Spacer Length Variation in Rice 5SrRNA Genes Revealed by Polymerase Chain Reaction

Polymerase chain reaction(PCR) was used to amplify 5S rRNA spacer from wild rice(Oryza rufipogon and O.nivara) and cultivated rice(indica and japonica varieties of O.sativa L).The results show that there is spacer length variation within and between species,and the typical indica and japonica varieties have their unique banding patterns of amplified 5S rRNA spacers,whereas intermediate showed no specific amplification profile of spacer regions.The 5S rRNA genes in intermediate are either identical with that of indica variety or that of japonica variety.These data suggest that the spacer length polymorphisms can be used to distinguish between closely ralated species and subspecies.

16S rRNA Gene Phylogenesis of Culturable Predominant Bacteria from Diseased Apostichopus japonicus(Holothuroidea,Echinodermata)

Cultured Apostichopus japonicus in China suffers from a kind of skin ulceration disease that has caused severe economic loss in recent years. The disease,pathogens of which are supposed to be bacteria by most researchers,is highly infectious and can often cause all individuals in the same culture pool to die in a very short time. The 16S rRNA gene phylogenesis of the culturable bacteria from the lesions of diseased individuals was conducted to study the biodiversity of the bacterial communities in the lesions and to identify probable pathogen(s) associated with this kind of disease. S. japonica samples were selected from a hatchery located in the eastern part of Qingdao,China. Bacterial universal primers GM5F and DS907R were used to amplify the 16S rRNA gene of bacteria colonies,and touchdown PCR was performed to amplify the target sequences. The results suggest that γ-proteobacteria(Alteromonadales and Vibrionales) of CFB group,many strains of which have been also determined as pathogens in other marine species,are the predominant bacterial genera of the diseased Apostichopus japonicus individuals.

Morphogenesis and Molecular Phylogeny of a Marine Urostylid Ciliate Apokeronopsis wrighti(Protista, Ciliophora, Hypotrichia)

Hypotrichs are one of the highly differentiated ciliated lineages which play important roles in ecological, environmental,evolutionary and basic biological studies. In the present study, we investigated the living characteristics, infraciliature, nuclear apparatus, ontogenesis and phylogenetic position of a marine hypotrichous ciliate, Apokeronopsis wrighti Long et al., 2008, which was isolated from coastal waters in Shenzhen, China. The new isolate resembles the type population in terms of morphological characteristics, morphometrics, and SSU rRNA gene sequence that is with a 99.7% similarity. Ontogenesis of A. wrighti is characterized by oral primordium for the proter as well as marginal and dorsal kineties anlagen in both filial products formed de novo, and the cirral row arranged along the paroral and endoral arises from several anterior frontoventral-transverse cirral streaks. Phylogenetic analyses based on SSU and concatenated gene data suggest that five species of Apokeronopsis form a monophyletic clade, and the genus Apokeronopsis is closely related to Thigmokeronopsis and Metaurostylopsis.

16S rRNA Gene-Based Analysis of Ileal Bacterial Community and Phylogeny in Nursing and Weaned Piglets

Weaning of piglets is generally considered as a stressor which changes intestinal ecosystem and leads to clinical implications. Microbiota inhabiting in small intestine （especially ileum） are assumed to promote health, but their functional properties are yet poody dascdbed. As indicated by the 16S rRNA gene sequences of ileal micrebiota in nursing piglets （at the age of 21 and 28 d） and 28-day-old weaned piglets （weaned at 21 d of age）, the microbiota were mainly comprised of gram-positive bacteria. There were 40 operational taxonomic units （OTUs） （from 171 clones） in the ileum of nursing piglets aged 21 d, 61 OTUs （from 194 clones） in the ileum of nursing piglets aged 28 d, and 56 OTUs （from 171 clones） in the ileum of weaned piglets aged 28 d. The flea of nursing piglets aged 21 d were dominantly occupied by Lactobacilli （87.7%） as well as Streptococ cus （ 3.5 % ）. Lactobacillus amy/ovorus （41.5 % ）, Lactobaci/lus sp. （ 19.3 % ）, Lactobaci/lus reuteri （ 12.3 % ）, Lactobacillus salivarius （ 9.4 % ） and L. mucosae （4.7%） were the predominant species among Lactobacil/L Similar results were obtained in the nursing piglets at 28 d of age ex- cept that Lactobaci/li decreased to 71.1% and Streptococcus increased to 21.1% significantly. Lactobacillus （52.0%） and Streptococcus （26.3%） were the two major groups in the ileum of weaned piglets aged 28 d. Lactobacillus amylovorus （31.6%） and Lactobaci/lus reuteri （ 16.4% ） was the two most important species in Lactobacillus. Therefore, Lactobacilli were predominant in the ileum of nursing and weaned piglets, and they had the highest diversity, followed by Streptococcus. The diversity of ileal microbiota was not different remarkably between the nursing piglets and the weaned piglets, but the composition changed significantly. These findings are helpful to understand ileal bacterial ecophysiology and further develop nutritional regimes to prevent or counteract complications during the weaning transition.

Cloning and Sequence Analysis of 16S rRNA and COI Gene in Mitochondrial DNA of Scortum barcoo

[Objective] The aim was to provide molecular biological basis for the researches on the genetic resources,genetic relationship among species and phyletic evolution of S.barcoo.[Method] PCR amplification and sequencing were used to study the 16S rRNA and COI gene fragments.[Result] As for 16S rRNA gene fragments,nucleotide sequences of 791 bp were obtained,and the A,T,G and C contents in this fragment were 31.6%,21.4%,20.4% and 26.7%respectively.As for the COI gene fragments,the size was 631 bp and the A,T,G And C contents were 27.7%,23.6%,29.8% and 18.9% respectively.Among these two gene fragments,the content of GC was lower than AT,and AT/GC of these two fragments was 1.13 and 1.05 respectively.[Conclusion] The genetic characteristics of gene fragments of 16S rRNA and COI of S.barcoo suggested that the variation in the same species was relatively low.The sequences of 16S rRNA gene in three samples the same,while the sequences of COI gene was also the same,indicating that these two gene of S.barcoo were conservative.

Identification of sika deer and red deer using partial cytochrome b and 12s ribosomal RNA genes

A study was conducted on the identifications of the degraded samples of sika deer （Cervus nippon） and red deer （Cervus elaphus） by phylogenetic and nucleotide distance analysis of partial Cytb and 12s rRNA genes sequences. 402 bp Cytb genes were achieved by PCR-sequencing using DNA extracted from 8 case samples, and contrasted with 27 sequences of Cytb gene downloaded from GenBank database. The values of three nucleotide distance between three suspected samples and sika deer were identical （0.026±0.006）, which was smaller than the smallest nucleotide distance between eastern red deer and sika deer （0.036）. Furthermore, phylogenetic analysis of sika deer and red deer indicated that the evidences located within the same cluster as sika deer. The evidences were sika deer materials. As the same way, other three suspected samples were derived from red deer. The results were further confirmed by phylogenetic and nucleotide distance analysis of 387 bp 12s rRNA gene. The method was powerful and less time-consuming and helpful to reduce the related cases with wildlife.

Genetic Diversity in Populations of Sepiella maindroni Using 16S rRNA Gene Sequence Analysis

Part of the 16S rRNA gene is amplified with PCR and sequenced for 5 populations of com-mon Chinese cuttlefish Sepiella maindroni: three from the South China Sea, one from East China Sea and one from Japan. The result shows that a total of 5 nucleotide positions are found to have gaps or insertions of base pairs among these individuals, and 13 positions are examined to be variable in all the sequences, which range from 494 to 509 base pairs. All of the individuals are grouped into 7 haplotypes (h1-h7). No marked genetic difference is observed among those populations. All of the individuals from Nagasaki belong to hl and the h3 haplotype is found only in the coastal waters of China. A(?)G transition in Nucleotide 255 is suggested to be taken as a kind of genetic marker to identify the populations distributed in East-South China Sea and the Nagasaki waters of Japan.

Preliminary study on mitochondrial 16S rRNA gene sequences and phylogeny of flatfishes (Pleuronectiformes)

A 605 bp section of mitochondrial 16S rRNA gene from Paralichthys olivaceus, Pseudorhombus cinnamomeus, Psetta maxima and Kareius bicoloratus, which represent 3 families of Order Pleuronectiformes was amplified by PCR and sequenced to show the molecular systematics of Pleuronectiformes for comparison with related gene sequences of other 6 flatfish downloaded from GenBank. Phylogenetic analysis based on ge- netic distance from related gene sequences of 10 flatfish showed that this method was ideal to explore the rela- tionship between species, genera and families. Phylogenetic trees set-up is based on neighbor-joining, maximum parsimony and maximum likelihood methods that accords to the general rule of Pleuronectiformes evolution. But they also resulted in some confusion. Unlike data from morphological characters, P. olivaceus clustered with K. bicoloratus, but P. cinnamomeus did not cluster with P. olivaceus, which is worth further studying.

Comparative evaluation of microscopy,OptiMAL® and 18S rRNA gene based multiplex PCR for detection of Plasmodium falciparum & Plasmodium vivax from field isolates of Bikaner,India

Objective:To evaluate microscopy,OptiMAL? and multiplex PCR for the identification of Plasmodium falciparum（P.falciparum） and Plasmodium vivax（P.vivax） from the field isolates of Bikaner,Rajasthan（Northwest India）.Methods:In this study,a multiplex PCR（P.falciparum and P.vivax） was further developed with the incorporation of Plasmodium malariae（P.malariae） specific primer and also a positive control.The performance of microscopy,plasmodium lactate dehydrogenase（pLDH） based malaria rapid diagnostic test OptiMAL? and 18S rRNA gene based multiplex PCR for the diagnosis of P.falciparum and P.vivax was compared.Results:The three species multiplex PCR if.falciparum,P.vivax and P.malariae) with an inbuilt positive control was developed and evaluated.In comparison with multiplex PCR,which showed the sensitivity and specificity of 99.36%（95%CI,98.11%-100.00%） and 100.00%（95%CI,100.00%-100.00%）,the sensitivity and specificity of microscopy was 90.44%（95%CI,88.849-95.04%） and 99.22%（95% CI,97.71%-100.00%）,and OptiMAL? was 93.58%（95%CI,89.75%-97.42%） and 97.69%（95%CI, 95.10%-100.00%）.The efficiencies were 99.65%,95.10%and 95.45%for multiplex PCK.microscopy and OptiMAL?.respectively.Conclusions:Our results raise concerns over the overall sensitivities of microscopy and OptiMAL?,when compared to the multiplex PCR and thus stress the need for new molecular interventions in the accurate detection of the malarial parasites.This further highlights the fact that further developments are needed to improve the performance of rapid diagnostic tests at field level.

Species identification and phylogenetic analysis of genus Nassarius(Nassariidae)based on mitochondrial genes

Genus Nassarius contains many subgenera, such as Zeuxis, Telasco, Niotha, Varicinassa, Plicarcularia, Nassarius s. str. and Reticunassa. On the basis of morphological characteristics of the shell and radula and sequences of mitochondrial cytochrome oxidase subunit I (COI) and 16S rRNA genes, Nassarius specimens collected from the South China Sea were identified and phylogenetically analyzed. Although Nassarius sp. and Nassarius (Varicinassa) variciferus were morphologically different in their shells, few variations were found among their radular teeth and sequences of mtCOI and mt16S RNA genes. Therefore, Nassarius sp. should be classified as N. (Varicinassa) variciferus. Nassarius (Zeuxis) sp. has only a subtle difference from Nassarius (Zeuxis) algidus on the shell, but it shows obvious differences in radular teeth and DNA sequence, indicating that they are two distinct species. Sequence divergence of mtCOI and mt16S RNA genes within Nassarius species was much lower than that between species, suggesting that these two genes are suitable for Nassarius species identification. Phylogenetic analysis (neighbor-joining and maximum parsimony) based on mtCOI and mt16S rRNA genes revealed the presence of two groups in genus Nassarius and a closest relationship between subgenera Zeuxis and Telasco. Species of subgenus Plicarcularia did not form a single clade. The molecular phylogeny was not congruent with the previous morphological phylogeny. The subgeneric divisions of genus Nassarius appear to be uncertain and unreliable.

First molecular identification of Cryptosporidium by 18S rRNA in goats and association with farm management in Terengganu

Objective:To identify the prevalence of Cryptosporidium from goats in three types of farm management systems in Terengganu,Malaysia and to determine the Cryptosporidium species infecting goats by using 18 S r RNA.Methods:A total of 478 fecal samples were randomly collected from goats in three farms;199 samples were collected from intensive farm,179 samples from semi-intensive farm and 100 samples from extensive farm.The samples were processed by using formolether concentration technique and stained by using modified Ziehl–Neelsen.Positive samples were performed by using nested PCR analysis by using 18 S r RNA.Results:Out of 478 goats,207(43.3%) were found to be infected with Cryptosporidium.Goats reared under the intensive farm management system reported the highest prevalence of infection(49.7%),followed by intensive farm management system(41%) and the lowest prevalence was reported in the goats reared under semi-intensive management system(37.4%).Conclusions:The identified species found in goat was Cryptosporidium parvum.Future study on the zoonotic transmission of Cryptosporidium parvum in goats needs to be done in order to find the source of transmission of this parasite.

Eukaryotic food sources analysis in situ of tropical common sea cucumber Holothuria leucospilota based on 18S rRNA gene high-throughput sequencing

Sea cucumber Holothuria leucospilota is one of the most widespread tropical holothurian species.In this study,eukaryotic organism composition in foregut and hindgut contents of H.leucospilota and surrounding sediments was assessed by 18S rRNA gene high-throughput sequencing.Eukaryon richness and diversity in the habitat sediments were significantly higher than those in foregut and hindgut contents of the sea cucumbers(P<0.05).The foregut content group,hindgut content group,and marine sediment group sequences were respectively assigned to 18.20±1.32,19.40±1.03,and 21.80±0.37 phyla.In the foregut contents,Nematoda(20.18%±9.59%),Mollusca(16.12%±10.49%),Chlorophyta(10.04%±4.85%),Annelida(8.72%±10.93%),Streptophyta(8.46%±4.65%),and Diatomea(5.99%±2.01%)were the predominant phyla,which showed the eukaryotic food sources of H.leucospilota were primarily belong to the above phyla.The predominant phyla in the hindgut contents were Streptophyta(45.55%±17.32%),Mollusca(4.93%±4.82%),Arthropoda(5.37%±3.08%),Diatomea(3.88%±2.34%),and Chlorophyta(3.79%±1.59%);and Annelida(37.80%±17.00%),Arthropoda(24.49%±12.53%),Platyhelminthes(7.14%±3.02%),Nematoda(4.14%±0.91%),and Diatomea(5.11%±1.35%)had large contents in the sediments.The comparatively high content of Paris genus in phylum Streptophyta in foregut contents indicated that land plants were one of the primary food sources of H.leucospilota,however the significantly higher contents of Streptophyta in hindgut contents than that in foregut contents might suggest a large part of the terrigenous detritus ingested might not be digested by H.leucospilota.UPGMA and PCoA analysis revealed that eukaryotic organism composition differed significantly between foregut contents of H.leucospilota and ambient sediments,indicating selective feeding feature of H.leucospilota.This study provided useful references for artificial feed of tropical sea cucumbers and enhanced understanding of the ecological roles of detritus-feeding macrobenthos.

Frequency of mitochondrial 12S rRNA gene A1555G and 961 insC mutations among children with sensorineural deafness in China

Objective： To investigate the frequency of mitochondrial 12S rRNA gene A1555G and 961 insC mutations among Chinese with sensorineural deafness. Methods： Blood samples from 78 sporadic cases with sensorineural deafness were obtained and DNA was extracted from the leukocytes, then the mitochondrial DNA target fragments were amplified by polymerase chain reaction（PCR）. The 1555G mutations were detected by BsmA 1 restriction endonuclease digestion, every fragment was analyzed by sequencing; All the 961 insC mutation were detected by direct sequencing. Results： The percent age of A1555G mutation and mt961C insertion were 6.4% and 2.6% in the hearing-impaired Chinese subjects respectively. Conclusion： A1555G and 961insC mutations in mitochondrial DNA 12S rRNA gene regions may play a role in the pathogenesis of hearing loss in the sporadic cases.

Phylogenetic studies of four species of ciliate inferred from 16s-like small subunit rRNA gene sequences

The phylogenetic relationships of four ciliate genera （Urostyla, Euplotes, Stylonychia and Pseudokeronopsis）, which also are the important environment inspection species, were analyzed by the comparison of small subunit ribosomal RNA gene sequences. Euplotes appeared as an early branching group whose divergence from the hypotrichous line at a deep level was strongly supported by parsimony and matrix analyses. The analyses also supported the hypothesis that there were closely relationship between species in Urostyla and Holosticha. The sibling species Stylonychia mytilus and S. lemnae could be separated by the evolutionary analyses. Furthermore, Pseudokeronopsis rubra had relatively more closely relationship with the species in Holostichidae than that in Urostylidae based on the evolutionary distance value.

16S rRNA Gene Sequence Analysis of Snow Leopard, Gray Wolf, Horse and Bactrian Camel in Mongolia

In this study, mitochondrial 16S rRNA sequences of snow leopard, gray wolf, domestic horse and Bactrian camel inhabited or domesticated in Mongolian territory were obtained by using polymerase chain reaction （PCR） based on universal primers for 16S rRNA （F-5＇-AACGAGCCTGGTGATA-3＇ and R-5＇-CTCCGGTCTGAACTCAGATCACGTA-3＇）. The 16S rRNA sequence was 1,048 bp to 1,086 bp in length, and each sequence was compared to other related species （Felidae, Camelidae, Equidae and Canidae） by using NCBI Basic Local Alignment Search Tool （BLAST）. Results showed that sequences were highly similar to sequences in GenBank database （93%-99%）. Then phylogenetic analysis was performed based on about 1,100 bp sequence of 16S rRNA for Panthera uncia, Canis lupus, Equus caballus, Camelus bactrianus and other related species. The result revealed that P. uncia and P. leo were sister species, C. bactrianus and C. ferus were more closely related species, and wolf and dog were the almost similar species. This finding could be important for designing species specific primers for PCR based analysis of animal species identification and forensic veterinary medicine.

16S rRNA Gene-Based Metagenomic Analysis of Soil Bacterial Diversity in Brazzaville, Republic of the Congo

Soil contains a great diversity of microorganisms, among which are bacteria. This study aimed to explore bacterial diversity in soil samples in Brazzaville in the Republic of the Congo. Environmental DNA was extracted. The illumina MiSeq sequencing was held and the diversity indices have been computed. Illumina MiSeq sequencing revealed 21 Phyla, four of which were abundant: Proteobacteria, Acidobacteria, Actinobacteria and Bacteroidetes. Soil microbial communities in the studied samples were phylogenetically diverse but with a stable community structure. 17 classes are represented with relative abundances of Rihzobiales, Bacillales, Actinomycetales and Acidobacteriales. 40 families, the Alphaproteobacteria, the Bacilli and the 12 Actinobacteria. 83 orders among which the Rhizobiales are the most abundant followed by Bacillales and the least abundant followed by the Flavobacteriaceae. Of the 28 genera listed, the Bradyrhizobium is the most dominant in Mw3 and Mw4. 25 listed species, Bradyrhizobium, Bacillus, Actinoplanes, and Candidatu coribacter Acidobacterium are the most abundant species. The Shannon indices of Mw3 and Mw4 are equal, the H’max of Mw4 is greater than the H’max of Mw3. The Simpson index of Mw4 is equal to the Simpson index of Mw3, and the Pielou index (J) of Mw4 is less than the R of Mw3, but very close. This study opens interesting perspectives on the knowledge and exploitation of telluric bacteria in several areas of life.

Phylogenetic Relationships among 12 Species of Tetrigidae (Orthoptera:Tetrigoidea) Based on Partial Sequences of 12S and 16S Ribosomal RNA

Mitochondrial 12S and 16S ribosomal RNA genes sequences were sequenced using dye-labeled terminator on an ABI 377 automated sequencer in 11 individuals and 1 species' sequences were gained from GenBank,representing 6 genera of family Tetrigidae.The collated sequences were aligned using Clustal X version 1.81 and then,the sequence variability and heredity distances based on Kimura 2-parameter model were calculated using Mega 2.1.In obtained sequences (736 bp),the average A+T content is 73.9%,ranging from 71.2% to 77.5%;the overall G+C content is 26.1%,ranging from 22.5% to 28.8%.Based on alignment of the combined sequences,185 parsimony-informative sites were revealed in 755 available base pairs.Phylogenetic trees were reconstructed using NJ,MP and ML methods with Cylindraustralia kochii as outgroup.The results indicated that the monophyletic nature of Tetrix is questioned and suggest that T.tubercarina may be given tribal rank.Our results also show that Coptltettix huanjiangensis and C.gongshanensis are the same species,i.e.Coptltettix gongshanensis Zheng,and C.huanjiangensis is the synonyms of C.gongshanensis.

PCR-DGGE法分析不同酒曲中细菌多样性

目的:研究不同酒曲中细菌的多样性.方法:利用DNA提取试剂盒,分别提取6种酒曲的总DNA,利用细菌16S rRNA gene V3区通用引物,采用变性梯度凝胶电泳(polymerase chain reaction-denatured gradient gelelectrophoresis,PCR-DGGE)方法,进行不同酒曲中细菌多样性的分析.结果:6种酒曲中共分离出12属的细菌,分别为:湖南韶山的酒曲中主要是Weissella,Streptophyta,Bacillus,Pantoea,Okibacterium这5个属的菌株,湖南湘潭酒曲中主要含有Weissella,Pediococcus,Lactococcus,Sphingomonas,Streptophyta,Pantoea这6属的菌株,湖南祁东酒曲中主要含有Weissella,Pediococcus,Bacillus,Klebsiella,Pantoea 5属的菌株,福建福州酒曲含有Weissella,Pediococcus,Pantoea,Pediococcus,Sphingomonas,Streptophyta 6属的菌株,浙江丽水酒曲含有Weissella,Pantoea,Pediococcus,Klebsiella,Enterobacter,Rhizobium,Sphingomonas,Thauera 8属的菌株,浙江兰溪酒曲含有Weissella,Streptophyta,Pantoea 3属的菌株.结论:这说明不同地区的酒曲中的细菌的种类有所不同,同时也说明酒曲中Weissella属的菌是发酵的常见菌群.

Distribution and diversity of microbial communities in meromictic soda Lake Doroninskoe(Transbaikalia,Russia) during winter

Meromictic soda and saline lakes are unique ecosystems characterized by the stability of physical,chemical and biological parameters,and they are distributed all over the world.Lakes located in regions with average annual negative air temperature are of particular interest because of the presence of two periods with intensive and dynamic processes:the so-called biological summer and the long ice season with the biological spring.Soda Lake Doroninskoe is located in Eastern Transbaikalia(51°14′N,112°14′E) in the permafrost zone in an extreme continental climate,and is covered by ice for seven months per year.The structure and diversity of the microbial communities throughout the water column of the lake was studied by 16 S r RNA gene amplicon metasequencing.Different species with specific functions were found to dominate at different depths.Metabolically flexible bacteria with a capacity to switch between anoxygenic photosynthesis and aerobic chemotrophic metabolism dominate in soda Lake Doroninskoe.

Isolation,Screening and Identification of High-temperature Cellulolytic Microbes in Pig Manure

[Objective] The research aimed to lay the foundation for high-efficiency biological degradation microbial inoculums. [Method] under the 60℃ temperature the cellulolytic microbes in pig manure were isolated and determined the CMCase and Fpase,then proceeded the 16S rRNA gene analysis. [Result]The results showed that:BC1 and BC3 strains characterized higher carboxymethyl cellulose enzyme activity and higher filter paper activity,but their difference was small,then the 16S rDNA sequence of BC1 and BC3 strains were related to pseudomonas sp. (98% and 99% similarities,respectively).[Conclusion] the experiment laid foundation for high-efficiency biological heating agent.