Identification of Cordyceps cicadae and Tolypocladium dujiaolongae based on ITS sequences and chemical pattern recognition method / 中国中药杂志

Based on ITS sequences, the molecular identification of Cordyceps cicadae and Tolypocladium dujiaolongae was carried out, and high-performance liquid chromatography(HPLC) fingerprint combined with chemical pattern recognition method was established to differentiate C. cicadae from its adulterant T. dujiaolongae. The genomic DNA from 10 batches of C. cicadae and five batches of T. dujiaolongae was extracted, and ITS sequences were amplified by PCR and sequenced. The stable differential sites of these two species were compared and the phylogenetic tree was constructed via MEGA 7.0. HPLC was used to establish the fingerprints of C. cicadae and T. dujiaolongae, and similarity evaluation, cluster analysis(CA), principal component analysis(PCA), and partial least squares discriminant analysis(PLS-DA) were applied to investigate the chemical pattern recognition. The result showed that the sources of these two species were different, and there were 115 stable differential sites in ITS sequences of C. cicadae and T. dujiao-longae. The phylogenetic tree could distinguish them effectively. HPLC fingerprints of 18 batches of C. cicadae and 5 batches of T. dujiaolongae were established. The results of CA, PCA, and PLS-DA were consistent, which could distinguish them well, indicating that there were great differences in chemical components between C. cicadae and T. dujiaolongae. The results of PLS-DA showed that six components such as uridine, guanosine, adenosine, and N~6-(2-hydroxyethyl) adenosine were the main differential markers of the two species. ITS sequences and HPLC fingerprint combined with the chemical pattern recognition method can serve as the identification and differentiation methods for C. cicadae and T. dujiaolongae.

Specific PCR Identification between Dendrobium huoshanense and Its Adulterants / 中国药学杂志

OBJECTIVE: To design specific identification primers based on ribosomal DNA ITS sequences and accurate identification of Dendrobium huoshanensis and its adultments. METHODS: Phylogenetic tree of D. huoshanense and D. officinale and D. henanense by maximum parsimony (MP) and Bayesian inference (BI) were constructed. Specific primers SH-CP9s /SH-CP9a, SHCP25s / SH-CP25a, SH-CP29s /SH-CP29a for PCR identification of D. huoshanensis based on mutation sites on ITS sequences were designed. RESULTS: In the phylogenetic tree, D. huoshanense and D. officinale and D. henanense were clustered into monophyletic group. The specific PCR reaction procedure of D. huoshanense was obtained by investigating the annealing temperature and the amount of DNA template, and different types of PCR and Taq enzymes. In the PCR product, D. huoshanense appearance of the band, and adultments with no band. CONCLUSION: The ITS sequences can be used as DNA Barcoding identification of D. huoshanensis, and identify D. huoshanense and its adultments by the designed primers.

Revision of the Lichen Genus Stereocaulon (Stereocaulaceae, Ascomycota) in South Korea

Lichen genus Stereocaulon (Schreb.) Hoffm is distributed throughout the world. Although 15 Stereocaulon species have been recorded in Korea, no detailed taxonomic or revisionary research has been conducted for nearly two decades. In this study, we collected 260 putative Stereocaulon spp. samples and identified the species based on morphological, chemical, and molecular characteristics. From the collected samples, 10 species of Stereocaulon were identified, nine of which had already been reported, although this was the first report for the tenth, S. octomerellum Hue, in Korea. General characteristics of Stereocaulon spp. include coralloid phyllocladia and tubercular cephalodia; however, the specimen first collected in Korea was a rare species with tomentum on the pseudopodetia. The specimen of S. octomerellum is characterized by the presence of a primary thallus, granule to short coralloid phyllocladia, and pseudopodetia up to 1 cm in size, with tubercular cephalodia. To determine the phylogeny of the specimens, we compared the ITS sequences of ribosomal DNA and the β-tubulin gene sequences. Phylogenetic analysis showed that the Korean Stereocaulon species were monophyletic and placed in the previous phylogenetic classification. Species of S. intermedium and S. exutum, however, were polyphyletic, and are morphologically variable and widespread species. Overall, we present here detailed morphological and chemical descriptions of each species identified and a revised key of all known Stereocaulon species in South Korea.

ITS1 and ITS2 sequences analysis of seven medicinal plants in Polygala L / 中草药

Objective: To provide the evidence for the molecular identification of seven medicinal plants in Polygala L. Methods: Sequencing of ITS1 and ITS2 genes from Polygala furcata, P. japonica, P. tenuifolia, P. glomerata, P. persicariifolia, P. sibirica, and P. arvensis was performed. Sequence features were compared and analyzed using ClustalX and MEGA 3.1 softwares. Results: In the seven medicinal plants of Polygala L., the length of ITS1 and ITS2 varied in 279-291 bp and 211-219 bp separately, with 232 variable sites and 53 parsimony information sites. The smallest genetic distance was observed between P. tenuifolia and P. sibirica, and the largest existed between P. furcata and P. glomerata revealing their farest genetic relationship. Conclusion: ITS sequences of the seven medicinal plants in Polygala L. can be used as the reference for the molecular identification.

Mushroom Flora of Ulleung-gun and a Newly Recorded Bovista Species in the Republic of Korea

We conducted five times surveys, in June, September and October in 2012; June and September 2013, to catalog the mushroom flora in Ulleung-gun, Republic of Korea. More than 400 specimens were collected, and 317 of the specimens were successfully sequenced using the ribosomal DNA internal transcribed spacer barcode marker. We also surveyed the morphological characteristics of the sequenced specimens. The specimens were classified into 2 phyla, 7 classes, 21 orders, 59 families, 122 genera, and 221 species, and were deposited in the herbarium of Korea National Arboretum. Among the collected species, 72% were saprophytic, 25% were symbiotic, and 3% were parasitic. The most common order was Agaricales (189 specimens, 132 species), followed by Polyporales (47 specimens, 27 species), Russulales (31 specimens, 22 species), Boletales (10 specimens, 7 species), and so on. Herein, we also reported the first Bovista species in Korea, which was collected from Dokdo, the far-eastern island of Korea.

Molecular Identification of Metacordyceps Liangshanensis, Its Adulterants and Its Relative Species Based on DNA Barcode / 世界科学技术-中医药现代化

This study was aimed to identify and distinguish Metacordyceps liangshanensis recorded by the Sichuan Province Traditional Chinese Medicine Standard from its adulterants and its relative species by combining ITS and COI barcode sequences in order to study the feasibility of this new method. After extracting DNA of 28 species of Cordyceps samples, DNA were amplified and sequenced. And then, ITS and COI sequences were received. Codon-Code Aligner V3.7.1 and Mega 5.0 were used to analyze the variable site and construct the N-J tree. The results showed that the minimum ITS inter-specific K-2P distance was relatively higher than the maximum intra-specific K-2P distance. The inter-specific sequence divergence between M. liangshanensis and its adulterants exhibited high while intra-specific sequence divergence exhibited low. And COI one was the same case. N-J tree of both ITS and COI indicated that same genus belonged together and each species belonged to relatively independent branch. It was concluded that based on the ITS and COI gene, the technology of DNA barcode can be an excellent identification of M. liangshanensis, its adulterants and its relative species. It provided technical support for the further research on species molecular identification and phylogenetics of Cordyceps .

Taxonomic Study on the Lichen Genus Xanthoparmelia (Ascomycotina, Parmeliaceae) in Korea

In previous studies investigating the genus Xanthoparmelia, thirteen different species have been reported from South Korea alone. However, there currently has been no revisional study performed until now. To explore the genus Xanthoparmelia, a phenotypic analysis was performed based on morphological, anatomical and chemical characters, while an investigation of Xanthoparmelia phylogeny was based on nuclear ribosomal (nr) DNA ITS sequences. A thorough examination of the specimens deposited in the Korean Lichen Research Institute (KoLRI) confirmed that eight species of Xanthoparmelia occur inside South Korea. Our analysis further confirmed the colors of the lower surface and medullar chemistry are important taxonomic characters in Xanthoparmelia. This study also presents a detailed description of each species and a key to the genus.

Taxonomic Study on the Lichen Genus Cetrelia (Lecanorales, Ascomycota) in South Korea

Seventy-two lichen specimens of Cetrelia collected in South Korea since 2003 were examined by both phenotypic and phylogenetic analyses. The phenotypic analysis was based on morphological and chemical characters, and the phylogenetic analysis was based on nrDNA ITS sequences. The result suggested that the presence and absence of isidia, soredia, lobules and medullar reaction C+ or C- are the important characters in the taxonomy of this genus. Four species of Cetrelia, C. chicitae, C. braunsiana, C. japonica, and C. pseudolivetorum have been identified in this study. Description of each species is presented with morphological and chemical characters. A key to the Cetrelia species is also presented.

Identification of Trapa L. plants along middle-low reaches of Changjiang River by analyzing their DNA sequences / 中草药

Object To analyze the rDNA ITS sequences between wi ld plants and cultivars of Trapa L. and study the utility in p hylogenesis and identification of these two groups. Methods The ITS gene fragments were PCR amplified and sequenced. The rDNA ITS regions w ere analyzed by means of the software of Clustal and Mega 2.0. Result s The rDNA sequences of 234-236 bp ITS1, 220-221 bp ITS2 gene fragment , and 5.8 S rDNA for 164 bp evenly were obtained from ten populations of Trapa L. The intraspecific substitution varies from 0.22% to 2. 94%. The variable sites are 16 while informative sites are six. The phylogenet ic tree based on ITS data was set up by NJ method. Conclusion ITS sequence is a pretty good molecular marker which can identify wild plants of Trapa L. from their cultivars. Diversity of ITS in differen t populations is less at intraspecific level. It is infered that the plants of Trapa L. may be derived from the same population of one species .

Analysis of rDNA ITS sequences of Radix et Rhizoma Salviae Miltiorrhizae and plants of Salvia L / 中草药

Objective To study the genetic diversity of ITS sequences of Radix et Rhizoma Salviae Miltiorrhizae (RRSM) and plants of Salvia L. and analyze the utility of ITS sequences in molecular authentication of RRSM and phylogenetic of plants of Salvia L. Methods The ITS gene fragment was amplified using a pair of primers. The PCR products were purified and sequenced by the methods of Sanger dideoxy. Results The DNA sequence of 222-224 bp ITS1, 220-223 bp ITS2 gene fragments and 5.8S rDNA were obtained from 13 samples of Salvia L. The interspecific substitution varied from 0% to 12.16% at ITS1 and 0.5% to 13.51% at ITS2. The intraspecific substitution of RRSM is 0% to 0.9% at ITS1 and 0% to 0.5% at ITS2. The substitution between group of Salvia L. and outgroup varied from 16.07% to 20.27% at ITS1 and 15.91% to 20.27% at ITS2. The phylogenetic tree based on ITS1 and ITS2 data was set up. Conclusion The ITS1 and ITS2 gene fragments are highly conservative at intraspecific level in RRSM, while they are less conservative at interspecific level in Salvia L. They are least conservative between group of Salvia L. and outgroup. Hence, the sequence of this fragment is a good molecular marker for authentication of RRSM and can be used for further study on the phylogenesis for plants of Salvia L.

Comparison on internal transcribed spacers (ITS) sequences of Tibetan medicine Saussurea medusa and its easily confusable species / 中草药

Object In order to identify the medicine at the molecular level, the internal transcribed spacers (ITS) of Saussurea medusa Maxim and its easily confusable species were sequenced Methods The double stranded DNA was amplified using PCR systems 9 600 kits and sequenced on an ABI 377 automated sequencer from both directions Results The ITS sequences of S medusa of different populations showed no variation, but there existed distinct variation between S medusa and its confusable species Conclusion ITS sequences can be used for the molecular authentication between S medusa and its confusable species