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。     

植物物种的快速鉴定与iFlora: DNA条形码在马先蒿属中的应用

DNA条形码技术就是利用一段较短的标准DNA序列对物种进行快速鉴定。与基于植物外部形态特征的传统分类鉴定方法相比, DNA条形码具有高效、准确,且易于实现自动化和标准化的特点。马先蒿属（Pedicularis L.）植物具对生（轮生）叶的种类70%以上分布在中国,近缘种间形态上非常相似,鉴定较为困难。研究选取马先蒿属具对生（轮生）叶类群43种164份样品,利用叶绿体基因（rbcL、matK、trnH psbA）和核基因（ITS）条形码片段,采用建树法和距离法检验4个条形码对这些物种的鉴定效果。结果表明,ITS片段用于建树法和距离法的鉴别率分别为81.40%和89.57%,其鉴别率高于3个叶绿体基因片段和任一基因片段的组合条码。另外,利用ITS成功解决了一些疑难种的分类问题。DNA条形码在马先蒿属研究中的实用性为新一代植物志（iFlora）实现物种的快速和准确鉴定提供了有力支持,并能为分类学、生态学、进化生物学、居群遗传学和保护遗传学等分支学科的研究提供重要信息。     

植物物种的快速鉴定与iFlora：DNA条形码在马先蒿属中的应用

DNA条形码技术就是利用一段较短的标准DNA序列对物种进行快速鉴定。与基于植物外部形态特征的传统分类鉴定方法相比,DNA条形码具有高效、准确,且易于实现自动化和标准化的特点。马先蒿属（PedicularisL．）植物具对生（轮生）叶的种类70％以上分布在中国．近缘种间形态上非常相似,鉴定较为困难。研究选取马先蒿属具对生（轮生）叶类群43种164份样品,利用叶绿体基因（rbcL、matK、trnH-psbA）和核基因（ITS）条形码片段,采用建树法和距离法检验4个条形码对这些物种的鉴定效果。结果表明,ITS片段用于建树法和距离法的鉴别率分别为81．40％和89．57％,其鉴别率高于3个叶绿体基因片段和任一基因片段的组合条码。另外,利用ITS成功解决了一些疑难种的分类问题。DNA条形码在马先蒿属研究中的实用性为新一代植物志（iFlora）实现物种的快速和准确鉴定提供了有力支持,并能为分类学、生态学、进化生物学、居群遗传学和保护遗传学等分支学科的研究提供重要信息。     

植物DNA条形码技术

DNA条形码技术是利用标准的、具有足够变异的、易扩增且相对较短的DNA片段在物种内的特异性和种间的多样性而创建的一种新的生物身份识别系统, 从而实现对物种的快速自动鉴定。尽管这一技术在理论上和具体应用上仍存在很多争论, 但DNA条形码概念自2003年由加拿大分类学家Paul Hebert首次提出后就在世界范围内受到了广泛关注。在植物类群中条形码的研究和应用尚处于探索阶段, 稍落后于对动物类群的研究, 这主要表现在: (1) DNA条形码的选择及其评价仍没有统一的标准; (2) 对类群较全面的形态分类学修订和植物DNA条形码研究的结合十分缺乏; (3) 以往研究在取样上尺度较大, 而对具体类群的研究较少, 一个科或一个属只用有限的种类作为代表, 同一种内的取样个体数量也不足, 这样虽然表面上看来利用选定的DNA条形码可以较容易地把代表物种区分开, 但实际上目前建议的植物DNA条形码(例如由生命条形码咨询委员会植物工作组最近提出的rbcL和matK)由于其分子进化速率较慢, 在种级水平上, 特别是对于那些经历了适应辐射或快速进化的属来说, 分辨率较低。而DNA条形码的应用主要集中在属内物种水平的鉴别, 因此只有针对具体类群进行探索研究, 发现进化速率较快、分辨率高且通用性好的条形码, 才可能为建立完整的条形码数据库起到积极有效的作用。     

Benchmarking DNA barcodes: An assessment using available primate sequences.

DNA barcoding has been recently promoted as a method for both assigning specimens to known species and for discovering new and cryptic species. Here we test both the potential and the limitations of DNA barcodes by analysing a group of well-studied organisms--the primates. Our results show that DNA barcodes provide enough information to efficiently identify and delineate primate species, but that they cannot reliably uncover many of the deeper phylogenetic relationships. Our conclusion is that these short DNA sequences do not contain enough information to build reliable molecular phylogenies or define new species, but that they can provide efficient sequence tags for assigning unknown specimens to known species. As such, DNA barcoding provides enormous potential for use in global biodiversity studies.     

Land plants and DNA barcodes: short-term and long-term goals

Land plants have had the reputation of being problematic for DNA barcoding for two general reasons: (i) the standard DNA regions used in algae, animals and fungi have exceedingly low levels of variability and (ii) the typically used land plant plastid phylogenetic markers (e.g. rbcL, trnL-F, etc.) appear to have too little variation. However, no one has assessed how well current phylogenetic resources might work in the context of identification (versus phylogeny reconstruction). In this paper, we make such an assessment, particularly with two of the markers commonly sequenced in land plant phylogenetic studies, plastid rbcL and internal transcribed spacers of the large subunits of nuclear ribosomal DNA (ITS), and find that both of these DNA regions perform well even though the data currently available in GenBank/EBI were not produced to be used as barcodes and BLAST searches are not an ideal tool for this purpose. These results bode well for the use of even more variable regions of plastid DNA (such as, for example, psbA-trnH) as barcodes, once they have been widely sequenced. In the short term, efforts to bring land plant barcoding up to the standards being used now in other organisms should make swift progress. There are two categories of DNA barcode users, scientists in fields other than taxonomy and taxonomists. For the former, the use of mitochondrial and plastid DNA, the two most easily assessed genomes, is at least in the short term a useful tool that permits them to get on with their studies, which depend on knowing roughly which species or species groups they are dealing with, but these same DNA regions have important drawbacks for use in taxonomic studies (i.e. studies designed to elucidate species limits). For these purposes, DNA markers from uniparentally (usually maternally) inherited genomes can only provide half of the story required to improve taxonomic standards being used in DNA barcoding. In the long term, we will need to develop more sophisticated barcoding tools, which would be multiple, low-copy nuclear markers with sufficient genetic variability and PCR-reliability; these would permit the detection of hybrids and permit researchers to identify the 'genetic gaps' that are useful in assessing species limits.     

Choosing and using a plant DNA barcode

The main aim of DNA barcoding is to establish a shared community resource of DNA sequences that can be used for organismal identification and taxonomic clarification. This approach was successfully pioneered in animals using a portion of the cytochrome oxidase 1 (CO1) mitochondrial gene. In plants, establishing a standardized DNA barcoding system has been more challenging. In this paper, we review the process of selecting and refining a plant barcode; evaluate the factors which influence the discriminatory power of the approach; describe some early applications of plant barcoding and summarise major emerging projects; and outline tool development that will be necessary for plant DNA barcoding to advance.     

Environmental barcoding: a next-generation sequencing approach for biomonitoring applications using river benthos

Timely and accurate biodiversity analysis poses an ongoing challenge for the success of biomonitoring programs. Morphology-based identification of bioindicator taxa is time consuming, and rarely supports species-level resolution especially for immature life stages. Much work has been done in the past decade to develop alternative approaches for biodiversity analysis using DNA sequence-based approaches such as molecular phylogenetics and DNA barcoding. On-going assembly of DNA barcode reference libraries will provide the basis for a DNA-based identification system. The use of recently introduced next-generation sequencing (NGS) approaches in biodiversity science has the potential to further extend the application of DNA information for routine biomonitoring applications to an unprecedented scale. Here we demonstrate the feasibility of using 454 massively parallel pyrosequencing for species-level analysis of freshwater benthic macroinvertebrate taxa commonly used for biomonitoring. We designed our experiments in order to directly compare morphology-based, Sanger sequencing DNA barcoding, and next-generation environmental barcoding approaches. Our results show the ability of 454 pyrosequencing of mini-barcodes to accurately identify all species with more than 1% abundance in the pooled mixture. Although the approach failed to identify 6 rare species in the mixture, the presence of sequences from 9 species that were not represented by individuals in the mixture provides evidence that DNA based analysis may yet provide a valuable approach in finding rare species in bulk environmental samples. We further demonstrate the application of the environmental barcoding approach by comparing benthic macroinvertebrates from an urban region to those obtained from a conservation area. Although considerable effort will be required to robustly optimize NGS tools to identify species from bulk environmental samples, our results indicate the potential of an environmental barcoding approach for biomonitoring programs.     

DNA barcoding: error rates based on comprehensive sampling

DNA barcoding has attracted attention with promises to aid in species identification and discovery; however, few well-sampled datasets are available to test its performance. We provide the first examination of barcoding performance in a comprehensively sampled, diverse group (cypraeid marine gastropods, or cowries). We utilize previous methods for testing performance and employ a novel phylogenetic approach to calculate intraspecific variation and interspecific divergence. Error rates are estimated for (1) identifying samples against a well-characterized phylogeny, and (2) assisting in species discovery for partially known groups. We find that the lowest overall error for species identification is 4%. In contrast, barcoding performs poorly in incompletely sampled groups. Here, species delineation relies on the use of thresholds, set to differentiate between intraspecific variation and interspecific divergence. Whereas proponents envision a “barcoding gap” between the two, we find substantial overlap, leading to minimal error rates of ~17% in cowries. Moreover, error rates double if only traditionally recognized species are analyzed. Thus, DNA barcoding holds promise for identification in taxonomically well-understood and thoroughly sampled clades. However, the use of thresholds does not bode well for delineating closely related species in taxonomically understudied groups. The promise of barcoding will be realized only if based on solid taxonomic foundations.     

After 7 years and 1000 citations: comparative assessment of the DNA barcoding and the DNA taxonomy proposals for taxonomists and non-taxonomists

In 2003, two different approaches-DNA taxonomy and DNA barcoding-were simultaneously proposed to overcome some of the perceived intrinsic weaknesses of the traditional morphology-based taxonomical system, and to help non-taxonomists to resolve their crucial need for accurate and rapid species identification tools. After 7 years, it seems unlikely that a completely new taxonomical system based on molecular characters only (DNA taxonomy) will develop in the future. It is more likely that both morphological and molecular data will be simultaneously analyzed, developing what has been coined as "integrative taxonomy". Concerning DNA barcoding, it is now clear that it does not focus on building a tree-of-life nor to perform DNA taxonomy, but rather to produce a universal molecular identification key based on strong taxonomic knowledge that is collated in the barcode reference library. The indisputable success of the DNA barcoding project is chiefly due to the fact that DNA barcoding standards considerably enhance current practices in the molecular identification field, and standardization offers virtually endless applications for various users.     

Evaluating next‐generation sequencing (NGS) methods for routine monitoring of wild bees: Metabarcoding,mitogenomics or NGS barcoding

Implementing cost‐effective monitoring programs for wild bees remains challenging due to the high costs of sampling and specimen identification. To reduce costs, next‐generation sequencing (NGS)‐based methods have lately been suggested as alternatives to morphology‐based identifications. To provide a comprehensive presentation of the advantages and weaknesses of different NGS‐based identification methods, we assessed three of the most promising ones, namely metabarcoding, mitogenomics and NGS barcoding. Using a regular monitoring data set (723 specimens identified using morphology), we found that NGS barcoding performed best for both species presence/absence and abundance data, producing only few false positives (3.4%) and no false negatives. In contrast, the proportion of false positives and false negatives was higher using metabarcoding and mitogenomics. Although strong correlations were found between biomass and read numbers, abundance estimates significantly skewed the communities' composition in these two techniques. NGS barcoding recovered the same ecological patterns as morphology. Ecological conclusions based on metabarcoding and mitogenomics were similar to those based on morphology when using presence/absence data, but different when using abundance data. In terms of workload and cost, we show that metabarcoding and NGS barcoding can compete with morphology, but not mitogenomics which was consistently more expensive. Based on these results, we advocate that NGS barcoding is currently the seemliest NGS method for monitoring of wild bees. Furthermore, this method has the advantage of potentially linking DNA sequences with preserved voucher specimens, which enable morphological re‐examination and will thus produce verifiable records which can be fed into faunistic databases.     

DNA条形码技术在海洋贝类鉴定中的实践: 以大亚湾生态监控区为例

为提高物种鉴定的准确性, 本研究采用DNA条形码技术对大亚湾生态监控区冬季采集的贝类样品进行了种类鉴定。结果表明, 26个形态种中, 有15个可以通过线粒体COI和16S rRNA基因的系统发育分析鉴定到种的水平。部分形态上难以鉴定的种类, 如线缝摺塔螺(Ptychobela suturalis)和区系螺(Funa sp.)可以通过条形码实现有效鉴定。锯齿巴非蛤(Paphia gallus)、西格织纹螺(Nassarius siquijorensis)、爪哇拟塔螺(Turricula javana)等种类存在相当大的种内遗传距离, 有存在隐存种的可能性。尽管基于线粒体COI和16S rRNA基因的种内遗传距离和属内种间的遗传距离发生重合, 无明显的条形码间隙, 但通过系统树的方法仍能有效鉴定物种。可见, DNA条形码技术能有效提高海洋贝类物种鉴定的准确性并发现隐存种。     

Estimating sample sizes for DNA barcoding

Sample size has long been one of the basic issues since the start of the DNA barcoding initiative and the global biodiversity investigation. As a contribution to resolving this problem, we propose a simple resampling approach to estimate several key sampling sizes for a DNA barcoding project. We illustrate our approach using both structured populations simulated under coalescent and real species of skipper butterflies. We found that sample sizes widely used in DNA barcoding are insufficient to assess the genetic diversity of a species, population structure impacts the estimation of the sample sizes, and hence will bias the species identification potentially.     

Identifying a “pseudogene” for the mitochondrial DNA COI region of the corixid aquatic insect,Hesperocorixa distanti (Heteroptera,Corixidae)

Limnology - DNA barcoding has been actively used as a method for species identification, and it will become an increasingly important method in the future. However, DNA barcoding can occasionally...     

DNA Barcoding is not enough: mismatch of taxonomy and genealogy in New Zealand grasshoppers (Orthoptera: Acrididae).

DNA barcoding has been touted as a program that will efficiently and relatively cheaply inform on biological diversity; yet many exemplars purporting to demonstrate the efficacy of the method have been undertaken by its principal proponents. Critics of DNA barcoding identify insufficient within-taxon sampling coupled with the knowledge that levels of haplotypic paraphyly are rather high as key reasons to be sceptical of the value of an exclusively DNA-based taxonomic. Here I applied a DNA barcoding approach using mtDNA sequences from the cytochrome oxidase I gene to examine diversity in a group of endemic New Zealand grasshoppers belonging to the genus Sigaus . The mtDNA data revealed high genetic distances among individuals of a single morpho-species, but this diversity was geographically partitioned. Phylogenetic analysis supported at least four haplogroups within one species ( Sigaus australis ) but paraphyly of this species with respect to several others. In some instances two morphologically and ecologically distinct species shared identical mtDNA haplotypes. The mismatch of genealogy and taxonomy revealed in the Sigaus australis complex indicates that, if used in isolation, DNA barcoding data can be highly misleading about biodiversity. Furthermore, failure to take into account evidence from natural history and morphology when utilizing DNA barcoding will tend to conceal the underlying evolutionary processes associated with speciation.
© The Willi Hennig Society 2007.     

The DNA Barcode Linker

DNA barcoding is based on the use of short DNA sequences to provide taxonomic tags for rapid, efficient identification of biological specimens. Currently, reference databases are being compiled. In the future, it will be important to facilitate access to these databases, especially for nonspecialist users. The method described here provides a rapid, web-based, user-friendly link between the DNA sequence from an unidentified biological specimen and various types of biological information, including the species name. Specifically, we use a customized, Google-type search algorithm to quickly match an unknown DNA sequence to a list of verified DNA barcodes in the reference database. In addition to retrieving the species name, our web tool also provides automatic links to a range of other information about that species. As the DNA barcode database becomes more populated, it will become increasingly important for the broader user community to be able to exploit it for the rapid identification of unknown specimens and to easily obtain relevant biological information about these species. The application presented here meets that need.     

真菌DNA条形码技术研究进展

DNA条形码(DNA barcoding)技术作为一门新兴的物种鉴定方法以其灵敏、精确、方便和客观的优势,在动植物和微生物的分类鉴定中已经得到广泛应用.真菌鉴定中常用作标准条形码的是核核糖体DNA内转录间隔区(Internal transcribed spacer,ITS),如今也有一些新型条形码被发现和应用到实际操作中,如微条形码、ND6、EF3.本文对DNA条形码技术的产生和发展做出了总结,通过研究其在真菌中应用的实际案例分析了DNA条形码技术的优缺点及发展趋势,并指出DNA条形码技术将以全新的视角来弥补传统分类学的不足,最终实现生物自身的序列变异信息与现有形态分类学的结合.     

A simulation study of sample size for DNA barcoding

For some groups of organisms, DNA barcoding can provide a useful tool in taxonomy, evolutionary biology, and biodiversity assessment. However, the efficacy of DNA barcoding depends on the degree of sampling per species, because a large enough sample size is needed to provide a reliable estimate of genetic polymorphism and for delimiting species. We used a simulation approach to examine the effects of sample size on four estimators of genetic polymorphism related to DNA barcoding: mismatch distribution, nucleotide diversity, the number of haplotypes, and maximum pairwise distance. Our results showed that mismatch distributions derived from subsamples of ≥20 individuals usually bore a close resemblance to that of the full dataset. Estimates of nucleotide diversity from subsamples of ≥20 individuals tended to be bell‐shaped around that of the full dataset, whereas estimates from smaller subsamples were not. As expected, greater sampling generally led to an increase in the number of haplotypes. We also found that subsamples of ≥20 individuals allowed a good estimate of the maximum pairwise distance of the full dataset, while smaller ones were associated with a high probability of underestimation. Overall, our study confirms the expectation that larger samples are beneficial for the efficacy of DNA barcoding and suggests that a minimum sample size of 20 individuals is needed in practice for each population.     

Applying DNA barcoding for the study of geographical variation in host-parasitoid interactions

Studies on the biogeography of host-parasitoid interactions are scarce, mainly because of technical difficulties associated with rearing and species identification. DNA barcoding is increasingly recognized as a valuable tool for taxon identification, allowing to link different life history stages of a species. We evaluate the usefulness of a protocol based on cytochrome oxidase I (COI) sequencing for the study of geographical variation of host-parasitoid interactions. Larvae of Acroclita subsequana (Lepidoptera: Tortricidae) were collected in Macaronesia and dissected to search for parasitoid larvae. Both hosts and parasitoids were sequenced and assigned to molecular operational taxonomic units (MOTUs) based on pairwise genetic distances, tree-based and similarity-based methods. Hosts were grouped into six MOTUs, usually with an allopatric distribution, while parasitoids clustered into 12 MOTUs, each of which was mostly found attacking a single host MOTU. Available COI sequence databases failed to provide identification to species level for these MOTUs. Three challenges related to the applicability of DNA barcoding in this type of studies are identified and discussed: (i) more suitable primers need to be developed for both parasitoids and hosts; (ii) the most commonly used approaches for inferring MOTUs have different limitations (e.g. arbitrary nature of defining a threshold to separate MOTUs) and need to be improved or replaced by other techniques; and (iii) for the identification of MOTUs, it is imperative to increase the range of sequenced taxa in the currently available reference databases. Finally, in spite of these difficulties, we discuss how DNA barcoding will help ecological and biogeographical studies of host-parasitoid interactions.     

DNA barcoding will frequently fail in complicated groups: An example in wild potatoes

DNA barcoding ("barcoding") has been proposed as a rapid and practical molecular method to identify species via diagnostic variation in short orthologous DNA sequences from one or a few universal genomic regions. It seeks to address in a rapid and simple way the "taxonomic impediment" of a greater need for taxonomic identifications than can be supplied by taxonomists. Using a complicated plant group, Solanum sect. Petota (wild potatoes), I tested barcoding with the most variable and frequently suggested plant barcoding regions: the internal nontranscribed spacer of nuclear ribosomal DNA (ITS) and the plastid markers trnH-psbA intergenic spacer and matK. These DNA regions fail to provide species-specific markers in sect. Petota because the ITS has too much intraspecific variation and the plastid markers lack sufficient polymorphism. The complications seen in wild potatoes are common in many plant groups, but they have not been assessed with barcoding. Barcoding is a retroactive procedure that relies on well-defined species to function, is based solely on a limited number of DNA sequences that are often inappropriate at the species level, has been poorly tested with geographically well-dispersed replicate samples from difficult taxonomic groups, and discounts substantial practical and theoretical problems in defining species.