DNA条形码技术在毒品原植物大麻鉴定中的应用

准确鉴定毒品原植物大麻的种属及品种具有重要的理论和实践意义。为了探讨DNA条形码技术用于毒品原植物大麻种属鉴定及品种鉴定的可行性,该研究以60份大麻原植物(分别采自内蒙、黑龙江、陕西延安、陕西榆林4个地区的栽培大麻雌雄各6株及新疆玛纳斯地区的野生大麻雌雄各6株)为材料,通过从其叶片中提取的DNA为模版,利用核糖体DNA基因间隔区的通用引物ITS2和叶绿体DNA的通用引物psbAtrnH进行PCR扩增,对扩增片段进行双向测序,将测序结果进行人工矫正和比对。结果显示:所有大麻样本的ITS2扩增片段序列没有变异完全一致,但psbA-trnH扩增片段变异较大共检测出8种cpDNA单倍型,用MEGE5.1软件计算种间遗传距离,并构建NJ系统聚类树可以有效把这五个地区的大麻样本区别开来,因此证明DNA条形码技术在毒品原植物大麻的种属鉴定方面具有可行性,但其用于大麻的种属鉴定的准确性、可靠性及在其来源地鉴定及品种鉴定中的可能性还有待进一步深入地研究。     

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 barcoding for the identification of smoked fish products

DNA barcoding was applied to the identification of smoked products from fish in 10 families in four orders and allowed identification to the species level, even among closely related species in the same genus. Barcoding is likely to become a standard tool for identification of fish specimens and products.     

Limited performance of DNA barcoding in a diverse community of tropical butterflies

DNA 'barcoding' relies on a short fragment of mitochondrial DNA to infer identification of specimens. The method depends on genetic diversity being markedly lower within than between species. Closely related species are most likely to share genetic variation in communities where speciation rates are rapid and effective population sizes are large, such that coalescence times are long. We assessed the applicability of DNA barcoding (here the 5' half of the cytochrome c oxidase I) to a diverse community of butterflies from the upper Amazon, using a group with a well-established morphological taxonomy to serve as a reference. Only 77% of species could be accurately identified using the barcode data, a figure that dropped to 68% in species represented in the analyses by more than one geographical race and at least one congener. The use of additional mitochondrial sequence data hardly improved species identification, while a fragment of a nuclear gene resolved issues in some of the problematic species. We acknowledge the utility of barcodes when morphological characters are ambiguous or unknown, but we also recommend the addition of nuclear sequence data, and caution that species-level identification rates might be lower in the most diverse habitats of our planet.     

DNA barcoding gap: reliable species identification over morphological and geographical scales

The philosophical basis and utility of DNA barcoding have been a subject of numerous debates. While most literature embraces it, some studies continue to question its use in dipterans, butterflies and marine gastropods. Here, we explore the utility of DNA barcoding in identifying spider species that vary in taxonomic affiliation, morphological diagnosibility and geographic distribution. Our first test searched for a ‘barcoding gap’ by comparing intra‐ and interspecific means, medians and overlap in more than 75 000 computed Kimura 2‐parameter (K2P) genetic distances in three families. Our second test compared K2P distances of congeneric species with high vs. low morphological distinctness in 20 genera of 11 families. Our third test explored the effect of enlarging geographical sampling area at a continental scale on genetic variability in DNA barcodes within 20 species of nine families. Our results generally point towards a high utility of DNA barcodes in identifying spider species. However, the size of the barcoding gap strongly depends on taxonomic groups and practices. It is becoming critical to define the barcoding gap statistically more consistently and to document its variation over taxonomic scales. Our results support models of independent patterns of morphological and molecular evolution by showing that DNA barcodes are effective in species identification regardless of their morphological diagnosibility. We also show that DNA barcodes represent an effective tool for identifying spider species over geographic scales, yet their variation contains useful biogeographic information.     

Re-integrating earthworm juveniles into soil biodiversity studies: species identification through DNA barcoding

Species identification of earthworms is usually achieved by careful observation of morphological features, often sexual characters only present in adult specimens. Consequently, juveniles or cocoons are often impossible to identify, creating a possible bias in studies that aim to document species richness and abundance. DNA barcoding, the use of a short standardized DNA fragment for species identification, is a promising approach for species discrimination. When a reference library is available, DNA-based identification is possible for all life stages. In this study, we show that DNA barcoding is an unrivaled tool for high volume identification of juvenile earthworms. To illustrate this advance, we generated DNA barcodes for specimens of Lumbricus collected from three temperate grasslands in western France. The analysis of genetic distances between individuals shows that juvenile sequences unequivocally match DNA barcode clusters of previously identified adult specimens, demonstrating the potential of DNA barcoding to provide exhaustive specimen identification for soil ecological research.     

Bayesian species identification under the multispecies coalescent provides significant improvements to DNA barcoding analyses

DNA barcoding methods use a single locus (usually the mitochondrial COI gene) to assign unidentified specimens to known species in a library based on a genetic distance threshold that distinguishes between‐species divergence from within‐species diversity. Recently developed species delimitation methods based on the multispecies coalescent (MSC) model offer an alternative approach to individual assignment using either single‐locus or multiloci sequence data. Here, we use simulations to demonstrate three features of an MSC method implemented in the program bpp . First, we show that with one locus, MSC can accurately assign individuals to species without the need for arbitrarily determined distance thresholds (as required for barcoding methods). We provide an example in which no single threshold or barcoding gap exists that can be used to assign all specimens without incurring high error rates. Second, we show that bpp can identify cryptic species that may be misidentified as a single species within the library, potentially improving the accuracy of barcoding libraries. Third, we show that taxon rarity does not present any particular problems for species assignments using bpp and that accurate assignments can be achieved even when only one or a few loci are available. Thus, concerns that have been raised that MSC methods may have problems analysing rare taxa (singletons) are unfounded. Currently, barcoding methods enjoy a huge computational advantage over MSC methods and may be the only approach feasible for massively large data sets, but MSC methods may offer a more stringent test for species that are tentatively assigned by barcoding.     

基于DNA条形码的物种丰富度估计: 以宿迁地区鳞翅目蛾类为例

为了探究基于DNA条形码方法量化物种多样性指标的可行性, 本研究以江苏省宿迁地区蛾类群落为例, 基于DNA条形码方法估计群落物种丰富度并绘制等级多度分布曲线(rank-abundance curves), 同时与基于传统形态学的对应指标进行比较。结果表明: (1)基于DNA条形码的物种丰富度估计与基于形态的物种丰富度估计之间没有显著差异; (2)基于形态和DNA条形码的等级多度分布曲线趋势一致, 通过K-S检测发现二者之间没有显著性差异(P > 0.05)。结果显示, 基于DNA条形码的物种丰富度估计能够在一定程度上补充基于形态学的方法, 可以尝试将其应用于蛾类群落生态学调查研究中。     

DNA barcodes identify Chinese medicinal plants and detect geographical patterns of Sinosenecio (Asteraceae)

DNA barcodes have proved to be efficient for plants species discrimination and identification using short and standardized genomic regions. The genus Sinosenecio(Asteraceae) is used for traditional medicinal purposes in China. Most species of the genus occur in restricted geographical regions and exhibit a wide range of morphological variations within species, making them difficult to differentiate in the field. Previously, taxonomic revisions have been made based on morphological and cytological evidence. In the present study, barcoding analysis was performed on 107 individuals representing 38 species in this genus to evaluate the performance of four candidate barcoding loci (matK, rbcL, trnH-psbA and internal transcribed spacer [ITS]) and detect geographical patterns. Three different methods based on genetic distance, sequence similarity, and the phylogenetic tree were used. Comparably high species discrimination power was detected in species-level taxonomic process by the ITS dataset alone or combined with other loci, which was suggested to be the most suitable barcode for Sinosenecio. Our results are congruent with previous taxonomic studies concerning the monophyly of the S. oldhamianus group. The present study provides an empirical paradigm for the identification of medicinal plant species and their geographical patterns, ascertaining the congruence between taxonomical studies and barcoding analysis inSinosenecio.     

DNA条形码在鳞翅目昆虫中的应用

2003年,Hebert等提出DNA条形码后,快速而精确的特点使它在物种鉴定中得到了广泛的应用。鳞翅目是昆虫纲中第二大目,其物种鉴定任务复杂而艰巨,因此DNA条形码具有广阔的应用前景。该文主要针对DNA条形码概况以及近年来它在鳞翅目昆虫中的研究情况予以综述。     

降香黄檀的DNA条形码鉴定研究

DNA条形码技术是利用基因组中一段短的标准序列进行物种的鉴定并探索其亲缘进化关系。本研究对采自海南不同地区降香黄檀五个居群24份样品的psbA-trnH,rbcL,核ITS及ITS2序列进行PCR扩增和测序,比较各序列扩增和测序效率。种间和种内变异,采用BLAST1和邻接 (NJ) 法构建系统聚类树方法评价不同序列的鉴定能力。结果表明ITS2在所研究的材料中具有最高的扩增和测序效率,而ITS扩增效率较低。ITS2完整序列在区分黄檀属不同种间差异具有较大优势。因此可利用ITS2从分子水平区分降香黄檀与其他混伪种。     

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.     

Identification of Belgian mosquito species (Diptera: Culicidae) by DNA barcoding

Since its introduction in 2003, DNA barcoding has proven to be a promising method for the identification of many taxa, including mosquitoes (Diptera: Culicidae). Many mosquito species are potential vectors of pathogens, and correct identification in all life stages is essential for effective mosquito monitoring and control. To use DNA barcoding for species identification, a reliable and comprehensive reference database of verified DNA sequences is required. Hence, DNA sequence diversity of mosquitoes in Belgium was assessed using a 658 bp fragment of the mitochondrial cytochrome oxidase I (COI) gene, and a reference data set was established. Most species appeared as well‐supported clusters. Intraspecific Kimura 2‐parameter (K2P) distances averaged 0.7%, and the maximum observed K2P distance was 6.2% for Aedes koreicus. A small overlap between intra‐ and interspecific K2P distances for congeneric sequences was observed. Overall, the identification success using best match and the best close match criteria were high, that is above 98%. No clear genetic division was found between the closely related species Aedes annulipes and Aedes cantans, which can be confused using morphological identification only. The members of the Anopheles maculipennis complex, that is Anopheles maculipennis s.s. and An. messeae, were weakly supported as monophyletic taxa. This study showed that DNA barcoding offers a reliable framework for mosquito species identification in Belgium except for some closely related species.     

Mitochondrial DNA barcoding detects some species that are real, and some that are not

Mimicry and extensive geographical subspecies polymorphism combine to make species in the ithomiine butterfly genus Mechanitis (Lepidoptera; Nymphalidae) difficult to determine. We use mitochondrial DNA (mtDNA) barcoding, nuclear sequences and amplified fragment length polymorphism (AFLP) genotyping to investigate species limits in this genus. Although earlier biosystematic studies based on morphology described only four species, mtDNA barcoding revealed eight well-differentiated haplogroups, suggesting the presence of four new putative 'cryptic species'. However, AFLP markers supported only one of these four new 'cryptic species' as biologically meaningful. We demonstrate that in this genus, deep genetic divisions expected on the basis of mtDNA barcoding are not always reflected in the nuclear genome, and advocate the use of AFLP markers as a check when mtDNA barcoding gives unexpected results.     

Existence of species complex largely reduced barcoding success for invasive species of Tephritidae: a case study in Bactrocera spp.

Fruit flies in the family Tephritidae are the economically important pests that have many species complexes. DNA barcoding has gradually been verified as an effective tool for identifying species in a wide range of taxonomic groups, and there are several publications on rapid and accurate identification of fruit flies based on this technique; however, comprehensive analyses of large and new taxa for the effectiveness of DNA barcoding for fruit flies identification have been rare. In this study, we evaluated the COI barcode sequences for the diagnosis of fruit flies using 1426 sequences for 73 species of Bactrocera distributed worldwide. Tree‐based [neighbour‐joining (NJ)]; distance‐based, such as Best Match (BM), Best Close Match (BCM) and Minimum Distance (MD); and character‐based methods were used to evaluate the barcoding success rates obtained with maintaining the species complex in the data set, treating a species complex as a single taxon unit, and removing the species complex. Our results indicate that the average divergence between species was 14.04% (0.00–25.16%), whereas within a species this was 0.81% (0.00–9.71%); the existence of species complexes largely reduced the barcoding success for Tephritidae, for example relatively low success rates (74.4% based on BM and BCM and 84.8% based on MD) were obtained when the sequences from species complexes were included in the analysis, whereas significantly higher success rates were achieved if the species complexes were treated as a single taxon or removed from the data set – BM (98.9%), BCM (98.5%) and MD (97.5%), or BM (98.1%), BCM (97.4%) and MD (98.2%).     

Diversity investigation by application of DNA barcoding: A case study of lepidopteran insects in Xinjiang wild fruit forests,China

To investigate the species diversity of lepidopteran insects in Xinjiang wild fruit forests, establish insect community monitoring systems, and determine the local species pool, we test the applicability of DNA barcoding based on cytochrome c oxidase subunit I (COI) gene for accurate and rapid identification of insect species. From 2017 to 2019, a total of 212 samples with ambiguous morphological identification were selected for DNA barcoding analysis. Five sequence‐based methods for species delimitation (ABGD, BINs, GMYC, jMOTU, and bPTP) were conducted for comparison to traditional morphology‐based identification. In total, 2,422 samples were recorded, representing 143 species of 110 genera in 17 families in Lepidoptera. The diversity analysis showed that the richness indices for Noctuidae was the highest (54 species), and for Pterophoridae, Cossidae, Limacodidae, Lasiocampidae, Pieridae, and Lycaenidae were the lowest (all with 1 species). The Shannon–Wiener species diversity index (H′) and Pielou''s evenness (J′) of lepidopteran insects first increased and then decreased across these 3 years, while the Simpson diversity index showed a trend of subtracted then added. For molecular‐based identification, 67 lepidopteran species within 61 genera in 14 families were identified through DNA barcoding. Neighbor‐joining (NJ) analysis showed that conspecific individuals were clustered together and formed monophyletic groups with a high support value, except for Lacanobia contigua (Denis & Schiffermüller, 1775) (Noctuidae: Hadeninae). Sixty‐seven morphospecies were classified into various numbers of MOTUs based on ABGD, BINs, GMYC, jMOTU, and bPTP (70, 96, 2, 71, and 71, respectively). In Xinjiang wild fruit forests, the family with the largest number of species is Noctuidae, followed by Geometridae, Crambidae, and the remaining families. The highest Shannon diversity index is observed for the family Noctuidae. Our results indicate that the distance‐based methods (ABGD and jMOTU) and character‐based method (bPTP) outperform GMYC. BINs is inclined to overestimate species diversity compared to other methods.     

Developing DNA barcodes for species identification in Podophylloideae (Berberidaceae)

Species of Podophyllum, Dysosma, Sinopodophyllum, and Diphylleia, genera from Podophylloideae of Berberidaceae, have long been used in traditional herbal medicine in East Asia and/or North America. Accurate identification of the species of these four genera is crucial to their medicinal uses. In this study, we tested the utility of nine barcodes (matK, rbcL, atpH-atpI, rpl32-trnL^UAG, rps18-clpp, trnL-trnF, trnL-ndhJ, trnS-trnfM, and internal transcribed spacer (ITS)) to discriminate different species of Podophylloideae. Thirty-six individuals representing 12 species of Podophylloideae were collected from different locations in China, Japan, and North America. We assessed the feasibility of amplification and sequencing of all markers, examined the levels of the barcoding gap based on DNA sequence divergence between ranges of intra- and interspecific variation using pairwise distances, and further evaluated successful identifications using each barcode by similarity-based and tree-based methods. Results showed that nine barcodes, except rps18-clpp, have a high level of primer universality and sequencing success. As a single barcode, ITS has the most variable sites, greater intra- and interspecific divergences, and the highest species discrimination rate (83%), followed by matKwhich has moderate variation and also high species discrimination rates. However, these species can also be discriminated by ITS alone, except Dysosma versipellis (Hance) M. Cheng ex T. S. Ying and D. pleiantha (Hance) Woodson. The combination of ITS + matK did not improve species resolution over ITS alone. Thus, we propose that ITS may be used as a sole region for identification of most species in Podophylloideae. The failure of ITS to distinguish D. versipellis and D. pleiantha is likely attributed to incomplete lineage sorting due to recent divergence of the two species.     

Multilocus species identification and fungal DNA barcoding: insights from blue stain fungal symbionts of the mountain pine beetle

There is strong community-wide interest in applying molecular techniques to fungal species delimitation and identification, but selection of a standardized region or regions of the genome has not been finalized. A single marker, the ribosomal DNA internal transcribed spacer region, has frequently been suggested as the standard for fungi. We used a group of closely related blue stain fungi associated with the mountain pine beetle (Dendroctonus ponderosae Hopkins) to examine the success of such single-locus species identification, comparing the internal transcribed spacer with four other nuclear markers. We demonstrate that single loci varied in their utility for identifying the six fungal species examined, while use of multiple loci was consistently successful. In a literature survey of 21 similar studies, individual loci were also highly variable in their ability to provide consistent species identifications and were less successful than multilocus diagnostics. Accurate species identification is the essence of any molecular diagnostic system, and this consideration should be central to locus selection. Moreover, our study and the literature survey demonstrate the value of using closely related species as the proving ground for developing a molecular identification system. We advocate use of a multilocus barcode approach that is similar to the practice employed by the plant barcode community, rather than reliance on a single locus.     

Assessment of the DNA barcoding for identification of Trigonostigma somphongsi,a critically endangered species in Thailand

Trigonostigma somphongsi, a critically endangered species, is a rare and endemic fish in Thailand. This species had disappeared from its natural habitat for 20 years until 2006. The DNA barcodes or the fragments of cytochrome c oxidase I (COI) of T. somphongsi were investigated for species identification. The remaining two native species in the genus Trigonostigma, T. heteromorpha and T. espei were also identified using Boraras urophthalmoides as an outgroup species. The 707-bp fragments were successfully amplified and sequenced in all fifteen fish samples. In the genus Trigonostigma, the genetic distance within and between species ranged from 0.000 to 0.005 and 0.016 to 0.039, respectively. The lowest genetic distance (0.016) was between T. heteromorpha and T. espei, while the highest genetic distance (0.039) was between T. somphongsi and T. espei, followed by T. somphongsi and T. heteromorpha (0.035). The phylogenetic analysis showed that the relationship between the three Trigonostigma species (T. somphongsi was clearly separated from T. heteromorpha and T. espei) agreed with the morphological characteristics. These results suggest that DNA barcoding is an effective approach to identify Trigonostigma species for use in the conservation and management of fisheries.