Phylogenetic analysis of six species of pacific abalone (Haliotidae) based on DNA sequences of 16s rRNA and cytochrome c oxidase subunit I mitochondrial genes

Six species of abalones (Haliotidae) are found on the Korean coasts. Identification and characterization of these abalones are usually based on morphologic characters. In this research we compared the partial sequences of the mitochondrial 16S ribosomal RNA and cytochrome c oxidase subunit I genes to identify species using molecular data and to determine their phylogenetic relationships. Sequence alignments and phylogenetic analysis revealed that the 6 species fell into 2 distinct groups which were genetically distant from each other and exhibited little internal phylogenetic resolution. One group included Haliotis discus hannai, H. discus discus, H. madaka, and H. gigantea, while the other group contained H. diversicolor supertexta and H. diversicolor diversicolor. The 16S rRNA sequences were relatively more conserved than to the COI sequences, but both gene sequences provided sufficient phylogenetic information to distinguish among the 6 species of Pacific abalone, and thus could be valuable molecular characters for species identification.     

Molecular phylogeny of musk deer: a genomic view with mitochondrial 16S rRNA and cytochrome b gene

The phylogenetic status of the infra order Pecora is controversial, even though it is supported by paleontological, morphological, and molecular evidence. We analyzed two mitochondrial genes (i.e., 16S rRNA and cytochrome b) to resolve the phylogenetic position of pecoran species, i.e., the Bovidae, Cervidae, and Moschidae endemic to the Indian subcontinent. We used phylogenetic analysis based on different algorithms, including neighbor joining, maximum parsimony, Bayesian inference, maximum likelihood, minimum evolution, median joining network, along with multidimensional scaling, and DNA word analysis. Our results established the basal position of Tragulidae and the monophyly of the infra order Pecora within the Suborder Ruminantia. Our results also demonstrated that Bovidae, Cervidae, and Moschidae are allied with the placement of musk deer as more closely related to bovids than to cervids. Molecular dating based on sequence analysis shows that the radiation of Pecora occurred during the early Oligocene and that the majority of the pecoran families radiated and dispersed rapidly during the Oligocene/Miocene transition.     

Identification of Comamonas species using 16S rRNA gene sequence

A bacterial strain Bz02 was isolated from a water sample collected from river Gomti at the Indian city of Lucknow. We characterized the strain using 16S rRNA sequence. Phylogenetic analysis showed that the strain formed a monophyletic clade with members of the genus Comamonas. The closest phylogenetic relative was Comamonas testosteroni with 95% 16S rRNA gene sequence similarity. It is proposed that the identified strain Bz02 be assigned as the type strain of a species of the genus Comamonas (Comamonas sp Bz02) based on 16S rRNA gene sequence search in Ribosomal Database Project, small subunit rRNA and large subunit rRNA databases together with the phylogenetic tree analysis. The sequence is deposted in GenBank with the accession number {"type":"entrez-nucleotide","attrs":{"text":"FJ211417","term_id":"209164434"}}FJ211417.     

Evaluation of the 16S and 12S rRNA genes as universal markers for the identification of commercial fish species in South Africa

The development of DNA-based methods for the identification of fish species is important for fisheries research and control, as well as for the detection of unintentional or fraudulent species substitutions in the marketplace. The aim of this study was to generate a comprehensive reference database of DNA sequences from the mitochondrial 16S and 12S ribosomal RNA (rRNA) genes for 53 commercial fish species in South Africa and to evaluate the applicability of these genetic markers for the identification of fish at the species level. The DNA extracted from all target species was readily amplified using universal primers targeting both rRNA gene regions. Sequences from the 16S and 12S rRNA genes were submitted to GenBank for the first time for 34% and 53% of the fish species, respectively. Cumulative analysis of the 16S rRNA gene sequences revealed mean conspecific, congeneric and confamilial Kimura two parameter (K2P) distances of 0.03%, 0.70% and 5.10% and the corresponding values at the 12S level were 0.03%, 1.00% and 5.57%. K2P neighbour-joining trees based on both sequence datasets generally clustered species in accordance with their taxonomic classifications. The nucleotide variation in both the 16S and 12S sequences was suitable for identifying the large majority of the examined fish specimens to at least the level of genus, but was found to be less useful for the explicit differentiation of certain congeneric fish species. It is recommended that one or more faster-evolving DNA regions be analysed to confirm the identities of closely-related fish species in South Africa.     

Identification of Escherichia coli through analysis of 16S rRNA and 16S-23S rRNA internal transcribed spacer region sequences

A bacterial strain, designated BzDS03 was isolated from water sample, collected from Dal Lake Srinagar. The strain was characterized by using 16S ribosomal RNA gene and 16S-23S rRNA internal transcribed spacer region sequences. Phylogenetic analysis showed that 16S rRNA sequence of the isolate formed a monophyletic clade with genera Escherichia. The closest phylogenetic relative was Escherichia coli with 99% 16S rRNA gene sequence similarity. The result of Ribosomal database project's classifier tool revealed that the strain BzDS03 belongs to genera Escherichia.16S rRNA sequence of isolate was deposited in GenBank with accession number FJ961336. Further analysis of 16S-23S rRNA sequence of isolate confirms that the identified strain BzDS03 be assigned as the type strain of Escherichia coli with 98% 16S-23S rRNA sequence similarity. The GenBank accession number allotted for 16S-23S rRNA intergenic spacer sequence of isolate is FJ961337.     

拟诺卡氏菌16S rRNA,gyrB,sod和rpoB基因的系统发育分析

为了更好地了解拟诺卡氏菌属(Nocardiopsis)各物种间的系统发育关系,该属现有有效描述种的gyrB,sod和rpoB基因的部分序列被测定,结合16S rRNA基因,对拟诺卡氏菌属进行了系统发育重建。研究发现拟诺卡氏菌属gyrB,sod和rpoB基因的平均相似性分别为87.7%、87.3%和94.1%,而16S rRNA基因的平均相似性则达到96.65%,3个看家基因均比16S rRNA具有更高的分歧度。比较基于不同基因的系统树发现,由gyrB基因得到的系统树拓扑结构与16S rRNA得到的结构在亚群上基本一致。因此,gyrB基因在拟诺卡氏菌属的系统分类上比16S rRNA基因更具优越性。     

A novel mutation in the mitochondrial 16S rRNA gene in a patient with MELAS syndrome, diabetes mellitus, hyperthyroidism and cardiomyopathy

Using RNase protection analysis, we found a novel C to G mutation at nucleotide position 3093 of mitochondrial DNA (mtDNA) in a previously reported 35-year-old woman exhibiting clinical features of mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome together with diabetes mellitus, hyperthyroidism and cardiomyopathy. The patient also had an A3243G mutation in the tRNA(Leu(UUR)) gene and a 260-base pair duplication in the D-loop of mtDNA. The fibroblasts of the patient were cultured and used for the construction of cybrids using cytoplasmic transfer of the patient's mtDNA to the mtDNA-less rho(0) cells. RNA isolated from the cybrids was subjected to RNase protection analysis, and a C3093G transversion at the 16S rRNA gene and a MELAS-associated A3243G mutation of mtDNA were detected. The novel C3093G mutation together with the A3243G transition were found in muscle biopsies, hair follicles and blood cells of this patient and also in her skin fibroblasts and cybrids. The proportion of the C3093G mutant mtDNA in muscle biopsies of the patient was 51%. In contrast, the mutation was not detected in three sons of the proband. To characterize the impact of the mtDNA mutation-associated defects on mitochondrial function, we determined the respiratory enzyme activities of the primary culture of fibroblasts established from the proband, her mother and her three sons. The proportions of mtDNA with the C3093G transversion and the A3243G transition in the fibroblasts of the proband were 45 and 58%, respectively. However, the fibroblasts of the proband's mother and children harbored lower levels of mtDNA with the A3243G mutation but did not contain the C3093G mutation. The complex I activity in the proband's fibroblasts was decreased to 47% of the control but those of the fibroblasts of the mother and three sons of the proband were not significantly changed. These findings suggest that the C3093G transversion together with the A3243G transition of mtDNA impaired the respiratory function of mitochondria and caused the atypical MELAS syndrome associated with diabetes mellitus, hyperthyroidism and cardiomyopathy in this patient.     

Molecular identification of pufferfish species using PCR amplification and restriction analysis of a segment of the 16S rRNA gene

This study amplified the mitochondrial 16S rRNA gene using polymerase chain reaction (PCR) with a template of total DNA from muscle tissues of nine pufferfish species collected from the coastal area of Okinawa Islands in Japan: Pleuranacanthus sceleratus, Triodon macropterus, Chelonodon patoca, Sphoeroides pachygaster, Arothron hispidus, A. stellatus, A. manilensis, A. mappa, and A. nigropunctatus. Then nucleotide sequence encoding a partial region of the 16S rRNA gene was compared among species. The sequenced fragment was also used to select restriction enzymes, yielding species-specific restriction fragment length polymorphisms (RFLP). The sequence of the segment of the 16S rRNA gene consisted of about 615 nucleotides and showed interspecies variations in the targeted region. After calculation of corresponding RFLP-patterns of nine species investigated with suitable restriction enzymes, three restriction enzymes – BanII, DdeI, and NlaIII – were found to be sufficient for identification of all nine species. Successful testing of this methodology in frozen and heated food samples suggests its utility for pufferfish species authentication in food products.     

Genetic diversity of cultured populations of giant freshwater prawn (Macrobrachium rosenbergii) in China using mtDNA COI and 16S rDNA markers

Partial sequences of mitochondrial DNA 16S rDNA and COI genes (395 bp and 498 bp respectively) were sequenced from samples of ten cultured populations of Macrobrachium rosenbergii (Giant Freshwater Prawn – GFP) in Zhejiang, Guangdong and Guangxi Provinces in China and two wild populations of GFP from Mekong River and Dongnai River in Viet Nam. Five haplotypes of 16S rDNA were identified in the 360 samples. The wild populations displayed nucleotide diversity (π) of 0.0008 and 0.0003, and genetic diversity (h) of 0.3030 and 0.1310 in the Mekong River and Dongnai River respectively. The cultured populations displayed no significant genetic diversity. COI sequences identified 17 haplotypes based on 21 polymorphic sites. At this marker, the 12 populations showed a range of h from 0.1290 to 0.6940 and π from 0.0003 to 0.0073. The largest genetic distance (Da) among the 12 populations was 0.0065 (between ZJB and BT/DN populations) and the lowest Da was 0.0003 (between GDD and GDA populations). The wild populations had higher genetic diversity than the cultured populations, but three of cultured populations from Zhejiang (ZJA, ZJB and ZJC) had π higher than wild populations, because they originated from Thailand, Bangladesh and the Mekong River in Viet Nam.     

Use of rpoB gene analysis for identification of nitrogen-fixing Paenibacillus species as an alternative to the 16S rRNA gene

AIM: To avoid the limitations of 16S rRNA-based phylogenetic analysis for Paenibacillus species, the usefulness of the RNA polymerase beta-subunit encoding gene (rpoB) was investigated as an alternative to the 16S rRNA gene for taxonomic studies. METHODS AND RESULTS: Partial rpoB sequences were generated for the type strains of eight nitrogen-fixing Paenibacillus species. The presence of only one copy of rpoB in the genome of P. graminis strain RSA19(T) was demonstrated by denaturing gradient gel electrophoresis and hybridization assays. A comparative analysis of the sequences of the 16S rRNA and rpoB genes was performed and the eight species showed between 91.6-99.1% (16S rRNA) and 77.9-97.3% (rpoB) similarity, allowing a more accurate discrimination between the different species using the rpoB gene. Finally, 24 isolates from the rhizosphere of different cultivars of maize previously identified as Paenibacillus spp. were assigned correctly to one of the nitrogen-fixing species. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The data obtained in this study indicate that rpoB is a powerful identification tool, which can be used for the correct discrimination of the nitrogen-fixing species of agricultural and industrial importance within the genus Paenibacillus.     

Phylogeny and phylogeography of the genus Patella based on mitochondrial DNA sequence data

Although some molecular phylogenies of Patella species have been published in recent years, unresolved questions concerning the phylogeny and taxonomy of this genus still remain. We sequenced the mitochondrial genes cytochrome c oxidase subunit I, 12S rRNA and 16S rRNA for all Patella species (Patella vulgata, Patella depressa, Patella candei, Patella caerulea, Patella lugubris, Patella ferruginea, Patella pellucida and the continental and Macaronesian forms of Patella ulyssiponensis and Patella rustica) and Cymbula safiana. Our results revealed the occurrence of five strongly supported clades, although relationships between these are not well supported. According to our data P. vulgata is a genetically distinct lineage and the close phylogenetic relationship between this species and P. depressa, found in previous mitochondrial DNA studies, is not supported. The Mediterranean Sea and the Macaronesian Islands seem to have played an important role in the speciation and diversification of this genus, although different clades show different phylogeographic patterns. Our dataset point to the necessity of a taxonomic revision, as P. candei is paraphyletic relative to P. lugubris.     

Genetic diversity and phylogenetic relationships of bacteria belonging to the Ochrobactrum-Brucella group by recA and 16S rRNA gene-based comparative sequence analysis

The genetic diversity and phylogenetic interrelationships among 106 Ochrobactrum strains (O. anthropi: 72, O. intermedium: 22, O. tritici: 5, O. oryzae: 2, O. grignonense: 2, O. gallinifaecis: 1, O. lupini: 2), the type strains of the eight Brucella species and other closely related taxa were studied by recA and rrs gene (16S rRNA) comparative sequence analysis. Both markers correctly delineated the various Ochrobactrum species; however, resolution at the subspecies level was considerably higher in the recA gene-based approach. Phylogenetic analyses using neighbor-joining, parsimony, and maximum likelihood algorithms generated trees with similar topologies but the overall branching order, and also the order of the subclades, were not stable in either assay, which could be explained by generally high recA and rrs sequence similarities. Ochrobactrum and Pseudochrobactrum formed separate clades distinct from other Alphaproteobacteria with Bartonella, Agrobacterium, and Rhizobium as the closest relatives. O. gallinifaecis was the most distinct member, when compared to the type species O. anthropi, with rrs and recA similarities of 96.2% and 81.4%. Brucella species were indistinguishable, exhibiting high rrs and recA gene similarities of 98.6% and 85.5% compared with Ochrobactrum intermedium. At the protein level, all RecA sequences among the various Ochrobactrum species and between Ochrobactrum and Brucella were highly similar with only a few amino acid substitutions. O. anthropi and O. tritici were indistinguishable by means of their RecA proteins. A set of initially biochemically classified strains did not cluster within their assigned species and they either grouped within other known species or grouped as potential novel Ochrobactrum species. In further investigations, these strains were reclassified and described as novel species. In summary, Ochrobactrum is a highly diverse genus comprising several novel species. We recommend recA- in addition to rrs gene-analysis for correct species allocation and subtyping of novel Ochrobactrum isolates.     

Molecular species identification of commercially important penaeid shrimp from the Gulf of Mexico using a multiplex haplotype-specific PCR assay

This study describes a multiplex PCR assay based on the 16S rRNA mitochondrial gene to identify the penaeid shrimp Farfantepenaeus aztecus, Farfantepenaeus duorarum, Farfantepenaeus brasiliensis and Litopenaeus setiferus, all native to the Gulf of Mexico, and the exotic Litopenaeus vannamei. The assay was validated using positively identified adult shrimp and confirmed by direct sequencing. Samples of postlarvae and early juveniles collected in the eastern and western Gulf of Mexico were tested yielding 119 F. aztecus, 78 F. duorarum and five L. setiferus. Reliable identification of the morphologically similar early life stages of F. aztecus and F. duorarum has important implications for management and conservation. Similarly, the ability to identify L. vannamei is relevant as early detection could help minimize the ecological impact if this species escapes to the wild.     

The identification of <Emphasis Type="Italic">Cryptosporidium</Emphasis> species in Isfahan,Iran by PCR-RFLP analysis of the 18S rRNA gene

Cryptosporidium is an important protozoan that causes diarrheal illness in humans and animals. Different species of Cryptosporidium have been reported and it is believed that species characteristics are an important factor to be considered in strategic planning for control. We therefore analyzed oocysts from human and animal isolates of Cryptosporidium by PCR-RFLP to determine strain variation in Isfahan. In total, 642 human fecal samples from children under five years of age, imunocompromised patients, and high-risk persons and 480 randomly selected rectal specimens of cows and calves in Isfahan were examined. Microscopic examination showed that 4.7% (30/642) of human samples and 6.2% (30/480) of animal samples were infected with Cryptosporidium. After identification of the samples infected with the parasite, oocysts were purified and their DNA was extracted. We used PCR-RFLP analysis of a 1750-bp region of the 18S rRNA gene to identify Cryptosporidium species. The human samples were infected with Cryptosporidium parvum II, C. muris, C. wrairi, and a new genotype of Cryptosporidium (GenBank accession no. DQ520951). The cattle samples were identified as C. parvum II, C. muris, C. wrairi, C. serpentis, C. baileyi, and a new genotype of Cryptosporidium (GenBank accession no. DQ520952). We also found a new genotype infecting both human and cattle samples (GenBank accession no. DQ520950). In addition to demonstrating the widespread occurrence of most species of Cryptosporidium, C. parvum, we also observed extensive polymorphism within species. Furthermore, the occurrence of the same species of parasite in both animal and human samples shows the importance of the animal and human cycle. Published in Russian in Molekulyarnaya Biologiya, 2007, Vol. 41, No. 5, pp. 934–939. The article was translated by the authors.     

Adding mitochondrial sequence data (16S rRNA and cytochrome c oxidase subunit I) to the phylogeny of centipedes (Myriapoda: Chilopoda): an analysis of morphology and four molecular loci

Relationships within Chilopoda (centipedes) are assessed based on 222 morphological characters, complete 18S rRNA sequences for 70 chilopod terminals, the D3 region of 28S rRNA for 65 terminals, 16S rRNA sequences for 54 terminals and cytochrome c oxidase subunit I sequences for 45 terminals. Morphological and molecular data for seven orders of Diplopoda are used to root cladograms for Chilopoda. Analyses use direct character optimization for 15 gap and substitution models. The Pleurostigmophora and Epimorpha s.l. hypotheses are largely stable to parameter variation for the combined data; the latter clade is formalized as the new taxon Phylactometria. The combined data include parameter sets that support either the monophyly of Epimorpha s.str. (=Scolopendromorpha + Geophilomorpha) or Craterostigmus + Geophilomorpha; the former derives its support from morphology and the nuclear ribosomal genes. Monophyly of Lithobiomorpha and the sister group relationship between Lithobiidae and Henicopidae are stable for morphological and combined data, and are also resolved for the molecular data for 14 of 15 parameter sets. The fundamental split in Scolopendromorpha is between Cryptopidae and Scolopendridae sensu Attems. Blind scolopendromorphs unite as a clade in most molecular and combined analyses, including those that minimize incongruence between data partitions. Geophilomorpha divides into Placodesmata and Adesmata under nine of 15 explored parameter sets.     

Bacterial diversity based on 16S rRNA and gyrB genes at Yinshan mine, China

The diversity of bacterial communities at three sites impacted by acid mine drainage (AMD) from the Yinshan Mine in China was studied using comparative sequence analysis of two molecular markers, the 16S rRNA and gyrB genes. The phylogenetic analyses retrieved sequences from six classes of bacteria, Nitrospira, Alphaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Acidobacteria, and Actinobacteria, as well as sequences related to the plastid of the cyanobacterium Cyanidium acidocaldarium and also some unknown bacteria. The results of phylogenetic analyses based on gyrB and 16S rRNA were compared. This confirmed that gyrB gene analysis may be a useful tool, in addition to the comparative sequence analysis of the 16S rRNA gene, for the analysis of microbial community compositions. Moreover, the Mantel test showed that the geochemical characteristics, especially the pH value and the concentration of iron, strongly influenced the composition of the microbial communities.     

Phylogenetic relationships among four species and a sub-species of Mullidae (Actinopterygii; Perciformes) based on mitochondrial cytochrome B, 12S rRNA and cytochrome oxidase II genes

DNA sequence comparisons of three mitochondrial DNA genes were used to reveal phylogenetic relationships among four species and a sub-species of Mullidae family. This is the first report using mitochondrial DNA sequence data to infer intraspecific relationship among different populations of Mullus barbatus and Mullus surmuletus; phylogenetic relationships between M. barbatus and its sub-species; M. barbatus ponticus. Cytochrome b, 12S ribosomal RNA, and cytochrome oxidase II regions of 242 individuals belonging to species M. barbatus, M. surmuletus, Upeneus moluccensis, Upeneus pori and sub-species M. barbatus ponticus were sequenced and phylogenetic trees were constructed using four different algorithms. The phylogenetic trees constructed support the existing taxonomical data of two mullid genera (Mullus, Upeneus). Molecular data shows no significant difference between same species of different geographical populations. The results suggest that the molecular difference is not large enough between M. barbatus and M. barbatus ponticus to consider them as sub-species.     

Multiplex 16S rRNA haplotype-specific PCR, a rapid and convenient method for fish species identification: an application to West African Clupeiform larvae

A multiplex haplotype-specific polymerase chain reaction (MHS-PCR) method was developed, which identified seven Clupeiform species living in the tropical Eastern Atlantic region: Sardinella aurita, Sardinella maderensis, Ethmalosa fimbriata, Sardina pilchardus, Engraulis encrasicholus, Pellonula leonensis and Ilisha africana. 16S rRNA fragments were amplified using a species-specific set of primers, yielding species-specific size fragments, and then separated using agarose gel electrophoresis, enabling direct visual identification of targeted species. This method provides an accurate, easy and rapid tool for identifying species within large Clupeiform samples. It is suitable for investigations on early Clupeiform stages, species and identification in fishery management in the tropical Eastern Atlantic area.     

基于ITS2和16S rRNA的西施舌群体遗传差异分析

目前,我国西施舌群体分子遗传差异研究结果存在争议。分析我国南北沿海（9个群体）、与广西北海毗邻的越南（1个群体）西施舌核DNA的内转录间隔区2（ITS2）和线粒体DNA的16S rRNA基因（16S）片段核苷酸序列及其二级结构,为解决争议问题提供分子生物学资料。扩增获得西施舌ITS2片段和16S序列,其长度分别为389-402 bp和306 bp,加之下载序列共147条;序列分析显示,74个ITS2序列共有17种基因型,73个16S序列有15种单倍型,其中,长乐（CL）群体独享9种ITS2基因型和5种16S单倍型,非长乐群体（nCL）多数为群体间交叉共享1种或几种基因（单倍）型;基因（单倍）型核苷酸变异位点占5.7%（ITS2）和11.8%（16S）;基于ITS2和16S的CL群体和nCL群体间的遗传距离与群体内遗传距离之比分别为2.42和11.08,nCL群体间的平均遗传距离均为0.007;二级结构显示CL群体ITS2的9种基因型和16S的5种单倍型均区别于nCL群体,nCL的ITS2和16S二级结构分别相似;ITS2和16S基因的系统发育分析显示,CL西施舌形成支持率很高（98,96）的单系支,而nCL群体则交叉聚为另一支（98,96）。研究结果揭示,福建西施舌是腔蛤蜊属（Coelomactra）的一个新种。