First Record of Elasmobranch Hosts for the Gnathiid Isopod Crustacean Thaumastognathia: Description of Thaumastognathia bicorniger sp. nov.

Gnathiid larvae (Crustacea; Isopoda; Gnathiidae) infesting elasmobranch and holocephalan fishes from mainly bathyal depths off Suruga Bay, off Kume-jima Island, and five sites from off Tokyo Bay to Shimoda City, Japan were examined. A total of 1460 gnathiid larvae were sampled from 87 host individuals belonging to seven families and 10 species. The morphology of these larvae was distinguishable from other gnathiid species by the head appendages. These larvae presented two pigmentation patterns, stripes or spots, on their dorsal thoraxes in live specimens. Furthermore, they were determined as the second and third stage praniza larvae on the basis of allometric variance of maximum head and abdomen widths. A third stage praniza with stripe pigmentation metamorphosed into an adult male and could be identified as a new species of the genus Thaumastognathia Monod, 1926. The duration between detachment from the host and metamorphosis into male adult required 204 days. This paper describes Thaumastognathia bicorniger sp. nov. on the basis of P3/stripe larvae and the male adult. This report is the first record of the larva and host information for a species of Thaumastognathia.

Description of a New Freshwater Ciliate Epistylis wuhanensis n. sp. (Ciliophora, Peritrichia) from China, with a Focus on Phylogenetic Relationships within Family Epistylididae.

Two populations of Epistylis wuhanensis n. sp., a new freshwater peritrich ciliate, were isolated from different freshwater ponds located in Hubei, China. Their morphological characteristics were investigated using live observation, protargol impregnation, and scanning electron microscopy (SEM). Specimens from the two populations showed identical arrangement of the infraciliature and identical small subunit ribosomal RNA (SSU rRNA) gene and ITS1-5.8S-ITS2 sequences. The zooids present bell-shaped and 90-175 × 27-54 µm in vivo. Macronucleus is variable in shape and located in the middle of cell. Pellicle is usually smooth with 139-154 and 97-105 striations above and below the trochal band, respectively. SSU rRNA gene and ITS1-5.8S-ITS2 sequences of E. wuhanensis n. sp. did not match any available sequences in GenBank. Phylogenetically, E. wuhanensis n. sp. clusters with the other Epistylis within the family Epistylididae, but is distinct from the major clades of Epistylis. Above all, the morphological characteristics and molecular analyses support that the present Epistylis is a new species. Expanded phylogenetic analyses of sessilids based on both SSU rRNA gene sequences and ITS1-5.8S-ITS2 sequences reveal that the genus Epistylis consists of Epistylis morphospecies and taxonomic revision of the genus is needed.

Exploring the northern limit of the distribution of Saccharomyces cerevisiae and Saccharomyces paradoxus in North America.

We examined the northern limit of Saccharomyces cerevisiae and Saccharomyces paradoxus in northeast America. We collected 876 natural samples at 29 sites and applied enrichment methods for the isolation of mesophilic yeasts. We uncovered a large diversity of yeasts, in some cases, associated with specific substrates. Sequencing of the ITS1, 5.8S and ITS2 loci allowed to assign 226 yeast strains at the species level, including 41 S. paradoxus strains. Our intensive sampling suggests that if present, S. cerevisiae is rare at these northern latitudes. Our sampling efforts spread across several months of the year revealed that successful sampling increases throughout the summer and diminishes significantly at the beginning of the fall. The data obtained on the ecological context of yeasts corroborate what was previously reported on Pichiaceae, Saccharomycodaceae, Debaryomycetaceae and Phaffomycetaceae yeast families. We identified 24 yeast isolates that could not be assigned to any known species and that may be of taxonomic, medical, or biotechnological importance. Our study reports new data on the taxonomic diversity of yeasts and new resources for studying the evolution and ecology of S. paradoxus.

Extensive Countrywide Molecular Identification and High Genetic Diversity of Haemonchus spp. in Domestic Ruminants in Greece.

The gastrointestinal nematode parasite Haemonchus spp. is one of the most pathogenic parasites of ruminants, due to its blood-sucking activity, which causes large economic losses in the ruminant industry. The latest epizootiological data recorded an increase in the infection, not only in Greece but also in other countries, mainly attributed to climatic changes. The study of the population structure and the investigation of the phylogenetic relationships of Haemonchus spp. are essential for the understanding of its biology and epizootiology to implement appropriate control and prevention strategies. In addition, the molecular approach allows the determination of evolutionary relationships between different species of this parasite, the diverse hosts they infect, as well as the different geographic compartments from which they originate. Therefore, the aim of the present study was to identify the species of the sympatric populations of the genus Haemonchus, a nematode parasite infecting ruminants (sheep, goats, cattle, and buffaloes) from different regions of Greece (continental and insular) using molecular methods. At the same time, an attempt was made to identify the possible subpopulations of Haemonchus spp. in Greece, to investigate their phylogenetic relationships, as well as to determine the genetic diversity of each population. A total of 288 worms of the genus Haemonchus were processed using molecular methods; of these, 96 were collected from sheep, 96 from goats, 48 from cattle, and finally, 48 from buffaloes. A fragment of 321 base pairs of the second internal transcribed spacer (ITS2) sequence of nuclear DNA was amplified for species identification, and, after basic local alignment search tool (Blast) analysis, it was revealed that they belonged to H. contortus. A fragment of 820 base pairs of subunit 4 of the nicotinamide dehydrogenase (ND4) gene of mitochondrial DNA was amplified for genetic diversity analysis. The Greek mitochondrial ND4 sequences of H. contortus were classified into 140 haplotypes, and the values of the average nucleotide and haplotype diversity were lower compared to the respective values derived from Italy, Malaysia, the USA, and China. The phylogenetic analysis of the ND4 gene revealed a clear grouping of the Greek haplotypes when compared with Asian ones, and, at the same time, there was no profound grouping of the same haplotypes with regard to their different hosts and geographical origin within different regions of Greece. The aforementioned findings confirmed that H. contortus prevails in our country and can infect all species of ruminants, without geographical boundaries, when the right conditions (i.e., common grazing) are created.

Updated distribution of anopheline mosquitoes in Hokkaido, Japan, and the first evidence of Anopheles belenrae in Japan.

BACKGROUND: In Hokkaido, northern island of Japan, at least seven cases of falciparum malaria were reported by 1951. A survey conducted at that time was unsuccessful in implicating any mosquito species as the possible vector. Although active anopheline mosquito surveillance continued until the middle of the 1980s, there is very limited information on their current status and distribution in Japan. Therefore, this study is an update on the current status and distribution of anopheline mosquitoes in Hokkaido based on a 15-year entomological surveillance between 2001 and 2015. METHODS: A survey of mosquitoes was conducted at 22 sites in Hokkaido, Japan, from 2001 to 2015. Adult mosquitoes were collected from cowsheds, lakesides, shrubs, and habitats ranging from open grassland to coniferous forest using a Centers for Disease Control and Prevention (CDC) miniature light trap enhanced with dry ice, aspirators, and sweeping nets. Larvae were collected from lakes, ponds, swamps, stagnant and flowing rivers, and paddy fields. All specimens were morphologically identified and subjected to polymerase chain reaction (PCR)-based sequence analysis of the internal transcribed spacer 2 ( ITS2) region of rDNA. Phylogenetic trees were reconstructed using the neighbor-joining method with the Kimura 2-parameter model on MEGA X version 10.2.2. RESULTS: A total of 46 anopheline specimens were used for the phylogenetic analysis. During the survey, a new member of the Anopheles hyrcanus group, An. belenrae, was discovered in eastern Hokkaido in 2004. Anopheles belenrae has since then been consistently found and confirmed to inhabit only this area of Japan. Four members of the An. hyrcanus group, namely An. belenrae, An. engarensis, An. lesteri, and An. sineroides, have been found in Hokkaido. The results also suggest that An. sinensis, formerly a dominant species throughout Japan, has become a rarely found species, at least currently in Hokkaido. CONCLUSION: The updated distribution of anopheline mosquitoes in Hokkaido, Japan, showed considerable differences from that observed in previous surveys conducted from 1969 to 1984. In particular, areas where An. sinensis was previously distributed may have been greatly reduced in Hokkaido. The phylogenetic analysis revealed a novel An. hyrcanus group member identified as An. belenrae, described in South Korea in 2005. It is interesting that An. belenrae was confirmed to inhabit only eastern Hokkaido, Japan.

Genetic diversity and biological activity of Curcuma longa ecotypes from Rapa Nui using molecular markers.

Curcuma Longa (CL) has been used for hundreds of years by native people from Rapa Nui for the treatment of different illness. Despite this plant was introduced from Polynesia or India, there is still scarce information about its origin. The objective of this study was to analyze the genetic variation of three CL ecotypes based on molecular phylogenetic and genotypification using internal transcribed spacer 2 (ITS2) and simple sequence repeats (SSR). Antioxidant and anti-inflammatory properties of rhizomes and leaves extracts of three CL plants were analyzed by spectrophotometric methods and cyclooxygenase 2 (COX-2) inhibition assay. Complementarily, we predicted the potential binding mode and binding energy of curcuminoids and nonsteroidals anti-inflammatory drugs (NSAIDs) into COX-2 via molecular docking. The ITS2 sequence shows two major clusters (I and II), group I consisted of Curcuma haritha and group II consisted of different species of Curcuma and Rapa Nui samples (MR-1, MR-2 and RK-2). Results of SSR markers show that genotype MR-2 was similar to MR-1 and RK-2 with 70.8 and 42.9% similarity, whereas genotype was similar to RK-2, MR-1 and MR-2 with 63.9, 43.2 and 42.9% similarity, respectively. MR-1 have better antioxidant and autoinflammatory activity than rest of CL samples due to its high concentration of polyphenols (33.68 mg/g) and curcumin (29.69 mg/g). Furthermore, docking results show that three curcuminoids of CL and selective NAIDs, as celecoxib, etodolac and meloxicam, share the same binding pocket into COX-2. However, three curcuminoids have a lower ΔGbinding than other COX-2 selective NAIDs as etodolac and meloxicam, except for Coxib family as valdecoxib, celecoxib and rofecoxib. Our findings suggest MR-1, MR-2 and MK-2 from Germplasm Bank (Mataveri Otai of CONAF) are closely related to Curcuma amada and Curcuma montana even though they have genetic variability.

Two new colonial peritrich ciliates (Ciliophora, Peritrichia, Sessilida) from China: With a note on taxonomic distinction between Epistylididae and Operculariidae.

Two new colonial sessilid species, Opercularia miaoxinensis spec. nov. and Epistylis conica spec. nov., were isolated from the pereopods of freshwater crayfish, Procambarus clarkii in Hubei Province, China from 2016 to 2017. Both species were investigated by living observation, protargol impregnation, and molecular methods. Opercularia miaoxinensis spec. nov. is morphologically characterized by the following characteristics: spindle-shaped zooid, zooid size of 48-74×20-35µm in vivo, contractile vacuole ventrally located above macronucleus, and dichotomously branched stalk. Epistylis conica spec. nov. is characterized by conical zooid shape, zooid size of 56-70×22-39µm in vivo, C-shaped macronucleus with transverse orientation, dorsally located contractile vacuole, and dichotomously branched stalk. To further identify both species, phylogenetic trees were constructed based on small subunit ribosomal DNA (SSU rDNA) and ITS1-5.8S-ITS2 sequences. Both results showed that E. conica spec. nov. was part of a clade consisting of the majority of Epistylis species. Surprisingly, O. miaoxinensis spec. nov. also clustered within this large clade of Epistylis species and had a distant relationship with the other Opercularia species. These findings challenged the distinguishing morphological characteristics between Epistylididae and Operculariidae.

Identification of seeds based on molecular markers and secondary metabolites in Senna obtusifolia and Senna occidentalis.

BACKGROUND: Senna obtusifolia and Senna occidentalis (Leguminosae), whose seeds have similar appearance and chemical constituents, are easily confused in using their seeds. To elucidate the similarities and differences between S. obtusifolia seeds and S. occidentalis seeds, three molecular markers and high performance liquid chromatography (HPLC) were employed to evaluate the seeds characteristics of these two medicinal herbs. RESULTS: The results showed that selected 3 ISSR and 7 SCoT primers could distinguish S. obtusifolia seeds from S. occidentalis seeds based on the specific band and UPGMA dendrogram. ITS2 sequence indicated that the intra-specific similarity of 20 S. obtusifolia and 16 S. occidentalis was 99.79 and 100.0%, respectively, while the inter-specific similarity between S . obtusifolia and S. occidentalis was 89.58%. Although phylogenetic analysis revealed that these two species had a close relationship, they were assigned to different branches. HPLC fingerprint results showed that seeds of S. obtusifolia and S. occidentalis shared some secondary metabolites, but aurantio-obtusin was not detected in S. occidentalis seeds which could differentiate S. obtusifolia seeds from S. occidentalis seeds. CONCLUSIONS: The present study not only compared the seeds characters of S. obtusifolia and S. occidentalis from molecular and secondary metabolites levels, but also provided a convenient method to identify S. obtusifolia seeds and S. occidentalis seeds effectively.

Characterization of brewing microorganisms isolated from Korean traditional nuruk for Cheongju production.

The objective of this study was to select and identify yeasts and molds isolated from traditional nuruk and to investigate their brewing characteristics for Cheongju production. The yeast strains Y190 (Accession ID-KACC 93251P), Y263 (Accession ID-KACC 93252P), and Y270 (Accession ID-KACC 93253P) showing high alcohol and flavor productivity were isolated and identified by phylogenetic inference based on an internal transcribed spacer 2 region sequence analysis. In addition, Aspergillus oryzae 83-10 (Accession ID-KACC 93254P) showing the highest enzyme activity was isolated. This study provides basic data for the production of Korean Cheongju and assesses the applicability of these three yeast strains and A. oryzae 83-10 isolated from traditional nuruk.

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.

Identification of Medicinal Aconitum Species Based on ITS2 Sequences and Analysis of Genetic Relationship / 中国实验方剂学杂志

ObjectiveThe internal transcribed spacer （ITS） 2 region of ribosomal gene， a DNA barcode， was employed to identify 12 medicinal Aconitum species and the genetic relationship among the species was analyzed. MethodA total of 30 samples of the 12 species were collected. The DNA was extracted with spin column plant genomic DNA kit and the universal primers of ITS2 sequence were used for polymerase chain reaction （PCR） amplification， followed by electrophoresis detection and bi-directional sequencing. The yielded sequences were aligned and spliced by CodonCode Aligner 17.0 and sequence variation was analyzed by MEGA 7.0. The secondary structure was predicted by ITS2 Database and the neighbor-joining （NJ） method was applied to generate the phylogenetic tree. ResultThe ITS2 sequences of the 12 species were 220-221 bp， with the average guanine and cytosine （GC） content of 64.09%， 140 variable sites， 137 informative sites， and 81 conservative sites. The intraspecific genetic distance （K2P） was smaller than the interspecific genetic distance. According to the secondary structures of ITS2 sequences and NJ cluster analysis， A. scaposum， A. sinomontanum， and A. barbatum had close genetic relationship， while the rest nine showed close kinship， particularly A. soongaricum and A. yinschanicum. ConclusionITS2 sequence is of great value for the molecular identification and genetic relationship determination of Aconitum， which provides a new method for the study of ethnomedicine.

Identification of Astragalus and its adulterants based on ITS2sequence and secondary structure / 药学学报

To effectively identify the Astragalus and its adulterants based on ITS2 sequence and secondary structure, in this study, 32 portions of Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Beg.) Hsiao and Astragalus membranaceus (Fisch.) Bge. collected were conducted ITS2 sequence amplification and bidirectional sequencing, whose results were then spliced by CExpress software remove the 5.8S and 28S sequences at both ends to obtain a complete ITS2 sequence. In addition, 3 ITS2 sequences for each of the adulterants of Astragalus, respectively, Oxytropis coerulea, Caragana sinica, Hedysarum polybotrys, Althaea rosea were downloaded from GenBank. The intra-specific and inter-specific genetic distances were calculated by the software MEGA7 to analyze the difference of each sequence; the Neighbor-joining (NJ) method was used to construct the phylogenetic tree based on ITS2 sequence (primary structure) as well as joint ITS2 sequence and its secondary structure. The results showed that the average ITS2 sequence length of both A. mongolicus and A. membranaceus was 216 bp, and their average GC content was 50.00% and 50.46%, respectively. The similarity of ITS2 sequence length and GC content between the two kind of Astragalus and Oxytropis coerulea was the highest, while the ITS2 sequence length and GC content of Althaea rosea showed great differences with those of Astragalus. The inter-specific distance between Astragalus and Oxytropis coerulea was the smallest, while that between the medicinal Astragalus and Hedysarum polybotrys, Caragana sinica as well as Althaea rosea was great. The phylogenetic trees constructed based on the ITS2 sequence (primary structure) and joint ITS2 sequence and its secondary structure showed that the topological relations of the two phylogenetic trees were basically the same, and both could effectively identify the Astragalus and its adulterants. What’s more, the addition of secondary structure information made end branch of the phylogenetic tree become more in its construction, and the distinguish ability and approval rating were also improved, which further reflected the genetic relationship of Astragalus and its adulterants. This provides some scientific basis for classification and accurate identification of Astragalus and its adulterants.

Molecular authentication of Sarcandra glabra and its adulterants using three DNA molecular markers / 中草药

Objective: To identify Sarcandra glabra and its adulterants using three DNA molecular markers including 18 S rRNA gene, ITS2 sequence and SCAR marker, and then provide the basis for its molecular authentication. Methods: 18 S rRNA gene sequence of S. glabra was obtained by PCR amplification, cloning and sequencing, and then Blast comparison was made in NCBI. The ITS2 sequence of S. glabra was obtained by PCR amplification, sequencing and annotation in ITS2 Database. In the meanwhile, the ITS2 sequences of adulterants and other plants were collected from GenBank. Using MEGA5.5, the genetic distance was calculated between species and then the ITS2 sequences were aligned to construct a phylogenetic clustering tree. SCAR molecular marker of S. glabra was obtained by RAPD. After cloning and sequencing, specific primers were designed to amplify S. glabra and its adulterants. Results: The length of 18 S rRNA obtained in our research was 1 820 bp. Blast comparison revealed that there was 99% homology between S. glabra and Chloranthaceae, which proved to be 18 S rRNA gene of S. glabra. The length of the ITS2 sequence in our research was 500 bp. Genetic distance between S.glabra and its adulterants ranged from 0.190 to 0.219, which was far more than genetic distance among adulterants (0.000-0.074). Cluster analysis showed that S. glabra and its adulterants respectively clustered into a different branch, which was far away from other plants. In our research, we obtained SCAR molecular marker of S. glabra and then a pair of specific primers were designed. Using the pair of specific primers, specific products were amplified from genomic DNA of S. glabra, but no specific products were obtained from that of its adulterants. Conclusion: We could authenticate S. glabra and its adulterants effectively with the combination of three molecular markers for establishing a novel method to identify S. glabra and its adulterants, which provides a new idea for the authentication of S. glabra.

Seeds identification of Panax ginseng and Panax quinquefolium using ITS2 DNA barcordes / 中草药

Objective Traditional identification methods of pharmacognosy is difficult to distinguish the seeds of Panax ginseng and Panax quinquefolius. In order to improve the efficacy and accuracy of identification and provide the scientific foundation for the establishment of Chinese herbal medicine seed quality standards, molecular identification methods of the seeds were established by DNA barcoding technology. Methods The pharmacognostical identification method was used to study the morphological identification and microscopic characters of different seeds. DNA barcodes and Chinese Pharmacopoeia species standard barcode database were employed to identify the seeds by ITS2 sequence comparison, genetic distance comparison and systematic NJ tree construction. Results Intraspecific genetic distances of individuals participating were smaller than interspecific genetic distances. Phylogenetic tree map showed that two species were repectively clustered into one. A total of 42 samples of seeds from Panax ginseng and Panax quinquefolius produced by nine areas were all top-quality and easy to distinguish. Conclusion ITS2 DNA barcodes can identify and differentiate the seeds of P. ginseng and P. quinquefolius germplasm resources quickly, accurately and efficiently.

DNA molecular identification of Chinese herbal materials of Dysosma by ITS2 barcode sequence / 中草药

Objective To explore a new identification method for medicinal materials of Dysosma, and analyze the second internal transcribed spacer (ITS2) barcode sequences of Diphylleia sinensis and Dysosma versipellis, Sinopodophyllum hexandrum, Dysosma difformis and Dysosma pleiantha in five kinds of podophyllum. Methods The ITS2 of ribosomal DNA of medicinal materials of podophyllum was amplified and sequenced by bi-directional sequencing of PCR products. Sequence assembly and consensus sequence generation were performed by using CodonCode Aligner. Phylogenetic study was performed using software MEGA 5.1 in accordance with Kimura-2-parameter (K2P) model. Genetic distances were calculated and analyzed and the phylogenetic tree was constructed by using the neighbor-joining (NJ) method. Results There were significant differences among five kinds of Dysosma. Their maximum intraspecific genetic distance (K2P distance) was far lower than their minimum interspecific genetic distance with the other species. In the cluster dendrogram, all species showed monophyletic. Conclusion ITS2 sequence as DNA barcoding technique can be used to identify Chinese herbal materials of Dysosma.

Identification of Bletilla Striata (Thunb.) Reichb.f.and Bletilla Formosana (Hayata) Schltr.Based on ITS2 Sequence / 世界科学技术-中医药现代化

This study was aimed to identify Bletilla Striata (Thunb.) Reichb.f.and Bletilla Formosana (Hayata) Schltr.by ITS2 sequence.The leaves of 38 samples of Bletilla striata and Bletillaformosana from Yunnan,Hubei,Guizhou,Hunan and Sichuan province were used as experiment materials.The total DNA was extracted.Internal transcribed spacer 2 (ITS2) sequences were obtained by PCR.All of the ITS2 sequences were checked.The 8 ITS2 sequences from two species were downloaded from GenBank.The intraspecific and interspecific Kimura-2-parameter (K2P) distances of Bletilla striata and Bletilla formosana were calculated by MEGAS.0.And neighbor-joining (NJ) tree was constructed.The results showed that the full-length sequences of ITS2 from Bletilla striata and Bletillaformosana were 259 bp,with a total of 14 variable sites.The maximum intraspecific K2P distance of Bletilla striata and Bletillaformosana was 0.008,while the minimum interspecific K2P distance was 0.040.The ITS2 secondary structure showed that different origins of Bletilla striata were gathered together and could be distinguished obviously from Bletilla formosana by NJ tree.It was concluded that ITS2 sequence was able to identify Bletilla striata and Bletillaformosana quickly and accurately.

Molecular Identification of Sparganium Stoloniferum and its Adulterants Based on ITS2 Sequence / 中国药师

Objective:To explore a new method to identify Sparganium stoloniferum and its adulterants by ITS2 regions. Methods:Eight samples of Sparganium stoloniferum and its adulterants were collected with five species, and 6 species with 23 ITS2 sequence of Sparganium stoloniferum and its adulterants were downloaded from Genbank. The intraspecific and interspecific K2P distances of Spar-ganium stoloniferum and its adulterants were calculated by MEGA5. 0, and the phylogenetic tree was constructed by MEGA 5. 0. Re-sults:The maximum intraspecific K2P distance of Sparganium stoloniferum was 0. 038,while the minimum interspecific K2P distance was 0. 697. The phylogenetic tree showed that Plantago asiatica was different obviously from its adulterants. The different samples of Sparganium stoloniferum were gathered together and could be distinguished from its adulterants by the NJ tree. Conclusion: ITS2 se-quence is able to identify Sparganium stoloniferum and its adulterants correctly, which provides a new method for the identification of Sparganium stoloniferum.

Identification of Bupleuri Radix and its adulterants by DNA barcoding / 中草药

Objective: To identify the authenticity of Bupleuri Radix in the market of Hebei province. Methods: In this research, the ITS2 sequence of 28 samples of Bupleuri Radix that were bought from the market in Hebei province was amplified by PCR, and the sequence of seven samples was downloaded from GenBank. Aligner CodonCode software was used for sequence alignment, the MEGA6.0 analysis was used to calculate the K2P distance, and the mutation sites of each sequence was analyzed. Results: Among the 28 Bupleuri Radix samples, there were 21 samples for Bupleurum chinese, 3 for B.smithii, and 4 for the false. Conclusion: ITS2 sequence can be used as an effective method to identify the authenticity of Bupleuri Radix, it is great value to the medicine market standard and the clinical medication safety.

ITS-2 sequences-based identification of Trichogramma species in South America / Identificação molecular de espécies de Trichogramma na América do Sul via sequenciamento da região ITS-2

Abstract ITS2 (Internal transcribed spacer 2) sequences have been used in systematic studies and proved to be useful in providing a reliable identification of Trichogramma species. DNAr sequences ranged in size from 379 to 632 bp. In eleven T. pretiosum lines Wolbachia-induced parthenogenesis was found for the first time. These thelytokous lines were collected in Peru (9), Colombia (1) and USA (1). A dichotomous key for species identification was built based on the size of the ITS2 PCR product and restriction analysis using three endonucleases (EcoRI, MseI and MaeI). This molecular technique was successfully used to distinguish among seventeen native/introduced Trichogramma species collected in South America.

Resumo Sequências do Espaço Transcrito Interno 2 (ITS2) têm sido utilizadas em estudos taxonômicos e sua utilidade constatada pela confiabilidade que o método confere à identificação das espécies de Trichogramma. Esta técnica molecular foi bem sucedida em distinguir dezessete espécies nativas e introduzidas de Trichogramma, coletadas na América do Sul. As sequências do DNAr variaram de 379 a 632 pb. Em 11 linhagens de T. pretiosum estudadas, o endosinbionte Wolbachia foi detectado pela primeira vez. Estas linhagens telítocas foram encontradas no Peru (9), Colômbia (1) e Estados Unidos (1). Uma chave dicotômica para identificação de espécies foi construída baseada no tamanho do produto da PCR do ITS2 e em análises de restrição utilizando-se três endonucleases (EcoRI, MseI and MaeI).

Identification of Pinelliae Rhizoma and Its Adulterants Based On ITS2 Sequence / 世界科学技术-中医药现代化

In this study, the ITS2 sequence was used to identify Pinelliae Rhizoma and its adulterants to ensure its market circulation, clinical effect and safety. All genomic DNA from 59 samples were extracted successfully. The Kimura 2-Parameter (K2P) distances and NJ tree were calculated using software MEGA 6.0. The length of the ITS2 sequence of Pinelliae Rhizoma was 251 bp. The intraspecific genetic distance was smaller than the interspecific ones;The NJ tree indicated that Pinelliae Rhizoma distinguished from its adulterants obviously. The results showed that the ITS2 sequence can be used to distinguish Pinelliae Rhizoma from its adulterants accurately and stably.