Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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    Evolution of Hyaloperonospora effectors: ATR1 effector homologs from sister species of the downy mildew pathogen H. arabidopsidis are not recognised by RPP1WsB
    Solovyeva, I. ; Schmuker, A. ; Cano, L.M. ; Damme, M. van; Ploch, S. ; Kamoun, S. ; Thines, M. - \ 2015
    Mycological Progress 14 (2015). - ISSN 1617-416X - 9 p.
    plant immune-system - phylogenetic-relationships - peronospora-parasitica - oomycete effector - resistance gene - proteins - sequences - thaliana - reveals - plasmopara
    Like other plant-pathogenic oomycetes, downy mildew species of the genus Hyaloperonospora manipulate their hosts by secreting effector proteins. Despite intense research efforts devoted to deciphering the virulence and avirulence activities of effectors in the H. arabidopsidis/Arabidopsis thaliana pathosystem, there is only a single study in this pathosystem on the variation of effectors and resistance genes in natural populations, and the evolution of these effectors in the context of pathogen evolution is studied even less. In this work, the identification of A rabidopsis t haliana recognised (ATR)1-homologs is reported in two sister species of H. arabidopsidis, H. thlaspeos-perfoliati, and H. crispula, which are specialized on the host plants Microthlaspi perfoliatum and Reseda lutea, respectively. ATR1-diversity within these sister species of H. arabidopsidis was evaluated, and the ATR1-homologs from different isolates of H. thlaspeos-perfoliati and H. crispula were tested to see if they would be recognised by the previously characterised RPP1-WsB protein from A. thaliana. None of the effectors from the sister species was recognised, suggesting that due to the adaptation to altered or new targets after a host jump, features of variable effectors might vary to a degree that recognition of orthologous Avr-causing effectors is no longer effective and probably does not contribute to non-host immunity.
    Distributions, ex situ conservation priorities, and genetic resource potential of crop wild relatives of sweetpotato [Ipomoea batatas (L.) Lam., I. series Batatas]
    Khoury, C.K. ; Heider, B. ; Castaneda-Alvarez, N.P. ; Achicanoy, H.A. ; Sosa, C.C. ; Miller, R.E. ; Scotland, R.W. ; Wood, J.R.I. ; Rossel, G. ; Eserman, L.A. ; Jarret, R.L. ; Yencho, G.C. ; Bernau, V. ; Juarez, H. ; Sotelo, S. ; Haan, S. de; Struik, P.C. - \ 2015
    Frontiers in Plant Science 6 (2015). - ISSN 1664-462X - 14 p.
    species distribution models - phylogenetic-relationships - beta-carotene - convolvulaceae - sequences - diversity - evolution - bias - challenges - tolerance
    Crop wild relatives of sweetpotato [Ipomoea batatas (L.) Lam., I. series Batatas] have the potential to contribute to breeding objectives for this important root crop. Uncertainty in regard to species boundaries and their phylogenetic relationships, the limited availability of germplasm with which to perform crosses, and the difficulty of introgression of genes from wild species has constrained their utilization. Here, we compile geographic occurrence data on relevant sweetpotato wild relatives and produce potential distribution models for the species. We then assess the comprehensiveness of ex situ germplasm collections, contextualize these results with research and breeding priorities, and use ecogeographic information to identify species with the potential to contribute desirable agronomic traits. The fourteen species that are considered the closest wild relatives of sweetpotato generally occur from the central United States to Argentina, with richness concentrated in Mesoamerica and in the extreme Southeastern United States. Currently designated species differ among themselves and in comparison to the crop in their adaptations to temperature, precipitation, and edaphic characteristics and most species also show considerable intraspecific variation. With 79% of species identified as high priority for further collecting, we find that these crop genetic resources are highly under-represented in ex situ conservation systems and thus their availability to breeders and researchers is inadequate. We prioritize taxa and specific geographic locations for further collecting in order to improve the completeness of germplasm collections. In concert with enhanced conservation of sweetpotato wild relatives, further taxonomic research, characterization and evaluation of germplasm, and improving the techniques to overcome barriers to introgression with wild species are needed in order to mobilize these genetic resources for crop breeding.
    Using RNA-Seq to assemble a rose transcriptome with more than 13,000 full-length expressed genes and to develop the WagRhSNP 68k Axiom SNP array for rose (Rosa L.)
    Koning, C.F.S. ; Esselink, G. ; Vukosavljev, M. ; Westende, W.P.C. van 't; Gitonga, V.W. ; Krens, F.A. ; Voorrips, R.E. ; Weg, W.E. van de; Schulz, D. ; Debener, T. ; Maliepaard, C.A. ; Arens, P.F.P. ; Smulders, M.J.M. - \ 2015
    Frontiers in Plant Science 6 (2015). - ISSN 1664-462X - 10 p.
    powdery mildew - markers - tool - identification - resistance - genome - diversity - sequences - platform - plant
    In order to develop a versatile and large SNP array for rose, we set out to mine ESTs from diverse sets of rose germplasm. For this RNA-Seq libraries containing about 700 million reads were generated from tetraploid cut and garden roses using Illumina paired-end sequencing, and from diploid Rosa multiflora using 454 sequencing. Separate de novo assemblies were performed in order to identify single nucleotide polymorphisms (SNPs) within and between rose varieties. SNPs among tetraploid roses were selected for constructing a genotyping array that can be employed for genetic mapping and marker-trait association discovery in breeding programs based on tetraploid germplasm, both from cut roses and from garden roses. In total 68,893 SNPs were included on the WagRhSNP Axiom array. Next, an orthology-guided assembly was performed for the construction of a non-redundant rose transcriptome database. A total of 21,740 transcripts had significant hits with orthologous genes in the strawberry (Fragaria vesca L.) genome. Of these 13,390 appeared to contain the full-length coding regions. This newly established transcriptome resource adds considerably to the currently available sequence resources for the Rosaceae family in general and the genus Rosa in particular.
    Composition and predicted functional ecology of mussel - associated bacteria in Indonesian marine lakes
    Cleary, D.F.R. ; Becking, L.E. ; Polonia, A. ; Freitas, R.M. ; Gomes, N. - \ 2015
    Antonie van Leeuwenhoek: : Nederlandsch tijdschrift voor hygiëne, microbiologie en serologie 107 (2015)3. - ISSN 0003-6072 - p. 821 - 834.
    microbial communities - brachidontes-exustus - scorched mussel - species complex - mytilidae - bivalvia - mycoplasma - diversity - islands - sequences
    In the present study, we sampled bacterial communities associated with mussels inhabiting two distinct coastal marine ecosystems in Kalimantan, Indonesia, namely, marine lakes and coastal mangroves. We used 16S rRNA gene pyrosequencing and predicted metagenomic analysis to compare microbial composition and function. Marine lakes are small landlocked bodies of seawater isolated to varying degrees from the open sea environment. They contain numerous endemic taxa and represent natural laboratories of speciation. Our primary goals were to (1) use BLAST search to identify closely related organisms to dominant bacterial OTUs in our mussel dataset and (2) to compare bacterial communities and enrichment in the predicted bacterial metagenome among lakes. Our sequencing effort yielded 3553 OTUs belonging to 44 phyla, 99 classes and 121 orders. Mussels in the largest marine lake (Kakaban) and the coastal mangrove habitat were dominated by bacteria belonging to the phylum Proteobacteria whereas smaller lakes, located on the island of Maratua, were dominated by bacteria belonging to the phyla Firmicutes and Tenericutes. The single most abundant OTU overall was assigned to the genus Mycoplasma. There were several significant differences among locations with respect to metabolic pathways. These included enrichment of xenobiotic biodegradation pathways in the largest marine lake and coastal mangrove. These locations were also the most enriched with respect to nitrogen metabolism. The presence of genes related to isoquinoline alkaloids, polyketides, hydrolases, mono and dioxygenases in the predicted analysis of functional pathways is an indication that the bacterial communities of Brachidontes mussels may be potentially important sources of new marine medicines and enzymes of industrial interest. Future work should focus on measuring how mussel microbial communities influence nutrient dynamics within the marine lake environment and isolating microbes with potential biotechnological applications.
    Small Homologous Blocks in Phytophthora Genomes Do Not Point to an Ancient Whole-Genome Duplication
    Hooff, J.J.E. van; Snel, B. ; Seidl, M.F. - \ 2014
    Genome Biology and Evolution 6 (2014)5. - ISSN 1759-6653 - p. 1079 - 1085.
    pathogen phytophthora - maximum-likelihood - evolution - genes - consequences - mechanisms - adaptation - repertoire - sequences - infestans
    Genomes of the plant-pathogenic genus Phytophthora are characterized by small duplicated blocks consisting of two consecutive genes (2HOM blocks) and by an elevated abundance of similarly aged gene duplicates. Both properties, in particular the presence of 2HOM blocks, have been attributed to a whole-genome duplication (WGD) at the last common ancestor of Phytophthora. However, large intraspecies synteny-compelling evidence for a WGD-has not been detected. Here, we revisited the WGD hypothesis by deducing the age of 2HOM blocks. Two independent timing methods reveal that the majority of 2HOM blocks arose after divergence of the Phytophthora lineages. In addition, a large proportion of the 2HOM block copies colocalize on the same scaffold. Therefore, the presence of 2HOM blocks does not support a WGD at the last common ancestor of Phytophthora. Thus, genome evolution of Phytophthora is likely driven by alternative mechanisms, such as bursts of transposon activity.
    The Colletotrichum gigasporum species complex
    Liu, F. ; Cai, L. ; Crous, P.W. ; Damm, U. - \ 2014
    Persoonia 33 (2014). - ISSN 0031-5850 - p. 83 - 97.
    sp-nov - primer sets - endophytes - pathogens - china - gloeosporioides - anthracnose - diversity - sequences - acutatum
    In a preliminary analysis, 21 Colletotrichum strains with large conidia preserved in the CBS culture collection clustered with a recently described species, C. gigasporum, forming a clade distinct from other currently known Colletotrichum species complexes. Multi-locus phylogenetic analyses (ITS, ACT, TUB2, CHS-1, GAPDH) as well as each of the single-locus analyses resolved seven distinct species, one of them being C. gigasporum. Colletotrichum gigasporum and its close allies thus constitute a previously unknown species complex with shared morphological features. Five of the seven species accepted in the C. gigasporum species complex are described here as novel species, namely C. arxii, C. magnisporum, C. pseudomajus, C. radicis and C. vietnamense. A species represented by a single sterile strain, namely CBS 159.50, was not described as novel species, and is treated as Colletotrichum sp. CBS 159.50. Furthermore, C. thailandicum is reduced to synonymy with C. gigasporum.
    Polycistronic expression of a ß-carotene biosynthetic pathway in Saccharomyces cerevisiae coupled to ß-ionone production
    Beekwilder, J. ; Rossum, H.M. ; Koopman, F. ; Sonntag, F. ; Buchhaupt, M. ; Schrader, J. ; Hall, R.D. ; Bosch, H.J. ; Pronk, J.T. ; Maris, A.J.A. van; Daran, J.M. - \ 2014
    Journal of Biotechnology 192 (2014)partB. - ISSN 0168-1656 - p. 383 - 392.
    cleavage dioxygenase - yeast - genes - sequences - transformation - translation - polyprotein - versatile - genome - strain
    The flavour and fragrance compound ß-ionone, which naturally occurs in raspberry and many other fruits and flowers, is currently produced by synthetic chemistry. This study describes a synthetic biology approach for ß-ionone production from glucose by Saccharomyces cerevisiae that is partially based on polycistronic expression. Experiments with model proteins showed that the T2A sequence of the Thosea asigna virus mediated efficient production of individual proteins from a single transcript in S. cerevisiae. Subsequently, three ß-carotene biosynthesis genes from the carotenoid-producing ascomycete Xanthophyllomyces dendrorhous (crtI, crtE and crtYB) were expressed in S. cerevisiae from a single polycistronic construct. In this construct, the individual crt proteins were separated by T2A sequences. Production of the individual proteins from the polycistronic construct was confirmed by Western blot analysis and by measuring the production of ß-carotene. To enable ß-ionone production, a carotenoid-cleavage dioxygenase from raspberry (RiCCD1) was co-expressed in the ß-carotene producing strain. In glucose-grown cultures with a second phase of dodecane, ß-ionone and geranylacetone accumulated in the organic phase. Thus, by introducing a polycistronic construct encoding a fungal carotenoid pathway and an expression cassette encoding a plant dioxygenase, a novel microbial production system has been established for a fruit flavour compound.
    Dynamics of the Microbiota in Response to Host Infection
    Belzer, C. ; Gerber, G.K. ; Roeselers, G. ; Delaney, M. ; DuBois, A. ; Liu, Q. ; Belavusava, V. ; Yeliseyev, V. ; Houseman, A. ; Onderdonk, A. ; Cavanaugh, C. ; Bry, L. - \ 2014
    PLoS ONE 9 (2014)7. - ISSN 1932-6203
    citrobacter-rodentium infection - gene-expression data - mucosal infection - gut microbiome - mice - inflammation - diversity - sequences - pathogen - colitis
    Longitudinal studies of the microbiota are important for discovering changes in microbial communities that affect the host. The complexity of these ecosystems requires rigorous integrated experimental and computational methods to identify temporal signatures that promote physiologic or pathophysiologic responses in vivo. Employing a murine model of infectious colitis with the pathogen Citrobacter rodentium, we generated a 2-month time-series of 16S rDNA gene profiles, and quantitatively cultured commensals, from multiple intestinal sites in infected and uninfected mice. We developed a computational framework to discover time-varying signatures for individual taxa, and to automatically group signatures to identify microbial sub-communities within the larger gut ecosystem that demonstrate common behaviors. Application of this model to the 16S rDNA dataset revealed dynamic alterations in the microbiota at multiple levels of resolution, from effects on systems-level metrics to changes across anatomic sites for individual taxa and species. These analyses revealed unique, time-dependent microbial signatures associated with host responses at different stages of colitis. Signatures included a Mucispirillum OTU associated with early disruption of the colonic surface mucus layer, prior to the onset of symptomatic colitis, and members of the Clostridiales and Lactobacillales that increased with successful resolution of inflammation, after clearance of the pathogen. Quantitative culture data validated findings for predominant species, further refining and strengthening model predictions. These findings provide new insights into the complex behaviors found within host ecosystems, and define several time-dependent microbial signatures that may be leveraged in studies of other infectious or inflammatory conditions.
    Binning metagenomic contigs by coverage and composition
    Alneberg, J. ; Bjarnason, B.S. ; Bruijn, I. de; Schirmer, M. ; Quick, J. ; Ijaz, U.Z. ; Lahti, L.M. ; Loman, N.J. ; Andersson, A.F. ; Quince, C. - \ 2014
    Nature Methods : techniques for life scientists and chemists 11 (2014). - ISSN 1548-7091 - p. 1144 - 1146.
    sequences - genomes
    Shotgun sequencing enables the reconstruction of genomes from complex microbial communities, but because assembly does not reconstruct entire genomes, it is necessary to bin genome fragments. Here we present CONCOCT, a new algorithm that combines sequence composition and coverage across multiple samples, to automatically cluster contigs into genomes. We demonstrate high recall and precision on artificial as well as real human gut metagenome data sets.
    An integrated catalog of reference genes in the human gut microbiome
    Li, J. ; Jia, H. ; Cai, X. ; Zhong, H. ; Feng, Q. ; Sunagawa, S. ; Arumugam, M. ; Kultima, J.R. ; Prifti, E. ; Nielsen, T. ; Juncker, A.S. ; Manichanh, C. ; Chen, B. ; Zhang, W. ; Levenez, F. ; Xu, X. ; Xiao, L. ; Liang, S. ; Zhang, D. ; Zhang, Z. ; Chen, W. ; Zhao, H. ; Al-Aama, J.Y. ; Edris, S. ; Yang, H. ; Hansen, H. ; Nielsen, H.B. ; Brunak, S. ; Kristiansen, K. ; Guarner, F. ; Pedersen, O. ; Doré, J. ; Ehrlich, S.D. ; Bork, P. ; Wang, J. ; Vos, W.M. de; Tims, S. ; Zoetendal, E.G. ; Kleerebezem, M. - \ 2014
    Nature Biotechnology 32 (2014)8. - ISSN 1087-0156 - p. 834 - 841.
    eukaryotic diversity - fecal microbiota - population-size - metagenome - sequences - genomes - tool - alignment - impact - twins
    Many analyses of the human gut microbiome depend on a catalog of reference genes. Existing catalogs for the human gut microbiome are based on samples from single cohorts or on reference genomes or protein sequences, which limits coverage of global microbiome diversity. Here we combined 249 newly sequenced samples of the Metagenomics of the Human Intestinal Tract (MetaHit) project with 1,018 previously sequenced samples to create a cohort from three continents that is at least threefold larger than cohorts used for previous gene catalogs. From this we established the integrated gene catalog (IGC) comprising 9,879,896 genes. The catalog includes close-to-complete sets of genes for most gut microbes, which are also of considerably higher quality than in previous catalogs. Analyses of a group of samples from Chinese and Danish individuals using the catalog revealed country-specific gut microbial signatures. This expanded catalog should facilitate quantitative characterization of metagenomic, metatranscriptomic and metaproteomic data from the gut microbiome to understand its variation across populations in human health and disease.
    BioHackathon series in 2011 and 2012: penetration of ontology and linked data in life science domains
    Katayama, T. ; Wilkinson, M.D. ; Aoki-Kinoshita, K.F. ; Prins, J.C.P. - \ 2014
    Journal of Biomedical Semantics 5 (2014). - ISSN 2041-1480
    genome analysis environment - metabolic pathways - web services - gene - sequences - software - biology - normalization - collection - glycomics
    The application of semantic technologies to the integration of biological data and the interoperability of bioinformatics analysis and visualization tools has been the common theme of a series of annual BioHackathons hosted in Japan for the past five years. Here we provide a review of the activities and outcomes from the BioHackathons held in 2011 in Kyoto and 2012 in Toyama. In order to efficiently implement semantic technologies in the life sciences, participants formed various sub-groups and worked on the following topics: Resource Description Framework (RDF) models for specific domains, text mining of the literature, ontology development, essential metadata for biological databases, platforms to enable efficient Semantic Web technology development and interoperability, and the development of applications for Semantic Web data. In this review, we briefly introduce the themes covered by these sub-groups. The observations made, conclusions drawn, and software development projects that emerged from these activities are discussed.
    Identification and assembly of genomes and genetic elements in complex metagenomic samples without using reference genomes
    Nielsen, H.B. ; Almeida, M. ; Sierakowska Juncker, A. ; Rasmussen, S. ; Li, J. ; Sunagawa, S. ; Plichta, D.R. ; Gautier, L. ; Pedersen, A.G. ; Chatelier, E. Le; Pelletier, E. ; Bonde, I. ; Nielsen, T. ; Manichanh, C. ; Arumugam, M. ; Batto, J.M. ; Quintanilha dos Santos, M.B. ; Blom, N. ; Borruel, N. ; Burgdorf, K.S. ; Boumezbeur, F. ; Casellas, F. ; Doré, J. ; Dworzynski, P. ; Guarner, F. ; Hansen, T. ; Hildebrand, F. ; Kaas, R.S. ; Kennedy, S. ; Kristiansen, K. ; Kultima, J.R. ; Leonard, P. ; Levenez, F. ; Lund, O. ; Moumen, B. ; Paslier, D. Le; Pons, N. ; Pedersen, O. ; Prifti, E. ; Qin, J. ; Raes, J. ; Sørensen, S. ; Tap, J. ; Tims, S. ; Ussery, D.W. ; Yamada, T. ; Jamet, A. ; Mérieux, A. ; Cultrone, A. ; Torrejon, A. ; Quinquis, B. ; Brechot, C. ; Delorme, C. ; M'Rini, C. ; Vos, W.M. de; Maguin, E. ; Varela, E. ; Guedon, E. ; Gwen, F. ; Haimet, F. ; Artiguenave, F. ; Vandemeulebrouck, G. ; Denariaz, G. ; Khaci, G. ; Blottière, H. ; Knol, J. ; Weissenbach, J. ; Hylckama Vlieg, J.E. van; Torben, J. ; Parkhil, J. ; Turner, K. ; Guchte, M. van de; Antolin, M. ; Rescigno, M. ; Kleerebezem, M. ; Derrien, M. ; Galleron, N. ; Sanchez, N. ; Grarup, N. ; Veiga, P. ; Oozeer, R. ; Dervyn, R. ; Layec, S. ; Bruls, T. ; Winogradski, Y. ; Zoetendal, E.G. ; Renault, D. ; Sicheritz-Ponten, ; Bork, P. ; Wang, J. ; Brunak, S. ; Ehrlich, S.D. - \ 2014
    Nature Biotechnology 32 (2014). - ISSN 1087-0156 - p. 822 - 828.
    short read alignment - sequences - systems - algorithms - microbiota - protein - life - sets - tree - tool
    Most current approaches for analyzing metagenomic data rely on comparisons to reference genomes, but the microbial diversity of many environments extends far beyond what is covered by reference databases. De novo segregation of complex metagenomic data into specific biological entities, such as particular bacterial strains or viruses, remains a largely unsolved problem. Here we present a method, based on binning co-abundant genes across a series of metagenomic samples, that enables comprehensive discovery of new microbial organisms, viruses and co-inherited genetic entities and aids assembly of microbial genomes without the need for reference sequences. We demonstrate the method on data from 396 human gut microbiome samples and identify 7,381 co-abundance gene groups (CAGs), including 741 metagenomic species (MGS). We use these to assemble 238 high-quality microbial genomes and identify affiliations between MGS and hundreds of viruses or genetic entities. Our method provides the means for comprehensive profiling of the diversity within complex metagenomic samples.
    Introducing Chaetothyriothecium, a new genus of Microthyriales
    Hongsanan, S. ; Chomnunti, P. ; Crous, P.W. ; Chukeatirote, E. ; Hyde, K.D. - \ 2014
    Phytotaxa 161 (2014)2. - ISSN 1179-3155 - p. 157 - 164.
    probability - sequences - bootstrap - inference - alignment - genera - trees - tools
    The order Microthyriales comprises foliar biotrophs, epiphytes, pathogens or saprobes that occur on plant leaves and stems. The order is relatively poorly known due to limited sampling and few in-depth studies. There is also a lack of phylogenetic data for these fungi, which form small black spots on plant host surfaces, but rarely cause any damage to the host. A "Microthyriaceae"-like fungus collected in central Thailand is described as a new genus, Chaetothyriothecium (type species Chaetothyriothecium elegans sp. nov.). Phylogenetic analyses of LSU gene data showed this species to cluster with other members of Microthyriales, where it is related to Microthyrium microscopicum the type of the order. The description of the new species is supplemented by DNA sequence data, which resolves its placement in the order. Little molecular data is available for this order, stressing the need for further collections and molecular data.
    New insights into domestication of carrot from root transcriptome analyses
    Rong, J. ; Lammers, Y. ; Strasburg, J.L. ; Schidlo, N.S. ; Ariyurek, Y. ; Jong, T.J. de; Klinkhamer, P.G.L. ; Smulders, M.J.M. ; Vrieling, K. - \ 2014
    BMC Genomics 15 (2014). - ISSN 1471-2164 - 15 p.
    daucus-carota l. - nucleotide polymorphism - wild - populations - inference - sativus - genome - diversity - sequences - selection
    Background - Understanding the molecular basis of domestication can provide insights into the processes of rapid evolution and crop improvement. Here we demonstrated the processes of carrot domestication and identified genes under selection based on transcriptome analyses. Results - The root transcriptomes of widely differing cultivated and wild carrots were sequenced. A method accounting for sequencing errors was introduced to optimize SNP (single nucleotide polymorphism) discovery. 11,369 SNPs were identified. Of these, 622 (out of 1000 tested SNPs) were validated and used to genotype a large set of cultivated carrot, wild carrot and other wild Daucus carota subspecies, primarily of European origin. Phylogenetic analysis indicated that eastern carrot may originate from Western Asia and western carrot may be selected from eastern carrot. Different wild D. carota subspecies may have contributed to the domestication of cultivated carrot. Genetic diversity was significantly reduced in western cultivars, probably through bottlenecks and selection. However, a high proportion of genetic diversity (more than 85% of the genetic diversity in wild populations) is currently retained in western cultivars. Model simulation indicated high and asymmetric gene flow from wild to cultivated carrots, spontaneously and/or by introgression breeding. Nevertheless, high genetic differentiation exists between cultivated and wild carrots (Fst =0.295) showing the strong effects of selection. Expression patterns differed radically for some genes between cultivated and wild carrot roots which may be related to changes in root traits. The up-regulation of water-channel-protein gene expression in cultivars might be involved in changing water content and transport in roots. The activated expression of carotenoid-binding-protein genes in cultivars could be related to the high carotenoid accumulation in roots. The silencing of allergen-protein-like genes in cultivated carrot roots suggested strong human selection to reduce allergy. These results suggest that regulatory changes of gene expressions may have played a predominant role in domestication. Conclusions - Western carrots may originate from eastern carrots. The reduction in genetic diversity in western cultivars due to domestication bottleneck/selection may have been offset by introgression from wild carrot. Differential gene expression patterns between cultivated and wild carrot roots may be a signature of strong selection for favorable cultivation traits.
    Ercella succinigenes gen. nov., sp. nov., an anaerobic succinate-producing bacterium
    Gelder, A.H. van; Sousa, D.Z. ; Rijpstra, W.I. ; Damsté, J.S. ; Stams, A.J.M. ; Sanchez Andrea, I. - \ 2014
    International Journal of Systematic and Evolutionary Microbiology 64 (2014)7. - ISSN 1466-5026 - p. 2449 - 2454.
    ribosomal-rna genes - acid - heterogeneity - fermentation - sequences - biofuels - glycerol - industry - operons - genomes
    A novel anaerobic succinate-producing bacterium, strain ZWBT, was isolated from sludge collected from a biogas desulfurization bioreactor (Eerbeek, the Netherlands). Cells were non-spore-forming, motile, slightly curved rods (0.4–0.5 µm in diameter and 2–3 µm in length), and stained Gram-negative. The temperature range for growth was 25–40 °C, with an optimum at 37 °C. The pH range for growth was 7.0–9.0, with an optimum at pH 7.5. Strain ZWBT was able to ferment glycerol and several carbohydrates mainly to H2, succinate and acetate. Sulfur and fumarate could be used as electron acceptors by strain ZWBT. The G+C content of the genomic DNA was 37.6 mol%. The most abundant fatty acids were iso-C14¿:¿0 and iso-C16¿:¿0 DMA. On the basis of 16S rRNA gene sequence similarity, strain ZWBT belongs to the family Ruminococcaceae and it is distantly related to Saccharofermentans acetigenes JCM 14006T (92.1¿%). Based on the physiological features and phylogenetic analysis, strain ZWBT represents a novel species of a new genus, for which the name Ercella succinigenes gen. nov., sp. nov. is proposed. The type strain of Ercella succinigenes is ZWBT (¿=¿DSM 27333T¿=¿JCM 19283T).
    Effects of selective digestive decontamination (SDD) on the gut resistome
    Buelow, E. ; Bello Gonzalez, T.D.G. ; Versluis, D. ; Oostdijk, E.A.N. ; Ogilvie, L.A. ; Mourik, M.S.M. van; Oosterink, L. ; Passel, M.W.J. van; Smidt, H. ; D’Andrea, M.M. ; Been, M. de; Jones, B.V. ; Willems, R.J.L. ; Bonten, M.J.M. ; Schaik, W. - \ 2014
    Journal of Antimicrobial Chemotherapy 69 (2014)8. - ISSN 0305-7453 - p. 2215 - 2223.
    intensive-care units - antimicrobial resistance - microbiota - tract - microarray - metagenome - sequences - bacterial - classification - generation
    Objectives Selective digestive decontamination (SDD) is an infection prevention measure for critically ill patients in intensive care units (ICUs) that aims to eradicate opportunistic pathogens from the oropharynx and intestines, while sparing the anaerobic flora, by the application of non-absorbable antibiotics. Selection for antibiotic-resistant bacteria is still a major concern for SDD. We therefore studied the impact of SDD on the reservoir of antibiotic resistance genes (i.e. the resistome) by culture-independent approaches. Methods We evaluated the impact of SDD on the gut microbiota and resistome in a single ICU patient during and after an ICU stay by several metagenomic approaches. We also determined by quantitative PCR the relative abundance of two common aminoglycoside resistance genes in longitudinally collected samples from 12 additional ICU patients who received SDD. Results The patient microbiota was highly dynamic during the hospital stay. The abundance of antibiotic resistance genes more than doubled during SDD use, mainly due to a 6.7-fold increase in aminoglycoside resistance genes, in particular aph(2¿)-Ib and an aadE-like gene. We show that aph(2¿)-Ib is harboured by anaerobic gut commensals and is associated with mobile genetic elements. In longitudinal samples of 12 ICU patients, the dynamics of these two genes ranged from a ~104 fold increase to a ~10-10 fold decrease in relative abundance during SDD. Conclusions ICU hospitalization and the simultaneous application of SDD has large, but highly individualized, effects on the gut resistome of ICU patients. Selection for transferable antibiotic resistance genes in anaerobic commensal bacteria could impact the risk of transfer of antibiotic resistance genes to opportunistic pathogens.
    Development and Validation of a Genotype 3 Recombinant Protein based Immunoassay for Hepatitis E Virus Serology in Swine
    Poel, W.H.M. van der; Pavio, N. ; Goot, J. van der; Es, M. van; Martin, M. ; Engel, B. - \ 2014
    Brazilian Journal of Medical and Biological Research 47 (2014)4. - ISSN 0100-879X - p. 334 - 339.
    sequences - zoonosis
    Hepatitis E virus (HEV) is classified within the family Hepeviridae, genus Hepevirus. HEV genotype 3 (Gt3) infections are endemic in pigs in Western Europe and in North and South America and cause zoonotic infections in humans. Several serological assays to detect HEV antibodies in pigs have been developed, at first mainly based on HEV genotype 1 (Gt1) antigens. To develop a sensitive HEV Gt3 ELISA, a recombinant baculovirus expression product of HEV Gt3 open reading frame-2 was produced and coated onto polystyrene ELISA plates. After incubation of porcine sera, bound HEV antibodies were detected with anti-porcine anti-IgG and anti-IgM conjugates. For primary estimation of sensitivity and specificity of the assay, sets of sera were used from pigs experimentally infected with HEV Gt3. For further validation of the assay and to set the cutoff value, a batch of 1100 pig sera was used. All pig sera were tested using the developed HEV Gt3 assay and two other serologic assays based on HEV Gt1 antigens. Since there is no gold standard available for HEV antibody testing, further validation and a definite setting of the cutoff of the developed HEV Gt3 assay were performed using a statistical approach based on Bayes' theorem. The developed and validated HEV antibody assay showed effective detection of HEV-specific antibodies. This assay can contribute to an improved detection of HEV antibodies and enable more reliable estimates of the prevalence of HEV Gt3 in swine in different regions.
    Comparison of two short DNA barcoding loci (COI and COII) and two longer ribosomal DNA genes (SSU & LSU rRNA) for specimen identification among quarantine root-knot nematodes (Meloidogyne spp.) and their close relatives
    Kiewnick, S. ; Holterman, M.H.M. ; Elsen, S.J.J. van den; Megen, H.H.B. van; Frey, J.E. ; Helder, J. - \ 2014
    European Journal of Plant Pathology 140 (2014)1. - ISSN 0929-1873 - p. 97 - 110.
    small-subunit rdna - molecular characterization - phylogenetic analysis - pellioditis-marina - globodera-pallida - complex nematoda - cyst nematodes - sequences - evolution - morphology
    Root-knot nematodes (Meloidogyne spp.) are important pests of numerous crops worldwide. Some members of this genus have a quarantine status, and accurate species identification is required to prevent further spreading. DNA barcoding is a method for organism identification in non-complex DNA backgrounds based on informative motifs in short DNA stretches (˜600 bp). As part of the EU 7th Framework project QBOL, 15 Meloidogyne species were chosen to compare the resolutions offered by two typical DNA barcoding loci, COI and COII, with the distinguishing signals produced by two ribosomal DNA genes (small and large subunit rDNA; SSU¿˜¿1,700 and LSU¿˜¿3,400 bp). None of the four markers distinguished between the tropical species Meloidogyne incognita, M. javanica and M. arenaria. Taking P ID (Liberal) values =0.93 as a measure for species delimitation, the four mtDNA and rDNA markers performed well for the tropical Meloidogyne species complex, M. enterolobii, M. hapla, and M. maritima. Within cluster III A (Holterman et al. Phytopathology, 99, 227–235, 2009), SSU rDNA did not offer resolution at species level. Both mtDNA loci COI and COII did, whereas for LSU rDNA a longer fragment (=700 bp) is required. The high level of mitochondrial heteroplasmy recently reported for M. chitwoodi (Humphreys-Pereira and Elling Nematology, 15, 315–327, 2013) was not found in the populations under investigation, suggesting this could be a regional phenomenon. For identification of RKNs, we suggest the combined use of SSU rDNA with one of three other markers presented here.
    Towards a new classification system for legumes: Progress report from the 6th International Legume Conference
    Pontes Coelho Borges, L.M. ; Bruneau, A. ; Cardoso, D. ; Crisp, M. ; Delgado-Salinas, A. ; Doyle, J.J. ; Egan, A. ; Herendeen, P.S. ; Hughes, C. ; Kenicer, G. ; Klitgaard, B. ; Koenen, E. ; Lavin, M. ; Lewis, G. ; Luckow, M. ; Mackinder, B. ; Malecot, V. ; Miller, J.T. ; Pennington, R.T. ; Queiroz, L.P. de; Schrire, B. ; Simon, M.F. ; Steele, K. ; Torke, B. ; Wieringa, J.J. ; Wojciechowski, M.F. ; Boatwright, S. ; Estrella, M. de la; Mansano, V.D. ; Prado, D.E. ; Stirton, C. ; Wink, M. - \ 2013
    South African Journal of Botany 89 (2013). - ISSN 0254-6299 - p. 3 - 9.
    caesalpinioid legumes - phylogeny - leguminosae - evolution - diversification - dipsacales - sequences - lineages - gene - rbcl
    Legume systematists have been making great progress in understanding evolutionary relationships within the Leguminosae (Fabaceae), the third largest family of flowering plants. As the phylogenetic picture has become clearer, so too has the need for a revised classification of the family. The organization of the family into three subfamilies and 42 tribes is outdated and evolutionarily misleading. The three traditionally recognized subfamilies, Caesalpinioideae, Mimosoideae, and Papilionoideae, do not adequately represent relationships within the family. The occasion of the Sixth International Legume Conference in Johannesburg, South Africa in January 2013, with its theme "Towards a new classification system for legumes," provided the impetus to move forward with developing a new classification. A draft classification, based on current phylogenetic results and a set of principles and guidelines, was prepared in advance of the conference as the basis for discussion. The principles, guidelines, and draft classification were presented and debated at the conference. The objectives of the discussion were to develop consensus on the principles that should guide the development of the classification, to discuss the draft classification's strengths and weaknesses and make proposals for its revision, and identify and prioritize phylogenetic deficiencies that must be resolved before the classification could be published. This paper describes the collaborative process by a large group of legume systematists, publishing under the name Legume Phylogeny Working Group, to develop a new phylogenetic classification system for the Leguminosae. The goals of this paper are to inform the broader legume community, and others, of the need for a revised classification, and spell out clearly what the alternatives and challenges are for a new classification system for the family. (C) 2013 SAAB. Published by Elsevier B.V. All rights reserved.
    The tropical African legume Scorodophloeus clade includes two undescribed Hymenostegia segregate genera and Micklethwaitia, a rare, monospecific genus from Mozambique.
    Mackinder, B.A. ; Saslis-Lagoudakis, H. ; Wieringa, J.J. ; Devey, D. ; Forest, F. ; Bruneau, A. - \ 2013
    South African Journal of Botany 89 (2013). - ISSN 0254-6299 - p. 156 - 163.
    odd man - leguminosae - caesalpinioideae - detarieae - patterns - phylogeny - sequences - forests
    Legume subfamily Caesalpinioideae accommodates approximately 2250 species in 171 genera which traditionally are placed in four tribes: Caesalpinieae, Cassieae, Cercideae and Detarieae. The monophyletic tribe Detarieae includes the Amherstieae subclade which contains about 55 genera. Our knowledge of the relationships among those genera is good in some cases but for many other genera phylogenetic relationships have been unclear. The non-monophyletic nature of at least two amherstioid genera, Cynometra and Hymenostegia has also complicated the picture. During the course of a multi-disciplinary study of Hymenostegia sensu lato, which includes phylogenetic analyses based on matK and trnL data, we have recovered the “Scorodophloeus clade”, an exclusively tropical African clade of four genera which includes the eponymous genus Scorodophloeus, two undescribed generic segregates of Hymenostegia sensu lato, and the previously unsampled rare monospecific genus Micklethwaitia from Mozambique. Zenkerella is suggested as a possible sister genus to the Scorodophloeus clade. A distribution map is presented of the seven species that belong to the Scorodophloeus clade.
    Comparative Genomics Analysis of Streptococcus Isolates from the Human Small Intestine Reveals their Adaptation to a Highly Dynamic Ecosystem
    Bogert, B. van den; Boekhorst, J. te; Herrmann, R. ; Smid, E.J. ; Zoetendal, E.G. ; Kleerebezem, M. - \ 2013
    PLoS ONE 8 (2013)12. - ISSN 1932-6203
    human gastrointestinal-tract - group-b streptococcus - prokaryotic genomes - lactococcus-lactis - bacterial samples - transport-system - microbiota - pneumoniae - competence - sequences
    The human small-intestinal microbiota is characterised by relatively large and dynamic Streptococcus populations. In this study, genome sequences of small-intestinal streptococci from S. mitis, S. bovis, and S. salivarius species-groups were determined and compared with those from 58 Streptococcus strains in public databases. The Streptococcus pangenome consists of 12,403 orthologous groups of which 574 are shared among all sequenced streptococci and are defined as the Streptococcus core genome. Genome mining of the small-intestinal streptococci focused on functions playing an important role in the interaction of these streptococci in the small-intestinal ecosystem, including natural competence and nutrient-transport and metabolism. Analysis of the small-intestinal Streptococcus genomes predicts a high capacity to synthesize amino acids and various vitamins as well as substantial divergence in their carbohydrate transport and metabolic capacities, which is in agreement with observed physiological differences between these Streptococcus strains. Gene-specific PCR-strategies enabled evaluation of conservation of Streptococcus populations in intestinal samples from different human individuals, revealing that the S. salivarius strains were frequently detected in the small-intestine microbiota, supporting the representative value of the genomes provided in this study. Finally, the Streptococcus genomes allow prediction of the effect of dietary substances on Streptococcus population dynamics in the human small-intestine
    Alternaria redefined
    Woudenberg, J.H.C. ; Groenewald, J.Z. ; Binder, M. ; Crous, P.W. - \ 2013
    Studies in Mycology 75 (2013)1. - ISSN 0166-0616 - p. 171 - 212.
    ribosomal dna - phylogenetic-relationships - species-group - ulocladium - embellisia - genus - identification - sequences - taxonomy - nuclear
    Alternaria is a ubiquitous fungal genus that includes saprobic, endophytic and pathogenic species associated with a wide variety of substrates. In recent years, DNA-based studies revealed multiple non-monophyletic genera within the Alternaria complex, and Alternaria species clades that do not always correlate to species-groups based on morphological characteristics. The Alternaria complex currently comprises nine genera and eight Alternaria sections. The aim of this study was to delineate phylogenetic lineages within Alternaria and allied genera based on nucleotide sequence data of parts of the 18S nrDNA, 28S nrDNA, ITS, GAPDH, RPB2 and TEF1-alpha gene regions. Our data reveal a Pleospora/Stemphylium clade sister to Embellisia annulata, and a well-supported Alternaria clade. The Alternaria clade contains 24 internal clades and six monotypic lineages, the assemblage of which we recognise as Alternaria. This puts the genera Allewia, Brachycladium, Chalastospora, Chmelia, Crivellia, Embellisia, Lewia, Nimbya, Sinomyces, Teretispora, Ulocladium, Undifilum and Ybotromyces in synonymy with Alternaria. In this study, we treat the 24 internal clades in the Alternaria complex as sections, which is a continuation of a recent proposal for the taxonomic treatment of lineages in Alternaria. Embellisia annulata is synonymised with Dendryphiella salina, and together with Dendryphiella arenariae, are placed in the new genus Paradendryphiella. The sexual genera Clathrospora and Comoclathris, which were previously associated with Alternaria, cluster within the Pleosporaceae, outside Alternaria s. str., whereas Alternariaster, a genus formerly seen as part of Alternaria, clusters within the Leptosphaeriaceae. Paradendryphiella is newly described, the generic circumscription of Alternaria is emended, and 32 new combinations and 10 new names are proposed. A further 10 names are resurrected, while descriptions are provided for 16 new Alternaria sections
    Habitat- and host-related variation in sponge bacterial symbiont communities in Indonesian waters
    Cleary, D.F.R. ; Becking, L.E. ; Voogd, N.J. de; Pires, A.C.C. ; Polonia, A. ; Egas, C. ; Gomes, N. - \ 2013
    FEMS microbiology ecology 85 (2013)3. - ISSN 0168-6496 - p. 465 - 482.
    enso-induced fires - marine sponges - vertical transmission - east kalimantan - kakaban-island - suberites-domuncula - butterfly diversity - microbial community - sp nov. - sequences
    Marine lakes are unique ecosystems that contain isolated populations of marine organisms. Isolated from the surrounding marine habitat, many lakes house numerous endemic species. In this study, microbial communities of sponges inhabiting these lakes were investigated for the first time using barcoded pyrosequencing of 16S rRNA gene amplicons. Our main goals were to compare the bacterial richness and composition of two sponge species (Suberites diversicolor and Cinachyrella australiensis) inhabiting both marine lakes and adjacent open coastal systems. Host species and habitat explained almost 59% of the variation in bacterial composition. There was a significant difference in composition between both host species. Within S. diversicolor, there was little discernible difference between bacterial communities inside and outside lakes. The bacterial community of this species was, furthermore, dominated (63% of all sequences) by three very closely related alphaproteobacterial taxa identified as belonging to the recently described order Kiloniellales. Cinachyrella australiensis, in contrast, hosted markedly different bacterial communities inside and outside lakes with very few shared abundant taxa. Cinachyrella australiensis in open habitat only shared 9.4% of OTUs with C. australiensis in lake habitat. Bacteria were thus both highly species specific and, in the case of C. australiensis, habitat specific.
    Computational protein design with electrostatic focusing: experimental characterization of a conditionally folded helical domain with a reduced amino acid alphabet
    Suarez Diez, M. ; Pujol, M. ; Matzapetakis, M. ; Jaramillo, A. ; Iranzo, O. - \ 2013
    Biotechnology Journal 8 (2013)7. - ISSN 1860-6768 - p. 855 - 864.
    solution nmr structure - structural basis - peptides - recognition - prediction - sequences - dynamics - energy - trifluoroethanol - optimization
    Automated methodologies to design synthetic proteins from first principles use energy computations to estimate the ability of the sequences to adopt a targeted structure. This approach is still far from systematically producing native-like sequences, due, most likely, to inaccuracies when modeling the interactions between the protein and its aqueous environment. This is particularly challenging when engineering small protein domains (with less polar pair interactions than with the solvent). We have re-designed a three-helix bundle, domain B, using a fixed backbone and a four amino acid alphabet. We have enlarged the rotamer library with conformers that increase the weight of electrostatic interactions within the design process without altering the energy function used to compute the folding free energy. Our synthetic sequences show less than 15% similarity to any Swissprot sequence. We have characterized our sequences in different solvents using circular dichroism and nuclear magnetic resonance. The targeted structure achieved is dependent on the solvent used. This method can be readily extended to larger domains. Our method will be useful for the engineering of proteins that become active only in a given solvent and for designing proteins in the context of hydrophobic solvents, an important fraction of the situations in the cell
    Detection and Quantification of Leveillula taurica Growth in Pepper Leaves
    Zheng, Z. ; Nonomura, T. ; Bóka, K. ; Matsuda, Y. ; Visser, R.G.F. ; Toyoda, H. ; Kiss, L. ; Bai, Y. - \ 2013
    Phytopathology 103 (2013)6. - ISSN 0031-949X - p. 623 - 632.
    internal transcribed spacer - powdery-mildew - genus leveillula - capsicum-annuum - resistance - pcr - infections - sequences - fungi - dna
    Leveillula taurica is an obligate fungal pathogen that causes powdery mildew disease on a broad range of plants, including important crops such as pepper, tomato, eggplant, onion, cotton, and so on. The early stage of this disease is difficult to diagnose and the disease can easily spread unobserved; for example, in pepper and tomato production fields and greenhouses. The objective of this study was to develop a detection and quantification method of L. taurica biomass in pepper leaves with special regard to the early stages of infection. We monitored the development of the disease to time the infection process on the leaf surface as well as inside the pepper leaves. The initial and final steps of the infection taking place on the leaf surface were consecutively observed using a dissecting microscope and a scanning electron microscope. The development of the intercellular mycelium in the mesophyll was followed by light and transmission electron microscopy. A pair of L. taurica-specific primers was designed based on the internal transcribed spacer sequence of L. taurica and used in real-time polymerase chain reaction (PCR) assay to quantify the fungal DNA during infection. The specificity of this assay was confirmed by testing the primer pair with DNA from host plants and also from another powdery mildew species, Oidium neolycopersici, infecting tomato. A standard curve was obtained for absolute quantification of L. taurica biomass. In addition, we tested a relative quantification method by using a plant gene as reference and the obtained results were compared with the visual disease index scoring. The real-time PCR assay for L. taurica provides a valuable tool for detection and quantification of this pathogen in breeding activities as well in plant-microbe interaction studies.
    Multi-locus phylogenies of the genus Barteria (Passifloraceae) protray complex patterns in the evolution of myrmecophytism.
    Peccoud, J. ; Piatscheck, F. ; Yockteng, R. ; Garcia, M. ; Sauve, M. ; Djieto-Lordon, C. ; Harris, D.J. ; Wieringa, J.J. ; Breteler, F.J. ; Born, C. ; McKey, D. ; Blatrix, R. - \ 2013
    Molecular Phylogenetics and Evolution 66 (2013)3. - ISSN 1055-7903 - p. 824 - 832.
    chloroplast dna - rain-forest - population - sequences - ants - pseudomyrmecinae - euphorbiaceae - divergence - protection - mutualism
    The four species of the central African genus Barteria show variation in habitat and in degree of association with ants. Whereas B. solida, restricted to submontane forests, attracts opportunistic ants to extrafloral nectar, the three other species, found in lowland rainforests (B. fistulosa, B. dewevrei) and in littoral scrub (B. nigritana), possess stem domatia of varying shapes and degrees of specialisation, hosting either non-specific arboreal ants (B. nigritana, some B. dewevrei) or two large species of ants of the genus Tetraponera Smith, 1852 that are specific to some species of Barteria (B. fistulosa, some B. dewevrei). We aimed to investigate whether this variation represents an evolutionary trend toward increasing specialisation of mutualism or the reduction or loss of myrmecophytic traits. For this, we determined phylogenetic relationships within the genus using DNA sequences (primarily nuclear ITS) and microsatellite genotypes (11 loci) on a large sample of individuals, mostly from Cameroon and Gabon. The two types of markers support an initial dichotomy that groups B. dewevrei with B. nigritana and B. fistulosa with B. solida respectively. Within these pairs, species do not appear reciprocally monophyletic. At microsatellite loci, B. nigritana forms a clade embedded within B. dewevrei; and within both B. solida and B. fistulosa, geographical populations show levels of differentiation similar to that observed between populations of B. solida and B. fistulosa. Geographic distance alone does not account for genetic differentiation between species, which indicates reproductive isolation. Divergence in each of the two pairs implies evolutionary transitions in habitat and in myrmecophytism. Specialised mutualism with specific ant species of the genus Tetraponera has been lost in species found in more marginal habitats.
    Pyrosequencing as a tool for the detection of Phytophthora species: error rate and risk of false Molecular Operational Taxonomic Units
    Vettraino, A.M. ; Bonants, P.J.M. ; Tomassini, A. ; Bruni, N. ; Vannini, A. - \ 2012
    Letters in Applied Microbiology 55 (2012)5. - ISSN 0266-8254 - p. 390 - 396.
    fungal communities - identification - phylogeny - diversity - sequences - reveals
    Aims: To evaluate the accuracy of pyrosequencing for the description of Phytophthora communities in terms of taxa identification and risk of assignment for false Molecular Operational Taxonomic Units (MOTUs). Methods and Results: Pyrosequencing of Internal Transcribed Spacer 1 (ITS1) amplicons was used to describe the structure of a DNA mixture comprising eight Phytophthora spp. and Pythium vexans. Pyrosequencing resulted in 16 965 reads, detecting all species in the template DNA mixture. Reducing the ITS1 sequence identity threshold resulted in a decrease in numbers of unmatched reads but a concomitant increase in the numbers of false MOTUs. The total error rate was 0_63% and comprised mainly mismatches (0_25%) Conclusions: Pyrosequencing of ITS1 region is an efficient and accurate technique for the detection and identification of Phytophthora spp. In environmental samples. However, the risk of allocating false MOTUs, even when demonstrated to be low, may require additional validation with alternative detection methods. Significance and Impact of the Study: Phytophthora spp. are considered among the most destructive groups of invasive plant pathogens, affecting thousands of cultivated and wild plants worldwide. Simultaneous early detection of Phytophthora complexes in environmental samples offers an unique opportunity for the interception of known and unknown species along pathways of introduction, along with the identification of these organisms in invaded environments.
    Genome-Wide Prediction and Validation of Sigma70 Promoters in Lactobacillus plantarum WCFS1
    Todt, T.J. ; Wels, M. ; Bongers, R.S. ; Siezen, R.J. ; Hijum, S.A.F.T. van; Kleerebezem, M. - \ 2012
    PLoS ONE 7 (2012)9. - ISSN 1932-6203
    transcription start sites - escherichia-coli - rna-polymerase - sequences - identification - dna - conservation - compilation - metabolism - initiation
    Background - In prokaryotes, sigma factors are essential for directing the transcription machinery towards promoters. Various sigma factors have been described that recognize, and bind to specific DNA sequence motifs in promoter sequences. The canonical sigma factor s70 is commonly involved in transcription of the cell's housekeeping genes, which is mediated by the conserved s70 promoter sequence motifs. In this study the s70-promoter sequences in Lactobacillus plantarum WCFS1 were predicted using a genome-wide analysis. The accuracy of the transcriptionally-active part of this promoter prediction was subsequently evaluated by correlating locations of predicted promoters with transcription start sites inferred from the 5'-ends of transcripts detected by high-resolution tiling array transcriptome datasets. Results - To identify s70-related promoter sequences, we performed a genome-wide sequence motif scan of the L. plantarum WCFS1 genome focussing on the regions upstream of protein-encoding genes. We obtained several highly conserved motifs including those resembling the conserved s70-promoter consensus. Position weight matrices-based models of the recovered s70-promoter sequence motif were employed to identify 3874 motifs with significant similarity (p-value
    Stagonosporopsis spp. associated with ray blight disease of Asteraceae
    Vaghefi, N. ; Pethybridge, S.J. ; Ford, R. ; Nicolas, M.E. ; Crous, P.W. ; Taylor, P.W.J. - \ 2012
    Australasian Plant Pathology 41 (2012)6. - ISSN 0815-3191 - p. 675 - 686.
    phoma-ligulicola - phylogenetic analyses - ribosomal dna - mixed models - host-range - pyrethrum - sequences - complex - genera - taxa
    Ray blight disease of pyrethrum (Tanacetum cinerariifolium) is shown to be caused by more than one species of Stagonosporopsis. The Australian pathogen, previously identified as Phoma ligulicola var. inoxydabilis, represents a new species described as Stagonosporopsis tanaceti based on morphological characters and a five-gene phylogeny employing partial sequences of the actin, translation elongation factor 1-alpha, internal transcribed spacers and 5.8S of the nrDNA, 28S large subunit and beta-tubulin 2 gene sequences. Furthermore, the two varieties of Stagonosporopsis ligulicola are elevated to species level as S. chrysanthemi and S. inoxydabilis based on their DNA phylogeny and morphology.
    Prediction of Altered 3'- UTR miRNA-Binding Sites from RNA-Seq Data: The Swine Leukocyte Antigen Complex (SLA) as a Model Region
    Endale, M.L. ; Fritz, E.R. ; Estelle, J. ; Hu, Z.L. ; Madsen, O. ; Groenen, M.A.M. - \ 2012
    PLoS ONE 7 (2012)11. - ISSN 1932-6203
    microrna target recognition - mediated gene-regulation - phenotypic variation - expression - accessibility - principles - sequences - evolution - disease - genome
    The SLA (swine leukocyte antigen, MHC: SLA) genes are the most important determinants of immune, infectious disease and vaccine response in pigs; several genetic associations with immunity and swine production traits have been reported. However, most of the current knowledge on SLA is limited to gene coding regions. MicroRNAs (miRNAs) are small molecules that post-transcriptionally regulate the expression of a large number of protein-coding genes in metazoans, and are suggested to play important roles in fine-tuning immune mechanisms and disease responses. Polymorphisms in either miRNAs or their gene targets may have a significant impact on gene expression by abolishing, weakening or creating miRNA target sites, possibly leading to phenotypic variation. We explored the impact of variants in the 3'-UTR miRNA target sites of genes within the whole SLA region. The combined predictions by TargetScan, PACMIT and TargetSpy, based on different biological parameters, empowered the identification of miRNA target sites and the discovery of polymorphic miRNA target sites (poly-miRTSs). Predictions for three SLA genes characterized by a different range of sequence variation provided proof of principle for the analysis of poly-miRTSs from a total of 144 M RNA-Seq reads collected from different porcine tissues. Twenty-four novel SNPs were predicted to affect miRNA-binding sites in 19 genes of the SLA region. Seven of these genes (SLA-1, SLA-6, SLA-DQA, SLA-DQB1, SLA-DOA, SLA-DOB and TAP1) are linked to antigen processing and presentation functions, which is reminiscent of associations with disease traits reported for altered miRNA binding to MHC genes in humans. An inverse correlation in expression levels was demonstrated between miRNAs and co-expressed SLA targets by exploiting a published dataset (RNA-Seq and small RNA-Seq) of three porcine tissues. Our results support the resource value of RNA-Seq collections to identify SNPs that may lead to altered miRNA regulation patterns.
    Cloning and characterization of a tuberous root-specific promoter from cassava (Manihot esculenta Crantz)
    Koehorst-van Putten, H.J.J. ; Wolters, A.M.A. ; Pereira-Bertram, I.J. ; Berg, H. ; Krol, A.R. van der; Visser, R.G.F. - \ 2012
    Planta 236 (2012)6. - ISSN 0032-0935 - p. 1955 - 1965.
    bound-starch-synthase - storage roots - transformation - expression - gene - agrobacterium - elements - sequences - database - program
    In order to obtain a tuberous root-specific promoter to be used in the transformation of cassava, a 1,728 bp sequence containing the cassava granule-bound starch synthase (GBSSI) promoter was isolated. The sequence proved to contain light- and sugar-responsive cis elements. Part of this sequence (1,167 bp) was cloned into binary vectors to drive expression of the firefly luciferase gene. Cassava cultivar Adira 4 was transformed with this construct or a control construct in which the luciferase gene was cloned behind the 35S promoter. Luciferase activity was measured in leaves, stems, roots and tuberous roots. As expected, the 35S promoter induced luciferase activity in all organs at similar levels, whereas the GBSSI promoter showed very low expression in leaves, stems and roots, but very high expression in tuberous roots. These results show that the cassava GBSSI promoter is an excellent candidate to achieve tuberous root-specific expression in cassava.
    Phyllosticta species associated with freckle disease of banana
    Wong, M.H. ; Crous, P.W. ; Henderson, J. ; Groenewald, J.Z. ; Drenth, A. - \ 2012
    Fungal Diversity 56 (2012)1. - ISSN 1560-2745 - p. 173 - 187.
    citrus black spot - ribosomal dna - mycosphaerella - sequences - musarum - plants
    The identity of the casual agent of freckle disease of banana was investigated. The pathogen is generally referred to in literature under its teleomorphic name, Guignardia musae, or that of its purported anamorph, Phyllosticta musarum. Based on morphological and molecular data from a global set of banana specimens, several species were found associated with freckle disease. Phyllosticta maculata (from Southeast Asia and Oceania) is introduced as a new name for Guignardia musae, and an epitype is designated from Australia. Phyllosticta musarum (from India and Thailand) is shown to represent a distinct species, and the name is fixed by designation of an epitype from India. Guignardia stevensii is confirmed as distinct species from Hawaii, while Guignardia musicola from northern Thailand is shown to contain different taxa and is regarded as nomen confusum. Phyllosticta cavendishii is described as a new, widely distributed species, appearing primarily on Cavendish, but also on non-Cavendish banana cultivars.
    A phylogenetic and taxonomic re-evaluation of the Bipolaris - Cochliobolus - Curvularia Complex
    Manamgoda, D.S. ; Cai, L. ; McKenzie, E.H.C. ; Crous, P.W. ; Madrid, H. ; Chukeatirote, E. ; Shivas, R.G. ; Tan, Y.P. ; Hyde, K.D. - \ 2012
    Fungal Diversity 56 (2012)1. - ISSN 1560-2745 - p. 131 - 144.
    species concepts - sequences - pseudocochliobolus - helminthosporium - pathogens - alignment - genes - fungi - genus - rdna
    Three genera, Cochliobolus, Bipolaris and Curvularia form a complex that contains many plant pathogens, mostly on grasses (Poaceae) with a worldwide distribution. The taxonomy of this complex is confusing as frequent nomenclatural changes and refinements have occurred. There is no clear morphological boundary between the asexual genera Bipolaris and Curvularia, and some species show intermediate morphology. We investigated this complex based on a set of ex-type cultures and collections from northern Thailand. Combined gene analysis of rDNA ITS (internal transcribed spacer), GPDH (glyceraldehyde 3-phosphate dehydrogenase), LSU (large subunit) and EF1-a (translation elongation factor 1-a) shows that this generic complex divides into two groups. Bipolaris and Cochliobolus species clustered in Group 1 along with their type species, whereas Curvularia species (including species named as Bipolaris, Cochliobolus and Curvularia) clustered in Group 2, with its generic type. The nomenclatural conflict in this complex is resolved giving priority to the more commonly used established generic names Bipolaris and Curvularia. Modern descriptions of the genera Bipolaris and Curvularia are provided and species resolved in this study are transferred to one of these genera based on their phylogeny.
    The development of a validated real-time (TaqMan) PCR for detection of Stagonosporopsis andigena and S. crystalliniformis in infected leaves of potato and tomato
    Gruyter, J. de; Gent-Pelzer, M.P.E. van; Woudenberg, J.H.C. ; Rijswick, P.C.J. van; Meekes, E.T.M. ; Crous, P.W. ; Bonants, P.J.M. - \ 2012
    European Journal of Plant Pathology 134 (2012)2. - ISSN 0929-1873 - p. 301 - 313.
    polymerase-chain-reaction - phoma-tracheiphila - didymella-bryoniae - complex - foveata - differentiation - phylogeny - multiplex - sequences - disease
    Stagonosporopsis andigena and S. crystalliniformis are serious foliage pathogens on potato (Solanum tuberosum) and tomato (Solanum lycopersicum). As both species have been recorded only in the Andes area, S. andigena is listed as an A1 quarantine organism in Europe. The actin region of isolates of Stagonosporopsis and allied species of Boeremia, Didymella, Peyronellaea and Phoma was amplified using generic primers. DNA sequence differences of the actin gene were utilised to develop species-specific real-time (TaqMan) PCR assays for the detection of S. andigena and S. crystalliniformis in leaves of potato or tomato. The specificity of the TaqMan PCR assays was determined on genomic DNA extracted from two S. andigena and two S. crystalliniformis isolates and 16 selected isolates of Stagonosporopsis, Phoma and Boeremia, which are the closest relatives. The validation of the methods developed included the DNA extraction and the TaqMan PCR assays. The performance criteria specificity, analytical sensitivity, reproducibility, repeatability and robustness of the TaqMan PCR assays demonstrated the reliability of both methods for the detection of S. andigena and S. crystalliniformis in leaf material. The TaqMan PCR assays were tested on symptomatic leaves of potato and tomato that were obtained after artificial inoculation of detached leaves with both pathogens under quarantine conditions. In the artificial inoculation experiments both S. andigena and S. crystalliniformis caused leaf infections on potato and tomato.
    Characterization of Hubera (Annonaceae), a new genus segregated from Polyalthia and allied to Miliusa.
    Chaowasku, T. ; Johnson, D.M. ; Ham, R.W.J.M. van der; Chatrou, L.W. - \ 2012
    Phytotaxa 69 (2012). - ISSN 1179-3155 - p. 33 - 56.
    molecular phylogenetics - uvaria annonaceae - pollen morphology - chloroplast dna - evolution - genera - reconstruction - hypotheses - sequences - revision
    On the basis of molecular phylogenetics, pollen morphology and macromorphology, a new genus of the tribe Miliuseae, Hubera, segregrated from Polyalthia and allied to Miliusa, is established and described. It is characterized by the combination of reticulate tertiary venation of the leaves, axillary inflorescences, a single ovule per ovary and therefore single-seeded monocarps, seeds with a flat to slightly raised raphe, spiniform(-flattened peg) ruminations of the endosperm, and pollen with a finely and densely granular infratectum. Twenty-seven species are accordingly transferred to this new genus.
    Efficiency of Agrobacterium rhizogenes-mediated root transformation of Parasponia and Trema is temperature dependent
    Cao, Q. ; Camp, R. Op den; Seifi Kalhor, M. ; Bisseling, T. ; Geurts, R. - \ 2012
    Plant Growth Regulation 68 (2012)3. - ISSN 0167-6903 - p. 459 - 465.
    medicago-truncatula - gene-transfer - non-legume - plants - nitrogen - andersonii - nodulation - rhizobium - phaseolus - sequences
    Parasponia trees are the only non-legume species that form nitrogen-fixing root nodules with rhizobium. Based on its taxonomic position in relation to legumes (Fabaceae), it is most likely that both lineages have gained this symbiotic capacity independently. Therefore, Parasponia forms a bridging species to understand the evolutionary constraints underlying this symbiosis. However, absence of key technologies to genetically modify Parasponia seriously impeded studies on these species. We employed Agrobacterium rhizogenes to create composite Parasponia andersonii plants that harbour transgenic roots. Here, we provide an optimized protocol to infect P. andersonii as well as its non-symbiotic sister species Trema tomentosa with A. rhizogenes. We show that the transformation efficiency is temperature dependent. Whereas the optimal growth temperature for both these species is 28 °C, the transformation is most efficient when co-cultivation with A. rhizogenes occurs at 21 °C. Using this optimized protocol up to 80 % transformation efficiency can be obtained. These robust transformation platforms will provide a strong tool to unravel the Parasponia–rhizobium symbiosis
    Scenario Analysis of Nutrient Removal from Municipal Wastewater by Microalgal Biofilms
    Boelee, N.C. ; Temmink, H. ; Janssen, M. ; Buisman, C.J.N. ; Wijffels, R.H. - \ 2012
    Water 4 (2012)2. - ISSN 2073-4441 - p. 460 - 473.
    afvalwaterbehandeling - biofilms - algen - biologische waterzuiveringsinstallaties - vergelijkend onderzoek - volgorden - haalbaarheidsstudies - heterotrofe micro-organismen - stikstof - fosfor - verwijdering - biomassa productie - biobased economy - waste water treatment - biofilms - algae - biological water treatment plants - comparative research - sequences - feasibility studies - heterotrophic microorganisms - nitrogen - phosphorus - removal - biomass production - biobased economy - marine-phytoplankton - chemical-composition - chlorella-vulgaris - nitrate uptake - algal biofilm - growth - photobioreactor - photosynthesis
    Microalgae can be used for the treatment of municipal wastewater. The application of microalgal biofilms in wastewater treatment systems seems attractive, being able to remove nitrogen, phosphorus and COD from wastewater at a short hydraulic retention time. This study therefore investigates the area requirement, achieved effluent concentrations and biomass production of a hypothetical large-scale microalgal biofilm system treating municipal wastewater. Three scenarios were defined: using microalgal biofilms: (1) as a post-treatment; (2) as a second stage of wastewater treatment, after a first stage in which COD is removed by activated sludge; and (3) in a symbiotic microalgal/heterotrophic system. The analysis showed that in the Netherlands, the area requirements for these three scenarios range from 0.32 to 2.1 m2 per person equivalent. Moreover, it was found that it was not possible to simultaneously remove all nitrogen and phosphorus from the wastewater, because of the nitrogen:phosphorus ratio in the wastewater. Phosphorus was limiting in the post-treatment scenario, while nitrogen was limiting in the two other scenarios. Furthermore, a substantial amount of microalgal biomass was produced, ranging from 13 to 59 g per person equivalent per day. These findings show that microalgal biofilm systems hold large potential as seasonal wastewater treatment systems and that it is worthwhile to investigate these systems further
    Promoter propagation in prokaryotes
    Matus-Garcia, M. ; Nijveen, H. ; Passel, M.W.J. van - \ 2012
    Nucleic acids research 40 (2012)20. - ISSN 0305-1048 - p. 10032 - 10040.
    horizontal gene-transfer - escherichia-coli - adaptive evolution - lactococcus-lactis - sequences - genomes - networks - identification - mutations - families
    Transcriptional activation or 'rewiring' of silent genes is an important, yet poorly understood, phenomenon in prokaryotic genomes. Anecdotal evidence coming from experimental evolution studies in bacterial systems has shown the promptness of adaptation upon appropriate selective pressure. In many cases, a partial or complete promoter is mobilized to silent genes from elsewhere in the genome. We term hereafter such recruited regulatory sequences as Putative Mobile Promoters (PMPs) and we hypothesize they have a large impact on rapid adaptation of novel or cryptic functions. Querying all publicly available prokaryotic genomes (1362) uncovered >4000 families of highly conserved PMPs (50 to 100 long with =80% nt identity) in 1043 genomes from 424 different genera. The genomes with the largest number of PMP families are Anabaena variabilis (28 families), Geobacter uraniireducens (27 families) and Cyanothece PCC7424 (25 families). Family size varied from 2 to 93 homologous promoters (in Desulfurivibrio alkaliphilus). Some PMPs are present in particular species, but some are conserved across distant genera. The identified PMPs represent a conservative dataset of very recent or conserved events of mobilization of non-coding DNA and thus they constitute evidence of an extensive reservoir of recyclable regulatory sequences for rapid transcriptional rewiring
    Relative entropy differences in bacterial chromosomes, plasmids, phages and genomic islands
    Bohlin, J. ; Passel, M.W.J. van - \ 2012
    BMC Genomics 13 (2012). - ISSN 1471-2164 - 35 p.
    usage patterns - sequences - prokaryotes - communication - reduction - diversity - ancestor
    Background: We sought to assess whether the concept of relative entropy (information capacity), could aid our understanding of the process of horizontal gene transfer in microbes. We analyzed the differences in information capacity between prokaryotic chromosomes, genomic islands (GI), phages, and plasmids. Relative entropy was estimated using the Kullback-Leibler measure. Results: Relative entropy was highest in bacterial chromosomes and had the sequence chromosomes >GI>phage>plasmid. There was an association between relative entropy and AT content in chromosomes, phages, plasmids and GIs with the strongest association being in phages. Relative entropy was also found to be lower in the obligate intracellular Mycobacterium leprae than in the related M. tuberculosis when measured on a shared set of highly conserved genes. Conclusions: We argue that relative entropy differences reflect how plasmids, phages and GIs interact with microbial host chromosomes and that all these biological entities are, or have been, subjected to different selective pressures. The rate at which amelioration of horizontally acquired DNA occurs within the chromosome is likely to account for the small differences between chromosomes and stably incorporated GIs compared to the transient or independent replicons such as phages and plasmids
    Analysis of the mating-type loci of co-occurring and phylogenetically related species of Ascochyta and Phoma
    Woudenberg, J.H.C. ; Gruyter, J. de; Crous, P.W. ; Zwiers, L.H. - \ 2012
    Molecular Plant Pathology 13 (2012)4. - ISSN 1464-6722 - p. 350 - 362.
    evolutionary relationships - sexual development - pyrenophora-teres - pisum-sativum - complex - genes - teleomorph - phylogeny - sequences - cloning
    Ascochyta and Phoma are fungal genera containing several important plant pathogenic species. These genera are morphologically similar, and recent molecular studies performed to unravel their phylogeny have resulted in the establishment of several new genera within the newly erected Didymellaceae family. An analysis of the structure of fungal mating-type genes can contribute to a better understanding of the taxonomic relationships of these plant pathogens, and may shed some light on their evolution and on differences in sexual strategy and pathogenicity. We analysed the mating-type loci of phylogenetically closely related Ascochyta and Phoma species (Phoma clematidina, Didymella vitalbina, Didymella clematidis, Peyronellaea pinodes and Peyronellaea pinodella) that co-occur on the same hosts, either on Clematis or Pisum. The results confirm that the mating-type genes provide the information to distinguish between the homothallic Pey. pinodes (formerly Ascochyta pinodes) and the heterothallic Pey. pinodella (formerly Phoma pinodella), and indicate the close phylogenetic relationship between these two species that are part of the disease complex responsible for Ascochyta blight on pea. Furthermore, our analysis of the mating-type genes of the fungal species responsible for causing wilt of Clematis sp. revealed that the heterothallic D. vitalbina (Phoma anamorph) is more closely related to the homothallic D. clematidis (Ascochyta anamorph) than to the heterothallic P. clematidina. Finally, our results indicate that homothallism in D. clematidis resulted from a single crossover between MAT1-1 and MAT1-2 sequences of heterothallic ancestors, whereas a single crossover event followed by an inversion of a fused MAT1/2 locus resulted in homothallism in Pey. pinodes.
    ProRepeat: an integrated repository for studying amino acid tandem repeats in proteins
    Luo, H. ; Lin, K. ; David, A. ; Nijveen, H. ; Leunissen, J.A.M. - \ 2012
    Nucleic acids research 40 (2012)D1. - ISSN 0305-1048 - p. D394 - D399.
    annotation resource - codon usage - sequences - database - evolution - genomes - selection - alanine - algorithm - proteomes
    ProRepeat (http://prorepeat.bioinformatics.nl/) is an integrated curated repository and analysis platform for in-depth research on the biological characteristics of amino acid tandem repeats. ProRepeat collects repeats from all proteins included in the UniProt knowledgebase, together with 85 completely sequenced eukaryotic proteomes contained within the RefSeq collection. It contains non-redundant perfect tandem repeats, approximate tandem repeats and simple, low-complexity sequences, covering the majority of the amino acid tandem repeat patterns found in proteins. The ProRepeat web interface allows querying the repeat database using repeat characteristics like repeat unit and length, number of repetitions of the repeat unit and position of the repeat in the protein. Users can also search for repeats by the characteristics of repeat containing proteins, such as entry ID, protein description, sequence length, gene name and taxon. ProRepeat offers powerful analysis tools for finding biological interesting properties of repeats, such as the strong position bias of leucine repeats in the N-terminus of eukaryotic protein sequences, the differences of repeat abundance among proteomes, the functional classification of repeat containing proteins and GC content constrains of repeats’ corresponding codons.
    Use of rbcL and trnL-F as a two-locus DNA barcode for identification of NW-European ferns: an ecological perspective
    Groot, G.A. de; During, H.J. ; Maas, J.W. ; Schneider, H. ; Erkens, R.H.J. - \ 2011
    PLoS ONE 6 (2011)1. - ISSN 1932-6203
    dryopteris-affinis group - land plants - noncoding regions - chloroplast dna - mononucleotide repeats - sequences - phylogeny - taxonomy - pcr - amplification
    Although consensus has now been reached on a general two-locus DNA barcode for land plants, the selected combination of markers (rbcL + matK) is not applicable for ferns at the moment. Yet especially for ferns, DNA barcoding is potentially of great value since fern gametophytes—while playing an essential role in fern colonization and reproduction—generally lack the morphological complexity for morphology-based identification and have therefore been underappreciated in ecological studies. We evaluated the potential of a combination of rbcL with a noncoding plastid marker, trnL-F, to obtain DNAidentifications for fern species. A regional approach was adopted, by creating a reference database of trusted rbcL and trnL-F sequences for the wild-occurring homosporous ferns of NW-Europe. A combination of parsimony analyses and distancebased analyses was performed to evaluate the discriminatory power of the two-region barcode. DNA was successfully extracted from 86 tiny fern gametophytes and was used as a test case for the performance of DNA-based identification. Primer universality proved high for both markers. Based on the combined rbcL + trnL-F dataset, all genera as well as all species with non-equal chloroplast genomes formed their own well supported monophyletic clade, indicating a high discriminatory power. Interspecific distances were larger than intraspecific distances for all tested taxa. Identification tests on gametophytes showed a comparable result. All test samples could be identified to genus level, species identification was well possible unless they belonged to a pair of Dryopteris species with completely identical chloroplast genomes. Our results suggest a high potential of the combined use of rbcL and trnL-F as a two-locus cpDNA barcode for identification of fern species. A regional approach may be preferred for ecological tests. We here offer such a ready-to-use barcoding approach for ferns, which opens the way for answering a whole range of questions previously unaddressed in fern gametophyte ecology
    First isolation of Hepatitis E virus genotype 4 in Europe through swine surveillance in the Netherlands and Belgium.
    Honing-Hakze, R.W. van der; Coillie, E. Van; Antonis, A.F.G. ; Poel, W.H.M. van der - \ 2011
    PLoS ONE 6 (2011)8. - ISSN 1932-6203 - 6 p.
    cross-species infection - united-states - rt-pcr - prevalence - hev - epidemiology - transmission - antibodies - sequences - humans
    Hepatitis E virus (HEV) genotypes 3 and 4 are a cause of human hepatitis and swine are considered the main reservoir. To study the HEV prevalence and characterize circulating HEV strains, fecal samples from swine in the Netherlands and Belgium were tested by RT-PCR. HEV prevalence in swine was 7–15%. The Dutch strains were characterized as genotype 3, subgroups 3a, 3c and 3f, closely related to sequences found in humans and swine earlier. The HEV strains found in Belgium belonged to genotypes 3f and 4b. The HEV genotype 4 strain was the first ever reported in swine in Europe and an experimental infection in pigs was performed to isolate the virus. The genotype 4 strain readily infected piglets and caused fever and virus shedding. Since HEV4 infections have been reported to run a more severe clinical course in humans this observation may have public health implications
    Investigation of Cyprinidherpesvirus-3 genetic diversity by a multi-locus variable number of tandem repeats analysis.
    Avarre, J.C. ; Madeira, J.P. ; Santika, A. ; Zainunb, Z. ; Baud, M. ; Cabon, J. ; Caruso, D. ; Castric, J. ; Bigarré, L. ; Engelsma, M.Y. ; Maskur, M. - \ 2011
    Journal of Virological Methods 173 (2011)2. - ISSN 0166-0934 - p. 320 - 327.
    mediated isothermal amplification - koi-herpesvirus - common carp - r-package - virus - khv - sequences - pcr - dna - mortality
    Cyprinid herpesvirus-3 (CyHV-3), or koi herpesvirus (KHV), is responsible for high mortalities in aquaculture of both common carp (Cyprinus carpio carpio) and koi carp (Cyprinus carpio koi) worldwide. The complete genomes of three CyHV-3 isolates showed more than 99% of DNA sequence identity, with the majority of differences located in short tandem repeats, also called VNTR (variable number of tandem repeats). By targeting these variations, eight loci were selected for genotyping CyHV-3 by multiple locus VNTR analysis (MLVA). CyHV-3 strains obtained after sequential in vivo infections exhibited identical MLVA profiles, whereas samples originating from a single isolate passaged 6 and 82 times in vitro exhibited mutations in two of the eight loci, suggesting a relatively slow genetic evolution rate of the VNTRs. The method was subsequently applied on 38 samples collected in Indonesia, France and the Netherlands. Globally, the isolates grouped in two main genetic clusters, each one divided in two subgroups including either CyHV-3-U/I or CyHV3-J. Interestingly, Indonesian strains were rather distant from CyHV-3-J isolate. The results of the present study indicate that these VNTR molecular markers are efficient in estimating the genetic diversity among CyHV-3 isolates and are therefore suitable for further molecular epidemiological studies.
    DNA damage in plant herbarium tissue.
    Staats, M. ; Cuenca, A. ; Richardson, J.E. ; Ginkel, R.V. ; Petersen, G. ; Seberg, O. ; Bakker, F.T. - \ 2011
    PLoS ONE 6 (2011)12. - ISSN 1932-6203 - 9 p.
    programmed cell-death - ancient dna - miscoding lesions - enzymatic amplification - specimens - repair - preservation - extraction - sequences - reveals
    Dried plant herbarium specimens are potentially a valuable source of DNA. Efforts to obtain genetic information from this source are often hindered by an inability to obtain amplifiable DNA as herbarium DNA is typically highly degraded. DNA post-mortem damage may not only reduce the number of amplifiable template molecules, but may also lead to the generation of erroneous sequence information. A qualitative and quantitative assessment of DNA post-mortem damage is essential to determine the accuracy of molecular data from herbarium specimens. In this study we present an assessment of DNA damage as miscoding lesions in herbarium specimens using 454-sequencing of amplicons derived from plastid, mitochondrial, and nuclear DNA. In addition, we assess DNA degradation as a result of strand breaks and other types of polymerase non-bypassable damage by quantitative real-time PCR. Comparing four pairs of fresh and herbarium specimens of the same individuals we quantitatively assess post-mortem DNA damage, directly after specimen preparation, as well as after long-term herbarium storage. After specimen preparation we estimate the proportion of gene copy numbers of plastid, mitochondrial, and nuclear DNA to be 2.4–3.8% of fresh control DNA and 1.0–1.3% after long-term herbarium storage, indicating that nearly all DNA damage occurs on specimen preparation. In addition, there is no evidence of preferential degradation of organelle versus nuclear genomes. Increased levels of C¿T/G¿A transitions were observed in old herbarium plastid DNA, representing 21.8% of observed miscoding lesions. We interpret this type of post-mortem DNA damage-derived modification to have arisen from the hydrolytic deamination of cytosine during long-term herbarium storage. Our results suggest that reliable sequence data can be obtained from herbarium specimens.
    Host status of false brome grass to the leaf rust fungus Puccinia brachypodii and the stripe rust fungus P. Striiformis
    Barbieri, M. ; Marcel, T.C. ; Niks, R.E. - \ 2011
    Plant Disease 95 (2011)11. - ISSN 0191-2917 - p. 1339 - 1345.
    agrobacterium-mediated transformation - model system - distachyon - genome - rice - nonhost - specificity - resistance - morphology - sequences
    Purple false brome grass (Brachypodium distachyon) has recently emerged as a model system for temperate grasses and is also a potential model plant to investigate plant interactions with economically important pathogens such as rust fungi. We determined the host status of five Brachypodium species to three isolates of Puccinia brachypodii, the prevalent rust species on Brachypodium sylvaticum in nature, and to one isolate each of three formae speciales of the stripe rust fungus P. striiformis. Two P. striiformis isolates produced sporulating lesions, both in only one of the tested interactions, suggesting a marginal host status of B. distachyon. P. brachypodii formed sporulating uredinia on the five Brachypodium species tested, and a range of reactions was observed. Surprisingly, the B. sylvaticum–derived rust isolates were more frequently pathogenic to B. distachyon than to their original host species. The B. distachyon diploid inbred lines, developed and distributed as reference material to the Brachypodium research community, include susceptible and resistant genotypes to at least three of the four P. brachypodii isolates tested. This creates the opportunity to use B. distachyon/P. brachypodii as a model pathosystem. In one B. distachyon accession, heavy infection by the loose smut fungus Ustilago bromivora occurred. That pathogen could also serve as a model pathogen of Brachypodium.
    A quantitative account of genomic island acquisitions in prokaryotes
    Roos, T.E. ; Passel, M.W.J. van - \ 2011
    BMC Genomics 12 (2011). - ISSN 1471-2164 - 34 p.
    art. no. 163 - pathogenicity islands - bacterial genomes - signature - sequences - dna - identification - amelioration - evolution - virulence
    BACKGROUND: Microbial genomes do not merely evolve through the slow accumulation of mutations, but also, and often more dramatically, by taking up new DNA in a process called horizontal gene transfer. These innovation leaps in the acquisition of new traits can take place via the introgression of single genes, but also through the acquisition of large gene clusters, which are termed Genomic Islands. Since only a small proportion of all the DNA diversity has been sequenced, it can be hard to find the appropriate donors for acquired genes via sequence alignments from databases. In contrast, relative oligonucleotide frequencies represent a remarkably stable genomic signature in prokaryotes, which facilitates compositional comparisons as an alignment-free alternative for phylogenetic relatedness. In this project, we test whether Genomic Islands identified in individual bacterial genomes have a similar genomic signature, in terms of relative dinucleotide frequencies, and can therefore be expected to originate from a common donor species. RESULTS: When multiple Genomic Islands are present within a single genome, we find that up to 28% of these are compositionally very similar to each other, indicative of frequent recurring acquisitions from the same donor to the same acceptor. CONCLUSIONS: This represents the first quantitative assessment of common directional transfer events in prokaryotic evolutionary history. We suggest that many of the resident Genomic Islands per prokaryotic genome originated from the same source, which may have implications with respect to their regulatory interactions, and for the elucidation of the common origins of these acquired gene clusters
    Base-pairing promotes leader selection to prime in vitro influenza genome transcription
    Geerts-Dimitriadou, C. ; Zwart, M.P. ; Goldbach, R.W. ; Kormelink, R.J.M. - \ 2011
    Virology 409 (2011)1. - ISSN 0042-6822 - p. 17 - 26.
    messenger-rna synthesis - cap-snatching mechanism - mosaic-virus rnas - viral-rna - 5' ends - complementary rna - a virus - polymerase - endonuclease - sequences
    The requirements for alignment of capped leader sequences along the viral genome during influenza transcription initiation (cap-snatching) have long been an enigma. In this study, competition experiments using an in vitro transcription assay revealed that influenza virus transcriptase prefers leader sequences with base complementarity to the 3'-ultimate residues of the viral template, 10 or 11 nt from the 5' cap. Internal priming at the 3'-penultimate residue, as well as prime-and-realign was observed. The nucleotide identity immediately 5' of the base-pairing residues also affected cap donor usage. Application to the in vitro system of RNA molecules with increased base complementarity to the viral RNA template showed stronger reduction of globin RNA leader initiated influenza transcription compared to those with a single base-pairing possibility. Altogether the results indicated an optimal cap donor consensus sequence of 7mG-(N)7–8-(A/U/G)-(A/U)-AGC-3'.
    Bivariate colour maps for visualizing climate data
    Teuling, A.J. ; Stöckli, R. ; Seneviratne, S.I. - \ 2011
    International Journal of Climatology 31 (2011)9. - ISSN 0899-8418 - p. 1408 - 1412.
    univariate - sequences
    The increasing availability of gridded, high-resolution, multivariate climatological data sets calls for innovative approaches to visualize inter-variable relations. In this study, we present a methodology, based on properties of common colour schemes, to plot two variables in a single colour map by using a two-dimensional colour legend for both sequential and diverging data. This is especially suited for climate data as the spatial distribution of the relation between different variables is often as important as the distribution of variables individually. Two example applications are given to illustrate the use of the method: one that shows the global distribution of climate based on observed temperature and relative humidity, and the other showing the distribution of recent changes in observed temperature and precipitation over Europe. A flexible and easy-to-implement method is provided to construct different colour legends for sequential and diverging data.
    Functional analysis and expression of HcrVf1 and HcrVf2 for development of scab resistant cisgenic and intragenic apples
    Joshi, S.G. ; Schaart, J. ; Groenwold, R. ; Jacobsen, E. ; Schouten, H.J. ; Krens, F.A. - \ 2011
    Plant Molecular Biology 75 (2011)6. - ISSN 0167-4412 - p. 579 - 591.
    receptor-like genes - real-time pcr - venturia-inaequalis - vf gene - plants - sequences - agrobacterium - promoters - linkage - cluster
    Apple scab resistance genes, HcrVf1 and HcrVf2, were isolated including their native promoter, coding and terminator sequences. Two fragment lengths (short and long) of the native gene promoters and the strong apple rubisco gene promoter (PMdRbc) were used for both HcrVf genes to test their effect on expression and phenotype. The scab susceptible cultivar ‘Gala’ was used for plant transformations and after selection of transformants, they were micrografted onto apple seedling rootstocks for scab disease tests. Apple transformants were also tested for HcrVf expression by quantitative RT-PCR (qRTPCR). For HcrVf1 the long native promoter gave significantly higher expression that the short one; in case of HcrVf2 the difference between the two was not significant. The apple rubisco gene promoter proved to give the highest expression of both HcrVf1 and HcrVf2. The top four expanding leaves were used initially for inoculation with monoconidial isolate EU-B05 which belongs to race 1 of V. inaequalis. Later six other V. inaequalis isolates were used to study the resistance spectra of the individual HcrVf genes. The scab disease assays showed that HcrVf1 did not give resistance against any of the isolates tested regardless of the expression level. The HcrVf2 gene appeared to be the only functional gene for resistance against Vf avirulent isolates of V. inaequalis. HcrVf2 did not provide any resistance to Vf virulent strains, even not in case of overexpression. In conclusion, transformants carrying the apple-derived HcrVf2 gene in a cisgenic as well as in an intragenic configuration were able to reach scab resistance levels comparable to the Vf resistant control cultivar obtained by classical breeding, cv. ‘Santana’.
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