DNA Barcoding Loci Research Articles

Diversity of Diptera and their parasitoids associated with Inga vera Willd. (1806) (Fabaceae) with new host record in Minas Gerais, Brazil

Diptera belonging to four different families, as well as four associated parasitoids were reared from fruits of Inga vera Willd. (Fabaceae) in Lavras, Minas Gerais, southeast Brazil. We collected 450 fruits from five trees of I. vera between 2020 and 2021 in various localities situated within the University Federal of Lavras. We reared specimens belonging to four different dipteran families: Anastrepha distincta Greene (Tephritidae), Asteromyia sp. (Cecidomyiidae), Drosophila zottii Vilela (Drosophilidae), and Neosilba pendula (Bezzi) (Lonchaeidae). Lopheucoilasp.(Hymenoptera: Figitidae) probably is a parasitoid of N. pendulawhereas Eupelmus sp. (Hymenoptera: Eupelmidae), and two species of Sycophila (Hymenoptera, Eurytomidae) appear to be parasitoids of Asteromyia sp.. We additionally conducted molecular characterization using the DNA barcoding locus for the majority of the species reared in this study. This study provides valuable insights into the biology and ecology of frugivorous flies in Brazil, and sheds light on potential biological control agents, enabling better management practices.

Comparison of four DNA barcoding loci to distinguish between some Apiaceae family species

BackgroundThe Apiaceae family is among the most significant plant families because it contains both beneficial and poisonous plants. Due to their morphological similarity, these harmless and lethal species are frequently confounded. Cumin, fennel, and anise are the most prevalent members of the family Apiaceae in Egypt. Members of this family are routinely used as medical surrogates, so it is crucial that they are correctly identified and distinguished. DNA barcoding is a molecular technique used for identifying species and reconstructing phylogenetic trees.ResultsSix plants from this family were chosen for this study due to their medicinal importance, and four DNA barcoding loci (rbcL, matK, trnH-psaA, and ITS) were used to identify them. The amplicons were sequenced, and the comparative analysis was conducted between the sequences evaluated and the most significant Blast results. The DNA rbcL, trnH-psaA, and ITS barcodes exhibited similar amplicons among the six species of Apiaceae, while the trnH-psaA barcode exhibited different amplicons among the Apiaceae. Maximum likelihood approach was used to calculate the genetic distance between the sex species of Apiaceae. The most significant findings were that the one from four DNA barcoding was able to distinguish between distinct species and confirm their evolutionary belonging to this family.ConclusionsThe current study concludes that trnH-psbA and ITS DNA identifiers can be used to accurately identify, differentiate, and record Apiaceae species, while the rbcl DNA barcode appears to have fallen short of its intended purpose. So, the data that come from DNA barcodes could be used for the biodiversity assessment and the similarities between hazardous and commercial plants to resolve some of these deficiencies.

DNA barcoding for the identification and authentication of medicinal deer (Cervus sp.) products in China.

Deer products from sika deer (Cervus nippon) and red deer (C. elaphus) are considered genuine and used for Traditional Chinese Medicine (TCM) materials in China. Deer has a very high economic and ornamental value, resulting in the formation of a characteristic deer industry in the prescription preparation of traditional Chinese medicine, health food, cosmetics, and other areas of development and utilization. Due to the high demand for deer products, the products are expensive and have limited production, but the legal use of deer is limited to only two species of sika deer and red deer; other wild deer are prohibited from hunting, so there are numerous cases of mixing and adulteration of counterfeit products and so on. There have been many reports that other animal (pig, cow, sheep, etc.) tissues or organs are often used for adulteration and confusion, resulting in poor efficacy of deer traditional medicine and trade fraud in deer products. To authenticate the deer products in a rapid and effective manner, the analysis used 22 deer products (antler, meat, bone, fetus, penis, tail, skin, and wool) that were in the form of blind samples. Total DNA extraction using a modified protocol successfully yielded DNA from the blind samples that was useful for PCR. Three candidate DNA barcoding loci, cox1, Cyt b, and rrn12, were evaluated for their discrimination strength through BLAST and phylogenetic clustering analyses. For the BLAST analysis, the 22 blind samples obtained 100% match identity across the three gene loci tested. It was revealed that 12 blind samples were correctly labeled for their species of origin, while three blind samples that were thought to originate from red deer were identified as C. nippon, and seven blind samples that were thought to originate from sika deer were identified as C. elaphus, Dama dama, and Rangifer tarandus. DNA barcoding analysis showed that all three gene loci were able to distinguish the two Cervus species and to identify the presence of adulterant species. The DNA barcoding technique was able to provide a useful and sensitive approach in identifying the species of origin in deer products.

Comparison of Machine Learning Algorithms for Species Family Classification using DNA Barcode

Classifying plant species within the Liliaceae and Amaryllidaceae families presents inherent challenges due to the complex genetic diversity and overlapping morphological traits among species. This study explores the difficulties in accurate classification by comparing 11 supervised learning algorithms applied to DNA barcode data, aiming to enhance the precision of species family classification in these taxonomically intricate plant families. The ribulose-1,5-bisphosphate carboxylase-oxygenase large sub-unit (rbcL) gene, selected as a DNA barcode locus for plants, is used to represent species within the Amaryllidaceae and Liliaceae families. The experimental results demonstrate that nearly all tested models achieve accurate species classification into the appropriate families, with an accuracy rate exceeding 97%, except for the Naïve Bayes model. Regarding computational time, the Random Forest model requires significantly more time for training than other models. Regarding memory usage, the Least Squares Support Vector Machine with a polynomial kernel, and Regularized Logistic Regression consume more memory than other models. These machine learning models exhibit strong concordance with NCBI's classifications when predicting families using the test dataset, effectively categorizing species into the Amaryllidaceae and Liliaceae families.

Conservation and molecular identification of traditional rice varieties collected from Kerala using matK and rbcL DNA barcode markers

Varietal identification of economically significant crops has acquired great importance in the global market especially in the terms of protection of plant varieties. Kerala is the home to several different specialty traditional rice varieties which is vital sources of nutritional and therapeutic properties. Traditional rice landraces are an integral part of the nutrition, medicine, and culture of Kerala. Most of these varieties possess similar morphological characteristics but differ in their nutritional and medicinal properties. It has become a challenge for a non-taxonomist to identify and recognize the traditional rice varieties accurately. DNA barcoding has been considered a standard method to identify species or variety which utilizes a unique pattern of variation in the DNA of the species. It can be adopted as a routine and faster approach to determine the molecular taxonomy when compared to morphological identification approaches. Currently, the most intensively applied two DNA barcode loci are maturase K (matK) and ribulose 1,5-biphosphate carboxylase (rbcL) for plant identification. But, the efficacy of these two loci in discriminating plant species may vary. Hence, in this study fifty-four traditional rice varieties collected from the Wayanad district of Kerala were used to generate and compare the two standard DNA barcode regions for plant matK and rbcL via PCR amplification. The varieties collected were varying in the husk color from black; red to brown and the brown rice varieties not display any morphological variation. The study identified that among the two selected markers rbcL was observed to have the potential to discriminate the intra and interspecific regions of the studied rice varieties and this would have been utilized to generate DNA barcodes for each variety. This study separates and identifies 28 traditional rice varieties among the 54 studied varieties as Oryza nivara, the wild progenitor of Asian cultivated rice. Along with other supporting information the data presented here could be used as a reference to check the authenticity and varietal purity of commercially significant rice varieties in the future.

IDENTIFICATION OF LENS CULTIVARS IN MARKET BY MOLECULAR TOOLS: DNA BARCODING AND SSRs

Substitution of plant cultivars of high commercial value with a cheaper, lower quality one is a common fraud committed against consumers and producers. Since it is one of the most widely grown legumes, lentil (Lens culinaris Medik.) is suitable for such frauds. This study aimed to identify lentil cultivars which are registered and authorized in the market in Türkiye by using current molecular methods. For this purpose, 26 lentil cultivars were analyzed for 15 SSR markers and two DNA barcode regions (trnH-psbA and matK). A high allele diversity was observed by 12 scorable SSR markers, and the average number of alleles was determined to be 16. One of the important findings was the presence of “cultivar-specific alleles” that can be used to identify each cultivar in the lentil market in Türkiye. At least one “cultivar-specific allele” was obtained for each cultivar. The lentil cultivars were also analyzed by two DNA barcode regions as trnH-psbA and matK. While it was observed that the rate of the intra-species variation for the trnH-psbA region was low and 26 varieties were divided into 7 groups, higher rate was found for matK and samples were distributed into 14 groups. Nevertheless, it was observed that intra-species discrimination can be made more effective when both loci are used together and 26 species were distributed into 18 different groups. We expect that the results of this study, especially the cultivar-specific SSR alleles and DNA barcoding sequence data may be used routinely to identify production and packaged products that are commercially available in markets.

What is Suksdorf’s hawthorn? Revision of the Western North American 20-stamen black-fruited hawthorns (Crataegus series Douglasianae, Rosaceae subtribe Malinae)

An agamic complex of 20- and 10-stamen, black-fruited hawthorns (Crataegus subg. Sanguineae, sect. Douglasianae) occurs in western North America, with a 10-stamen taxon disjunct in the upper Great Lakes basin. Here, we recircumscribe the 20-stamen taxa at the core of this complex (C. ser. Douglasianae). This is needed in order to distinguish between a presumptively ancestral diploid and its allo- and autopolyploid derivatives, all differing in breeding system, distribution, morphology, and pattern of genetic variation. The earliest name for these 20-stamen taxa, Crataegus gaylussacia A. Heller, was given to distinctive plants of Marin and Sonoma counties in California that have recently been shown to be autotriploids. In Flora North America, however, this name was applied to all 20-stamen, black-fruited hawthorns. We recircumscribe C. gaylussacia, and also recircumscribe and typify a slightly younger name, C. suksdorfii (Sarg.) Kruschke, with a specimen from southern Washington with the diminished pollen fertility found in allopolyploid, apomictic black-fruited hawthorns occurring east of the Cascades, from northern California north to southernmost Alaska. Finally, we recognize the diploid, self-incompatible, sexual black-fruited hawthorns found west of the Cascades from northern California to southwestern Washington as Crataegus rhodamae-loveae sp. nov. Together with the 10-stamen, black-fruited hawthorns in predominantly tetraploid, apomictic, and self-fertile C. douglasii Lindl. and its segregates (discussed in detail in a companion paper), these species are linked by whole genome duplications associated in most cases with hybridization, with members of red-fruited C. subg. Americanae, and with each other. We suggest that this complex provides a model for understanding other such groups of Crataegus species that are related by whole genome duplications resulting from the fertilization of unreduced gametes (facilitated by but not necessarily dependent on occurrence of gametophytic apomixis), often together with hybridization. We note that an earlier effort at DNA barcoding these and other hawthorn species that provided limited support for our taxonomic decisions here also demonstrated some limited utility of the original plant DNA barcoding loci in groups like Crataegus. The taxonomic decisions we advocate will warrant consideration when other groups of hawthorns are revised in the light of data like those employed here.

Molecular identification and mapping of mixed root samples to species and individuals.

This article is a Commentary on Osborne et al. (2023), 238: 1305–1317.

Skimming for barcodes: rapid production of mitochondrial genome and nuclear ribosomal repeat reference markers through shallow shotgun sequencing.

DNA barcoding is critical to conservation and biodiversity research, yet public reference databases are incomplete. Existing barcode databases are biased toward cytochrome oxidase subunit I (COI) and frequently lack associated voucher specimens or geospatial metadata, which can hinder reliable species assignments. The emergence of metabarcoding approaches such as environmental DNA (eDNA) has necessitated multiple marker techniques combined with barcode reference databases backed by voucher specimens. Reference barcodes have traditionally been generated by Sanger sequencing, however sequencing multiple markers is costly for large numbers of specimens, requires multiple separate PCR reactions, and limits resulting sequences to targeted regions. High-throughput sequencing techniques such as genome skimming enable assembly of complete mitogenomes, which contain the most commonly used barcoding loci (e.g., COI, 12S, 16S), as well as nuclear ribosomal repeat regions (e.g., ITS1&2, 18S). We evaluated the feasibility of genome skimming to generate barcode references databases for marine fishes by assembling complete mitogenomes and nuclear ribosomal repeats. We tested genome skimming across a taxonomically diverse selection of 12 marine fish species from the collections of the National Museum of Natural History, Smithsonian Institution. We generated two sequencing libraries per species to test the impact of shearing method (enzymatic or mechanical), extraction method (kit-based or automated), and input DNA concentration. We produced complete mitogenomes for all non-chondrichthyans (11/12 species) and assembled nuclear ribosomal repeats (18S-ITS1-5.8S-ITS2-28S) for all taxa. The quality and completeness of mitogenome assemblies was not impacted by shearing method, extraction method or input DNA concentration. Our results reaffirm that genome skimming is an efficient and (at scale) cost-effective method to generate all mitochondrial and common nuclear DNA barcoding loci for multiple species simultaneously, which has great potential to scale for future projects and facilitate completing barcode reference databases for marine fishes.

Identification and Characterization of Fungal Pathogens Associated with Boxwood Diseases in the Republic of Korea.

Boxwood is a representative ornamental shrub that is widely used in landscaping horticulture. After pruning, damaged leaves or stems of boxwoods are unavoidably vulnerable to infection by various plant pathogens. Several boxwood diseases caused by fungi, such as Volutella blight and Macrophoma leaf spot, have been reported worldwide including Republic of Korea. In this study, we isolated and identified fungal pathogens of boxwood diseases that occurred in Korea and characterized their morphological and taxonomic characteristics. Boxwood samples showing blight symptoms were collected in Seoul, Republic of Korea, and the putative fungal pathogens Pseudonectria buxi, P. foliicola, and Neofusicoccum buxi were successfully identified. Investigation of the morphological features of the field isolates, including mycelial growth and conidial morphology, and phylogenetic analysis of multiple DNA barcode loci revealed that there were some morphological and genetic variations among isolates, but all of the analyzed isolates were closely related to the corresponding reference strains. We also found that P. foliicola strains were more virulent than P. buxi, and the N. buxi strains isolated in this study were weak pathogens or saprophytes. The results of our study will contribute to the development of control strategies for boxwood diseases caused by fungi and accelerate research on the complex ecology of boxwood diseases.

Identification and phylogenetic analysis of the genus Syringa based on chloroplast genomic DNA barcoding.

DNA barcoding is a supplementary tool in plant systematics that is extensively used to resolve species-level controversies. This study assesses the significance of using two DNA barcoding loci (e.g., psbA-trnH and trnC-petN) in distinguishing 33 plant samples of the genus Syringa. Results showed that the average genetic distance K2P of psbA-trnH DNA marker was 0.0521, which is much higher than that of trnC-petN, which is 0.0171. A neighbor-joining phylogenetic tree based on psbA-trnH and trnC-petN indicated that the identification rate of psbA-trnH and trnC-petN alone were 75% and 62.5%, respectively. The barcode combination of psbA-trnH+trnC-petN could identify 33 samples of the genus Syringa accurately and effectively with an identification rate of 87.5%. The 33 Syringa samples were divided into four groups: Group I is series Syringa represented by Syringa oblata; Group II is series Villosae represented by Syringa villosa; Group III is series Pubescentes represented by Syringa meyeri; and Group IV is section Ligustrina represented by Syringa reticulata subsp. pekinensis. These research results provided strong evidence that the combinatorial barcode of psbA-trnH+trnC-petN had high-efficiency identification ability and application prospects in species of the genus Syringa.

Two new species of the braconid wasp genus Bracon (Braconinae) from Los Tuxtlas region in Veracruz, Mexico, reared from fruits of three species of Lauraceae.

Two new species belonging to the braconid genus Bracon (Braconinae) are described from the tropical rainforest of Los Tuxtlas in the state of Veracruz, Mexico, B. laurae sp. nov. and B. rosamondae sp. nov. These species are morphologically similar and were reared from fruits of three Lauraceae species, Damburneya ambigens, D. salicifolia and Nectandra turbacensis. However, comparison of their DNA barcoding locus and a fragment of the nuclear ribosomal 28S gene confirmed their allospecificity. These two species share a number of morphological features with the two described Neotropical Bracon species that are known to be phytophagous (seed predators), B. phytophagus Quicke and B. zuleideae Perioto Lara. We therefore propose a new species-group for the above four species, the B. phytophagus Quicke species-group, and suggest that the two newly described species also have a phytophagous feeding strategy.

TAXONOMIC ASSESSMENT OF CURCULIGO ORCHIOIDES USING matK AND rbcL DNA BARCODES

Curculigo orchioides is most commonly used as an imperative medicinal plant in Vietnam. The plant roots are mainly used to treat sexual dysfunction, back pain, arthritis, nephritis, jaundice, and infertility. Given the high market demand in the herbal market, C. orchioides traders commonly adulterate with other similar plants for illegitimate benefits. Maturase K (matK) and ribulose 1,5-biphosphate carboxylase (rbcL) DNA barcode loci are presently used to identify the blends and counterfeit the medicinal herbs, as well as, identify the propagating conservation material. Nevertheless, the identification accuracy is highly dependent on NCBI Genbank or Barcode of Life Database (BOLD). In the recent study, the efficiency of DNA barcode loci, matK and rbcL for the classification of C. orchioides populations, was investigated during 2020–2021. After examining 11 accessions of C. orchioides collected from different locations in Vietnam, the obtained results revealed that using NCBI database is more effective for classifying C. orchioides. In addition, the matK locus also showed higher identification power than rbcL. The obtained findings could be helpful in the trading management, conservation, and development of C. orchioides in Vietnam.

Distribution analysis, updated checklist, and DNA barcodes of the endemic vascular flora of the Altai mountains, a Siberian biodiversity hotspot

The Altai Mountains of central Asia are biologically rich and comprise a wide diversity of ecosystems and lineages, including numerous endemic vascular plant species. Here we provide an updated checklist of the endemic vascular flora of the Altai Mountains with more taxa and higher geographic resolution than previously reported, as well as first molecular data and specimen images for many of these species. This flora is now known to contain 321 endemic species distributed in 34 families, many of which are narrowly restricted to one subregion of the Altai. The Fabaceae has given rise to the most endemic species in the Altai (74 spp.), and most of this diversity is found in the large and ecologically important genera Astragalus and Oxytropis. Approximately 60% of the endemic flora was imaged and successfully barcoded with at least one of three common DNA barcoding loci, and a phylogenetic tree based upon these loci is also presented to display the evolutionary breadth of endemism in the Altai. The distribution of each endemic species is presented in terms of a standard geographic subdivision of the Altai region, with general conservation priorities discussed based on areas currently afforded protected status.

Application of DNA barcoding tool for authentication of traditionally important medicinal Dendrobium (Orchidaceae) species from Sikkim Himalaya

Dendrobium, the second largest genus of the Orchidaceae family, is equally important for ornamental value and traditional medicine. Sikkim Himalaya region is rich in traditional knowledge of medicinal plants and their diversity. However, these regions were least explored through modern molecular tools. Reference databases association with DNA Barcoding technique is a new tool for species authentication. Here, we tested the efficiency of DNA barcoding loci (ITS, matK, rbcL, trnH-psbA) based on similarity search, characters based and phylogenetic methods to authenticate the medicinally important Dendrobium species. DNA from young leaves of studied species were isolated, and ITS (∼700 bp), rbcL(∼650 bp), matK (∼ 800 bp), trnH-psbA (∼ 450 bp) were amplified and sequenced for species-level identification. ITS, rbcL and trnH-psbA regions showed easy amplification and sequencing. Among the 54 sequences, 9 sequences were first time submitted to GenBank. In case of similarity search, ITS showed most efficient to identify all Dendrobium species except two species Dendrobium thyrsiflorum and Dendrobium densiflorum. However, the characters-based method easily differentiated between Dendrobium thyrsiflorum and Dendrobium densiflorum. The similarity-based methods and ITS sequence information together may help correct species identification for medicinal plants of Dendrobium.

Evaluation of Four Commonly Used DNA Barcoding Loci for Ardisia Species Identification.

Ardisia plants have been used as medicinal plants for a long time in China. Traditional techniques such as morphological, microscopic, and chemical identification methods all have limitations in the species identification of Ardisia. For the sake of drug safety, four DNA barcodes (psbA-trnH, ITS, rbcL, and matK) were assessed for Chinese Ardisia plants using a total of 121 individuals from 33 species. Four criteria (The success rates of PCR amplification, DNA barcoding gap, DNA sequence similarity analysis and NJ tree clustering analysis) were used to evaluate the species identification ability of these four DNA barcodes. The results show that ITS had the highest efficiency in terms of PCR and sequencing and exhibited the most apparent inter- and intra-specific divergences and the highest species identification efficiency. There was no significant increase in species identification after combining the three cpDNA fragments with the ITS fragment. Considering the cost and experimental effectiveness, we recommend ITS as the core barcode for identifying Chinese Ardisia plants.

<i>Corrigendum to:</i> Extensive species diversification and marked geographic phylogenetic structure in the Mesoamerican genus <i>Stenopelmatus</i> (Orthoptera: Stenopelmatidae: Stenopelmatinae) revealed by mitochondrial and nuclear 3RAD data

The Jerusalem cricket subfamily Stenopelmatinae is distributed from south-western Canada through the western half of the United States to as far south as Ecuador. Recently, the generic classification of this subfamily was updated to contain two genera, the western North American <i>Ammopelmatus</i>, and the Mexican, and central and northern South American <i>Stenopelmatus</i>. The taxonomy of the latter genus was also revised, with 5, 13 and 14 species being respectively validated, declared as nomen dubium and described as new. Despite this effort, the systematics of <i>Stenopelmatus</i> is still far from complete. Here, we generated sequences of the mitochondrial DNA barcoding locus and performed two distinct DNA sequence-based approaches to assess the species’ limits among several populations of <i>Stenopelmatus</i>, with emphasis on populations from central and south-east Mexico. We reconstructed the phylogenetic relationships among representative species of the main clades within the genus using nuclear 3RAD data and carried out a molecular clock analysis to investigate its biogeographic history. The two DNA sequence-based approaches consistently recovered 34 putative species, several of which are apparently undescribed. Our estimates of phylogeny confirmed the recent generic update of Stenopelmatinae and revealed a marked phylogeographic structure within <i>Stenopelmatus</i>. Based on our results, we propose the existence of four species-groups within the genus (the <i>faulkneri</i>, <i>talpa</i>, Central America and <i>piceiventris</i> species-groups). The geographic distribution of these species-groups and our molecular clock estimates are congruent with the geological processes that took place in mountain ranges along central and southern Mexico, particularly since the Neogene. Our study emphasises the necessity to continue performing more taxonomic and phylogenetic studies on <i>Stenopelmatus</i> to clarify its actual species richness and evolutionary history in Mesoamerica.

Extensive species diversification and marked geographic phylogenetic structure in the Mesoamerican genus

The Jerusalem cricket subfamily Stenopelmatinae is distributed from south-western Canada through the western half of the United States to as far south as Ecuador. Recently, the generic classification of this subfamily was updated to contain two genera, the western North American Ammopelmatus, and the Mexican, and central and northern South American Stenopelmatus. The taxonomy of the latter genus was also revised, with 5, 13 and 14 species being respectively validated, declared as nomen dubium and described as new. Despite this effort, the systematics of Stenopelmatus is still far from complete. Here, we generated sequences of the mitochondrial DNA barcoding locus and performed two distinct DNA sequence-based approaches to assess the species’ limits among several populations of Stenopelmatus, with emphasis on populations from central and south-east Mexico. We reconstructed the phylogenetic relationships among representative species of the main clades within the genus using nuclear 3RAD data and carried out a molecular clock analysis to investigate its biogeographic history. The two DNA sequence-based approaches consistently recovered 34 putative species, several of which are apparently undescribed. Our estimates of phylogeny confirmed the recent generic update of Stenopelmatinae and revealed a marked phylogeographic structure within Stenopelmatus. Based on our results, we propose the existence of four species-groups within the genus (the faulkneri, talpa, Central America and piceiventris species-groups). The geographic distribution of these species-groups and our molecular clock estimates are congruent with the geological processes that took place in mountain ranges along central and southern Mexico, particularly since the Neogene. Our study emphasises the necessity to continue performing more taxonomic and phylogenetic studies on Stenopelmatus to clarify its actual species richness and evolutionary history in Mesoamerica.

RbcL, a potential candidate DNA barcode loci for aconites: conservation of himalayan aconites.

Aconitum heterophyllum Wall. ex Royle and Aconitum balfourii Stapf, are two highly important, threatened medicinal plants of the Indian Himalayan Region. Root-tubers of Aconites have occupied an important place in Indian pharmacopoeia from very ancient times. India is a hub of the wild-collected medicinal herbs industry in Asia and these two aconites are known to have been heavily traded from the region in illicit manner. Prosecution of these illegal trading crimes is hampered by lack of pharma-forensic expertise and tools. Present study was conducted to evaluate the discriminatory potential of rbcL, a Chloroplast based DNA barcode marker for the authentication of these two Himalayan Aconites. Fresh plant samples were collected from their natural distributional range as well as raw materials were procured from herbal market and a total of 32 sequences were generated for the rbcL region. Analysis demonstrated that rbcL region can successfully be used for authentication and importantly, both the aconites, were successfully discriminated by rbcL locus with high bootstrap support (> 50%). Molecular markers could certainly be relied upon morphological and chemical markers being tissue specific, having a higher discriminatory power and not age dependent. Phylogenetic analysis using Maximum Likelihood Method revealed that the rbcL gene could successfully discriminate Himalayan Aconites to species level and have potential to be used in pharma-forensic applications as well as to curb illicit trade of these invaluable medicinal plants.

Identification of Tinomiscium petiolare from Vietnam using the DNA barcode

Background. Tinomiscium petiolare Hook.f. & Thomson is a medicinal species of the family Menispermaceae. This species is currently being intensively exploited for therapeutic purposes. Precise and rapid identification of T. petiolare is critical and essential for the classification, propagation, use and conservation of its genetic resources. In recent years, DNA barcoding has been known to be a fast and sensitive method for identifying species at any stage of development, using short DNA sequences. In this study we have performed the identification of T. petiolare specimens in Vietnam based on the sequence analysis of 4 DNA barcode loci: ITS, matK, rbcL and rpoC.Materials and methods. Total DNA was extracted from leaf samples using DNeasy Plant Mini Kit. PCR amplification of the ITS, matK, rbcL and rpoC regions was carried out on the GeneAmp PCR System 9700 with specific primers. The purified PCR products were sequenced on the ABI 3500 Genetic Analyzer system, using BigDye®Terminator v3.1 Cycle Sequencing Kit. These genetic sequences were analyzed and compared, and a phylogenetic tree was constructed using BioEdit, BLAST, and MEGA 6 programs.Results and conclusion. The success rate of amplification and sequencing was 100% for all 4 DNA barcode loci (ITS, matK, rbcL and rpoC) in the studied specimens. The produced sequence sizes of ITS, matK, rbcL and rpoC in the specimens were 574 bp, 810 bp, 527 bp and 488 bp, respectively. Further, we identified that all studied specimens were genetically related to each other and associated with the same species T. petiolare. Overall, the results of the study generated the most complete DNA barcode database of T. petiolare collected in Vietnam, contributing to the taxonomy and identification of this species.