Phylogenetic and Mutation Points Analysis of Sophora japonica cv. jinhuai Based on DNA Barcoding Sequences / 中国实验方剂学杂志

ObjectiveTo conduct phylogenetic analysis of internal transcribed spacer 2 （ITS2） and chloroplast gene segments including psbA-trnH， rbcL， and matK of Sophora japonica cv. jinhuai resource samples from different geographical sources， and to explore the genetic diversity of S. japonica cv. jinhuai. MethodPolymerase chain reaction （PCR） method was used to amplify the nucleic acid sequences of ITS2， psbA-trnH， rbcL， and matK of S. japonica cv. jinhuai. Neighbor joining （NJ） method was used to construct phylogenetic trees， and Kimura 2-Parameter （K2P） model was used to calculate the genetic distance of different samples. MEGA and BIOEDIT softwares were applied for mutiple alignment and analysis of ITS2， psbA-trnH， rbcL， and matK sequences of S. japonica cv. jinhuai. ResultThe lengths of ITS2 sequence were 278-279 bp. The lengths of psbA-trnH were 289 bp. The lengths of rbcL sequence were 673 bp. The lengths of matK sequences were 786-792 bp. There were 3 mutation points in ITS2 and psbA-trnH， no mutation point in rbcL， and 13 mutation points in matK. The samples of S. japonica cv. jinhuai were clustered into two groups based on the phylogenetic tree constructed by ITS2 sequences. The sample of seedling tree in Baibao was clustered into one group， while the other 25 samples were clustered into another group. For the psbA-trnH sequence， the success rate of PCR amplification of 28 samples of S. japonica cv. jinhuai was 100%. The 28 samples of S. japonica cv. jinhuai were clustered into three groups based on the clustering results of psbA-trnH sequence. The sample of seedling tree in Shaoshui was clustered into one group. The five samples of grafting tree and seedling tree in Miaotou， grafting trees in Jiantang， Wenqiao， and Daxu， and seeding tree in Xianshui were clustered into one group. The other 21 samples were clustered into another group. The 26 samples of S. japonica cv. jinhuai were clustered into two groups based on the phylogenetic tree constructed by matK sequences. The sample of seedling tree in Xianshui was clustered into one group， while the other 25 samples were clustered into another group. The clustering results of the rbcL sequence of S. japonica cv. jinhuai could not distinguish 28 resource samples. The phylogenetic tree constructed by the combined sequence of ITS2+psbA-trnH+rbcL+matK divided S. japonica cv. jinhuai resource samples into 4 groups. The 13 samples of seedling trees in Qiyang， Daoxian， Miaotou， Shaoshui， Shitang， Xianshui， Jiantang， and Xiangli， and grafting trees in Qiyang， Miaotou， Yongsui， Wenqiao， and Yangtang were clustered into one group. The sample of seedling tree in Wenqiao was clustered into one group. The sample of seedling tree in Daxu was clustered into one group. The remaining samples were clustered into another group. ConclusionPhylogenetic and mutation analysis provide the theoretic foundation to investigate the evolution of the resources of S. japonica cv. jinhuai， and evaluate their genuineness. The results of mutation points can be used to identify the related S. japonica cv. jinhuai resources. The findings of this study show that the combination of different gene sequences has an optimal effect on plant identification.

Molecular Identification of 22 Species of Lauraceae by DNA Barcoding / 中国实验方剂学杂志

Objective:DNA barcodes suitable for Lauraceae plants were screened，and 22 Lauraceae plants were identified and classified. Method:The DNA of 22 species of Lauraceae was extracted and amplified by polymerase chain reaction（PCR） with different DNA barcoding primers，followed by electrophoresis and sequencing. Codon Code Aligner was used to proofread，splice， and remove the forward and reverse primer sequences. The sequence was imported into DNAMAN for multiple sequence alignment，and the base mutation sites were analyzed. The Kimura 2-Parameter（K2P） distance of different plants was calculated by MEGA，and the variation degree was analyzed according to the genetic distance. The phylogenetic tree was constructed based on the adjacency method. Result:Three pairs of DNA barcoding primers were used to amplify and sequence the DNA of 22 species of Lauraceae，and 20 species were identified by comparing the specific base sites of gene sequences<bold>.</bold> Conclusion:Four <italic>Litsea</italic> plants could be identified by <italic>matK</italic>， three <italic>Phoebe</italic> plants except for<italic> Cinnamomum validinerve </italic>by<italic> rbcL</italic>， and 20 Lauraceae plants by the combination of<italic> matK</italic>， <italic>rbcL</italic>， and <italic>rpoB</italic>，which provided a theoretical basis for the identification and development of Lauraceae plants. Among them，<italic>matK</italic> was predominant in the identification of Lauraceae plants，and the results also showed that the combination of sequences for plant identification could achieve a better result in DNA barcoding. This study investigated the genetic relationship between Lauraceae plants at the molecular level，aiming at providing a basis for the investigation，cultivation，development，protection， and utilization of medicinal plant resources.

Identification of Cannabis Sativa L. Based on rbcL Sequence / 法医学杂志

Objective To assess the feasibility of the rbcL sequence of chloroplast DNA as a genetic marker to identify Cannabis sativa L. Methods The rbcL sequences in 62 Cannabis sativa L. samples, 10 Humulus lupulus samples and 10 Humulus scandens DNA samples were detected, and 96 rbcL sequences of the Cannabaceae family were downloaded from Genbank. Sequence alignment was performed by MEGA X software, the intraspecific and interspecific Kimura-2-Parameter （K2P） genetic distances were calculated, and the system clustering tree was constructed. Results The rbcL sequence length acquired by sequencing of Cannabis sativa L. and Humulus scandens were 617 bp and 649 bp, respectively, and two haplotypes of Cannabis sativa L. were observed in the samples. The BLAST similarity search results showed that the highest similarity between the sequences acquired by sequencing and Cannabis sativa L. rbcL sequences available from Genbank was 100%. The genetic distance analysis showed that the maximum intraspecific genetic distance （0.004 9） of Cannabis sativa L. was less than the minimum interspecific genetic distance （0.012 9）. The results of median-joining network and system clustering tree analysis showed that Cannabis sativa L. and other members of the Cannabaceae family were located in different branches. Conclusion The rbcL sequence could be used as a DNA barcode for identifying Cannabis sativa L., and combined with comparative analysis of the rbcL sequence and system cluster analysis could be a reliable and effective detection method for Cannabis sativa L. identification in forensic investigation.

Phylogenetic relationships of plant species from the flowering desert of the Atacama Region / Relaciones filogenéticas de especies de plantas que crecen en el desierto florido de la Región de Atacama

Every 3 to 7 year angiosperms species of the flowering desert appear in the Atacama Region of Chile, as a result of the climatic phenomenon "El Niño". Our objective was to evaluate the universality of matK and rbcL barcode markers of these species, and validate their taxon through phylogenetic relationships. Argemone hunnemannii, Oenothera coquimbensis, Malesherbia humilis, Leucocoryne appendiculata, Loasa elongata, Nicotiana solanifolia, Stachys grandidentata, Aristolochia chilensis, Alstroemeria kingii and Adesmia eremophila, almost all classified as endemic to Chile, were collected in Pan de Azúcar and Llanos de Challe National Park (Atacama Region, Chile) at the end of October 2017. The phylogeny of these ten angiosperm species from the flowering desert was analyzed using rbcL and matK markers with the maximum likelihood and Bayesian inference methods. The results showed that 70% of the species can be distinguished with the matK or rbcL locus, however, 100% were distinguished using both loci. The phylogenetic results showed that the species formed clades with high reliability and high support with both the matK and rbcL genes, when comparing our results with sequences obtained from GenBank. The matK and rbcL genes are efficient markers for analyzing phylogenetic relationships and validating the taxonomy of flowering species.

Las especies de angiospermas del Desierto Florido de la Región de Atacama de Chile aparecen cada 3 a 7 años, influenciado por el fenómeno climático "El Niño". Nuestro objetivo fue evaluar la universalidad de los marcadores de código de barra matK y rbcL de estas especies, y validar su taxón por medio de relaciones filogenéticas. Las especies Argemone hunnemannii, Oenothera coquimbensis, Malesherbia humilis, Leucocoryne appendiculata, Loasa elongata, Nicotiana solanifolia, Stachys grandidentata, Aristolochia chilensis, Alstroemeria kingii y Adesmia eremophila son clasificadas la mayoría endémicas de Chile. Estas especies fueron colectadas en el Parque Nacional Pan de Azúcar y Llanos de Challe, Región de Atacama, Chile. La colecta se realizó a fines de octubre de 2017. Con los marcadores rbcL y matK se analizó la filogenia con los métodos máxima verosimilitud e inferencia bayesiana en diez especies de angiosperma del Desierto Florido. Los resultados mostraron que el 70% de las especies pueden ser distinguidas con un locus matK o rbcL, sin embargo, el 100% se distinguió usando ambos locus. Los resultados filogenéticos mostraron que las especies formaron clados con alta fiabilidad y alto soporte tanto con los genes matK y rbcL, al comparar con accesos de secuencias obtenidas de GenBank. Lo genes matK y rbcL son marcadores eficientes para analizar relaciones filogenéticas y validar el taxón de las especies de flor.

Fingerprint and molecular identification of rbcL sequence of Mongolian medicine Althaeae Roseae Flos by HPLC / 中草药

Objective: To establish the fingerprint of Althaeae Roseae Flos by HPLC and the molecular identification method of DNA barcode of rbcL sequence. Methods: The fingerprint establishment of Althaeae Roseae Flos was performed on Welchrom Column C18 (300 mm × 4.6 mm, 5 μm) with acetonitrile - 0.1% formic acid solution as mobile phase for gradient elution, with flow rate of 1.0 mL/min, column temperature of 35 ℃, detection wavelength of 365 nm, injection volume of 10 μL. DNA barcode molecular identification method was used for PCR amplification and determination of rbcL sequence. Results: The fingerprints of 11 samples were established, 21 common peaks were obtained, their similarities were calculated, and four components (hyperoside, quercetin, apigenin and kaempferide) were determined. The total length of rbcL sequence of 11 samples was measured, and the G + C content was 44.10%-44.40% and genetic distance (K2P) was 0.001 4. There were 10 ectopic points and the similarity was 99.00%. Conclusion: The two methods are stable and reliable, which can provide basis for the identification and quality control of A. rosea.

Evaluación de dos secuencias de código de barras de ADN en germoplasmas de Arracacia xanthorrhiza (Apiaceae) de Ecuador / Evaluation of two sequences of DNA barcodes in germplasms of Arracacia xanthorrhiza (Apiaceae) from Ecuador

El presente estudio evalua el gen de cloroplasto rbcL y la región espaciadora no codificante psbA-trnH de Arracacia xanthorrhiza como posible secuencia de código de barra. Se colectó material vegetal de A. xanthorrhiza en huertos de las provincias de Pichincha, Tungurahua y Cotopaxi, las cuales fueron sembradas en condiciones homogéneas en la Facultad de Ciencias Agropecuarias de la Universidad Técnica. El análisis del locus rbcL identificó los cinco materiales de A. xanthorrhiza con entre 97 y 99% de homología. La alineación de secuencias del locus rbcL y de psbA-trnH permitió diferenciar dos grupos, el primer grupo con SJ, QU, PP y B, observándose poca diversidad entre ellos, mientras que el segundo grupo está conformado por el material CH cultivado a 3260 m de altitud. En el segundo árbol, se demostró la divergencia entre los materiales colectados en diferentes provincias de la Sierra ecuatoriana, separándolos de acuerdo a su localidad, así como al color de la pulpa de la raíz. La región intergénica no codificadora (psbA-trnH) permitió identificar y obtener la diversidad genética de materiales cultivados de A. xanthorrhiza, provenientes de diversas zonas geográficas de la sierra ecuatoriana, con características morfológicas distintivas. Adicionalmente, esta secuencia pudo diferenciar a A. xanthorrhiza de otras especies de la familia Apiaceae, con lo cual se recomienda como código de barra.

The present study aimed to evaluate the Arracacia xanthorrhiza rbcL chloroplast gene and the non-coding spacer region psbA-trnH as a possible barcode sequence. Plant material of A. xanthorrhiza was collected in orchards of Pichincha, Tungurahua and Cotopaxi provinces. This material were cultivated in standard conditions in the la Facultad de Ciencias Agropecuarias de la Universidad Técnica. The rbcL locus analysis identified the five materials of A. xanthorrhiza with between 97 and 99% homology. The sequence alignment of rbcL locus and psbA-trnH allowed to differentiate two groups, the first group with SJ, QU, PP and B, showing low diversity among them, while the second group consisted of the CH material grown in 3260 m of altitude. In the second tree, the divergence between the materials collected in different provinces of the Ecuadorian Sierra was demonstrated, separating them according to their locality, as well as the color of the root pulp The non-coding intergenic region (psbA-trnH) allowed identify and obtain the genetic diversity of cultivated materials of A. xanthorrhiza, from various geographical areas of the Ecuadorian Sierra, with distinctive morphological characteristics. Additionally, this sequence was able to differentiate A. xanthorrhiza from other species of the Apiaceae family, which is recommended as a bar code.

Phylogenetic relationship analysis of common medicinal submetals and multiple real-time fluorescence PCR identification of Fritillariae Cirrhosae Bulbus / 中草药

Objective To investigate the feasibility of multiple real-time PCR for the detection of Fritillariae Cirrhosae Bulbus and adulterants. Methods Based on the analysis of interspecies variation, genetic distance and phylogenetic relationship of ITS, psbA-trnH, rbcL and matK gene sequences, the genes with fast evolution rate, big interspecies variation and small intraspecies variation were selected as target genes. Fritillariae Cirrhosae Bulbus and adulterants specific primers and Taqman probes were designed to establish a multiplex real-time PCR assay. Methods were evaluated by comparison of specificity, sensitivity and mixed sample detection and sequencing. Results The ITS and psbA-trnH mutations were higher than rbcL and matK, and rbcL and matK were significantly lower than ITS and psbA-trnH genes by genetic distance analysis. And the sensitivity of the establish multiple real-time PCR using ITS as the target gene was 0.01 ng. Four samples of adulterants were detected in 18 samples, and the results were consistent with the results of NJ tree clustering analysis. Conclusion Based on the IIS region sequence as the target gene to establish multiple real-time fluorescence PCR detection method can successfully identify Fritillariae Cirrhosae Bulbus and its counterfeit goods, which provides a new basis for the authenticity of identification.

Effect of calcium on ion contents and expression of photosynthetic related genes in honeysuckle under salt stress / 中国中药杂志

Exogenous calcium can enhance the resistance of certain plants to abiotic stress. Research have demonstrated that exogenous calcium could enhances the resistance of honeysuckle under salt stress by promoting the transmission of photosynthetic electrons.The aim of this study was to investigate the effects of exogenous calcium on the contents of Na~+,K~+,Ca~(2+),Mg~(2+)and the expression of photosynthetic related genes Cab and rbc L. In this study,we used ICP-OES to analysis ion contents and used qRT-PCR to analysis the expression patterns of Cab and rbc L. The results showed that CaCl_2 significantly enhanced the K~+-Na~+,Ca~(2+)-Na~+,Mg~(2+)-Na+ratio of honeysuckle treated with 50 and 100 mmol·L~(-1) NaCl. Meanwhile,Cab and rbc L were significantly up-regulated under short-term salt stress,and CaCl_2 promoted this trend. From the two gene expression patterns,rbc L rapidly up-regulated on the first day of stress and then decreased,and was more sensitive to environmental changes. In summary,exogenous calcium could alleviate salt stress and increase plant development by increasing intracellular K~+-Na~+,Ca~(2+)-Na~+,Mg~(2+)-Na+ratio,and the transient overexpression of Cab and rbc L.

DNA barcoding identification of Dendrobium huoshanense and its adulterants / 中国中药杂志

This research preliminarily discusses the relations of Dendrobium system growth through chloroplast gene rbcL, matK and the nuclear genome ITS2. The DNA barcoding universal sequence for authentication of the Dendrobium medical plants was slected and the possibility concerning utilizing the DNA barcoding to distinguish the D. huoshanenseand its adulterants was analyzed. Using the universal primer pair of ITS2, rbcL and matK, series of extended sequencing in the Dendrobium were conducted. Meanwhile, considering the different index about amplification and sequencing success rate of each sequence, the intraspecific and interspecific aberrance, the employment of BioEdit and MEGA 5.0 software were applied to establish the systematic tree of the NJ molecular and evaluate the diversified authentication capability of various sequences. The consequence demonstrates that the sequence of ITS2 is not only the largest one both in the intraspecific and interspecific aberrance of the Dendrobium but also has obvious barcoding gap. Considering the few overlap between the intraspecific and interspecific aberrance and the highest percentage regarding the formation of unilateral branch in diverse Dendrobium which have different ITS2 sequences, it can differentiate the species of Dendrobium. Furthermore, due to the inferior success rate of the rbcL and thematK and the lower reliability of NJ systematic tree, the percentage of the unilateral species which are generated by the systematic tree of rbcL and matK sequences is deficient. Therefore, the sequence of ITS2 can serves as DNA barcoding to distinguish the D. huoshanense, the D. moniliform and the D. officinale.

Identification of uighur medicine Cichorium glandulosum and its adulterants by Allele-Specific PCR / 中草药

Objective To establish molecular identification method of Cichorium glandulosum and its adulterants Cichorium intybus by Allele-Specific PCR. Methods The samples of C. glandulosum and C. intybus were collected in different geographical areas. The DNA was extracted, and rbcL gene segments were amplified and sequenced directionally. The multiple sequences were aligned by using Clustal W. Specific primers were designed and amplified according to its variable sites, and PCR reaction system was optimized to determine detection limits and establish Allele-Specific PCR identification method. Results According to Allele-Specific PCR system established in this study for C. glandulosum, the optimization results was a total of 30 μL reaction system containing TaqDNA polymerase 0.25 μL, 10 × buffer 2.5 μL, dNTP 2.0 μL, primer 0.5 μL, template DNA 2 μL, and ddH2O 22.25 μL. The most suitable PCR amplification procedure is one cycle of predegeneration at 94 ℃ for 3 min; 32 cycles of denaturing at 94 ℃ for 30 s, annealing at the primer temperature 55 ℃ for 30 s and extending at 72 ℃ for 1 min, and extending at 72 ℃ for 7min. Through the detection of 20 medicinal materials of C. glandulosum and C. intybus, the result showed that 230 bp amplified band of target fragment was identified for C. glandulosum but no amplified band was observed for its adulterants. Conclusion In this study, we established and optimized the Allele-Specific PCR identification technology of C. glandulosum and its adulterants C. intybus, which can accurately, reliably, and effectively identify these two medicinal materials.

Adulteration detection of tea samples based on plant rbcL gene sequencing / 生物工程学报

Tea is one of the most popular drinks in the world, but counterfeit or adulterated tea can be found now and then on the tea market. The traditional methods dependent on sensory, physical and chemical tests cannot identify the composition of adulterated plant species accurately. We developed therefore a method for identification of adulterated plants in tea based on qualitative detection of plant rbcL (Ribulose 1,5-bisphosphate carboxylase-oxygenase large subunit) fragments, which involved amplification, sequencing and sequence analyses of rbcL fragments. Seven tea samples were analyzed with the established method. The results showed that Yueyanghuangcha (yellow tea) and Xinyangmaojian (green tea) were pure with only detection of the tea plant Camellia sinensis; Zhengshan Souzhong (black tea), Tieguanyin (oolong tea), Tailaoyinzhen (white tea), Liupao and Pu-erh (dark tea) were, to a certain extent, adulterated with non-Camellia sinensis plants. The method introduced in this study only requires a small amount of tea samples, easy to operate and reliable. It can be used to determine if any tea samples are adulterated.

Molecular identification of six species in Aloe L. based on DNA barcoding / 中草药

Objective: To screen out a proper DNA barcode for suitable of six species in Aloe L. Methods: The genomic DNA from 18 samples of six species in Aloe L. was extracted by Plant Genomic DNA Kit, then nuclear ITS2 and the chloroplast psbA-trnH, rbcL, and matK of the samples were amplified using the universal primers and sequenced bidirectionally. The obtained sequences were assembled using Sequencher 4.1.4. The characteristics of four candidate DNA barcodes were analyzed and the Kimura 2-Parameter (K2P) distances were calculated based on the sequences using MEGA 5.0. DNA barcoding gaps were evaluated by the analysis of the intra-and inter-specific divergences. The phylogenetic trees were constructed on the basis of Neighbor-Joining method. Results: Totally 72 sequences of species in Aloe L. were acquired altogether, with each of ITS2, psbA-trnH, rbcL, and matK regions having 18 sequences respectively. The successful sequencing rate was 100% of all the four candidate barcodes. The alignment length and GC contents ranged from 255 to 723 bp and 30.6% to 68.2%. The maximum K2P intra-specific genetic distances were much smaller than the minimum inter-specific genetic distance of all samples in terms of psbA-trnH sequence and thus forming into a prominent barcoding gap. Due to some overlaps of intra-and inter-specific variation, there was no obvious barcoding gap in the aspect of ITS2, rbcL, and matK sequences. The phylogenetic tree constructed by psbA-trnH sequence was capable to identify the six species of Aloe L. while there existed fuzzy identification as to the other three candidate barcodes. Conclusion: DNA barcoding technology can identify species in Aloe L. accurately and psbA-trnH sequence can be the optimal barcode to identify the species of Aloe L.

Identification of plants in Mussaenda L. based on DNA barcoding / 中草药

This study aims at developing fast and accurate species identification methods for the plants of Mussaenda L. In the present study, DNA barcoding analysis was carried out on 89 individuals representing 20 species of Mussaenda in order to evaluate the performance of the four candidate barcoding loci (matK, rbcL, trnH-psbA, and ITS) and ITS2 region. Based on sequence similarity and Neighbor-joining (NJ) tree reconstruction, we detected inter-and intra-specific genetic distances using Kimura 2-parameter (K2P). Inter-specific genetic distance of species in Mussaenda was significantly higher than intra-specific genetic distance. The region of ITS2 showed the highest discrimination power among the independent sequences. Comparably high species discrimination power was also revealed by the matK and ITS data set. The candidate barcode of rbcL displayed the lowest identification rate among the others. However, each individual candidate barcode demonstrated significantly lower discrimination power than the barcode of combined data set. Comparable discrimination power was revealed between the two barcodes of combined sequences matK + rbcL + ITS and matK + rbcL + trnH-psbA + ITS, which showed the values around 77% and 75% based on sequence similarity and NJ tree method. Totally 15 species were identified based on NJ analysis of matK + rbcL + ITS. Consequently, the combined sequence of matK + rbcL + ITS provides an effective and fast tool for the identification and authentication of medicinal plant species in the genus Mussaenda L.

Identification of DNA barcoding on medicinal plants in Flemingia Roxb. ex Ait. et Ait. f / 中草药

Objective: To screen a DNA barcoding sequence that can identify the medicinal plants of Flemingia Roxb. ex Ait. et Ait. f. accurately and efficiently. Methods: Four species and 14 individuals of Flemingia Roxb. ex Ait. et Ait. f. were collected, the (second internal transcribed spacer, ITS2) of ribosomal DNA, rbcL, psbA-trnH of chloroplast DNA were amplified and sequenced. Meanwhile, the NCBI data were retrieved and the according sequence was downloaded. The total numbers of species and individuals were six and twenty. The PCR amplification and sequencing efficiency, barcoding gap, and NJ trees were used to evaluate the efficiency of species identification. Results: The sequencing success rates of ITS2, rbcL, and psbA-trnH were 100%, 100%, and 85.71%, respectively; Among the three DNA barcoding sequences, only ITS2 has remarkable barcoding gap; ITS2 could distinguish every species of Flemingia Roxb. ex Ait. et Ait. f. (except F. philippinensis and F. stricta). Conclusion: ITS2 could identify the medicinal plants of Flemingia Roxb. ex Ait. et Ait. f. accurately and efficiently, and could be used as an ideal DNA barcoding of species identification for medicinal materials of Flemingia Roxb. ex Ait. et Ait. f.

Identification of Gardenia Based on cpDNA Barcoding / 中国中医药信息杂志

Objective To test and eva1uate the abi1ity of three potential chloroplast DNA (cpDNA) barcoding sequences;To find new methods to identify the species of gardenia. Methods Three cpDNA sequences were amplified and sequenced by universal primers of matK, rbcL and psbA. By comparing PCR amplification efficiency, length, intra-and inter-specific divergence, and barcoding gap, BLAST and DNA MAN were used to evaluate these loci. Results The amplification efficiency of 5 samples from 3 gardenia species was 100%. Analysis of the intra-and inter-specific divergence of matK among the sequences showed that barcoding gap was superior to psbA and rbcL, with higher identification efficiency. Conclusion Gardenia jasminoides Ellis can be better identified by matK sequence.

Identification of traditional chinese medicine rubiae radix et and its adulterants using DNA barcodes / 中国药学杂志

OBJECTIVE: To identify traditional Chinese medicine Rubiae Radix et Rhizoma and its adulterants using DNA barcodes. METHODS: A total of 317 sequences of psbA-trnH, matK and rbcL from 13 species were analized. The intra- and inter-specific K2P genetic distances and neighbor-joining tree were calculated using MEGA5. 1 program. RESULTS: The DNAs were successfully extracted from 76 original plant samples. The amplification rates of psbA-trnH, matK and rbcL were 100%, 96%, and 99%, respectively. The adulterants could be identified from R. cordifolia by the single marker and the two combinations except for those adulterants in the Rubia genus. Fortunately, R. cordifolia could be identified from all the adulterants by psbA-trnH + matK + rbcL combination marker. The intra- and inter-specific K2P genetic distances of psbA-trnH + matK + rbcL were 0-0.001 4 and 0.000 7-0.630 3. R. cordifolia could be identified from alTthe adulterants by the psbA-trnH + matK + rbcL combination using NJ tree method. Among the 30 unidentified samples of Rubiae Radix et Rhizoma which were collected from medicinal herb markets, all three markers, including psbA-trnH, matK and rbcL, could be amplified from 22 samples. The 11 samples of Rubiae Radix et Rhizoma clustered with R. cordifolia and the others were divided into three clusters by the psbA-trnH + matK + rbcL combination using NJ tree method. CONCLUSION: psbA-trnH + matK + rbcL combination can be used as DNA barcode to identify R. cordifolia from adulterants.

Rapid molecular identification of saffron (Crocus sativus L.) based on specific PCR / 中国药学杂志

OBJECTIVE: To establish a rapid molecular method for identifying saffron (Crocus sativus L.) and its adulterants by PCR: amplification using specific primers and fluorescent dyes detection. METHODS: The chloroplast barcode was sequenced and analyzed to find the SNPs between saffron and its adulterants. Specific primers were designed for the SNPs, the PCR reaction systems were built and optimized, and fluorescent dyes method was used to identify PCR products. RESULTS: A 421 bp saffron identification band based on the psbA-trnH barcode sequence was screened when 100 × SYBR Green I was added into the optimized PCR product under the following condition; initial denaturation at 90℃ for 1 min, denaturation at 90℃ for 5 s, annealing at 58℃ for 5 s, 26 cycles; the saffron (Crocus sativus L.) showed strong green fluorescence under 365 nm UV lamp whereas adulterants did not. CONCLUSION: Fast site-specific PCR can rapidly identify saffron and its adulteration.

Differentiation on Original Plants of Xi-Huang-Cao through DNA Barcodes / 世界科学技术-中医药现代化

This study was aimed to identify original plants of Xi-Huang-Cao (XHC) through DNA barcodes. The nu-cleotide sequence information of rbcL, psbA-trnH, matK and ITS2 regions were abstracted using standardized man-ners from 41 samples (including Rabdosia serra, R. lophanthoides and R. lophanthoides var. graciliflora). Sequencing efficiency of each marker was calculated. Species identification capability was tested on the basis of TaxonGap. The results showed that sequence success rates were 100% for rbcL, 90.2% for psbA-trnH, 87.8% for ITS2, and 70.7%for matK. Three markers (rbcL, psbA-trnH and ITS2) were competent for species discrimination (not for subspecies). It was concluded that rbcL can be the preferred barcode for XHC because of its convenience and efficacy.

Prospects and Problems for Identification of Poisonous Plants in China using DNA Barcodes / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>Poisonous plants are a deadly threat to public health in China. The traditional clinical diagnosis of the toxic plants is inefficient, fallible, and dependent upon experts. In this study, we tested the performance of DNA barcodes for identification of the most threatening poisonous plants in China.</p><p><b>METHODS</b>Seventy-four accessions of 27 toxic plant species in 22 genera and 17 families were sampled and three DNA barcodes (matK, rbcL, and ITS) were amplified, sequenced and tested. Three methods, Blast, pairwise global alignment (PWG) distance, and Tree-Building were tested for discrimination power.</p><p><b>RESULTS</b>The primer universality of all the three markers was high. Except in the case of ITS for Hemerocallis minor, the three barcodes were successfully generated from all the selected species. Among the three methods applied, Blast showed the lowest discrimination rate, whereas PWG Distance and Tree-Building methods were equally effective. The ITS barcode showed highest discrimination rates using the PWG Distance and Tree-Building methods. When the barcodes were combined, discrimination rates were increased for the Blast method.</p><p><b>CONCLUSION</b>DNA barcoding technique provides us a fast tool for clinical identification of poisonous plants in China. We suggest matK, rbcL, ITS used in combination as DNA barcodes for authentication of poisonous plants.</p>

ITS2 and rbcL sequence analysis on plants of Marsdenia R. Br. and its related species / 中草药

Objective: To identify and preliminarily analyze the plants of Marsdenia R. Br. and its related species by ITS2 and rbcL sequences. Methods: The ITS2 and rbcL regions were amplified and sequenced directly. The sequence was manually calibrated using CodonCode Aligner, and splited and jointed by Contig software. The genetic distance analysis was carried out by MEGA 4.0 and the phylogenetic tree was constructed using the Neighbor-joining method. Results: The samples analyzed were well identified by ITS2 and rbcL sequences, and the authentication ability of ITS2 was superior to rbcL. The genetic relationship among the plants of Marsdenia R. Br., Dregea E. Mey., and Gymnema R. Br. was very close. Conclusion: ITS2 and rbcL sequences could distinguish Marsdenia R. Br. species very well, and the authentication ability of ITS2 is superior to rbcL. The preliminary studies suggest that the Dregea E. Mey. and Gymnema R. Br. should be classified into genus Marsdenia R. Br.