The molecular investigations on the subgenus Artemisia Less. of the genus Artemisia L. (Asteraceae) in Turkey / Investigações moleculares sobre o subgênero Artemisia Less. do gênero Artemisia L. (Asteraceae) na Turquia

The genus Artemisia L. of the family Asteraceae is systematically very complex. The aim of this study was to evaluate taxonomic positions of taxa of the subgenus Artemisia belonging to the genus Artemisia in Turkey using some molecular techniques. In this molecular study, 44 individuals belong to 14 species of the subgenus Artemisia were examined. Analyses were performed on the combined dataset using maximum parsimony, maximum likelihood and Bayesian inference and Molecular parameters obtained from co-evaluations of sequences of the psbA-trnH, ITS and ETS regions of examined individuals were used in the phylogenetic tree drawing. According to the results of this study, two molecular groups have been formed based on the DNA sequence similarity of the species, but there are no obvious morphological characters corresponding to two molecular groups. There is no also agreement between the two molecular groups and the two morphological groups formed according to the hairiness condition of the receptacle of species. Due to the lack of molecular significance of their receptacles with or without hair, dividing of the subgenus Artemisia species into new subgenera or sections was not considered appropriate. Likewise, it has been found that with or without hair on the corolla lobes of the central hermaphrodite disc flowers have no molecular significance. It was found that there were no gene flow and hybridization between the 14 species of the subgenus Artemisia and these 14 species were found completed their speciation. This study is important as it is the first molecular based study relating with belong to subgenus Artemisia species growing naturally in Turkey. In addition, new haplotypes related to the populations of Turkey belonging to the subgenus Artemisia taxa were reported by us for the first time and added to the GenBank database.

O gênero Artemisia L. da família Asteraceae é sistematicamente muito complexo. O objetivo deste estudo foi avaliar as posições taxonômicas de táxons do subgênero Artemisia pertencentes ao gênero Artemisia na Turquia usando algumas técnicas moleculares. Neste estudo molecular, 44 indivíduos pertencentes a 14 espécies do subgênero Artemisia foram examinados. As análises foram realizadas no conjunto de dados combinado usando máxima parcimônia, máxima verossimilhança e inferência bayesiana e parâmetros moleculares obtidos a partir de coavaliações de sequências das regiões psbA-trnH, ITS e ETS de indivíduos examinados foram usados no desenho da árvore filogenética. De acordo com os resultados deste estudo, dois grupos moleculares foram formados com base na similaridade da sequência de DNA das espécies, mas não há caracteres morfológicos óbvios correspondentes a dois grupos moleculares. Também não há concordância entre os dois grupos moleculares e os dois grupos morfológicos formados de acordo com a condição de pilosidade do receptáculo da espécie. Devido à falta de significado molecular de seus receptáculos com ou sem cabelo, a divisão das espécies do subgênero Artemisia em novos subgêneros ou seções não foi considerada apropriada. Da mesma forma, verificou-se que com ou sem cabelo nos lobos da corola das flores do disco hermafrodita central não tem significado molecular. Constatou-se que não houve fluxo gênico e hibridização entre as 14 espécies do subgênero Artemisia e essas 14 espécies concluíram sua especiação. Este estudo é importante porque é o primeiro estudo de base molecular relacionado com espécies pertencentes ao subgênero Artemisia crescendo naturalmente na Turquia. Além disso, novos haplótipos relacionados às populações da Turquia pertencentes ao subgênero Artemisia taxa foram relatados por nós pela primeira vez e adicionados ao banco de dados do GenBank.

The molecular investigations on the subgenus Artemisia Less. of the genus Artemisia L. (Asteraceae) in Turkey

Abstract The genus Artemisia L. of the family Asteraceae is systematically very complex. The aim of this study was to evaluate taxonomic positions of taxa of the subgenus Artemisia belonging to the genus Artemisia in Turkey using some molecular techniques. In this molecular study, 44 individuals belong to 14 species of the subgenus Artemisia were examined. Analyses were performed on the combined dataset using maximum parsimony, maximum likelihood and Bayesian inference and Molecular parameters obtained from co-evaluations of sequences of the psbA-trnH, ITS and ETS regions of examined individuals were used in the phylogenetic tree drawing. According to the results of this study, two molecular groups have been formed based on the DNA sequence similarity of the species, but there are no obvious morphological characters corresponding to two molecular groups. There is no also agreement between the two molecular groups and the two morphological groups formed according to the hairiness condition of the receptacle of species. Due to the lack of molecular significance of their receptacles with or without hair, dividing of the subgenus Artemisia species into new subgenera or sections was not considered appropriate. Likewise, it has been found that with or without hair on the corolla lobes of the central hermaphrodite disc flowers have no molecular significance. It was found that there were no gene flow and hybridization between the 14 species of the subgenus Artemisia and these 14 species were found completed their speciation. This study is important as it is the first molecular based study relating with belong to subgenus Artemisia species growing naturally in Turkey. In addition, new haplotypes related to the populations of Turkey belonging to the subgenus Artemisia taxa were reported by us for the first time and added to the GenBank database.

Resumo O gênero Artemisia L. da família Asteraceae é sistematicamente muito complexo. O objetivo deste estudo foi avaliar as posições taxonômicas de táxons do subgênero Artemisia pertencentes ao gênero Artemisia na Turquia usando algumas técnicas moleculares. Neste estudo molecular, 44 indivíduos pertencentes a 14 espécies do subgênero Artemisia foram examinados. As análises foram realizadas no conjunto de dados combinado usando máxima parcimônia, máxima verossimilhança e inferência bayesiana e parâmetros moleculares obtidos a partir de coavaliações de sequências das regiões psbA-trnH, ITS e ETS de indivíduos examinados foram usados no desenho da árvore filogenética. De acordo com os resultados deste estudo, dois grupos moleculares foram formados com base na similaridade da sequência de DNA das espécies, mas não há caracteres morfológicos óbvios correspondentes a dois grupos moleculares. Também não há concordância entre os dois grupos moleculares e os dois grupos morfológicos formados de acordo com a condição de pilosidade do receptáculo da espécie. Devido à falta de significado molecular de seus receptáculos com ou sem cabelo, a divisão das espécies do subgênero Artemisia em novos subgêneros ou seções não foi considerada apropriada. Da mesma forma, verificou-se que com ou sem cabelo nos lobos da corola das flores do disco hermafrodita central não tem significado molecular. Constatou-se que não houve fluxo gênico e hibridização entre as 14 espécies do subgênero Artemisia e essas 14 espécies concluíram sua especiação. Este estudo é importante porque é o primeiro estudo de base molecular relacionado com espécies pertencentes ao subgênero Artemisia crescendo naturalmente na Turquia. Além disso, novos haplótipos relacionados às populações da Turquia pertencentes ao subgênero Artemisia taxa foram relatados por nós pela primeira vez e adicionados ao banco de dados do GenBank.

The molecular investigations on the subgenus Artemisia Less. of the genus Artemisia L. (Asteraceae) in Turkey / Investigações moleculares sobre o subgênero Artemisia Less. do gênero Artemisia L. (Asteraceae) na Turquia

Abstract The genus Artemisia L. of the family Asteraceae is systematically very complex. The aim of this study was to evaluate taxonomic positions of taxa of the subgenus Artemisia belonging to the genus Artemisia in Turkey using some molecular techniques. In this molecular study, 44 individuals belong to 14 species of the subgenus Artemisia were examined. Analyses were performed on the combined dataset using maximum parsimony, maximum likelihood and Bayesian inference and Molecular parameters obtained from co-evaluations of sequences of the psbA-trnH, ITS and ETS regions of examined individuals were used in the phylogenetic tree drawing. According to the results of this study, two molecular groups have been formed based on the DNA sequence similarity of the species, but there are no obvious morphological characters corresponding to two molecular groups. There is no also agreement between the two molecular groups and the two morphological groups formed according to the hairiness condition of the receptacle of species. Due to the lack of molecular significance of their receptacles with or without hair, dividing of the subgenus Artemisia species into new subgenera or sections was not considered appropriate. Likewise, it has been found that with or without hair on the corolla lobes of the central hermaphrodite disc flowers have no molecular significance. It was found that there were no gene flow and hybridization between the 14 species of the subgenus Artemisia and these 14 species were found completed their speciation. This study is important as it is the first molecular based study relating with belong to subgenus Artemisia species growing naturally in Turkey. In addition, new haplotypes related to the populations of Turkey belonging to the subgenus Artemisia taxa were reported by us for the first time and added to the GenBank database.

Resumo O gênero Artemisia L. da família Asteraceae é sistematicamente muito complexo. O objetivo deste estudo foi avaliar as posições taxonômicas de táxons do subgênero Artemisia pertencentes ao gênero Artemisia na Turquia usando algumas técnicas moleculares. Neste estudo molecular, 44 indivíduos pertencentes a 14 espécies do subgênero Artemisia foram examinados. As análises foram realizadas no conjunto de dados combinado usando máxima parcimônia, máxima verossimilhança e inferência bayesiana e parâmetros moleculares obtidos a partir de coavaliações de sequências das regiões psbA-trnH, ITS e ETS de indivíduos examinados foram usados ​​no desenho da árvore filogenética. De acordo com os resultados deste estudo, dois grupos moleculares foram formados com base na similaridade da sequência de DNA das espécies, mas não há caracteres morfológicos óbvios correspondentes a dois grupos moleculares. Também não há concordância entre os dois grupos moleculares e os dois grupos morfológicos formados de acordo com a condição de pilosidade do receptáculo da espécie. Devido à falta de significado molecular de seus receptáculos com ou sem cabelo, a divisão das espécies do subgênero Artemisia em novos subgêneros ou seções não foi considerada apropriada. Da mesma forma, verificou-se que com ou sem cabelo nos lobos da corola das flores do disco hermafrodita central não tem significado molecular. Constatou-se que não houve fluxo gênico e hibridização entre as 14 espécies do subgênero Artemisia e essas 14 espécies concluíram sua especiação. Este estudo é importante porque é o primeiro estudo de base molecular relacionado com espécies pertencentes ao subgênero Artemisia crescendo naturalmente na Turquia. Além disso, novos haplótipos relacionados às populações da Turquia pertencentes ao subgênero Artemisia taxa foram relatados por nós pela primeira vez e adicionados ao banco de dados do GenBank.

An investigation on the chloroplast and nuclear genomes of taxa belong to the subgenus Dracunculus (Bess) Rydb. of Artemisia L (Asteraceae) in Turkey / Uma investigação sobre o cloroplasto e os genomas nucleares de táxons pertencentes ao subgênero Dracunculus (Bess) Rydb. de Artemisia L (Asteraceae) na Turquia

Artemisia is one of the biggest genera in the family Asteraceae, with around 500-600 taxa at specific and sub specific levels and organised in 5 subgenera. Due to the high number of taxa, a lot taxonomists are trying to solve the problem of its classification and phylogeny but its natural classification still hasn't been achieved. In this research, 60 individuals belonging to 4 taxa of the subgenus Dracunculus of Artemisia L. in Turkey were examined. For all the examined individuals from both the same and different populations belonging to the taxa of the subgenus Dracunculus, the sequences of the regions both psbA-trnH of chloroplast DNA and ITS of nuclear DNA were determined. Also, the gene regions obtained were recorded in the NCBI GenBank database and an accession number was taken. It was found that there was no gene flow and hybridization between the four studied taxa of the subgenus Dracunculus, and these 4 taxa also completed their speciation. According to the results of this molecular study, A. campestris var. campestris, A. campestris var. marschalliana and A. campestris var. araratica were proposed to be raised from the variety level to the species level. This research is important as it is the first molecular based study relating with the subgenus Dracunculus growing in Turkey.

Artemisia é um dos maiores gêneros da família Asteraceae, com cerca de 500 a 600 táxons em níveis específicos e subespecíficos e organizados em cinco subgêneros. Em razão do grande número de táxons, muitos taxonomistas estão tentando resolver o problema de sua classificação e filogenia, mas sua classificação natural ainda não foi alcançada. Nesta pesquisa, 60 indivíduos pertencentes a quatro táxons do subgênero Dracunculus de Artemisia L. na Turquia foram examinados. Para todos os indivíduos examinados de populações iguais e diferentes pertencentes aos táxons do subgênero Dracunculus, foram determinadas as sequências das regiões psbA-trnH do DNA do cloroplasto e ITS do DNA nuclear. Além disso, as regiões gênicas obtidas foram registradas no banco de dados do NCBI GenBank e um número de acesso foi obtido. Foi constatado que não houve fluxo gênico nem hibridização entre os quatro táxons estudados do subgênero Dracunculus, os quais também completaram sua especiação. De acordo com os resultados deste estudo molecular, A. campestris var. campestris, A. campestris var. marschalliana e A. campestris var. araratica foram propostos para ser elevados do nível de variedade para o nível de espécie. Esta pesquisa é importante porque é o primeiro estudo de base molecular relacionado com o subgênero Dracunculus em crescimento na Turquia.

An investigation on the chloroplast and nuclear genomes of taxa belong to the subgenus Dracunculus (Bess.) Rydb. of Artemisia L. (Asteraceae) in Turkey

Abstract Artemisia is one of the biggest genera in the family Asteraceae, with around 500-600 taxa at specific and sub-specific levels and organised in 5 subgenera. Due to the high number of taxa, a lot taxonomists are trying to solve the problem of its classification and phylogeny but its natural classification still hasnt been achieved. In this research, 60 individuals belonging to 4 taxa of the subgenus Dracunculus of Artemisia L. in Turkey were examined. For all the examined individuals from both the same and different populations belonging to the taxa of the subgenus Dracunculus, the sequences of the regions both psbA-trnH of chloroplast DNA and ITS of nuclear DNA were determined. Also, the gene regions obtained were recorded in the NCBI GenBank database and an accession number was taken. It was found that there was no gene flow and hybridization between the four studied taxa of the subgenus Dracunculus, and these 4 taxa also completed their speciation. According to the results of this molecular study, A. campestris var. campestris, A. campestris var. marschalliana and A. campestris var. araratica were proposed to be raised from the variety level to the species level. This research is important as it is the first molecular based study relating with the subgenus Dracunculus growing in Turkey.

Resumo Artemisia é um dos maiores gêneros da família Asteraceae, com cerca de 500 a 600 táxons em níveis específicos e subespecíficos e organizados em cinco subgêneros. Em razão do grande número de táxons, muitos taxonomistas estão tentando resolver o problema de sua classificação e filogenia, mas sua classificação natural ainda não foi alcançada. Nesta pesquisa, 60 indivíduos pertencentes a quatro táxons do subgênero Dracunculus de Artemisia L. na Turquia foram examinados. Para todos os indivíduos examinados de populações iguais e diferentes pertencentes aos táxons do subgênero Dracunculus, foram determinadas as sequências das regiões psbA-trnH do DNA do cloroplasto e ITS do DNA nuclear. Além disso, as regiões gênicas obtidas foram registradas no banco de dados do NCBI GenBank e um número de acesso foi obtido. Foi constatado que não houve fluxo gênico nem hibridização entre os quatro táxons estudados do subgênero Dracunculus, os quais também completaram sua especiação. De acordo com os resultados deste estudo molecular, A. campestris var. campestris, A. campestris var. marschalliana e A. campestris var. araratica foram propostos para ser elevados do nível de variedade para o nível de espécie. Esta pesquisa é importante porque é o primeiro estudo de base molecular relacionado com o subgênero Dracunculus em crescimento na Turquia.

An investigation on the chloroplast and nuclear genomes of taxa belong to the subgenus Dracunculus (Bess.) Rydb. of Artemisia L. (Asteraceae) in Turkey / Uma investigação sobre o cloroplasto e os genomas nucleares de táxons pertencentes ao subgênero Dracunculus (Bess.) Rydb. de Artemisia L. (Asteraceae) na Turquia

Artemisia is one of the biggest genera in the family Asteraceae, with around 500-600 taxa at specific and subspecific levels and organised in 5 subgenera. Due to the high number of taxa, a lot taxonomists are trying to solve the problem of its classification and phylogeny but its natural classification still hasn't been achieved. In this research, 60 individuals belonging to 4 taxa of the subgenus Dracunculus of Artemisia L. in Turkey were examined. For all the examined individuals from both the same and different populations belonging to the taxa of the subgenus Dracunculus, the sequences of the regions both psbA-trnH of chloroplast DNA and ITS of nuclear DNA were determined. Also, the gene regions obtained were recorded in the NCBI GenBank database and an accession number was taken. It was found that there was no gene flow and hybridization between the four studied taxa of the subgenus Dracunculus, and these 4 taxa also completed their speciation. According to the results of this molecular study, A. campestris var. campestris, A. campestris var. marschalliana and A. campestris var. araratica were proposed to be raised from the variety level to the species level. This research is important as it is the first molecular based study relating with the subgenus Dracunculus growing in Turkey.

Artemisia é um dos maiores gêneros da família Asteraceae, com cerca de 500 a 600 táxons em níveis específicos e subespecíficos e organizados em cinco subgêneros. Em razão do grande número de táxons, muitos taxonomistas estão tentando resolver o problema de sua classificação e filogenia, mas sua classificação natural ainda não foi alcançada. Nesta pesquisa, 60 indivíduos pertencentes a quatro táxons do subgênero Dracunculus de Artemisia L. na Turquia foram examinados. Para todos os indivíduos examinados de populações iguais e diferentes pertencentes aos táxons do subgênero Dracunculus, foram determinadas as sequências das regiões psbA-trnH do DNA do cloroplasto e ITS do DNA nuclear. Além disso, as regiões gênicas obtidas foram registradas no banco de dados do NCBI GenBank e um número de acesso foi obtido. Foi constatado que não houve fluxo gênico nem hibridização entre os quatro táxons estudados do subgênero Dracunculus, os quais também completaram sua especiação. De acordo com os resultados deste estudo molecular, A. campestris var. campestris, A. campestris var. marschalliana e A. campestris var. araratica foram propostos para ser elevados do nível de variedade para o nível de espécie. Esta pesquisa é importante porque é o primeiro estudo de base molecular relacionado com o subgênero Dracunculus em crescimento na Turquia.

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 Tibetan Codonopsis Plants Based on ITS2 and psbA-trnH Barcode / 中国实验方剂学杂志

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.

DNA barcoding identification of commercial decoctions in traditional Chinese medicine / 药学学报

Although the guiding principles for molecular identification of traditional Chinese medicines (TCM) using DNA barcoding have been recorded in the Chinese Pharmacopoeia, there is still a lack of systematic research on its application to commercial TCM decoctions. In this study, a total of 212 commercial TCM decoctions derived from different medicinal parts such as root and rhizome, fruit and seed, herb, flower, leaf, cortex, and caulis were collected to verify applicability and accuracy of the method. DNA barcodes were successfully obtained from 75.9% (161/212) of the samples, while other samples failed to be amplified due to genomic DNA degradation. Among the 161 samples, 85.7% of them were identified as recorded species in the Chinese Pharmacopoeia (2020 edition). In addition, 14 samples could be identified as species recorded in the Chinese Pharmacopoeia and their closely related species in the same genus. Morphological identification for the unconfirmed samples showed that eight were genuine species and three were adulterants, while the other three were unidentifiable due to lack of morphological characteristics. Furthermore, the DNA barcodes of seven samples accurately mapped to the sequences of adulterants. Remarkably, counterfeit products were detected in two samples. These results demonstrate that DNA barcoding is suitable for the identification of commercial TCM decoctions. The method can effectively detect adulterants and is appropriate for use throughout the industrial chain of TCM production and distribution, and by the supervisory agencies as well.

Molecular identification of Spatholobi Caulis and its adulterants based on DNA barcoding / 中草药

Objective: Molecular biology identification technology was used to screen the appropriate DNA barcoding to establish a fast and accurate method for identifying Spatholobi Caulis. Methods: A total of 72 samples of Spatholobi Caulis and its adulterants were collected, the sample DNA was extracted, the ITS2, matK, psbA-trnH, ITS, and rbcL sequences were amplified and sequenced, and the amplification success rate and sequencing success rate of each sequence were calculated. The alignment of all sequences was determined by MEGA 7.0 software, and the interspecies and intraspecific genetic distance of them were analyzed to evaluate the Barcoding gap, based on the Kimura-2-Parameter (K2P) two-parameter model. Phylosuite software was used to construct the ITS2, matK and psbA-trnH and multi-gene (I-M-P) phylogenetic tree. Results: The amplification success rate and sequencing success rate of ITS2 were the highest (100%), and the sequencing success rates of matK and psbA-trnH were 94.4% and 91.7% respectively, while rbcL and ITS were only 69.4% and 61.1%. Compared with other barcoding, ITS2 has obvious Barcoding gap, and there was less overlap tree showed that ITS2 and psbA-trnH can obviously cluster Spatholobi Caulis and its adulterants into different branches, while matK cannot separate Kadsurae Caulis and Schisandrae Sphenantherae Fructus. I-M-P phylogenetic tree had the same result as ITS2 and psbA-trnH. between species. Conclusion: The identification method based on ITS2 and supplemented by the psbA-trnH sequence can quickly and accurately identify S. Caulis and its adulterants, which can provide the basis for the safety and the accuracy of the clinical application.

Phylogenetic and mutation point analysis of DNA barcoding sequences in Polygonatum cyrtonema / 中草药

Objective: In this work, phylogenetic analysis was used to compare the ITS2 and psbA-trnH sequences of Polygonatum cyrtonema samples from different geographical sources, so as to explore the genetic diversity and genetic relationship of these resources. Methods: PCR method was used to amplify the regions of ITS and psbA-trnH, and the sequences of ITS2 and psbA-trnH were obtained after the amplified fragment sequences were blasted in NCBI database. The neighbor joining (NJ) and maximum parsimony (MP) methods were used to construct phylogenetic trees and Kimura two-parameter (K2-P) model was used to calculate the genetic distance of different samples. Mega and DNAman softwares were applied for mutiple alignment of ITS2 and psbA-trnH sequences of 25 samples of P. cyrtonema. Results: The lengths of ITS2 and psbA-trnH sequences of Anhui Qingyang and Fujian Taining samples of P. cyrtonema were 224 bp and 620 bp, respectively. The lengths of ITS2 and psbA-trnH of the remaining 24 samples were 225 bp and 621 bp, respectively. ITS2 and psbA-trnH had seven and four mutation points, respectively. These 25 samples were clustered into two groups based on ITS2 sequences. Five samples in Hunan and Guizhou were clustered into one group, while the other 20 samples were clustered into another group. The genetic distance showed that the samples from Huaxi and Jianhe in Guizhou Province and Jianyang in Fujian Province had the largest genetic distance. Phylogenetic tree constructed by psbA-trnH sequences were unable to distinguish 25 samples from different geographical sources. Conclusion: Phylogenetic and mutation analysis will provide the theoretic foundation to utilize the resources of P. cyrtonema, investigate their evolution, and evaluate their genuineness. The results of mutation point will also be used in the identification of related P. cyrtonema resources.

Identification of medicinal plants within the Apocynaceae family using ITS2 and psbA-trnH barcodes / 中国天然药物

To ensure the safety of medications, it is vital to accurately authenticate species of the Apocynaceae family, which is rich in poisonous medicinal plants. We identified Apocynaceae species by using nuclear internal transcribed spacer 2 (ITS2) and psbA-trnH based on experimental data. The identification ability of ITS2 and psbA-trnH was assessed using specific genetic divergence, BLAST1, and neighbor-joining trees. For DNA barcoding, ITS2 and psbA-trnH regions of 122 plant samples of 31 species from 19 genera in the Apocynaceae family were amplified. The PCR amplification for ITS2 and psbA-trnH sequences was 100%. The sequencing success rates for ITS2 and psbA-trnH sequences were 81% and 61%, respectively. Additional data involved 53 sequences of the ITS2 region and 38 sequences of the psbA-trnH region were downloaded from GenBank. Moreover, the analysis showed that the inter-specific divergence of Apocynaceae species was greater than its intra-specific variations. The results indicated that, using the BLAST1 method, ITS2 showed a high identification efficiency of 97% and 100% of the samples at the species and genus levels, respectively, via BLAST1, and psbA-trnH successfully identified 95% and 100% of the samples at the species and genus levels, respectively. The barcode combination of ITS2/psbA-trnH successfully identified 98% and 100% of samples at the species and genus levels, respectively. Subsequently, the neighbor joining tree method also showed that barcode ITS2 and psbA-trnH could distinguish among the species within the Apocynaceae family. ITS2 is a core barcode and psbA-trnH is a supplementary barcode for identifying species in the Apocynaceae family. These results will help to improve DNA barcoding reference databases for herbal drugs and other herbal raw materials.

Use of ITS2 and psbA-trnH DNA barcode sequences of wild Phytolacca resources in Guanzhong area of Shaanxi Province / 药学学报

In order to explore the use of DNA barcode in the identification of wild Phytolacca resources in the Shaanxi Guanzhong area, 29 DNA samples were amplified and sequenced by using the universal primers ITS2 and psbA-trnH. The sequences were spliced and proof-read by Codon CodeA aligner V3.0, followed by blast comparison and identification analysis; mega 6.0 was used to analyze sequence characteristics, Kimura 2-Parameter (K2P) was used to analyze distance and intraspecific or interspecific variation, and Neighbor-Joining trees were established to evaluate the ability of two pairs of candidate sequences to distinguish Phytolaccae Radix from its adulterants. The results showed that the success rate of PCR amplification and sequencing of ITS2 and psbA-trnH was 100%; the NJ tree showed that both ITS2 and psbA-trnH sequences could separate P. acinosa, P. americana, other species of the same genus like P. japonica, P. exiensis and two adulterant species into a single clade; primer ITS2 had an advantage over psbA-trnH in determining interspecific genetic distances. Therefore, both ITS2 and psbA-trnH sequences can be used for identification of Phytolacca and their adulterants, which provides a theoretical basis for the distribution of wild Phytolacca resources and their rational development and utilization.

Cluster Analysis of Ziziphora bungeana from Different Geographical Populations Based on ITS2 and psbA-trnH Sequences / 中国实验方剂学杂志

Objective:To analyze the internal transcribed spacer（ITS）2 and psbA-trnH sequences of Ziziphora bungeana in 18 different geographic populations,in order to provide reference about evaluation of germplasm resources and analysis of genetic diversity of medicinal plants. Method:Genomic DNAs of the Z. bungeana were extracted by kit method. Polymerase chain reaction（PCR）was used to amplify ITS2 and psbA-trnH interstitial region sequences，bidirectional sequencing，splicing，and constructing Neighbor-joining（NJ） based on Kimura 2-parameter（K2P） model. Result:All of sequences of ITS2 and psbA-trnH of Z. bungeana in different geographic populations showed intraspecific variations. The average ITS2 sequence length of Z. bungeana was 236 bp，9 haplotypes were detected，and the genetic distance was 0-0.022. Z. bungeana of different geographical groups gathered into two branches，10 geographic populations,including XTH3，XTH6 and XTH9,were considered as one branch，while 8 geographic populations, including XTH4，XTH5 and XTH10,were the other branch. In addition to XTH6 that lacked 6 in bp psbA-trnH sequence，all of the other geographic populations had a 355 bp sequence of psbA-trnH，4 haplotypes were detected，and the genetic distance was 0-0.023. 12 geographic populations,such as XTH1，XTH3，XTH4,gathered into one branch，while XTH14，XTH17 and XTH18 gathered into the other branch. NJ tree based on ITS2+psbA-trnH combination sequence showed that Z. bungeana of different geographical populations could be divided into two branches，with 12 geographical populations,like XTH11，XTH12，XTH16 as one branch, and XTH14，XTH17 and XTH18 as the other branch. Conclusion:Near or similar geographical locations of different geographical populations implies relatively short genetic distance and relatively close genetic relationship，which indicates that genetic relationship and genetic diversity of Z. bungeana in different geographical populations are related to geographical locations.

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.

Molecular identification and genetic relationship of Fritillaria cirrhosa and related species based on DNA barcode / 药学学报

Based on the ITS2 and psbA-trnHsequences, molecular biological identification and genetic relationship of Fritillaria cirrhosa with its relative species were carried out. In this paper, the PCR-RFLP method specified by the Chinese Pharmacopoeia was performed on all samples at first. Secondly, the ITS2 and psbA-trnH sequences of all samples were amplified. Then, the amplified products were used to analyze the genetic distance, construct the phylogenetic tree, assess the identification efficiency, and evaluate the genetic relationship as well. The result showed that all the samples were divided into two groups by PCR-RFLP method. The samples in the first group, including Fritillaria ussuriensis, Fritillaria thunbergii and Fritillaria pallidiflora, could not be digested by SmaI, while the other samples in the second group, including Fritillaria mellea, Fritillaria sinica, Fritillaria cirrhosa var. ecirrhosa Franch, Fritillaria unibracteata var. longinectarea and Fritillaria cirrhosa, could be digested by SmaI. Then, ITS2 and psbA-trnH sequences of all samples were obtained. The length of various ITS2 sequences were distributed from 235 to 239 bp, and the average intra- and inter-specific genetic distance were 0.001 and 0.022, respectively. NJ tree showed that all samples were separated into "Northern Fritillaria" group (Fritillaria ussuriensis and Fritillaria pallidiflora) and "Southern Fritillaria" group (Fritillaria thunbergii, Fritillaria mellea, Fritillaria sinica, Fritillaria cirrhosa var. ecirrhosa Franch, Fritillaria unibracteata var. longinectarea and Fritillaria cirrhosa). The latter group could be further divided into Fritillaria thunbergii and Fritillaria cirrhosa subgroup, and the species in Fritillaria cirrhosa subgroup had close phylogenetic relationships. The length of psbA-trnH sequences was distributed from 337 to 373 bp, and the intra- and inter-specific genetic distance were 0.263 and 0.329, respectively. The samples in this paper could not be clustered effectively by NJ tree. This indicated that the ITS2 sequences were not only able to identify Fritillaria cirrhosa with its partial relative species quickly and accurately, but also clarify the relationship between different Fritillaria species. Therefore, it provided an important theoretical foundation for the development of molecular markers, effective protection, and rational development and utilization of Fritillaria resources.

Identification of 23 unknown Li minority medicinal plants based on DNA barcoding / 中国中药杂志

DNA barcode molecular biological technique is used to identify the species of 23 unknown Li minority medicinal plants.DNA was extracted from 23 unknown medicines using the Plant Genomic DNA Extraction kit. The ITS2 and psbA-trnH regions were amplified and sequenced bi-directionally. The Codon Code Aligner V 7. 0. 1 was used to proofread and assemble the contigs and generated consensus sequences. All the sequences were submitted to Traditional Chinese Medicine DNA Barcode Database and NCBI Gen Bank to get information of the species identifications. If the maximum similarity of the identification result is ≥ 97%,exact species can be known. If it is between 97% and 90%,samples' genus can be confirmed; If it is <90%,then we can only confirm its family. Finally there are 17 samples can be identified to species level,5 can be identified to genus level and 1 can be identified to family level. This shows that DNA barcoding used in medicinal plants molecular identification,can identify unknown species rapidly and accurately.

Screening and evaluation of three excellent Paris resources from mountain area of Sichuan Basin / 中草药

Objective Mining Paris resources with medicinal value could underlay the breeding selection and relieve the pressure on wild resources. Methods Twenty-one Paris resources in the mountain area of Sichuan Basin were collected and selected according to their phenotype and rhizome features. Two resources GQ, MH with bigger rhizome and one resource (GK) with polygemmic feature were screened. After preliminary identification, based on Kimura-2-parameter model, molecular phylogenetic trees were constructed based on the different sequence of ITS and psbA-trnH between the screened resources and the homologous sequences from NCBI using the Maximum Like (ML) method. Main active saponin was determinated by HPLC method to predict its potential medicinal value. Results GQ, GK, and MH were special resources of P. polyphylla var. chinensis, P. polyphylla var. yunnanensis, and P. forrestii, respectively, in mountains around Sichuan Basin. The content and proportion of polyphyllin I, II, VI, VII in GQ, GK, MH were different. The total content was GQ > MH > CK > GK. The proportion of polyphyllin I in GQ and MH was 67.61% and 73.25% higher than CK, respectively. While the proportion of polyphyllin VII was most in GK (56.38%). Conclusion This study specified three Sichuan local Paris resources with excellent rhizome features. And they performed well after introduced to Chengdu Plain providing the material basis for the follow-up breeding study of Paris. Three resources have medicinal potential, especially the polyphyllin I and polyphyllin content in MH (P. forrestii) is higher, which can provide a new choice for screening substitution materials of P. polyphylla var. chinensis and P. polyphylla var. yunnanensis.

Sequence analysis of psbA-trnH gene in chloroplast of Lycoris aurea populations from different regions / 中草药

Objective System evolution relationship and molecular identification method of the germplasm resources of Lycoris aurea from different regions was analyzed based on the sequence of psbA-trnH chloroplast gene. Methods DNA samples of 52 L. aurea populations were extracted from 15 provinces or cities in China. The psbA-trnH sequences of the populations were amplified by PCR, and the purified PCR products were sequenced and analyzed by Mega 5.0 software etc. Results The length of psbA-trnH sequences were 544-656 bp, and GC content of them was 35.8%-37.0%, and the genetic distances among the populations were 0-0.009 47. There were 33 variable (polymorphic) sites, including nine parsimony informative sites and 18 singleton variable sites and six insertion/deletion gaps. Ten haplotypes (H) were identified. Values of haplotype diversity (Hd) and nucleotide diversity (π) were 0.749 and 0.002 63, respectively. The genetic diversity of the populations of L. aurea were very high. In the maximum parsimony phylogenetic tree, 52 populations of L. aurea were clustered into four branches, which was almost consistent with their geographical distributions. Conclusion The genetic variation of L. aurea populations from different regions is significant and the psbA-trnH sequence could be used as a molecular evidence for identifying the germplasm resources of L. aurea from different regions. There is very obvious regional characteristics in evolution for germplasm resources of L. aurea in China.

Identification of Fallopia multiflora by allele-specific polymerase chain reaction / 中草药

Objective :To establish a rapid molecular identification method for Fallopia multiflora and its adulterants. Methods: Based on psbA-trnH sequences of F. multiflora and its adulterants, the SNP site was searched and the specific primers were designed. The allele-specific PCR amplification of F. multiflora and its adulterants from different producing areas was carried out and the reaction system was optimized. Results: When the annealing temperature was raised to 48 ℃ with 30 cycle number, only the template DNA of F. multiflora could be amplified to obtain the specific 191 bp band whereas the diagnostic PCRs of the other adulterants were all negative. Conclusion: It’s simple and reliable to identify the authenticity of F. multifloa by allele loci specific PCR.