Barcodes matK Research Articles

Morphological and molecular characterization of two species of genus Ageratum.

The species of genus Ageratum (family Asteraceae) are distributed in various parts of the world. Ageratum conyzoides and A. houstonianum are the most commonly occurring species in India. These species are quite similar in their morphology thus creating a challenge in identification during the field survey and taxonomic validation. The accurate identification of the species is highly significant especially when those are of medicinal interest. To overcome the barriers in morphological based identification, DNA barcoding has been employed during the present investigation. Morphological and DNA barcodes matK and ITS genes, were employed to differentiate between Ageratum conyzoides and A. houstonianum. The obtained matK and ITS gene sequences were submitted to GenBank and BOLD system to obtain accession numbers. The DNA sequences were aligned with database sequences using BLAST and phylogenetic trees were constructed through neighbor-joining algorithm in MEGA 11 software. The distinguish features of A. conyzoides include ovate to elliptic-oblong leaves with a cuneate base and inflorescence heads forming domed to flat-topped clusters. However, A. houstonianum has triangular to ovate leaves with a cordate to truncate base, cymose clusters in the inflorescence and stipulate glandular involucre bracts. The matK gene has shown the highest identity percentages (100%) for A. houstonianum and 99.87% for A. conyzoides. The phylogenetic tree analysis has demonstrated a close association of A. conyzoides and A. houstonianum with their respective species, supported by bootstrap values in the matK and ITS trees. This study revealed that morphological and molecular data can be successfully utilized in the identification of A. conyzoides and A. houstonianum. The matK and ITS barcodes provide promising results in the identification of Ageratum species, with their phylogeny supporting classification within the family asteraceae.

Testing plastomes and nuclear ribosomal DNA sequences as the next-generation DNA barcodes for species identification and phylogenetic analysis in Acer

BackgroundAcer is a taxonomically intractable and speciose genus that contains over 150 species. It is challenging to distinguish Acer species only by morphological method due to their abundant variations. Plastome and nuclear ribosomal DNA (nrDNA) sequences are recommended as powerful next-generation DNA barcodes for species discrimination. However, their efficacies were still poorly studied. The current study will evaluate the application of plastome and nrDNA in species identification and perform phylogenetic analyses for Acer.ResultBased on a collection of 83 individuals representing 55 species (c. 55% of Chinese species) from 13 sections, our barcoding analyses demonstrated that plastomes exhibited the highest (90.47%) species discriminatory power among all plastid DNA markers, such as the standard plastid barcodes matK + rbcL + trnH-psbA (61.90%) and ycf1 (76.19%). And the nrDNA (80.95%) revealed higher species resolution than ITS (71.43%). Acer plastomes show abundant interspecific variations, however, species identification failure may be due to the incomplete lineage sorting (ILS) and chloroplast capture resulting from hybridization. We found that the usage of nrDNA contributed to identifying those species that were unidentified by plastomes, implying its capability to some extent to mitigate the impact of hybridization and ILS on species discrimination. However, combining plastome and nrDNA is not recommended given the cytonuclear conflict caused by potential hybridization. Our phylogenetic analysis covering 19 sections (95% sections of Acer) and 128 species (over 80% species of this genus) revealed pervasive inter- and intra-section cytonuclear discordances, hinting that hybridization has played an important role in the evolution of Acer.ConclusionPlastomes and nrDNA can significantly improve the species resolution in Acer. Our phylogenetic analysis uncovered the scope and depth of cytonuclear conflict in Acer, providing important insights into its evolution.

Identification of toxic Gelsemium elegans in processed food and honey based on real-time PCR analysis

Gelsemium elegans (GE) is a widely distributed hypertoxic plant that has caused many food poisoning incidents. Its pollen can also be collected by bees to produce toxic honey, posing a great threat to the health and safety of consumers. However, for the complex matrices such as cooked food and honey, it is challenging to perform composition analysis. It is necessary to establish more effective strategies for investigating GE contamination. In this study, the real-time PCR (qPCR) analysis combined with DNA barcode matK was proposed for the identification and detection of GE. Fifteen honey samples along with twenty-eight individuals of GE and the common confusable objects Lonicera japonica, Ficus hirta, Stellera chamaejasme and Chelidonium majus were gathered. Additionally, the food mixtures treated with 20-min boiling and 30-min digestion were prepared. Specific primers were designed, and the detection capability and sensitivity of qPCR in honey and boiled and digested food matrices were tested. The results demonstrated that the matK sequence with sufficient mutation sites was an effective molecular marker for species differentiation. GE and the confusable species could be clearly classified by the fluorescence signal of qPCR assay with a high sensitivity of 0.001 ng/μl. In addition, this method was successfully employed for the detection of deeply processed food materials and honey containing GE plants which even accounted for only 0.1 %. The sequencing-free qPCR approach undoubtedly can serve as a robust support for the quality supervision of honey industry and the prevention and diagnosis of food poisoning.

Molecular taxonomy via DNA barcodes for species identification in selected genera of Fabaceae

Fabaceae is an invaluable plant family with considerable ecological and economic importances for example as food sources, bio-fertilizer, and medicinal plants. However, Fabaceae has been overexploited in Indonesia and several species belong to this family are critically endangered. Due to morphological similarity, rapid and accurate identification of Fabaceae species is essential to support its conservation efforts. Nowadays, species identification through DNA barcoding has become an effective taxonomic classification tool. This study was aimed to evaluate the effectiveness of chloroplast markers, matK and rbcL and the combination (matK+rbcL), as DNA barcodes for the identification of seleted genera in Fabaceae. The result showed that matK+rbcL and matK had the highest level of identification (90% and 82.05%) of the investigated genera, respectively. Additionally, matK had the highest mean of interspesific distance (0.134) and intraspecific distance (0.003). The combined barcode matK+rbcL had the highest correct identification rate when comparing the morphological with molecular identification. Furthermore, the best phylogenetic tree was obtained using Neighbor Joining method. Based on the overall performance, matK and matK+rbcL were the best barcodes for the selected genera in this study.

DNA barcodes and microsatellites: How they complement for species identification in the complex genus Tamarix (Tamaricaceae)

AbstractDNA barcoding allows the identification of an organism by comparing the sequence of selected DNA regions (barcodes) with a previously compiled database, and it can be useful for taxonomic identification of species in complex genera, such as Tamarix. Many species of this genus show convergent morphology, which leads to frequent errors in their identification. Highly variable genetic markers, such as microsatellites or short sequence repeats (SSR), could be used to differentiate species where DNA barcodes fail. Here, we tested the ability of both, 5 different marker regions (rbcL, matK, ITS, trnH‐psbA, and ycf1), and 14 microsatellites, to properly identify Tamarix species, especially those from the Mediterranean Basin, and compared the pros and cons of the different analytical methods for species identification. DNA barcoding allows the genetic identification of certain species in Tamarix. The two‐locus barcodes matK + ITS and ITS + ycf1 were the best‐performing combinations, allowing up to 69% and 70%, respectively, correct identification. However, DNA barcoding failed in phylogenetically close groups, such as many Mediterranean species. The use of SSR can aid the identification of species, and the combination of both types of data (DNA barcoding and SSR) improved the success. The combination of data was especially relevant in detecting the presence of hybridization processes, which are common in the genus. However, caution must be exercised when choosing the clustering methods for the SSR data since different methods can lead to very different results.

Evaluation of Arabian Vascular Plant Barcodes (rbcL and matK): Precision of Unsupervised and Supervised Learning Methods towards Accurate Identification

Arabia is the largest peninsula in the world, with >3000 species of vascular plants. Not much effort has been made to generate a multi-locus marker barcode library to identify and discriminate the recorded plant species. This study aimed to determine the reliability of the available Arabian plant barcodes (>1500; rbcL and matK) at the public repository (NCBI GenBank) using the unsupervised and supervised methods. Comparative analysis was carried out with the standard dataset (FINBOL) to assess the methods and markers’ reliability. Our analysis suggests that from the unsupervised method, TaxonDNA’s All Species Barcode criterion (ASB) exhibits the highest accuracy for rbcL barcodes, followed by the matK barcodes using the aligned dataset (FINBOL). However, for the Arabian plant barcode dataset (GBMA), the supervised method performed better than the unsupervised method, where the Random Forest and K-Nearest Neighbor (gappy kernel) classifiers were robust enough. These classifiers successfully recognized true species from both barcode markers belonging to the aligned and alignment-free datasets, respectively. The multi-class classifier showed high species resolution following the two classifiers, though its performance declined when employed to recognize true species. Similar results were observed for the FINBOL dataset through the supervised learning approach; overall, matK marker showed higher accuracy than rbcL. However, the lower rate of species identification in matK in GBMA data could be due to the higher evolutionary rate or gaps and missing data, as observed for the ASB criterion in the FINBOL dataset. Further, a lower number of sequences and singletons could also affect the rate of species resolution, as observed in the GBMA dataset. The GBMA dataset lacks sufficient species membership. We would encourage the taxonomists from the Arabian Peninsula to join our campaign on the Arabian Barcode of Life at the Barcode of Life Data (BOLD) systems. Our efforts together could help improve the rate of species identification for the Arabian Vascular plants.

Genetical and morphological studies on Ficus trees

In this current study, DNA barcoding and fifteen plant species of Ficus genus belongs to family moracea were used. The plant species were collected from the botanical garden of Antoniadis Alexandria government, Egypt. To authenticate the morphological identification and measure the evolutionary rate among them, rbcL and matK genes were used as genes universal DNA barcoding. The obtained results for rbcL gene amplification success were 93% and the matK gene amplification 80%. The DNA sequencing was got for 14 species of rbcL gene and 11 species for matK gene in addition, the matK and rbcL sequences for all samples were checked by GenBank databases and the accession numbers were detected for all the studied species. Finally, the rbcL was suggested as a plant barcode for its discriminatory power at high taxonomic levels than matK barcode.

Genetic Diversity among Selected Medicago sativa Cultivars Using Inter-Retrotransposon-Amplified Polymorphism, Chloroplast DNA Barcodes and Morpho-Agronomic Trait Analyses.

Alfalfa (Medicago sativa L.) is a major forage crop of family Fabaceae and is frequently cultivated in Egypt. The present study is concerned with the genetic discrimination of fifteen alfalfa cultivars from three different countries (Egypt, Australia, and USA) using two molecular approaches: inter-retrotransposon-amplified polymorphism (IRAP) markers and two chloroplast DNA barcodes matK and the trnH in addition to the analysis of fifteen morpho-agronomic traits. The genetic relatedness, based on analysis of IRAP marker polymorphism and produced using eleven primers by clustering via principal component analysis (PCA) and multivariate heatmap biostatistical methods differentiated the two Egyptian cultivars EGY1-Ismailia1 and EGY2-Nubaria1 from the three Australian and seven American cultivars, with some distinction of the cv. USA6-SW9720 and cv. AUS4-SuperFast. The results were also supported by the sequence analysis of the matK and the trnH genes on the genetic relatedness between eight cultivars. Moreover, it might be suggested that breeding lines from M. sativa cultivars may provide novel insights and a better understanding of the domestication of M. sativa genetic diversity. The classification of the eight cultivars, as revealed by morpho-agronomic traits, confirmed the close genetic relationship between the two Egyptian cultivars and indicated some resemblance between them and the AUS2-Siri Nafa, whereas the two American cultivars, USA1-Super supreme and USA4-Cuf101, were clearly isolated from a cluster of other three cultivars USA7-SW9628, USA8-Magna901, and USA9-Perfect. The results are useful sources of genetic information for future breeding programs in crop development and open new possibilities of producing M. sativa lines harboring high forage quality, productivity, and resistance to biotic and abiotic stresses.

Barcoding of estuarine macrophytes and phylogenetic diversity of estuaries along the South African coastline.

Plant DNA barcoding serves as an effective approach to building community phylogenies and increasing our understanding of the factors that determine plant community assemblages. The aims of the study were to (i) barcode macrophytes with high estuarine fidelity and (ii) to determine the phylogenetic diversity (PD) of selected South African estuaries for conservation prioritisation. Three DNA barcoding gene regions (rbcLa, matK, and trnH-psbA) were assessed, and community phylogenies were constructed for 270 estuaries. Generally, the matK barcode had the greatest discrimination success rate of 67.4% (parsimony informative sites = 418). Closely related species formed clades that also represent estuarine habitat types. Estuaries with high phylogenetic diversity along the southeast coast were associated with a combination of mangrove and salt marsh habitats. Species richness was strongly and significantly correlated with PD (r = 0.93; p < 0.000). Based on mean pairwise distance (MPD), more temperate estuaries (56) showed significant phylogenetic clustering compared to subtropical estuaries (24) (p < 0.05). Similarly, based on mean nearest taxon distance (MNTD), significant phylogenetic clustering was highest in temperate estuaries (50) compared to subtropical estuaries (12) (p < 0.05). This suggests that the coexistence of plant species in estuaries is structured by both biotic and abiotic interactions.

DNA barcoding of flowering plants in Sumatra, Indonesia.

The rapid conversion of Southeast Asian lowland rainforests into monocultures calls for the development of rapid methods for species identification to support ecological research and sustainable land‐use management. Here, we investigated the utilization of DNA barcodes for identifying flowering plants from Sumatra, Indonesia. A total of 1,207 matK barcodes (441 species) and 2,376 rbcL barcodes (750 species) were successfully generated. The barcode effectiveness is assessed using four approaches: (a) comparison between morphological and molecular identification results, (b) best‐close match analysis with TaxonDNA, (c) barcoding gap analysis, and (d) formation of monophyletic groups. Results show that rbcL has a much higher level of sequence recoverability than matK (95% and 66%). The comparison between morphological and molecular identifications revealed that matK and rbcL worked best assigning a plant specimen to the genus level. Estimates of identification success using best‐close match analysis showed that >70% of the investigated species were correctly identified when using single barcode. The use of two‐loci barcodes was able to increase the identification success up to 80%. The barcoding gap analysis revealed that neither matK nor rbcL succeeded to create a clear gap between the intraspecific and interspecific divergences. However, these two barcodes were able to discriminate at least 70% of the species from each other. Fifteen genera and twenty‐one species were found to be nonmonophyletic with both markers. The two‐loci barcodes were sufficient to reconstruct evolutionary relationships among the plant taxa in the study area that are congruent with the broadly accepted APG III phylogeny.

DNA barcoding evaluation of Vicia (Fabaceae): Comparative efficacy of six universal barcode loci on abundant species

AbstractVicia L. is an invaluable genus with considerable agricultural and economic importance due to its high value as feed, green manure, and medicine. However, most of this genus is not well known and remains underutilized. Due to the imprecise circumscription and delineation of Vicia species, the taxonomic history of this genus is controversial, which has hindered the identification of species with high economic potential. Therefore, rapid and accurate identification of Vicia species is essential. Simultaneously, species identification through DNA barcoding has become an effective taxonomic classification tool. Here, the species‐discrimination abilities of matK, rbcL, trnH‐psbA, trnL‐trnF, ITS1, and ITS2 were tested in 161 Vicia species with both sequence similarity (nearest neighbor, best match, and best close match) and tree‐based (maximum likelihood tree) methods. Among the single barcode sites, trnH‐psbA had the highest level of polymorphism (52.4% variable sites; nucleotide diversity, 0.1338). Additionally, trnH‐psbA had the highest mean interspecific distance (0.1352) and intraspecific distance (0.0071). The combined barcode matK+trnH‐psbA had the highest correct identification rate by the sequence similarity method. Both trnH‐psbA (75.38%) and matK (70.73%) showed higher species discrimination rates than the other barcodes when using the tree‐based method. Based on overall performance, matK and trnH‐psbA, alone or in combination, were the best barcodes for Vicia. Internal transcribed spacer (ITS1) also showed good performance and provided essential information regarding nuclear DNA, so this site is also recommended as a backup barcode for Vicia. These Vicia barcodes can be used to identify species and to evaluate germplasm resource collections.

DNA barcoding of the genus Nepenthes (Pitcher plant): a preliminary assessment towards its identification

BackgroundDNA barcoding is impending towards the generation of universal standards for species discrimination with a standard gene region that can be sequenced accurately and within short span of time. In this study, we were successful in developing efficient barcode locus in the Nepenthes genus. A total of 317 accessions were retrieved from GenBank of NCBI which represent 140 different species Nepenthes and evaluated the efficacy of ITS, rbcl and matK barcode candidates using barcode gap, applied distance similarity, and tree-based methods.ResultOur result indicates that single-locus ITS or combined with plastid regions (matK) showed the best species discrimination with distinctive barcoding gaps. Therefore, we tentatively proposed the combination of ITS+matK as a core barcode for Nepenthes genus.ConclusionThis study provides a report on DNA barcoding for unique insectivores’ Nepenthes genus. As the different species of Nepenthes are higly endemic and endangered, it would be a useful study to understand the evolutionary relationship, sketched in emigration, mislabeling and can be a probable assessment for its biodiversity.

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 and Identification of Medicinal Plants in the Kingdom of Bahrain

Authentication of medicinally important plants is essential for increasingly demands of herbal remedies worldwide. DNA barcoding technology is currently gaining importance as a reliable tool for plant species identification, although one barcode gene is not enough in the exceptions. Short sequence diversity of standardized specific coding gene regions of rbcLa and matK of plastid genome together with noncoding ribosomal internal transcribed spacer 2 (ITS2) marker is used as barcode to compare and differentiate plant species. The success of obtaining sequences of the 29 analyzed plants distributed in 21 families using three different barcode genes rbcLa, matK and ITS2 were 97%, 79% and 75% respectively. Multiple sequence alignment confirmed the medicinal plants at species level by 89.28%, 86.32% and 60.86% obtained through rbcLa, ITS2 and matK barcodes sequences respectively. The genetic distance between sequence pairs (GD) and percentage identity (PI) is compared to analyze the plant identity at species level. The phylogenic trees constructed to show the relatedness and distance of the analyzed plants in the history of evolution by the analysis of richness of clades. The construction of DNA barcode library of desert medicinal plants is an introductory research arena in Kingdom of Bahrain in helping the routine identification of plants, and developing guidelines for detection of adulterants in herbal medicines as well as protection of biodiversity.

Replacing Sanger with Next Generation Sequencing to improve coverage and quality of reference DNA barcodes for plants

We estimate the global BOLD Systems database holds core DNA barcodes (rbcL + matK) for about 15% of land plant species and that comprehensive species coverage is still many decades away. Interim performance of the resource is compromised by variable sequence overlap and modest information content within each barcode. Our model predicts that the proportion of species-unique barcodes reduces as the database grows and that ‘false’ species-unique barcodes remain >5% until the database is almost complete. We conclude the current rbcL + matK barcode is unfit for purpose. Genome skimming and supplementary barcodes could improve diagnostic power but would slow new barcode acquisition. We therefore present two novel Next Generation Sequencing protocols (with freeware) capable of accurate, massively parallel de novo assembly of high quality DNA barcodes of >1400 bp. We explore how these capabilities could enhance species diagnosis in the coming decades.

Efficient Identification of the Forest Tree Species in Aceraceae Using DNA Barcodes.

Aceraceae is a large forest tree family that comprises many economically and ecologically important species. However, because interspecific and/or intraspecific morphological variations result from frequent interspecific hybridization and introgression, it is challenging for non-taxonomists to accurately recognize and identify the tree species in Aceraceae based on a traditional approach. DNA barcoding is a powerful tool that has been proposed to accurately distinguish between species. In this study, we assessed the effectiveness of three core standard markers (matK, rbcL and ITS) plus the chloroplast locus trnS-trnG as Aceraceae barcodes. A total of 231 sequences representing 85 species in this forest family were collected. Of these four barcode markers, the discrimination power was highest for the ITS (I) region (50%) and was progressively reduced in the other three chloroplast barcodes matK (M), trnS-trnG (T) and rbcL (R); the discrimination efficiency of the ITS marker was also greater than any two-locus combination of chloroplast barcodes. However, the combinations of ITS plus single or combined chloroplast barcodes could improve species resolution significantly; T+I (90.5% resolution) and R+M+T+I (90.5% resolution) differentiated the highest portion of species in Aceraceae. Our current results show that the nuclear ITS fragment represents a more promising DNA barcode marker than the maternally inherited chloroplast barcodes. The most efficient and economical method to identify tree species in Aceraceae among single or combined DNA barcodes is the combination of T+I (90.5% resolution).

Identification of species adulteration in traded medicinal plant raw drugs using DNA barcoding

Plants are the major source of therapeutic ingredients in complementary and alternative medicine (CAM). However, species adulteration in traded medicinal plant raw drugs threatens the reliability and safety of CAM. Since morphological features of medicinal plants are often not intact in the raw drugs, DNA barcoding was employed for species identification. Adulteration in 112 traded raw drugs was tested after creating a reference DNA barcode library consisting of 1452 rbcL and matK barcodes from 521 medicinal plant species. Species resolution of this library was 74.4%, 90.2%, and 93.0% for rbcL, matK, and rbcL + matK, respectively. DNA barcoding revealed adulteration in about 20% of the raw drugs, and at least 6% of them were derived from plants with completely different medicinal or toxic properties. Raw drugs in the form of dried roots, powders, and whole plants were found to be more prone to adulteration than rhizomes, fruits, and seeds. Morphological resemblance, co-occurrence, mislabeling, confusing vernacular names, and unauthorized or fraudulent substitutions might have contributed to species adulteration in the raw drugs. Therefore, this library can be routinely used to authenticate traded raw drugs for the benefit of all stakeholders: traders, consumers, and regulatory agencies.

Applying DNA Barcodes to Identify Closely Related Species of Ferns: A Case Study of the Chinese Adiantum (Pteridaceae)

DNA barcoding is a fast-developing technique to identify species by using short and standard DNA sequences. Universal selection of DNA barcodes in ferns remains unresolved. In this study, five plastid regions (rbcL, matK, trnH-psbA, trnL-F and rps4-trnS) and eight nuclear regions (ITS, pgiC, gapC, LEAFY, ITS2, IBR3_2, DET1, and SQD1_1) were screened and evaluated in the fern genus Adiantum from China and neighboring areas. Due to low primer universality (matK) and/or the existence of multiple copies (ITS), the commonly used barcodes matK and ITS were not appropriate for Adiantum. The PCR amplification rate was extremely low in all nuclear genes except for IBR3_2. rbcL had the highest PCR amplification rate (94.33%) and sequencing success rate (90.78%), while trnH-psbA had the highest species identification rate (75%). With the consideration of discriminatory power, cost-efficiency and effort, the two-barcode combination of rbcL+ trnH-psbA seems to be the best choice for barcoding Adiantum, and perhaps basal polypod ferns in general. The nuclear IBR3_2 showed 100% PCR amplification success rate in Adiantum, however, it seemed that only diploid species could acquire clean sequences without cloning. With cloning, IBR3_2 can successfully distinguish cryptic species and hybrid species from their related species. Because hybridization and allopolyploidy are common in ferns, we argue for including a selected group of nuclear loci as barcodes, especially via the next-generation sequencing, as it is much more efficient to obtain single-copy nuclear loci without the cloning procedure.

The nuclear internal transcribed spacer (ITS2) as a practical plant DNA barcode for herbal medicines

With the development of DNA barcoding methods and databases, misidentification may be easily caught. However, this technique has presented challenges with regard to the tissue type and marker locus variation. In this study, DNA barcoding was used to investigate the taxonomic accuracy of herbal medicines sold in the New York City (NYC) area using the plastid markers rbcL, matK and the nuclear ribosomal region ITS2. Forty-seven (47) samples (single-ingredient herbal medicinal products or HMPs) were purchased from eight herbal stores, and represented different structures: 22 leaf samples, 11 roots, 9 barks, and 5 samples that were ground beyond recognition. MEGABLAST sequence comparison was performed to verify the taxonomic identity of the samples. Maximum likelihood trees were constructed for sequence queries with equivocal multiple high-scoring MEGABLAST hits. Of the barcodes, ITS2 had the most success being amplified and sequenced (barcoded) in 38 of the 47 HMPs (81%), mostly leaf and root samples as barks were difficult to barcode. This is in contrast to 26/47 (55%) and 15/47 (32%) successful barcoding for rbcL and matK, respectively. ITS2 alone was accurate enough to match 88% of ITS2 sequences to the expected species, while only 59% of sequenced rbcL and 45% of matK was able to match with the expected species. All four HMPs obtained from one store did not match the declared species on the label based on ITS2, and this result was echoed by the rbcL and matK barcodes. However, all other herbal stores appeared to be selling accurately labeled HMPs. Our study showed that DNA barcoding is a worthwhile effort to test HMP authenticity, which may help build reputation of quality brands and ensure consumer safety. ITS2 is a practical DNA barcode that regulatory agencies may propose to NYC herbal medicine manufacturers and merchants to ensure quality.

Comparative efficacy of four candidate DNA barcode regions for identification of Vicia species

AbstractThe genus Vicia L., one of the earliest domesticated plant genera, is a member of the legume tribe Fabeae of the subfamily Papilionoideae (Fabaceae). The taxonomic history of this genus is extensive and controversial, which has hindered the development of taxonomic procedures and made it difficult to identify and share these economically important crop resources. Species identification through DNA barcoding is a valuable taxonomic classification tool. In this study, four DNA barcodes (ITS2, matK, rbcL and psbA-trnH) were evaluated on 110 samples that represented 34 taxonomically best-known species in the Vicia genus. Topologies of the phylogenetic trees based on an individual locus were similar. Individual locus-based analyses could not discriminate closely related Vicia species. We proposed a concatenated data approach to increase the resolving power of ITS2. The DNA barcodes matK, psbA-trnH and rbcL were used as an additional tool for phylogenetic analysis. Among the four barcodes, three-barcode combinations that included psbA-trnH with any two of the other barcodes (ITS2, matK or rbcL) provided the best discrimination among Vicia species. Species discrimination was assessed with bootstrap values and considered successful only when all the conspecific individuals formed a single clade. Through sequencing of these barcodes from additional Vicia accessions, 17 of the 34 known Vicia species could be identified with varying levels of confidence. From our analyses, the combined barcoding markers are useful in the early diagnosis of targeted Vicia species and can provide essential baseline data for conservation strategies, as well as guidance in assembling germplasm collections.