OBJECTIVETo find the patterns of the rDNA ITS sequence variation in Gentiana, and establish the molecular biological method for the identification of the four kinds wild Gentiana from different regions in Gansu.

METHODThe ITS gene fragments were PCR amplified and sequenced. The rDNA ITS regions were analyzed by means of the software of Clustal X, MEGA3.

RESULTThe Complete ITS sequence of G. macrophylla, G. straminea, G. dahurica and G. officinale was 800 bp. The sequences of ITS1, ITS2 and 5.8S were 290, 340, 170 bp, respectively. Phylogenetic tree based on ITS1 and ITS2 sequences data was constrcuted by Neighbor-joining method.

CONCLUSIONITS sequence could be as the evidence for the molecular authentication of Gentiana.

To identify Herba Dendrobii and its adulterant species on molecular level, the rDNA ITS sequences of 17 species of Herba Dendrobii were studied. Genomic DNA of Dendrobium was extracted using the modified cetyltrimethyl ammonium bromide (CTAB) method. The PCR products of the rDNA ITS sequences of Dendrobium (32 materials) were purified and then sequenced. The characteristic of the sequences and the genetic distance were compared between Bulbophyllum odoratissimum and Dendrobium, Dendrobium interspecies and different populations. Phylogenetic trees were constructed using the UPGMA method by the biology softwares including BioEdit, MEGA4.0 etc. The PCR products were purified and then sequenced. It was built up that the database of rDNA ITS sequences of 17 species of Herba Dendrobii (32 materials). The ITS1 was 228-234 bp, the GC content accounting for 45.7%-53.0%. Its variable sites were 167, accounting for 67.34%. The Parsim-Informative positions were 106, accounting for 42.74%. The ITS2 was 241-247 bp, the GC accounting for 44.8% - 55.7%. The variable sites were 165, accounting for 66.27%. The Parsim-Informative positions were 115, accounting for 46.18%. The genetic distance between B. odoratissimum and Dendrobium was 0.295. The average genetic distance was 0.142 between Dendrobium species, and there were 2-156 variable nucleotides. The average genetic distance between different populations was 0.002, and there were 2-156 variable nucleotides. The genetic distance between B. odoratissimum and Dendrobium was greater than that of Denrobium interspecies. Meanwhile, the genetic distance between Denrobium species was also greater than that of different populations (varieties). The molecular phylogeny tree was constructed on the database of rDNA ITS the sequences of 17 species of Herba Dendrobii using the biology softwares. Then 10 materials on molecular level were authenticated. It is concluded that using of the whole sequences database of 17 species of Herba Dendrobii and heredity analysis softwares, and measuring the sequences of rDNA ITS of the inspected species, can authenticate the Dendrobium on molecular level, and provide basis for molecular authentication.
China ; DNA, Plant ; genetics ; DNA, Ribosomal ; genetics ; DNA, Ribosomal Spacer ; genetics ; Dendrobium ; classification ; genetics ; Molecular Sequence Data ; Phylogeny

The paper is aimed to identify SNP in Sarcandra glabra and Chloranthus spicatus, and authenticate S. glabra from Ch. spicatus and the mixture by using PCR amplification of specific alleles. SNPs in the ITS sequences of S. glabra and Ch. spicatus were found by ClustulX 2. 1 program and Bioedit software. Primers for authentic S. glabra and Ch. spicatus was designed according to the SNP site, and ITS sequence universal primers plus to the authentic primer to construct a multi-PCR reaction system, and then optimized the PCR reaction system. Five hundred and eighty band special for S. glabra and 470 bp band special for Ch. spicatus were found by using multi-PCR reaction. The multi-PCR reaction system could be applied to identify S. glabra and Ch. spicatus's leaves.
DNA, Plant ; analysis ; genetics ; DNA, Ribosomal ; genetics ; DNA, Ribosomal Spacer ; analysis ; genetics ; Magnoliopsida ; classification ; genetics ; Plant Leaves ; genetics ; Polymerase Chain Reaction ; Polymorphism, Single Nucleotide ; RNA, Ribosomal ; genetics ; RNA, Ribosomal, 18S ; genetics ; RNA, Ribosomal, 5.8S ; genetics ; Species Specificity

Root rot was occurred widely in the production area of Rehmannia glutinosa, and which result in serious influence on the yield and quality of R. glutinosa. In the present work, a new phytopathogen was isolated from roots with root rot symptom in the production area of R. glutinosa. The colony of the pathogen growing on PDA medium was gray-black, the structure of hyphae was compact, the aerial hyphae was less developed, and the back of the colony was black. The hyphae of the pathogen were uneven in size, about 2 to 3 μm in diameter and twined with each other, the conidia of the pathogen were small, nearly round and about 1 μm in diameter. The healthy roots of R. glutinosa were inoculated with the pathogen in vitro, black-brown rot was observed at the inoculate sites after a few days' incubation. The rhizosphere soil of healthy R. glutinosa seedlings were inoculated in vivo, the leaves were wilted and the roots were black-brown rotted after several days' normal culture, the symptoms were consistent with those observed in the field. The genomic DNA of the pathogen was amplified by fungus rDNA-ITS universal primer ITS1/ITS4 and homologous analyzed, the pathogen was in a branch with Heterophoma sp., Phoma sp., P. novae-verbascicola and P. herbarum with the nuclear acid homology of 99.21% to 99.43%. The pathogen shown 97.00% to 98.02% nuclear acid homology with H. verbascicola, H. novae-verbascicola, H. poolensis, P. herbarum, H. sylvatica, H. verbascicola and H. verbasci-densiflori when amplified by the tub2 gene special primer Btub2 fd/Btub4 rd, and H. novae-verbascicola was the highest. The pathogen was in a branch with H. novae-verbascicola when amplified by the lsu gene special primer LR0 R/LR7. Based on the morphological characteristics, nucleotide sequence analysis and Koch's test results, the isolated pathogen causing root rot of R. glutinosa was identified as H. novae-verbascicola. This study is of great significance for the further theoretical research on root rot of R. glutinosa and root rot control in field.
DNA, Ribosomal ; Fungi/genetics* ; Plant Leaves ; Rehmannia/genetics* ; Seedlings

The taxonomy of Acanthamoeba spp., an amphizoic amoeba which causes granulomatous amoebic encephalitis and chronic amoebic keratitis, has been revised many times. The taxonomic validity of some species has yet to be assessed. In this paper, we analyzed the morphological characteristics, nuclear 18s rDNA and mitochondrial 16s rDNA sequences and the Mt DNA RFLP of the type strains of four Acanthamoeba species, which had been previously designated as A. divionensis, A. parasidionensis, A. mauritaniensis, and A. rhysodes. The four isolates revealed characteristic group II morphology. They exhibited 18S rDNA sequence differences of 0.2-1.1% with each other, but more than 2% difference from the other compared reference strains. Four isolates formed a different clade from that of A. castellanii Castellani and the other strains in morphological group II on the phylogenetic tree. In light of these results, A. paradivionensis, A. divionensis, and A. mauritaniensis should be regarded as synonyms for A. rhysodes.
Acanthamoeba/*classification/*genetics ; Animals ; DNA, Mitochondrial/genetics ; DNA, Ribosomal/genetics ; Phylogeny ; Polymorphism, Restriction Fragment Length ; RNA, Ribosomal, 18S/genetics

Objective To detect the diatom population diversity in Dianchi by constructing a 18S rDNA clone library. Methods DNA from diatoms in 6 water samples of Dianchi was amplified with diatom 18S rDNA specific primer.The 18S rDNA clone library was constructed, and clones were randomly selected for sequence. Sequence alignment was performed by BLAST. The diatom population distribution in Dianchi was analyzed and the phylogenetic tree of diatom 18S rDNA in Dianchi waters was established with the MEGA v7.0.14 software. Results Two hundred and forty clones were sequenced, with 167 diatom sequences obtained, including 11 diatom species such as Stephanodiscus, Diatoma, and Melosira. There were certain differences in diatom population distribution among the 6 samples. Conclusion The population distribution of diatom species in Dianchi shows unique features and the sequence analysis of diatom 18S rDNA has a certain reference value to the inference of forensic drowning sites.
China ; DNA, Ribosomal/genetics* ; Diatoms/classification* ; Drowning ; Forensic Sciences ; Humans ; Phylogeny ; RNA, Ribosomal, 18S/genetics*

Objective: To explore the differences in subsequent analysis between metagenomic and 16Sr DNA sequencing in compositionally characterizing gut microbiota of healthy elderly. Methods: By using a panel study design, five monthly repeated measurements were performed among 76 healthy older people in Jinan City, Shandong Province. Their fecal samples were collected, and genomic DNA was extracted and analyzed through metagenomic and 16Sr DNA sequencing to compare the composition and diversity of gut microbiota. The correlation between species abundance and α diversity was analyzed by Pearson correlation analysis, and the correlation between species abundance and β diversity was determined by Procrustes analysis. Results: The age of 76 participants was (65.07±2.75), and the body mass index was (25.03±2.40) kg/m². There were 38 males and 38 females. A total of 345 fecal samples were obtained from five monthly repeated measurements . Compared with 16S rDNA sequencing, metagenomic sequencing showed more annotated species at each level. The difference in the number of two sequencing species increased with the decrease of the level. Although there were significant differences in species richness between the two sequencing methods. Their species richness was highly correlated at both phylum (r=0.88, P<0.001) and genus (r=0.77, P<0.001) levels. Bacteroidetes and Firmicutes were the common dominant species. Gut microbiota diversity analysis further showed that there was a significantly positive correlation between α diversity (r=0.70, P<0.001) and β diversities (M²=0.84, P<0.05) in the two groups. Conclusion: The annotation efficiency of metagenomic sequencing is much higher than that of 16S rDNA sequencing. The two sequencing methods are consistent in phylum abundance as well as α diversity.
Male ; Female ; Humans ; Aged ; Gastrointestinal Microbiome/genetics* ; DNA, Ribosomal/genetics* ; Feces ; Sequence Analysis, DNA ; Metagenomics ; RNA, Ribosomal, 16S/genetics*

Objective To identify the species of common necrophagous flies in Fujian Province by gene fragment sequences of mitochondrial cytochrome c oxidase subunit Ⅰ （COⅠ） and 16S ribosomal deoxyribonucleic acid （16S rDNA）, and to explore the identification efficacy of these two molecular markers. Methods In total 22 common necrophagous flies were collected from the death scenes in 9 different regions in Fujian Province and DNA was extracted from the flies after morphological identification. The gene fragments of COⅠ and 16S rDNA were amplified and sequenced. All the sequences were uploaded to GeneBank and BLAST and MEGA 10.0 software were used to perform sequence alignment, homology analysis and intraspecific and interspecific genetic distance analysis. The phylogenetic trees of DNA fragment sequences of COⅠ and 16S rDNA of common necrophagous flies in Fujian Province were established by unweighted pair-group method with arithmetic means （UPGMA）, respectively. Results The flies were classified into 6 species, 5 genera and 3 families by morphological identification. The results of gene sequence analysis showed that the average number of interspecific and intraspecific genetic distance of 16S rDNA ranged from 1.8% to 8.9% and 0.0% to 2.4%, respectively. The average number of interspecific and intraspecific genetic distance of COⅠ ranged from 7.2% to 13.6% and 0.0% to 6.3%, respectively. Conclusion The gene sequences of COⅠ and 16S rDNA can accurately identify the species of different necrophagous flies, and 16S rDNA showed higher value in species identification of common calliphoridae necrophagous flies in Fujian Province.
Animals ; DNA, Ribosomal/genetics* ; Diptera/genetics* ; Humans ; Phylogeny ; RNA, Ribosomal, 16S/genetics* ; Sequence Analysis, DNA ; Species Specificity

The important role of intestinal microorganisms in human health has been widely confirmed. At present, most of the studies on intestinal microorganisms are based on amplification of the V3-V4 region of bacterial 16S rRNA gene, and little attention has been paid to archaea. In this study, a primer set which can amplify 16S rRNA gene of both bacteria and archaea at the same time was used. By comparing the community changes before and after probiotics intake, it showed that this primer set is suitable for analyzing the changes of human intestinal bacteria and archaea communities. The fecal samples of volunteers were collected, and the amplification and high-throughput sequencing were carried out by using bacterial primer set (B primer) and bacterial and archaeal universal primer (AB primer); several commonly used rRNA databases were used to determine the amplification ability of the primer set to bacteria and archaea. The results showed that AB primer could display the bacterial community amplified by B primer, and could obtain the sequence of common methanogenic archaea in intestinal tract. AB primer set can analyze the bacteria and archaea in the intestinal tract at the same time by only one amplification and sequencing, which can show the structure of intestinal microbial community more comprehensively, which is suitable for the research of intestinal microorganisms.
Archaea/genetics* ; Bacteria/genetics* ; DNA Primers ; DNA, Bacterial ; Humans ; Microbiota/genetics* ; Phylogeny ; RNA, Ribosomal, 16S/genetics*

The community structure and diversity of the gut microbiota are associated with human diseases. However, the analysis of different community structure might be influenced by experimental approaches such as the quality of DNA extraction. Therefore, evaluating the efficiency of different DNA extraction methods for specific intestinal species is a guideline for obtaining a comprehensive human gut microbial profile, which may assist the in-depth investigation into the structure of the gut microbial community. The aim of this study was to perform a comparative analysis of five different DNA extraction methods. With the aid of qPCR, the efficiency of five DNA extraction kits was evaluated in terms of the purity of the extracted DNA, the DNA concentration, and the abundance of genomic DNA extracted from specific intestinal species. The results showed that the kit Q gave the best extraction results, especially for Gram-positive bacteria such as Lactobacillus and Bifidobacterium. The average DNA concentration of the N kit was lower than that of the Q kit, but there was no significant difference between the two in terms of the purity. Compared to the other three commercial kits (M, PSP, TG), the efficiency of the N kit in extracting the genomic DNA of the specified microorganisms were the least different from those of the Q kit. In contrast, the DNA extracted by the M kit was of higher quality but of lower concentration, and was not very efficient for Gram-positive bacteria. The DNA extracted by the TG and PSP kits was inferior to the other validated kits in terms of the concentration, quality and bacterial abundance. These results provide a basis for the selection of genomic DNA extraction methods in microecological research experiments.
DNA/genetics* ; DNA, Bacterial/genetics* ; Feces/microbiology* ; Humans ; Microbiota/genetics* ; RNA, Ribosomal, 16S/genetics*