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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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Investigating the fruit texture genetic control in apple and its interplay with the production of volatile compounds using multi-family based analysis and genome wide association mapping
Guardo, Mario Di - \ 2017
Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Eric van de Weg; F. Costa. - Wageningen : Wageningen University - ISBN 9789463432054 - 177
malus domestica - apples - fruit - fruit growing - genetics - plant breeding - genome analysis - appels - fruitteelt - genetica - plantenveredeling - genoomanalyse

Although varying with context, quality of fresh fruits includes several properties such as color, texture, flavor and health promoting compounds. This thesis focused on two important quality aspects, namely texture and aroma in apple, and defining the genomic regions involved in the control of these two features. The genetic control of texture and VOCs production have been investigated using two marker-trait association analysis approaches: Pedigree Based Analysis (PBA) and Genome Wide Association Study (GWAS). In chapter 2, ASSIsT (Automatic SNP ScorIng Tool), a software dedicated for the efficient calling and filtering of SNPs from Illumina InfiniumÒ arrays is presented. ASSIsT builds on GenomeStudio® derived data and identifies markers showing reliable genotype calls (bi-allelic segregation pattern). In addition, ASSIsT identifies and re-edits SNP calls of markers showing additional alleles (null alleles or additional SNPs in the probe annealing site). Chapter 3 aimed to dissect the genetic control of fruit firmness in apple during storage through PBA and employing 24 bi-parental families (1216 individuals) connected by a common pedigree structure. Ten QTLs were identified encompassing eight linkage groups, which unravelled a QTL dynamics over storage shedding light on the specific genetic control at each time-point. Chapter 4: aimed to comprehensively decipher the genetic control of fruit texture. Two complementing QTL mapping approaches were employed together with a novel and high sophisticated phenotyping device for fruit texture. The PBA was carried out on six full-sib pedigreed families (416 individuals), while the GWAS was performed on a collection of 233 apple accessions. The texture analyser employed (TAXT-AED texture analyser) allowed the measurement of both the mechanical properties (firmness) and the acoustic properties (crispness) of fruit texture. The QTL results indicated chromosome 10 being associated in changes of the mechanical properties of fruit texture, while chromosomes 2 and 14 were more associated to the acoustic response. In Chapter 5 the interplay between texture and volatile organic compounds (VOCs) was investigated in 162 apple accessions. The array of volatile compounds phenotyped was implemented into a GWAS identifying seven chromosomes harbouring important candidate genes for aroma, such as MdAAT1 and MdIGS. Next, volatilome and fruit texture data were integrated revealing a negative correlation between these two features.

Physiology and application of sulfur-reducing microorganisms from acidic environments
Florentino, Anna Patrícya - \ 2017
Wageningen University. Promotor(en): Fons Stams, co-promotor(en): Irene Sanchez Andrea; Jan Weijma. - Wageningen : Wageningen University - ISBN 9789463430975 - 264
bacteria - desulfurella - metabolism - sulfur - reduction - genome analysis - proteomes - bacteriën - metabolisme - zwavel - reductie - genoomanalyse - proteomen

Sulfur cycle is one of the main geochemical cycles on Earth. Oxidation and reduction reactions of sulfur are mostly biotic and performed by microorganisms. In anaerobic conditions – marine and some freshwater systems, dissimilatory sulfur- and sulfate-reducing bacteria and archaea are key players in the decomposition of organic carbon releasing sulfide as the product of their metabolism. Sulfide can then be used as terminal reductant by anoxygenic photosynthetic microorganisms or it can be used as electron donor for aerobic or nitrate-reducing bacteria, etc.

One particular case of the sulfur cycle is the naturally occurring oxidation of metallic sulfide-ores, which produce sulfur-rich waters with low pH and high heavy metals content. Extremophilic sulfur-reducing microorganisms are of scientific and technological interest. They are abundant in natural conditions in extreme environments, so they are environmentally relevant. Although hydrogen sulfide is corrosive and odorous, its production can be beneficial for industrial activities such as the precipitation and recovery of heavy metals. Therefore, sulfur reducers have also potential for extending the range of operating conditions of metal precipitation. This thesis describes the isolation and characterization of acidotolerant sulfur-reducing bacteria, providing a first understanding on their metabolism of sulfur compounds and insights on the beneficial microbial interactions for biotechnological purposes.

In Chapter 2, the ecology and physiology of sulfur-reducing prokaryotes is investigated. The ability of sulfur reduction is wide-spread phylogenetically over the microbial tree of life, found in more than 70 genera. Elemental sulfur reduction can occur via direct cell attachment to the solid substrate or with polysulfide as an intermediate. At least four different enzymes are described to be involved in sulfur reduction pathways, and these enzymes were also detected in several microorganisms that are potential sulfur reducers, but were not reported as such in literature so far. The ecological distribution of sulfur respiration seems to be more widespread at high temperatures with neutral pH values. However, some sulfur reducers can grow at pH as low as 1 and the strategies adopted by microorganisms to face high proton concentrations in the environment were commented in this chapter. The sulfide produced from sulfur reduction can be used to selectively precipitate metals by varying the pH values from 2 to 7, depending on the target metal. Economic calculations were presented to show that sulfur reduction is more advantageous then sulfate reduction due to the cost savings of the electron donor needed. Therefore, acidophilic sulfur reducers are of particular interest for application in selective precipitation and recovery of heavy metals from metalliferous waste streams and the suitable technologies for that purpose are also discussed.

Enrichments for sulfur reducers with various electron donors at low pH and mesophilic conditions were performed from sediments of the acidic Tinto river (Spain). A solid-media with colloidal sulfur was developed to facilitate the isolation of true elemental sulfur reducers at low pH. This strategy resulted in the isolation of a sulfur-reducing bacterium, strain TR1, belonging to the Desulfurella genus. The enrichment and isolation procedure were described in Chapter 3. The growth and activity of the isolate was tested at different pH values, temperature conditions, utilization of electron donors, and growth in the presence of heavy metals in solution. The isolate showed tolerance to metals, and growth in a broad temperature and pH, revealing its feasibility to precipitate and recover heavy metals from acidic wastewater and mining water, without the need to neutralize the water before treatment. In Chapter 4, the morphological, biochemical and physiological characterization of the isolate is provided, for which the name Desulfurella amilsii TR1 sp. nov. was proposed. D. amilsii is affiliated to the Deltaproteobacteria class showing 97% of 16S rRNA gene identity to the four species described in the Desulfurella genus. In the presence of elemental sulfur, D. amilsii utilized acetate, formate, lactate, pyruvate, stearate, arginine and H2/CO2 as substrates, completely oxidizing them to H2S and CO2. Besides elemental sulfur, thiosulfate was used as an electron acceptor and the isolate also grew in the absence of external electron donor, by disproportionation of elemental sulfur into sulfide and sulfate.

The draft genome sequence of Desulfurella amilsii TR1 and a comparative genomic analysis with the members of Desulfurellaceae family are reported in Chapter 5. Based on average nucleotide identity and in silico DNA hybridization values, D. multipotens and D. acetivorans were revealed to belong to the same species. Reclassification was therefore suggested. Regarding sulfur metabolism, the analysed genomes encode different sulfur-reducing enzymes per genus. Hippea species encode polysulfide reductase and a sulfide dehydrogenase. The analysed genomes of Desulfurella especies do not possess the polysulfide reductase but possess the sulfide dehydrogenase. D. amilsii is the only member of the family encoding sulfur reductase. Since D. amilsii is able to grow at the lowest pH value, this enzyme was suggested to play a role in sulfur reduction when the microorganism grows in acidic conditions. Genes encoding resistance to acidic conditions were reported for all Desulfurellaceae members, countering physiological tests that showed ability to grow at low pH only for D. amilsii and D. acetivorans. Sulfur respiration by D. amilsii was studied in more detail in Chapter 6, in which the requirement for cell-sulfur interaction at acidic (pH 3.5) and circumneutral (pH 6.5) conditions was evaluated. D. amilsii was shown to benefit from contact with the insoluble substrate, as activity and number of cells decreased when sulfur was sequestered from the medium in dialysis bags of 6-8 kDa pore size. Besides, the abundance of enzymes possibly involved in sulfur respiration, acid resistance and chemolithotrophic growth were investigated by proteomics. Sulfur reductases were not detected in the dataset, but the limitations of the method might leave membrane-bound proteins underrepresented in the study. Different rhodanese-like proteins were detected in high abundance at low and neutral pH, while sulfide dehydrogenase seems to function as a ferredoxin:NADP oxidoreductase. We suggest that the sulfurtransferases might play a key role in sulfur/polysulfide reduction in D. amilsii. Proteomic data also showed that genes involved in acid resistance are constitutively expressed in this microorganism. Some proteins were exclusively detected at low pH, but with very few overlapping with proteins reported to be involved in acid resistance. Moreover, analysis of the proteome revealed the involvement of the hydrogenase HydABC for oxidation of hydrogen during chemolitotrophic growth, as well as the complete pathway for CO2 fixation via the reductive TCA cycle.

More aspects of the sulfur metabolism by D. amilsii were investigated in Chapter 7. Cultures grown on acetate with sulfur or thiosulfate as electron acceptor and cultures grown by disproportionation of elemental sulfur, all at pH 6.5, had their proteomes compared. Rhodanese-like sulfurtransferases were abundant in all the analyzed conditions, with specific differences in the sequences. In sulfur respiration and disproportionation, sulfurtransferases were the only sulfur enzymes detected and so, they are likely to play a central role in the process. The respiration of thiosulfate is likely to happen via a thiosulfate reductase and a dissimilatory sulfite reductase, highly abundant in this specific condition. Analysis on the heterotrophic cultures revealed the ability of D. amilsii to activate acetate to acetyl-CoA via the acetyl-CoA synthetase enzyme and its oxidation via the TCA cycle being this the first report of acetate activation happening via acetyl-CoA synthetase in sulfur-reducing bacteria.

The isolation and characterization of another acidotolerant sulfur respirer, Lucifera butyrica strain ALE, and its growth in co-culture with D. amilsii were described in Chapter 8. L. butyrica was shown to use a wide range of substrate, such as glucose, lactose, ethanol, glycerol glycogen, peptone, etc. When growing on glycerol, a cheap substrate, by fermentation or by respiration of elemental sulfur, L. butyrica produced acetate, ethanol and 1,3-propanediol as major products. Elemental sulfur reduction by this bacterium, however, was not efficient and led to the production of maximum 2.5 mM of sulfide. When L. butyrica grew in a co-culture with D. amilsii, the acetate produced by the first was consumed by the latter and the production of sulfide was boosted in the culture. As D. amilsii is not able to degrade glycerol, the co-culture represents a strategy to broaden the applicability of sulfur reduction at low pH with different sources of electron donors.

Development and application of a 20K SNP array in potato
Vos, Peter - \ 2016
Wageningen University. Promotor(en): Richard Visser; Fred van Eeuwijk, co-promotor(en): Herman van Eck. - Wageningen : Wageningen University - ISBN 9789462579569 - 166
solanum tuberosum - potatoes - genotypes - single nucleotide polymorphism - data analysis - plant breeding - linkage disequilibrium - genome analysis - tetraploidy - aardappelen - genotypen - gegevensanalyse - plantenveredeling - verstoord koppelingsevenwicht - genoomanalyse - tetraploïdie

In this thesis the results are described of investigations of various application of genome wide SNP (single nucleotide polymorphism) markers. The set of SNP markers was identified by GBS (genotyping by sequencing) strategy. The resulting dataset of 129,156 SNPs across 83 tetraploid varieties was used directly to map traits, but also as a basis for the development of a 20K SNP array in Potato (Solanum tuberosum L.). Subsequently this array, named SolSTW, was used to collect genotypic data from 569 potato genotypes. This dataset offered insight in the breeding history of potato, population structure, linkage disequilibrium (LD) and the potential of GWAS (genome wide association studies) in potato.

In Chapter 2 we describe to development of the SolSTW 20K Infinium SNP array. One third of the SNPs on this array originate from the well-known SolCAP 8303 SNP array. The other SNPs are a subset from a targeted re-sequencing project of 83 tetraploid potato varieties. Because of the high SNP density in potato only a limited number of SNPs is suitable for assay development on a SNP array. An obvious outcome is that flanking SNPs contribute to assay failure, particularly for assays with SNPs located in introns. We used fitTetra software to cluster the distribution of captured signals of each marker into the expected five genotypic classes (nulliplex, simplex, duplex, triplex, quadruplex), resulting in a dataset with 14,530 SNP markers. Subsequently the genotypic data obtained with the SolSTW array was used to characterize a set of 569 potato varieties, advanced breeding clones and progenitors. This resulted in the identification of several footprints of potato breeding. Firstly SNPs were dated i.e. the year of market release of the first variety showing polymorphism for a SNP locus is an indication of the ancestry of a SNP. In such a way we identified SNPs with an ancestry tracing back to heirloom varieties, and SNPs (post-1945 SNPs) tracing back to wild species used in modern introgression breeding. Secondly, the changes in allele frequency were calculated over time. Most SNPs show a relative stable allele frequency over time, and very limited genetic variation is removed from the gene-pool of potato i.e genetic erosion is almost absent. Therefore we conclude that 100 years of breeding has not been able to get rid of non-beneficial genetic variation. Only a limited number of SNPs show a rapid increased in allele frequency, which can be explained by positive selection for disease resistance by breeders, or the more frequent use of several founders.

Better understanding of the genome wide decay of Linkage Disequilibrium (LD) and population structure offers relevant knowledge to perform and interpret the results of a genome wide association study (GWAS) (Chapter 3). Linkage disequilibrium (LD) is a complex phenomenon, and the influence of the factors shaping LD in tetraploids is hardly studied. Therefore we used simulated data to disentangle and therewith understand often-confounded factors underlying LD-decay. We simulated datasets differing in number of haplotypes in a population, and differing in percentage of haplotype specific SNPs. In these simulations we observed that the choice of an estimator of LD-decay has a major effect on the outcome of an LD-decay estimate, while the true LD-decay remains the same. Based on the simulation we conclude that a 90% percentile and a so-called D1/2 (the distance where 50% of the initial LD is decayed) performed best to estimate and compare LD-decay in potato. To understand the various aspects of LD-decay in the variety panel of 537 varieties, the panel was subdivided in several groups based on the age of a variety and the population structure groups. This resulted in the identification of LD-decay over time, i.e in relatively young varieties the average size of the LD-blocks is smaller. The differences between subpopulations were smaller and are most likely the effect of the population structure. We also observed that there are very long LD-blocks caused by introgression breeding and that different a priori MAF-thresholds also can influence the outcome of LD-decay estimation.

Having both LD-decay and population structure defined a genome wide association study (GWAS) was conducted (Chapter 4). For this purpose α-solanine and α-chaconine were measured in potato tubers. Subsequently the sum of both (total SGA) and the ratio between the two were used to discover QTLs for these traits in a GWAS. Additionally we used three bi-parental populations to validate the GWAS results. Total SGA content was confounded with population structure and therefore it was difficult to explain all phenotypic variation with SNP markers. Two QTLs (Sgt1.1 and Sgt11.1) were identified which could be validated in one of the segregating populations. The ratio between α-solanine and α-chaconine was not confounded with population structure, resulted in the identification of two major-effect QTLs (Sgr7.1 & Sgr8.1) located near the candidate genes SGT1 and SGT2, which are known for being responsible in the final steps towards either α-solanine or α-chaconine. The QTL Sgr8.1 could be validated, however similar phenotypes were explained by different haplotypes in two populations. We show that population structure, low frequent alleles and genetic heterogeneity may explain to some degree the missing heritability in GWAS in potato.

In Chapter 5 we describe how the method of graphical genotyping, which is widely used in diploid bi-parental populations, can be applied in a variety panel of tetraploid varieties. We show that a few discrete filtering steps in Excel can be used to display patterns that are visual representations of introgression segments and the locations of historical recombination events. Using this method we identified introgression segments from Solanum vernei including the Gpa5 locus on chromosome 5 and Solanum stoloniferum introgression segment including a gene involved in resistance to Potato Virus Y on chromosome 11. This method requires that the haplotypes that cause the phenotypic effect have to be identical by descent (IBD).

In the final chapter 6 the results of chapter 2 to 5 are discussed. We look forward on how our results can be used in future research and applied in marker-assisted breeding. Additionally some new GWAS results are presented for tuber flesh colour, foliage maturity and resistance to Globodera pallida pathotype 3.

The allo-octoploid strawberry: simply complex
Dijk, Thijs van - \ 2016
Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Eric van de Weg. - Wageningen : Wageningen University - ISBN 9789462579637 - 185
fragaria ananassa - strawberries - polyploidy - microsatellites - linkage mapping - genome analysis - quantitative trait loci - genetic mapping - flowering - plant breeding - aardbeien - polyploïdie - microsatellieten - koppelingskartering - genoomanalyse - loci voor kwantitatief kenmerk - genetische kartering - bloei - plantenveredeling

The garden strawberry (Fragaria x ananassa) is a fruit species that was developed through human intervention less than 300 years ago. Currently, it is the most important soft fruit in both production as well as value and renowned for its deliciousness. There are many challenges in growing such a delicate fruit, many of which have been overcome through improved cultivation techniques and breeding. The perishability of the product is, however, not the only challenge faced by strawberry breeders. In terms of genome composition, strawberry appears to have accumulated a wonderful array of obstacles to genetic studies. It is a vegetatively propagated allo-octoploid outbreeder, and only few crop species are worse of in this respect. Many of the molecular genetic ground work is therefore performed in its diploid ancestor, the woodland strawberry Fragaria vesca, which was sequenced in 2011. However, since nearly all strawberries that are eaten are octoploid, genetic research can’t linger at the wild diploids forever. In this thesis we developed new tools and analysis methods for genetic studies in the allo-octoploid strawberry and subsequently applied these methods in the detection of marker-trait associations.

The purpose of Chapter 2 was to develop a method to interpret the complex peak patterns generated by microsatellites in octoploid strawberry in such a way that we ended up with as much information as one would expect to retrieve from a microsatellite in a diploid system. This information could then be used to generate high quality linkage maps for the different sub-genomes and allow for easy alignment and comparison. We named the method MADCE, which stands for Microsatellite Allele Dose & Configuration Establishment. In the MADCE methodology, we first need to determine the dose of each allele present in an individual. For this we used the signal of fluorescent microsatellite peaks in relation to the total fluorescent signal generated by all peaks for that microsatellite. We then used the disomic inheritance of strawberry to establish the allelic configuration of each different homoeologue (subgenome). The repulsion of alleles from the same subgenome in offspring allowed us to form subgenomic pairs of alleles. We found that in single cross mapping populations, the deployment of our method was fairly easy due to the high number of offspring that can be used to establish repulsion between alleles. However, for pedigreed breeding germplasm this was another matter, as generally only few offspring were available. For this we added some additional tricks to the MADCE method, although some uncertainty about the configuration would remain for problematic lines and alleles.

In Chapter 3 we used the MADCE method from Chapter 2 to generate a genome wide linkage map for the Holiday x Korona (HxK) mapping population. This linkage map was to be used in subsequent experiments for QTL discovery as well as provide the strawberry community with a highly detailed map consisting not only of marker distances, but allele and haplotype configuration of the parents Holiday and Korona as well. The haplotype information revealed that inbreeding (homozygosity) levels in Holiday were similar to the levels expected from its pedigree, but that inbreeding levels of Korona were more than three times higher than expected, which could be resultant from selection pressure enacted by breeders. Selection pressure could also be causal to our discovery that the kinship between the two cultivars was twice as high as expected from their shared ancestry. Another discovery was a large inversion on one of the subgenomes of linkage group 2 (D). Up until the publication of our linkage map this inversion had not been reported in other linkage maps. Another innovation was our attempt at giving a biological or evolutionary meaning to the denomination of the linkage groups by arranging them according to similarity to the diploid ancestor F. vesca, based on F. vesca derived primer amplification efficiencies. The HxK map has been used in several (ongoing) research projects outside of our research group and has contributed to the development of the 90k Axiom SNP array for cultivated strawberry.

In Chapter 4 we performed a QTL mapping study for disease resistance against the problematic pathogen Phytophthora cactorum, which causes crown rot in strawberry plants. In this study we used two different mapping populations: the Holiday x Korona (HxK) population from the previous chapter as well as E1998-142 x Elsanta (ExEls), developed more specifically for the purpose of finding resistance against P. cactorum. The HxK and ExEls populations were phenotyped over three years (2008, 2010 and 2011) under different seasons and conditions. The correlation between years for was quite low for both populations (ranging from 0.18 to 0.47), indicating a large environmental effect on disease pressure. Results from the QTL analysis showed that most QTLs were small in effect and only just above the statistical significance threshold. Only for ExEls we uncovered two QTLs with relatively high significance levels, but none were significant in all three years. Because of the high environmental influence, and the desire to have QTLs that are robust over environments, we used the average of all three years (AOTY) as an additional phenotype. When we used the AOTY trait, the QTL on LG7D became stronger than for any of the individual years. Whereas for the LG7C QTL the significance dropped to just below threshold levels. These results indicated that removing environmental noise through averaging over experiments is a good way to uncover the most reliable and therefore more valuable to a breeding program.

In Chapter 5 we investigated the genetics behind two different flowering habits that are grown commercially worldwide: seasonal flowering habit (SF) and perpetual flowering (PF) These varieties initiate flowering under long days, and can therefore produce fruit for a much longer period: throughout the summer and early fall. Evidence from literature and practical breeding suggested that PF is under dominant control. We decided to treat PF as a qualitative trait and divided two small mapping populations into PF and SF individuals. After screening several microsatellites, we found one locus that completely cosegregated with the PF trait at the bottom of LG4D. At the moment of mapping, a paper was published which mapped the same trait to the same location. We found that there were two very clear candidate genes within our QTL interval, FaCDF2 and FaFT2, which were homologous to genes that are major factors in the flowering pathway of Arabidopsis and many other plant species. We then sequenced the FaCDF2 gene from a number of distinct PF and SF cultivars. This resulted in the discovery of two quite distinct allelic variants, one of which was present in all PF cultivars. However this variant was also present in some of the SF cultivars, indicating that either FaCDF2 is not the causal gene, or that other loci can have a qualitative effect on the switch from SF to PF. We then performed microsatellite haplotyping on hundreds of cultivars and this revealed that all PF varieties of all origins carry the same haplotype in the PF QTL region, and that there weren’t any recombinations between the candidate genes FaCDF2 and FaFT2, which are 250kb apart on the physical genome. This makes it still undecided which of these two candidate genes are causal to the PF trait. Another interesting result from the haplotyping was that the complete PF haplotype was present with moderate frequency in SF varieties as well. Not only does this suggest a common origin, it also complicates the establishment of a theory for the mechanisms behind perpetual flowering in cultivated strawberry. So far we have not been able to establish whether the PF haplotype that is present in SF cultivars is functionally distinct from the PF haplotype in PF cultivars. All we know is that it does not confer perpetual flowering in these SF cultivars, and further experiments would be needed to find out the exact mechanism behind perpetual flowering.

In the general discussion (Chapter 6), the results of this thesis were placed in the broader context of science in general and plant breeding in particular.

Host-plant resistance to western flower thrips in Arabidopsis
Thoen, Manus P.M. - \ 2016
Wageningen University. Promotor(en): Marcel Dicke; Harro Bouwmeester, co-promotor(en): Maarten Jongsma. - Wageningen : Wageningen University - ISBN 9789462578807 - 191
arabidopsis thaliana - host plants - insect pests - frankliniella occidentalis - defence mechanisms - pest resistance - genomics - genome analysis - host-seeking behaviour - optical tracking - data analysis - insect plant relations - waardplanten - insectenplagen - verdedigingsmechanismen - plaagresistentie - genomica - genoomanalyse - gedrag bij zoeken van een gastheer - optisch sporen - gegevensanalyse - insect-plant relaties

Western flower thrips is a pest on a large variety of vegetable, fruit and ornamental crops. The damage these minute slender insects cause in agriculture through feeding and the transmission of tospoviruses requires a sustainable solution. Host-plant resistance is a cornerstone of Integrated Pest Management (IPM). Plants have many natural defense compounds and morphological features that aid in the protection against herbivorous insects. However, the molecular and physiological aspects that control host-plant resistance to thrips are largely unknown.

A novel and powerful tool to study host-plant resistance to insects in natural populations is genome-wide association (GWA) mapping. GWA mapping provides a comprehensive untargeted approach to explore the whole array of plant defense mechanisms. The development of high-throughput phenotyping (HTP) systems is a necessity when large plant panels need to be screened for host-plant resistance to insects. An automated video-tracking platform that could screen large plant panels for host-plant resistance to thrips, and dissect host-plant resistance to thrips in component traits related to thrips behavior, was developed. This phenotyping platform allows the screening for host-plant resistance against thrips in a parallel two-choice setup using EthoVision tracking software. The platform was used to establish host-plant preference of thrips with a large plant population of 345 wild Arabidopsis accessions (the Arabidopsis HapMap population) and the method was optimized with two extreme accessions from this population that differed in resistance to thrips. This method can be a reliable and effective high throughput phenotyping tool to assess host-plant resistance to thrips in large plant populations. EthoAnalysis, a novel software package was developed to improve the analyses of insect behavior. There were several benefits from using EthoAnalysis to analyze EthoVision data. The detailed event statistics that could be extracted from EthoAnalysis allows researchers to distinguish detailed differences in moving and feeding behavior of thrips. The potential of this additional information is discussed in the light of quantitative genetic studies.

Stress resistance was studied in the HapMap population on a total of 15 different biotic and abiotic stresses ranging from biotic stresses like insects and nematodes, to abiotic stresses like drought and salt. A multi-trait GWA study to unravel the genetic architecture underlying plant responses to the different stresses was performed. A genetic network in this study revealed little correlation between the plant responses to the different insect herbivores studied (aphids, whiteflies, thrips and caterpillars). For thrips resistance a weak positive correlation with resistance to drought stress and Botrytis, and a negative correlation with resistance to parasitic plants were observed. One of the surprising outcomes of this study was the absence of shared major QTLs for host-plant resistance and abiotic stress tolerance mechanisms. RESISTANCE METHYLATED GENE 1 (RMG1) was one of the candidate genes in this multi-trait GWA study that could be controlling shared resistance mechanisms against many different stresses in Arabidopsis. RMG1 is a nucleotide-binding site Leucine-rich repeat (NB-LRR) disease resistance protein and its potential relation to several resistance/tolerance traits was successfully demonstrated with T-DNA insertion lines.

The 15 stresses were used in a comparison with a metabolomics dataset on this Arabidopsis HapMap population. It was discovered that levels of certain aliphatic glucosinolates correlated positively with the levels of resistance to thrips. This correlation was further investigated with the screening of a RIL (Recombinant Inbred Line) population for resistance to thrips, several knockout mutants and the analysis of co-localization of GWA mapping results between glucosinolates genes and thrips resistance. In a GWA analysis, the C4 alkenyl glucosinolates that correlated the strongest with thrips resistance mapped to the genomic regions containing genes known to regulate the biosynthesis of these compounds. However, thrips resistance did not co-localize with any of the GSL genes, unless a correction for population stratification was omitted. Additional screening of a Cvi x Ler RIL population showed a QTL for thrips resistance on chromosome 2, but no co-localization with any known glucosinolate genes, nor with thrips resistance loci identified by GWA mapping. Knock-out mutants and overexpressors of glucosinolate synthesis genes could also not confirm a causal link between glucosinolates and resistance to thrips. It is possible that the crucial factors that control resistance to thrips may not have been present in sufficient quantities or in the right combinations in the mutants, RILs and NIL screened in this study. Alternatively, the correlation between thrips feeding damage and glucosinolate profiles could be based on independent geographical clines. More research should be conducted to assess which of these explanations is correct.

In the general discussion, the results from this thesis are discussed in a broader perspective. Some prototypes of new phenotyping platforms that could further aid screening for resistance to thrips in the future are presented. Natural variation in host-plant resistance to thrips is compared to the variation in host-plant resistance to aphids and caterpillars. The geographic distribution of host-plant resistance to thrips is not evident in the other insects, in line with the distribution of glucosinolate profiles and other climate factors. The chapter concludes with some suggestions for future research in the field of host-plant resistance to thrips.

Dietary epicatechnin and quercetin in cardiovascular health and disease
Dower, J.I. - \ 2016
Wageningen University. Promotor(en): Daan Kromhout; Marianne Geleijnse, co-promotor(en): Peter Hollman. - Wageningen : Wageningen University - ISBN 9789462577862 - 164 p.
cardiovascular disorders - cardiovascular diseases - epicatechin - quercetin - epidemiological surveys - genome analysis - chocolate - hart- en vaatstoornissen - hart- en vaatziekten - epicatechine - quercetine - epidemiologische onderzoeken - genoomanalyse - chocolade

Epidemiological studies showed that the consumption of flavonoid-rich foods such as cocoa and tea is associated with a lower risk of cardiovascular disease (CVD). Randomised controlled trials (RCTs) showed that cocoa and tea improved markers of cardiometabolic health including blood pressure, endothelial function, insulin resistance, arterial stiffness and inflammation.

Cocoa is particularly rich in the flavan-3-ol epicatechin and tea is the main dietary source of epicatechin and of the major flavonol quercetin. However, evidence on the individual roles of epicatechin and quercetin in the health effects of cocoa and tea is still scarce. Therefore, we estimated the strength of the association between epicatechin intake and CVD mortality in a prospective cohort study. Furthermore, we also investigated the effects of epicatechin and quercetin on markers of cardiometabolic health and gene expression, by means of two RCTs.

In Chapter 2, the association between epicatechin intake and CVD mortality was studied using data from the Zutphen Elderly Study, a cohort of 744 elderly Dutch men. During 25 years of follow-up, 329 men died from CVD and 148 from coronary heart disease (CHD). Results from this study showed that men in the highest tertile of epicatechin intake had a 38% lower risk of CHD mortality compared to men in the lowest tertile. For men with prevalent CVD, the risk of CVD mortality was 46% lower for men in the highest tertile of intake, compared to men in the lowest tertile. This is the first epidemiological study to have investigated the association between epicatechin intake and CVD mortality. Hence, more and larger cohort studies are required to confirm this association, possibly with a focus on populations with a high risk of CVD.

In Chapter 3, the chronic effects of pure epicatechin and quercetin on markers of cardiometabolic health were investigated by means of a RCT. Thirty-seven apparently healthy men and women aged 40–80 years consumed (-)-epicatechin (100 mg/d), quercetin-3-glucoside (160 mg/d), or placebo capsules for 4 weeks, in random order. Markers of cardiometabolic health were measured before and after each 4-week intervention. The results of this study showed that epicatechin improved insulin resistance and had a borderline significant effect on endothelial function. This suggests that epicatechin contributes to the cardioprotective effects of cocoa and tea, however, larger long-term RCTs are required to confirm these effects. Pure quercetin supplementation did not affect any of these markers of cardiometabolic health.

Using data from the same study, we investigated the effects of supplementation of pure epicatechin and quercetin on a comprehensive set of biomarkers of endothelial dysfunction and inflammation (Chapter 4). With the exception of sE-selectin (a biomarker of endothelial dysfunction), epicatechin supplementation did not beneficially influence any of the biomarkers, suggesting a lack of evidence for a role of epicatechin in inflammation. Quercetin also lowered sE-selectin as well as the inflammatory biomarker IL-1β and the overall z-score for inflammation. This suggests that quercetin may contribute to the cardioprotective effects of tea by reducing inflammation and possibly by improving endothelial function.

In the same study, the effects of pure epicatechin supplementation on whole genome gene expression profiles of circulating immune cells were also assessed (Chapter 5). Pure epicatechin supplementation modestly reduced gene expression related to inflammation signalling routes in circulating immune cells – routes which are known to play a role in cardiovascular health. However, there was no evidence that epicatechin affected pathways related to insulin resistance or endothelial function.

To directly compare the acute effects of pure epicatechin and epicatechin from dark chocolate on vascular function, we carried out an acute RCT in 20 apparently healthy men aged 40-80 years (Chapter 6). On three separate occasions, subjects consumed: 1) 70g dark chocolate (150 mg epicatechin) with two placebo capsules; 2) two pure epicatechin capsules (100 mg epicatechin) with 75g white chocolate and 3) two placebo capsules with 75g white chocolate (0 mg epicatechin). Endothelial function and arterial stiffness were measured before and two hours after each intervention. To determine epicatechin bioavailability, epicatechin metabolites were measured in blood samples taken at repeated intervals over a period of 8 hours. There was no significant difference in improvement in endothelial function or arterial stiffness between pure epicatechin and dark chocolate. There was also no difference in bioavailability of pure epicatechin and epicatechin from dark chocolate, when standardised per 100 mg of epicatechin. This suggests that epicatechin may contribute to the vascular effects of cocoa and that the bioavailability of pure epicatechin and epicatechin from dark chocolate is similar.

In the general discussion, the main findings of this thesis were first summarised. Methodological considerations related to cohort studies, such as the assessment of flavonoid intake and the possibility of residual confounding were also discussed. Issues related to the relevance of cardiometabolic markers in RCTs and the effect of cocoa flavan-3-ol bioavailability were addressed. Finally, suggestions for future research were put forward.

In conclusion, the results of this thesis suggest that epicatechin contributes to the cardioprotective effects of cocoa and tea. Epicatechin intake was inversely related to CHD mortality in elderly men, and to CVD mortality in men with prevalent CVD. The cardioprotective effects of epicatechin are likely mediated through improvements in insulin resistance and possibly endothelial function. In contrast, quercetin is unlikely to play a major role in the cardioprotective effects of tea. Results for quercetin from cohort studies are inconclusive, and based on the results of our chronic RCT, quercetin did not affect vascular function or insulin resistance, but may help to lower inflammation. Evidence of the role that individual flavonoids play in the aetiology of CVD is still limited. More studies with pure flavonoids are required to elucidate their role.

Quantitative and ecological aspects of Listeria monocytogenes population heterogeneity
Metselaar, K.I. - \ 2016
Wageningen University. Promotor(en): Marcel Zwietering; Tjakko Abee, co-promotor(en): Heidy den Besten. - Wageningen : Wageningen University - ISBN 9789462577664 - 173 p.
listeria - listeria monocytogenes - stress - stress tolerance - ribosomes - proteins - lactobacillus plantarum - behaviour - ecological assessment - genome analysis - dna sequencing - resistance - heterogeneity - stresstolerantie - ribosomen - eiwitten - gedrag - ecologische beoordeling - genoomanalyse - dna-sequencing - weerstand - heterogeniteit

Bacterial stress response and heterogeneity therein is one of the biggest challenges posed by minimal processing. Heterogeneity and resulting tailing representing a more resistant fraction of the population, can have several causes and can be transient or stably in nature. Stable increased stress resistance is caused by alterations in the genome and therefore inheritable and is referred to as stable stress resistant variants. Also L. monocytogenes exhibits a heterogeneous response upon stress exposure which can be partially attributed to the presence of stable stress resistant variants. Adverse environments were shown to select for stable stress resistant variants. The objective of the research described in this thesis was to evaluate if L. monocytogenes population diversity and the presence of stable resistant variants is a general phenomenon that is observed upon different types of stress exposure, to get more insight in the mechanisms leading to increased resistance and to evaluate the ecological behaviour and potential impact on food safety of these stable resistant variants. Acid stress was chosen as it is an important hurdle both in food preservation, as well as in stomach survival.

First, the non-linear inactivation kinetics of L. monocytogenes upon acid exposure were quantitatively described. A commonly used biphasic inactivation model was reparameterized, which improved the statistical performance of the model and resulted in more accurate estimation of the resistant fraction within L. monocytogenes WT populations. The observed tailing suggested that stable stress resistant variants might also be found upon acid exposure. Indeed, 23 stable acid resistant variants of L. monocytogenes LO28 were isolated from the tail after exposure of late-exponential phase cells to pH 3.5 for 90 min, with different degrees of acid resistance amongst them. Increased acid resistance showed to be significantly correlated to reduced growth rate. Studying the growth boundaries of the WT and a representative set of variants indicated that the increased resistance of the variants was only related to survival of severe pH stress but did not allow for better growth or survival at mild pH stress.
A set of variants were further characterized phenotypically and cluster analysis was performed. This resulted in three clusters and four individual variants and revealed multiple-stress resistance, with both unique and overlapping features related to stress resistance, growth, motility, biofilm formation and virulence indicators. A higher glutamate decarboxylase (GAD) activity correlated with increased acid resistance. Whole genome sequencing of a set of variants was performed and revealed mutations in rpsU, encoding ribosomal protein S21. This rpsU mutation was found in all 11 variants comprising the largest phenotypic cluster, indicating a potential role of this ribosomal protein in stress resistance. Mutations in ctsR, which were previously shown to be responsible for increased resistance of heat and HHP resistant variants, were not found in the acid resistant variants. This underlined that large population diversity exists within one L. monocytogenes strain and that different adverse conditions drive selection for different variants.

Next, the performance in mixed species biofilms with Lactobacillus plantarum was evaluated, as well as their benzalkonium chloride (BAC) resistance in these biofilms. It was hypothesized that the acid resistant variants might also show better survival in biofilms with L. plantarum, which provide an acidic environment by lactose fermentation with pH values below the growth boundary of L. monocytogenes when biofilms mature. L. monocytogenes LO28 WT and eight acid resistant variants were capable of forming mixed biofilms with L. plantarum at 20°C and 30°C in BHI supplemented with manganese and glucose. Some of the variants were able to withstand the low pH in the mixed biofilms for a longer time than the WT and there were clear differences in survival between the variants which could not be correlated to (lactic) acid resistance alone. Adaptation to mild pH of liquid cultures during growth to stationary phase increased the acid resistance of some variants to a greater extent than of others, which could be correlated to increased survival in the mixed biofilms. There were no clear differences in BAC resistance between the wild type and variants in mixed biofilms.

Lastly, a set of robustness and fitness parameters of WT and variants was obtained and used to model their growth behaviour under combined mild stress conditions and to model their performance in a simulated food chain. This gave more insight in the trade-off between increased stress resistance and growth capacity. Predictions of performance were validated in single and mixed cultures by plate counts and by qPCR in which WT and an rpsU deletion variant were distinguished by specific primers. Growth predictions for WT and rpsU deletion variant were matching the experimental data generally well. Globally, the variants are more robust than the WT but the WT grows faster than most variants. Validation of performance in a simulated food chain consisting of subsequent growth and inactivation steps, confirmed the trend of higher growth fitness and lower stress robustness for the WT compared to the rpsU variant. This quantitative data set provides insights into the conditions which can select for stress resistant variants in industrial settings and their potential persistence in food processing environments.

In conclusion, the work presented in this thesis highlights the population diversity of L. monocytogenes and the impact of environmental conditions on the population composition, which is of great importance for minimal processing. The work of this thesis resulted in more insight in the mechanisms underlying increased resistance of stress resistant variants and quantitative data on the behaviour of stress resistant variants which can be implemented in predictive microbiology and quantitative risk assessments aiming at finding the balance between food safety and food quality.

Isolation, characterization and engineering of Bacillus smithii : a novel thermophilic platform organism for green chemical production
Bosma, E.F. - \ 2015
Wageningen University. Promotor(en): Willem de Vos; John van der Oost, co-promotor(en): Richard van Kranenburg. - Wageningen : Wageningen University - ISBN 9789462575073 - 220
bacillus smithii - bacillus (bacteria) - biobrandstoffen - chemicaliën uit biologische grondstoffen - thermofielen - metabolische profilering - genoomanalyse - mutaties - isolatie - bioengineering - karakterisering - biofuels - biobased chemicals - thermophiles - metabolic profiling - genome analysis - mutations - isolation - characterization

Due to the globally increasing demand for chemicals and fuels and the high environmental impact and limited amount of fossil resources, there is a growing interest in green chemicals and fuels derived from renewable resources. As described in Chapter 1, one of the most feasible alternatives on the short term is microbial conversion of the sugars in biomass to fuels and chemicals in a biorefinery. To be economically and ethically feasible, non-food biomass should be used as a resource, which is often difficult with currently used production organisms. Also, to be economically feasible, the costs of green chemicals and fuels need to be further reduced to be below the costs of products based on fossil resources. To do so, other organisms than the currently most-used platform organisms such as Escherichia coli and Saccharomyces cerevisiae should be used. Ideally, this alternative organism is genetically accessible, has high productivity, titre and yield, is flexible in carbon source, robust, moderately thermophilic, acidophilic, facultatively anaerobic and has little nutritional requirements. The organisms that come closest to these criteria are thermophilic bacilli, which form a diverse class of organisms in the family of Bacillaceae. This thesis describes the isolation, characterization and metabolic engineering of Bacillus smithii, a novel potential thermophilic platform organism.

Chapter 2 provides more detail on the use of thermophilic microorganisms as platform organisms for green chemical production in a biorefinery concept. As commercially available enzyme mixtures used in the simultaneous saccharification and fermentation (SSF) of biomass have their optimum temperature around 50-60°C, using a moderately thermophilic organism would reduce the costs of the SSF process compared to when using mesophiles by reducing the amount of required enzyme. Also, thermophilic processes are less prone to contaminations, and substrate and product solubility are increased. Several successful examples of the application of facultatively anaerobic thermophiles for green chemical production from lignocellulose in an SSF setting are for example Bacillus coagulans for lactic acid production and Bacillus licheniformis for 2,3-butanediol production. However, whereas strongly developed genetic toolboxes are available for current mesophilic production organisms, these tools are still in their infancy for thermophilic organisms. Such tools are required to optimize production and to study metabolism. Thermophilic organisms show a wide variety in metabolism and in many cases the metabolism of these organisms is still poorly understood, hampering full optimization. Chapter 2 furthermore provides an overview of transformation, integration and counter-selection methods currently used for thermophiles. Although several deletion mutants have been constructed using these methods, not all of them are entirely markerless and most are not suited as high-throughput engineering tools, stressing the need for further research in this area.

Despite several facultatively anaerobic thermophiles being described as genetically accessible, this feature is still one the major bottlenecks in developing these organisms into platform organisms. Therefore, in Chapter 3, we set out to isolate a facultatively anaerobic, moderately thermophilic bacterium that was genetically accessible and produced high titers of organic acids. A total of 267 strains of different thermophilic bacilli species were isolated from compost and screened for C5 and C6 sugar utilization and acid production. The 44 best strains were screened for genetic accessibility via electroporation. Only 3 strains tested positive for this, namely Geobacillus thermodenitrificans strains ET 144-2 and ET 251 and B. smithii strain ET 138. In subsequent evaluations in lab-scale bioreactors at 55°C and pH 6.5 on glucose, the two G. thermodenitrificans strains performed poorly whereas B. smithii performed well with high titers, yields and productivity of mainly lactate. In similar lab-scale reactors, this strain also performed well on xylose and at pH 5.5 and was still able to perform for 48 at pH 4.5. The electroporation protocol for this strain was optimized, resulting in a maximum efficiency of 5x103 colonies per µg plasmid pNW33n. Two other B. smithii strains, among which the type strain DSM 4216T, were also shown to be transformable with pNW33n. This is the first time that genetic accessibility is described for B. smithii and it is the first step towards developing it into a platform organism, for which it appears to be suitable based on its efficient C5 and C6 sugar utilization and acid production profile.

In order to become a platform organism and to study its atypical metabolism, a genetic toolbox needs to be established for B. smithii. Chapter 5 describes the development of a markerless gene deletion method for B. smithii. For strains ET 138 and DSM 4216T, the ldhL gene was markerlessly removed via double homologous recombination using plasmid pNW33n. Despite the replicative nature of this plasmid at 55°C, mixtures of single and double crossovers were readily obtained. A pure double crossover deletion mutant was obtained after several transfers on a more defined medium containing acetate or lactate and PCR-based screenings. To eliminate the possibility of mixed genotypes, we subsequently developed a lacZ-counter-selection system, which is based on the toxicity of high X-gal concentrations in the presence of the plasmid-encoded lacZ gene. Using this method, the sporulation-specific sigma factor sigF and pyruvate dehydrogenase complex E1-α pdhA were consecutively removed from the B. smithii ET 138 genome in a markerless way. An initial evaluation of the growth and production profiles of the mutant strains in tubes showed that removal of the ldhL gene eliminates l-lactate production and causes a severe decrease in anaerobic growth and production capacities. B. smithii mutants lacking the sigF gene were unable to sporulate and removal of the pdhA gene eliminated acetate production and rendered the strains auxotrophic for acetate.

Kaf van het koren scheiden
Calus, Mario - \ 2014
dairy farming - ai bulls - dairy bulls - animal breeding - breeding value - selective breeding - genome analysis - genomes
Molecular characterization of the alloherpesvirus anguillid herpesvirus 1
Beurden, S.J. van - \ 2012
Utrecht University. Promotor(en): Olga Haenen. - Utrecht : Gildeprint drukkerijen - ISBN 9789461083258 - 205
herpesviridae - european eels - virusziekten - moleculaire genetica - genoomanalyse - genexpressie - moleculaire virologie - viral diseases - molecular genetics - genome analysis - gene expression - molecular virology
All herpesviruses belong to the order Herpesvirales, which consists of the families Herpesviridae, Alloherpesviridae and Malacoherpesviridae. Although herpesviruses share unique morphological characteristics, only the gene encoding the ATPase subunit of terminase is detectably conserved throughout the order. The family Herpesviridae, which comprises mammalian, avian and reptilian herpesviruses, has been studied extensively, but much less knowledge is available for members of the families Alloherpesviridae and Malacoherpesviridae, which respectively comprise amphibian and fish, and invertebrate herpesviruses. Anguillid herpesvirus 1 (AngHV1) frequently causes disease in wild and cultured European eel, a traditionally important fish species in the Netherlands. Hence, in this study AngHV1 was chosen as a model for the family Alloherpesviridae. The aim of the study was to characterize AngHV1 at the molecular level, and to determine its similarities and differences as compared with other herpesviruses. AngHV1 has a genome of close to 250 kbp, including an 11 kbp terminal direct repeat. The genome contains a total of 129 protein-coding genes, five of which are duplicated in the terminal repeat. Since only a dozen genes are detectably conserved among fish and amphibian herpesviruses, the family Alloherpesviridae appears to be more divergent than the family Herpesviridae, among which more than 40 genes are conserved. Taxonomically, AngHV1 is most closely related to the cyprinid herpesviruses. High-resolution transcriptome analysis based on deep sequencing revealed that RNA splicing is much more abundant than had been assumed. A total of 58 functional splice junctions were identified. Eleven genes contain integral, spliced protein-coding exons, and nine contain 5’-untranslated exons or, in instances of alternative splicing, 5’-untranslated or -translated exons. In contrast to mammalian herpesviruses, overall levels of antisense transcription in AngHV1 were low, and no abundant, non-overlapping non-coding RNAs were identified. A genome-wide expression study using qPCR showed that gene expression is regulated in a temporal fashion, similar to mammalian herpesviruses. The putative regulatory immediate-early genes of AngHV1 were identified, and appeared to be located within and near the terminal repeats. The remaining open reading frames were classified into early, early-late and late genes. Most early genes encode enzymes and proteins involved in DNA replication, and most late genes encode structural proteins. The structural proteins of AngHV1 were identified using a combination of classical virus purification and fractionation techniques and modern mass spectrometry analyses. A total of 40 different structural proteins were identified, of which 7 could be assigned to the capsid, 11 to the envelope, and 22 to the tegument. Although no convincing sequence homology is apparent between the herpesvirus families for any of the structural proteins, virion composition shows many similarities. AngHV1 encodes several viral homologs of components of the host immune system, including an interleukin-10-like open reading frame. Although amino acid sequence homology between the European eel interleukin-10 and the AngHV1 interleukin-10 homolog is low, the three-dimensional structures as predicted by modelling are highly similar, suggesting functionality. Overall, despite the virtual absence of detectable genetic similarities, AngHV1 and the other alloherpesviruses resemble members of the family Herpesviridae in many fundamental aspects.
Animal breeding for food security : opportunities in the genome sequencing era
Veerkamp, R.F. - \ 2012
Wageningen : Wageningen University, Wageningen UR - ISBN 9789461733245 - 23
dierveredeling - genomica - genoomanalyse - dna-sequencing - fokkerijmethoden - animal breeding - genomics - genome analysis - dna sequencing - animal breeding methods
Inaugural lecture upon taking up the position of Special Professor of Numerical Genetics and Genomics at Wageningen University.
Characterization of the SpaCBA Pilus Fibers in the Probiotic Lactobacillus rhamnosus GG
Reunanen, J. ; Ossowski, I. von; Hendrickx, A.P. ; Palva, A. ; Vos, W.M. de - \ 2012
Applied and Environmental Microbiology 78 (2012)7. - ISSN 0099-2240 - p. 2337 - 2344.
gram-positive bacteria - corynebacterium-diphtheriae - stabilizing isopeptide - actinomyces-naeslundii - genome analysis - reveals pili - streptococcus - surface - bonds - identification
Lactobacillus rhamnosus GG is a human intestinal isolate that has been studied intensively because of its probiotic properties. We have previously shown that L. rhamnosus GG produces proteinaceous pili that earlier had been observed only in Gram-positive pathogens (M. Kankainen et al., Proc. Natl. Acad. Sci. U. S. A. 106:17193-17198, 2009). These pili were found to be encoded by the spaCBA gene cluster, and the pilus-associated SpaC pilin was shown to confer on the cells a mucus-binding ability. In addition to the spaCBA cluster, another putative pilus cluster, spaFED, was predicted from the L. rhamnosus GG genome sequence. Herein, we show that only SpaCBA pili are produced by L. rhamnosus, and we describe a detailed analysis of cell wall-associated and affinity-purified SpaCBA pili by Western blotting and immunogold electron microscopy. Our results indicate that SpaCBA pili are heterotrimeric protrusions with a SpaA subunit as the shaft-forming major pilin. Only a few SpaB subunits could be observed in pilus fibers. Instead, SpaB pilins were found at pilus bases, as assessed by immunogold double labeling of thin sections of cells, suggesting that SpaB is involved in the termination of pilus assembly. The SpaC adhesin was present along the whole pilus length at numbers nearly equaling those of SpaA. The relative amount and uniform distribution of SpaC within pili not only makes it possible to exert both long-distance and intimate contact with host tissue but also provides mucus-binding strength, which explains the prolonged intestinal residency times observed for L. rhamnosus GG compared to that of nonpiliated lactobacilli
Genetic analysis of potato tuber quality traits
Werij, J.S. - \ 2011
Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Christian Bachem. - [S.l.] : S.n. - ISBN 9789461730923 - 125
solanum tuberosum - aardappelen - plantenveredeling - knollen - kwaliteit - kenmerken - genetische kartering - genoomanalyse - genetische analyse - potatoes - plant breeding - tubers - quality - traits - genetic mapping - genome analysis - genetic analysis

In this thesis the results of the four year project P5 within the Centre for BioSystems Genomics ( are described. The intention of this project was to gain understanding of the genetic principles underlying a series of different potato tuber quality traits and the development of molecular markers linked to the traits of interest. Molecular markers that can be used in breeding programs with the aim of producing potato varieties with improved quality. To provide a window in which the research of this thesis can be placed, a review of the current status of quality genetics in potato is given in chapter 1. Here the different quality traits applicable for potato are described and different breeding methodologies discussed. Enzymatic discoloration (ED) of potato tubers was investigated in chapter 2. QTL analysis of the trait itself an it’s potential substrates was performed in combination with an allele specificity study (including expression analysis) on the candidate genes PPO. In chapter 3, potato starch related traits and the underlying starch biosynthesis and degradation are investigated by a QTL analysis in combination with a candidate gene approach. Furthermore, in chapter 4 we analyzed potato tuber metabolic content through liquid chromatography-time of flight mass spectrometry (LC-QTOF MS). This resulted in a large set of mass peaks. Clustering of the signals through a multivariate mass spectra reconstruction strategy revealed reconstructed metabolites (ea centrotypes). Quantitative Trait Locus (QTL) analysis of the centrotypes revealed over 800 metabolite QTLs (mQTLs), distributed over all 12 chromosomes. To investigate the contribution of the metabolome to phenotypic traits, the mQTL results were compared with the QTL results for tuber flesh colour (raw and after-cooking) and total tuber protein content. Finally in chapter 5, the general discussion, the results obtained in the preceding chapters are discussed and placed in the bigger perspective of finding tools for breeding for quality traits in potato. Prospects on the basis of these results and an outlook for breeding for quality are presented as well.

Functional identification in Lactobacillus reuteri of a PocR-like transcription factor regulating glycerol utilization and vitamin B12 synthesis
Santos, F. dos; Molenaar, D. ; Teusink, B. ; Vos, W.M. de - \ 2011
Microbial Cell Factories 10 (2011)1. - ISSN 1475-2859
folate production - genome analysis - biosynthesis - lactococcus - promoters - alignment - bacteria - database - plasmid - single
BACKGROUND: Lactobacillus reuteri harbors the genes responsible for glycerol utilization and vitamin B12 synthesis within a genetic island phylogenetically related to gamma-Proteobacteria. Within this island, resides a gene (lreu_1750) that based on its genomic context has been suggested to encode the regulatory protein PocR and presumably control the expression of the neighboring loci. However, this functional assignment is not fully supported by sequence homology, and hitherto, completely lacks experimental confirmation. RESULTS: In this contribution, we have overexpressed and inactivated the gene encoding the putative PocR in L. reuteri. The comparison of these strains provided metabolic and transcriptional evidence that this regulatory protein controls the expression of the operons encoding glycerol utilization and vitamin B12 synthesis. CONCLUSIONS: We provide clear experimental evidence for assigning Lreu_1750 as PocR in Lactobacillus reuteri. Our genome-wide transcriptional analysis further identifies the loci contained in the PocR regulon. The findings reported here could be used to improve the production-yield of vitamin B12, 1,3-propanediol and reuterin, all industrially relevant compounds
Agrobacterium-mediated transformation of Mycosphaerella fijiensis, the devastating Black Sigatoka pathogen of bananas
Díaz-Trujillo, C. ; Adibon, H. ; Kobayashi, K. ; Zwiers, L.H. ; Souza, M.T. ; Kema, G.H.J. - \ 2010
Gewasbescherming 41 (2010)3. - ISSN 0166-6495 - p. 151 - 151.
mycosphaerella fijiensis - fungiciden - bananen - genotypen - fenotypen - rhizobium - genetische transformatie - genoomanalyse - fungicides - bananas - genotypes - phenotypes - genetic transformation - genome analysis
Mycosphaerella fijiensis, M. musicola en M. eumusae veroorzaken de Sigatoka-ziekte in banaan. Op dit moment is de toepassing van fungiciden de enige optie om deze ziekte te bestrijden. Het PRPB (Pesticide Reduction Program for Banana) investeert in de ontwikkeling van technieken voor de genotype- en fenotypebepaling van M. fijiensis. Hierbij wordt gebruikt gemaakt van ATMT (Agrobacterium tumefaciens-mediated transformation).
Functional Analysis of Cladosponum fulvum Effector Catalog
Ökmen, B. ; Hollander, M. de; Stergiopoulos, I. ; Burg, H.A. van den; Wit, P.J.G.M. de - \ 2010
Gewasbescherming 41 (2010)3. - ISSN 0166-6495 - p. 149 - 150.
pathogenesis-gerelateerde eiwitten - dna-sequencing - genoomanalyse - passalora fulva - solanum lycopersicum - bio-informatica - genen - plant-microbe interacties - pathogenesis-related proteins - dna sequencing - genome analysis - bioinformatics - genes - plant-microbe interactions
Onlangs is de DNA-sequentie van het genoom van Cladosporium fulvum bepaald. Het voornaamste doel daarvan is de identificatie en karakterisering van nieuwe effectors.
Biosynthesis and regulation of cyclic lipopeptides in Pseudomonas fluorescens
Bruijn, I. de - \ 2009
Wageningen University. Promotor(en): Pierre de Wit, co-promotor(en): Jos Raaijmakers. - [S.l. : S.n. - ISBN 9789085853589 - 212
pseudomonas fluorescens - plantenziekteverwekkende bacteriën - antagonisten - pathogenesis-gerelateerde eiwitten - cyclische peptiden - antibiotica - biosynthese - genetische regulatie - genoomanalyse - plant pathogenic bacteria - antagonists - pathogenesis-related proteins - cyclic peptides - antibiotics - biosynthesis - genetic regulation - genome analysis
Cyclic lipopeptides (CLPs) are surfactant and antibiotic metabolites produced by a variety of bacterial
genera. For the genus Pseudomonas, many structurally different CLPs have been identified. CLPs play an
important role in surface motility of Pseudomonas strains, but also in virulence and
attachment/detachment to and from surfaces. In this Ph.D. thesis project, two new CLP biosynthesis
clusters were identified in Pseudomonas fluorescens and fully sequenced. In P. fluorescens strain SBW25, the
viscosin biosynthesis cluster was identified by bioinformatic analyses of the genome followed by genetic
and chemical analyses. For P. fluorescens strain SS101, the genes for massetolide biosynthesis were
identified via random mutagenesis followed by cloning, sequencing and chemical analyses. Biosynthesis
of viscosin and massetolide is governed by three nonribosomal peptide synthetase (NRPS) genes,
designated viscABC and massABC, respectively. The viscosin and massetolide biosynthesis gene clusters
are very similar, but different from CLP gene clusters described for other Pseudomonas as the viscA and
massA genes are physically disconnected from viscBC and massBC, respectively. Viscosin differs from
massetolide A only at position number four in the peptide moiety, which is a valine in viscosin and an
isoleucine in massetolide A. Because of the modular structure of the NRPSs and the co-linearity of the
assembly process, transfer of the mass genes of strain SS101 into strain SBW25 resulted in the
production of both massetolide A and viscosin, demonstrating that the assembly line for CLP
biosynthesis in Pseudomonas can be altered leading to the production of non-native products.
Compared to the understanding of CLP biosynthesis, not so much is known about the
regulation. This thesis shows that the GacA/GacS two-component system regulates massetolide and
viscosin biosynthesis in strains SS101 and SBW25, respectively. No indications were found that
massetolide or viscosin biosynthesis is regulated by quorum sensing via N-acylhomoserine lactones.
Site-directed mutagenesis of the LuxR-type regulator genes luxR-vA and luxR-vBC flanking the viscosin
biosynthesis cluster resulted in a loss of viscosin production, indicating that both LuxR-type
transcriptional regulators are important for viscosin biosynthesis in strain SBW25. Phylogenetic analyses
further suggested that these LuxR-type transcriptional regulators do not contain the autoinducerbinding
domain found for the quorum sensing-associated LuxR regulator in Vibrio fischeri. Instead, the
LuxR-type regulator genes flanking the massetolide and viscosin biosynthesis genes are closely related
to the LuxR-type regulators identified for syringomycin/ syringopeptin biosynthesis and appear to
belong to a separate LuxR-type regulator subfamily, different from the autonomous effector domain
protein GerE. Via random mutagenesis and subsequent screening for massetolide-deficient mutants,
also other regulator genes were identified including clpP. ClpP is a serine protease that plays a crucial
role in intracellular refolding and degradation of proteins, which is an essential process for the viability
of cells. ClpP was shown to affect transcription of luxR-mA, thereby regulating transcription of the
massetolide biosynthesis genes. Results further suggested that, at the transcriptional level, ClpPmediated
regulation of massetolide biosynthesis operates independently from regulation by the
GacA/GacS two-component system. In conclusion, the results of this thesis led to the identification of
several genes and previously unknown pathways involved in regulation of CLP biosynthesis and
highlighted the complexity of the signaling cascades underlying CLP biosynthesis in Pseudomonas.
CLPs have diverse functions for the producing bacterial strains, including a role in motility,
biofilm formation, antimicrobial activity and virulence. Also in establishment and persistence in plant
environments, CLPs were shown to confer a competitive advantage. A new function of CLPs,
identified in a collaboration with Mark Mazzola (USDA) and presented in this thesis, is their
protective effects against predation by protozoa. In vitro assays showed that both massetolide and
viscosin can lyse the trophozoites of Naeglaria americana and that wild type strains SS101 and SBW25
were substantially less sensitive to protozoan grazing than their CLP-deficient mutants. Also in soil
containing N. americana, population densities of wild type strains SS101 and SBW25 were significantly
higher compared to the massetolide and viscosin-deficient mutants, showing that CLP production
confers a competitive advantage in survival in complex environments. Moreover, transcription of the
CLP-biosynthesis genes increased significantly upon protozoan grazing, indicating that the
Pseudomonas strains sense the protozoa and react by producing CLPs as defense compounds. Which
signal triggers the induction of the CLP biosynthesis genes is not known yet and currently under
investigation. Based on these results, we postulate that CLPs are an important component of the preingestional
defense mechanisms of bacteria against protozoan predation, not only due to their lytic
effects on protozoa, but also because CLPs contribute to evasion of protozoan grazing via altered
cell surface properties, swimming and swarming, and microcolony and biofilm formation.
Genome-wide investigation into roles of Arabidosis receptor-like proteins in pathogen defense
Ellendorff, U. - \ 2009
Wageningen University. Promotor(en): Pierre de Wit, co-promotor(en): Bart Thomma. - [S.l. : S.n. - ISBN 9789085853206 - 141
planten - arabidopsis thaliana - verdedigingsmechanismen - plantenziekteverwekkers - planteiwitten - pathogenesis-gerelateerde eiwitten - genoomanalyse - mutanten - genexpressie - plant-microbe interacties - gene silencing - plants - defence mechanisms - plant pathogens - plant proteins - pathogenesis-related proteins - genome analysis - mutants - gene expression - plant-microbe interactions
Receptor-like proteins (RLPs) are receptors on the surface of plant cells that are important for the activation of disease resistance. Furthermore, some RLPs are important for plant development. The Arabidopsis genome contains 57 genes encoding RLPs. A genome wide collection of RLP gene knock-out mutants was assembled and functionally analyzed for defects in defense and development. This resulted in the identification of an RLP that plays a role in hormone perception, and two RLPs that play a role in non-host resistance, the phenomenon that a plant species is typically resistant to pathogens of other plant species.
RNA silencing is a regulatory mechanism by which the expression of genes is downregulated or entirely suppressed. In this thesis, it is demonstrated for the first time that this mechanism is important for defense of Arabidopsis against a fungal pathogen; the vascular wilt fungus Verticillium. This is an extremely important pathogen of over 200 plant species including economically important crops.

Genoom-analyse van ziektewerende bodems
Speksnijder, A.G.C.L. ; Overbeek, L.S. van - \ 2008
dna - detectie - antibiotica - genen - genomen - bodembiologie - biodiversiteit - genoomanalyse - bodemkwaliteit - agrobiodiversiteit - detection - antibiotics - genes - genomes - soil biology - biodiversity - genome analysis - soil quality - agro-biodiversity
Onderzoek naar DNA-detectietechnieken, die zijn opgezet en uitgevoerd om antibiotica-genen en diversiteit van de microbiële gemeenschap in bodems te meten
From living system to DNA
Animal Sciences Group (ASG), - \ 2007
animal breeding - genomics - dna - genome analysis - dna sequencing - cattle husbandry - livestock farming
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