Analysis of Whole-Genome as a Novel Strategy for Animal Species Identification.

Survival crises stalk many animals, especially endangered and rare animals. Accurate species identification plays a pivotal role in animal resource conservation. In this study, we developed an animal species identification method called Analysis of whole-GEnome (AGE), which identifies species by finding species-specific sequences through bioinformatics analysis of the whole genome and subsequently recognizing these sequences using experimental technologies. To clearly demonstrate the AGE method, Cervus nippon, a well-known endangered species, and a closely related species, Cervus elaphus, were set as model species, without and with published genomes, respectively. By analyzing the whole genomes of C. nippon and C. elaphus, which were obtained through next-generation sequencing and online databases, we built specific sequence databases containing 7,670,140 and 570,981 sequences, respectively. Then, the species specificities of the sequences were confirmed experimentally using Sanger sequencing and the CRISPR-Cas12a system. Moreover, for 11 fresh animal samples and 35 commercially available products, our results were in complete agreement with those of other authoritative identification methods, demonstrating AGE's precision and potential application. Notably, AGE found a mixture in the 35 commercially available products and successfully identified it. This study broadens the horizons of species identification using the whole genome and sheds light on the potential of AGE for conserving animal resources.

The genome of Corydalis reveals the evolution of benzylisoquinoline alkaloid biosynthesis in Ranunculales.

Species belonging to the order Ranunculales have attracted much attention because of their phylogenetic position as a sister group to all other eudicot lineages and their ability to produce unique yet diverse benzylisoquinoline alkaloids (BIAs). The Papaveraceae family in Ranunculales is often used as a model system for studying BIA biosynthesis. Here, we report the chromosome-level genome assembly of Corydalis tomentella, a species of Fumarioideae, one of the two subfamilies of Papaveraceae. Based on comparisons of sequenced Ranunculalean species, we present clear evidence of a shared whole-genome duplication (WGD) event that has occurred before the divergence of Ranunculales but after its divergence from other eudicot lineages. The C. tomentella genome enabled us to integrate isotopic labeling and comparative genomics to reconstruct the BIA biosynthetic pathway for both sanguinarine biosynthesis shared by papaveraceous species and the cavidine biosynthesis that is specific to Corydalis. Also, our comparative analysis revealed that gene duplications, especially tandem gene duplications, underlie the diversification of BIA biosynthetic pathways in Ranunculales. In particular, tandemly duplicated berberine bridge enzyme-like genes appear to be involved in cavidine biosynthesis. In conclusion, our study of the C. tomentella genome provides important insights into the occurrence of WGDs during the early evolution of eudicots, as well as into the evolution of BIA biosynthesis in Ranunculales.

Progress of Nanomaterials-Based Photothermal Therapy for Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) is one of the top 15 most prevalent cancers worldwide. However, the current treatment models for OSCC (e.g., surgery, chemotherapy, radiotherapy, and combination therapy) present several limitations: damage to adjacent healthy tissue, possible recurrence, low efficiency, and severe side effects. In this context, nanomaterial-based photothermal therapy (PTT) has attracted extensive research attention. This paper reviews the latest progress in the application of biological nanomaterials for PTT in OSCC. We divide photothermal nanomaterials into four categories (noble metal nanomaterials, carbon-based nanomaterials, metal compounds, and organic nanomaterials) and introduce each category in detail. We also mention in detail the drug delivery systems for PTT of OSCC and briefly summarize the applications of hydrogels, liposomes, and micelles. Finally, we note the challenges faced by the clinical application of PTT nanomaterials and the possibility of further improvement, providing direction for the future research of PTT in OSCC treatment.

Tandem gene duplications drive divergent evolution of caffeine and crocin biosynthetic pathways in plants.

BACKGROUND: Plants have evolved a panoply of specialized metabolites that increase their environmental fitness. Two examples are caffeine, a purine psychotropic alkaloid, and crocins, a group of glycosylated apocarotenoid pigments. Both classes of compounds are found in a handful of distantly related plant genera (Coffea, Camellia, Paullinia, and Ilex for caffeine; Crocus, Buddleja, and Gardenia for crocins) wherein they presumably evolved through convergent evolution. The closely related Coffea and Gardenia genera belong to the Rubiaceae family and synthesize, respectively, caffeine and crocins in their fruits. RESULTS: Here, we report a chromosomal-level genome assembly of Gardenia jasminoides, a crocin-producing species, obtained using Oxford Nanopore sequencing and Hi-C technology. Through genomic and functional assays, we completely deciphered for the first time in any plant the dedicated pathway of crocin biosynthesis. Through comparative analyses with Coffea canephora and other eudicot genomes, we show that Coffea caffeine synthases and the first dedicated gene in the Gardenia crocin pathway, GjCCD4a, evolved through recent tandem gene duplications in the two different genera, respectively. In contrast, genes encoding later steps of the Gardenia crocin pathway, ALDH and UGT, evolved through more ancient gene duplications and were presumably recruited into the crocin biosynthetic pathway only after the evolution of the GjCCD4a gene. CONCLUSIONS: This study shows duplication-based divergent evolution within the coffee family (Rubiaceae) of two characteristic secondary metabolic pathways, caffeine and crocin biosynthesis, from a common ancestor that possessed neither complete pathway. These findings provide significant insights on the role of tandem duplications in the evolution of plant specialized metabolism.

[Research progress in original species identification in industry chain of Rhei Radix et Rhizoma].

Rhei Radix et Rhizoma is a kind of commonly used Chinese medicinal materials. Due to the overharvesting, the wild resource is endangering. Large market demand caused severely adulterant of commercial Rhei Radix et Rhizoma medicinal materials and decoction pieces. This manuscript reviewed the advances of the original species authentication in the industrial chain of Rhei Radix et Rhizoma during the latest decade, including characteristics and microscopic features, phytochemical analysis on anthraquinones, and molecular authentication based on DNA barcoding. Accordingly, an original species authentication route for the industrial chain of Rhei Radix et Rhizoma was summarized:(1)the identification of seeds and seedlings by DNA barcoding;(2) the selection of high variable sites based on the chloroplast genome;(3)biomonitoring of the Rhei Radix et Rhizoma medicinal materials and decoction pieces by two-dimensional DNA barcode;(4)traceability of Chinese patent medicines by third-generation sequencing. In conclusion, the combination of molecular identification and traditional identification methods provides a new idea for the identification of the original species of Rhei Radix et Rhizoma in the industrial chain and a essential guidance for the research of drug safety and efficacy of Rhei Radix et Rhizoma.

Idelalisib inhibits vitreous-induced Akt activation and proliferation of retinal pigment epithelial cells from epiretinal membranes.

Proliferative vitreoretinopathy (PVR) is a blinding fibrotic eye disease that develops in 8-10% of patients who undergo primary retinal detachment-reparative surgery and in 40-60% of patients with open-globe injury. At present, there is no pharmacological treatment for this devastating disease. Vitreal growth factors activate their respective receptors of cells in the vitreous, trigger their downstream signaling transduction (e.g. phosphoinositide 3 kinases (PI3Ks)/Akt), and drive cellular responses intrinsic to the pathogenesis of PVR. PI3Ks play a central role in experimental PVR. However, which isoform(s) are involved in PVR pathogenesis remain unknown. Herein, we show that p110δ, a catalytic subunit of receptor-regulated PI3K isoform δ, is highly expressed in epiretinal membranes from patients with PVR, and that idelalisib, a specific inhibitor of PI3Kδ, effectively inhibits vitreous-induced Akt activation, proliferation, migration and contraction of retinal pigment epithelial cells derived from an epiretinal membrane of a PVR patient. Small molecules of kinase inhibitors have shown great promise as a class of therapeutics for a variety of human diseases. The data herein suggest that idelalisib is a promising PVR prophylactic.

[Identification of antler powder components based on DNA barcoding technology].

In order to authenticate the components of antler powder in the market, DNA barcoding technology coupled with cloning method were used. Cytochrome c oxidase subunit I (COI) sequences were obtained according to the DNA barcoding standard operation procedure (SOP). For antler powder with possible mixed components, the cloning method was used to get each COI sequence. 65 COI sequences were successfully obtained from commercial antler powders via sequencing PCR products. The results indicates that only 38% of these samples were derived from Cervus nippon Temminck or Cervus elaphus Linnaeus which is recorded in the 2010 edition of "Chinese Pharmacopoeia", while 62% of them were derived from other species. Rangifer tarandus Linnaeus was the most frequent species among the adulterants. Further analysis showed that some samples collected from different regions, companies and prices, contained adulterants. Analysis of 36 COI sequences obtained by the cloning method showed that C. elaphus and C. nippon were main components. In addition, some samples were marked clearly as antler powder on the label, however, C. elaphus or R. tarandus were their main components. In summary, DNA barcoding can accurately and efficiently distinguish the exact content in the commercial antler powder, which provides a new technique to ensure clinical safety and improve quality control of Chinese traditional medicine

[Identification of peucedani radix, peucedani decursivi radix and its adulterants using ITS2 sequence].

In order to identify Peucedani Radix, Peucedani Decursivi Radix and their adulterants, the internal transcribed spacer 2 (ITS2) regions of Peucedani Radix, Peucedani Decursivi Radix and their adulterants were amplified and bidirectionally sequenced based on the Principles for Molecular Identification of Traditional Chinese Materia Medica Using DNA Barcoding, which has been promulgated by Chinese Pharmacopoeia Commission. Sequences were analyzed and assembled by Codon Code Aligner V3. 7.1. The relevant data were analyzed by MEGA 5. 0. Species identification analyses were performed by using the nearest distance methods and neighbor-joining (NJ) methods. The result showed that the ITS2 sequence lengths of Peucedani Radix were 229-230 bp and the average intra-specific genetic distances were 0.005. The ITS2 sequence lengths of Peucedani Decursivi Radix were 227 bp and the sequences contained no variation site. The average inter-specific K2P genetic distance of Peucedani Radix, Peucedani Decursivi Radix and their adulterants species were 0.044 and 0.065 respectively. The minimum inter-specific divergence is larger than the maximum intra-specific divergence of Peucedani Decursivi Radix. The nearest distance methods and NJ trees results indicated that Peucedani Radix, Peucedani Decursivi Radix and their adulterants species could be identification clearly. The ITS2 regions can stably and accurately distinguish Peucedani Radix, Peucedani Decursivi Radix and their adulterants.

[Identification of Placenta hominis and its adulterants using COI barcode].

In order to provide a new method for the identification of Placenta hominis, the COI barcode has been employed to identify the P. hominis medicinal materials and its adulterants. Genomic DNA was extracted from the experimental samples. The COI sequences were amplified and sequenced bi-directionally. Sequence assembly and consensus sequence generation were performed using the CodonCode Aligner. NJ tree was constructed by MEGA6.0 software. COI sequences can be successfully obtained from all experimental samples. The intra-specific variation and inter-specific divergence were calculated. The average intra-specific K2P distance of P. hominis was 0.001 and the maximum intra-specific distance was 0.008. The cluster dendrogram constructed can be seen that the same genus is together, and distinguished from its adulterants. It is concluded that P. hominis and its adulterants can be correctly identified by DNA barcoding method.

Identification of closely related species in Aspergillus through Analysis of Whole-Genome.

The challenge of discriminating closely related species persists, notably within clinical diagnostic laboratories for invasive aspergillosis (IA)-related species and food contamination microorganisms with toxin-producing potential. We employed Analysis of the whole-GEnome (AGE) to address the challenges of closely related species within the genus Aspergillus and developed a rapid detection method. First, reliable whole genome data for 77 Aspergillus species were downloaded from the database, and through bioinformatic analysis, specific targets for each species were identified. Subsequently, sequencing was employed to validate these specific targets. Additionally, we developed an on-site detection method targeting a specific target using a genome editing system. Our results indicate that AGE has successfully achieved reliable identification of all IA-related species (Aspergillus fumigatus, Aspergillus niger, Aspergillus nidulans, Aspergillus flavus, and Aspergillus terreus) and three well-known species (A. flavus, Aspergillus parasiticus, and Aspergillus oryzae) within the Aspergillus section. Flavi and AGE have provided species-level-specific targets for 77 species within the genus Aspergillus. Based on these reference targets, the sequencing results targeting specific targets substantiate the efficacy of distinguishing the focal species from its closely related species. Notably, the amalgamation of room-temperature amplification and genome editing techniques demonstrates the capacity for rapid and accurate identification of genomic DNA samples at a concentration as low as 0.1 ng/µl within a concise 30-min timeframe. Importantly, this methodology circumvents the reliance on large specialized instrumentation by presenting a singular tube operational modality and allowing for visualized result assessment. These advancements aptly meet the exigencies of on-site detection requirements for the specified species, facilitating prompt diagnosis and food quality monitoring. Moreover, as an identification method based on species-specific genomic sequences, AGE shows promising potential as an effective tool for epidemiological research and species classification.

Analysis of Whole-Genome facilitates rapid and precise identification of fungal species.

Fungal identification is a cornerstone of fungal research, yet traditional molecular methods struggle with rapid and accurate onsite identification, especially for closely related species. To tackle this challenge, we introduce a universal identification method called Analysis of whole GEnome (AGE). AGE includes two key steps: bioinformatics analysis and experimental practice. Bioinformatics analysis screens candidate target sequences named Targets within the genome of the fungal species and determines specific Targets by comparing them with the genomes of other species. Then, experimental practice using sequencing or non-sequencing technologies would confirm the results of bioinformatics analysis. Accordingly, AGE obtained more than 1,000,000 qualified Targets for each of the 13 fungal species within the phyla Ascomycota and Basidiomycota. Next, the sequencing and genome editing system validated the ultra-specific performance of the specific Targets; especially noteworthy is the first-time demonstration of the identification potential of sequences from unannotated genomic regions. Furthermore, by combining rapid isothermal amplification and phosphorothioate-modified primers with the option of an instrument-free visual fluorescence method, AGE can achieve qualitative species identification within 30 min using a single-tube test. More importantly, AGE holds significant potential for identifying closely related species and differentiating traditional Chinese medicines from their adulterants, especially in the precise detection of contaminants. In summary, AGE opens the door for the development of whole-genome-based fungal species identification while also providing guidance for its application in plant and animal kingdoms.

Lysine demethylase 3A promotes chondrogenic differentiation of aged human dental pulp stem cells.

Background/purpose: Aging severely impairs the beneficial effects of human dental pulp stem cells (hDPSCs) on cartilage regeneration. Lysine demethylase 3A (KDM3A) is involved in regulating mesenchymal stem cells (MSCs) senescence and bone aging. In this study, we investigated the role of KDM3A in hDPSCs aging and whether KDM3A could rejuvenate aged hDPSCs to enhance their chondrogenic differentiation capacity. Materials and methods: The cellular aging of hDPSCs was evaluated by senescence-associated ß-galactosidase (SA-ß-gal) staining. Protein levels were determined using Western blot analysis. KDM3A was overexpressed in aged hDPSCs by lentivirus infection. Quantitative reverse-transcription polymerase chain reaction (RT-qPCR) were used to determine the mRNA levels of stemness markers. Toluidine blue staining was used to evaluate the effect of KDM3A overexpression on the chondrogenic differentiation of aged hDPSCs. Results: hDPSCs at passage 12 or treated with etoposide exhibited augmented cellular senescence as evidenced by increased SA-ß-gal activity. KDM3A was significantly increased during senescence of hDPSCs. Overexpression of KDM3A did not affect the stemness properties but significantly promoted the chondrogenic differentiation of aged hDPSCs. Conclusion: Our findings indicate that KDM3A plays an important role in the maintenance of the chondrogenic differentiation capacity of aged hDPSCs and suggest that therapies targeting KDM3A may be a novel strategy to rejuvenate aged hDPSCs.

Crocus genome reveals the evolutionary origin of crocin biosynthesis.

Crocus sativus (saffron) is a globally autumn-flowering plant, and its stigmas are the most expensive spice and valuable herb medicine. Crocus specialized metabolites, crocins, are biosynthesized in distant species, Gardenia (eudicot) and Crocus (monocot), and the evolution of crocin biosynthesis remains poorly understood. With the chromosome-level Crocus genome assembly, we revealed that two rounds of lineage-specific whole genome triplication occurred, contributing important roles in the production of carotenoids and apocarotenoids. According to the kingdom-wide identification, phylogenetic analysis, and functional assays of carotenoid cleavage dioxygenases (CCDs), we deduced that the duplication, site positive selection, and neofunctionalization of Crocus-specific CCD2 from CCD1 members are responsible for the crocin biosynthesis. In addition, site mutation of CsCCD2 revealed the key amino acids, including I143, L146, R161, E181, T259, and S292 related to the catalytic activity of zeaxanthin cleavage. Our study provides important insights into the origin and evolution of plant specialized metabolites, which are derived by duplication events of biosynthetic genes.

Lineage-Specific CYP80 Expansion and Benzylisoquinoline Alkaloid Diversity in Early-Diverging Eudicots.

Menispermaceae species, as early-diverging eudicots, can synthesize valuable benzylisoquinoline alkaloids (BIAs) like bisbenzylisoquinoline alkaloids (bisBIAs) and sinomenines with a wide range of structural diversity. However, the evolutionary mechanisms responsible for their chemo-diversity are not well understood. Here, a chromosome-level genome assembly of Menispermum dauricum is presented and demonstrated the occurrence of two whole genome duplication (WGD) events that are shared by Ranunculales and specific to Menispermum, providing a model for understanding chromosomal evolution in early-diverging eudicots. The biosynthetic pathway for diverse BIAs in M. dauricum is reconstructed by analyzing the transcriptome and metabolome. Additionally, five catalytic enzymes - one norcoclaurine synthase (NCS) and four cytochrome P450 monooxygenases (CYP450s) - from M. dauricum are responsible for the formation of the skeleton, hydroxylated modification, and C-O/C-C phenol coupling of BIAs. Notably, a novel leaf-specific MdCYP80G10 enzyme that catalyzes C2'-C4a phenol coupling of (S)-reticuline into sinoacutine, the enantiomer of morphinan compounds, with predictable stereospecificity is discovered. Moreover, it is found that Menispermum-specific CYP80 gene expansion, as well as tissue-specific expression, has driven BIA diversity in Menispermaceae as compared to other Ranunculales species. This study sheds light on WGD occurrences in early-diverging eudicots and the evolution of diverse BIA biosynthesis.

[Principles for molecular identification of traditional Chinese materia medica using DNA barcoding].

Since the research of molecular identification of Chinese Materia Medica (CMM) using DNA barcode is rapidly developing and popularizing, the principle of this method is approved to be listed in the Supplement of the Pharmacopoeia of the People's Republic of China. Based on the study on comprehensive samples, the DNA barcoding systems have been established to identify CMM, i.e. ITS2 as a core barcode and psbA-trnH as a complementary locus for identification of planta medica, and COI as a core barcode and ITS2 as a complementary locus for identification of animal medica. This article introduced the principle of molecular identification of CMM using DNA barcoding and its drafting instructions. Furthermore, its application perspective was discussed.

Application of third-generation sequencing to herbal genomics.

There is a long history of traditional medicine use. However, little genetic information is available for the plants used in traditional medicine, which limits the exploitation of these natural resources. Third-generation sequencing (TGS) techniques have made it possible to gather invaluable genetic information and develop herbal genomics. In this review, we introduce two main TGS techniques, PacBio SMRT technology and Oxford Nanopore technology, and compare the two techniques against Illumina, the predominant next-generation sequencing technique. In addition, we summarize the nuclear and organelle genome assemblies of commonly used medicinal plants, choose several examples from genomics, transcriptomics, and molecular identification studies to dissect the specific processes and summarize the advantages and disadvantages of the two TGS techniques when applied to medicinal organisms. Finally, we describe how we expect that TGS techniques will be widely utilized to assemble telomere-to-telomere (T2T) genomes and in epigenomics research involving medicinal plants.

Lysine 68 Methylation-Dependent SOX9 Stability Control Modulates Chondrogenic Differentiation in Dental Pulp Stem Cells.

Dental pulp stem cells (DPSCs), characterized by easy availability, multi-lineage differentiation ability, and high proliferation ability, are ideal seed cells for cartilage tissue engineering. However, the epigenetic mechanism underlying chondrogenesis in DPSCs remains elusive. Herein, it is demonstrated that KDM3A and G9A, an antagonistic pair of histone-modifying enzymes, bidirectionally regulate the chondrogenic differentiation of DPSCs by controlling SOX9 (sex-determining region Y-type high-mobility group box protein 9) degradation through lysine methylation. Transcriptomics analysis reveals that KDM3A is significantly upregulated during the chondrogenic differentiation of DPSCs. In vitro and in vivo functional analyses further indicate that KDM3A promotes chondrogenesis in DPSCs by boosting the SOX9 protein level, while G9A hinders the chondrogenic differentiation of DPSCs by reducing the SOX9 protein level. Furthermore, mechanistic studies indicate that KDM3A attenuates the ubiquitination of SOX9 by demethylating lysine (K) 68 residue, which in turn enhances SOX9 stability. Reciprocally, G9A facilitates SOX9 degradation by methylating K68 residue to increase the ubiquitination of SOX9. Meanwhile, BIX-01294 as a highly specific G9A inhibitor significantly induces the chondrogenic differentiation of DPSCs. These findings provide a theoretical basis to ameliorate the clinical use of DPSCs in cartilage tissue-engineering therapies.

A comprehensive analysis of the Bencao (herbal) small RNA Atlas reveals novel RNA therapeutics for treating human diseases.

Cross-kingdom herbal miRNA was first reported in 2012. Using a modified herbal extraction protocol, we obtained 73,677,287 sequences by RNA-seq from 245 traditional Chinese Medicine (TCM), of which 20,758,257 were unique sequences. We constructed a Bencao (herbal) small RNA (sRNA) Atlas ( http://bencao.bmicc.cn ), annotated the sequences by sequence-based clustering, and created a nomenclature system for Bencao sRNAs. The profiles of 21,757 miRNAs in the Atlas were highly consistent with those of plant miRNAs in miRBase. Using software tools, our results demonstrated that all human genes might be regulated by sRNAs from the Bencao sRNA Atlas, part of the predicted human target genes were experimentally validated, suggesting that Bencao sRNAs might be one of the main bioactive components of herbal medicines. We established roadmaps for oligonucleotide drugs development and optimization of TCM prescriptions. Moreover, the decoctosome, a lipo-nano particle consisting of 0.5%-2.5% of the decoction, demonstrated potent medical effects. We propose a Bencao (herbal) Index, including small-molecule compounds (SM), protein peptides (P), nucleic acid (N), non-nucleic and non-proteinogenic large-molecule compounds (LM) and elements from Mendeleev's periodic table (E), to quantitatively measure the medical effects of botanic medicine. The Bencao sRNA Atlas is a resource for developing gene-targeting oligonucleotide drugs and optimizing botanical medicine, and may provide potential remedies for the theory and practice of one medicine.

[Identification of gentianae macrophyllae radix using the ITS2 barcodes].

DNA barcoding is a rapidly developing frontier technology in the world and will be useful in promoting the quality control and standardization of traditional Chinese medicine. Until now, many studies concerning DNA barcoding have focused on leaf samples but rarely on Chinese herbal medicine. There are three issues involved in DNA barcoding for traditional Chinese medicinal materials: (1) the extraction methods for total DNA of the rhizomes of the medicinal materials; (2) intra-specific variation among samples from different places of origin; (3) accuracy and stability of this method. In this study, Gentianae Macrophyllae Radix was used to verify the stability and accuracy of DNA barcoding technology. Five regions (ITS2, psbA-trnH, matK, rbcL, and ITS) were tested for their ability to identify 86 samples of Gentianae Macrophyllae Radix and their adulterants. After improving the DNA extraction method, genomic DNA from all samples was successfully obtained. To evaluate each barcode's utility for species authentication, PCR amplification efficiency, genetic divergence, and species authentication were assessed. Among all tested regions only ITS2 locus showed 100% of PCR amplification and identification efficiencies. Based on the established method, we successfully identified two samples of Gentianae Macrophyllae Radix bought in pharmacy to the original species.

[Stability and accuracy of the identification of Notopterygii Rhizoma et Radix using the ITS/ITS2 barcodes].

In this study, Notopterygii Rhizoma et Radix was used to verify the stability and accuracy of DNA barcodes in identification of Chinese materia medica for the first time. All genomic DNAs from thirty one samples were extracted. The ITS (internal transcribed spacer) regions were amplified and sequenced bi-directionally. Obtained sequences were assembled using the CodonCode Aligner. And the sequences of the ITS regions were aligned through Clustal-W and the genetic distances were computed using MEGA 5.0 in accordance with the kimura 2-parameter (K2P) model. The neighbor-joining (NJ) phylogenetic trees were constructed. The ITS2 regions were obtained by using the hidden Markov model (HMM)-based annotation methods from the ITS sequences. Results indicated that the lengths of ITS regions of Notopterygii Rhizoma et Radix were 603-604 bp, while the lengths of ITS2 regions were 228 bp. The haplotypes of ITS/ITS2 regions of Notopterygii Rhizoma et Radix were the same as those of the original plant leaves. The intra-specific genetic distances were smaller than inter-specific ones in ITS/ITS2 regions of Notopterygium incisum and N. franchetii. The NJ trees showed that N. incisum, N. franchetii and its adulterants can be easily differentiated according to their monophyly. Therefore, ITS/ITS2 regions as DNA barcodes can stably and accurately distinguish Notopterygii Rhizoma et Radix from its adulterants and could provide a new technique to ensure clinical safety in utilization of traditional Chinese medicines.