Identification of 24 Rana Species Including Rana dybowskii and Rana chensinensis Based on COⅠ Sequences / 中国实验方剂学杂志

Objective:To identify 24 <italic>Rana</italic> species such as <italic>Rana dybowskii</italic> by mitochondrial cytochrome C oxidase subunit I （<italic>CO</italic>Ⅰ） gene-based DNA barcoding and build the neighbour-joining （NJ） tree for hierarchical cluster analysis， so as to provide a basis for the identification and classification of <italic>Rana</italic> species as well as the discovery of new species. Method:<italic>R. dybowskii</italic>， <italic>R. chensinensis</italic>， <italic>R. amurensis</italic>， <italic>R. culaiensi</italic>s， and <italic>R. huanrenesis</italic>， ten for each species， were collected for DNA extraction and polymerase chain reaction （PCR） amplification<italic> </italic>and sequencing. A total of 50 <italic>CO</italic>Ⅰ gene sequences were obtained. Then 163 <italic>CO</italic>Ⅰ gene sequences for 24 species of <italic>Rana</italic> and one <italic>CO</italic>Ⅰ gene sequence for <italic>Pelophylax</italic>，<italic> Odorrana</italic>， <italic>Nidirana</italic>， <italic>Hylarana</italic>， and <italic>Amolops</italic> were harvested from GenBank. After sequence alignment by MEGA X， the parsimony-informative sites of <italic>CO</italic>Ⅰ gene sequences were analyzed and the intraspecific and interspecific genetic distances were calculated， followed by the built of NJ tree and hierarchical cluster analysis. Result:The <italic>CO</italic>Ⅰ gene sequences of 24<italic> Rana</italic> species including <italic>R. dybowskii</italic> were 554 bp in length and there were 210 parsimony-informative sites in total. The intraspecific genetic distance of each species was smaller than 2%. Except that the interspecific genetic distance between <italic>R. sangzhiensis</italic> and <italic>R. zhengi</italic> was 0.004， the genetic distances between the other species ranged from 0.024 to 0.228. <italic>R. sangzhiensis</italic> and <italic>R. zhengi</italic> were clustered into one branch and some <italic>R. dybowskii</italic> and <italic>R. uenoi</italic> into one branch. There were two separate branches for <italic>R. chensinensis</italic> and the other species were all clustered independently. Conclusion:<italic>CO</italic>Ⅰ-based DNA barcoding enabled the identification of 24 species of <italic>Rana</italic> including <italic>R.dybowskii</italic>. The findings supported that <italic>R. sangzhiensis</italic>， <italic>R. zhengi</italic>， <italic>R. coreana</italic>， and <italic>R. kunyuensis</italic> were the same species. One branch of <italic>R. chensinensis </italic>might be one of the four undownloaded species in Ranidae or a new species. The results have demonstrated that <italic>CO</italic>Ⅰ-based DNA barcoding allows not only the identification of 24 species of Rana including <italic>R. dybowskii </italic>but also the classification of ranidae species and the discovery of new species or subspecies.

COⅠ and 16S rDNA Sequence Identification of Common Necrophagous Flies in Fujian Province / 法医学杂志

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.

Analysis of 28S rRNA and COⅠ Gene Sequence of Nine Necrophagous Calliphorid Flies from Luoyang / 法医学杂志

Objective To assess the feasibility of using 28S ribosomal RNA （28S rRNA） and mitochondrial cytochrome c oxidase subunit Ⅰ （COⅠ） gene sequences of nine necrophagous Calliphorid flies for the identification of common necrophagous Calliphorid flies, and to provide technical support for postmortem interval （PMI） estimation. Methods Twenty-three Calliphorid flies were collected and identified morphologically, and DNA were extracted from legs. The gene fragments of 28S rRNA and COⅠ were amplified and sequenced, then the sequence alignment was performed with BLAST. The composition of obtained sequences was analyzed and evolutionary divergence rate between species and intraspecies were established. The phylogeny tree was constructed with neighbor-joining method. Results The 23 necrophagous Calliphorid flies were identified to 9 species of 5 genera. The 715 bp from 28S rRNA and 637 bp from COⅠ gene were obtained and the online BLAST result showed more than 99% of similarity. The phylogeny tree showed that the necrophagous flies could cluster well into 9 groups, which was consistent with morphological identification results. The intraspecific difference in 28S rRNA was 0 and the interspecific difference was 0.001-0.033. The intraspecific difference in COⅠ was 0-0.008 and the interspecific difference was 0.006-0.101. Conclusion Combined use of 28S rRNA and COⅠ gene sequence fragments can effectively identify the nine Calliphorid flies in this study. However, for closely related blowfly species, more genetic markers should be explored and used in combination in future.

Genotyping and polymorphism analysis of cytochrome c oxidase subunit Ⅰgene of Pomacea canaliculata from Lincang City in Yunnan Province / 中国血吸虫病防治杂志

Objective To analyze the genetic diversity of Pomacea canaliculata based on the mitochondria DNA cytochrome c oxidase subunitⅠ(mtDNA COⅠ)gene as a molecular marker in Lincang City of Yunnan Province,so as to provide the scien-tific data for monitoring Angiostrongylus cantonensis in local areas. Methods The genotypes and polymorphisms of 38 speci-mens of P.canaliculata collected from Mengding Town of Lincang City were analyzed by sequencing COⅠgene.The phylogenet-ic tree and genetic distances were produced based on the haplotypes from GenBank and the present study by using the neighbour-joining method with the software MEGA version 6.06. Results Totally 31 sequences were acquired in the present study and they produced 3 unique haplotypes.Haplotype 1 showed a higher frequency compared to the others and it accounted for 83.9 % (26/31).The data showed that the least genetic distances ranged from 0 to 0.052 between P.canaliculata and 3 haplotypes,as well as the largest genetic distances ranged from 0.021 to 0.239 between Pila conica and 3 haplotypes.Otherwise,the analysis of the phylogenetic trees based on COⅠgene sequences of P.canaliculata indicated that all of 3 haplotypes clustered into one big clade with that from Japan(GenBank accession number: AB433769),China(GenBank accession number: KT313034)and USA(GenBank accession number:EU523129),which owned the closet relationship amongst them.Their genetic relationships were distantly related to the GenBank's reference sequences of P.insularum(GenBank accession number:EF514942),P.cam-ena(GenBank accession number: EF515059)and so on. Conclusion There is a P. canaliculata species in Lincang City of Yunnan Province as well as a high genetic diversity amongst the acquired 3 haplotypes in this study.