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

This study was directed to determine the nucleotide sequence of the ITS (internal transcribed spacer) gene of Leishmania donovani isolates from desert foci (L. d XJ771), hill foci (L. d SC10) and plain foci (L. d SD2), and to find out the differences of the sequences of ITS gene among these three isolates. The specific ITS fragments from nuclear DNA of three Leishmania isolates were amplified by PCR, cloned into PMD18-T vector, and then sequenced by the dideoxy chain termination method. Sequence analysis showed that the amplified DNA fragments of the three isolates were 1 086 bp (L. d XJ771), 1 027 bp (L. d SC10) and 1 028 bp (L. d SD2). There were obvious sequence differences among L. d XJ771, L. d SC10 and L. d SD2. The differences between L. d XJ771 (desert foci isolate) and L. d SC10 (hill foci isolate) were less than the differences between L. d XJ771 (desert foci isolate ) and L. d SD2. (plain foci isolate).
China ; Cloning, Molecular ; DNA, Protozoan ; genetics ; DNA, Ribosomal Spacer ; genetics ; Environment ; Leishmania donovani ; genetics ; Polymerase Chain Reaction ; Sequence Analysis, DNA

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 study the correlation between ITS sequence of Arctium lappa and Fructus Arctii quality of different origin.

METHODThe samples of Fructu arctii materials were collected from 26 different producing areas. Their ITS sequence were determined after polymerase chain reaction (PCR) and quality were evaluated through the determination of arctiin content by HPLC. Genetic diversity, genotype and correlation were analyzed by ClustalX (1.81), Mage 4.0, SPSS 13.0 statistical software.

RESULTITS sequence of A. was obtained from 26 samples, and was registered in the GenBank. Corresponding arctiin content of Fructus arctii and 1000-grain weight were determined. A. lappa genotype correlated with Fructus arctii quality by statistical analysis.

CONCLUSIONThe research provided a foundation for revealing the molecular mechanism of Fructus arctii geoherbs.

Arctium ; chemistry ; genetics ; Computational Biology ; DNA, Ribosomal Spacer ; genetics ; Drugs, Chinese Herbal ; standards ; Furans ; analysis ; Genotype ; Glucosides ; analysis ; Polymorphism, Genetic ; genetics ; Quality Control ; Sequence Analysis, DNA ; Software

Molecular systematic techniques were applied to reveal the genetic diversity of medicinal plants and their related species in Salvia. The internal transcribed spacer (ITS) as well as 5.8S rDNA sequences of 27 samples of Salvia were amplified using PCR method and sequenced. Mega 3.1 was used to analyze the genetic diversity within genus. The complete sequences of ITS plus 5.8S rDNA are about 612-617 bp. A phylogenetic tree generated by Neighbor-Joining method partly supported the morphological classification within Salvia, but incompatible results were also obtained in the treatment of phylogenetic positions of some species such as Salvia trijuga, Salvia flava var. flava and Salvia flava var. megalentha. The ITS regions of present Salria species showed considerable variation between subgenera in contrast with the conservative 5.8S rDNA sequences. The native Salvia species might have a different origin from the foreign species. The phylogenetic positions of subgenera and sections inferred by ITS analysis were comparable with that of traditional classification, while the phylogeny within sections is still doubtful due to limited information in ITS sequence and need to be further proved by other evidence. ITS analysis in this study supports the rationality of using species from Drymosphace section as substitute drug resources of Dan shen, but also reveals significant genetic differences between high mountain Dan shen species such as Salvia przewalskii with traditional Dan shen origins.
Base Sequence ; DNA, Plant ; genetics ; DNA, Ribosomal ; genetics ; DNA, Ribosomal Spacer ; genetics ; Genetic Variation ; Phylogeny ; Plants, Medicinal ; genetics ; Polymerase Chain Reaction ; methods ; RNA, Ribosomal, 5.8S ; genetics ; Salvia ; classification ; genetics ; Sequence Alignment ; Sequence Analysis, DNA

Freshwater snails of the family Lymnaeidae play an important role in the transmission of fascioliasis worldwide. In Vietnam, 2 common lymnaeid species, Lymnaea swinhoei and Lymnaea viridis, can be recognized on the basis of morphology, and a third species, Lymnaea sp., is known to exist. Recent studies have raised controversy about their role in transmission of Fasciola spp. because of confusion in identification of the snail hosts. The aim of this study is, therefore, to clarify the identities of lymnaeid snails in Vietnam by a combination of morphological and molecular approaches. The molecular analyses using the second internal transcribed spacer (ITS2) of the nuclear ribosomal DNA clearly showed that lymnaeids in Vietnam include 3 species, Austropeplea viridis (morphologically identified as L. viridis), Radix auricularia (morphologically identified as L. swinhoei) and Radix rubiginosa (morphologically identified as Lymnaea sp.). R. rubiginosa is a new record for Vietnam. Among them, only A. viridis was found to be infected with Fasciola spp. These results provide a new insight into lymnaeid snails in Vietnam. Identification of lymnaeid snails in Vietnam and their role in the liver fluke transmission should be further investigated.
Animals ; Cluster Analysis ; DNA, Ribosomal Spacer/chemistry/genetics ; Fasciola/*isolation & purification ; Lymnaea/*anatomy & histology/genetics/*parasitology ; Molecular Sequence Data ; Phylogeny ; Sequence Analysis, DNA ; Sequence Homology, Nucleic Acid ; Vietnam

Phylogenetic relationship between Paecilomyces hepiali and Cordyceps sinensis was studied by analyzing the sequence of rDNA-ITS. The samples of C. sinensis were collected from Hualong County in Qinghai Province and Kangding County in Sichuan Province in May and June, respectively. The rDNA-ITS fragments were obtained by PCR amplification with the template genomic DNA of the fresh stroma or caterpillar body of the collected samples and the cultured mycelium of P. hepiali, with the universal fungal primers ITS1/ITS4. The amplified fragments were cloned into pMD18-T Vector and sequenced. Phylogenetic analysis was performed with these sequences and those from GenBank. The result showed that all of the 46 clones randomly chosen from the amplification of C. sinensis shared identical or almost identical rDNA-ITS regions and had over 99% identity with some rDNA-ITS sequences of Hirsutella sinensis and C. sinensis registered in GenBank, but all of them had only about 72% identity with that of P. hepiali. Two pairs of specific primers were designed based on the rDNA-ITS sequence of P. hepiali, then PCR and Nest-PCR were performed with the template genomic DNA of the stroma or caterpillar body of C. sinensis samples mentioned above. The apparent bands amplified by Nest-PCR were obtained from all of the samples, and the sequences showed 100% identity with the rDNA-ITS sequence of P. hepiali. In addition, another pair of specific primers were designed based on the rDNA-ITS sequence registered in GenBank as the marker of C. sinensis (accession no. AB067740) but the latter only shared 87.3% identity with that of H. sinensis (accession no. AJ309353). This pair of primers was used to amplify the C. sinensis samples by PCR, and the amplified sequence showed 100% identity with that of AB067740. The result indicated that H. sinensis is the main body of C. sinensis, while some other endoparasitic fungi such as P. hepiali commonly exist in the natural C. sinensis.
Base Sequence ; Cordyceps ; classification ; genetics ; DNA, Fungal ; genetics ; DNA, Ribosomal Spacer ; genetics ; Molecular Sequence Data ; Paecilomyces ; classification ; genetics ; Phylogeny ; Polymerase Chain Reaction ; methods ; Sequence Alignment ; Sequence Analysis, DNA

OBJECTIVETo analyze the nuclear ribosome DNA (nrDNA) internal transcribed spacer (ITS) sequences of 4 Leonurus species, and the possibility of using them for molecular authentication of the crude drugs from the genus.

METHODSThe nrDNA ITS sequence (including ITS1, 5.8S rDNA, ITS2, and partial 18S rDNA and 26S rDNA) of L. japonicus and its 3 adulterant species were amplified and sequenced, and CLUSTRAL X and MEGA software was employed for analysis.

RESULTSThe variation of ITS1 and ITS2 between L. japonicus and its adulterant species ranged between 7.2% and 18.8% and between 14.2% and 27%, respectively. The phylogenic tree derived from the dendrograms based on the ITS sequence data contained some discrepancy from the traditional classification.

CONCLUSIONThe nrDNA ITS sequences can be used potentially as efficient markers for identification of L. japonicus and its adulterants, and further study is needed for studying the phylogeny of Leonurus.

DNA, Plant ; chemistry ; genetics ; DNA, Ribosomal Spacer ; chemistry ; genetics ; Leonurus ; classification ; genetics ; Molecular Sequence Data ; Phylogeny ; Sequence Analysis, DNA ; Species Specificity

OBJECTIVEThe study the patterns of rDNA ITS sequence variation of 9 medicinal species of genus Bupleurum in China to find the DNA molecular markers for identification of these crude drugs.

METHODThe ITS regions of these species were amplified by PCR and sequenced, and their sequences were analyzed by DNAssist Version 2.2.

RESULTSHomologous alignment indicated that the consanguinity of the ITS sequences between genus Bupleurum and outgroups was lower than 75%, and that within genus Bupleurum was higher than 88%. For the same species, the consanguinity was higher than 99%.

CONCLUSIONThe ITS sequences may serve as reliable molecular markers for identification of Radix Bupleuri.

Bupleurum ; classification ; genetics ; DNA, Plant ; chemistry ; genetics ; DNA, Ribosomal Spacer ; chemistry ; genetics ; Molecular Sequence Data ; Phylogeny ; Plant Roots ; genetics ; Sequence Analysis, DNA ; Species Specificity

AIMTo study the difference of rDNA ITS sequences between Zanthexylum bungeanum populations and their adulterants in main habitants of China so as to provide molecular markers for identifying Zanthexylum bungeanum populations against adulterants.

METHODSrDNA ITS regions (including ITS-1, 5.8S and ITS-2) of 7 populations of Zanthexylum bungeanum which are separate located in Gansu, Shanxi, Sichuan, Hebei provinces, and 3 adulterants were sequenced by PCR products sequencing method or clone sequencing method.

RESULTSThe sequences of rDNA ITS region of Zanthexylum bungeanum were reported for the first time, and the sequences of ITS region ranged from 619 to 620 bp, and the length difference amoung Zanthexylum bungeanum and their adulterants is 4 bp. There are 15 variable sites, 12 informative sites and 3 authenticable sites among Zanthexylum bungeanum populations. The difference of rDNA ITS regions amoung Zanthexylum bungeanum and their adulterants is obvious, the number of variable sites is 71.

CONCLUSIONThe difference of rDNA ITS sequences can be used to authenticate accurately the populations of Zanthexylum bungeanum and their adulterants. These populations of Z. bungeanum which have close relationship always distribute in near geographic areas. The characteristics of rDNA ITS sequence can be used as good markers for authenticating Zanthexylum bungeanum populations form their adulterants.

Base Sequence ; China ; DNA, Plant ; genetics ; DNA, Ribosomal Spacer ; genetics ; Drug Contamination ; Ecosystem ; Molecular Sequence Data ; Phylogeny ; Plants, Medicinal ; genetics ; Sequence Analysis, DNA ; Species Specificity ; Zanthoxylum ; classification ; genetics