ABSTRACT
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.
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Objective:To accurately identify Bupleurum seeds by traditional morphological identification method combined with DNA barcoding technique. Method:A total of 41 seed samples on the market were collected and 75 ribosomal DNA internal transcribed spacer 2 (ITS2) sequences of 15 varieties were downloaded from the GenBank database as experimental materials. The seeds were measured and observed by stereomicroscope and vernier caliper, and their 1 000-grain weights were calculated. Genomic DNA was extracted from the seeds and used as a template, and ITS2 sequences were amplified using polymerase chain reaction (PCR) and bidirectional sequencing. Species identification was conducted based on BLAST method, neighbor-joining (NJ) phylogenetic tree method, Kimura two-parameter model (K2P) genetic distance method, and secondary structure of ITS2 sequence. Result:There were slight differences in the length, width, cross-section, and 1 000-grain weight among Bupleurum seeds from different origins. The ITS2 sequences of B. chinense seeds had 2 intraspecific variable sites and 3 haplotypes, the maximum intraspecific genetic distance (0.009) was far smaller than the minimum interspecific genetic distance (0.032). B. chinense and B. scorzonerifolium in the NJ phylogenetic tree were clustered into independent branches with good monophyletic property. The secondary structure of ITS2 sequences could make up for the shortcomings of NJ tree in identifying variants. The collected 41 seeds included 30 B. chinense seeds, 3 B. scorzonerifolium seeds, 5 B. falcatum seeds, 2 B. marginatum var. stenophyllum seeds, and 1 B. smithii var. parvifolium seeds. Conclusion:The B. chinense seeds on the market have problems of diverse sources and chaotic origins. Based on the combination of ITS2 gentic barcoding and seed morphological identification, the Bupleurum seeds can be accurately identified, which provides scientific bases for establishing the quality standard of Bupleurum seeds, standardizing the cultivation of B. chinense, and solving the quality problems of B. chinense from the source, and provides a reference for the accurate identification of other medicinal plant seeds or seed medicinal materials.
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Objective:To construct a systematic identification system of Anemonis Flaccidae Rhizoma, and to evaluate the comprehensive quality of Anemonis Flaccidae Rhizoma from 16 regions in China, so as to lay a foundation for its origin selection and clinical medication safety. Method:The authenticity of Anemonis Flaccidae Rhizoma was quickly identified by traditional identification method and DNA barcode molecular identification technology, and HPLC-UV was used to determine the contents of 5 active ingredients in Anemonis Flaccidae Rhizoma. All high pressure chromatographic separations were performed with a Welch Ultimate XB-C18 column (4.6 mm×250 mm, 5 μm), the mobile phase consisted of acetonitrile-0.01% trifluoroacetic acid aqueous solution (30∶70) at a flow rate of 1.0 mL·min-1. The detection wavelength was set at 210 nm and the column temperature was maintained at 30 ℃. Result:The authenticity of Anemonis Flaccidae Rhizoma could be precisely and rapidly identified by ribosomal DNA internal transcribed spacer 2 (ITS2) sequence and traditional identification methods. BLAST comparative analysis found that medicinal materials from 16 areas were all Anemone flaccida. Based on the contents of multi-index components, it was shown that the total content of 5 triterpenoid saponins in Anemonis Flaccidae Rhizoma from Banqiao, Enshi, Hubei was the highest (10.59%), followed by Hezhang, Bijie, Guizhou (6.28%) and Duzhenwan, Changyang, Hubei (5.64%). Conclusion:DNA barcoding can be used as an effective supplement to the traditional identification technology, it can ensure the authenticity of Anemonis Flaccidae Rhizoma and the safety of clinical use. The comprehensive evaluation of multi-index components of HPLC and cluster analysis show that the quality of medicinal materials in Enshi, Changyang, Wufeng of Hubei, Bijie of Guizhou and Jinfoshan of Chongqing is superior, which can be considered as important origin of Anemonis Flaccidae Rhizoma.
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Paragonimiasis is an important food-borne parasitic zoonosis caused by infection with lung flukes of the genus Paragonimus. Of the 7 members of the genus known in Thailand until recently, only P. heterotremus has been confirmed as causing human disease. An 8th species, P. pseudoheterotremus, has recently been proposed from Thailand, and has been found in humans. Molecular data place this species as a sister species to P. heterotremus, and it is likely that P. pseudoheterotremus is not specifically distinct from P. heterotremus. In this study, we collected metacercariae of both nominal species (identification based on metacercarial morphology) from freshwater crabs from Phetchabun Province in northern Thailand, Saraburi Province in central Thailand, and Surat Thani Province in southern Thailand. In addition, we purchased freshwater crabs imported from Myanmar at Myawaddy Province, western Thailand, close to the Myanmar-Thailand border. The DNAs extracted from excysted metacercariae were PCR-amplified and sequenced for ITS2 and cox1 genes. The ITS2 sequences were nearly identical among all samples (99-100%). Phylogenies inferred from all available partial cox1 sequences contained several clusters. Sequences from Indian P. heterotremus formed a sister group to sequences from P. pseudoheterotremus-type metacercariae. Sequences of P. heterotremus from Thailand, Vietnam, and China formed a separate distinct clade. One metacercaria from Phitsanulok Province was distinct from all others. There is clearly considerable genetic variation in the P. heterotremus complex in Thailand and the form referred to as P. pseudoheterotremus is widely distributed in Thailand and the Thai-Myanmar border region.