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Ribosomal DNA Internal Transcribed Spacer (ribosomal + dna_internal_transcribed_spacer)
Selected AbstractsMolecular diagnosis of Phytophthora lateralis in trees, water, and foliage baits using multiplex polymerase chain reactionFOREST PATHOLOGY, Issue 5 2001L. M. Winton A polymerase chain reaction (PCR)-based protocol for detection of Phytophthora lateralis in plant tissues and water is described. Base-pair (bp) deletions in both of the ribosomal DNA internal transcribed spacer (ITS) regions in P. lateralis were used to design complementary PCR primer sequences that amplify a 738 bp fragment only if P. lateralis DNA is present in the sample. Universal control primers based on conserved sequences of the nuclear ribosomal small subunit are included in a multiplexed reaction, providing an internal check on the procedure. The universal primers amplify an approximately 550 bp fragment that is common to plants, protists, and true fungi. The procedure reliably detects P. lateralis in cedar stem tissues and in roots. Positive reactions were obtained with as few as 200 P. lateralis zoospores in water. Diagnostic moléculaire par PCR multiplex pour détecter Phytophthora lateralis dans les arbres, l'eau et le feuillage utilisé comme piège Un protocole basé sur la PCR est décrit pour détecter Phytophthora lateralis dans les tissus végétaux et l'eau. Des délétions de paires de bases dans chacune des régions ITS de l'ADN ribosomal de P. lateralis ont été utilisées pour définir des amorces de PCR qui n'amplifient un fragment de 738 paires de bases que si l'ADN de P. lateralis est présent dans l'échantillon. Des amorces universelles basées sur des régions conservées de la petite sous-unité de l'ADN ribosomal nucléaire ont été incluses dans une réaction de PCR multiplex, fournissant ainsi un témoin interne de la réaction. Ces amorces universelles amplifient un fragment de 550 pb qui est commun aux plantes, aux protistes et aux champignons vrais. Ce protocole permet la détection de P. lateralis dans les tiges et dans les racines du Chamaecyparis. Des réactions positives ont été obtenues avec seulement 200 zoospores de P. lateralis dans l'eau. Molekulare Diagnose von Phytophthora lateralis in Bäumen, Wasser und als Köder benutzten Blättern mittels Multiplex-PCR Eine auf der PCR beruhende Methode zum Nachweis von Phytophthora lateralis in Pflanzengeweben und Wasser wird beschrieben. Deletionen in den beiden ITS Regionen der ribosomalen DNA von P. lateralis wurden zur Synthese von PCR-Primern ausgenutzt, die ein 738 Basenpaare langes Fragment nur dann amplifizieren, wenn P. lateralis in der Probe vorhanden ist. Universelle Primer, die konservierten Sequenzen der kleinen Unterheit der ribosomalen Kern-DNA entsprechen, wurden als interne Kontrollen in die Multiplex-PCR miteinbezogen. Diese Primer amplifizieren ein ungefähr 550 Basenpaare langes Fragment, das sowohl bei Pflanzen als auch bei Protisten und höheren Pilzen vorkommt. Mit der Methode liess sich P. lateralis im Stamm und in den Wurzeln von Lawsons Scheinzypresse verlässlich nachweisen. Für den Nachweis von P. lateralis im Wasser waren mindestens 200 Zoosporen nötig. [source] Phylogeny of the subgenus Culicoides and related species in Italy, inferred from internal transcribed spacer 2 ribosomal DNA sequencesMEDICAL AND VETERINARY ENTOMOLOGY, Issue 2 2006L. M. Gomulski Abstract., Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) include vectors for the economically important animal diseases, bluetongue (BT) and African horse sickness (AHS). In the Mediterranean Basin, these diseases are transmitted by four species of Culicoides: the first three belong in the subgenus Avaritia Fox and are Culicoides imicola Kieffer, Culicoides obsoletus (Meigen) and Culicoides scoticus Downes and Kettle; the fourth is Culicoides pulicaris (Linnaeus) in the subgenus Culicoides Latreille. In the Palaearctic Region, this subgenus (usually referred to as the C. pulicaris group) now includes a loose miscellany of some 50 taxa. The lack of clarity surrounding its taxonomy stimulated the present morphological and molecular study of 11 species collected in Italy. Phylogenetic analysis of nuclear ribosomal DNA internal transcribed spacer 2 (ITS2) sequence variation demonstrated a high degree of divergence. These results, combined with those from a parallel morphological study, disclosed: (1) that some previously described taxa should be resurrected from synonymy; (2) that there are new species to be described; (3) that the subgenus Culicoides (as currently employed) is a polyphyletic assemblage of four lineages , the subgenus Culicoides sensu stricto, the subgenus Silvicola Mirzaeva and Isaev, the subgenus Hoffmania Fox and the hitherto unrecognized Fagineus species complex. Each is discussed briefly (but not defined) and its constituent Palaearctic taxa listed. Strong congruence between morphological and molecular data holds promise for resolving many of the difficult taxonomic issues plaguing the accurate identification of vector Culicoides around the world. [source] Diversity of algal endosymbionts (zooxanthellae) in octocorals: the roles of geography and host relationshipsMOLECULAR ECOLOGY, Issue 8 2005M. J. H. VAN OPPEN Abstract The presence, genetic identity and diversity of algal endosymbionts (Symbiodinium) in 114 species from 69 genera (20 families) of octocorals from the Great Barrier Reef (GBR), the far eastern Pacific (EP) and the Caribbean was examined, and patterns of the octocoral,algal symbiosis were compared with patterns in the host phylogeny. Genetic analyses of the zooxanthellae were based on ribosomal DNA internal transcribed spacer 1 (ITS1) region. In the GBR samples, Symbiodinium clades A and G were encountered with A and G being rare. Clade B zooxanthellae have been previously reported from a GBR octocoral, but are also rare in octocorals from this region. Symbiodinium G has so far only been found in Foraminifera, but is rare in these organisms. In the Caribbean samples, only Symbiodinium clades B and C are present. Hence, Symbiodinium diversity at the level of phylogenetic clades is lower in octocorals from the Caribbean compared to those from the GBR. However, an unprecedented level of ITS1 diversity was observed within individual colonies of some Caribbean gorgonians, implying either that these simultaneously harbour multiple strains of clade B zooxanthellae, or that ITS1 heterogeneity exists within the genomes of some zooxanthellae. Intracladal diversity based on ITS should therefore be interpreted with caution, especially in cases where no independent evidence exists to support distinctiveness, such as ecological distribution or physiological characteristics. All samples from EP are azooxanthellate. Three unrelated GBR taxa that are described in the literature as azooxanthellate (Junceella fragilis, Euplexaura nuttingi and Stereonephthya sp. 1) contain clade G zooxanthellae, and their symbiotic association with zooxanthellae was confirmed by histology. These corals are pale in colour, whereas related azooxanthellate species are brightly coloured. The evolutionary loss or gain of zooxanthellae may have altered the light sensitivity of the host tissues, requiring the animals to adopt or reduce pigmentation. Finally, we superimposed patterns of the octocoral,algal symbiosis onto a molecular phylogeny of the host. The data show that many losses/gains of endosymbiosis have occurred during the evolution of octocorals. The ancestral state (azooxanthellate or zooxanthellate) in octocorals remains unclear, but the data suggest that on an evolutionary timescale octocorals can switch more easily between mixotrophy and heterotrophy compared to scleractinian corals, which coincides with a low reliance on photosynthetic carbon gain in the former group of organisms. [source] Spawning times, reproductive compatibilities and genetic structuring in the Acropora aspera group: evidence for natural hybridization and semi-permeable species boundaries in coralsMOLECULAR ECOLOGY, Issue 8 2002Madeleine J. H. Van Oppen Abstract Species boundaries among five sympatric coral species of the Indo-Pacific Acropora aspera group were examined by a combination of in vitro breeding trials, comparisons of spawning times and DNA sequence analysis of ribosomal DNA internal transcribed spacer (rDNA ITS) and 5.8S regions. The breeding trials showed that reproductive compatibility exists between at least some colonies of all the species pairs tested, suggesting a large potential for natural hybridization and introgression. The Acropora ITS regions exhibited extremely high levels of variability (up to ,62% for ITS1, ,11% for 5.8S and ,43% for ITS2), but most of the variation was shared among four of the five species, A. millepora, A. papillare, A. pulchra and A. spathulata, consistent with extensive introgression. Phylogenetic analyses did not resolve these four species as distinct clusters across a wide biogeographic region stretching from the southern Great Barrier Reef to Papua New Guinea. However, most colonies of the fifth species, A. aspera, constituted a distinct clade in phylogenetic analyses. This is consistent with our observations of a semi-permeable temporal barrier involving differences in spawning times between this and the other four species. Although the majority of colonies of all five species generally spawned within 90 min of each other, in two out of four years, gametes were absent prior to mass spawning episodes from at least some A. aspera colonies. Hence, our data suggest that transient reproductive barriers may be the result of year-to-year variation in the date of spawning and that this difference in spawning time contributes to the genetic structure detected among Acropora species in this group. Occasional leakage through the reproductive barrier was confirmed by the observation of A. aspera ×A. pulchra F1 hybrids, identified based on additivity of ITS sequences. [source] Phylogenetics and biogeography of eastern Asian,North American disjunct genus Pachysandra (Buxaceae) inferred from nucleotide sequencesJOURNAL OF SYSTEMATICS EVOLUTION, Issue 3 2009Zhihua JIAO Abstract Pachysandra is an eastern Asian,North American disjunct genus with three species, two in eastern Asia (Pachysandra axillaris and Pachysandra terminalis) and one in eastern North America (Pachysandra procumbens). Although morphological and cytological studies suggest a close affinity of P. procumbens with P. axillaris, molecular data from nuclear and chloroplast DNA regions have provided conflicting signals. In this study, we tested previous phylogenetic hypotheses using sequences of nuclear ribosomal DNA internal transcribed spacers and chloroplast ndhF gene from multiple individuals of each of the three species. We also estimated the time of divergence between eastern Asia and eastern North America. Our results support the morphological and cytological conclusion that P. procumbens is more closely related to P. axillaris than to P. terminalis. The estimated time of divergence of P. axillaris and P. procumbens was 14.6±5.5 mya, consistent with estimates from many other eastern Asian,North American disjunct genera. The migration of Pachysandra populations from eastern Asia to North America might have occurred by way of the North Atlantic land bridge. [source] Characterization of Aspergillus flavus strains from Brazilian Brazil nuts and cashew by RAPD and ribosomal DNA analysisLETTERS IN APPLIED MICROBIOLOGY, Issue 1 2008G.E.O. Midorikawa Abstract Aims:, The aim of this study was to determine the genetic variability in Aspergillus flavus populations from Brazil nut and cashew and develop a polymerase chain reaction (PCR) detection method. Methods and Results:, Chomatography analysis of 48 isolates identified 36 as aflatoxigenic (75%). One hundred and forty-one DNA bands were generated with 11 random amplified polymorphic DNA (RAPD) primers and analysed via unweighted pair group analysis, using arithmetic means (UPGMA). Isolates grouped according to host, with differentiation of those from A. occidentale also according to geographical origin. Aspergillus flavus -specific PCR primers ASPITSF2 and ASPITSR3 were designed from ribosomal DNA internal transcribed spacers (ITS 1 and 2), and an internal amplification control was developed, to prevent false negative results. Specificity to only A. flavus was confirmed against DNA from additional aspergilli and other fungi. Conclusions:, RAPD-based characterization differentiated isolates according to plant host. The PCR primer pair developed showed specificity to A. flavus, with a detection limit of 10 fg. Significance and Impact of the Study:, Genetic variability observed in A. flavus isolates from two Brazilian agroecosystems suggested reproductive isolation. The PCR detection method developed for A. flavus represents progress towards multiplex PCR detection of aflatoxigenic and nonaflatoxigenic strains in Hazard Analysis Critical Control Point systems. [source] | ||||||||||