Ribosomal Gene Spacer Research Articles

Phylogenetic study of Aliinostoc species (Cyanobacteria) using pc-igs, nifH and mcy as markers for investigation of horizontal gene transfer

Selection of genes that have not been horizontally transferred for prokaryote phylogenetic studies is regarded as a challenging task. Internal transcribed spacer of ribosomal genes (16S–23S ITS), microcystin synthetase genes (mcy), nitrogenase (nifH) and phycocyanin intergenic spacer (PC-IGS) are among the most used markers in cyanobacteria. The region of the ribosomal genes has been considered stable, whereas the nifH, mcyG and PC-IGS may have undergone horizontal transfer. To investigate the occurrence of horizontal transfer of nifH, mcyG and PC-IGS, phylogenetic trees of Aliinostoc strains Ay1375 and Me1355 were generated and compared. Phylogenetic trees based on the markers were mostly congruent for PC-IGS, indicating a common evolutionary history among ribosomal and phycocyanin genes with no evidence for horizontal transfer of PC-IGS. Phylogenetic trees constructed from the nifH and 16S rRNA genes were incongruent. Our results suggest that nifH has been transferred from one cyanobacterium to another. Moreover, the low non-synonymous/synonymous mutation ratio (Ka/Ks) was consistent with an ancient origin of the mcyG.

Morpho-molecular characterization of Discosia ravennica sp. nov. and a new host record for Sporocadus rosigena.

Collections of fungal samples from two dead leaf specimens from Italy were subjected to morphological examination and phylogenetic analyses. Two coelomycetous taxa belonging to two different genera in Xylariomycetidae, Sordariomycetes, namely Discosia and Sporocadus, were identified. The Discosia taxon is revealed as a new species and is herein introduced as Discosia ravennicasp. nov. while the Sporocadus taxon is identified as Sporocadus rosigena. Multi-locus phylogeny based on DNA sequence data of the large subunit (LSU) and internal transcribed spacer (ITS) of nuclear ribosomal genes, β-tubulin (β-tub) and RNA polymerase II second largest subunit (rpb2) showed that D. ravennica is related to D. neofraxinea but it forms an independent lineage that supports its new species status. The new taxon also differs from other Discosia species by its unilocular to bilocular, superficial and applanate conidiomata with basal stroma composed of cells of textura angularis, elongate-ampulliform conidiogenous cells and conidia smaller in size. Sporocadus rosigena is here reported as a new host record from Quercus ilex from Italy. Descriptions, illustrations and molecular data for both species are provided in this paper.

Rapid pathogen detection by metagenomic next-generation sequencing of infected body fluids.

We developed a metagenomic next-generation sequencing (mNGS) test using cell-free DNA from body fluids to identify pathogens. The performance of mNGS testing of 182 body fluids from 160 patients with acute illness was evaluated using two sequencing platforms in comparison to microbiological testing using culture, 16S bacterial PCR and/or 28S-internal transcribed ribosomal gene spacer (28S-ITS) fungal PCR. Test sensitivity and specificity of detection were 79 and 91% for bacteria and 91 and 89% for fungi, respectively, by Illumina sequencing; and 75 and 81% for bacteria and 91 and 100% for fungi, respectively, by nanopore sequencing. In a case series of 12 patients with culture/PCR-negative body fluids but for whom an infectious diagnosis was ultimately established, seven (58%) were mNGS positive. Real-time computational analysis enabled pathogen identification by nanopore sequencing in a median 50-min sequencing and 6-h sample-to-answer time. Rapid mNGS testing is a promising tool for diagnosis of unknown infections from body fluids.

Detecting and molecular typing of Vibrio parahaemolyticus

Vibrio parahaemolyticus is a prevalent food-borne pathogen in many Asian countries and has been spreading rapidly following the appearance of pandemic strains. This article reviews conventional and molecular methods for culturing, identifying, enumerating and rapidly detecting V. parahaemolyticus and its virulence factors, such as thermostable direct hemolysin (TDH), TDH-related hemolysin and thermolabile hemolysin. It also discusses various molecular typing methods, namely, pulsed-field gel electrophoresis, ribotyping, random amplified polymorphic DNA and three other polymerase chain reaction methods based on specific conserved nucleotide sequences, such as ribosomal gene spacer, repetitive extragenic palindromic and enterobacterial repetitive intergenic consensus sequences, for use in epidemiological investigations of this pathogen.

Optimization and characterization of red pigment production from an endophytic fungus, Nigrospora aurantiaca CMU-ZY2045, and its potential source of natural dye for use in textile dyeing.

Some of the most important natural pigments have been produced from fungi and used for coloring in food, cosmetics, textiles, and pharmaceutical products. Forty-seven isolates of endophytic fungi were isolated from Cinnamomum zeylanicum in northern Thailand. Only one isolate, CMU-ZY2045, produced an extracellularly red pigment. This isolate was identified as Nigrospora aurantiaca based on morphological characteristics and the molecular phylogenetic analysis of a combined four loci (large subunit and internal transcribed spacer of ribosomal DNA, β-tubulin, and translation elongation factor 1-alpha genes). The optimum conditions for red pigment production from this fungus were investigated. The results indicated that the highest red pigment yield was observed in the liquid medium containing glucose as a carbon source and yeast extract as a nitrogen source, at a pH value of 5.0 and at 27°C with shaking for 5days. The crude red pigment revealed the highest level of solubility in methanol. A fungal red pigment was found to have high stability at temperatures ranging from 20 to 50°C and pH values at a range of 5.0-6.0. Based on liquid chromatography-mass spectrometry analyses, the red pigment was characterized as bostrycin. The extracted pigment was used for the textile dyeing process. Crude fungal red pigment revealed the highest staining ability in cotton fabrics and displayed excellent fastness to washing, which showing negative cytotoxicity at the concentrations used to cell culture. This is the first report on bostrycin production from N. aurantiaca.

First Report of Ixodes nipponensis Infection in Goats in China.

Ticks are obligate blood-sucking ectoparasites that infect a wide range of animals and humans, causing a variety of both human and animal diseases around the world. Ixodes nipponensis is the most commonly reported tick in Korea and Japan, but it is very rare in China. In this study, six I. nipponensis samples were collected from three black goats in Hunan province, China. Ticks identified morphologically as I. nipponensis were then examined by PCR with two different molecular markers: mitochondrial cox1 and the second internal transcribed spacer of ribosomal DNA genes. Sequence comparison and phylogenetic analysis of the cox1 sequences confirmed that all of the examined hard Ixodes ticks represented I. nipponensis. This finding indicates a potential risk of zoonotic I. nipponensis infection in humans and animals in China. To our knowledge, this is the first report documenting the occurrence of I. nipponensis infection in goats in China.

Genetic diversity among eight Dendrolimus species in Eurasia (Lepidoptera: Lasiocampidae) inferred from mitochondrial COI and COII, and nuclear ITS2 markers.

BackgroundMoths of genus Dendrolimus (Lepidoptera: Lasiocampidae) are among the major pests of coniferous forests worldwide. Taxonomy and nomenclature of this genus are not entirely established, and there are many species with a controversial taxonomic position. We present a comparative evolutionary analysis of the most economically important Dendrolimus species in Eurasia.ResultsOur analysis was based on the nucleotide sequences of COI and COII mitochondrial genes and ITS2 spacer of nuclear ribosomal genes. All known sequences were extracted from GenBank. Additional 112 new sequences were identified for 28 specimens of D. sibiricus, D. pini, and D. superans from five regions of Siberia and the Russian Far East to be able to compare the disparate data from all previous studies. In total, 528 sequences were used in phylogenetic analysis. Two clusters of closely related species in Dendrolimus were found. The first cluster includes D. pini, D. sibiricus, and D. superans; and the second, D. spectabilis, D. punctatus, and D. tabulaeformis. Species D. houi and D. kikuchii appear to be the most basal in the genus.ConclusionGenetic difference among the second cluster species is very low in contrast to the first cluster species. Phylogenetic position D. tabulaeformis as a subspecies was supported. It was found that D. sibiricus recently separated from D. superans. Integration of D. sibiricus mitochondrial DNA sequences and the spread of this species to the west of Eurasia have been established as the cause of the unjustified allocation of a new species: D. kilmez. Our study further clarifies taxonomic problems in the genus and gives more complete information on the genetic structure of D. pini, D. sibiricus, and D. superans.Electronic supplementary materialThe online version of this article (doi:10.1186/s12863-016-0463-5) contains supplementary material, which is available to authorized users.

Phylogenetic study of Geitlerinema and Microcystis (Cyanobacteria) using PC-IGS and 16S-23S ITS as markers: investigation of horizontal gene transfer.

Selection of genes that have not been horizontally transferred for prokaryote phylogenetic inferences is regarded as a challenging task. The markers internal transcribed spacer of ribosomal genes (16S-23S ITS) and phycocyanin intergenic spacer (PC-IGS), based on the operons of ribosomal and phycocyanin genes respectively, are among the most used markers in cyanobacteria. The region of the ribosomal genes has been considered stable, whereas the phycocyanin operon may have undergone horizontal transfer. To investigate the occurrence of horizontal transfer of PC-IGS, phylogenetic trees of Geitlerinema and Microcystis strains were generated using PC-IGS and 16S-23S ITS and compared. Phylogenetic trees based on the two markers were mostly congruent for Geitlerinema and Microcystis, indicating a common evolutionary history among ribosomal and phycocyanin genes with no evidence for horizontal transfer of PC-IGS. Thus, PC-IGS is a suitable marker, along with 16S-23S ITS for phylogenetic studies of cyanobacteria.

Genetic heterogeneity of non-O1 and non-O139 Vibrio cholerae isolates from shrimp aquaculture system: a comparison of RS-, REP- and ERIC-PCR fingerprinting approaches

The genetic diversity of Vibrio cholerae isolated from black tiger shrimp (Penaeus monodon) aquaculture farms was determined using three PCR typing methods based on enterobacterial repetitive intergenic consensus (ERIC) sequences, ribosomal gene spacer (RS) sequence and repetitive extragenic palindromic (REP) sequences. Non-O1 and non-O139 V. cholerae isolates were obtained from shrimp pond water, pond sediment, shrimp head and shrimp muscle. RS-PCR yielded fewer bands than REP-PCR and ERIC-PCR. Higher similarity was observed in RS-PCR (75-100%) than in REP-PCR (60-95%) and ERIC-PCR (40-95%). A 100% similarity between V. cholerae isolates was only noticed in RS-PCR. The choleratoxigenic V. cholerae (non-O1 and non-O139) showed greater genetic similarity with ctx-negative V. cholerae than among ctx-positive V. cholerae. The greater similarity of ctx-positive V. cholerae with ctx-negative V. cholerae isolates indicates that the ctx-positive strains (non-O1 and non-O139) might have originated from autochthonous V. cholerae in the aquatic niche.

Isolation of Trypanosoma (Megatrypanum) theileri from Dairy Cattle in Taiwan

Although Trypanosoma (Megatrypanum) theileri, a blood parasite of bovid species, is spread widely throughout the world, it has never been reported in Taiwan. When an anti-coagulated blood sample from febrile dairy cattle was directly smeared, no parasite was observed. However, a highly distinctive morphological feature of trypanosome appeared in baby hamster kidney (BHK) cell culture inoculated with non-thrown blood buffy coat. The different stages and typical ultrastructures of trypanosome were observed in our isolate. The isolate was subsequently identified as T. theileri by species-specific PCR assay (Tth625), 18S rDNA sequencing alignment and internal transcribed spacer of ribosomal genes (ITS) as a marker for molecular phylogenetic analysis. The first T. theileri isolate in Taiwan (TWTth1) could be periodically passaged in BHK cell culture for more than one year and retained good re-cryopreservation viability. The BHK culture method would be excellent for diagnostic isolation and maintenance long-term development of this parasite.

Incidence and Molecular Typing of Vibrio parahaemolyticus from Tiger Shrimp Culture Environments along the Southwest Coast of India

Vibrio parahaemolyticus is one of the most prevalent food-borne pathogens along the southwest coast of India, where marine foods are frequently consumed. Shrimp (Penaeus monodon) and environmental samples were collected from aquaculture farms located in and around Cochin. Confirmation of the biochemically identified strains with species-specific toxR gene and detection of virulent genes viz., tdh and trh was performed by polymerase chain reaction (PCR). The phenotypic markers for the presence of tdh and trh genes were assayed by Kanagawa phenomenon and urease activity, respectively. Protease activity was examined to identify other potential virulence factors. After phenotypic characterization of bacterial strains fingerprinting of genomic DNA was carried by various typing methods, viz., random amplified polymorphic DNA (RAPD), enterobacterial repetitive intergenic consensus sequence (ERIC), repetitive extragenic palindromic sequence (REP), and ribosomal gene spacer sequence (RS) PCR methods to assess the genetic diversity within the isolates. Eighteen percent of the samples were found positive for the incidence of V. parahaemolyticus by biochemical protocols and toxR (368 bp) targeted PCR. PCR analyses revealed 1% of the samples positive for tdh (269 bp) and trh (500 bp) gene. RAPD analysis revealed clustering of toxigenic strains into a single group. Cluster analysis revealed the conglomeration of isolates into two, five, and seven major groups using RS, ERIC, and REP PCR methods, respectively. RS PCR generated fewer amplified bands compared to REP and ERIC PCR methods, thus giving scope for higher discrimination. Moreover, RS PCR patterns were more discernible visually from other patterns, suggesting RS PCR as a considerably practical method for routine use.

Detection of Marteilia refringens using nested PCR and in situ hybridisation in Chamelea gallina from the Balearic Islands (Spain)

In the course of a histopathological survey performed to discover the cause of mass mortality of the striped clam Chamelea gallina in the Balearic Islands (Spain, Mediterranean Sea), we detected a Marteilia-like parasite in 3 clams. Molecular methods were applied to identify the parasite. DNA extracted from a paraffin block was used to carry out a PCR assay for Marteilia refringens detection based on a rDNA sequence of the parasite (the intergenic spacer of ribosomal genes, IGS). The nucleotide sequence of the IGS amplified fragment and the positive signal obtained by in situ hybridisation analysis with a M. refringens-specific probe allowed us to confirm the presence of this parasite in the digestive gland tissue of C. gallina.

Molecular and chromosomal characterization of repeated and single-copy DNA sequences in the genome of Dasypyrum villosum

Restriction fragment length polymorphism of ribosomal DNA repeated unit and single-copy DNA fragments and chromosomal distribution of a highly repeated sequence, have been studied to assess molecular markers and the extent of their heterogeneity in Dasypyrum villosum. Substantial variation has been found for the length of the intergenic spacer of ribosomal genes clustered in different alleles at Nor- VI locus of heterozygous individuals, but not within the cluster of rDNA of homozygous individuals. After Southern blots and hybridization to an intergenic spacer probe, each cluster of rDNA was detected as a single band with at least four variants differing for the number of 130 bp subrepeats in the intergenic spacer. One recombinant plasmid contained a 2270 bp DNA insert from the D. villosum genome that upon Sph I restriction endonuclease digestion was cleaved in three 380 bp repeat elements and one 1090 bp fragment. When Southern blots of Sph 1 digested D. villosum DNAs of different genotypes were hybridized to the 32P-labelled 380 bp repeat, a distinct ladder consisting of multiples of a basic repeat unit of about 380 bp in length was revealed on autoradiograms. The in situ hybridization of the 3H-labelled 380 bp repeat element showed that one chromosome pair (7V) was not labelled. In the other pairs, silver grains remained clustered at or near the telomeres. Dot-blot hybridization analysis of DNAs from a range of diploid, tetraploid, and hexaploid Triticeae species indicated that the 380 bp repeated element was a specific feature of the D. villosum genome. Other cloned DNA sequences of D. villosum showed a large restriction length polymorphism and one was located on V chromosomes.

Rapid, Sensitive, and Discriminating Identification ofNaegleriaspp. by Real-Time PCR and Melting-Curve Analysis

The free-living amoeboflagellate genus Naegleria includes one pathogenic and two potentially pathogenic species (Naegleria fowleri, Naegleria italica, and Naegleria australiensis) plus numerous benign organisms. Monitoring of bathing water, water supplies, and cooling systems for these pathogens requires a timely and reliable method for identification, but current DNA sequence-based methods identify only N. fowleri or require full sequencing to identify other species in the genus. A novel closed-tube method for distinguishing thermophilic Naegleria species is presented, using a single primer set and the DNA intercalating dye SYTO9 for real-time PCR and melting-curve analysis of the 5.8S ribosomal DNA gene and flanking noncoding spacers (ITS1, ITS2). Collection of DNA melting data at close temperature intervals produces highly informative melting curves with one or more recognizable melting peaks, readily distinguished for seven Naegleria species and the related Willaertia magna. Advantages over other methods used to identify these organisms include its comprehensiveness (encompassing all species tested to date), simplicity (no electrophoresis required to verify the product), and sensitivity (unambiguous identification from DNA equivalent to one cell). This approach should be applicable to a wide range of microorganisms of medical importance.

Identification of highly prized commercial fish using a PCR‐based methodology

We report a practical class designed for undergraduate students of Marine Sciences as a part of the Genetics course. The class can also be included in undergraduate studies of food technology. The exercise was designed to emphasize the application of molecular biology techniques to fish species authentication and traceability. After a simple and rapid protocol for DNA extraction, PCR was used to analyze variation in size of individual 5S ribosomal gene spacer sequences in eight commercially important fishes. The resulting fragments containing these sequences were separated on agarose gels and visualized under a UV lamp. Differences in fragment length can be used to differentiate between species. This method is particularly useful in the identification of larvae, eggs, and processed food including frozen and canned products.

(293) Single Nucleotide Polymorphism in matK and Ribosomal ITS of Astilbe

The genus Astilbe (Saxifragaceae) comprises about 13 species and is ranked consistently among the top 10 landscape perennials. Through extensive hybridization, selection and marketing, the lineage of many Astilbehas been lost. Subdioecious Astilbebiternatais the only species in the genus native to North America while other members of the genus are endemic to Asia and monoecious. Due to the unusual geographic distribution of the species and the variation in floral development among them, development of genetic markers using single nucleotide polymorphisms (SNPs) would confirm phylogenetic relationships and establish lineage within the genus. Astilbespecies, hybrids, and cultivars were obtained from plant nurseries and botanical gardens across the country. To elucidate relationships among the genus, we conducted phylogenetic analysis of DNA sequences of the chloroplast gene matKand the internal transcriber spacer (ITS) of ribosomal rDNA genes. DNA was extracted, and gene primers trnK3914 and trnK2R were used to amplify matK, and primers 1406F and ITS2 were used to amplify the ITS1 region between 18S and 5.8S ribosomal DNA units. Both matKand ITS were sequenced for each plant specimen and sequences were aligned to identify nucleotide diversity and detect SNPs. Variation in nucleotide sequence for either gene yielded similar dendrograms. Nucleotide variation among the Astilbeutilized in this study has allowed the development of SNP markers that may be useful for fingerprinting unknown hybrids or cultivars in the industry, and may be used for species alignment within the genus.

Analysis of nucleotide sequence polymorphism of internal transcribed spacers of ribosomal genes in diploid Aegilops (L.) species

The nucleotide sequence of the fragment of the internal transcribed spacer (ITS) of rDNA comprising the full-length ITS1, the gene encoding 5.8S rRNA, and part of the ITS2 sequence was determined in 22 samples of five diploid Aegilops species. The full alignment length of compared sequences was 524 bp. Species-specific substitutions were found in the ITS nucleotide sequence of rDNA of different Aegilops species. Intraspecific differences in ITS structure in diploid Aegilops species were detected for the first time. Polymorphism of the ITS nucleotide sequence within the same sample was revealed, which might be due either to differences between the genomes of individual plants comprising the sample or to the presence of several types of ribosomal genes in the genome of one plant. In general, both interspecific and intraspecific variability of the ITS nucleotide sequences of rDNA is extremely low. In total, 26 variable sites, twelve of which were informative, were identified.

Efficient polymerase chain reaction detection of the second internal transcribed spacer of mucosa-derived larvae is dependent on the larval extraction method.

Methods for estimating abundance of arrested gastrointestinal larvae in large mammal hosts by digestion of the gastrointestinal mucosa are well established. The effects of digestion on the success of species identification using the polymerase chain reaction (PCR) are, however, unknown. In this study, the relationship between numerical recovery of arrested larvae and the success of PCR-typing for the second internal transcribed spacer of ribosomal genes was characterized. Fresh and prefrozen mucosa of 4 sheep yielded very similar rates of recovery and PCR detection. When sheep mucosa were digested with neutral N-acetyl cysteine, recovery increased, whereas PCR detection remained constant (60-80%) with digest duration (1-16 hr). In contrast, when sheep and Svalbard reindeer mucosa were digested with acid-pepsin, recovery increased, whereas PCR detection declined to 0 with digest duration. Thus, to optimize recovery and PCR analysis of arrested gastrointestinal nematode larvae, acid-pepsin digestion of 1-2 hr for PCR detection and 16 hr for recovery, or neutral N-acetyl cysteine digestion of 8-16 hr for both assays, should be used.

Novel strategies to study the role of mutation and nucleic acid structure in evolution

Natural selection processes tune genomes in the edge of the chaos imposed by mutation and drift, allowing an enduring exploration of fitter genetic networks within the constraints imposed by self-organization and the interactions of genotype and phenotype. Alternatively, evolution can be viewed from thermodynamic, kinetic or cybernetic perspectives. Regardless of insight, there is need to understand structure-function relationships at the molecular and holistic evolutionary levels. Strategies are here described that analyze genetic variation in time and trace the evolution of nucleic acid structure. Nucleic acid scanning techniques were used to measure sequence divergence and provide a direct inference of genome-wide mutation rate. This was tested for the first time in vegetatively propagating plants. The method is general and was also used in a study of mutational patterns in phytopathogenic fungi, showing there was a link between sequence and structural diversification of ribosomal gene spacers. In order to determine if this was a general phenomenon, the origin and diversification of nucleic acid secondary structure was traced using a cladistic method capable of producing rooted phylogenetic trees. Phylogenies reconstructed from primary and secondary RNA structure were congruent at all taxonomical levels, providing evidence of a strong link between phenotype and genotype favoring thermodynamic stability and dissipation of Gibbs free energy. Overall results suggest that thermodynamic principles are important driving forces of the evolutionary processes of the living world.

Reticulate evolution and the origins of ribosomal internal transcribed spacer diversity in apomictic Meloidogyne.

Among root knot nematodes of the genus Meloidogyne, the polyploid obligate mitotic parthenogens M. arenaria, M. javanica, and M. incognita are widespread and common agricultural pests. Although these named forms are distinguishable by closely related mitochondrial DNA (mtDNA) haplotypes, detailed sequence analyses of internal transcribed spacers (ITSs) of nuclear ribosomal genes reveal extremely high diversity, even within individual nematodes. This ITS diversity is broadly structured into two very different groups that are 12%-18% divergent: one with low diversity (< 1.0%) and one with high diversity (6%-7%). In both of these groups, identical sequences can be found within individual nematodes of different mtDNA haplotypes (i.e., among species). Analysis of genetic variance indicates that more than 90% of ITS diversity can be found within an individual nematode, with small but statistically significant (5%-10%; P < 0.05) variance distributed among mtDNA lineages. The evolutionarily distinct parthenogen M. hapla shows a similar pattern of ITS diversity, with two divergent groups of ITSs within each individual. In contrast, two diploid amphimictic species have only one lineage of ITSs with low diversity (< 0.2%). The presence of divergent lineages of rDNA in the apomictic taxa is unlikely to be due to differences among pseudogenes. Instead, we suggest that the diversity of ITSs in M. arenaria, M. javanica, and M. incognita is due to hybrid origins from closely related females (as inferred from mtDNA) and combinations of more diverse paternal lineages.