Current understanding of an Emerging Coronavirus usingin silico approach: Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) / Compreensão atual de um coronavírus emergente usando abordagem in silico: síndrome respiratória aguda grave - coronavírus-2 (SARS-CoV-2)

Novel coronavirus (nCoV) namely "SARS-CoV-2" is being found responsible for current PANDEMIC commenced from Wuhan (China) since December 2019 and has been described with epidemiological linkage to China in about 221 countries and territories until now. In this study we have characterized the genetic lineage of SARS-CoV-2 and report the recombination within the genus and subgenus of coronaviruses. Phylogenetic relationship of thirty nine coronaviruses belonging to its four genera and five subgenera was analyzed by using the Neighbor-joining method using MEGA 6.0. Phylogenetic trees of full length genome, various proteins (spike, envelope, membrane and nucleocapsid) nucleotide sequences were constructed separately. Putative recombination was probed via RDP4. Our analysis describes that the "SARS-CoV-2" although shows great similarity to Bat-SARS-CoVs sequences through whole genome (giving sequence similarity 89%), exhibits conflicting grouping with the Bat-SARS-like coronavirus sequences (MG772933 and MG772934). Furthermore, seven recombination events were observed in SARS-CoV-2 (NC_045512) by RDP4. But not a single recombination event fulfills the high level of certainty. Recombination mostly housed in spike protein genes than rest of the genome indicating breakpoint cluster arises beyond the 95% and 99% breakpoint density intervals. Genetic similarity levels observed among "SARS-CoV-2" and Bat-SARS-CoVs advocated that the latter did not exhibit the specific variant that cause outbreak in humans, proposing a suggestion that "SARS-CoV-2" has originated possibly from bats. These genomic features and their probable association with virus characteristics along with virulence in humans require further consideration.

O novo coronavírus (nCoV), nomeadamente "SARS-CoV-2", foi considerado responsável pela pandemia atual iniciada em Wuhan (China) desde dezembro de 2019 e foi descrito com ligação epidemiológica à China em cerca de 221 países e territórios até agora. Neste estudo, caracterizamos a linhagem genética do SARS-CoV-2 e relatamos a recombinação dentro do gênero e subgênero dos coronavírus. A relação filogenética de 39 coronavírus pertencentes a seus quatro gêneros e cinco subgêneros foi analisada usando o método de Neighbour-joining usando MEGA 6.0. Árvores filogenéticas do genoma de comprimento total, várias proteínas (espícula, envelope, membrana e nucleocapsídeo), sequências de nucleotídeos foram construídas separadamente. A recombinação putativa foi testada via RDP4. Nossa análise descreve que o "SARS-CoV-2", embora mostre grande semelhança com as sequências de Bat-SARS-CoVs em todo o genoma (dando semelhança de sequência de 89%), exibe agrupamento conflitante com as sequências de coronavírus do tipo Bat-SARS (MG772933 e MG772934) Além disso, sete eventos de recombinação foram observados em SARS-CoV-2 (NC045512) por RDP4. Mas nem um único evento de recombinação preenche o alto nível de certeza. A recombinação está alojada mais em genes de proteína de pico, principalmente, do que no resto do genoma, indicando que o cluster de ponto de interrupção surge além dos intervalos de densidade de ponto de interrupção de 95% e 99%. Os níveis de similaridade genética observados entre "SARS-CoV-2" e Bat-SARS-CoVs defendem que o último não exibe a variante específica que causa surto em humanos, sugerindo que "SARS-CoV-2" tenha se originado possivelmente de morcegos. Essas características genômicas e sua provável associação com as características do vírus, juntamente com a virulência em humanos, requerem uma consideração mais aprofundada.

Current understanding of an Emerging Coronavirus using in silico approach: Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2)

Abstract Novel coronavirus (nCoV) namely SARS-CoV-2 is being found responsible for current PANDEMIC commenced from Wuhan (China) since December 2019 and has been described with epidemiological linkage to China in about 221 countries and territories until now. In this study we have characterized the genetic lineage of SARS-CoV-2 and report the recombination within the genus and subgenus of coronaviruses. Phylogenetic relationship of thirty nine coronaviruses belonging to its four genera and five subgenera was analyzed by using the Neighbor-joining method using MEGA 6.0. Phylogenetic trees of full length genome, various proteins (spike, envelope, membrane and nucleocapsid) nucleotide sequences were constructed separately. Putative recombination was probed via RDP4. Our analysis describes that the SARS-CoV-2 although shows great similarity to Bat-SARS-CoVs sequences through whole genome (giving sequence similarity 89%), exhibits conflicting grouping with the Bat-SARS-like coronavirus sequences (MG772933 and MG772934). Furthermore, seven recombination events were observed in SARS-CoV-2 (NC_045512) by RDP4. But not a single recombination event fulfills the high level of certainty. Recombination mostly housed in spike protein genes than rest of the genome indicating breakpoint cluster arises beyond the 95% and 99% breakpoint density intervals. Genetic similarity levels observed among SARS-CoV-2 and Bat-SARS-CoVs advocated that the latter did not exhibit the specific variant that cause outbreak in humans, proposing a suggestion that SARS-CoV-2 has originated possibly from bats. These genomic features and their probable association with virus characteristics along with virulence in humans require further consideration.

Resumo O novo coronavírus (nCoV), nomeadamente SARS-CoV-2, foi considerado responsável pela pandemia atual iniciada em Wuhan (China) desde dezembro de 2019 e foi descrito com ligação epidemiológica à China em cerca de 221 países e territórios até agora. Neste estudo, caracterizamos a linhagem genética do SARS-CoV-2 e relatamos a recombinação dentro do gênero e subgênero dos coronavírus. A relação filogenética de 39 coronavírus pertencentes a seus quatro gêneros e cinco subgêneros foi analisada usando o método de Neighbour-joining usando MEGA 6.0. Árvores filogenéticas do genoma de comprimento total, várias proteínas (espícula, envelope, membrana e nucleocapsídeo), sequências de nucleotídeos foram construídas separadamente. A recombinação putativa foi testada via RDP4. Nossa análise descreve que o SARS-CoV-2, embora mostre grande semelhança com as sequências de Bat-SARS-CoVs em todo o genoma (dando semelhança de sequência de 89%), exibe agrupamento conflitante com as sequências de coronavírus do tipo Bat-SARS (MG772933 e MG772934) Além disso, sete eventos de recombinação foram observados em SARS-CoV-2 (NC045512) por RDP4. Mas nem um único evento de recombinação preenche o alto nível de certeza. A recombinação está alojada mais em genes de proteína de pico, principalmente, do que no resto do genoma, indicando que o cluster de ponto de interrupção surge além dos intervalos de densidade de ponto de interrupção de 95% e 99%. Os níveis de similaridade genética observados entre SARS-CoV-2 e Bat-SARS-CoVs defendem que o último não exibe a variante específica que causa surto em humanos, sugerindo que SARS-CoV-2 tenha se originado possivelmente de morcegos. Essas características genômicas e sua provável associação com as características do vírus, juntamente com a virulência em humanos, requerem uma consideração mais aprofundada.

Current understanding of an Emerging Coronavirus using in silico approach: Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) / Compreensão atual de um coronavírus emergente usando abordagem in silico: síndrome respiratória aguda grave - coronavírus-2 (SARS-CoV-2)

Abstract Novel coronavirus (nCoV) namely "SARS-CoV-2" is being found responsible for current PANDEMIC commenced from Wuhan (China) since December 2019 and has been described with epidemiological linkage to China in about 221 countries and territories until now. In this study we have characterized the genetic lineage of SARS-CoV-2 and report the recombination within the genus and subgenus of coronaviruses. Phylogenetic relationship of thirty nine coronaviruses belonging to its four genera and five subgenera was analyzed by using the Neighbor-joining method using MEGA 6.0. Phylogenetic trees of full length genome, various proteins (spike, envelope, membrane and nucleocapsid) nucleotide sequences were constructed separately. Putative recombination was probed via RDP4. Our analysis describes that the "SARS-CoV-2" although shows great similarity to Bat-SARS-CoVs sequences through whole genome (giving sequence similarity 89%), exhibits conflicting grouping with the Bat-SARS-like coronavirus sequences (MG772933 and MG772934). Furthermore, seven recombination events were observed in SARS-CoV-2 (NC_045512) by RDP4. But not a single recombination event fulfills the high level of certainty. Recombination mostly housed in spike protein genes than rest of the genome indicating breakpoint cluster arises beyond the 95% and 99% breakpoint density intervals. Genetic similarity levels observed among "SARS-CoV-2" and Bat-SARS-CoVs advocated that the latter did not exhibit the specific variant that cause outbreak in humans, proposing a suggestion that "SARS-CoV-2" has originated possibly from bats. These genomic features and their probable association with virus characteristics along with virulence in humans require further consideration.

Resumo O novo coronavírus (nCoV), nomeadamente "SARS-CoV-2", foi considerado responsável pela pandemia atual iniciada em Wuhan (China) desde dezembro de 2019 e foi descrito com ligação epidemiológica à China em cerca de 221 países e territórios até agora. Neste estudo, caracterizamos a linhagem genética do SARS-CoV-2 e relatamos a recombinação dentro do gênero e subgênero dos coronavírus. A relação filogenética de 39 coronavírus pertencentes a seus quatro gêneros e cinco subgêneros foi analisada usando o método de Neighbour-joining usando MEGA 6.0. Árvores filogenéticas do genoma de comprimento total, várias proteínas (espícula, envelope, membrana e nucleocapsídeo), sequências de nucleotídeos foram construídas separadamente. A recombinação putativa foi testada via RDP4. Nossa análise descreve que o "SARS-CoV-2", embora mostre grande semelhança com as sequências de Bat-SARS-CoVs em todo o genoma (dando semelhança de sequência de 89%), exibe agrupamento conflitante com as sequências de coronavírus do tipo Bat-SARS (MG772933 e MG772934) Além disso, sete eventos de recombinação foram observados em SARS-CoV-2 (NC045512) por RDP4. Mas nem um único evento de recombinação preenche o alto nível de certeza. A recombinação está alojada mais em genes de proteína de pico, principalmente, do que no resto do genoma, indicando que o cluster de ponto de interrupção surge além dos intervalos de densidade de ponto de interrupção de 95% e 99%. Os níveis de similaridade genética observados entre "SARS-CoV-2" e Bat-SARS-CoVs defendem que o último não exibe a variante específica que causa surto em humanos, sugerindo que "SARS-CoV-2" tenha se originado possivelmente de morcegos. Essas características genômicas e sua provável associação com as características do vírus, juntamente com a virulência em humanos, requerem uma consideração mais aprofundada.

Spatial Scale Patterns of Genetic Diversity and Gene Flow in Populations of Sweet Detar (Detarium microcarpum Guill. & Perr.; Fabaceae)

The main objective of this study is to investigate the patterns of genetic diversity and phylogenetic relationships within populations of Detarium microcarpum (Fabaceae) relative to different spatial conditions. Seventy-eight (78) accessions of D. microcarpum belonging to six populations (Phytogeographic districts) were sampled. In order to have very good quality DNA for molecular analysis, an optimization of the DNA isolation protocol was made. The molecular analysis of the accessions was carried out using 7 chloroplast microsatellite markers. The polymorphism rate (P) is 85.71% and the Polymorphism Information Content (PIC) was in the range of 0.43 (Ntcp_9) to 0.73 (Ccmp_2) with an average of 0.59. Allelic richness (A) ranged from 1.41 to 2.85 with an average of 2.04. The observed heterozygosity (Ho) ranged from 0.23 to 0.60 with an average of 0.39. The expected heterozygosity (He) ranged from 0.43 to 0.60 with a mean of 0.50. Wright's fixation index (FIS) ranged from -0.17 to 0.47. The effective allele (Ae) is between 1.77 and 2.53 with an average of 2.02. Wright differentiation index (FST) was 0.024. Phylogenetic analysis revealed that the NST value was significantly higher than the GST value (NST = 0.452; GST = 0.190; p <0.05). A relatively low hd haplotype diversity is obtained (Hd = 0.320). AMOVA analysis showed that 17.35% of the variation existed within populations but 45.80% among populations within the species. Neighbor-Joining phylogenetic tree of D. microcarpum revealed three non-distinct clusters haplotypes showing the existence of gene flow between populations of the species. Our findings of genetic structure and gene flow of D. microcarpum populations based on different spatial conditions is caused by evolutionary forces such as scattering and pollination.

Re-evaluation of the phylogenetic relationships and species delimitation of two closely related families (Lamiaceae and Verbenaceae) using two DNA barcode markers

The families Lamiaceae and Verbenaceae comprise several closely related species that possess high morphological synapomorphic traits. Hence, there is a tendency of species misidentification using only the morphological characters. Herein, we evaluated the discriminatory power of the universal DNA barcodes (matKand rbcL) for 53 species spanning the two families. Using these markers, we inferred phylogenetic relationships and conducted species delimitation analysis using four delimitation methods: Automated Barcode GapDiscovery (ABGD), TaxonDNA, Bayesian Poisson Tree Processes (bPTP) and General Mixed Yule Coalescent(GMYC). The phylogenetic reconstruction based on the matK gene resolved the relationships between thefamilies and further suggested the expansion of the Lamiaceae to include some core Verbanaceae genus, e.g.,Gmelina. The rbcL marker using the TaxonDNA method displayed high species delimitation resolutions, whilethe ABGD, GMYC, and bPTP generated different number of Operational Taxonomic Units/genetic clusters.Our results underscored the efficiency of the matK and rbcL genes as reliable markers for resolving phylogenetic relationships and species delimitation of both families, respectively. The current study provides insightsinto the DNA barcode applications in these families, at the same time contributing to the current understandingof genetic divergence patterns in angiosperms.

Molecular structure and phylogenetic analyses of the complete chloroplast genomes of three original species of Pyrrosiae Folium / 中国天然药物

Pyrrosia petiolosa, Pyrrosia lingua and Pyrrosia sheareri are recorded as original plants of Pyrrosiae Folium (PF) and commonly used as Chinese herbal medicines. Due to the similar morphological features of PF and its adulterants, common DNA barcodes cannot accurately distinguish PF species. Knowledge of the chloroplast (cp) genome is widely used in species identification, molecular marker and phylogenetic analyses. Herein, we determined the complete cp genomes of three original species of PF via high-throughput sequencing technologies. The three cp genomes exhibited a typical quadripartite structure with sizes ranging from 158 165 to 163 026 bp. The cp genomes of P. petiolosa and P. lingua encoded 130 genes, whilst that of P. sheareri encoded 131 genes. The complete cp genomes were compared, and five highly divergent regions of petA-psbJ, matK-rps16, ndhC-trnM, psbM-petN and psaC-ndhE were screened as potential DNA barcodes for identification of Pyrrosia genus species. The phylogenetic tree we obtained indicated that P. petiolosa and P. lingua are clustered in a single clade and, thus, share a close relationship. This study provides invaluable information for further studies on the species identification, taxonomy and phylogeny of Pyrrosia genus species.

Comparative plastomic analysis of three Bulbophyllum medicinal plants and its significance in species identification / 药学学报

italic>Bulbophyllum orchids are popular for its ornamental appearance and great medicinal values. However, there is still a lack of research on phylogenetic relationship and species identification for this genus. In this study, the plastome sequences of three medicinal Bulbophyllum orchids (Bulbophyllum affine, Bulbophyllum pectinatum, Bulbophyllum funingense) were sequenced and analyzed. After assembly and annotation, it was found that the plastomes of Bulbophyllum plants encoded a total of 108 genes, including 74 protein-coding genes, 30 tRNA genes and 4 rRNA genes. Based on the analysis of mVISTA and comparison between junctions, it was found that the plastome structure of Bulbophyllum orchids was relatively conserved, and the variation mainly existed in the non-coding regions. Phylogenetic analysis showed that Bulbophyllum orchids were closely related to Dendrobium orchids. SSR analysis of Bulbophyllum showed that most SSRs were located in the intergenic spacer and had the most single nucleotide repeats. In addition, based on the comparative analysis of non-coding sequences, a total of 10 high-variability sequences were screened out, among which the combination of five non-coding region sequences, including psbI-trnS, psbC-trnS, clpP-ex1-psbB, psaJ-rpl33, rpl33-rps18, had the highest sequence variability and could be used in the species identification study of medicinal plants of Bulbophyllum. In conclusion, this study provides a theoretical basis for phylogenetic relationship and species identification of Bulbophyllum orchids through the comparative analysis of plastome sequences of three medicinal plants of the genus Bulbophyllum.

Genetic distance and phylogenetic studies of two Indian snakehead and one carp fish species

Aim: To study genetic distance and phylogenetic relationship of two different stock of three important fish species, i.e., Channa stewartii, C. striatus and Labeo gonius of Assam. Methodology: Three fish species, namely Channa stewartii, C. striatus and Labeo gonius were collected from two beels, i.e., Thekera and Samaguri beels of Assam, India. Standard procedures were followed for isolation of their DNA and sequencing. Thereafter, genetic distance and diversity and phylogenetic studies of three species were calculated using computer based softwares ClustalW and MEGA6. Results: In the present study, the pairwise genetic distance of three fish species ranged from 0 to 20.436. No pairwise distance was found between two stocks at different locations Thekera and Samaguri beels of Assam for each of three species C. stewartii, C. striatus and L. gonius. The highest values (20.436) of pairwise distance were found between C. striatus and L. gonius population of Thekera beel; Thekera and Samaguri beels; and L. gonius population of the Thekera and Samaguri beels. The overall average distance for the two populations of three fish species was 15.387. No significant intraspecific difference was observed in the phylogenetic studies of two stocks of three fish species. Interpretation: The overall average distance (15.387) of three fish species could be attributed to genetic distance from each other. The high value of coefficient of differentiation (1.000) for three fish species from two stocks indicated that all the species were different from each other. Overall genetic and phylogenetic studies revealed that C. stewartii, C. striatus and L. gonius. were less differentiated from each other.

Diversity and Phylogenetic Relationships of Full Genome Sequences of Cassava Brown Streak Viruses in Kenya

Cassava brown streak disease is caused by cassava brown streak virus (CBSV) and Uganda cassava brown streak virus (UCBSV). Many of the CBSV and UCBSV diversity studies utilize partial coat protein sequences due to the unavailability of representative full genome sequences. Hence, there is little information on the diversity of cassava brown streak viruses in the rest of the genomes of the two species that are present in the farmers’ fields. The aim of this study was to determine Kenyan full CBSV and UCBSV genomes, and their sequence diversity and phylogenetic relationships within various genome and genome segments. Twenty four CBSVs positive samples tested by RT PCR from major cassava producing regions in Kenya were sequenced using Illumina MiSeq. Quality assessment of the output reads was done using the CLC Genomics 5.5.1 software programs. Genome assembly was done by de novo and reference guided assembly. Nucleotide sequence similarity of CBSV and UCBSV was determined. Phylogenetic relationships between CBSV and UCBSV were determined by performing the neighbour-joining analysis using MEGA 6.0 software. Six CBSV and 9 UCBSV genomes were generated from this study. The coat protein of the CBSV sequences had nucleotide sequence similarity of 92-100% while P1 and P3 gene had 75-100% and 76-100%, respectively. The coat protein of the UCBSV sequences had nucleotide sequence similarity of 91-99%. P1 and P3 gene had 83-100% and 86-99%, respectively. The phylogenetic analysis of full genomes revealed two distinct clusters one for UCBSV and another cluster for CBSV. Individual gene segments phylogenetic tree resembled that of the whole genome by clustering the nucleotide sequences into two clusters, one belonging to UCBSV and the other CBSV. The study revealed an average genome nucleotide diversity of 21.5% and 15.8% in CBSV and UCBSV respectively. A vast genetic diversity observed in CBSV and UCBSV in this study portends a lot of challenges in developing molecular diagnostic techniques as well as control strategies against the viruses.

Molecular identification of Tibetan medicinal plants based on ITS2 sequence / 中草药

Objective: The barcoded ITS2 DNA sequence was used to identify 44 Tibetan medicinal plants. Methods: Genomic DNA of Tibetan medicinal plants were extracted with high salt and low pH method. The PCR technique was performed to obtain the ITS2. A total of 145 ITS2 sequences was obtained belonging to 24 families, 39 genera, and 44 species. Some homologous sequences were also selected according to sequence alignment from Genbank database. The ITS2 sequences were aligned using Bioedit and the intraspecific and interspecific Kimura 2-parameter genetic distance was calculated using MEGA, and the neighbor-joining (NJ) phylogenetic trees were constructed to analyze phylogenetic relationship. Results: ITS2 regions have significant intra-and inter-specific difference, phylogenetic analysis based on ITS2 regions concurred with the result of morphological classification, and it could also determine the phylogenetic relationship between species. In addition, the secondary structure of the ITS2 sequence of Tibetan medicinal plants is different, providing another method for species identification. Conclusion: ITS2 can be used as a very effective single-locus barcode in the identification and phylogenetic study of Tibetan medicinal plants. The barcoding technique provides a scientific baseline for the utilization of resources and conservation of Tibetan medicinal plants.

Analysis of the Phylogenetic Relationships of 3 Basic Plants of Tibetan Medicine “Dida”Based on ISSR Technology / 中国药房

OBJECTIVE： To study the phylogenetic relationships of 3 basic plants of Tibetan medicine “Dida”， such as Swertia puricea， Wertia mileensis， Halenia elliptica. METHODS： ISSR technology was used for PCR amplification of 9 samples of S. puricea （ZT-1 to ZT-5 from Gantongsi in Dali Cangshan， ZC-1 to ZC-4 from Binchuan county of Dali）， 2 samples of W. mileensis （QYD-1 to QYD-2） and 2 samples of H. elliptica （HM-1 to HM-2）. Using DNA genome of S. puricea as template， 8 primers were screened and used for PCR reaction. The PCR amplification products were read by hand， the original data matrix was established， and the polymorphic band ratio was calculated. At the same time， genetic similarity coefficient was calculated by using NTSYS 2.1 software， and UPGMA method was used to draw cluster diagram. RESULTS： A total of 113 clear and identifiable amplification product bands were obtained by 8 ISSR primers. The rate of polymorphic site was 100％. The genetic similarity coefficients for totally 13 samples of S. puricea， W. mileensis and H. elliptica ranged 0.301-0.500. Intraspecific genetic similarity coefficients for 9 samples of S. puricea ranged from 0.752 to 0.929. The cluster analysis showed， when the range line was 0.410， 13 samples could be divided into three groups， i.e. S. puricea， W. mileensis， H. elliptica； when the range line was 0.780， 9 samples of S. purpurea could be divided into 2 subgroups， one of which was only sample ZT-1 collected from Gantongsi in Cangshan， and the other contained the remaining 8 samples. CONCLUSIONS： ISSR technology can be used to identify S. punicea， S. glabra and H. elliptica at the molecular level. S. punicea has some genetic relationship with S. glabra and H. elliptica， but the genetic relationship is relatively distant and the genetic difference is large. S. punicea from two different locations in Dali area has little genetic difference and close relationship， but it shows abundant genetic diversity.

Discovery of differential sequences for improving breeding and yield of cultivated Ophiocordyceps sinensis through ITS sequencing and phylogenetic analysis / 中国天然药物

To accelerate the breeding process of cultivated Ophiocordyceps sinensis and increase its yield, it is important to identify molecular fingerprint of dominant O. sinensis. In the present study, we collected 3 batches of industrially cultivated O. sinensis product with higher yield than the others and compared their internal transcribed spacer (ITS) sequences with the wild and the reported. The ITS sequence was obtained by bidirectional sequencing and analyzed with molecular systematics as a DNA barcode for rapid and accurate identification of wild and cultivated O. sinensis collected. The ITS sequences of O. sinensis with detailed collection loci on NCBI were downloaded to construct a phylogenetic tree together with the sequences obtained from the present study by using neighbor-joining method based on their evolution relationship. The information on collection loci was analyzed with ArcGIS 10.2 to demonstrate the geographic distribution of these samples and thus to determine the origin of the dominant samples. The results showed that all wild and cultivated samples were identified as O. sinensis and all sequences were divided into seven phylogenetic groups in the tree. Those groups were precisely distributed on the map and the process of their system evolution was clearly presented. The three cultivated samples were clustered into two dominant groups, showing the correlation between the industrially cultivated samples and the dominant wild samples, which can provide references for its optimized breeding in the future.

Discovery of differential sequences for improving breeding and yield of cultivated Ophiocordyceps sinensis through ITS sequencing and phylogenetic analysis / 中国天然药物

To accelerate the breeding process of cultivated Ophiocordyceps sinensis and increase its yield, it is important to identify molecular fingerprint of dominant O. sinensis. In the present study, we collected 3 batches of industrially cultivated O. sinensis product with higher yield than the others and compared their internal transcribed spacer (ITS) sequences with the wild and the reported. The ITS sequence was obtained by bidirectional sequencing and analyzed with molecular systematics as a DNA barcode for rapid and accurate identification of wild and cultivated O. sinensis collected. The ITS sequences of O. sinensis with detailed collection loci on NCBI were downloaded to construct a phylogenetic tree together with the sequences obtained from the present study by using neighbor-joining method based on their evolution relationship. The information on collection loci was analyzed with ArcGIS 10.2 to demonstrate the geographic distribution of these samples and thus to determine the origin of the dominant samples. The results showed that all wild and cultivated samples were identified as O. sinensis and all sequences were divided into seven phylogenetic groups in the tree. Those groups were precisely distributed on the map and the process of their system evolution was clearly presented. The three cultivated samples were clustered into two dominant groups, showing the correlation between the industrially cultivated samples and the dominant wild samples, which can provide references for its optimized breeding in the future.

Study of mitochondrial genomes and molecular genetic marker of microsatellite in chinese hamster / 中国比较医学杂志

Chinese hamster is an important laboratory animal in medical and biological researches,but the molecular genetic markers research was rarely reported.In our study the base composition,gene structure,genetic evolution and other characteristics of mitochondrial genome of Chinese hamster were analyzed using the methods of bioinformatics and comparative genomics,genetic quality detection system of Chinese hamster were also established.These results would supply genome data for animal models of human diseases,and lay the foundation for scientific evaluation and reasonable utilization.

Comparison on DNA barcodes of nuclear and chloroplast gene fragments in Rehmannia Libosch. ex Fisch. et Mey. (Rehmanniaceae) / 中草药

Objective: To compare the usage of single-copy nuclear genes (SCNGs), chloroplast gene fragments and internal transcribed spacer (ITS) as DNA barcodes in plants of Rehmannia Libosch. ex Fisch. et Mey. and elucidate the interspecific relationships in the genus. Methods: Distance and tree-based methods were performed to analyze 12 SCNGs, six cpDNA regions, and ITS fragment in 13 populations of Rehmannia Libosch. ex Fisch. et Mey. Neighbor-joining tree was constructed to investigate the relationships among the species. Results: CpDNA regions (matK and rbcL) and SCNGs had lower identification rates than others, especially ITS. Multiple DNA barcodes combination would be helpful to improve the species identification rate. The NJ tree indicated that R. glutinosa and R. solanifolia were clustered together, other species in the genus were monophyletic, and R. piasezkii was the basal group. Conclusion: ITS should be used as the core barcode in the genus while psbA-trnH, trnL-F, trnM-V, and trnS-G would be considered as the candidates, and nuclear genes R255 and R257 might be utilized as unique barcode for the identification of R. glutinosa. The origin of tetraploid species (R. glutinosa and R. solanifolia) still remains unknown and further works should be done to solve the question.

Morphological Characteristics and Phylogenetic Trends of Trematode Cercariae in Freshwater Snails from Nakhon Nayok Province, Thailand

The prevalence of cercarial infection in freshwater snails and their evolutionary trends were studied in Nakhon Nayok province, Thailand. A total of 2,869 individual snails were examined for parasitic infections. The results showed that 12 snail species were found to host larval stages of trematodes with an overall prevalence of 4.7%. The infected specimens included 7 types at the cercarial stage; cercariae, megalurous cercariae, echinostome cercariae, furcocercous cercariae, parapleurolophocercous cercariae, virgulate cercariae, and xiphidiocercariae. Regarding molecular identification, ITS2 sequence data of each larval trematode were analyzed, and a dendrogram was constructed using the neighbor-joining method with 10,000 replicates. The dendrogram was separated into 6 clades (order/family), including Echinostomatida/Echinostomatidae, Echinostomatida/Philophthalmidae, Opisthorchiida/Heterophyidae, Plagiorchiida/Prosthogonimidae, Plagiorchiida/Lecithodendriidae, and Strigeatida/Cyathocotylidae. These findings were used to confirm morphological characteristics and evolutionary trends of each type of cercariae discovered in Nakhon Nayok province. Furthermore, this investigation confirmed that the ITS2 data of cercariae could be used to study on phylogenetic relationships or to determine classification of this species at order and/or family level when possible.

ISSR analysis on genetic diversities and phylogenetic relationships of Gynostemma pentaphyllun germplasm reseources / 中草药

Objective: To analyze the genetic diversities and phylogenetic relationships of Gynostemma pentaphyllun germplasm. Methods: Genetic diversities and phylogenetic relationships of 48 G. pentaphyllun germplasm from 12 provinces in different ecological environments were analyzed by inter simple sequence repeat (ISSR) molecular makers. Results: (1) Fifteen primers were selected from 100 primers which had clear and stable polymorphic bands. A total of 214 loci were obtained, including 206 loci, on average, amplification site of each primer 14.27 and polymorphic loci (PPL) was 96.26%. The averagely observed number of alleles (Na), effective number of alleles (Ne), Shannon information index (I) and Nei's genetic index (H) were 1.9626, 1.3358, 0.2211, and 0.3598, respectively. The enetic similarity coefficient (GS) ranged from 0.57 to 0.96. Cluster annlysis with UPGMA method showed that the 48 testing materials were divided into four groups at the level GS 0.72. Conclusion: The results of ISSR analysis reveal that G. pentaphyllun germplasm has a higher genetic diversity level and the genetic relationship is not entirely consistent with geographical distribution and ecological environment.

Molecular phylogeny of trematodes in Family Heterophyidae based on mitochondrial cytochrome c oxidase subunit I (mCOI)

OBJECTIVE@#To analyze a phylogenetic tree for understanding the molecular systematic of trematode in Family Heterophyidae, which are highly distributed in Thailand.@*METHODS@#Based on thirteen sequences of mitochondrial cytochrome c oxidase subunit I (mCOI) gene from six genera of heterophyid trematodes, viz. Haplorchis, Stellantchasmus, Centrocestus, Metagonimus, Pygidopsis, and Haplorchoides were aligned automatically using the Clustal × 2.0 program. A phylogenetic tree was constructed by maximum likeinghood (ML) and neighbor-joining (NJ) methods, with 1 000 bootstrap using the 5.0 program.@*RESULTS@#The phylogenetic relationship from both methods was similar and separated into three groups consisting of Haplorchoides pumilio group, Haplorchoides taichui group and another heterophyid genera.@*CONCLUSIONS@#The sequence data of mtCOI can be used to investigate the phylogenetic relationships of trematodes at the genus level. Each clade of different genera of heterophyid trematodes can be separated into sister groups that correlated with the morphological characteristic, kind of secondary intermediate host and geographic distribution.

Phylogeny of Qinghai Plateau Echinococcus g ranulosus isolates inferred by cytochrome oxidase Ⅰ gene sequences / 中国人兽共患病学报

Cystic echinococcosis is one of the most widespread and severe zoonotic helminthic diseases .To understand the phylogeny and genetic polymorphism Echinococcus granulosus (E .granulosus) prevailed in south region of Qinghai Prov-ince ,partial fragment of cytochrome oxidase Ⅰ (COX Ⅰ ) gene sequences were used to infer the phylogenetic relationship of 59 collected samples of E .granulosus in Qinghai Province .Total 72 sequences (13 sequences from GenBank) were aligned using CLUSTAL X ,and then ,Bayesian analyses were performed in Mrbayes-3 .1 .2 .The results revealed that Echinococcus spp .isolates did not form a monophyletic group .The most samples clustered with E . granulosus strain (G1) (AB297617) , but showed high genetic polymorphism .Another three samples clustered with E .multilocularis (AB018440) ,while they showed complex phylogenetic relationships among them ,further indicating that Echinococcus spp .isolates from Qinghai Prov-ince may has a more complex evolutionary history than expected .

Molecular authentication and phylogenetic relationship of Bidens pilosa and its relative species / 中草药

Objective: To construct the phylogenetic relationship of Bidens pilosa and its relative species, and to accurately identify B. pilosa and its relative species. Methods: Total genomic DNA was isolated from B. pilosa and its relative species. Nuclear DNA internal transcibed spacer (ITS) and chloroplast gene psbA-trnH sequences were amplified and sequenced. The Kimura 2-parameter (K2P) distances were calculated. Authentication analyses were performed using Nearest Distance, BLAST1, and Neighbor-joining (NJ) methods. Results: The NJ trees (ITS and psbA-trnH data) indicated that the different populations of B. pilosa form a monophyletic clade [Bootstrap (BS) = 79% and 87%]. B. pilosa and B. pilosa var. radiata formed one monophyletic clade, and B. biternata was sister to B. bipinnata (ITS and combined data: BS = 100%). B. tripartita, B. cernua, and B. frondosa formed one monophyletic clade (ITS and combined data: BS = 100%; psbA-trnH data: BS = 99%). The inter-specific genetic distances (ITS and psbA-trnH data) between B. pilosa and its relative species were (0.00794-0.12880) and (0.005 18-0.074 52) which were far higher than intra-specific genetic distances of B. pilosa (0-0.00159) and (0-0.00252). Conclusion: The closest relative of B. pilosa is B. pilosa var. radiata. B. tripartita is closely relative to B. cernua and B. frondosa. The sister relationship between B. biternata and B. bipinnata is corroborated. ITS and psbA-trnH are two efficient barcodes for the authentication of B. pilosa and its relative species.