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.

DNA, Plant ; chemistry ; DNA, Ribosomal ; chemistry ; DNA, Ribosomal Spacer ; chemistry ; Gentiana ; genetics ; Phylogeny

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

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.

DNA Barcoding, Taxonomic ; methods ; DNA, Ribosomal Spacer ; genetics ; Plants, Medicinal ; classification ; genetics ; Sequence Analysis, DNA ; methods

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

OBJECTIVETo study the genetic diversity of rDNA ITS sequences in different species of Bai Shouwu, utilize the molecular diversity of ITS sequences to authenticate the different species of Bai Shouwu.

METHODFirstly, total DNA was extracted from the different species of Bai Shouwu. Secondly, the ITS sequence was amplified by PCR with universal primer of ITS and sequenced after cloning and purification.

RESULTFrom four species the complete sequence of ITS and 5.8 S rDNA, the partial sequences of 18S rDNA and 26S rDNA were obtained. The rDNA ITS sequences of Cynanchum bungei (sign in No. GU198970 and No. GU479037) were obtained. Ten variable sites among the sequences were found.

CONCLUSIONITS sequence could be used to authenticate the species. The method could be used to identify germplasm resources and authenticate.

Cynanchum ; classification ; genetics ; DNA, Plant ; genetics ; DNA, Ribosomal Spacer ; genetics ; Molecular Sequence Data

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.

DNA Barcoding, Taxonomic ; methods ; DNA, Plant ; DNA, Ribosomal Spacer ; Plants, Medicinal ; classification ; genetics

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.

DNA Barcoding, Taxonomic ; methods ; DNA, Ribosomal Spacer ; genetics ; Ephedra sinica ; classification ; genetics

In this paper, the chloroplast psbK-psbI intergenic spacers of 18 species of Dendrobium and their adulterants were amplified and sequenced, and then the sequence characteristics were analyzed. The sequence lengths of chloroplast psbK-psbI regions of Dendrobium ranged from 474 to 513 bp and the GC contents were 25.4%-27.6%. The variable sites were 71 while the informative sites were 46. The inter-specific genetic distances calculated by Kimura 2-parameter (K2P) of Dendrobium were 0.006 1-0.058 1, with an average of 0.028 4. The K2P genetic distances between Dendrobium species and Bulbophyllum odoratissimum were 0.093 2-0.120 4. The NJ tree showed that the Dendrobium species can be easily differentiated from each other and 6 samples of the inspected Dendrobium species were identified successfully through sequencing the psbK-psbI intergenic spacer. Therefore, the chloroplast psbK-psbI intergenic spacer can be used as a candidate marker to identify Dendrobium species and its adulterants.
Chloroplasts ; DNA, Chloroplast ; genetics ; DNA, Plant ; genetics ; DNA, Ribosomal Spacer ; genetics ; Dendrobium ; classification ; genetics ; Plants, Medicinal ; classification ; genetics

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 evaluate the ability of several hotspot candidate sequence of DNA barcodes for identifying medicinal plant species in Verbenaceae.

METHODUsing universal primers, three chloroplast sequences, psbA-trnH, rbcL, matK, two nuclear ribosomal DNA ITS2 and ITS were amplified and sequenced. PCR amplification and sequencing efficiency, intra- and inter-specific variation, barcoding gap and identification efficiency (with BLAST 1 and Nearest Distance methods) were used to evaluate these loci.

RESULTThe rate of successful amplification using matK was too low to further analyze, and the rate of successful amplification and sequencing using psbA-trnH, ITS, and ITS2 and rbcL sequence was 83.6%, 83.6%, 96.4%, 98.2%, respectively. The rate of successful identification using psbA-trnH, ITS, and ITS2 was 100% at the species level except that rbcL was 77.8%, 75.9% for 55 samples belonging to 32 species, but ITS2 did better in intra- and inter-specific variation, barcoding gap than the other loci. The rate of successful identification of ITS2 was 89.5%, 87.6% even when joining the date of 165 samples from GenBank.

CONCLUSIONIt proposes that the combination of ITS2 and psbA-trnH senquence is promising for the identification of the species in Verbenaceae.

DNA Barcoding, Taxonomic ; methods ; DNA, Plant ; genetics ; DNA, Ribosomal Spacer ; genetics ; Plants, Medicinal ; classification ; genetics ; Verbenaceae ; classification ; genetics