Metabarcoding技术在植物鉴定和多样性研究中的应用

目前植物学研究已进入后植物志时代,iFlora的实现需要以传统植物分类学及相关研究为基础,整合基于高通量测序的DNA条形码（DNAbareoding）技术并开发便携式快速鉴定仪器及构建信息平台。传统植物鉴定多基于形态学分类,而近年来快速发展的DNA条形码快速鉴定技术被各界分类学家认可,在植物鉴定中也被广泛应用。但DNA条形码技术仍存在一些问题亟待解决,如种的鉴定需要多个条形码的解析、Sanger测序平台无法处理混合样品。本文介绍了传统植物分类技术和DNA条形码技术在植物研究中的应用和遇到的瓶颈;并重点介绍了基于高通量测序的metabarcoding技术在植物鉴定及多样性研究中的应用及前景,及其与iFlora的关系。     

Some ‘ant’swers: Application of a layered barcode approach to problems in ant taxonomy

DNA barcoding has emerged as a routine tool in modern taxonomy. Although straightforward, this approach faces new challenges, when applied to difficult situation such as defining cryptic biodiversity. Ants are prime examples for high degrees of cryptic biodiversity due to complex population differentiation, hybridization and speciation processes. Here, we test the DNA barcoding region, cytochrome c oxidase 1 and two supplementary markers, 28S ribosomal DNA and long‐wavelength rhodopsin, commonly used in ant taxonomy, for their potential in a layered, character‐based barcoding approach across different taxonomic levels. Furthermore, we assess performance of the character‐based barcoding approach to determine cryptic species diversity in ants. We found (i) that the barcode potential of a specific genetic marker varied widely among taxonomic levels in ants; (ii) that application of a layered, character‐based barcode for identification of specimens can be a solution to taxonomical challenging groups; (iii) that the character‐based barcoding approach allows us to differentiate specimens even within locations based on pure characters. In summary, (layered) character‐based barcoding offers a reliable alternative for problematic species identification in ants and can be used as a fast and cost‐efficient approach to estimate presence, absence or frequency of cryptic species.     

植物DNA条形码与生物多样性数据共享平台构建

DNA条形码基于较短的DNA序列实现物种的快速、准确鉴定, 不仅加快了全球生物物种的鉴定和分类步伐, 也为生物多样性的管理、保护和可持续利用提供了新思路和研究方法。植物DNA条形码标准数据库的不断完善, 将使植物多样性信息的快速获取成为可能; 将不同类型数据资源整合、共享和利用, 构建植物DNA条形码数据共享平台, 是满足公众对物种准确鉴定和快速认知的重要支撑。本文介绍了近年来植物DNA条形码的研究进展; 植物DNA条形码参考数据库的研发现状和存在的问题。结合上述问题, 围绕“大数据”时代背景, 对如何管理和使用好海量的植物信息, 如何构建数据共享平台提出了一些设想: (1)数据共享平台的元数据应尽可能翔实、丰富、准确和多关联; (2)数据标准应统一规范; (3)查询入口方便、迅速、多样, 易于管理, 便于实现更大程度的数据共享和全球化的合作交流。     

关于植物DNA条形码研究技术规范

DNA条形码是利用标准的基因片段对物种进行快速鉴定的技术,已经成功用于生物物种分类和鉴定、生态学调查和生物多样性评估等研究领域。尽管生命条形码数据（BOLD）系统提供了主要针对动物类群DNA条形码研究的技术规范,但由于植物本身的生物学特性与所使用的条形码不同,因此已有技术规范并不完全适用于植物DNA条形码的研究。本文根据植物DNA条形码研究的特点与我国的实际情况,编写了植物DNA条形码研究技术标准和规范指南,具体包括十个方面的内容,即植物DNA条形码研究的样品采集策略;植物标本和野外数据的采集规范;植物标本图像信息的采集规范;植物DNA材料的采集规范;植物DNA材料的干燥与保存规范;植物总DNA的质量标准及保存规范;植物标准DNA条形码的选择与通用引物;DNA条形码的扩增与测序;DNA条形码数据的命名、编辑和提交规范;以及DNA条形码数据分析。我们期望通过这些标准规范的实施和在实践中的不断修订和完善,能为我国学者开展植物DNA条形码和iFlora研究提供参考和借鉴。
关键词：植物DNA条形码;技术规范;物种鉴定;标准;新一代植物志     

A regional approach to plant DNA barcoding provides high species resolution of sedges (Carex and Kobresia, Cyperaceae) in the Canadian Arctic Archipelago

Previous research on barcoding sedges (Carex) suggested that basic searches within a global barcoding database would probably not resolve more than 60% of the world’s some 2000 species. In this study, we take an alternative approach and explore the performance of plant DNA barcoding in the Carex lineage from an explicitly regional perspective. We characterize the utility of a subset of the proposed protein-coding and noncoding plastid barcoding regions (matK, rpoB, rpoC1, rbcL, atpF-atpH, psbK-psbI) for distinguishing species of Carex and Kobresia in the Canadian Arctic Archipelago, a clearly defined eco-geographical region representing 1% of the Earth’s landmass. Our results show that matK resolves the greatest number of species of any single-locus (95%), and when combined in a two-locus barcode, it provides 100% species resolution in all but one combination (matK + atpFH) during unweighted pair-group method with arithmetic mean averages (UPGMA) analyses. Noncoding regions were equally or more variable than matK, but as single markers they resolve substantially fewer taxa than matK alone. When difficulties with sequencing and alignment due to microstructural variation in noncoding regions are also considered, our results support other studies in suggesting that protein-coding regions are more practical as barcoding markers. Plastid DNA barcodes are an effective identification tool for species of Carex and Kobresia in the Canadian Arctic Archipelago, a region where the number of co-existing closely related species is limited. We suggest that if a regional approach to plant DNA barcoding was applied on a global scale, it could provide a solution to the generally poor species resolution seen in previous barcoding studies.     

植物DNA条形码研究领域文献计量学及可视化分析

为全面了解植物DNA条形码研究领域的发展和最新动态,探讨中国DNA条形码发展的状态和前景,该文利用Web of Science数据库对该研究领域进行文献计量学统计,并对引用频次、研究热点和研究前沿进行了可视化分析。结果表明:(1)中国、美国、加拿大学者在该领域文献贡献率最大,中国研究机构发文量领先,但美国、加拿大科研机构论文质量较高,影响力较大。(2) 2009年是该领域研究的高峰期,该研究领域的前沿和研究热点主要集中在物种的识别和生物多样性应用、DNA条形码候选序列筛选和鉴定技术的规范化。(3)中国学者在植物DNA条形码领域研究具有领军作用和很高的影响力,国家提倡中药产业的发展也推动了我国DNA条形码蓬勃发展,但论文的质量和影响力与美国、英国、加拿大等发达国家研究还有一定差距,应加大与发达国家科研机构合作,提高研究能力,DNA条形码技术在植物的鉴定、分类和生物多样性的保护起到非常重要的作用。这表明建立一个更全面、通用的全球植物DNA条码库以及开发新的标记并采用新的测序技术是植物DNA条形码研究的未来前景。     

Plant DNA barcoding: from gene to genome

DNA barcoding is currently a widely used and effective tool that enables rapid and accurate identification of plant species; however, none of the available loci work across all species. Because single‐locus DNA barcodes lack adequate variations in closely related taxa, recent barcoding studies have placed high emphasis on the use of whole‐chloroplast genome sequences which are now more readily available as a consequence of improving sequencing technologies. While chloroplast genome sequencing can already deliver a reliable barcode for accurate plant identification it is not yet resource‐effective and does not yet offer the speed of analysis provided by single‐locus barcodes to unspecialized laboratory facilities. Here, we review the development of candidate barcodes and discuss the feasibility of using the chloroplast genome as a super‐barcode. We advocate a new approach for DNA barcoding that, for selected groups of taxa, combines the best use of single‐locus barcodes and super‐barcodes for efficient plant identification. Specific barcodes might enhance our ability to distinguish closely related plants at the species and population levels.     

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。     

Are plant DNA barcodes a search for the Holy Grail?

In a recent study, Kress et al. compared two plant genomes to seek out plant DNA barcodes. Two promising markers balanced the variability that is needed to distinguish species with conserved primer regions that enable universal amplification. Although this study is the most rigorous effort to date, problems from earlier barcoding efforts, such as the use of non-evolutionary species concepts and differential sorting of genes and species, could reemerge. Single-gene barcoding might not be universally effective owing to inherent inaccuracies. Kress et al. suggest the use of multiple genes, reflecting an integrated approach that is likely to be the best answer to identifying species quickly and accurately.     

Diet analysis by next‐generation sequencing indicates the frequent consumption of introduced plants by the critically endangered red‐headed wood pigeon (Columba janthina nitens) in oceanic island habitats

Oceanic island ecosystems are vulnerable to the introduction of alien species, and they provide a habitat for many endangered species. Knowing the diet of an endangered animal is important for appropriate nature restoration efforts on oceanic islands because introduced species may be a major component of the diets of some endangered species. DNA barcoding techniques together with next‐generation sequencing may provide more detailed information on animal diets than other traditional methods. We performed a diet analysis using 48 fecal samples from the critically endangered red‐headed wood pigeon that is endemic to the Ogasawara Islands based on chloroplast trnL P6 loop sequences. The frequency of each detected plant taxa was compared with a microhistological analysis of the same sample set. The DNA barcoding approach detected a much larger number of plants than the microhistological analysis. Plants that were difficult to identify by microhistological analysis after being digested in the pigeon stomachs were frequently identified only by DNA barcoding. The results of the barcoding analysis indicated the frequent consumption of introduced species, in addition to several native species, by the red‐headed wood pigeon. The rapid eradication of specific introduced species may reduce the food resources available to this endangered bird; thus, balancing eradication efforts with the restoration of native food plants should be considered. Although some technical problems still exist, the trnL approach to next‐generation sequencing may contribute to a better understanding of oceanic island ecosystems and their conservation.     

Assessment of candidate plant DNA barcodes using the Rutaceae family

DNA barcoding is a rapidly developing frontier technology that is gaining worldwide attention.Here,seven regions (psbA-trnH,matK,ycf5,rpoC1,rbcL,ITS2,and ITS) with potential for use as DNA barcodes were tested for their ability to identify 300 samples of 192 species from 72 genera of the family Rutaceae.To evaluate each barcode’s utility for species authentication,PCR amplification efficiency,genetic divergence,and barcoding gaps were assessed.We found that the ITS2 region exhibited the highest inter-specific divergence,and that this was significantly higher than the intra-specific variation in the "DNA barcoding gap" assessment and Wilcoxon two-sample tests.The ITS2 locus had the highest identification efficiency among all tested regions.In a previous study,we found that ITS2 was able to discriminate a wide range of plant taxa,and here we confirmed that ITS2 was also able to discriminate a number of closely related species.Therefore,we propose that ITS2 is a promising candidate barcode for plant species identification.     

DNA Barcoding as an Effective Tool in Improving a Digital Plant Identification System: A Case Study for the Area of Mt. Valerio, Trieste (NE Italy)

Background

Identification keys are decision trees which require the observation of one or more morphological characters of an organism at each step of the process. While modern digital keys can overcome several constraints of classical paper-printed keys, their performance is not error-free. Moreover, identification cannot be always achieved when a specimen lacks some morphological features (i.e. because of season, incomplete development or miss-collecting). DNA barcoding was proven to have great potential in plant identification, while it can be ineffective with some closely related taxa, in which the relatively brief evolutionary distance did not produce differences in the core-barcode sequences.

Methodology/Principal Findings

In this paper, we investigated how the DNA barcoding can support the modern digital approaches to the identification of organisms, using as a case study a local flora, that of Mt. Valerio, a small hill near the centre of Trieste (NE Italy). The core barcode markers (plastidial rbcL and matK), plus the additional trnH-psbA region, were used to identify vascular plants specimens. The usefulness of DNA barcoding data in enhancing the performance of a digital identification key was tested on three independent simulated scenarios.

Conclusions/Significance

Our results show that the core barcode markers univocally identify most species of our local flora (96%). The trnH-psbA data improve the discriminating power of DNA barcoding among closely related plant taxa. In the multiparametric digital key, DNA barcoding data improves the identification success rate; in our simulation, DNA data overcame the absence of some morphological features, reaching a correct identification for 100% of the species. FRIDA, the software used to generate the digital key, has the potential to combine different data sources: we propose to use this feature to include molecular data as well, creating an integrated identification system for plant biodiversity surveys.     

The changing epitome of species identification – DNA barcoding

The discipline taxonomy (the science of naming and classifying organisms, the original bioinformatics and a basis for all biology) is fundamentally important in ensuring the quality of life of future human generation on the earth; yet over the past few decades, the teaching and research funding in taxonomy have declined because of its classical way of practice which lead the discipline many a times to a subject of opinion, and this ultimately gave birth to several problems and challenges, and therefore the taxonomist became an endangered race in the era of genomics. Now taxonomy suddenly became fashionable again due to revolutionary approaches in taxonomy called DNA barcoding (a novel technology to provide rapid, accurate, and automated species identifications using short orthologous DNA sequences). In DNA barcoding, complete data set can be obtained from a single specimen irrespective to morphological or life stage characters. The core idea of DNA barcoding is based on the fact that the highly conserved stretches of DNA, either coding or non coding regions, vary at very minor degree during the evolution within the species. Sequences suggested to be useful in DNA barcoding include cytoplasmic mitochondrial DNA (e.g. cox1) and chloroplast DNA (e.g. rbcL, trnL-F, matK, ndhF, and atpB rbcL), and nuclear DNA (ITS, and house keeping genes e.g. gapdh). The plant DNA barcoding is now transitioning the epitome of species identification; and thus, ultimately helping in the molecularization of taxonomy, a need of the hour. The ‘DNA barcodes’ show promise in providing a practical, standardized, species-level identification tool that can be used for biodiversity assessment, life history and ecological studies, forensic analysis, and many more.     

Identification of Amazonian Trees with DNA Barcodes

Background

Large-scale plant diversity inventories are critical to develop informed conservation strategies. However, the workload required for classic taxonomic surveys remains high and is particularly problematic for megadiverse tropical forests.

Methodology/Principal Findings

Based on a comprehensive census of all trees in two hectares of a tropical forest in French Guiana, we examined whether plant DNA barcoding could contribute to increasing the quality and the pace of tropical plant biodiversity surveys. Of the eight plant DNA markers we tested (rbcLa, rpoC1, rpoB, matK, ycf5, trnL, psbA-trnH, ITS), matK and ITS had a low rate of sequencing success. More critically, none of the plastid markers achieved a rate of correct plant identification greater than 70%, either alone or combined. The performance of all barcoding markers was noticeably low in few species-rich clades, such as the Laureae, and the Sapotaceae. A field test of the approach enabled us to detect 130 molecular operational taxonomic units in a sample of 252 juvenile trees. Including molecular markers increased the identification rate of juveniles from 72% (morphology alone) to 96% (morphology and molecular) of the individuals assigned to a known tree taxon.

Conclusion/Significance

We conclude that while DNA barcoding is an invaluable tool for detecting errors in identifications and for identifying plants at juvenile stages, its limited ability to identify collections will constrain the practical implementation of DNA-based tropical plant biodiversity programs.     

A test of seven candidate barcode regions from the plastome in Picea (Pinaceae)

DNA barcoding, as a tool for species discrimination, has been used efficiently in animals, algae and fungi, but there are still debates on which DNA region(s) can be used as the standard barcode(s) for land plants. Gymnosperms, especially conifers, are important components of forests, and there is an urgent need for them to be identified through DNA barcoding because of their high frequency of collection in the field. However, the feasibility of DNA barcoding in gymnosperms has not been examined based on a dense species sampling. Here we selected seven candidate DNA barcodes from the plastome (matK, rbcL, rpoB, rpoC1, atpF-atpH, psbA-trnH, and psbK-psbI) to evaluate their suitability in Picea (spruce). The results showed that none of them or their different combinations has sufficient resolution for spruce species, although matK+rbcL might be used as a two-locus barcode. The low efficiency of these candidate barcodes in Picea might be caused by the paternal inheritance of the chloroplast genome, long generation time, recent radiation, and frequent inter-specific hybridization aided by wind pollination. Some of these factors could also be responsible for the difficulties in barcoding other plant groups. Furthermore, the potential of the nuclear LEAFY gene as a land plant barcode was discussed.     

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。     

Deciphering amphibian diversity through DNA barcoding: chances and challenges

Amphibians globally are in decline, yet there is still a tremendous amount of unrecognized diversity, calling for an acceleration of taxonomic exploration. This process will be greatly facilitated by a DNA barcoding system; however, the mitochondrial population structure of many amphibian species presents numerous challenges to such a standardized, single locus, approach. Here we analyse intra- and interspecific patterns of mitochondrial variation in two distantly related groups of amphibians, mantellid frogs and salamanders, to determine the promise of DNA barcoding with cytochrome oxidase subunit I (cox1) sequences in this taxon. High intraspecific cox1 divergences of 7-14% were observed (18% in one case) within the whole set of amphibian sequences analysed. These high values are not caused by particularly high substitution rates of this gene but by generally deep mitochondrial divergences within and among amphibian species. Despite these high divergences, cox1 sequences were able to correctly identify species including disparate geographic variants. The main problems with cox1 barcoding of amphibians are (i) the high variability of priming sites that hinder the application of universal primers to all species and (ii) the observed distinct overlap of intraspecific and interspecific divergence values, which implies difficulties in the definition of threshold values to identify candidate species. Common discordances between geographical signatures of mitochondrial and nuclear markers in amphibians indicate that a single-locus approach can be problematic when high accuracy of DNA barcoding is required. We suggest that a number of mitochondrial and nuclear genes may be used as DNA barcoding markers to complement cox1.     

DNA barcoding exposes a case of mistaken identity in the fern horticultural trade

Using cheilanthoid ferns, we provide an example of how DNA barcoding approaches can be useful to the horticultural community for keeping plants in the trade accurately identified. We use plastid rbcL, atpA, and trnG-R sequence data to demonstrate that a fern marketed as Cheilanthes wrightii (endemic to the southwestern USA and northern Mexico) in the horticultural trade is, in fact, Cheilanthes distans (endemic to Australia and adjacent islands). Public and private (accessible with permission) databases contain a wealth of DNA sequence data that are linked to vouchered plant material. These data have uses beyond those for which they were originally generated, and they provide an important resource for fostering collaborations between the academic and horticultural communities. We strongly advocate the barcoding approach as a valuable new technology available to the horticulture industry to help correct plant identification errors in the international trade.     

Molecular identification of species in Juglandaceae:A tiered method

DNA barcoding is a method of species identification and recognition using DNA sequence data. A tiered or multilocus method has been recommended for barcoding plant species. In this study, we sampled 196 individuals representing 9 genera and 54 species of Juglandaceae to investigate the utility of the four potential barcoding loci (rbcL, matK, trnH-psbA, and internal transcribed spacer (ITS)). Our results show that all four DNA regions are easy to amplify and sequence. In the four tested DNA regions, ITS has the most variable information, and rbcL has the least. At generic level, seven of nine genera can be efficiently identified by matK. At species level, ITS has higher interspecific p-distance than the trnH-psbA region. Difficult to align in the whole family, ITS showed heterogeneous variability among different genera. Except for the monotypic genera (Cyclocarya, Annamocarya, Platycarya), ITS appeared to have limited power for species identification within the Carya and Engelhardia complex, and have no power for Juglans or Pterocarya. Overall, our results confirmed that a multilocus tiered method for plant barcoding was applicable and practicable. With higher priority, matK is proposed as the first-tier DNA region for genus discrimination, and the second locus at species level should have enough stable variable characters.