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。     

药用植物DNA条形码鉴定研究进展

DNA条形码是利用相对较短的标准DNA片段对物种进行快速准确鉴定的一门技术。DNA条形码技术可以从分子水平弥补传统鉴定方法的一些不足。该技术具有良好的通用性,使得物种鉴定过程更加快速,已经广泛应用于动物物种的鉴定研究中。近年来,随着药用植物DNA条形码鉴定研究的快速发展,逐渐形成了药用植物和植物源中药材鉴定的完善体系。本文综述了DNA条形码技术鉴定药用植物的原理,介绍了中草药传统鉴定方法及其缺陷、使用DNA条形码技术鉴定植物源药材的意义以及DNA条形码在药用植物鉴定中的应用,对其应用前景进行了展望。     

DNA条形码在入侵植物鉴定中的应用进展

生物入侵对世界经济、环境造成了巨大的影响,已经成为世界关注的焦点。传统的海关检验方法存在鉴定缓慢、准确率低、鉴定专家稀缺等问题,因此急需一种鉴定率高、操作简单和快速的方法对入侵植物的繁殖体进行精确的鉴别。DNA条形码是一种基于DNA序列差异进行物种鉴定的技术,鉴定结果只受样品组织内DNA保存状况影响,不受形态学性状保存状态影响,只需掌握简单分子生物学技术的工作人员即可实现对未知样品的鉴定,在入侵植物检疫鉴定中有很大的应用潜力。根据入侵植物进化快、变异多的特点,可优先考虑种间、种内差异度高的ITS基因作为核心条形码,再以mat K和rbc L基因为辅助条形码。本文分析了植物DNA条形码技术及其衍生出的超级DNA条形码和metabarcoding技术在入侵植物鉴定中的应用潜力,提出构建入侵植物DNA条形码参考数据库与智能植物志(i Flora)相结合,为利用DNA条形码技术对入侵植物进行快速鉴定和相关研究提供参考。     

线粒体COⅠ基因在昆虫DNA条形码中的研究与应用

自2003年DNA条形码(DNA barcodes)概念出现以来,DNA条形码技术(DNA barcoding)受到生物分类学领域普遍关注,线粒体细胞色素氧化酶亚基I(mtDNACOⅠ)被用作动物类群的主要条形码序列,基于该基因片段的昆虫条形码研究在国内外广泛开展。本文在概述DNA条形码、条形码技术及已开展的昆虫条形码研究计划的基础上,总结了昆虫mtDNACOⅠ条形码及其技术在发现和描述隐种、种类分子鉴定以及系统发育等方面的研究进展,分析了细胞核线粒体假基因(Numts)对mtDNACOⅠ条形码扩增的影响,提出检测和避免Numts的方法,并对DNA条形码技术的进一步研究和应用进行了讨论和展望。     

DNA条形码技术及其在微生物资源鉴别保护中的应用研究

DNA条形码作为一种新型的物种鉴定、分类、鉴别和溯源方法,通过生物信息学分析将标准DNA片段进行分析比对实现。经过多年的发展,该技术现已成为物种鉴定和分类的研究热点。回顾十余年来DNA条形码技术的发展历程,介绍了这一技术的进展与成果,分析了该技术的优势及局限,并展望了DNA条形码的应用前景,为后续研究提供参考。     

DNA条形码在生态学研究中的应用与展望

DNA条形码是利用标准的DNA片段对物种进行快速鉴定的技术,已在生物学各相关领域得到广泛应用。随着DNA条形码技术的不断发展和完善,已成功应用于生态学领域的相关研究中。本文综述了DNA条形码在物种快速鉴定和隐存种发现、群落系统发育重建和生态取证、群落内物种间相互关系研究等方面的应用,并介绍了DNAmetabarcoding技术和环境DNA条形码在生物多样性和生态学研究领域中的应用。最后,结合新的测序技术和未来大科学装置的发展,在相关数据库逐渐完善,新分析方法和计算模型不断开发使用的情景下,对DNA条形码在生态学相关领域的应用前景进行了展望。     

应用DNA条形码对植物标本的核查

DNA条形码主要目的是物种鉴定和新物种或隐存种的发现,而DNA条形码参考数据库是物种快速鉴定的重要基础。目前中国维管植物DNA条形码参考数据库正在建设之中,借助于公共数据库（NCBI）和初步建立的中国植物DNA条形码参考数据库,运用DNA条形码数据开展了植物标本鉴定的核查工作：（1）比较DNA序列信息与标本鉴定信息,从科、属、种级水平查找鉴定错误的标本;（2）基于有较好研究基础的DNA条形码参考数据库,开展未知标本的鉴定;（3）通过对标本核查的总结,提出DNA条形码参考数据库建设过程中的几点建议。     

DNA宏条形码技术在食草动物食性研究中的应用

随着测序技术的快速发展,整合DNA条形码和高通量测序的DNA宏条形码技术已经成为当前研究热点之一,在食草动物的食性鉴定中有很大潜力。放牧动物食性研究是动物营养学和草地生态学领域的重要研究内容。而与传统食性研究方法相比,宏条形码技术可通过对植物DNA条形码的高通量测序,获得样本中的物种组成进而分析动物食性。介绍了传统食性分析手段的局限,重点综述了DNA宏条形码技术的产生、操作原理以及在食草类动物食性鉴定领域中的应用,同时还简述了可能存在的挑战,并对该技术今后的发展方向进行了展望。     

DNA条形码在鞘翅目昆虫分子系统学研究中的应用

近年来,DNA条形码(DNA Barcoding)技术已经成为生物分类学研究中备受关注的新型技术,并在鞘翅目昆虫系统发育研究中得到广泛应用。本文总结了鞘翅目昆虫DNA条形码研究所用COⅠ基因序列,概述了DNA条形码在鞘翅目昆虫的物种分类鉴定、发现新种和隐存种、系统发育关系研究等方面的应用,并对DNA条形码研究技术新进展和标准序列筛选需要注意的问题进行了讨论。     

DNA条形码技术的研究进展及其应用

DNA条形码技术(DNA Barcod ing)是通过对一个标准目的基因的DNA序列进行分析从而进行物种鉴定的技术。这个概念的原理与零售业中对商品进行辨认的商品条形码是一样的。简单地说,DNA条形码技术的关键就是对一个或一些相关基因进行大范围的扫描,进而来鉴定某个未知的物种或者发现新种[1—3]。自从提出DNA条形码的概念以来,这种新兴分类学技术已经引起了越来越多的生物学家的关注。DNA条形码技术是分类学中辅助物种鉴定的新技术,它代表了生物分类学研究的一个新方向[4],因此它在生态、环境、食品等诸多领域都将会有广泛的应用[5]。本文概括综述了DNA条形码技术的发展历史、原理与操作,分析了其在生物分类中的应用及应用上的优势与限制,对DNA条形码技术在鱼类学研究的意义与可行性进行了探讨。1 DNA条形码技术的发展历史2003年,Herbert研究发现利用线粒体细胞色素C氧化酶亚基Ⅰ(M itochondrial cytochrom ecoxidase subun itⅠ,COⅠ)基因一段长度为648bp的片段,能够在DNA水平上成功的区分物种,并且认为利用COⅠ基因从分子演化的角度,将提供一种快速、简便、可信的分...     

Detection of invasive and cryptic species in marine mussels (Bivalvia,Mytilidae): A chromosomal perspective

Marine mussels illustrate a stunning variability in shape and color. Such variability, added to the scarcity of reliable morphological characters for their identification, can mislead recognition prompting the assignation of specimens of a single species to different ones or incorporate specimens belonging to different taxa into a single one. DNA barcoding is widely used for species identification; however, as this method relies on the previous morphological identification of the specimens, some of the DNA sequences stored in DNA databases are incorrectly assigned to a given species. In view of this uncertainty, further criteria beyond morphological characters and DNA sequences in databases are required to more reliably and accurately identify marine mussels. In this work we mapped ribosomal RNA and histone gene clusters to chromosomes of four species of marine mussels and compared them with those from another eight marine mussel taxa. Specimens of these twelve taxa were also DNA barcoded. Our results clearly demonstrated that the chromosomal analysis of marine mussels could shed light on their identification and, therefore, solve contradictions posed by morphological and molecular data.     

DNA barcoding herbaceous and woody plant species at a subalpine forest dynamics plot in Southwest China

Although DNA barcoding has been widely used to identify plant species composition in temperate and tropical ecosystems, relatively few studies have used DNA barcodes to document both herbaceous and woody components of forest plot. A total of 201 species (72 woody species and 129 herbaceous species) representing 135 genera distributed across 64 families of seed plants were collected in a 25 ha CForBio subalpine forest dynamics plot. In total, 491 specimens were screened for three DNA regions of the chloroplast genome (rbcL, matK, and trnH‐psbA) as well as the internal transcribed spacers (ITS) of nuclear ribosomal DNA. We quantified species resolution for each barcode separately or in combination using a ML tree‐based method. Amplification and sequencing success were highest for rbcL, followed by trnH‐psbA, which performed better than ITS and matK. The rbcL + ITS barcode had slightly higher species resolution rates (88.60%) compared with rbcL + matK (86.60%) and rbcL + trnH‐psbA (86.01%). The addition of trnH‐psbA or ITS to the rbcL + matK barcode only marginally increased species resolution rates, although in combination the four barcodes had the highest discriminatory power (90.21%). The situations where DNA barcodes did not discriminate among species were typically associated with higher numbers of co‐occurring con‐generic species. In addition, herbaceous species were much better resolved than woody species. Our study represents one of the first applications of DNA barcodes in a subalpine forest dynamics plot and contributes to our understanding of patterns of genetic divergence among woody and herbaceous plant species.     

Applying plant DNA barcodes to identify species of Parnassia (Parnassiaceae)

DNA barcoding is a technique to identify species by using standardized DNA sequences. In this study, a total of 105 samples, representing 30 Parnassia species, were collected to test the effectiveness of four proposed DNA barcodes (rbcL, matK, trnH-psbA and ITS) for species identification. Our results demonstrated that all four candidate DNA markers have a maximum level of primer universality and sequencing success. As a single DNA marker, the ITS region provided the highest species resolution with 86.7%, followed by trnH-psbA with 73.3%. The combination of the core barcode regions, matK+rbcL, gave the lowest species identification success (63.3%) among any combination of multiple markers and was found unsuitable as DNA barcode for Parnassia. The combination of ITS+trnH-psbA achieved the highest species discrimination with 90.0% resolution (27 of 30 sampled species), equal to the four-marker combination and higher than any two or three marker combination including rbcL or matK. Therefore, matK and rbcL should not be used as DNA barcodes for the species identification of Parnassia. Based on the overall performance, the combination of ITS+trnH-psbA is proposed as the most suitable DNA barcode for identifying Parnassia species. DNA barcoding is a useful technique and provides a reliable and effective mean for the discrimination of Parnassia species, and in combination with morphology-based taxonomy, will be a robust approach for tackling taxonomically complex groups. In the light of our findings, we found among the three species not identified a possible cryptic speciation event in Parnassia.     

生命条形码新秀：DNA微型条形码技术

近些年来,DNA条形码技术为便捷的物种鉴定提供了很大的帮助,但随着该技术的发展,也出现了一系列的问题。微型条形码技术是作为DNA条形码技术的补充而出现的一项新兴技术,具体是指通过通用引物扩增出比细胞色素c氧化酶I号基因全序列更短的一段序列,并通过该序列进行物种鉴定、分类等研究工作。作为一项新兴技术,其优点包括,适用于部分降解的DNA样品的目的基因扩增,能够很好地解决环境混合样品多样性的调查等。但是,该技术所选DNA片段非常短,因此标记序列包含的遗传信息有限,在鉴定的精确度方面和COI全条形码存在一定的差距。本文在总结前人研究的基础上,简要概述了微型条形码技术的优缺点,并对其未来在害虫分子识别方面的应用做了初步探讨。     

Assessing DNA for fish identifications from reference collections: the good,bad and ugly shed light on formalin fixation and sequencing approaches

Natural history collections are repositories of biodiversity and are potentially used by molecular ecologists for comparative taxonomic, phylogenetic, biogeographic and forensic purposes. Specimens in fish collections are preserved using a combination of methods with many fixed in formalin and then preserved in ethanol for long-term storage. Formalin fixation damages DNA, thereby limiting genetic analyses. In this study, the authors compared the DNA barcoding and identification success for frozen and formalin-fixed tissues obtained from specimens in the CSIRO Australian National Fish Collection. They studied 230 samples from fishes (consisting of >160 fish species). An optimized formalin-fixed, paraffin-embedded DNA extraction method resulted in usable DNA from degraded tissues. Four mini barcoding assays of the mitochondrial DNA (mtDNA) were characterized with Sanger and Illumina amplicon sequencing. In the good quality DNA (without exposure to formalin), up to 88% of the specimens were correctly matched at the species level using the cytochrome oxidase subunit 1 (COI) mini barcodes, whereas up to 58% of the specimens exposed to formalin for less than 8 weeks were correctly identified to species. In contrast, 16S primers provided higher amplification success with formalin-exposed tissues, although the COI gene was more successful for identification. Importantly, the authors found that DNA of a certain size and quality can be amplified and sequenced despite exposure to formalin, and Illumina sequencing provided them with greater power of resolution for taxa identification even when there was little DNA present. Overall, within parameter constraints, this study highlights the possibilities of recovering DNA barcodes for identification from formalin-fixed fish specimens, and the authors provide guidelines for when successful identification could be expected.     

Quantitative microarray-based DNA-DNA hybridization assay for measuring genetic distances among bacterial species and its application to the identification of family Enterobacteriaceae

Quantitative DNA-DNA hybridization to measure the genetic distances among bacterial species is indispensable for taxonomical determination. In the current studies, we developed a method to determine bacterial DNA relatedness on a glass microarray. Reference DNAs representing a total 93 species of Enterobacteriaceae were arrayed on a glass microplate, and signal intensities were measured after 2 hr of hybridization with Cy3-labeled bacterial DNAs. All immobilized DNAs from members of the family Enterobacteriaceae were identified by this method except for DNAs from Yersinia pseudotuberculosis and Y. pestis. These results suggest that quantitative microarray hybridization could be an alternative to conventional DNA-DNA hybridization for measuring chromosome relatedness among bacterial species.     

Differentiation of Chlamydia Species by Combined Use of Polymerase Chain Reaction and Restriction Endonuclease Analysis

To differentiate Chlamydia spp., a primer pair designed to generate a genus-specific region of the major outer membrane protein (MOMP) gene was used in a PCR to amplify a single DNA fragment of 245-259 bp. In the PCR, the expected single DNA fragment was amplified from strains of Chlamydia trachomatis, C. psittaci, C. pneumoniae and C. pecorum, respectively. By restriction endonuclease analysis with AluI and PvuII, the amplified products exhibited four distinct patterns, corresponding to the four species. It is, therefore, concluded that one-step PCR followed by restriction endonuclease analysis as described in this study could be a valuable method for the detection and differentiation of Chlamydia species.     

Identification of species within Tetrastigma (Miq.) Planch.(Vitaceae) based on DNA barcoding techniques

Many species of Tetrastigma (Miq.) Planch. (Vitaceae) have long been used as medicinal plants in China, and some are endangered due to overexploitation. Although adulterants are often added to traditional Chinese medicines, there is no reliable or practical method for identifying them. In this study, we used four markers (rbcL, matK, trnH-psbA and internal transcribed spacer [ITS]) as DNA barcodes to test their ability to distinguish species of Tetrastigma. The results indicated that the best barcode was ITS, which showed significant inter-specific genetic variability, and thus its potential as a DNA barcode for identifying Tetrastigma. Multiple loci provided a greater ability to distinguish species than single loci. We recommend using the combined rbcL+matK+ITS barcode for the genus. Phylogenetic trees from each barcode were compared. Analyses using the unweighted pair group method with arithmetic mean discriminated an equal or greater percentage of resolvable species than did neighbor joining, maximum likelihood, or maximum parsimony analyses. Additionally, five medicinal species of Tetrastigma, especially T. Hemsleyanum, could be identified precisely using DNA barcoding.     

Genotyping single nucleotide polymorphisms (SNPs) across species in Old World Monkeys

The development of DNA markers is becoming increasingly useful in the field of primatology for studies on paternity, population history, and biomedical research. In this study, we determine the efficacy of using cross-species amplification to identify single nucleotide polymorphisms (SNPs) in closely related species. The DNA of 93 individuals representing seven Old World Monkey species was analyzed to identify SNPs using cross-species amplification and genotyping. The loci genotyped were 653 SNPs identified and validated in rhesus macaques. Of the 653 loci analyzed, 27% were estimated to be polymorphic in the samples studied. SNPs identified at the same locus among different species (coincident SNPs) were found in six of the seven species studied with longtail macaques exhibiting the highest number of coincident SNPs (84). The distribution of coincident SNPs among species is not biased based on proximity to genes in the samples studied. In addition, the frequency of coincident SNPs is not consistent with expectations based on their phylogenetic relationships. This study demonstrates that cross-species amplification and genotyping using the Illumina Golden Gate Array is a useful method to identify a large number of SNPs in closely related species, although issues with ascertainment bias may limit the type of studies where this method can be applied.     

环境DNA metabarcoding及其在生态学研究中的应用

环境DNA metabarcoding(eDNA metabarcoding)是指利用环境样本(如土壤、水、粪便等)中分离的DNA进行高通量的多个物种(或高级分类单元)鉴定的方法。近年来,该方法引起了学者的广泛关注,逐渐应用于生物多样性研究、水生生物监测、珍稀濒危物种和外来入侵物种检测等生态学领域。介绍环境DNA metabarcoding的含义和研究方法;重点介绍环境DNA metabarcoding在物种监测、生物多样性研究和食性分析等生态学领域中的应用;总结环境DNA metabarcoding应用于生态学研究领域面临的挑战并对该方法的发展进行展望。