OBJECTIVETo find the patterns of the rDNA ITS sequence variation in Gentiana, and establish the molecular biological method for the identification of the four kinds wild Gentiana from different regions in Gansu.

METHODThe ITS gene fragments were PCR amplified and sequenced. The rDNA ITS regions were analyzed by means of the software of Clustal X, MEGA3.

RESULTThe Complete ITS sequence of G. macrophylla, G. straminea, G. dahurica and G. officinale was 800 bp. The sequences of ITS1, ITS2 and 5.8S were 290, 340, 170 bp, respectively. Phylogenetic tree based on ITS1 and ITS2 sequences data was constrcuted by Neighbor-joining method.

CONCLUSIONITS sequence could be as the evidence for the molecular authentication of Gentiana.

To identify Herba Dendrobii and its adulterant species on molecular level, the rDNA ITS sequences of 17 species of Herba Dendrobii were studied. Genomic DNA of Dendrobium was extracted using the modified cetyltrimethyl ammonium bromide (CTAB) method. The PCR products of the rDNA ITS sequences of Dendrobium (32 materials) were purified and then sequenced. The characteristic of the sequences and the genetic distance were compared between Bulbophyllum odoratissimum and Dendrobium, Dendrobium interspecies and different populations. Phylogenetic trees were constructed using the UPGMA method by the biology softwares including BioEdit, MEGA4.0 etc. The PCR products were purified and then sequenced. It was built up that the database of rDNA ITS sequences of 17 species of Herba Dendrobii (32 materials). The ITS1 was 228-234 bp, the GC content accounting for 45.7%-53.0%. Its variable sites were 167, accounting for 67.34%. The Parsim-Informative positions were 106, accounting for 42.74%. The ITS2 was 241-247 bp, the GC accounting for 44.8% - 55.7%. The variable sites were 165, accounting for 66.27%. The Parsim-Informative positions were 115, accounting for 46.18%. The genetic distance between B. odoratissimum and Dendrobium was 0.295. The average genetic distance was 0.142 between Dendrobium species, and there were 2-156 variable nucleotides. The average genetic distance between different populations was 0.002, and there were 2-156 variable nucleotides. The genetic distance between B. odoratissimum and Dendrobium was greater than that of Denrobium interspecies. Meanwhile, the genetic distance between Denrobium species was also greater than that of different populations (varieties). The molecular phylogeny tree was constructed on the database of rDNA ITS the sequences of 17 species of Herba Dendrobii using the biology softwares. Then 10 materials on molecular level were authenticated. It is concluded that using of the whole sequences database of 17 species of Herba Dendrobii and heredity analysis softwares, and measuring the sequences of rDNA ITS of the inspected species, can authenticate the Dendrobium on molecular level, and provide basis for molecular authentication.
China ; DNA, Plant ; genetics ; DNA, Ribosomal ; genetics ; DNA, Ribosomal Spacer ; genetics ; Dendrobium ; classification ; genetics ; Molecular Sequence Data ; Phylogeny

The paper is aimed to identify SNP in Sarcandra glabra and Chloranthus spicatus, and authenticate S. glabra from Ch. spicatus and the mixture by using PCR amplification of specific alleles. SNPs in the ITS sequences of S. glabra and Ch. spicatus were found by ClustulX 2. 1 program and Bioedit software. Primers for authentic S. glabra and Ch. spicatus was designed according to the SNP site, and ITS sequence universal primers plus to the authentic primer to construct a multi-PCR reaction system, and then optimized the PCR reaction system. Five hundred and eighty band special for S. glabra and 470 bp band special for Ch. spicatus were found by using multi-PCR reaction. The multi-PCR reaction system could be applied to identify S. glabra and Ch. spicatus's leaves.
DNA, Plant ; analysis ; genetics ; DNA, Ribosomal ; genetics ; DNA, Ribosomal Spacer ; analysis ; genetics ; Magnoliopsida ; classification ; genetics ; Plant Leaves ; genetics ; Polymerase Chain Reaction ; Polymorphism, Single Nucleotide ; RNA, Ribosomal ; genetics ; RNA, Ribosomal, 18S ; genetics ; RNA, Ribosomal, 5.8S ; genetics ; Species Specificity

OBJECTIVETo identify Menthae Haplocalycis Herba and its closely related species using DNA barcoding technique.

METHODTotal genomic DNA was isolated from Mentha canadensis and its closely related species. Nuclear DNA ITS2 sequences were amplified, and purified PCR products were sequenced. Sequence assembly and consensus sequence generation were performed using the CodonCode Aligner V3.0. The Kimura 2-Parameter (K2P) distances were calculated using software MEGA 5.0. Identification analyses were performed using BLAST1, Nearest Distance and neighbor-joining (NJ) methods.

RESULTThe intra-specific genetic distances of M. canadensis were ranged from 0 to 0.006, which were lower than inter-specific genetic distances between M. canadensis and its closely related species (0.071-0.231). All the three methods showed that ITS2 could discriminate M. canadensis from its closely related species correctly.

CONCLUSIONThe ITS2 region is an efficient barcode for identification of Menthae Haplocalycis Herba, which provides a scientific basis for fast and accurate identification of the herb.

OBJECTIVETo identify Junci Medulla using the ITS2 barcode.

METHODThe ITS2 regions of Juncus effuses and its closely related species were PCR amplified and sequenced. Sequence assembly and consensus sequence generation were performed using the CodonCode Aligner. The Kimura 2-Parameter (K2P) distances were calculated using software MEGA 5.0. Identification analyses were performed using BLAST1, Nearest Distance, PWG Distance and neighbor-joining (NJ) methods.

RESULTThe intra-specific genetic distances of J. effuses were ranged from 0 to 0.005, which were far lower than inter-specific genetic distances between J. effuses and its closely related species (0.215-0.614). All the four methods showed that ITS2 could discriminate J. effuses from its closely related species correctly.

CONCLUSIONThe ITS2 region is an efficient barcode for authentication of Junci Medulla, and our study further confirmed the ability of ITS2 to identify traditional Chinese medicinal materials.

To establish a new method for identifying genus of Lilium by DNA barcoding technology, ITS, ITS2, psbA-trnH, matK and rbcL sequences were analyzed in term of variation of inter- and intra-species, barcoding gap, neighbor-joining tree to distinguish genus of Lilium based on 978 sequences from experimental and GenBank database, and identification efficiency was evaluated by Nearest distance and BLAST1 methods. The results showed that DNA barcoding could identify different species in genus of Lilium. ITS sequence performed higher identification efficiency, and had significant difference between intra- and inter-species. And NJ tree could also divide species into different clades. Results indicate that DNA barcoding can identify genus of Lilium accurately. ITS sequence can be the optimal barcode to identify species of Lilium.
DNA Barcoding, Taxonomic ; DNA, Plant ; genetics ; DNA, Ribosomal Spacer ; genetics ; Lilium ; classification

DNA barcode technology was used to establish a rapid identification method of Chrysanthemum indicum based on ITS2 sequences. The total DNA was extracted from 22 collected samples,and the ITS2 sequence was amplified by PCR and sequenced,and the information of ITS2 sequence was obtained. Another 14 items of the same family or the same genus were downloaded from Gen Bank.We aligned all 36 sequences,calculated the intraspecific and interspecific distances,and constructed Neighbor Joining( NJ) phylogenetic tree,using MEGA 7. 0. The difference of the secondary structure between the ITS2 sequences was compared. The results showed that the genetic distance of Ch. indicum and Ch. morifolium was overlapped,but the maximum intraspecific distance was far less than the minimum interspecific distance between and among Ch. indicum and other species,with an obvious barcoding gap. The NJ tree showed that Ch. indicum and Ch. morifolium shared a clade,and most of Ch. morifolium with some Ch. indicum were shared a subclade,while Inula lineariifolia,Sinosenecio oldhamianus and Senecio scandens belonged to one clade separately. ITS2 secondary structures for I. lineariifolia,S. oldhamianus and S. scandens were significantly different enough to identify completely but Ch. indicum and Ch. morifolium shared two secondary structures of A and B. It was proved that Ch. indicum was one of the evolutionary sources of Ch.morifolium. Therefore ITS2 sequence as DNA barcode can identify Ch. indicum and its adulterants accurately and quickly. The study provides an important basis for Ch. indicum for the identification of germplasm resources and the safety of clinical medication.
Chrysanthemum ; DNA Barcoding, Taxonomic ; DNA, Plant ; DNA, Ribosomal Spacer ; Drugs, Chinese Herbal ; Phylogeny ; Quality Control

By exploring additional phylogenetic information hidden in ITS2 secondary structure,the possibility of identifying Chrysanthemum indicum and its related species with DNA barcode of ITS2 nucleic acid sequence and its structure information were discussed.The genomic DNA was extracted from 12 samples. The ITS2 fragments were amplified by PCR and sequenced bidirectionally to obtain ITS2 sequence information. 28 sequences of related species for Ch. indicum were downloaded from Gen Bank. Until all 40 ITS2 sequences were aligned,ITS2 secondary structure prediction and structure comparison were finished. Then ITS2 secondary structure information was coded. After comparing ITS2 structure information and nucleic acid information,MP phylogenetic trees were built. The results showed that the secondary structures of ITS2 shared the same structure model--a four-fingered hand. They not only have the common characteristics of ITS2 secondary structures in plants,but also have many other conservative sequences,and their overall conservativeness is high. Among all species used in this study,their ITS2 secondary structures had obvious difference. In addition,the number of mutation sites in the joint matrix compared with the nucleic acid sequences increased by nearly 90%,which greatly enriched the number of mutation sites. This method of information analysis distinguished Ch. indicum from its related species. At the same time,the support rate of the branches of evolutionary trees and the identification rate of species were significantly improved. Although there was no distinction between Ch. zawadskii and Ch. morifolium,it effectively distinguished the three species,namely,Ch. hypargyrum,Ch.oreastrum,and Ch. dichrum. Therefore,the authors suggest that the ITS2 sequence combined with its structural data information should be applied to the identification of Ch. indicum and its related species,and be widely applied to DNA barcode research.
Chrysanthemum ; DNA Barcoding, Taxonomic ; DNA, Plant ; DNA, Ribosomal Spacer ; Phylogeny ; Plants

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.
Apiaceae ; classification ; genetics ; DNA Barcoding, Taxonomic ; methods ; DNA, Ribosomal Spacer ; Drug Contamination

OBJECTIVETo identify Ephedrae Herba using the ITS2 barcode and to secure its quality and safety in medication.

METHODTotal genomic DNA was isolated from Ephedrae Herba and its closely related species. Nuclear DNA ITS2 sequences were amplified, and purified PCR products were sequenced. Sequence assembly and consensus sequence generation were performed using the CodonCode Aligner. The Kimura 2-Parameter (K2P) distances were calculated using software MEGA 5.0. Identification analyses were performed using BLAST1, Nearest Distance and neighbor-joining (NJ) methods.

RESULTThe intra-specific genetic distances of Ephedrae Herba were ranged from 0 to 0.002. The inter-specific genetic distances between Ephedrae Herba and its closely related species were ranged from 0.004 to 0.034. All the three methods showed that ITS2 could discriminate Ephedrae Herba from its closely related species correctly.

CONCLUSIONThe ITS2 region is suitable to be used for authentication of Ephedrae Herba, and our study further confirmed the effectiveness of ITS2 to identify traditional Chinese medicinal materials.