Rickettsial Agents from Parasitic Dermanyssoidea (Acari: Mesostigmata)

Mites are often overlooked as vectors of pathogens, but have been shown to harbor and transmit rickettsial agents such as Rickettsia akari and Orientia tsutsugamushi. We screened DNA extracts from 27 mites representing 25 species of dermanyssoids for rickettsial agents such as Anaplasma, Bartonella, Rickettsia, and Wolbachia by PCR amplification and sequencing. DNA from Anaplasma spp., a novel Bartonella sp., Spiroplasma sp., Wolbachia sp., and an unclassified Rickettsiales were detected in mites. These could represent mite-borne bacterial agents, bacterial DNA from blood meals, or novel endosymbionts of mites.     

Registration of S alleles in Brassica campestris L by the restriction fragment sizes of SLGs

Polymorphism of SLG (the S-locus glycoprotein gene) in Brassica campestris was analyzed by PCR-RFLP using SLG-specific primers. Nucleotide sequences of PCR products from 15 S genotypes were determined in order to characterise the exact DNA fragment sizes detected in the PCR-RFLP analysis. Forty-seven lines homozygous for 27 S-alleles were used as plant material. One combination of primers, PS5 + PS 15, which had a nucleotide sequence specific to a class-I SLG, gave amplification of a single DNA fragment of approximately 1.3kb from the genomic DNA of 15 S genotypes. All the DNA fragments showed different electrophroetic profiles from each other after digestion with MboI or MspI. Different lines having the same S genotype had an identical electrophoretic profile even between the lines collected in Turkey and in Japan. Another class-I SLG-specific primer, PS 18, gave amplification of a 1.3-kb DNA fragment from three other S genotypes in combination with PS 15, and the PCR product also showed polymorphism after cleavage with the restriction endonucleases. Genetic analysis, Southern-hybridization analysis, and determination of the nucleotide sequences of the PCR products suggested that the DNA fragments amplified with these combinations of primers are class-I SLGs. Expected DNA fragment sizes in the present PCR-RFLP condition were calculated from the determined nucleotide sequence of SLG PCR products. A single DNA fragment was also amplified from six S genotypes by PCR with a combination of primers, PS3 + PS21, having a nucleotide sequence specific to a class-II SLG. The amplified DNA showed polymorphisnm after cleavage with restriction endonucleases. The cleaved fragments were detected by Southern-hybridization analysis using a probe of S ⁵ SLG cDNA, a class-IISLG. Partial sequencing revealed a marked similarity of these amplified DNA fragments to a class-II SLG, demonstrating the presence of class-I and class-II S alleles also in B. campestris. The high SLG polymorphism detected by the present investigation suggests the usefulness of the PCR-RFLP method for the identification of S alleles in breeding lines and for listing S alleles in B. campestris.     

Genotyping of Trichophyton rubrum by analysis of ribosomal-DNA intergenic spacer regions

An attempt was made to explore the genotyping of Trichophyton rubrum (T. rubrum) and the relationship between genotype and geographical origin using ribosomal restriction endonuclease polymorphic analysis. The total DNA was extracted by cetyltrimethyl ammonium bromide (CTAB). The probe was amplified from part of the 18S, ITSI, 5.8S, and ITSII region of T. rubrum standard strain with the universal fungal primers NS5 [5′-AACTT AAAGG AATTG ACGGA AG-3′] and ITS4 [5′-TCCTC CGCTT ATTGA TATGC-3′]. The genomic DNA of 49 clinical T. rubrum isolates digested by EcoR1 were hybridized with this probe, and the hybridization patterns were used as the basis of genotyping. Of the data from 49 strains of T. rubrum studied (21 from Nanjing, 26 from Dalian, and two from Beijing), 20 individual patterns (DNA Type A–T) were identified, among which Type A–C accounted for 48.98% of all the strains. The DNA patterns of Nanjing strains were represented by three bands, those of Dalian strains were represented by four bands. The DNA typing of T. rubrum by Southern blotting was highly sensitive and highly distinguishable. The DNA patterns of Nanjing strains were obviously different from those of Dalian strains.     

Relationships between species ofLeymus,Psathyrostachys, andHordeum (Poaceae,Triticeae) inferred from Southern hybridization of genomic and cloned DNA probes

We have used total genomic DNA as a probe to size-fractionated restriction enzyme digests of genomic DNA from a range ofTriticeae species from the generaLeymus Hochst.,Psathyrostachys Nevski, andHordeum L., and hybrids betweenHordeum andLeymus to investigate their taxonomic relationships. Genomic Southern hybridization was found to be an effective and simple way to assess the distribution and diversity of essentially species-specific and common, repetitive DNA sequences, and is hence especially useful in evolutionary studies. The DNA sequences ofH. vulgare seem to diverge substantially from those ofH. brachyantherum, H. lechleri, H. procerum, andH. depressum. The genome ofThinopyron bessarabicum shows little homology to those of theLeymus species investigated, confirming thatT. bessarabicum is not an ancestral genome inLeymus. Although the genomes ofLeymus andPsathyrostachys share substantial proportions of DNA sequences, they include divergent repeated sequences as well. Hybridization with a ribosomal DNA probe (pTa 71) showed that the coding regions containing structural genes encoding the 18 S, 5.8 S, and 26 S ribosomal RNA were conserved among the species investigated, whereas the intergenic spacer region was more variable, presenting different sizes of restriction fragments and enabling a classification of the species. The rye heterochromatin probe pSc 119.2 hybridized to DNA fromH. lechleri andT. bessarabicum, but not to DNA from the other species investigated.     

Detection and quantification of Entomophaga maimaiga resting spores in forest soil using real-time PCR

Environmental sampling to monitor entomopathogen titre in forest soil, a known reservoir of insect pathogens such as fungi and viruses, is important in the evaluation of conditions that could trigger epizootics and in the development of strategies for insect pest management. Molecular or PCR-based analysis of environmental samples provides a sensitive method for strain- or species-based detection, and real-time PCR, in particular, allows quantification of the organism of interest. In this study we developed a DNA extraction method and a real-time PCR assay for detection and quantification of Entomophaga maimaiga (Zygomycetes: Entomophthorales), a fungal pathogen of the gypsy moth, in the organic layer of forest soil. DNA from fungal resting spores (azygospores) in soil was extracted using a detergent and bead mill homogenization treatment followed by purification of the crude DNA extract using Sephadex–polyvinylpolypyrrolidone microcolumns. The purification step eliminated most of the environmental contaminants commonly co-extracted with genomic DNA from soil samples but detection assays still required the addition of bovine serum albumin to relieve PCR inhibition. The real-time PCR assay used primers and probe based on sequence analysis of the nuclear ribosomal ITS region of several E. maimaiga and two E. aulicae strains. Comparison of threshold cycle values from different soil samples spiked with E. maimaiga DNA showed that soil background DNA and remaining co-extracted contaminants are critical factors determining detection sensitivity. Based on our results from comparisons of resting spore titres among different forest soils, estimates were best for organic soils with comparatively high densities of resting spores.     

<Emphasis Type="Italic">Drosophila</Emphasis><Emphasis Type="Italic">P</Emphasis> transposons of the urochordata <Emphasis Type="Italic">Ciona intestinalis</Emphasis>

P transposons belong to the eukaryotic DNA transposons, which are transposed by a cut and paste mechanism using a P-element-coded transposase. They have been detected in Drosophila, and reside as single copies and stable homologous sequences in many vertebrate species. We present the P elements Pcin1, Pcin2 and Pcin3 from Ciona intestinalis, a species of the most primitive chordates, and compare them with those from Ciona savignyi. They showed typical DNA transposon structures, namely terminal inverted repeats and target site duplications. The coding region of Pcin1 consisted of 13 small exons that could be translated into a P-transposon-homologous protein. C. intestinalis and C. savignyi displayed nearly the same phenotype. However, their P elements were highly divergent and the assumed P transposase from C. intestinalis was more closely related to the transposase from Drosophila melanogaster than to the transposase of C. savignyi. The present study showed that P elements with typical features of transposable DNA elements may be found already at the base of the chordate lineage. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Production and analysis of intergeneric somatic hybrids between <Emphasis Type="Italic">Brassica oleracea</Emphasis> and<Emphasis Type="Italic"> Matthiola incana</Emphasis>

The gene pool of Brassica oleracea was enriched via intergeneric somatic hybridization between B. oleracea (2n = 18) and Matthiola incana (2n = 14). One hundred and eighteen plants were obtained from 96 calli. Only four plants (H1, H2, H3 and H4) showed an intermediate phenotype from the parents; among these, H1 and H3 arose from the same callus. Random amplified polymorphic DNA (RAPD), sequence-related amplified polymorphism (SRAP), and cytological analyses confirmed that H1 and H3 were hybrids. The nuclear DNA content of the regenerated plants was determined by flow cytometry. More than half of the plants contained a nuclear DNA content of 1.3 to 3.9 pg/cell, which was higher than the content of B. oleracea but lower than that of M. incana. H1 contained 4.89 ± 0.02 pg of DNA per cell, while H3 nuclear DNA content was estimated at 4.87 ± 0.06 pg/cell. Cytological study of the root-tip cells revealed that the majority of the H1 and H3 hybrid cells contained 28 chromosomes.     

Larvae of chigger mites Neotrombicula spp. (Acari: Trombiculidae) exhibited Borrelia but no Anaplasma infections: a field study including birds from the Czech Carpathians as hosts of chiggers

Chigger mites were collected from 1,080 wild birds of 37 species at Certak (Czech Republic), in the western Carpathian Mountains, from 29 July to 24 September 2005. The prevalence of infestation with chigger larvae was 7%. A total of 325 chigger specimens from 10 bird species was identified and three chigger species were found: Neotrombicula autumnalis, N. carpathica, and N. inopinata, the latter two species being reported on new hosts. Neotrombicula carpathica is reported in the Czech Republic for the first time. A total of 509 chigger larvae found on 79 host specimens were examined by polymerase chain reaction (PCR) for the presence of Borrelia burgdorferi s.l. DNA (fragments of the rrf (5S)—rrl (23S) intergenic spacer), and Anaplasma phagocytophilum DNA (epank1 gene). A fragment of specific Borrelia DNA was amplified through PCR in one sample, and the PCR product was further analyzed by reverse line blotting assay, whereby both genospecies of B. garinii and B. valaisiana were proved. This sample pooled five chigger larvae collected from one Sylvia atricapilla on 11 August 2005. No A. phagocytophilum DNA was amplified. We conclude that larvae of the genus Neotrombicula can be infected with Borrelia genospecies originated from their present or former hosts.     

TrnL-trnF sequence data imply paraphyly of Aquilaria and Gyrinops (Thymelaeaceae) and provide new perspectives for agarwood identification

The genera Aquilaria and Gyrinops (Thymelaeaceae, Malvales) are well known for the production of agarwood which is a highly wanted forest product of substantial economic value. The taxonomic status of Aquilaria and Gyrinops as separate genera is doubted as they are only distinguished by the number of stamens. We investigated their status by conducting phylogenetic analyses of DNA sequences from the plastid trnL-trnF spacer. Control of international trade of agarwood is currently hampered by the failure of traditional methods such as microscopy to identify samples to species level. We therefore evaluated the potential of molecular identification of agarwood by searching for species- and region-specific plastid DNA polymorphisms. DNA sequences were obtained from 31 Thymelaeaceae accessions encompassing 20 different species in six genera. Aquilaria and Gyrinops appear to be paraphyletic. Success in sequencing wood samples demonstrates that molecular markers provide new perspectives for agarwood identification.     

Lack of evidence on hybrid swarm in the sympatric population of Pinus mugo and P. sylvestris

Natural hybridisation between the taxa from Pinus mugo complex and P. sylvestris was postulated in several sympatric populations of the species in Europe. However, due to the absence of precise methods for identification of hybrid seeds and hybrid trees, the frequency of hybridisation and its influence on the genetic structure of relict P. mugo populations has not been clarified so far. In the present study, the species-specific chloroplast DNA (cpDNA) marker and isozymes were applied to test the hybridisation hypothesis in a postulated hybrid swarm population of the species from “Bór na Czerwonem” reserve at the northern foothills of Tatra Mts., Poland. The cpDNA marker was used to identify hybrids within two groups of polycormic (multi-stemmed) and monocormic (single-stemmed) pines from this population. Allelic frequencies at isozyme loci from both groups were compared to frequencies found in allopatric populations of the species. Additionally, cpDNA haplotypes of seedlings derived from open pollinated seeds were studied to detect the possibility of successful cross-pollination. The mixture of seedlings with P. sylvestris and P. mugo cpDNA haplotypes was derived from one parental tree that indicates hybridisation. However, all the mature polycormic pines had cpDNA haplotypes species specific to P. mugo and the isozyme frequencies were similar to frequencies found in three allopatric populations of P. mugo from Tatra Mts. (mean genetic distance, Dn=.027). The differences were much larger in comparisons with monocormic pines from this area (Dn=.085) and two P. sylvestris samples from distant allopatric populations (Dn=.077). Nearly all monocormic pines had cpDNA species specific to P. sylvestris and isozyme frequencies similar to other populations of this species (mean Dn=.004). Only one P. sylvestris-like monocormic tree had cpDNA of P. mugo and can be considered as a hybrid. The results do not provide evidence supporting the hybrid swarm hypothesis. Rather, the results suggest that mature hybrids have a low frequency within this population and (rare) hybridisation is not reciprocal but unidirectional with P. mugo as pollen donor.     

Phylogenetic investigations of Sordariaceae based on multiple gene sequences and morphology

The family Sordariaceae incorporates a number of fungi that are excellent model organisms for various biological, biochemical, ecological, genetic and evolutionary studies. To determine the evolutionary relationships within this group and their respective phylogenetic placements, multiple-gene sequences (partial nuclear 28S ribosomal DNA, nuclear ITS ribosomal DNA and partial nuclear β-tubulin) were analysed using maximum parsimony and Bayesian analyses. Analyses of different gene datasets were performed individually and then combined to generate phylogenies. We report that Sordariaceae, with the exclusion Apodus and Diplogelasinospora, is a monophyletic group. Apodus and Diplogelasinospora are related to Lasiosphaeriaceae. Multiple gene analyses suggest that the spore sheath is not a phylogenetically significant character to segregate Asordaria from Sordaria. Smooth-spored Sordaria species (including so-called Asordaria species) constitute a natural group. Asordaria is therefore congeneric with Sordaria. Anixiella species nested among Gelasinospora species, providing further evidence that non-ostiolate ascomata have evolved from ostiolate ascomata on several independent occasions. This study agrees with previous studies that show heterothallic Neurospora species to be monophyletic, but that homothallic ones may have a multiple origins. Although Gelasinospora and Neurospora are closely related and not resolved as monophyletic groups, there is insufficient evidence to place currently accepted Gelasinospora and Neurospora species into the same genus.     

Cloning and partial characterization of the putative rifamycin biosynthetic gene cluster from the actinomycete Amycolatopsis mediterranei DSM 46095

The actinomycete Amycolatopsis mediterranei produces the commercially and medically important polyketide antibiotic rifamycin, which is widely used against mycobacterial infections. The rifamycin biosynthetic (rif) gene cluster has been isolated, cloned and characterized from A. mediterranei S699 and A. mediterranei LBGA 3136. However, there are several other strains of A. mediterranei which also produce rifamycins. In order to detect the variability in the rif gene cluster among these strains, several strains were screened by PCR amplification using oligonucleotide primers based on the published DNA sequence of the rif gene cluster and by using dEBS II (second component of deoxy-erythronolide biosynthase gene) as a gene probe. Out of eight strains of A. mediterranei selected for the study, seven of them showed the expected amplification of the DNA fragments whereas the amplified DNA pattern was different in strain A. mediterranei DSM 46095. This strain also showed striking differences in the banding pattern obtained after hybridization of its genomic DNA against the dEBS II probe. Initial cloning and characterization of the 4-kb DNA fragment from the strain DSM 46095, representing a part of the putative rifamycin biosynthetic cluster, revealed nearly 10% and 8% differences in the DNA and amino acid sequence, respectively, as compared to that of A. mediterranei S699 and A. mediterranei LBGA 3136. The entire rif gene cluster was later cloned on two cosmids from A. mediterranei DSM 46095. Based on the partial sequence analysis of the cluster and sequence comparison with the published sequence, it was deduced that among eight strains of A. mediterranei, only A. mediterranei DSM 46095 carries a novel rifamycin biosynthetic gene cluster.     

DNA divergency and speciation inSymbiodinium (Dinophyceae)

Relationship among symbiotic dinoflagellates of the genusSymbiodinium derived from seven different host species has been studied by means of DNA/DNA hybridization. DNA homologies range from about 70 to 30%D. Highest homology is regarded as subspecific level. Based on the characteristics of DNA and phenotypes,Symbiodinium microadriaticum subsp.microadriaticum is separated fromSymbiodinium microadriaticum subsp.condylactis. Lowest homology occurs at the methodical background, and is similar to that obtained with DNA of algae belonging to different classes. The data are in excellent agreement with DNA base composition, karyotypes, and morpohological as well as biochemical markers, emphasizing speciation among these gymnodinioid zooxanthellae.Dedicated to DrRobert K. Trench, Professor of Biology and Geology, University of California at Santa Barbara, whose work initiated recognition of speciation in zooxanthellae.     

Analysis of nuclear and organellar DNA of somatic hybrid calli and plants between Lycopersicon spp. and Nicotiana spp.

Protoplast fusion experiments between Lycopersicon esculentum or L. peruvianum and Nicotiana tabacum or N. plumbaginifolia were performed to investigate the possibility of producing symmetric and asymmetric somatic hybrids between these genera. These fusions, which involved 1.7 × 10⁸ protoplasts, yielded 35 viable hybrid calli. Plant regeneration was successful with two calli. One of these regenerants flowered, but developed no fruits. Analysis of the nuclear DNA by means of dot blot hybridization with species-specific repetitive DNA probes combined with flow cytometry, revealed that the nuclei of most hybrid calli contained the same absolute amount of Nicotiana DNA as the Nicotiana parent or (much) less, whereas the amount of Lycopersicon DNA per nucleus was 2–5 times that of the parental genotype. Eighteen of the 34 hybrids analyzed possessed Lycopersicon chloroplast DNA (cpDNA), whereas the other 16 had DNA from Nicotiana chloroplasts. The cpDNA type was correlated with the nuclear DNA composition; hybrids with more than 2C Nicotiana nuclear DNA possessed Nicotiana chloroplasts, whereas hybrids with 2C or less Nicotiana nuclear DNA contained Lycopersicon chloroplasts. Mitochondrial DNA (mtDNA) composition was correlated with both nuclear DNA constitution and chloroplast type. Hybrids possessed only or mainly species-specific mtDNA fragments from the parent predominating in the nucleus and often providing the chloroplasts. The data are discussed in relation to somatic incompatibility which could explain the low frequency at which hybrids between Lycopersicon and Nicotiana species are obtained and the limited morphogenetic potential of such hybrids.     

Cybrids of Nicotiana tabacum and Petunia hybrida have an intergeneric mixture of chloroplasts from P. hybrida and mitochondria identical or similar to N. tabacum

Summary The mitochondrial genomes of cybrids of Nicotiana tabacum containing chloroplasts of Petunia hybrida were characterized by restriction endonuclease digestion and agarose gel electrophoresis. Cybrids that displayed normal growth and development contained mitochondrial DNA indistinguishable from N. tabacum mitochondrial DNA. Cybrids that displayed abnormal growth and development contained mitochondrial DNA that differed from N. tabacum either by possessing a few additional fragments, by lacking a few fragments, or both. In spite of these differences, the mitochondrial DNA of cybrids showing abnormal growth and development was much more similar to N. tabacum than to P. hybrida mitochondrial DNA. In those cybrids that contained P. hybrida chloroplasts and N. tabacum mitochondria, cotransfer of cytoplasmic organelles did not occur. Although P. hybrida chloroplasts can interact compatibly with the N. tabacum nucleus, no cybrids were found in which P. hybrida mitochondria coexisted with the N. tabacum nucleus.     

Nuclear DNA content of Vitis species,cultivars, and other genera of the Vitaceae

The nuclear DNA content was analyzed in Vitis species, hybrid cultivars, and genera of the Vitaceae using flow cytometry. Significant variation was found among Vitis species, hybrids, and other genera of the Vitaceae (Ampelopsis and Parthenocissus). DNA content was estimated to range from 0.98 to 1.05 pg/2C within V. labrusca (ns) and 0.86 to 1.00 pg/2C within V. vinifera (ns). Genotypes from Vitis and Parthenocissus were similar in nuclear DNA content (approximately 1.00 pg/2C) whereas they differed significantly from Ampelopsis (1.39 pg/2C). No correlation between DNA content and the center of origin of genotypes of the Vitaceae was noted. Based on the present study, the Vitis genome size is 475 Mbp, 96% of which is non-coding. Knowledge of DNA content is useful in order to understand the complexity of the Vitis genome and to establish a relationship between the genetic and physical map for map-based cloning.     

Molecular phylogeny of the genus Stereocaulon (Stereocaulaceae, lichenized ascomycetes)

In the present study phylogenetic relationships of the genus Stereocaulon (lichenized ascomycetes) were examined using DNA sequences from the ITS1–5.8 S–ITS2 rDNA gene cluster and from the protein-coding β-tubulin gene. In addition to the fruticose species traditionally classified in Stereocaulon, representatives of the crustose species that have recently been transferred to the genus were included. Muhria, a monotypic genus that is morphologically similar to Stereocaulon, differing only in apothecia ontogeny, was also incorporated. The analyses included 101 specimens from the ingroup representing 49 taxa. Sequences from both DNA regions were analysed simultaneously using direct optimization under the parsimony optimality criterion. The results support the inclusion of the crustose species and Muhria in Stereocaulon, while the current infrageneric classification is not supported. As Muhria is securely nested within Stereocaulon the new combination Stereocaulon urceolatum comb. nov. (syn. Muhria urceolata) is made. Further, species concepts need to be re-examined, as some species do not appear as monophyletic entities in the phylogeny.     

Progress on the genetics of the N2-fixing actinorhizal symbiont Frankia

The actinomycete Frankia is of fundamental and ecological interests for several reasons including its wide distribution, its ability to fix nitrogen, differentiate into sporangium and vesicle (specialized cell for nitrogen-fixation), and to nodulate plants from about 24 genera. Here, we present a review on the genetics performed so far on Frankia. At the end of July 2001, 293 kbp of Frankia DNA sequences were found in the databases. Thirty five percent of these sequences corresponded to full gene or gene cluster sequences. These genes could be divided according to their role into 6 key activities: gene translation (rrnA and tRNA ^pro gene), proteolysis (pcr genes), assimilation of ammonium (glnA and glnII), protection against superoxide ions (sodF), nitrogen fixation (nif cluster), and plasmid replication. We present a review of these genetic islands; their function, expression, localization and particular properties are discussed. A comparative analysis of Frankia nif genes from various strains and species is presented. An improved nomenclature for some of these genes is suggested to avoid conflicts. Frankia plasmids DNA sequences are also presented. The novel trends in Frankia genetics are described.     

Two translesion synthesis DNA polymerase genes, AtPOLH and AtREV1, are involved in development and UV light resistance in Arabidopsis

Plants are continually exposed to external and internal DNA-damaging agents. Although lesions can be removed by different repair processes, damages often remain in the DNA during replication. Synthesis of template damages requires the replacement of replicative enzymes by translesion synthesis polymerases, which are able to perform DNA synthesis opposite specific lesions. These proteins, in contrast to replicative polymerases, operate at low processivity and fidelity. DNA polymerase η and Rev 1 are two proteins found in eukaryotes that are involved in translesion DNA synthesis. In Arabidopsis, DNA polymerase η and Rev 1 are encoded by AtPOLH and AtREV1 genes, respectively. Transgenic plants over-expressing AtPOLH showed increased resistance to ultraviolet light. Only plants with moderate AtREV1 over-expression were obtained, indicating that this enzyme could be toxic at high levels. Transgenic plants that over-expressed or disrupted AtREV1 showed reduced germination percentage, but the former exhibited a higher stem growth rate than the wild type during development.