[Site-specific PCR identification of animal species and formula particles of Scorpio].

Scorpio, a commonly used animal medicine in China, is derived from Buthus martensii as recorded in the Chinese Pharmacopoeia. China harbors rich species of Scorpionida and adulterants exist in the raw medicinal material and deep-processed products of Scorpio. The microscopic characteristics of the deep-processed products may be incomplete or lost during processing, which makes the identification difficult. In this study, the maximum likelihood(ML) tree was constructed based on the morphology and cytochrome C oxidase subunit I(COⅠ) to identify the species of Scorpio products. The results showed that the main adulterant of Scorpio was Lychas mucronatus. According to the specific SNP sites in the COⅠ sequence of B. martensii, the stable primers were designed for the identification of the medicinal material and formula granules of Scorpio. The polymerase chain reaction(PCR) at the annealing temperature of 61 ℃ and 30 cycles produced bright specific bands at about 150 bp for both B. martensii and its formula particles and no band for adulterants. The adaptability of the method was investigated, which showed that the bands at about 150 bp were produced for Scorpio medicinal material, lyophilized powder, and formula granules, and commercially available formula granules. The results showed that the established method could be used to identify the adulterants of Scorpio and its formula granules, which could help to improve the quality control system and ensure the safe clinical application of Scorpio formula granules.

Species Identification and Genetic Diversity Analysis of Medicinal Plants Aconitum pendulum Busch and Aconitum flavum Hand.-Mazz.

The classification system for the genus Aconitum is highly complex. It is also the subject of ongoing debate. Aconitum pendulum Busch and Aconitum flavum Hand.-Mazz. are perennial herbs of the genus Aconitum. Dried roots of these two plants are used in traditional Chinese medicine. In this study, morphological observations and ISSR molecular markers were employed to discriminate between A. flavum and A. pendulum, with the objective of gaining insights into the interspecies classification of Aconitum. The pubescence on the inflorescence of A. flavum was found to be appressed, while that on the inflorescence of A. pendulum was spread. UPGMA (unweighted pair-group method with arithmetic average) cluster analysis, PCoA (principal coordinates analysis), and Bayesian structural analysis divided the 199 individuals (99 individuals from DWM population and 100 individuals from QHL population) into two main branches, which is consistent with the observations of the morphology of pubescence on the inflorescence. These analyses indicated that A. flavum and A. pendulum are distinct species. No diagnostic bands were found between the two species. Two primer combinations (UBC808 and UBC853) were ultimately selected for species identification of A. flavum and A. pendulum. This study revealed high levels of genetic diversity in both A. flavum (He = 0.254, I = 0.395, PPB = 95.85%) and A. pendulum (He = 0.291, I = 0.445, PPB = 94.58%). We may say, therefore, that ISSR molecular markers are useful for distinguishing A. flavum and A. pendulum, and they are also suitable for revealing genetic diversity and population structure.

Comparative analysis of medicinal plants Scutellaria baicalensis and common adulterants based on chloroplast genome sequencing.

BACKGROUND: Scutellaria baicalensis Georgi has been extensively used as a medicinal herb in China for over 2000 years. They may be intentionally or inadvertently substituted or blended with comparable species in the local market, threatening clinical medication safety. Molecular markers are effective tools to prevent misidentification and eliminate doping and falsification among Scutellaria plants. This study screened four highly variable regions to identify Scutellaria and its adulterants. In addition, a phylogenetic analysis was performed using the complete cp genome combined with published Scutellaria species samples. Moreover, a comparative analysis of the cp genomes was conducted to investigate the cp genome evolution of S. baicalensis. RESULTS: The complete cp genome of five species of Scutellaria was sequenced for the first time, and four previously published Scutellaria species were re-sequenced. They all exhibited a conserved quadripartite structure in their cp genomes, including two distinct regions, namely a small and large single copy region, respectively, and two inverted repeats encompassing the majority of ribosomal RNA genes. Furthermore, the nine species exhibited high conservation from aspects of the genome structure, codon usage, repeat sequences, and gene content. Four highly variable regions (matK-rps16, ndhC-trnV-UAC, psbE-petL, and rps16-trnQ-UUG) may function as potential molecular markers for differentiating S. baicalensis from its adulterants. Additionally, the monophyly of Scutellaria was ascertained and could be reclassified into two subgenera, subgenus Anaspis and subgenus Scutellaria, as evidenced by the phylogenetic analyses on sequences of cp genome and shared protein-coding sequences. According to the molecular clock analysis, it has been inferred that the divergence of Scutellaria occurred at approximately 4.0 Mya during the Pliocene Epoch. CONCLUSION: Our study provides an invaluable theoretical basis for further Scutellaria species identification, phylogenetics, and evolution analysis.

Assessing deep convolutional neural network models and their comparative performance for automated medicinal plant identification from leaf images.

Medicinal plants have got notable attention in recent years in the field of pharmaceutical and drug research. The high demand of herbal medicine in the rural areas of developing countries and drug industries necessitates correct identification of the medicinal plant species which is challenging in absence of expert taxonomic knowledge. Against this backdrop, we attempted to assess the performance of seven advanced deep learning algorithms in the automated identification of the plants from their leaf images and to suggest the best model from a comparative study of the models. We meticulously trained VGG16, VGG19, DenseNet201, ResNet50V2, Xception, InceptionResNetV2, and InceptionV3 deep neural network models. This training utilized a dataset comprising 5878 images encompassing 30 medicinal species distributed among 20 families. Our approach involved two avenues: the utilization of public data (PI) and a blend of public and field data (PFI), the latter featuring intricate backgrounds. Our study elucidates the robustness of these models in accurately identifying and classifying both interfamily and interspecies variations. Despite variations in accuracy across diverse families and species, the models demonstrated adeptness in these classifications. Comparing the models, we unearthed a crucial insight: the Normalized leverage factor (γω) for DenseNet201 stands at 0.19, elevating it to the pinnacle position for PI with a remarkable 99.64 % accuracy and 98.31 % precision. In the PFI scenario, the same model achieves a γω of 0.15 with a commendable 97 % accuracy. These findings serve as a guiding beacon for shaping future application tools designed to automate medicinal plant identification at the user level.

DNA barcoding of herbal supplements on the US commercial market associated with the purported treatment of COVID-19.

INTRODUCTION: The COVID-19 pandemic was associated with an increased global use of traditional medicines, including Ayurvedic herbal preparations. Due to their growing demand, their processed nature, and the complexity of the global supply chain, there is an increased risk of adulteration in these products. OBJECTIVES: The objective of this study was to assess the use of DNA barcoding for species identification in herbal supplements on the US market associated with the Ayurvedic treatment of respiratory symptoms. METHODS: A total of 54 commercial products containing Ayurvedic herbs were tested with four DNA barcoding regions (i.e., rbcL, matK, ITS2, and mini-ITS2) using two composite samples per product. Nine categories of herbs were targeted: amla, ashwagandha, cinnamon, ginger, guduchi, tribulus, tulsi, turmeric, and vacha. RESULTS: At least one species was identified in 64.8% of products and the expected species was detected in 38.9% of products. Undeclared plant species, including other Ayurvedic herbs, rice, and pepper, were detected in 19 products, and fungal species were identified in 12 products. The presence of undeclared plant species may be a result of intentional substitution or contamination during harvest or processing, while fungal DNA was likely associated with the plant material or the growing environment. The greatest sequencing success (42.6-46.3%) was obtained with the matK and rbcL primers. CONCLUSION: The results of this study indicate that a combination of genetic loci should be used for DNA barcoding of herbal supplements. Due to the limitations of DNA barcoding in identification of these products, future research should incorporate chemical characterization techniques.

Plastid phylogenomics and species discrimination in the "Chinese" clade of Roscoea (Zingiberaceae).

Roscoea is an alpine or subalpine genus from the pan-tropical family Zingiberaceae, which consists of two disjunct groups in geography, namely the "Chinese" clade and the "Himalayan" clade. Despite extensive research on the genus, Roscoea species remain poorly defined and relationships between these species are not well resolved. In this study, we used plastid genomes of nine species and one variety to resolve phylogenetic relationships within the "Chinese" clade of Roscoea and as DNA super barcodes for species discrimination. We found that Roscoea plastid genomes ranged in length from 163,063 to 163,796 bp, and encoded 113 genes, including 79 protein-coding genes, 30 tRNA genes, four rRNA genes. In addition, expansion and contraction of the IR regions showed obvious infraspecific conservatism and interspecific differentiation. Plastid phylogenomics revealed that species belonging to the "Chinese" clade of Roscoea can be divided into four distinct subclades. Furthermore, our analysis supported the independence of R. cautleoides var. pubescens, the recovery of Roscoea pubescens Z.Y. Zhu, and a close relationship between R. humeana and R. cautloides. When we used the plastid genome as a super barcode, we found that it possessed strong discriminatory power (90%) with high support values. Intergenic regions provided similar resolution, which was much better than that of protein-coding regions, hypervariable regions, and DNA universal barcodes. However, plastid genomes could not completely resolve Roscoea phylogeny or definitively discriminate species. These limitations are likely related to the complex history of Roscoea speciation, poorly defined species within the genus, and the maternal inheritance of plastid genomes.

Qualitative identification of lonicerae japonicae flos in traditional chinese medicine using metabarcoding combined with specific mini-barcodes.

BACKGROUND: Lonicerae japonicae flos, also known as Jinyinhua (JYH), is an important component of traditional Chinese patent medicine (TCPM) products. However, the potential for adulteration and substitution with low-quality materials highlights the need for a reliable and sensitive approach to identify the species composition of TCPM products for consumer safety. METHODS AND RESULTS: We used universal ITS2 primers to amplify TCPMs containing JYH. However, the results were inconclusive, as only one operational taxonomic unit (OTU) was identified as Lonicera sp., which could not be identified at the species level. To confirm the species identification of Lonicera sp. in TCPM, we developed a short mini-barcode primer based on the psbA-trnH region, which, in combination with DNA metabarcoding technology, allowed for qualitative and quantitative analysis of artificially mixed samples. We applied the mini-barcode to distinguish TCPMs containing JYH and demonstrated its relatively accurate quantitative ability in identifying two Lonicera species. CONCLUSIONS: Our study presents a method for qualitative and quantitative identification of JYH, providing a promising application of DNA metabarcoding technology in the quality control of TCPM products.

Development of a tetra-primer ARMS-PCR for identification of sika and red deer and their hybrids.

Accurate identification of deer-derived components is significant in food and drug authenticity. Over the years, several methods have been developed to authenticate these products; however, identifying whether female deer products are hybrids is challenging. In this study, the zinc finger protein X-linked (ZFX) gene sequences of sika deer (Cervus nippon), red deer (Cervus elaphus) and their hybrid offspring were amplified and sequenced, the X221 and X428 species-specific single nucleotide polymorphisms (SNP) loci were verified, and a tetra-primer amplification refractory mutation system (T-ARMS-PCR) assay was developed to identify the parent-of-origin of female sika deer, red deer, and their hybrid deer. The T-ARMS-PCR developed based on the X221 locus could identify sika deer, red deer, and their hybrid offspring according to the presence or absence of PCR product sizes of 486 bp, 352 bp, and 179 bp, respectively, just as X428 locus could identify sika deer, red deer, and their hybrid offspring according to the presence or absence of PCR product sizes of 549 bp, 213 bp, and 383 bp, respectively. Forty products labeled deer-derived ingredients randomly purchased were tested using this assay, and the results showed that the identification results based on the two SNP loci were utterly consistent with the actual sources. In addition, this method was found to be accurate, simple, convenient, and with high specificity, thus providing an essential technical reference for deer product species identification. It is also an important supplement to the identification methods of the original ingredients of existing deer products.

Sweet Basil between the Soul and the Table-Transformation of Traditional Knowledge on Ocimum basilicum L. in Bulgaria.

The study tracks the utilization of Ocimum basilicum L. (sweet basil)-a garden plant popular for its ritual and ornamental value in the past, that is currently applied in various forms and ways as medicine, food, insect repellent, etc.-in Bulgaria. Previous data for Bulgarian rural home gardens showed a significant number of preserved local landraces; however, it remained unclear how people perceive the large varietal diversity of this species and how the traditions related to its use are preserved. We combined a literature review on the cultural value of sweet basil and the breeding of local genetic resources with an online questionnaire, directed to adult laypeople, that sought to access different aspects of past (recalled) and present use and related knowledge. The identification skills of the participants were tested using images of local plant landraces and foreign varieties. Responses from 220 participants showed that potted "Genovese"-type individual was most frequently identified as sweet basil (89.9%), followed by two examples of local landraces in flower. Participants who grow sweet basil or used it in more varied ways had significantly better identification skills. Ocimum basilicum was most frequently reported as food, while ritual/symbolic use was preserved while devalued during the Communism regime (1945-1989). Food and religious uses were negatively associated in the past, but presently, the tendency is completely reversed. Preferences for the informal exchange of seeds and seed-saving practices were discussed.

Characterization and Comparative Analysis of Chloroplast Genomes of Medicinal Herb Scrophularia ningpoensis and Its Common Adulterants (Scrophulariaceae).

Scrophularia ningpoensis, a perennial medicinal plant from the Scrophulariaceae family, is the original species of Scrophulariae Radix (SR) in the Chinese Pharmacopoeia. This medicine is usually deliberately substituted or accidentally contaminated with other closely related species including S. kakudensis, S. buergeriana, and S. yoshimurae. Given the ambiguous identification of germplasm and complex evolutionary relationships within the genus, the complete chloroplast genomes of the four mentioned Scrophularia species were sequenced and characterized. Comparative genomic studies revealed a high degree of conservation in genomic structure, gene arrangement, and content within the species, with the entire chloroplast genome spanning 153,016-153,631 bp in full length, encoding 132 genes, including 80 protein-coding genes, 4 rRNA genes, 30 tRNA genes, and 18 duplicated genes. We identified 8 highly variable plastid regions and 39-44 SSRs as potential molecular markers for further species identification in the genus. The consistent and robust phylogenetic relationships of S. ningpoensis and its common adulterants were firstly established using a total of 28 plastid genomes from the Scrophulariaceae family. In the monophyletic group, S. kakudensis was determined to be the earliest diverging species, succeeded by S. ningpoensis. Meanwhile, S. yoshimurae and S. buergeriana were clustered together as sister clades. Our research manifestly illustrates the efficacy of plastid genomes in identifying S. ningpoensis and its counterfeits and will also contribute to a deeper understanding of the evolutionary processes within Scrophularia.

Genome skimming as an efficient tool for authenticating commercial products of the pharmaceutically important Paris yunnanensis (Melanthiaceae).

BACKGROUND: Paris yunnanensis (Melanthiaceae) is a traditional Chinese medicinal plant of significant pharmaceutical importance. Due to previous taxonomic confusion, a congeneric species, Paris liiana, has been mistaken for P. yunnanensis and cultivated on a large scale, leading to the mixing of commercial products (i.e., seedlings and processed rhizomes) of P. yunnanensis with those of P. liiana. This may have adverse effects on quality control in the standardization of P. yunnanensis productions. As the lack of PCR amplifiable genomic DNA within processed rhizomes is an intractable obstacle to the authentication of P. yunnanensis products using PCR-based diagnostic tools, this study aimed to develop a PCR-free method to authenticate commercial P. yunnanensis products, by applying genome skimming to generate complete plastomes and nrDNA arrays for use as the molecular tags. RESULTS: Based on a dense intraspecies sampling of P. liiana and P. yunnanensis, the robustness of the proposed authentication systems was evaluated by phylogenetic inferences and experimental authentication of commercial seedling and processed rhizome samples. The results indicate that the genetic criteria of both complete plastomes and nrDNA arrays were consistent with the species boundaries to achieve accurate discrimination of P. yunnanensis and P. liinna. Owing to its desirable accuracy and sensitivity, genome skimming can serve as an effective and sensitive tool for monitoring and controlling the trade of P. yunnanensis products. CONCLUSION: This study provides a new way to solve the long-standing problem of the molecular authentication of processed plant products due to the lack of PCR amplifiable genomic DNA. The proposed authentication system will support quality control in the standardization of P. yunnanensis products in cultivation and drug production. This study also provides molecular evidence to clarify the long-standing taxonomic confusion regarding the species delimitation of P. yunnanensis, which will contribute to the rational exploration and conservation of the species.

[Species identification of Ligustrum lucidum].

Ligustrum lucidum is a woody perennial plant of genus Ligustrum in family Oleaceae. Its dried fruit has high medicinal value. In this study, the authors evaluated the variability and species identification efficiency of three specific DAN barcodes(rbcL-accD, ycf1a, ycf1b) and four general DAN barcodes(matK, rbcL, trnH-psbA, ITS2) for a rapid and accurate molecular identification of Ligustrum species. The results revealed that matK, rbcL, trnH-psbA, ITS2 and ycf1a were inefficient for identifying the Ligustrum species, and a large number of insertions and deletions were observed in rbcL-accD sequence, which was thus unsuitable for development as specific barcode. The ycf1b-2 barcode had DNA barcoding gap and high success rate of PCR amplification and DNA sequencing, which was the most suitable DNA barcode for L. lucidum identification and achieved an accurate result. In addition, to optimize the DNA extraction experiment, the authors extracted and analyzed the DNA of the exocarp, mesocarp, endocarp and seed of L. lucidum fruit. It was found that seed was the most effective part for DNA extraction, where DNAs of high concentration and quality were obtained, meeting the needs of species identification. In this study, the experimental method for DNA extraction of L. lucidum was optimized, and the seed was determined as the optimal part for DNA extraction and ycf1b-2 was the specific DNA barcode for L. lucidum identification. This study laid a foundation for the market regulation of L. lucidum.

A useful species identification and quality control using volatile patterns of ssajuari-ssuk and sajabal-ssuk (Artemisia princeps Pamp. cv. ssajuari and Artemisia princeps Pamp. cv. sajabal; Korean mugwort) according to air-drying term by fast gas chromatography with uncoated surface acoustic wave sensor.

INTRODUCTION: Ssajuari-ssuk and sajabal-ssuk have many clinical benefits. It is difficult to discriminate between these two species based on general characteristics aside from the shapes of the leaves. Thus, species identification and quality control between ssajuari-ssuk and sajabal-ssuk are of great concern in plant science and clinical therapy. OBJECTIVE: The aim of this study is to determine whether fast gas chromatography with uncoated surface acoustic wave sensor (GC-SAW) can be a useful technique for performing species identification and quality control using volatile patterns of ssajuari-ssuk and sajabal-ssuk air-dried for 4 months and 2 years and 4 months. METHODOLOGY: Fast GC-SAW sensor provides second unit analysis, simple, on-line measurements that do not require pretreatment of the sample and rapid sensory information. Headspace solid-phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS) was employed to confirm the identification of the volatiles and compared to fast GC-SAW sensor. RESULTS: In air-dried sajabal-ssuk, the concentration of 1,8-cineole was higher than that in air-dried ssajuari-ssuk, while the level of α-thujone was considerably lower than that of air-dried ssajuari-ssuk. Each of ssajuari-ssuk and sajabal-ssuk air-dried for 4 months and 2 years and 4 months has its own characteristic volatile pattern owing to its individual chemotypes or chemical compositions. CONCLUSION: Consequently, the fast GC-SAW sensor can be a useful technique for species identification and quality control using volatile patterns of ssajuari-ssuk and sajabal-ssuk air-dried for 4 months and 2 years and 4 months. This method can be used for the standardisation of quality control using volatile patterns of herbal medicines.

Rapid identification of chemical compositions from three species of Siegesbeckiae Herba by ultra-performance liquid chromatography-electrospray ionization-quadrupole time of flight-mass spectrometry in combination with deoxyribonucleic acid barcoding.

Siegesbeckiae Herba, a traditional Chinese medicine, originates from Siegesbeckia orientalis, S. glabrescens, and S. pubescens in the Pharmacopoeia of the People's Republic of China. However, accurate identification of decoction pieces from the three plants remains a challenge. In this study, 26 batches of Siegesbeckiae Herba were identified by deoxyribonucleic acid barcoding, and their chemical compositions were determined using ultra-performance liquid chromatography-electrospray ionization-quadrupole time of flight-mass spectrometry. The results showed that the internal transcribed spacer 2 and internal transcribed spacer 1-5.8 S- internal transcribed spacer 2 sequences could distinguish three species. In total, 48 compounds were identified including 12 marker compounds screened for three species using the partial least square discriminant analysis. Among these, two diterpenoids 16-O-malonylkirenol and 15-O-malonylkirenol, and a novel diterpenoid 15,16-di-O-malonylkirenol were isolated and identified. A convenient method for the identification of Siegesbeckiae Herba was established using kirenol and 16-O-acetlydarutoside as control standards by thin-layer chromatography. Unexpectedly, none of the batches of S. orientalis contained kirenol, which did not meet the quality standards of Siegesbeckiae Herba, suggesting that the rationality of kirenol as a quality marker for S. orientalis should be further investigated. The results of this study will contribute to the quality control of Siegesbeckiae Herba.

Comparison of Semen Aesculi species from different geographical origins and horse chestnut based on metabolomics and chemometrics analysis.

BACKGROUND: Semen Aesculi, a traditional Chinese herbal medicine, has a long history of use for treating chest and abdominal pain with distension. In addition, the horse chestnut (Aesculus hippocastanum L.) is another species of Aesculus in Europe and has notable clinical significance in alleviating chronic venous insufficiency, hemorrhoids, and postoperative edema. Thus, highlighting the comparative study of Semen Aesculi and horse chestnut may broaden clinical applications. OBJECTIVES: To conduct a comprehensive comparative analysis on the chemical profiling of these two varieties and determine whether they have equivalent clinical efficacy by integrating plant metabolomics and multivariate statistical methods. METHODS: Initially, a comprehensive characterisation was performed using ultra-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (UPLC-QTOF-MS/MS) platform, and in total 44 active ingredients were identified. Then, untargeted metabolomics combined with principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) was applied for the discrimination of a German species and three official Chinese species. Next, 24 marker compounds responsible for the discrimination of different species were screened out and used to predict the species of unknown samples by genetic algorithm-optimised support vector machine (GA-SVM) with a high prediction accuracy. Finally, a heatmap visualisation was employed for clarifying the distribution of the identified active ingredients. RESULTS: The three species of Chinese Semen Aesculi showed distinct separation from each other, while European horse chestnut and Aesculus chinensis Bunge were similar in chemical composition. CONCLUSIONS: This work provided experimental evidence for further expanding the clinical application of Chinese Semen Aesculi and promoted the species identification and quality control of Semen Aesculi.

Establishing DNA markers to differentiate Agastache rugosa and Pogostemon cablin, which are confusedly used as medicinal herbs, using real-time PCR.

Agastache rugosa and Pogostemon cablin are used as medicinal herbs and aromatic plants and belong to the family Lamiaceae. Despite differences in composition and physicochemical properties, both plants are frequently sold as the medical substance "Kwakhyang" in some Asian countries. Molecular markers were established to distinguish between the two plants using quantitative real-time PCR. Species-specific primers were designed in the nuclear internal transcribed spacer region of ribosomal DNA and in the chloroplast genes matK, rbcL, and rpoB. Six primer sets were tested, the correlation coefficient was higher than 0.99, and the slope was approximately - 3.36 to - 3.58. Efficiency ranged from 90.13 to 98.52%. The developed real-time PCR assay was validated with 14 off-target species, and its reliability was verified through blind testing of 14 commercial products. The assay developed here may help protect consumer rights, and the designed primers can be used to distinguish between the target species. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-022-01176-y.

Development of real-time PCR based molecular markers for two medicinal herb Artemisia species A. capillaris and A. iwayomogi.

Artemisia capillaris and Artemisia iwayomogi are well-known herbal medicines which are used as hepatotherapeutic drugs. These two herbal species can be confused with each other, owing to their morphological similarity and similar Korean common names of "Injinho" and "Haninjin," respectively. Molecular markers to distinguish between the two plants were developed. Six primer sets were designed and verified, and their efficiencies were found to range from 90.28 to 98.29%. The developed primer sets had significant correlation coefficient values between the cycle threshold values and the logarithm of DNA concentration for their target species (R2 > 0.98), with slopes ranged from - 3.3637 to - 3.5793. The specificity of the quantitative polymerase chain reaction (qPCR) was confirmed with 14 other species. Additionally, 16 commercial medicinal herbs and 40 blind samples were tested to evaluate their reliability. Collectively, the findings indicate that developed qPCR-based target-specific primer sets have potential applicability toward protection of consumers' rights. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-022-01166-0.

Identification of Apiaceae using ITS, ITS2 and psba-trnH barcodes.

Apiaceae plants are used as medicinal herbs, pesticides, spices, and vegetables; thus, accurately identifying Apiaceae species is important. The grassland ecosystem of Heilongjiang Province in northern China has huge reserves of wild Apiaceae plants, but few reports have systematically documented their diversity. In this study, 275 Apiaceae plants of 23 species in 18 genera were collected from this area. We identified Apiaceae species by using nuclear internal transcribed spacer (ITS/ITS2) and psbA-trnH (chloroplast non-coding region) sequences based on experimental data. The identification efficiency of ITS, ITS2 and psbA-trnH sequences was determined and evaluated by sequence alignment and analysis, intraspecific and interspecific genetic distance analyses, and phylogenetic tree construction. ITS, ITS2 could distinguish 21 species from 17 genera of Apiaceae with good identification effect. When identifying species in the Apiaceae family, ITS2 can be used as the core barcode and psbA-trnH can be used as the supplementary barcode. These results can enrich the reference Apiaceae DNA barcode database.

Comparative analysis of medicinal plant Isodon rubescens and its common adulterants based on chloroplast genome sequencing.

Isodon rubescens (Hemsley) H. Hara is the source of Donglingcao under the monograph Rabdosiae Rubescentis Herba in Chinese Pharmacopoeia. In the local marketplace, this medicine can be accidentally contaminated, deliberately substituted, or mixed with other related species. The contaminants of herbal products are a threat to consumer safety. Due to the scarcity of genetic information on Isodon plants, more molecular markers are needed to avoid misidentification. In the present study, the complete chloroplast (cp) genome of seven species of Isodon was sequenced, de novo assembled and characterized. The cp genomes of these species universally exhibited a conserved quadripartite structure, i.e., two inverted repeats (IRs) containing most of the ribosomal RNA genes and two unique regions (large single copy and small single copy). Moreover, the genome structure, codon usage, and repeat sequences were highly conserved and showed similarities among the seven species. Five highly variable regions (trnS-GCU-trnT-CGU, atpH-atpI, trnE-UUC-trnT-GGU, ndhC-trnM-CAU, and rps15-ycf1) might be potential molecular markers for identifying I. rubescens and its contaminants. These findings provide valuable information for further species identification, evolution, and phylogenetic research of Isodon.

Comparative and phylogenetic analysis of complete chloroplast genomes from five Artemisia species.

Artemisia Linn. is a large genus within the family Asteraceae that includes several important medicinal plants. Because of their similar morphology and chemical composition, traditional identification methods often fail to distinguish them. Therefore, developing an effective identification method for Artemisia species is an urgent requirement. In this study, we analyzed 15 chloroplast (cp) genomes, including 12 newly sequenced genomes, from 5 Artemisia species. The cp genomes from the five Artemisia species had a typical quadripartite structure and were highly conserved across species. They had varying lengths of 151,132-151,178 bp, and their gene content and codon preferences were similar. Mutation hotspot analysis identified four highly variable regions, which can potentially be used as molecular markers to identify Artemisia species. Phylogenetic analysis showed that the five Artemisia species investigated in this study were sister branches to each other, and individuals of each species formed a monophyletic clade. This study shows that the cp genome can provide distinguishing features to help identify closely related Artemisia species and has the potential to serve as a universal super barcode for plant identification.