DNA barcoding of medicinal plant material for identification

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Phylogenetic relationships and DNA barcoding of nine endangered medicinal plant species endemic to Saint Katherine protectorate

A high degree of endemism has been recorded for several plant groups collectively in Saint Katherine Protectorate (SKP) in the Sinai Peninsula. Nine endangered endemic plant species in SKP were selected to test the variable abilities of three different DNA barcodes; Riboluse-1,5- Biphosphate Carboxylase/Oxygenase Large subunit (rbcL), Internal Transcribed Spacer (ITS), and the two regions of the plastid gene (ycf1) as well as Start Codon Targeted (SCoT) Polymorphism to find the phylogenetic relationships among them. The three barcodes were generally more capable of finding the genetic relationships among the plant species under study, new barcodes were introduced to the National Centre for Biotechnology Information (NCBI) for the first time through our work. The barcode sequences were efficient in finding the genetic relationships between the nine species. However, SCoT polymorphism could only cluster plant species belonging to the same genus together in one group, but it could not cluster plant species belonging to the same families except for some primers solely. RbcL was the most easily amplified and identified barcode in eight out of the nine species at the species level and the ninth barcode to the genus level. ITS identified all the species to the genus level. Finally, ycf1 identified six out of the eight species, but it could not identify two of the eight species to the genus level.     

DNA barcoding of catfish: species authentication and phylogenetic assessment

As the global market for fisheries and aquaculture products expands, mislabeling of these products has become a growing concern in the food safety arena. Molecular species identification techniques hold the potential for rapid, accurate assessment of proper labeling. Here we developed and evaluated DNA barcodes for use in differentiating United States domestic and imported catfish species. First, we sequenced 651 base-pair barcodes from the cytochrome oxidase I (COI) gene from individuals of 9 species (and an Ictalurid hybrid) of domestic and imported catfish in accordance with standard DNA barcoding protocols. These included domestic Ictalurid catfish, and representative imported species from the families of Clariidae and Pangasiidae. Alignment of individual sequences from within a given species revealed highly consistent barcodes (98% similarity on average). These alignments allowed the development and analyses of consensus barcode sequences for each species and comparison with limited sequences in public databases (GenBank and Barcode of Life Data Systems). Validation tests carried out in blinded studies and with commercially purchased catfish samples (both frozen and fresh) revealed the reliability of DNA barcoding for differentiating between these catfish species. The developed protocols and consensus barcodes are valuable resources as increasing market and governmental scrutiny is placed on catfish and other fisheries and aquaculture products labeling in the United States.     

Extraction of DNA from milligram amounts of fresh,herbarium and mummified plant tissues

Summary We have developed a DNA extraction procedure for milligram amounts of plant tissue. Yields ranged from 0.3–200 nanograms of DNA per milligram of tissue. The factors affecting yield are discussed. Fresh tissue, as well as herbarium specimens (22–118 years old) and mummified seeds and embryos (500 to greater than 44 600 years old) were used. All tissues attempted (57 types from 29 species) yielded measurable amounts of DNA. In no case tested was inhibition observed for restriction enzymes BamHI or EcoRI.     

DNA barcoding in medicinal plants: Testing the potential of a proposed barcoding marker for identification of Uncaria species from China

DNA barcoding, an increasingly popular mean of species identification, has been widely used for global species identification despite a consensus not being reached regarding which DNA sequences can be used as the best plant barcodes. In this study, we tested the feasibility of five candidate DNA barcodes (nrITS, nrITS2, matk, rbcL and trnH-psbA) for identifying Uncaria species. We collected a total of 54 specimens of 10 Uncaria species across its distributional range. BLAST, barcoding gaps, tree-based methods and TAXONDNA analysis were used to investigate the molecular identification capability of the candidate DNA barcodes. The results showed that the ITS2 is most suitable as a candidate DNA barcode for identification of medicinal plants of the genus Uncaria.     

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。     

From species to individuals: combining barcoding and microsatellite analyses from non-invasive samples in plant ecology studies

By means of DNA barcoding and microsatellite analyses, we studied the species and individuals of legitimate seed dispersers of the Mediterranean shrub Pistacia lentiscus, a keystone species that represents the main source of food in winter for frugivorous birds. We collected dropping of birds containing seeds, and after DNA extraction we amplified and sequenced a fragment of the mitochondrial COI gene. Through BLASTN queries of the sequenced fragments against registered sequences in the GenBank database we identified the bird species that are currently dispersing P. lentiscus seeds. Further, through the amplification of specific nuclear microsatellite loci we calculated standard genetic diversity parameters of the population of birds from the genus Sylvia (the blackcap and Sardinian warbler), the most important dispersers of P. lentiscus. Five bird species were identified as seed dispersers through their barcode match. Further, we found that S. melanocephala displayed slightly lower levels of genetic diversity than S. atricapilla. In this study we show how the genetic analyses of environmental faecal samples can be a useful and convenient tool for the study of plant-frugivore interactions through the ascertainment of the identity of the species involved and through the analyses of genetic variability of their populations.     

DNA barcoding of the main cultivated yams and selected wild species in the genus Dioscorea

Distinguishing yam species based on morphological traits is extremely difficult and unreliable, posing a challenge to breeders and genebank curators. Development of a molecular assay based on DNA barcoding can facilitate rapid and accurate identification of important Dioscorea species. To develop a DNA barcoding system forDioscorea species identification, the rbcL and matK loci (in unison and in combination), the non-coding intergenic spacer trnH-psbA of the chloroplast genome, and the nuclear ITS regions were investigated using criteria for developing candidate DNA barcodes. All DNA barcoding sequences were assessed for ease of PCR amplification, sequence quality and species discriminatory power. Amongst the markers investigated, the matK locus performed well in terms of species identification (63.2%), in addition to detecting high interspecific variation with mean divergence of 0.0196 (SD=0.0209). The combination of the two coding regions (rbcL + matK) was determined to be the optimal (76.2%) DNA barcoding approach as 16 out of 21 species could be defined. While the rbcL exhibited good PCR amplification efficiency and sequence quality, its species discriminatory power was relatively poor with 47.6% identification. Similarly, the trnH-psbA region had a weak discrimination efficiency of only 36.8%. While the development of more robust DNA barcoding systems is an ongoing challenge, our results indicate that therbcL + matK combination can be utilized as multi-locus DNA barcode regions for Dioscorea species identification.     

DNA barcoding of fresh seafood in Australian markets reveals misleading labelling and sale of endangered species

Flake and shark samples were purchased from outlets in several coastal Australian regions and genetically barcoded using the cytochrome oxidase subunit 1 (CO1) gene to investigate labelling reliability and species-specific sources of ambiguously labelled fillets. Of the 41 shark fillet samples obtained, 23 yielded high-quality CO1 sequences, out of which 57% (n = 13) were labelled ambiguously (misleading) and 35% (n = 8) incorrectly. In contrast, barramundi fillets, which are widely available and sought after in Australian markets, were shown to be accurately labelled. Species identified from shark samples, including the shortfin mako (n = 3) and the scalloped hammerhead (n = 1), are assessed by the IUCN as endangered and critically endangered, respectively, with several others classified as vulnerable and near threatened.     

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.     

DNA barcoding of some medicinally important plant species of Lamiaceae family in India

Molecular Biology Reports - Several species of the Lamiaceae family are the primary source of bioactive aromatic oils and secondary metabolites, having broader applications in the cosmetics,...     

Utility of DNA barcoding to identify rare endemic vascular plant species in Trinidad

The islands of the Caribbean are considered to be a “biodiversity hotspot.” Collectively, a high level of endemism for several plant groups has been reported for this region. Biodiversity conservation should, in part, be informed by taxonomy, population status, and distribution of flora. One taxonomic impediment to species inventory and management is correct identification as conventional morphology‐based assessment is subject to several caveats. DNA barcoding can be a useful tool to quickly and accurately identify species and has the potential to prompt the discovery of new species. In this study, the ability of DNA barcoding to confirm the identities of 14 endangered endemic vascular plant species in Trinidad was assessed using three DNA barcodes (matK, rbcL, and rpoC1). Herbarium identifications were previously made for all species under study. matK, rbcL, and rpoC1 markers were successful in amplifying target regions for seven of the 14 species. rpoC1 sequences required extensive editing and were unusable. rbcL primers resulted in cleanest reads, however, matK appeared to be superior to rbcL based on a number of parameters assessed including level of DNA polymorphism in the sequences, genetic distance, reference library coverage based on BLASTN statistics, direct sequence comparisons within “best match” and “best close match” criteria, and finally, degree of clustering with moderate to strong bootstrap support (>60%) in neighbor‐joining tree‐based comparisons. The performance of both markers seemed to be species‐specific based on the parameters examined. Overall, the Trinidad sequences were accurately identified to the genus level for all endemic plant species successfully amplified and sequenced using both matK and rbcL markers. DNA barcoding can contribute to taxonomic and biodiversity research and will complement efforts to select taxa for various molecular ecology and population genetics studies.     

药用昆虫神农洁蜣螂炮制品DNA条形码序列的测定与分析

当药用昆虫被加工炮制成碎块或粉末状态时,其昆虫类别和真伪的鉴别就显得十分困难。未经准确鉴定的药用昆虫会影响临床用药安全。为寻找准确、灵敏且快速的检测方法,采用DNA条形码技术,分别以药用神农洁蜣螂(Catharsius molossus)炮制品药材的完整和破碎个体为研究材料,对其mtCOI基因进行提取、扩增及测序分析,探讨DNA条形码技术对炮制后药用蜣螂鉴定的可行性。通过对不同DNA提取方法的比较,成功从炮制后干燥药材虫体内提取出总DNA并扩增出mtCOI基因片段。测序分析结果表明mtCOI基因序列能够区分神农洁蜣螂和其形态近似种。作为药用神农洁蜣螂DNA条形码标准片段的mtCOI基因序列已投递至GenBank。研究还表明,碎块状药用蜣螂药材中混杂有一定比例的其他昆虫。该结果有助于建立适合加工炮制后药用蜣螂的DNA条形码分子鉴定标准,并为保证临床用药安全提供支持。     

Identification of herbarium whole-leaf samples of Epilobium species by ATR-IR spectroscopy

A simple, high-accuracy FT-IR method based on attenuated total reflection (ATR) was developed for the rapid determination of leaf samples of Epilobium species. The method is superior to other analytical techniques, since there is no need of laborious sample preparation such as grinding or extraction and solvent removal. A total of 70 herbarium specimens, belonging to all 13 Epilobium and to 2 Chamerion species growing in Slovenia, were analyzed. With the 100 most-informative wavenumbers in the range 700-1800 cm(-1), we obtained over 90% accuracy of species identification, with discriminant multivariate statistical analysis on the measurements made on whole dried leaves.     

DNA barcoding meets molecular scatology: short mtDNA sequences for standardized species assignment of carnivore noninvasive samples

Although species assignment of scats is important to study carnivore biology, there is still no standardized assay for the identification of carnivores worldwide, which would allow large-scale routine assessments and reliable cross-comparison of results. Here, we evaluate the potential of two short mtDNA fragments [ATP6 (126 bp) and cytochrome oxidase I gene (COI) (187 bp)] to serve as standard markers for the Carnivora. Samples of 66 species were sequenced for one or both of these segments. Alignments were complemented with archival sequences and analysed with three approaches (tree-based, distance-based and character-based). Intraspecific genetic distances were generally lower than between-species distances, resulting in diagnosable clusters for 86% (ATP6) and 85% (COI) of the species. Notable exceptions were recently diverged species, most of which could still be identified using diagnostic characters and uniqueness of haplotypes or by reducing the geographic scope of the comparison. In silico analyses were also performed for a 110-bp cytochrome b (cytb) segment, whose identification success was lower (70%), possibly due to the smaller number of informative sites and/or the influence of misidentified sequences obtained from GenBank. Finally, we performed case studies with faecal samples, which supported the suitability of our two focal markers for poor-quality DNA and allowed an assessment of prey DNA co-amplification. No evidence of prey DNA contamination was found for ATP6, while some cases were observed for COI and subsequently eliminated by the design of more specific primers. Overall, our results indicate that these segments hold good potential as standard markers for accurate species-level identification in the Carnivora.     

DNA barcoding of endangered Indian Paphiopedilum species

The indiscriminate collections of Paphiopedilum species from the wild for their exotic ornamental flowers have rendered these plants endangered. Although the trade of these endangered species from the wild is strictly forbidden, it continues unabated in one or other forms that elude the current identification methods. DNA barcoding that offers identification of a species even if only a small fragment of the organism at any stage of development is available could be of great utility in scrutinizing the illegal trade of both endangered plant and animal species. Therefore, this study was undertaken to develop DNA barcodes of Indian species of Paphiopedilum along with their three natural hybrids using loci from both the chloroplast and nuclear genomes. The five loci tested for their potential as effective barcodes were RNA polymerase-β subunit (rpoB), RNA polymerase-β' subunit (rpoC1), Rubisco large subunit (rbcL) and maturase K (matK) from the chloroplast genome and nuclear ribosomal internal transcribed spacer (nrITS) from the nuclear genome. The intra- and inter-specific divergence values and species discrimination rates were calculated by Kimura 2 parameter (K2P) method using mega 4.0. The matK with 0.9% average inter-specific divergence value yielded 100% species resolution, thus could distinguish all the eight species of Paphiopedilum unequivocally. The species identification capability of these sequences was further confirmed as each of the matK sequences was found to be unique for the species when a blast analysis of these sequences was carried out on NCBI. nrITS, although had 4.4% average inter-specific divergence value, afforded only 50% species resolution. DNA barcodes of the three hybrids also reflected their parentage.     

Efficient distinction of invasive aquatic plant species from non‐invasive related species using DNA barcoding

Biological invasions are regarded as threats to global biodiversity. Among invasive aliens, a number of plant species belonging to the genera Myriophyllum, Ludwigia and Cabomba, and to the Hydrocharitaceae family pose a particular ecological threat to water bodies. Therefore, one would try to prevent them from entering a country. However, many related species are commercially traded, and distinguishing invasive from non‐invasive species based on morphology alone is often difficult for plants in a vegetative stage. In this regard, DNA barcoding could become a good alternative. In this study, 242 samples belonging to 26 species from 10 genera of aquatic plants were assessed using the chloroplast loci trnH‐psbA, matK and rbcL. Despite testing a large number of primer sets and several PCR protocols, the matK locus could not be amplified or sequenced reliably and therefore was left out of the analysis. Using the other two loci, eight invasive species could be distinguished from their respective related species, a ninth one failed to produce sequences of sufficient quality. Based on the criteria of universal application, high sequence divergence and level of species discrimination, the trnH‐psbA noncoding spacer was the best performing barcode in the aquatic plant species studied. Thus, DNA barcoding may be helpful with enforcing a ban on trade of such invasive species, such as is already in place in the Netherlands. This will become even more so once DNA barcoding would be turned into machinery routinely operable by a nonspecialist in botany and molecular genetics.     

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.     

DNA barcoding is a new approach in comparative genomics of plants

DNA barcoding was proposed as a method for recognition and identification of eukaryotic species through comparison of sequences of a standard short DNA fragment—DNA barcode—from an unknown specimen to a library of reference sequences from known species. This allows identifying an organism at any stage of development from a very small tissue sample, fresh or conserved many years ago. Molecular identification of plant samples can be used in various scientific and applied fields. It would also help to find new species, which is particularly important for cryptogamic plants. An optimal DNA barcode region is a small fragment presented in all species of a major taxonomic group, having invariable nucleotide sequence in all members of the same species, but with sufficient variation to discriminate among the species. This fragment should be flanked by low-variable regions for use of universal primers in PCR for amplification and sequencing. The DNA barcode that is well established in animals is a sequence of a fragment of the mitochondrial cytochrome c oxidase gene CO1. However, searching for DNA barcode in plants proved to be a more challenging task. No DNA region universally suitable for all plants and meeting all of the necessary criteria has been found. Apparently, a multilocus or two-stage approach should be applied for this purpose. Several fragments of the chloroplast genome (trnH-psbA, matK, rpoC, rpoB, rbcL) in combinations of two or three regions were suggested as candidate regions with highest potential, but more representative samples should be examined to choose the best candidate. The possibility is discussed to use as DNA barcode internal transcribed spacers (ITS) of nuclear rRNA genes, which are highly variable, widely employed in molecular phylogenetic studies at the species level, but also have some limitations.