状。     

DNA条形码技术在植物中的研究现状

DNA条形码技术（DNA barcoding）是用短的DNA片段对物种进行识别和鉴定的分子生物学技术。在动物研究中该技术已经成功应用于利用线粒体细胞色素c氧化酶亚基I（COI）进行物种鉴定和发现隐种或新物种。相对于动物, COI基因在高等植物中进化速率较慢, 因此植物条形码研究以叶绿体基因组作为重点, 但目前还处于寻找合适的基因片段阶段。许多学者对此进行了积极的探索, 报道了多种植物条形码的候选片段或组合, 但还没有获得满足所有标准的特征位点片段。该文介绍了DNA条形码的标准、优点、工作流程及数据分析方法, 总结了DNA条形码在植物中的研究现状。     

环境DNA宏条形码在生物多样性研究与监测中的应用

环境DNA宏条形码(eDNA metabarcoding)技术通过提取水体、土壤、空气中的环境DNA,使用引物PCR扩增与高通量测序,进行物种鉴定与生物多样性评估.作为一种新的监测技术,相比于传统监测技术更加快捷、准确以及对自然环境的破坏小,因此在一定程度上改变了我们调查地球生物多样性的方式.本文综述了环境DNA宏条形...     

甘肃省鱼类资源现状及DNA条形码在鱼类物种鉴定中的应用

为了摸清甘肃省土著鱼类资源与分布现状, 探索DNA条形码在鱼类辅助物种鉴定中的适用性, 2012年6-9月对甘肃境内黄河水系、嘉陵江水系和河西内陆河水系进行了较全面的鱼类调查。共采集鱼类标本3,087尾, 隶属于5目10科38属64种, 以鲤科种类最多, 为30种, 占总种数的46.88%。物种多样性分析表明, 在黄河水系的夏河和庄浪河多样性指数是所有调查点中最低的, 分别为1.38和1.09。嘉陵江水系各河段的多样性指数较高(H = 2.15-3.27), 其次为河西内陆河水系(H = 2.01-2.83)。在河西内陆河水系中, 疏勒河的均匀度指数最高, 为1.10, 黑河最低(0.68)。庄浪河的优势度指数最高, 为0.34, 而嘉陵江干流两当段的优势度指数在所有调查点中最低, 为0.04。利用DNA条形码分析了49种662尾标本的COI基因部分序列, 大部分种类在neighbor-joining系统树中形成各自的单系, 种内平均遗传距离0.88%, 种间平均遗传距离为9.99%, 在种内和种间COI序列遗传距离之间形成明显的条形码间隙, 斯氏高原鳅(Triplophysa stoliczkae)与达里湖高原鳅(T. dalaica), 甘肃高原鳅(T. robusta)与似鲇高原鳅(T. siluroides), 嘉陵裸裂尻鱼(Schizopygopsis kialingensis)与黄河裸裂尻鱼(S. pylzovi)之间的遗传距离低于2%, 甘肃高原鳅与似鲇高原鳅不能通过COI基因片段区分开, 其他两对物种可以采用核苷酸诊断法来进一步区分。斯氏高原鳅和拉氏鱼岁(Phoxinus lagowskii)种内遗传分歧较大, 揭示种内可能存在隐存种。结果表明, 对某些近缘种和不同地理种群差异较大的物种, 要将分子、形态和地理分布特点结合起来才能准确鉴定。     

DNA宏条形码技术在海洋浮游动物多样性和生态学研究中的应用

浮游动物是海洋生态系统的关键类群,其覆盖门类广泛,多样性高。传统形态鉴定技术需要检测人员具备专业的形态鉴定知识,且费时费力。宏条形码技术无需分离生物个体,而是提取拖网采集到的浮游动物混合样本的总DNA,或者水体中的环境DNA （eDNA）,依托高通量测序平台测序,能够实现对大规模样本快速、准确、经济的分析,在海洋浮游动物生态学研究中得到越来越广泛的应用。分析了DNA宏条形码技术常用的核糖体和线粒体分子标记,在浮游动物多样性和数量研究中的可靠性和不足,并给出在海洋浮游动物群落监测,食物关系分析及生物入侵早期预警等研究中的应用。未来,开发多基因片段组合条形码,发展完备的参考数据库及实现准确的量化研究是DNA宏条形码技术发展的重要方向。     

DNA条形码技术及其在保护生物学中的应用

物质文明及生活水平的提升为人类带来诸多好处的同时,也使人们越来越清晰地意识到保护生物多样性及生态系统稳定性的重大意义.DNA条形码技术作为现今生物分类学中重要的分子技术,可以快速准确地鉴定物种.多国科学家都在致力于对DNA条形码的研究,以便能共同组建数据库.将现有物种正确分类并期望用于发现新种,为生物的保护及生态系统的维护作出巨大贡献.细胞色素C氧化酶亚单位I(COI)是现在动物种类鉴定中最常用的基因之一.综述DNA条形编码技术的产生、原理、发展概况与操作及其在保护动物分类中的应用.阐明该技术在保护生物学中应用的意义与可行性,并讨论DNA条形编码在生物分类应用中可能存在的问题.     

DNA条形码及其在生物多样性研究中的应用

DNA条形码是利用生物体内标准的、有足够变异的、易扩增且相对较短的DNA片段对物种进行快速准确鉴定的技术。自2003年DNA条形码相关概念提出以来广受关注,国内外相继开展了DNA条形码及信息系统建设研究,为DNA条形码技术的发展提供了坚实的研究基础和生物信息学分析平台。DNA条形码技术弥补了传统分类学的不足,为生物多样性研究提供了新的思路和方法。本文介绍了DNA条形码的产生与发展过程,国内外DNA条形码技术与信息系统建设研究进展,重点阐述了DNA条形码技术在物种鉴定、濒危物种保护、隐存种发现、生物多样性评估等研究领域中的应用。最后结合DNA条形码技术目前存在的问题,对其在相关研究领域的应用前景进行了展望。     

DNA条形码试剂盒检测技术在大小蠹属种类鉴定中的应用

[目的]DNA条形码技术已成为生物分类鉴定的有力工具.DNA条形码技术的相关问题,如物种种内和种间的遗传距离出现重叠区域,将直接影响到物种鉴定的准确性.我们应用DNA条形码试剂盒检测技术来快速、准确地鉴定口岸截获的检疫性大小蠹属种类.[方法]针对大小蠹昆虫设计引物以提高PCR扩增效率.运用自主研发的基因条码分析软件找出基因片段上区分每个物种的多态位点规律,作为该物种的鉴定特征并建立数据库,应用于物种鉴定.[结果]使用针对大小蠹属昆虫设计的引物成功扩增出325 bp的COI基因片段.将大小蠹属12种昆虫的COI基因片段上的核苷酸诊断位点的组合作为物种的鉴定特征,可以准确地区分近似种.通过比对植物检疫鉴定系统数据库里的鉴定特征,将6个大小蠹属的未知样品成功鉴定到种(核苷酸序列一致性为100％),与形态鉴定结果一致.[结论]结果表明DNA条形码试剂盒检测技术可以准确鉴定大小蠹属的种类.该检测技术可以应用于其他经济重要性有害生物的检测鉴定.     

Molecular phylogenetics of Triculine snails (Gastropoda: Pomatiopsidae) from southern China

Triculine (Gastropoda: Rissooidea: Pomatiopsidae) snails are involved in the transmission of schistosomiasis and paragonimiasis; their distributions are mainly across southeastern Asia and southern China. In the present investigation, partial sequences of COI, 16S, and 28S were examined to infer the phylogenetic relationships among the species rich and poorly understood gastropod. Samples were collected from 12 geographic locations in six provinces of southern China. Several methods such as maximum parsimony, maximum likelihood and distance analysis were used in phylogenetic analyses among these taxa. The resultant phylogenetic trees showed a similar topology irrespective of the phylogenetic methods used. The taxa fell into two clades, with those from Fujian, Guangxi, and Zhejiang Provinces in one clade and those from Hunan, Sichuan and Hubei in the other. Among the taxa in Hubei Province, five formed a monophyletic clade, but Tricula sp. H-SHY fell into a sister clade of Tricula hortensis of Sichuan, whilst Tricula hongshanensis formed a single clade. Sister taxa Tricula pingi and Tricula hsiangi formed well-supported clade within almost all the trees. These results, while preliminary, represent the first attempt to reconstruct a phylogeny for Triculinae across China.     

Salenthydrobia gen. nov. (Rissooidea: Hydrobiidae): a potential relict of the Messinian salinity crisis

The Messinian salinity crisis (MSC) occurred synchronously throughout the Mediterranean basin about 5.96 ± 0.02 Mya and represents one of the most dramatic oceanic changes since the early Miocene. It is thought that the concomitant environmental changes brought about isolation of faunas and the development of endemism. As part of the search for possible speciation events triggered by the MSC, the author studied 38 populations of hydrobiine snails from the Mediterranean and Black Sea, including three populations from the Salentina Peninsula, Italy. Partial sequences (COI, 16S) and anatomical data were used to test the taxonomic and phylogenetic status of the peninsular populations. A maximum likelihood analysis of 11 hydrobiine taxa revealed five clades and lineages, four of which corresponded to previously recognized genera: Adriohydrobia , Hydrobia , Peringia , Ventrosia . The fifth clade was formed by haplotypes of the peninsular populations, which are characterized by distinct male and female reproductive systems. Based on molecular and anatomical data, these populations are considered to represent a new species, Salenthydrobia ferrerii , belonging to a new genus, Salenthydrobia . Ecological and biogeographical data for S. ferrerii strongly support a correlation between its origin and the MSC. Based on an island age of 5.33 Myr and a population divergence of 0.0973 ± 0.0114, the COI molecular clock rate for the Salenthydrobia and Peringia clades would be 1.83 ± 0.21% population divergence per Myr. The genetic diversity of S. ferrerii , its phylogenetic relationships, and the validity of the proposed local molecular clock rate are discussed.     

Identification of Cultured Brachionus Rotifers Based on RFLP and SSCP Screening

The marine finfish industry worldwide depends greatly on the mass culture of Brachionus rotifers. Recently, molecular data have revealed a more complicated view about the species status of Brachionus rotifers than previous mainly morphological assessments. Under this view, Brachionus rotifers are comprised of many morphologically similar, albeit genetically differentiated, cryptic members of larger groups. A redefinition of the cultured rotifer species/biotypes is therefore needed if aquaculture is to reach higher levels of standardization and predictability. In this work, restriction fragment length polymorphism (RFLP) and single-strand conformational polymorphism (SSCP) methods are applied to the COI and 16S rRNA mitochondrial genes. A detailed COI restriction map was constructed, using sequence data from all known representatives of Brachionus phylogroups. Therefore, it is the first time that such an extended restriction database has been produced. Several restriction endonucleases are proposed for the discrimination of the different Brachionus species/biotypes. Furthermore, eight different SSCP gel alleles are described for the 16S region. Using these data, five Brachionus species/biotypes were identified in 78 samples collected from laboratories and hatcheries around the world. Spiros Papakostas and Stefania Dooms contributed equally to this work.     

Scorpions of the genus Odontobuthus Vachon, 1950 (Scorpiones: Buthidae) from Iran: Phylogenetic relationships inferred from mitochondrial DNA sequence data

The molecular phylogeny of the genus Odontobuthus Vachon, 1950 (Scorpiones: Buthidae) in Iran was evaluated using two mitochondrial DNA genes, cytochrome c oxidase, subunit I (COI) and 16S ribosomal RNA (16S rRNA). The molecular phylogenetic analyses were performed using Maximum Parsimony, Maximum Likelihood and Bayesian inference methods. The resulting topologies supported two main clades: the clade comprising Odontobuthus doriae, O. bidentatus, and O. tavighiae, and another one which is the O. tirgari clade. The results clearly presented additional support for the taxonomic validity of the recently described species, O. tirgari and O. tavighiae. In addition, the monophyly of two previously described species O. doriae and O. bidentatus was confirmed. According to the data presented here, three taxonomically valid species belonging to the genus Odontobuthus occur in Iran.     

Polymorphism of mtDNA regions in brown hare (Lepus europaeus) populations from Vojvodina (Serbia and Montenegro)

It is well known that there is heterogeneity of mitochondrial DNA (mtDNA) within and among natural populations of same species. The polymorphism level of particular regions of mtDNA gives valuable results in detection of population genetic structure. The aim of this paper was to detect polymorphism of three mtDNA regions: cytochrome oxidase I (COI), Control region, and 12S/16S rRNA, by the mtDNA RFLP-PCR method, in three Lepus europaeus populations from Vojvodina province (Serbia and Montenegro). Polymorphism was detected within the two regions, COI and Control region, while 12S/16S rRNA region was monomorphic in all 77 individuals. Eight haplotypes were detected in the brown hare population in Vojvodina, and three were unique for the Srem brown hare population.     

Evolution of Mesobuthus gibbosus (Brullé, 1832) (Scorpiones: Buthidae) in the northeastern Mediterranean region

Sequence data derived from two mitochondrial markers, 16S rRNA and COI genes, were used to infer the evolutionary history of 47 insular and mainland populations covering most of the distributional range of the northeastern Mediterranean scorpion species Mesobuthus gibbosus. Based on the estimated divergence times of Mesobuthus lineages, the temporal frame of the genus differentiation in the northeastern Mediterranean region is placed in middle Miocene (15 million years ago). The biogeographic affinities of M. gibbosus populations point towards a mainly vicariant pattern of differentiation of the species which is consistent with the geological events that transformed the Aegean region during the period from 12 to 5 million years ago. M. gibbosus is an old northeastern Mediterranean species that has retained valuable bits of genetic information, reflecting some of the oldest vicariant events that have occurred in the area. Most importantly, the history witnessed by M. gibbosus has not been obscured by more recent palaeoevents of the region. Therefore, the case of M. gibbosus is in favour of a taxon-oriented 'perception' of the natural history of a given area.