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ObjectiveThe internal transcribed spacer （ITS） 2 region of ribosomal gene， a DNA barcode， was employed to identify 12 medicinal Aconitum species and the genetic relationship among the species was analyzed. MethodA total of 30 samples of the 12 species were collected. The DNA was extracted with spin column plant genomic DNA kit and the universal primers of ITS2 sequence were used for polymerase chain reaction （PCR） amplification， followed by electrophoresis detection and bi-directional sequencing. The yielded sequences were aligned and spliced by CodonCode Aligner 17.0 and sequence variation was analyzed by MEGA 7.0. The secondary structure was predicted by ITS2 Database and the neighbor-joining （NJ） method was applied to generate the phylogenetic tree. ResultThe ITS2 sequences of the 12 species were 220-221 bp， with the average guanine and cytosine （GC） content of 64.09%， 140 variable sites， 137 informative sites， and 81 conservative sites. The intraspecific genetic distance （K2P） was smaller than the interspecific genetic distance. According to the secondary structures of ITS2 sequences and NJ cluster analysis， A. scaposum， A. sinomontanum， and A. barbatum had close genetic relationship， while the rest nine showed close kinship， particularly A. soongaricum and A. yinschanicum. ConclusionITS2 sequence is of great value for the molecular identification and genetic relationship determination of Aconitum， which provides a new method for the study of ethnomedicine.
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ObjectiveTo identify the molecular biology of various species of Tibetan Codonopsis plants based on internal transcribed spacer（ITS）2 and psbA-trnH sequence barcode technology. MethodThe genomic DNA of 28 Tibetan Codonopsis plant samples from four species （Codonopsis canescens，C. foetens subsp. nervosa，C. pilosula， and C. thalictrifolia var. mollis） were extracted，and the ITS2 and psbA-trnH sequences were amplified and sequenced. The related sequences of 81 Tibetan Codonopsis plant samples belonging to 15 species were downloaded from GenBank， and MEGA 6.0 was used for sequence comparison and mutation site analysis. The GC content and genetic distance within and between species were calculated. Additionally， phylogenetic trees were constructed by maximum likelihood （ML） method， neighbor-joining （NJ） method，and unweighted pair-group method with arithmetic means （UPGMA） . ResultAccording to the mutation site，C. canescens， C. pilosula，C. pilosula subsp. tangshen， C. pilosula var. modesta，C. bhutanica，C. clematidea，C. lanceolata，C. subglobosa and C. foetens were distinguished. In the phylogenetic trees，the optimal clustering effects for ITS2 and psbA-trnH sequences were obtained using the ML method and the UPGMA method， respectively， and 12 species were effectively clustered. ConclusionITS2 and psbA-trnH sequences have a high identification rate for species of single origin，but there are still some limitations in identifying variants and original variants. This study provides basis for the identification of affinity relationship and clinical safety of Tibetan Codonopsis plants.
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Objective: To identify Sarcandra glabra and its adulterants using three DNA molecular markers including 18 S rRNA gene, ITS2 sequence and SCAR marker, and then provide the basis for its molecular authentication. Methods: 18 S rRNA gene sequence of S. glabra was obtained by PCR amplification, cloning and sequencing, and then Blast comparison was made in NCBI. The ITS2 sequence of S. glabra was obtained by PCR amplification, sequencing and annotation in ITS2 Database. In the meanwhile, the ITS2 sequences of adulterants and other plants were collected from GenBank. Using MEGA5.5, the genetic distance was calculated between species and then the ITS2 sequences were aligned to construct a phylogenetic clustering tree. SCAR molecular marker of S. glabra was obtained by RAPD. After cloning and sequencing, specific primers were designed to amplify S. glabra and its adulterants. Results: The length of 18 S rRNA obtained in our research was 1 820 bp. Blast comparison revealed that there was 99% homology between S. glabra and Chloranthaceae, which proved to be 18 S rRNA gene of S. glabra. The length of the ITS2 sequence in our research was 500 bp. Genetic distance between S.glabra and its adulterants ranged from 0.190 to 0.219, which was far more than genetic distance among adulterants (0.000-0.074). Cluster analysis showed that S. glabra and its adulterants respectively clustered into a different branch, which was far away from other plants. In our research, we obtained SCAR molecular marker of S. glabra and then a pair of specific primers were designed. Using the pair of specific primers, specific products were amplified from genomic DNA of S. glabra, but no specific products were obtained from that of its adulterants. Conclusion: We could authenticate S. glabra and its adulterants effectively with the combination of three molecular markers for establishing a novel method to identify S. glabra and its adulterants, which provides a new idea for the authentication of S. glabra.
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To effectively identify the Astragalus and its adulterants based on ITS2 sequence and secondary structure, in this study, 32 portions of Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Beg.) Hsiao and Astragalus membranaceus (Fisch.) Bge. collected were conducted ITS2 sequence amplification and bidirectional sequencing, whose results were then spliced by CExpress software remove the 5.8S and 28S sequences at both ends to obtain a complete ITS2 sequence. In addition, 3 ITS2 sequences for each of the adulterants of Astragalus, respectively, Oxytropis coerulea, Caragana sinica, Hedysarum polybotrys, Althaea rosea were downloaded from GenBank. The intra-specific and inter-specific genetic distances were calculated by the software MEGA7 to analyze the difference of each sequence; the Neighbor-joining (NJ) method was used to construct the phylogenetic tree based on ITS2 sequence (primary structure) as well as joint ITS2 sequence and its secondary structure. The results showed that the average ITS2 sequence length of both A. mongolicus and A. membranaceus was 216 bp, and their average GC content was 50.00% and 50.46%, respectively. The similarity of ITS2 sequence length and GC content between the two kind of Astragalus and Oxytropis coerulea was the highest, while the ITS2 sequence length and GC content of Althaea rosea showed great differences with those of Astragalus. The inter-specific distance between Astragalus and Oxytropis coerulea was the smallest, while that between the medicinal Astragalus and Hedysarum polybotrys, Caragana sinica as well as Althaea rosea was great. The phylogenetic trees constructed based on the ITS2 sequence (primary structure) and joint ITS2 sequence and its secondary structure showed that the topological relations of the two phylogenetic trees were basically the same, and both could effectively identify the Astragalus and its adulterants. What’s more, the addition of secondary structure information made end branch of the phylogenetic tree become more in its construction, and the distinguish ability and approval rating were also improved, which further reflected the genetic relationship of Astragalus and its adulterants. This provides some scientific basis for classification and accurate identification of Astragalus and its adulterants.
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Objective Traditional identification methods of pharmacognosy is difficult to distinguish the seeds of Panax ginseng and Panax quinquefolius. In order to improve the efficacy and accuracy of identification and provide the scientific foundation for the establishment of Chinese herbal medicine seed quality standards, molecular identification methods of the seeds were established by DNA barcoding technology. Methods The pharmacognostical identification method was used to study the morphological identification and microscopic characters of different seeds. DNA barcodes and Chinese Pharmacopoeia species standard barcode database were employed to identify the seeds by ITS2 sequence comparison, genetic distance comparison and systematic NJ tree construction. Results Intraspecific genetic distances of individuals participating were smaller than interspecific genetic distances. Phylogenetic tree map showed that two species were repectively clustered into one. A total of 42 samples of seeds from Panax ginseng and Panax quinquefolius produced by nine areas were all top-quality and easy to distinguish. Conclusion ITS2 DNA barcodes can identify and differentiate the seeds of P. ginseng and P. quinquefolius germplasm resources quickly, accurately and efficiently.
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Objective To explore a new identification method for medicinal materials of Dysosma, and analyze the second internal transcribed spacer (ITS2) barcode sequences of Diphylleia sinensis and Dysosma versipellis, Sinopodophyllum hexandrum, Dysosma difformis and Dysosma pleiantha in five kinds of podophyllum. Methods The ITS2 of ribosomal DNA of medicinal materials of podophyllum was amplified and sequenced by bi-directional sequencing of PCR products. Sequence assembly and consensus sequence generation were performed by using CodonCode Aligner. Phylogenetic study was performed using software MEGA 5.1 in accordance with Kimura-2-parameter (K2P) model. Genetic distances were calculated and analyzed and the phylogenetic tree was constructed by using the neighbor-joining (NJ) method. Results There were significant differences among five kinds of Dysosma. Their maximum intraspecific genetic distance (K2P distance) was far lower than their minimum interspecific genetic distance with the other species. In the cluster dendrogram, all species showed monophyletic. Conclusion ITS2 sequence as DNA barcoding technique can be used to identify Chinese herbal materials of Dysosma.
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This study was aimed to identify Bletilla Striata (Thunb.) Reichb.f.and Bletilla Formosana (Hayata) Schltr.by ITS2 sequence.The leaves of 38 samples of Bletilla striata and Bletillaformosana from Yunnan,Hubei,Guizhou,Hunan and Sichuan province were used as experiment materials.The total DNA was extracted.Internal transcribed spacer 2 (ITS2) sequences were obtained by PCR.All of the ITS2 sequences were checked.The 8 ITS2 sequences from two species were downloaded from GenBank.The intraspecific and interspecific Kimura-2-parameter (K2P) distances of Bletilla striata and Bletilla formosana were calculated by MEGAS.0.And neighbor-joining (NJ) tree was constructed.The results showed that the full-length sequences of ITS2 from Bletilla striata and Bletillaformosana were 259 bp,with a total of 14 variable sites.The maximum intraspecific K2P distance of Bletilla striata and Bletillaformosana was 0.008,while the minimum interspecific K2P distance was 0.040.The ITS2 secondary structure showed that different origins of Bletilla striata were gathered together and could be distinguished obviously from Bletilla formosana by NJ tree.It was concluded that ITS2 sequence was able to identify Bletilla striata and Bletillaformosana quickly and accurately.
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Objective: To identify the authenticity of Bupleuri Radix in the market of Hebei province. Methods: In this research, the ITS2 sequence of 28 samples of Bupleuri Radix that were bought from the market in Hebei province was amplified by PCR, and the sequence of seven samples was downloaded from GenBank. Aligner CodonCode software was used for sequence alignment, the MEGA6.0 analysis was used to calculate the K2P distance, and the mutation sites of each sequence was analyzed. Results: Among the 28 Bupleuri Radix samples, there were 21 samples for Bupleurum chinese, 3 for B.smithii, and 4 for the false. Conclusion: ITS2 sequence can be used as an effective method to identify the authenticity of Bupleuri Radix, it is great value to the medicine market standard and the clinical medication safety.
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Objective:To explore a new method to identify Sparganium stoloniferum and its adulterants by ITS2 regions. Methods:Eight samples of Sparganium stoloniferum and its adulterants were collected with five species, and 6 species with 23 ITS2 sequence of Sparganium stoloniferum and its adulterants were downloaded from Genbank. The intraspecific and interspecific K2P distances of Spar-ganium stoloniferum and its adulterants were calculated by MEGA5. 0, and the phylogenetic tree was constructed by MEGA 5. 0. Re-sults:The maximum intraspecific K2P distance of Sparganium stoloniferum was 0. 038,while the minimum interspecific K2P distance was 0. 697. The phylogenetic tree showed that Plantago asiatica was different obviously from its adulterants. The different samples of Sparganium stoloniferum were gathered together and could be distinguished from its adulterants by the NJ tree. Conclusion: ITS2 se-quence is able to identify Sparganium stoloniferum and its adulterants correctly, which provides a new method for the identification of Sparganium stoloniferum.
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Abstract ITS2 (Internal transcribed spacer 2) sequences have been used in systematic studies and proved to be useful in providing a reliable identification of Trichogramma species. DNAr sequences ranged in size from 379 to 632 bp. In eleven T. pretiosum lines Wolbachia-induced parthenogenesis was found for the first time. These thelytokous lines were collected in Peru (9), Colombia (1) and USA (1). A dichotomous key for species identification was built based on the size of the ITS2 PCR product and restriction analysis using three endonucleases (EcoRI, MseI and MaeI). This molecular technique was successfully used to distinguish among seventeen native/introduced Trichogramma species collected in South America.
Resumo Sequências do Espaço Transcrito Interno 2 (ITS2) têm sido utilizadas em estudos taxonômicos e sua utilidade constatada pela confiabilidade que o método confere à identificação das espécies de Trichogramma. Esta técnica molecular foi bem sucedida em distinguir dezessete espécies nativas e introduzidas de Trichogramma, coletadas na América do Sul. As sequências do DNAr variaram de 379 a 632 pb. Em 11 linhagens de T. pretiosum estudadas, o endosinbionte Wolbachia foi detectado pela primeira vez. Estas linhagens telítocas foram encontradas no Peru (9), Colômbia (1) e Estados Unidos (1). Uma chave dicotômica para identificação de espécies foi construída baseada no tamanho do produto da PCR do ITS2 e em análises de restrição utilizando-se três endonucleases (EcoRI, MseI and MaeI).
Subject(s)
Animals , Wasps/classification , Wasps/physiology , DNA, Ribosomal Spacer/genetics , Molecular Sequence Data , Parthenogenesis , Sequence Analysis, DNA , South America , Wasps/genetics , Wasps/microbiology , Wolbachia/physiologyABSTRACT
The misuse of toxic drugsisseriousone of the threats of public health. In this study, toxic Hyoscyami Semen and its adulterants were identified by DNA barcoding. The genomic DNA was extracted from 61 samples including Hyoscyami Semen and its adulterants by reagent kit method. Their ITS2 sequences were amplified, and purified PCR products were sequenced. Sequence assembly and consensus sequence generation were performed using CodonCode Aligner v 4.25. The genetic distances, variable sites and the neighbor-joning (NJ) phylogenetic tree were computed by MEGA 6.0 in accordance with the Kimura 2-parameter(K2P) model. The results showed that the intra-specific genetic distances of Hyoscyamusniger were 0.005 which were smaller than inter -specific ones (0.360) of H. niger and their adulterants. The NJ tree showed that H. niger was clustered into one monophyletic branch, and clearly separated with other species. Therefore, ITS2 sequence was able to identify Hyoscyami Semen and its adulterants to ensure the safty of medicines.
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Objective: The Phenomenon of different species with the same name has created potential threats to cinical safety of medication. Because of that, two kinds of “gonglao leaf” and their sibling adulterants have been identified by molecular methods in this study, to secure its safety in medication. Methods: Ilexcornuta, Mahoniabealei, Mahoniafortuneiand their sibling adulterants total of 9 species 45 samples were collected in this experiment,Total genomic DNA was extracted from them by the method of improved CTAB, ITS2 sequences were amplified, and the PCR products were sequenced. Sequence assembly and consensus sequence generation were performed by the CodonCode Aligner V 4.2.4.. The genetic distances were computed by MEGA 6.0 in accordance with the Kimura 2-Parameter (K2P) model and the phylogenetic tree constructed by the neighbor-joining (NJ) method. Results: The analysis results of the genetic distances, variable sites and the NJ phylogenetic tree showed that Ilexcornuta, Mahoniabealei, Mahoniafortunei were seperated from their sibling adulterants obviously. Conclusion: ITS2 sequence was able to identify two kinds of “gonglao leaf” and their sibling adulterants which can provide a basis for clinical accurate medication.
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In this study, the ITS2 sequence was used to identify Pinelliae Rhizoma and its adulterants to ensure its market circulation, clinical effect and safety. All genomic DNA from 59 samples were extracted successfully. The Kimura 2-Parameter (K2P) distances and NJ tree were calculated using software MEGA 6.0. The length of the ITS2 sequence of Pinelliae Rhizoma was 251 bp. The intraspecific genetic distance was smaller than the interspecific ones;The NJ tree indicated that Pinelliae Rhizoma distinguished from its adulterants obviously. The results showed that the ITS2 sequence can be used to distinguish Pinelliae Rhizoma from its adulterants accurately and stably.
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This study aimed to identify Inulae Herba, Inulae Flos and their closely related species using the ITS2 bar-code, and secure the quality and clinical curative effect of these medicinal materials. DNA was extracted from Inula linariifolia, I. japonica, I. britanica, which are the original species of Inulae Herba and Inulae Flos, together with their closely related species. The ITS2 sequence was amplified by PCR and sequenced bidirectionally. Sequence assembly and generation of consensus sequence were conducted by the CodonCode Aligner. The genetic distances were comput-ed using MEGA 5.0 in accordance with the Kimura-2-parameter (K2P) model, and the phylogenetic tree constructed by the neighbor-joining (NJ) method. The results showed that the ITS2 sequences of the various species have stable variable sites. The intraspecific genetic distances among Inulae Herba and Inulae Flos were obviously lower than the interspecific genetic distance among the above two medicinal materials and its adulterants. The NJ tree based on ITS2 sequences can clearly differ from Inulae Herba, Inulae Flos and their closely related species. It is concluded that ITS2 sequence can be used as DNA barcode to identify Inulae Herba, Inulae Flos and their closely related species.
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Descurainiae, Lepidii Semen and their it adulterants were identified by analysising their ITS2 sequences. The genomic DNA was extracted from 46 samples including Descurainiae and Lepidii Semen and their it adulterants. Their ITS2 sequences were amplified, and purified PCR products were sequenced. Sequence assembly and consensus sequence generation were performed using CodonCode Aligner v 4.25. The genetic distances, variable sites and the neighbor-joning (NJ) phylogenetic tree were computed by MEGA 6.0 in accordance with the Kimura 2-parameter (K2P) model. The results showed that the intra-specific genetic distances of Descurainia sophia and Lepidium apetalum were 0.021 and 0.010, which were smaller than inter-specific ones of D. sophia, L. apetalum and their adulterants. The NJ tree showed that both D. sophia and L. apetalum were clustered into one monophyletic branch, and clearly separated with their sibling species. Therefore ITS2 sequence was able to identify Descurainiae and Lep-idii Semen and its adulterants to ensure the quality of medicines and clinical efficacy.
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This study was aimed to explore a new method to identify the original plant of Plantaginis Semen and its adulterants by the ITS2 regions. The second internal transcribed spacer (ITS2) of ribosomal DNA was amplified and sequenced by bidirectional sequencing of PCR products. Sequence assembly and consensus sequence generation were performed by using CodonCode Aligner. The ITS2 secondary structure was predicted using ITS2 database and web-sites. The phylogenetic tree was constructed by MEGA5. The results showed that the maximum intraspecific K2P dis-tance of Plantago asiatica was 0.009 9, while the minimum interspecific K2P distance was 0.497 6; the maximum in-traspecific K2P distance of P. depressa was 0.005 2, while the minimum interspecific K2P distance was 0.519 1. The ITS2 secondary structure showed that P. asiatica and P. depressa can be differentiated obviously from its adulterants. Different samples of P. asiatica and P. depressa were gathered together and can be distinguished from its adulterants by NJ tree. It was concluded that the ITS2 sequence was able to identify original plant of Plantaginis Semen and its adulterants correctly. It provided a new method for the identification of original plant of Plantaginis Semen.
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Objective: This study aimed to discriminate between Eupatorii Herba and its adulterants in order to guarantee the quality and clinical curative effect of this medicinal material. Methods: Genomic DNA extracted from Eupatorii Herba was used as templates. The internal transcribed spacer 2 (ITS2) of nuclear ribosomal DNA was amplified. Sequence assembly and consensus sequence generation were performed by CodonCode Aligner. The intraspecific and interspecific genetic distances of Eupatorii Herba and its adulterants were computed by MEGA5 and the phylogenetic tree was constructed using the neighbor-joining (NJ) method. Results: The length of ITS2 sequence of Eupatorii Herba was 218 bp. The maximum intraspecific genetic distance (K2P distance) of Eupatorii Herba was 0.0092. The minimum interspecific genetic distance of Eupatorii Herba and its adulterants was 0.024. The NJ trees showed that the ITS2 sequence would be used to identify Eupatorii Herba and its adulterants. Con-clusion: ITS2 sequence was able to identify Eupatorii Herba and its adulterants correctly and it provided a new technique to ensure clinical safety in utilization of traditional Chinese medicines.