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.

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Phylogenetic analysis of highly pathogenic avian influenza A (H5N8) virus outbreak strains provides evidence for four separate introductions and one between-poultry farm transmission in the Netherlands, November 2014
Bouwstra, R.J. ; Koch, G. ; Heutink, C.G. ; Harders, F.L. ; Spek, A. van der; Elbers, A.R.W. ; Bossers, A. - \ 2015
EuroSurveillance 20 (2015)26. - ISSN 1025-496X
mitochondrial-dna - a viruses - sequence - amplification - china - h5
Phylogenetic analysis of highly pathogenic avian influenza A(H5N8) virus strains causing outbreaks in Dutch poultry farms in 2014 provides evidence for separate introduction of the virus in four outbreaks in farms located 16–112 km from each other and for between-farm transmission between the third and fourth outbreak in farms located 550 m from each other. In addition, the analysis showed that all European and two Japanese H5N8 virus strains are very closely related and seem to originate from a calculated common ancestor, which arose between July and September 2014. Our findings suggest that the Dutch outbreak virus strain ‘Ter Aar’ and the first German outbreak strain from 2014 shared a common ancestor. In addition, the data indicate that the Dutch outbreak viruses descended from an H5N8 virus that circulated around 2009 in Asia, possibly China, and subsequently spread to South Korea and Japan and finally also to Europe. Evolution of the virus seemed to follow a parallel track in Japan and Europe, which supports the hypothesis that H5N8 virus was exchanged between migratory wild waterfowl at their breeding grounds in Siberia and from there was carried by migrating waterfowl to Europe.
Detection of Single-Nucleotide Polymorphisms in Plasmodium falciparum by PCR Primer Extension and Lateral Flow Immunoassay
Moers, A.P.H.A. ; Hallett, R.L. ; Borrow, R. ; Schallig, H.D.F.H. ; Sutherland, C.J. ; Amerongen, A. van - \ 2015
Antimicrobial Agents and Chemotherapy 59 (2015)1. - ISSN 0066-4804 - p. 365 - 371.
real-time pcr - carbon nanoparticles - malaria - dna - chloroquine - assay - amplification - sensitivity - resistance - travelers
The resistance of Plasmodium falciparum to some antimalarial drugs is linked to single-nucleotide polymorphisms (SNPs). Currently, there are no methods for the identification of resistant parasites that are sufficiently simple, cheap, and fast enough to be performed at point-of-care, i.e., in local hospitals where drugs are prescribed. Primer extension methods (PEXT) were developed to identify 4 SNPs in P. falciparum positioned at amino acids 86, 184, and 1246 of the P. falciparum multidrug resistance 1 gene (pfmdr1) and amino acid 76 of the chloroquine resistance transporter gene (pfcrt). The PEXT products were visualized by a nucleic acid lateral flow immunoassay (NALFIA) with carbon nanoparticles as the detection labels. PCR-PEXT-NALFIAs showed good correlation to the reference methods, quantitative PCR (qPCR) or direct amplicon sequence analysis, in an initial open-label evaluation with 17 field samples. The tests were further evaluated in a blind study design in a set of 150 patient isolates. High specificities of 98 to 100% were found for all 4 PCR-PEXT genotyping assays. The sensitivities ranged from 75% to 100% when all PEXT-positive tests were considered. A number of samples with a low parasite density were successfully characterized by the reference methods but failed to generate a result in the PCR-PEXT-NALFIA, particularly those samples with microscopy-negative subpatent infections. This proof-of principle study validates the use of PCR-PEXT-NALFIA for the detection of resistance-associated mutations in P. falciparum, particularly for microscopy-positive infections. Although it requires a standard thermal cycler, the procedure is cheap and rapid and thus a potentially valuable tool for point-of-care detection in developing countries
The Role of the Mean State of Arctic Sea Ice on Near-Surface Temperature Trends
Linden, E.C. van der; Bintanja, R. ; Hazeleger, W. ; Katsman, C.A. - \ 2014
Journal of Climate 27 (2014)8. - ISSN 0894-8755 - p. 2819 - 2841.
climate model sensitivity - albedo feedback - amplification - future - predictability - variability - inversion - thickness - extent - gcm
Century-scale global near-surface temperature trends in response to rising greenhouse gas concentrations in climate models vary by almost a factor of 2, with greatest intermodel spread in the Arctic region where sea ice is a key climate component. Three factors contribute to the intermodel spread: 1) model formulation, 2) control climate state, and 3) internal climate variability. This study focuses on the influence of Arctic sea ice in the control climate on the intermodel spread in warming, using idealized 1% yr(-1) CO2 increase simulations of 33 state-of-the-art global climate models, and combining sea ice-temperature relations on local to large spatial scales. On the Arctic mean scale, the spread in temperature trends is only weakly related to ice volume or area in the control climate, and is probably not dominated by internal variability. This suggests that other processes, such as ocean heat transport and meteorological conditions, play a more important role in the spread of long-term Arctic warming than control sea ice conditions. However, on a local scale, sea ice-warming relations show that in regions with more sea ice, models generally simulate more warming in winter and less warming in summer. The local winter warming is clearly related to control sea ice and universal among models, whereas summer sea ice-warming relations are more diverse, and are probably dominated by differences in model formulation. To obtain a more realistic representation of Arctic warming, it is recommended to simulate control sea ice conditions in climate models so that the spatial pattern is correct.
Invasive alien species under attack: natural enemies of Harmonia axyridis in the Netherlands
Raak-van den Berg, C.L. ; Wielink, P. van; Jong, P.W. de; Gort, G. ; Haelewaters, D. ; Helder, J. ; Lenteren, J.C. van - \ 2014
BioControl 59 (2014)2. - ISSN 1386-6141 - p. 229 - 240.
coleoptera-coccinellidae - ladybird beetle - pallas coleoptera - fungus - laboulbeniales - europe - north - amplification - populations - prevalence
The aphid predator Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae) is an invasive alien species in Europe and North America with negative effects on non-target species (including a decline of native ladybird populations), as well as fruit production, and human health. It is, therefore, important to find out which natural enemies could be used to reduce their numbers. Knowledge of H. axyridis’ natural enemies is summarised and data collected from the Netherlands over the past ten years are presented. Beetles were sampled from winter aggregations and from spring through to autumn with illuminated screens at night. Natural enemies were not found in samples of H. axyridis from 2003–2007. From 2008 onward H. axyridis adults were infested by: Hesperomyces virescens Thaxt. fungi (summer and winter), Parasitylenchus bifurcatus Poinar and Steenberg nematodes (winter), Coccipolipus hippodamiae (McDaniel and Morrill) mites (winter), and Dinocampus coccinellae (Schrank) parasitoids (summer and winter). Our results indicate that these natural enemies are starting to use H. axyridis as a host, but are as yet not sufficiently abundant to control the population.
The northward shifting neophyte Tragopogon dubius is just as effective in forming mycorrhizal associations as the native T. pratensis
Grunsven, R.H.A. van; Yuwati, T. ; Kowalchuk, G.A. ; Putten, W.H. van der; Veenendaal, E. - \ 2014
Plant Ecology & Diversity 7 (2014)4. - ISSN 1755-0874 - p. 533 - 539.
plant-soil feedback - functional diversity - fungal communities - ammophila-arenaria - climate-change - sand dunes - range - grass - identification - amplification
Background: As a consequence of climate warming, many organisms are shifting their range towards higher latitudes and altitudes. As not all do so at the same speed, this may disrupt biotic interaction. Release from natural enemies through range expansion can result in invasiveness, whereas loss of mutualists can reduce plant vigour and fitness. One of the most important groups of plant symbiotic mutualists is the arbuscular mycorrhizal fungi (AMF). Aims: We used greenhouse experiments to test whether Tragopogon dubius, a species that has recently expanded its range northward and colonised the Netherlands, is able to associate with the same AMF as the native congener T. pratensis. Methods: In soils collected from four locations in the new range of T. dubius we compared the density of infective AMF propagules associating with both plant species, as well as AMF colonisation of the roots. The AMF community structure in the roots of these species was also analysed using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). Results: Tragopogon dubius and T. pratensis did not differ in any of these characteristics. Conclusions: We therefore conclude that the range-shifting T. dubius is as effective in the formation of mycorrhiza as the native congener.
A multiplex bead-based suspension array assay for interrogation of phylogenetically informative single nucleotide polymorphisms for Bacillus anthracis
Thierry, S. ; Hamidjaja, R.A. ; Girault, G. ; Lofstrom, C. ; Ruuls-van Stalle, E.M.F. ; Sylviane, D. - \ 2013
Journal of Microbiological Methods 95 (2013)3. - ISSN 0167-7012 - p. 357 - 365.
tandem-repeat analysis - large-scale - listeria-monocytogenes - pathogen detection - genotyping assays - dna - probes - discrimination - amplification - pcr
Single nucleotide polymorphisms (SNPs) are abundant in genomes of all species and represent informative DNA markers extensively used to analyze phylogenetic relationships between strains. Medium to high throughput, open methodologies able to test many SNPs in a minimum time are therefore in great need. By using the versatile Luminex (R) xTAG technology, we developed an efficient multiplexed SNP genotyping assay to score 13 phylogenetically informative SNPs within the genome of Bacillus anthracis. The Multiplex Oligonucleotide Ligation-PCR procedure (MOL-PCR) described by Deshpande et al., 2010 has been modified and adapted for simultaneous interrogation of 13 biallelic canonical SNPs in a 13-plex assay. Changes made to the originally published method include the design of allele-specific dual-priming-oligonucleotides (DPOs) as competing detection probes (MOLigo probes) and use of asymmetric PCR reaction for signal amplification and labeling of ligation products carrying SNP targets. These innovations significantly reduce cross-reactivity observed when initial MOLigo probes were used and enhance hybridization efficiency onto the microsphere array, respectively. When evaluated on 73 representative samples, the 13-plex assay yielded unambiguous SNP calls and lineage affiliation. Assay limit of detection was determined to be 2 ng of genomic DNA. The reproducibility, robustness and easy-of-use of the present method were validated by a small-scale proficiency testing performed between four European laboratories. While cost-effective compared to other singleplex methods, the present MOL-PCR method offers a high degree of flexibility and scalability. It can easily accommodate newly identified SNPs to increase resolving power to the canSNP typing of B. anthracis. (C) 2013 Elsevier B.V. All rights reserved.
Single-enzyme analysis in a droplet-based micro- and nanofluidic system
Arayanarakool, R. ; Shui, L. ; Kengen, S.W.M. ; Berg, A. van den; Eijkel, J.C. - \ 2013
Lab on a Chip 13 (2013)10. - ISSN 1473-0197 - p. 1955 - 1962.
fluorescence microscopy - pyrococcus-furiosus - beta-glucosidase - propyl gallate - amplification - purification - evolution - kinetics - assays
The kinetic activity of individual enzyme molecules was determined in aqueous droplets generated in a nano- and microfluidic device. To avoid high background noise, the enzyme and substrate solution was confined into femtoliter carriers, achieving high product concentrations from single-molecule encapsulation. The tiny droplets (f ~ 2.5-3 µm) generated from this fluidic system were highly monodisperse, beneficial for an analysis of single enzyme activity. The method presented here allows to follow large numbers of individual droplets over time. The instrumental requirements are furthermore modest, since the small droplet size allows to use of standard microscope and standard Pyrex glass chips as well as the use of relatively high enzyme concentrations (nM range) for single molecule encapsulation
Genomic Treasure Troves: Complet Genome Sequencing of Herbarium and Insect Museum Specimens
Staats, M. ; Erkens, R.H.J. ; Vossenberg, B. van de; Wieringa, J.J. ; Kraaijeveld, K. ; Stielow, B. ; Geml, J. ; Richardson, J.E. ; Bakker, F.T. - \ 2013
PLoS One 8 (2013)7. - ISSN 1932-6203
ancient dna-sequences - miscoding lesions - extraction - amplification - patterns - fungi - plant - enrichment - evolution - alignment
Unlocking the vast genomic diversity stored in natural history collections would create unprecedented opportunities for genome-scale evolutionary, phylogenetic, domestication and population genomic studies. Many researchers have been discouraged from using historical specimens in molecular studies because of both generally limited success of DNA extraction and the challenges associated with PCR-amplifying highly degraded DNA. In today's next-generation sequencing (NGS) world, opportunities and prospects for historical DNA have changed dramatically, as most NGS methods are actually designed for taking short fragmented DNA molecules as templates. Here we show that using a standard multiplex and paired-end Illumina sequencing approach, genome-scale sequence data can be generated reliably from dry-preserved plant, fungal and insect specimens collected up to 115 years ago, and with minimal destructive sampling. Using a reference-based assembly approach, we were able to produce the entire nuclear genome of a 43-year-old Arabidopsis thaliana (Brassicaceae) herbarium specimen with high and uniform sequence coverage. Nuclear genome sequences of three fungal specimens of 22–82 years of age (Agaricus bisporus, Laccaria bicolor, Pleurotus ostreatus) were generated with 81.4–97.9% exome coverage. Complete organellar genome sequences were assembled for all specimens. Using de novo assembly we retrieved between 16.2–71.0% of coding sequence regions, and hence remain somewhat cautious about prospects for de novo genome assembly from historical specimens. Non-target sequence contaminations were observed in 2 of our insect museum specimens. We anticipate that future museum genomics projects will perhaps not generate entire genome sequences in all cases (our specimens contained relatively small and low-complexity genomes), but at least generating vital comparative genomic data for testing (phylo)genetic, demographic and genetic hypotheses, that become increasingly more horizontal. Furthermore, NGS of historical DNA enables recovering crucial genetic information from old type specimens that to date have remained mostly unutilized and, thus, opens up a new frontier for taxonomic research as well.
Development, validation and evaluation of a rapid PCR-nucleic acid lateral flow immuno-assay for the detection of Plasmodium and the differentiation between Plasmodium falciparum and Plasmodium vivax
Mens, P.F. ; Moers, A.P.H.A. ; Bes, L.M. de; Flint, J. ; Sak, J.R.S. ; Keereecharoen, L. ; Overmeir, C. ; Verweij, J.J. ; Hallett, R.L. ; Wihokhoen, B. ; Proux, S. ; Schallig, H.D.F.H. ; Amerongen, A. van - \ 2012
Malaria Journal 11 (2012). - ISSN 1475-2875
malaria diagnosis - amplification - parasite - microscopy - accuracy - tests - care
Background: Molecular tools are very sensitive and specific and could be an alternative for the diagnosis of malaria. The complexity and need for expensive equipment may hamper implementation and, therefore, simplifications to current protocols are warranted. Methods: A PCR detecting the different Plasmodium species and differentiating between Plasmodium falciparum and Plasmodium vivax was developed and combined with a nucleic acid lateral flow immuno-assay (PCR-NALFIA) for amplicon detection. The assay was thoroughly evaluated for the analytical sensitivity and specificity in the laboratory, the robustness and reproducibility in a ring trial and accuracy and predictive value in a field trial. Results: The analytical sensitivity and specificity were 0.978 (95% CI: 0.932-0.994) and 0.980 (95% CI: 0.924-0.997), respectively, and were slightly less sensitive for the detection of P. vivax than for P. falciparum. The reproducibility tested in three laboratories was very good (k = 0.83). This evaluation showed that the PCR machine used could influence the results. Accuracy was evaluated in Thailand and compared to expert microscopy and rapid diagnostic tests (RDTs). The overall and P. falciparum-specific sensitivity and specificity was good ranging from 0.86-1 and 0.95-0.98 respectively, compared to microscopy. Plasmodium vivax detection was better than the sensitivity of RDT, but slightly less than microscopy performed in this study. Conclusion: PCR-NALFIA is a sensitive, specific and robust assay able to identify Plasmodium species with good accuracy. Extensive testing including a ring trial can identify possible bottlenecks before implementation and is therefore essential to perform in additon to other evaluations.
Direct blood PCR in combination with Nucleic Acid Laterla Flow Immuno-Assay for the detection of Plasmodium species in malaria endemic settings.
Mens, P.F. ; Bes, H.M. ; Sondo, P. ; Amerongen, A. van - \ 2012
Journal of Clinical Microbiology 50 (2012)11. - ISSN 0095-1137 - p. 3520 - 3525.
real-time pcr - rapid diagnostic-tests - molecular diagnosis - parasite detection - amplification - microscopy - kenya - field - assay
Declining malaria transmission and known difficulties with current diagnostic tools for malaria, such as microscopy and rapid diagnostic tests (RDTs) in particular at low parasite densities, still warrant the search for sensitive diagnostic tests. Molecular tests need substantial simplification before implementation in clinical settings in countries where malaria is endemic. Direct blood PCR (db-PCR), circumventing DNA extraction, to detect Plasmodium was developed and adapted to be visualized by nucleic acid lateral flow immunoassay (NALFIA). The assay was evaluated in the laboratory against samples from confirmed Sudanese patients (n = 51), returning travelers (n = 214), samples from the Dutch Blood Bank (n = 100), and in the field in Burkina Faso (n = 283) and Thailand (n = 381) on suspected malaria cases and compared to RDT and microscopy. The sensitivity and specificity of db-PCR-NALFIA compared to the initial diagnosis in the laboratory were 94.4% (95% confidence interval [CI] = 0.909 to 0.969) and 97.4% (95% CI = 0.909 to 0.969), respectively. In Burkina Faso, the sensitivity was 94.8% (95% CI = 0.88.7 to 97.9%), and the specificity was 82.4% (95% CI = 75.4 to 87.7%) compared to microscopy and 93.3% (95% CI = 87.4 to 96.7%) and 91.4% (95% CI = 85.2 to 95.3%) compared to RDT. In Thailand, the sensitivity and specificity were 93.4% (CI = 86.4 to 97.1%) and 90.9 (95% CI = 86.7 to 93.9%), respectively, compared to microscopy and 95.6% (95% CI = 88.5 to 98.6%) and 87.1% (95% CI = 82.5 to 90.6) compared to RDT. db-PCR-NALFIA is highly sensitive and specific for easy and rapid detection of Plasmodium parasites and can be easily used in countries where malaria is endemic. The inability of the device to discriminate Plasmodium species requires further investigation.
Relationships between declining summer sea ice, increasing temperatures and changing vegetation in the Siberian Arctic tundra from MODIS time series (2000–11)
Dutrieux, L.P. ; Bartholomeus, H. ; Herold, M. ; Verbesselt, J. - \ 2012
Environmental Research Letters 7 (2012)4. - ISSN 1748-9326 - 12 p.
climate-change - shrub expansion - high-latitudes - ndvi - responses - amplification - ecosystems - community - carbon - alaska
The concern about Arctic greening has grown recently as the phenomenon is thought to have significant influence on global climate via atmospheric carbon emissions. Earlier work on Arctic vegetation highlighted the role of summer sea ice decline in the enhanced warming and greening phenomena observed in the region, but did not contain enough details for spatially characterizing the interactions between sea ice, temperature and vegetation photosynthetic absorption. By using 1 km resolution data from the Moderate Resolution Imaging Spectrometer (MODIS) as a primary data source, this study presents detailed maps of vegetation and temperature trends for the Siberian Arctic region, using the time integrated normalized difference vegetation index (TI-NDVI) and summer warmth index (SWI) calculated for the period 2000-11 to represent vegetation greenness and temperature respectively. Spatio-temporal relationships between the two indices and summer sea ice conditions were investigated with transects at eight locations using sea ice concentration data from the Special Sensor Microwave/Imager (SSM/I). In addition, the derived vegetation and temperature trends were compared among major Arctic vegetation types and bioclimate subzones. The fine resolution trend map produced confirms the overall greening (+1% yr(-1)) and warming (+0.27% yr(-1)) of the region, reported in previous studies, but also reveals browning areas. The causes of such local decreases in vegetation, while surrounding areas are experiencing the opposite reaction to changing conditions, are still unclear. Overall correlations between sea ice concentration and SWI as well as TI-NDVI decreased in strength with increasing distance from the coast, with a particularly pronounced pattern in the case of SWI. SWI appears to be driving TI-NDVI in many cases, but not systematically, highlighting the presence of limiting factors other than temperature for plant growth in the region. Further unravelling those limiting factors constitutes a priority in future research. This study demonstrates the use of medium resolution remotely sensed data for studying the complexity of spatio-temporal vegetation dynamics in the Arctic.
How to Open the Treasure Chest? Optimising DNA Extraction from Herbarium Specimens
Särkinen, T. ; Staats, M. ; Richardson, J.E. ; Cowan, R.S. ; Bakker, F.T. - \ 2012
PLoS One 7 (2012)8. - ISSN 1932-6203
ancient dna - land plants - amplification - barcode - bones - pcr - preservation - limitations
Herbarium collections are potentially an enormous resource for DNA studies, but the use of herbarium specimens in molecular studies has thus far been slowed down by difficulty in obtaining amplifiable DNA. Here we compare a set of commercially available DNA extraction protocols and their performance in terms of DNA purity and yield, and PCR amplification success as measured by using three differentially sized markers, the rbcL barcoding marker (cpDNA), the LEAFY exon 3 (nrDNA), and the trnL(UAA) P6 loop (cpDNA). Results reveal large differences between extraction methods, where DNA purity rather than yield is shown to be strongly correlated with PCR success. Amplicon size shows similarly strong correlation with PCR success, with the shortest fragment showing the highest success rate (78%, P6 loop, 10–143 base pairs (bp)) and the largest fragment the lowest success (10%, rbcL, 670 bp). The effect of specimen preparation method on PCR success was also tested. Results show that drying method strongly affects PCR success, especially the availability of fragments longer than 250 bp, where longer fragments are more available for PCR amplification in air dried material compared to alcohol dried specimens. Results from our study indicate that projects relying on poor-quality starting material such as herbarium or scat samples should focus on extracting pure DNA and aim to amplify short target regions (
Detection of Prion Protein Particles in Blood Plasma of Scrapie Infected Sheep
Bannach, O. ; Birkmann, E. ; Reinartz, E. ; Karl-Erich, J. ; Langeveld, J.P.M. ; Rohwer, R.G. ; Gregori, L. ; Terry, L.A. ; Willbold, D. ; Riesner, D. - \ 2012
PLoS One 7 (2012)5. - ISSN 1932-6203 - 8 p.
transmissible spongiform encephalopathy - creutzfeldt-jakob-disease - infectivity - removal - amplification - components - conversion - antibody - models - cattle
Prion diseases are transmissible neurodegenerative diseases affecting humans and animals. The agent of the disease is the prion consisting mainly, if not solely, of a misfolded and aggregated isoform of the host-encoded prion protein (PrP). Transmission of prions can occur naturally but also accidentally, e.g. by blood transfusion, which has raised serious concerns about blood product safety and emphasized the need for a reliable diagnostic test. In this report we present a method based on surface-FIDA (fluorescence intensity distribution analysis), that exploits the high state of molecular aggregation of PrP as an unequivocal diagnostic marker of the disease, and show that it can detect infection in blood. To prepare PrP aggregates from blood plasma we introduced a detergent and lipase treatment to separate PrP from blood lipophilic components. Prion protein aggregates were subsequently precipitated by phosphotungstic acid, immobilized on a glass surface by covalently bound capture antibodies, and finally labeled with fluorescent antibody probes. Individual PrP aggregates were visualized by laser scanning microscopy where signal intensity was proportional to aggregate size. After signal processing to remove the background from low fluorescence particles, fluorescence intensities of all remaining PrP particles were summed. We detected PrP aggregates in plasma samples from six out of ten scrapie-positive sheep with no false positives from uninfected sheep. Applying simultaneous intensity and size discrimination, ten out of ten samples from scrapie sheep could be differentiated from uninfected sheep. The implications for ante mortem diagnosis of prion diseases are discussed
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
Extracting a century of preserved molecular and population demographic data from archived otoliths in the endangered European eel (Anguilla anguilla L.)
Schaerlaekens, D.G. ; Dekker, W. ; Wickstrom, H. ; Volckaert, F.A.M. ; Maes, G.E. - \ 2011
Journal of Experimental Marine Biology and Ecology 398 (2011)1-2. - ISSN 0022-0981 - p. 56 - 62.
cod gadus-morhua - old scale samples - age-determination - dna extraction - ancient dna - microsatellite loci - genetic analyses - north-sea - amplification - conservation
Archived otolith collections represent an invaluable source of information to study demographic and genetic changes in commercially important fish populations. Studies combining both approaches are however rare and reliable extraction of molecular and population demographic data from the same collection of otoliths has never been assessed in the endangered European eel (Anguilla anguilla L). Here we evaluate various DNA extraction protocols to compare DNA yield, microsatellite amplification success, genotype integrity and precision of age determination for eel otoliths that have been archived for 4 weeks, and for 28 and 48 years. Our results show a high amplification success and an equal genotype integrity for DNA fragments extracted from both recently sampled otoliths and high quality reference DNA tissue. Although historical samples yielded low amounts of DNA. PCR amplification was successful and genotyping reliable for short fragments, but decreased significantly with PCR fragment size. None of the extraction protocols caused physical damage to the otoliths and precision of age determination was high for both treated and untreated otoliths. Hence, the methodology can be applied as a standard for the further joint analysis of past demographic and genetic changes during the last century in the highly exploited European eel and in other fish requiring urgent conservation measures.
Single-cell genomics: unravelling the genomes of unculturable microorganisms
Jager, V.C.L. de; Siezen, R.J. - \ 2011
Microbial Biotechnology 4 (2011)4. - ISSN 1751-7907 - p. 431 - 437.
genetic-analysis - marine sponges - flow-cytometry - amplification - heterogeneity - communities - polymerase
The effect of design parameters on temperature of a heated microsystem
Swarts, J.W. ; Janssen, A.E.M. ; Boom, R.M. - \ 2011
Chemical Engineering Research & Design 89 (2011)3. - ISSN 0263-8762 - p. 310 - 317.
total analysis systems - chip - amplification - reactors - gradient
This paper demonstrates the effect of microfluidic system design on temperature profiles of the whole system and more specifically of the microfluidics. Computational fluid dynamics models were used to estimate the effect of some parameters on the temperature of a microsystem with a relatively small heater. The system consisted of a Poly-Ether-Ether-Ketone (PEEK) chipholder with a heater, and a glass chip, surrounded by air. The material and design of the chipholder had a dominant effect on the temperature. Bringing the heater from 22 to 80 ° C resulted in a temperature gradient of over 40 ° C over the length of the chip at fluid level. Due to slow heating, quick switching of temperature is not possible. The steady state temperature profile at fluid level can be changed by adapting the geometry and material of the chipholder. By including the system’s thermal properties in microfluidic system design desired temperature profiles can be obtained. Computer models, such as described in this paper can be used to design a system which thermal behaviour matches the process requirements for the intended use
Genetic diversity and population structure of Iranian wild Pleurotus eryngii species-complex strains revealed by URP-PCR markers
Behnamian, Mahdi ; Mohammadi, Seyed A. ; Sonnenberg, A.S.M. ; Goltapeh, Ebrahim M. ; Hendrickx, P.M. - \ 2010
Journal of Food, Agriculture & Environment 8 (2010)3&4. - ISSN 1459-0255 - p. 1203 - 1207.
rapd analysis - mushroom - dna - polymorphisms - amplification - primers - fungi
In the present study, a set of 68 P. eryngii wild strains collected from nine locations in northwest and west of Iran along with six commercial strains were studied using universal rice primers (URP). The wild strains were isolated from Ferula ovina, F. haussknechtii, Cachrys ferulacea, Kellusia odoratissima and Smyrniopsis aucheri plant species. Eleven URP primers amplified 188 polymorphic fragments. A total of 3, 2 and 7 bands were specific to the strains collected from F. ovina and C. ferulacea plant species and commercial strains, respectively. The highest and lowest polymorphisms were identified in populations B (66.49%) and F (24.47%), respectively. Genetic distances among populations ranged from 0.027 (between populations A and B) to 0.393 (between populations C and F) with an average of 0.210. The closest and furthest wild populations to commercial strains were populations B (0.102) and F (0.234), respectively. Analysis of molecular variance (AMOVA) revealed significant among regions, among populations within regions and within population diversity, whereas within population variation (61.6%) accounted for most of the total molecular variance.
An efficient method for DNA extraction from Cladosporioid fungi
Moslem, M.A. ; Bahkali, A.H. ; Abd-Elsalam, K.A. ; Wit, P.J.G.M. de - \ 2010
Genetics and Molecular Research 9 (2010)4. - ISSN 1676-5680 - p. 2283 - 2291.
candida-albicans - pcr - amplification - pathogens - diagnosis
We developed an efficient method for DNA extraction from Cladosporioid fungi, which are important fungal plant pathogens. The cell wall of Cladosporioid fungi is often melanized, which makes it difficult to extract DNA from their cells. In order to overcome this we grew these fungi for three days on agar plates and extracted DNA from mycelium mats after manual or electric homogenization. High-quality DNA was isolated, with an A260/A280 ratio ranging between 1.6 and 2.0. Isolated genomic DNA was efficiently digested with restriction enzymes and produced distinct banding patterns on agarose gels for the different Cladosporium species. Clear DNA fragments from the isolated DNA were amplified by PCR using small and large subunit rDNA primers, demonstrating that this method provides DNA of sufficiently high quality for molecular analyses
Towards a multiplex cereal traceability tool using padlock probe ligation on genomic DNA
Prins, T.W. ; Dijk, J.P. van; Hoef, A.M.A. van; Voorhuijzen, M.M. ; Broeders, S. ; Trapmann, S. ; Seyfarth, R. ; Pardigol, A. ; Schoen, C.D. ; Aarts, H.J.M. ; Kok, E.J. - \ 2010
Food Chemistry 118 (2010)4. - ISSN 0308-8146 - p. 966 - 973.
nearest-neighbor thermodynamics - bread wheat - durum-wheat - amplification - quantification - semolina - pasta - pcr
Current EU regulations on the protection of products with certain characteristics (geographical indications and designations of origin) aim to ensure fair competition for producers and increased consumers¿ trust. Within the European integrated research project TRACE analytical methods are being developed to allow the maintenance of specific regulations for PGIs (products of protected geographical indication) and PDOs (products of designated origin). An example within the project is the PGI wheat variety Farro della Garfagnana. The aim of the research was to develop a method to establish the purity of Farro della Garfagnana DNA in complex cereal mixtures. The combined approach of padlock probe ligation and multiplex microarray detection can identify possible admixtures. One undesired `contaminant¿ for Farro della Garfagnana is common bread wheat (Triticum aestivum), containing the BBAuAuDD genome. Since Farro harbours the BBAuAu genome, absence of the D-genome rules out the presence of bread wheat. The current detection limit of this multimethod is at least 2.5% bread wheat in Farro
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