Staff Publications

Staff Publications

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

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

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

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

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    Hoe kies je de beste stieren voor je bedrijf?
    Hoving, A.H. ; Ducro, B.J. - \ 2019
    Wageningen : Centre for Genetic Resources, the Netherlands (CGN), Wageningen University & Research
    animal breeding - dairy cattle - genetics - teaching materials - intermediate vocational training - animal welfare - animal production - animal health
    Docentenhandleiding bij lesmateriaal ‘Hoe kies je de beste stieren voor je bedrijf?’ Powerpoint ‘Hoe kies je de beste stieren voor je bedrijf?’ Excel file ‘Een selectie van de stierenkaart’.
    Reverse Breeding : Creating parental lines for a heterozygous plant
    Schaart, J.G. ; Wiel, C.C.M. van de - \ 2017
    Wageningen : Wageningen University
    plants - cultivation - genetics - crossing
    Video about breeding and genetics
    Reverse Breeding : Complications
    Schaart, J.G. ; Wiel, C.C.M. van de - \ 2017
    Wageningen : Wageningen University
    cultivation - genetics - plants - crossbreds
    Video recording about breeding and genetics
    Unravelling the genetic base of the meiotic recombination landscapes in two varieties of the button mushroom, Agaricus bisporus
    Sedaghat Telgerd, Narges - \ 2017
    Wageningen University. Promotor(en): R.G.F. Visser, co-promotor(en): A.S.M. Sonnenberg. - Wageningen : Wageningen University - ISBN 9789463436953 - 142
    fungi - agaricus bisporus - mushrooms - genetics - breeding - meiosis - recombination - schimmels - agaricus bisporus - paddestoelen - genetica - veredelen - meiose - recombinatie

    The button mushroom, Agaricus bisporus var. bisporus, is one of the most cultivated mushrooms worldwide. Even though wild isolates of this variety have a broad genetic variation, the traditional and present-day hybrids only have a very narrow genetic base. The button mushroom has a typical meiotic recombination landscape (MRL) in which crossover (CO) events are predominantly restricted to the extreme ends of the chromosomes. This has been one of the main obstacles for mushroom breeders in improving or generating new mushroom hybrids due to a considerable linkage drag. A wild variety of A. bisporus, i.e., burnettii appeared to have CO spread more evenly across the genome. The existence of two extremely different MRLs in two compatible A. bisporus varieties offers an excellent opportunity to study the genetic basis for positioning CO in meiosis. The main objective of the research presented in this thesis initially was to examine meiosis of the var. burnettii in more detail and subsequently to identify genomic regions revealing the difference in MRL of the two A. bisporus varieties. The availability of genome sequences in the bisporus variety has produced many more informative markers such as SNP. We aimed to de novo sequence one of the haplotypes of a heterokaryotic strain of the burnettii variety using the PacBio sequencing technique and resequencing the other haplotype using Illumina HiSeq. In parallel to this, we used Genotyping by Sequencing (GBS) to construct the first linkage map of the burnettii variety, showing a more or less even distribution of COs across the genome. The constructed linkage map has also proved to be a useful tool for de novo assembly of the burnettii variety genome sequence. In addition, we performed comparative genome sequence studies between the burnettii variety and the previously sequenced genomes of two of the bisporus variety homokaryons, indicating high levels of collinearity between all three genomes. The only chromosomal rearrangement to be found was on chromosome 10, where an inversion of ~ 800 kb in the burnettii variety was detected compared to the var. bisporus genomes. As a starting point for unravelling the genetic basis underlying MRL in the A. bisporus, we performed quantitative trait loci (QTL) analysis using bisporus and burnettii varieties. An inter-varietal population was developed from a cross between a constituent nucleus of the bisporus and the burnettii variety. This population contains 178 haploid progenies which were genotyped by 210 SNP markers to construct a genetic linkage map, which proves to be a solid foundation for exploring the genetic control of MRL of A. bisporus. In addition, we performed a comparative genetic mapping study using the genetic maps of the bisporus variety Horst U1, the burnettii variety Bisp119/9 and the inter-varietal hybrid by selecting markers having similar positions in these three maps. In contrast to the bisporus variety where CO events are mainly restricted to chromosome ends, the burnettii variety shows a more or less equal distribution of CO events across the entire genome. The recombination landscape of the inter-varietal hybrid shows an intermediate pattern to that of both varieties. The MRL trait is expressed as a CO event in the offspring of each individual of the inter-varietal mapping population. For this reason, the individuals of the inter-varietal mapping population were intercrossed and outcrossed to generate three types of second generation hybrids. Two compatible tester homokaryons derived from the bisporus and burnettii varieties were used for outcrossing. Subsequently, the haploid progenies from each type of second generation hybrids were isolated to generate three types of segregating populations. The haploid progenies from segregating populations were genotyped with SNP markers covering the whole length of all the chromosomes. Recombination frequencies were determined at distal ends and elsewhere on the chromosomes and used to compare recombination frequencies between chromosomes within each population as well as between segregating populations across all chromosomes. A prerequisite for successful QTL mapping the MRL is to select segregating populations in which the segregation of MRL is clear. We observed that segregating populations outcrossed with the bisporus tester homokaryon were the most useful populations to generate haploid offspring in which COs are assessed for further QTL study of MRL at the time when this research was carried out. To map genomic regions involved in the different MRLs of A. bisporus, 71 homokaryotic offspring of the inter-varietal hybrid were outcrossed with an unrelated tester homokaryon of the bisporus variety. Subsequently, the haploid progenies were isolated from each hybrid and genotyped with SNP markers. Marker pairs were generated for the end regions of chromosomes to assess CO there or anywhere else on the chromosomes for each segregating population. QTL mapping analysis revealed two QTLs located on chromosome l and three others located on chromosomes IV, VI and VII. The QTLs identified span large parts of their respective chromosomes; therefore further strategies are needed for a more precise assessment and localisation of MRL.

    On the genetic mechanisms of nutrient-dependent lifespan and reproduction
    Zandveld, Jelle - \ 2017
    Wageningen University. Promotor(en): B.J. Zwaan, co-promotor(en): A.J.M. Debets. - Wageningen : Wageningen University - ISBN 9789463436861 - 209
    genetics - lifespan - reproduction - nutrients - drosophila melanogaster - fungi - diet - evolution - genetica - levensduur - voortplanting - voedingsstoffen - drosophila melanogaster - schimmels - dieet - evolutie

    Dietary restriction (DR), a moderate reduction in nutrient intake, improves health or extends lifespan across many species. Moreover, recent insights have shown that also the effects of specific nutrients are of importance for the beneficial effects of DR rather than intake alone. However, we still lack much insight through what mechanisms the lifespan increase through diet changes is exactly mediated.

    To further increase our understanding of the genetic mechanisms of nutrient-dependent lifespan, in Chapter 2, 3, 4, and 5 I employed different methods of genetic interventions (i.e. a genetic knockout, natural genetic variation and experimental evolution) using the model species Drosophila melanogaster and Podospora anserina. To test whether the genetic interventions affected the diet response, a broad range of diets was applied, thereby taking the recent insights of nutritional geometry into account. Furthermore, the response of the fly’s whole-genome transcription to different dietary treatments were assessed in Chapter 6 and 7 to identify and potentially disentangle genetic mechanisms for lifespan from those for reproduction.

    Chapter 2 addressed the effects of a triple knockout in the insulin-IGF signalling (IIS) pathway, namely for three genes encoding insulin-like peptides in Drosophila (dilp2-3,5). The mutant showed a strong elevation of lifespan that was irrespective of food type, but also a strong reduction of the female fly fecundity. In addition, this assay also revealed that the same knockout can yield different interpretations for its function in the fly’s diet response, which was strongly dependent per diet dimension under consideration (i.e. varying yeast, sugar, or its ratio in the diet). This observation set the stage for other experimental chapters in this thesis, where a broad range of diets was applied to depict the exact genotypic effects that are involved in the lifespan response to diet. For example, in Chapter 2, interactive effects were observed between dilp2-3,5 knockout and the lifespan response to dietary sugar, but however, not for the yeast component of the diet.

    In Chapter 3, for the same experimental diets, gene expression responses in dilp2-3,5 knockout flies were measured to describe the general dynamics on the pathway level. Interestingly, expression of the remaining fly head-expressed dilp, dilp6, was elevated on higher yeast levels upon dilp2-3,5 knockout. Therefore, compensatory mechanisms within IIS might still partly mediate the lifespan response to yeast.

    In Chapter 4 the natural genetic variation for the response to DR was explored in wild-derived strains of the fungus Podospora anserina. By applying a broad range of glucose concentrations in a synthetic medium, we constructed reaction norms for 62 natural occurring strains and showed considerable natural variation in the shape of the reaction norms, including the glucose concentration at which lifespan increased and how steeply the fungus’ lifespan responds to diet (the slope S). Furthermore, I identified a significant correlation between a strain’s general lifespan and both parameters, suggesting that the lifespan response to diet partly acts through a mechanism involved in the fungus’ lifespan determination under high nutrient, growth and reproduction permissive, conditions. On moderate glucose restriction levels we showed that a reduced reproduction was not always associated with lifespan extension, which indicates that decoupling of these traits (that often trade-off) can be achieved.

    An evolutionary perspective on diet response and the connection between reproduction and lifespan, two often interconnected traits in lifespan research, was provided in Chapter 5. Here, experimental evolution (EE) was performed in Drosophila melanogaster to test whether improved reproductive capacity (i.e. local adaptation) to three nutritionally distinct diets directly affected the lifespan response. Adaptation to the distinct nutritional conditions, had no consistent effect on the lifespan response to diet. Other life-history traits that I assessed could more consistently be associated with the evolutionary nutritional treatments, which together suggested that the adaptive genetic mechanisms increasing the fly’s reproduction were not necessarily interconnected singly with a change of lifespan, but rather with a change in the whole life-history strategy.

    By exploring the fly’s whole-genome transcription response in a continuously changing environ­ment, Chapter 6 continued on the evolutionary relevance of lifespan responses to diet. This type of fluctuations may better reflect the fly’s natural ecological setting than the continuous diets typically applied in whole-genome transcription laboratory studies. This revealed that flies were able to respond quickly to diet fluctuations throughout lifespan by drastically changing their transcription pattern and, moreover, my results indicated that a large part of the whole-genome transcription response could be attributed to the female fly’s reproduction. Because I measured the response of multiple life-history traits to the fluctuating diet changes, I was able to decouple groups of genes associated with lifespan from those associated with reproduction. This is an important step in the direction of unravelling the genetic architecture that specifically mediates the lifespan response to diet, which can be especially useful in whole-genome transcription studies.

    In Chapter 7, the consistencies between studies for their whole-genome transcription responses upon DR were investigated. This revealed large transcriptomic variations on different regulatory levels, i.e. the level of whole-genome transcription, most significant genes, and also gene ontology. To test whether the observed inconsistent whole-genome transcription responses were primarily a reflection of the fly’s reproduction, such as observed in Chapter 6, a new cohort of flies was subjected to different regimes that resulted in very different age-dependent reproduction patterns. By assessing whole-genome transcription in this cohort at two time points, the gene expression changes reflected the age-dependent reproduction patterns observed, rather than the lifespan phenotypes. Similar to Chapter 6, this again highlighted the importance of measuring multiple life-history traits for associating whole-genome transcription responses to lifespan effects of dietary restriction.

    In Chapter 8 the acquired insights across the experimental chapters were synthesized, discussing the importance of assessing a broad range of nutrients for the interpretation of any genotypic effect, and in addition discussing the value of measuring multiple life-history traits for genetic associations. In this chapter I also suggested directions for future research in Drosophila and Podospora that may be valuable for further unravelling and understanding the mechanisms of diet responses in other organisms, including in humans.

    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): R.G.F. Visser, co-promotor(en): W.E. van de Weg; F. Costa. - Wageningen : Wageningen University - ISBN 9789463432054 - 177
    malus domestica - apples - fruit - fruit growing - genetics - plant breeding - genome analysis - malus domestica - appels - fruit - 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.

    Farewell symposium Kor Oldenbroek
    Windig, Jack - \ 2017
    animal welfare - pets - dogs - animal breeding methods - genetics - animal health
    Breeding healthy dogs with genomics / Presentation
    Genetische monitoring van de Nederlandse otterpopulatie : ontwikkeling van populatieomvang en genetische status 2015/2016
    Kuiters, A.T. ; Groot, G.A. de; Lammertsma, D.R. ; Jansman, H.A.H. ; Bovenschen. J., Jan - \ 2016
    Wageningen : Wettelijke Onderzoekstaken Natuur & Milieu (WOt-technical report 81) - 47
    otters - lutra - populatiebiologie - monitoring - populatiegroei - inteelt - genetica - nederland - otters - lutra - population biology - monitoring - population growth - inbreeding - genetics - netherlands
    Jaarlijks wordt in opdracht van het ministerie van Economische Zaken de Nederlandse otterpopulatiegenetisch gemonitord. Daarmee wordt een vinger aan de pols gehouden voor de ontwikkeling van degenetische status van de populatie. Deze vorm van monitoring, waarbij gebruik wordt gemaakt van DNAgeïsoleerd uit uitwerpselen en doodvondsten, maakt het tevens mogelijk veranderingen in de ruimtelijkeverspreiding en de populatieomvang te volgen. De monitoringsronde van 2015/2016 laat zien dat depopulatie verder is gegroeid naar ca. 185 individuen. Op populatieniveau is de genetische variatie weer wattoegenomen doordat op steeds meer plekken otters van Duitse origine in de Nederlandse populatie opduikendie hier op eigen kracht komen. De genetische variatie binnen individuen is niet verder afgenomen zoals deeerste periode van het herintroductieprogramma. Onderdeel van deze monitoring is ook autopsie van dodeotters, waarbij wordt gekeken naar de doodsoorzaak en de belangrijkste lichaamskenmerken. Verkeer isverreweg de belangrijkste doodsoorzaak. Het aantal verkeersslachtoffers neemt nog ieder jaar toe, waarbijde toename evenredig is aan de toename in de populatieomvang. Locaties waar otters worden doodgereden,worden geregistreerd en toegevoegd aan een database. Deze informatie is belangrijk om knelpuntlocatiesveiliger te maken om zo het aantal verkeersslachtoffers te beperken---The Ministry of Economic Affairs requires that the Dutch otter population is surveyed each year to monitorthe genetic status of the population using DNA isolated from spraints and tissue from dead individuals. Theresulting information is also used to detect changes in the spatial range and population size. The 2015/2016survey showed that the population size has further increased to about 185 individuals. The amount ofgenetic variation at the population level has further increased, mainly as the result of migration of otters ofGerman origin to the Dutch population. In contrast to previous years, genetic variation within individuals hasnot declined further. The survey includes autopsies of dead otters to assess body condition and the mostlikely cause of death. Traffic is by far the most important cause of mortality. The annual number of road killsis still increasing in proportion to the increase in population size. Locations where road kills occur areregistered and added to a database. This information is important to localise places where mitigatingmeasures have to be taken to improve the safety for otters
    Statistical methods for QTL mapping and genomic prediction of multiple traits and environments: case studies in pepper
    Alimi, Nurudeen Adeniyi - \ 2016
    Wageningen University. Promotor(en): Fred van Eeuwijk, co-promotor(en): Marco Bink. - Wageningen : Wageningen University - ISBN 9789462579361 - 153
    capsicum - statistical analysis - statistics - genomics - quantitative trait loci - quantitative traits - quantitative methods - genetics - crop yield - capsicum - statistische analyse - statistiek - genomica - loci voor kwantitatief kenmerk - kwantitatieve kenmerken - kwantitatieve methoden - genetica - gewasopbrengst

    In this thesis we describe the results of a number of quantitative techniques that were used to understand the genetics of yield in pepper as an example of complex trait measured in a number of environments. Main objectives were; i) to propose a number of mixed models to detect QTLs for multiple traits and multiple environments, ii) to extend the multi-trait QTL models to a multi-trait genomic prediction model, iii) to study how well the complex trait yield can be indirectly predicted from its component traits, and iv) to understand the ‘causal’ relationships between the target trait yield and its component traits.

    The thesis is part of an EU-FP7 project “Smart tools for Prediction and Improvements of Crop Yield” (SPICY- This project generated phenotypic data from four environments using 149 individuals from the sixth generation of recombinant inbred lines obtained from intraspecific cross between large – fruited inbred pepper cultivar ‘Yolo Wonder’ (YW) and the hot pepper cultivar ‘Criollo de Morelos 334’ (CM 334). A total of 16 physiological traits were evaluated across the four trials and various types of genetic parameters were estimated. In a first analysis, the traits were univariately analyzed using linear mixed model. Trait heritabilities were generally large (ranging between 0.43 – 0.96 with an average of 0.86) and mostly comparable across trials while many of the traits displayed heterosis and transgression. The same QTLs were detected across the four trials, though QTL magnitude differed for many of the traits. We also found that some QTLs affected more than one trait, suggesting QTL pleiotropy (a QTL region affecting more than one trait). We discussed our results in the light of previously reported QTLs for these and similar traits in pepper.

    We addressed the presence of genotype-by-environment interaction (GEI) in yield and the other traits through a multi-environment (ME) mixed model methodology with terms for QTL-by-environment interaction (QEI). We opined that yield would benefit from joint analysis with other traits and so deployed two other mixed model based multi-response QTL approaches: a multi-trait approach (MT) and a multi-trait multi-environment approach (MTME). For yield as well as the other traits, MTME was superior to ME and MT in the number of QTLs, the explained variance and accuracy of predictions. Many of the detected QTLs were pleiotropic and showed quantitative QEI. The results confirmed the feasibility and strengths of novel mixed model QTL methodology to study the architecture of complex traits.

    The QTL methods considered thus far are not well suited for prediction purposes as only a limited set of QTL-related markers are used. Since the main interest of this research includes improvement of yield prediction, we explored both single-trait and multi-trait versions of genomic prediction (GP) models as alternatives to the QTL-based prediction (QP) models. This was termed direct prediction. The methods differed in their predictive accuracies with GP methods outperforming QP methods in both single and multi-traits situations. We borrowed ideas from crop growth model (CGM) to dissect complex trait yield into a number of its component traits. Here, we integrated QTL/genomic prediction and CGM approaches and showed that the target trait yield can be predicted via its component traits together with environmental covariables. This was termed indirect prediction. The CGM approach seemed to work well at first sight, but this is especially due to the fact that yield appeared to be strongly driven by just one of its components, the partitioning to fruit.

    An alternative representation of the biological knowledge of a complex target trait such as yield is provided by network type models. We constructed both conditional and unconditional networks across the four environments to understand the ‘causal’ relationships between target trait yield and its component traits. The final networks for each environment from both conditional and unconditional methods were used in a structural equation model to assess the causal relationships. Conditioning QTL mapping on network structure improved detection of refined genetic architecture by distinguishing between QTL with direct and indirect effects, thereby removing non-significant effects found in the unconditional network and resolving QTL pleiotropy. Similar to the CGM topology, yield was established to be downstream to its component traits, indicating that yield can be studied and predicted from its component traits. Thus, the genetic improvements of yield would benefit from improvements on the component traits.

    Finally, complex trait prediction can be enhanced by a full integration of the methods described in the different chapters. Recent research efforts have been channelled to incorporating both multivariate whole genome prediction models and crop growth models. Further research is required, but we hope that the present thesis presents useful steps towards better prediction models for complex traits exhibiting genotype by environment interaction.

    Exploring the genetics underlying the responses to consecutive combinations of biotic stresses and drought in Arabidopsis thaliana
    Huang, Pingping - \ 2016
    Wageningen University. Promotor(en): Maarten Koornneef, co-promotor(en): Mark Aarts. - Wageningen : Wageningen University - ISBN 9789462578593 - 291
    arabidopsis thaliana - genetic models - stress - stress response - drought - botrytis - pieris (lepidoptera) - genetics - gene expression - arabidopsis thaliana - genetische modellen - stress - stressreactie - droogte - botrytis - pieris (lepidoptera) - genetica - genexpressie

    Plants growing in natural environments are exposed to a broad range of biotic (pathogen attack, insect herbivory, etc.) and abiotic factors (drought, extreme temperatures, UV radiation, salinity, etc.) that are known to cause stress symptoms in many species (Pareek et al., 2010; Robert-Seilaniantz et al., 2010). Biotic and abiotic stress-inducing determinants often adversely impact plant growth and development, frequently leading to severe annual yield losses in agricultural production (Pierik et al., 2013; Pieterse et al., 2012; Stam et al., 2014). In the research endeavors described in this thesis, Arabidopsis thaliana was used as a model organism to study plant responses to different sequential combinations of biotic factors (infection with Botrytis or herbivory by Pieris) and drought. The main objective was to identify genes that contribute to tolerance to the aforementioned sequential stress combinations. Genome-wide association (GWA) mapping and RNA sequencing (RNA-seq) approaches were used to identify combinatorial stress responsive genes. A number of candidate genes to combinatorial stress responses were identified by GWA analysis and RNA-seq. The physiological function of some candidate genes in different stress conditions were characterized using T-DNA insertion mutants and gene expression study. However, the physiological function of many allelic variants in stress conditions remain to be discovered. The study highlights the importance of an array of genes, crucial to the underlying defense processes, as targets for breeding by allele mining, ultimately aimed at improvement of crop tolerance to frequent combinations of stress factors.

    Genetic constraints that determine rhizobium-root nodule formation in Parasponia andersonii
    Seifi Kalhor, M. - \ 2016
    Wageningen University. Promotor(en): Ton Bisseling, co-promotor(en): Rene Geurts. - Wageningen : Wageningen University - ISBN 9789462579118 - 160
    parasponia - rhizobium - root nodules - rhizobium rhizogenes - temperature - nitrates - symbiosis - genetics - parasponia - rhizobium - wortelknolletjes - rhizobium rhizogenes - temperatuur - nitraten - symbiose - genetica

    Bacteria of the genus Rhizobium play a very important role in agriculture by inducing nitrogen-fixing nodules on the roots of legumes. Root nodule symbiosis enables nitrogen‐fixing bacteria (Rhizobium) to convert atmospheric nitrogen into a form that is directly available for plant growth. This symbiosis can relieve the requirements for added nitrogenous fertilizer during the growth of leguminous crops. Research on legume-rhizobium symbioses has emphasized fitness benefits to plants but in our research, we take a different vantage point, focusing on the Parasponia-rhizobium symbiosis. Parasponia is the only non-legume plant capable of establishing mutualistic relation with rhizobia. This study will provide background knowledge for use in applied objectives as well as yielding a wealth of fundamental knowledge with wide implications from rhizobium symbiosis evolution. This thesis describes my research on genetic constrains that determine rhizobium-root nodule formation. To identify these constraints we used Parasponia anadersnii as only non-legume capable to establish nitrogen fixing rhizobium symbiosis. Our main attempt in this thesis was to find the genetic constraints using Parasponia as a key and reconstruct an auto active symbiotic signaling cascade in the non- legume plants. In line with this, a simple and efficient hairy root transformation method was established in this thesis. To determine the genetic elements that underlie the rhizobium symbiosis, we aimed to compare Parasponia with closest non nodulating specious, Trema tomentosa. To do so, we also developed an efficient genetic transformation method for Trema mediated by Agrobacterium tumefaciens. In different attempt we implemented in a physiological study on symbiotic response of Parasponia to nitrate. This research opened a novel view on the Parasponia-rhizobium symbiosis by discovering a different mechanism that control root nodule formation in Parasponia in compare with legumes. We discovered that Parasponai-rhizbium symbiosis is not evolved to regulate the nodule number in presence of the nitrate. According to the fact that Parasponia and legumes are remotely related, it was hypothesized that, Parasponia-rhizobium symbiosis evolved independently. Therefore we put forward our attempt to determine the genes required for nodule formation in Parasponia, by extending our research on symbiotic genes which are available in non nodulating plants with different function, namely NSP1 and NSP2. We showed that NSP1 and NSP2 are involved in both nodulation and mycorrhization. This result highlight the idea that RN and AM symbiosis are conserved in part of the pathway and probably bifurcates into two branches by NSP transcription factor allowing specific activation of nodulation or mycorrhization. Aiming to know the role of hormones in symbiotic behavior, we focused on ethylene as a negative regulator of nodule formation in legumes. We found the negative effect of ethylene on root nodulation of Parasponia. For the first time we reported a hyper nodulation (20 fold nodule number in compare with control plants) phenotype in Parasponia by performing knocked down mutant of EIN2 gene. Finally, the results obtained in this study provide new insight into the fact that rhizobium symbiosis are under tight genetic constraints that guide endosymbiosis in remotely evolved host plants, legumes and Parasponia.

    Mixed culture engineering for steering starter functionality
    Spuś, Maciej - \ 2016
    Wageningen University. Promotor(en): E.J. Smid; Tjakko Abee. - Wageningen : Wageningen University - ISBN 9789462578333 - 170
    bacteriophages - predation - microorganisms - starters - genetics - diversity - bacteriofagen - predatie - micro-organismen - zuursels - genetica - diversiteit

    Undefined mixed complex starter cultures are broadly used in Gouda-type cheese production due to their robustness to phage predation, resilience for changes in environmental conditions and aroma compounds production ability during ripening. These microbial communities of lactic acid bacteria prior their isolation and deposition in starter culture collections were continuously used at the farm-level production facilities. Thus, one can consider undefined mixed complex starters as domesticated microbial communities. The process of domestication was facilitated by humans who have been continuously repeating successful fermentations using part of previous batch as inoculum (i.e. back-slopping). Therefore, a term ‘community breeding’ can describe this human-driven domestication of microbial communities. Community breeding of a model complex starter Ur led to establishment of a simple two-species composition of Lactococcus lactis and Leuconostoc mesenteroides represented by, in total, 8 genetic lineages. At the same time, this simple microbial community displays a high degree of intraspecies diversity, presumably caused by evolutionary processes of horizontal gene transfer, genome decay and mutations. Such diversity at strain level is particularly interesting in the context of continuous bacteriophage predation pressure present in this microbial community. It is thought that constant-diversity (CD) dynamics, based on the ‘kill-the-winner’ principles, is operational in Ur starter at the strain level. According to CD model, the fittest strain(s), which feed on the most abundant substrate, will be selected against due to density-dependent phage predation. The control of the fittest strain abundance by bacteriophages opens space for differentiation of strains via eco-evolutionary feedbacks. In particular, strains of complex starter culture not only adapted to quickly acidify milk (via efficient consumption of lactose and protein to peptides degradation), but concurrently, to consume other substrates present in milk. In addition, throughout the process of community breeding microbe-microbe interactions between community members have evolved. These interactions have led to division of metabolic labor among strains present in the culture, and eventually to better starter microbial community functioning.

    The aim of this thesis was to investigate the factors impacting the formation of compositionally and functionally stable undefined mixed complex starter cultures to further use this knowledge in steering its functionality, and potentially in developing new strategies for robust starter culture design. To facilitate this study, well-characterized Ur culture strain isolates were used to systematically reconstitute the starter culture into multi-strain blends with increasing level of strain and genetic lineage diversity. The investigation of factors such as phage predation, level of strain and genetic lineage diversity as well as environmental conditions, was performed during experimental evolution studies in milk. The functionality of the (evolved) starter cultures was tested in an adapted lab-scale MicroCheese model system. The specific approach used in each of the research chapters is described below in more detail.

    Strains isolated from Ur starter culture were characterized in terms of their resistance against bacteriophages isolated from the same starter (Chapter 2). This test confirmed high diversity in phage resistance among strains belonging to different genetic lineages as well as among strains of the same lineage. Next, selected strains, which represented different levels of bacteriophage predation: resistant, moderately resistant, sensitive and no detectable sensitivity, were mixed in simple blends containing 4 strains representing 3 genetic lineages of Ur starter (3 such blends were designed). These blends were exposed to phage predation (one phage per blend) at the onset of prolonged sequential propagation experiment or propagated without phage addition (control). Throughout the serial propagation the genetic lineage composition was monitored. During the propagation of control blends we detected quick domination of a single lineage. This dominating lineage contained strains sensitive to phages. Genetic lineage level composition of the phage-challenged blends was much more dynamic suggesting the impact of phage predation. The relatively low strain diversity introduced in these blends was not high enough to sustain maximal diversity at the level of lineages.

    Chapter 3 describes a study using defined blends with higher complexity by extending the number of strains used. In total, 24 strains representing all 8 Ur starter lineages were exposed in sequential propagation experiment to a cocktail of 3 phages isolated from Ur starter. The propagation in milk of this multi-strain blend was executed for more than 500 generations and the abundance of genetic lineages was monitored throughout. Similarly as in the simple blends experiment, control blends were not exposed to bacteriophages. In control blends we observed a domination of one genetic lineage upon serial propagation, which resembles a periodic-selection-like (PS) behavior, where the fittest strains are dominating the microbial community and in result genetic-lineage diversity is being substantially reduced. In contrast, the composition of phage-challenged blends was again more dynamic than in control blends. In one of the phage-challenged blends behavior characteristic for a constant-diversity dynamics model was observed; throughout the serial transfer experiment, genetic lineage diversity was maintained by the presence of phage predation at relatively high level. In case of the second phage-challenged blend, due to a stochastic event, which likely caused a reduction in phage pressure, we observed a gradual recovery of the fittest strains, which again resembled a periodic-selection behavior. Therefore, phage predation, among other factors, can lead to shifts in microbial community population dynamics resulting in alternative stable states.

    The experimental evolution approach, resembling traditional process of back-slopping, was used in a Long-term experimental evolution of Undefined Mixed Starter Culture (LUMSC) study described in Chapter 4. The aim of this study was to investigate the compositional and functional stability ascribed to the undefined mixed Ur starter during enclosed prolonged propagation without any possible external influx of bacterial or phage material. Surprisingly, during this 1000-generation long experiment the enforced conditions of specific incubation temperature and propagation regime resulted in enrichment of previously not detected strain of Lactococcus laudensis. This strain was found to consume a by-product of metabolism of another strain present in the community, in particular, D-mannitol produced by Le. mesenteroides. Thus, a new putative interaction in the microbial community of the complex starter culture was found. This new interaction and the possible ability of L. laudensis to efficiently use peptides released by caseinolytic L. lactis ssp. cremoris resulted in a relatively high abundance of L. laudensis in all evolved LUMSC cultures. The high abundance of L. laudensis had a certain effect on the functionality of the cultures. The aroma profiles of model lab-scale milli-cheeses manufactured with LUMSC cultures, showed significant differences in formation of esters and alcohols when compared to cheeses produced with the original Ur starter. Moreover, L. laudensis strain was not only under the radar of previously used culture-dependent and culture-independent methods, but as well, under the radar of phage predation continuously present throughout the LUMSC experiment. This observation sheds new light on the possibility of how a strain can emerge to relatively high abundance in an enclosed serially propagated microbial community operating in accordance with CD dynamics model.

    Finally, the aspect of adaptation to environmental conditions was addressed by the study of an adjunct strain of Lactobacillus helveticus DSM 20075 described in Chapter 5. The aim was to develop a strain with increased autolytic capacity in conditions resembling the cheese matrix to possibly improve cheese ripening. The approach used here was based on a previously reported study, where the incubation of Lactococcus lactis MG1363 at high temperature resulted in spontaneous mutations causing stable heat-resistant and, in some cases, salt-hypersensitive phenotypes. In present study, after incubation of the Lb. helveticus DSM 20075 adjunct at different high temperatures (45-50 °C), heat-sensitive variants were recovered from plates. These variants were further characterized in terms of their growth rates at elevated temperatures (42-45 °C) and their autolytic capacity in low pH buffer with addition of NaCl. One of the variants (V50) showed substantially increased intracellular lactate dehydrogenase enzyme activity in the buffer suggesting its increased autolytic capacity. Next, both wild type and variant V50 were tested as adjuncts in lab-scale model milli-cheeses to determine their possible impact on the cheese aroma profiles. Indeed, adjunct strains, both WT and the variant, impacted the aroma profiles by producing benzaldehyde. In case of the variant strain the relative abundance of this compound was 3-fold higher. The applied strategy of incubating Lb. helveticus DSM20075 at high temperature resulted in specific, but different than in case of L. lactis MG1363, mutations suggesting another, yet to be elucidated, mechanisms for increasing the autolytic capacity of industrially-relevant strains. The approach of high-temperature incubation can be applied in dairy industry for the selection of (adjunct) cultures targeted at accelerated cheese ripening and aroma formation.

    In conclusion, the work presented in this thesis highlights the importance of co-evolution of strains in compositional and functional stability of the complex undefined mixed starter culture. In particular, the factors such as heterogeneity of bacteriophage resistance among highly related strains, microbe-microbe interactions and division of metabolic labor are crucial for optimal functioning of a complex starter microbial community. Further investigation of the factors impacting the composition of starter cultures is crucial to steer the functionality in a desired direction. With straightforward methods, such as changing the incubation temperature or the propagation regime it is possible to induce shifts in strain composition and thereby obtain cultures with new characteristics. Moreover, experimental evolution studies with microbial communities used in food fermentation can lead to the discovery of new strains with potentially new characteristics. Additionally, the study of microbial communities of starter cultures not only delivers industrially applicable knowledge but also reveals the action of basic principles in microbial ecology and evolution.

    Conservation genetics of the frankincense tree
    Bekele, A.A. - \ 2016
    Wageningen University. Promotor(en): Frans Bongers, co-promotor(en): Rene Smulders; K. Tesfaye Geletu. - Wageningen : Wageningen University - ISBN 9789462576865 - 158
    boswellia - genomes - dna sequencing - tropical forests - genetic diversity - genetic variation - genetics - forest management - plant breeding - boswellia - genomen - dna-sequencing - tropische bossen - genetische diversiteit - genetische variatie - genetica - bosbedrijfsvoering - plantenveredeling

    Boswellia papyrifera is an important tree species of the extensive Combretum-Terminalia dry tropical forests and woodlands in Africa. The species produces a frankincense which is internationally traded because of its value as ingredient in cosmetic, detergent, food flavor and perfumes productions, and because of its extensive use as incense during religious and cultural ceremonies in many parts of the world. The forests in which B. papyrifera grows are increasingly overexploited at the expense of the economic benefit and the wealth of ecological services they provide. Populations of B. papyrifera have declined in size and are increasingly fragmented. Regeneration has been blocked for the last 50 years in most areas and adult productive trees are dying. Projections showed a 90% loss of B. papyrifera trees in the coming 50 years and a 50% loss of frankincense production in 15 years time.

    This study addressed the conservation genetics of B. papyrifera. Forty six microsatellite (SSR) markers were developed for this species, and these genetic markers were applied to characterize the genetic diversity pattern of 12 B. papyrifera populations in Ethiopia. Next to this, also the generational change in genetic diversity and the within-population genetic structure (FSGS) of two cohort groups (adults and seedlings) were studied in two populations from Western Ethiopia. In these populations seedlings and saplings were found and natural regeneration still takes place, a discovery that is important for the conservation of the species.

    Despite the threats the populations are experiencing, ample genetic variation was present in the adult trees of the populations, including the most degraded populations. Low levels of population differentiation and isolation-by-distance patterns were detected. Populations could be grouped into four genetic clusters: the North eastern (NE), Western (W), North western (NW) and Northern (N) part of Ethiopia. The clusters corresponded to environmentally different conditions in terms of temperature, rainfall and soil conditions. We detected a low FSGS and found that individuals are significantly related up to a distance of 60-130 m.

    Conservation of the B. papyrifera populations is urgently needed. The regeneration bottlenecks in most existing populations are an urgent prevailing problem that needs to be solved to ensure the continuity of the genetic diversity, species survival and sustainable production of frankincense. Local communities living in and around the forests should be involved in the use and management of the forests. In situ conservation activities will promote gene flow among fragmented populations and scattered remnant trees, so that the existing level of genetic diversity may be preserved. Geographical distance among populations is the main factor to be considered in sampling for ex situ conservation. A minimum of four conservation sites for B. papyrifera is recommended, representing each of the genetic clusters. Based on the findings of FSGS analyses, seed collection for ex situ conservation and plantation programmes should come from trees at least 100 m, but preferably 150 m apart.

    Robot meet fotosynthese
    Aarts, Mark ; Harbinson, Jeremy - \ 2016
    photosynthesis - robots - measurement - agricultural research - plant breeding - genetics
    Fokken, wat is dat?
    Oldenbroek, Kor - \ 2016
    animal welfare - pets - dogs - animal health - animal behaviour - animal breeding - genetics
    Populatieanalyse Groninger Paard
    Hoving, A.H. ; Vernooij, Kelly ; berg, Rozemarijn van den; Windig, Jack - \ 2015
    Zeldzaam huisdier 40 (2015)4. - ISSN 0929-905X - p. 18 - 19.
    paardenrassen - nederland - fokdoelen - groninger paard - populaties - genetica - inteelt - verwantschap - genenbanken - horse breeds - netherlands - breeding aims - groningen horse - populations - genetics - inbreeding - kinship - gene banks
    We prijzen Nederlandse paardenrassen zoals het Groninger paard niet alleen omdat ze onderdeel van ons cultuurhistorisch erfgoed zijn maar ook vanwege hun veelzijdigheid en betrouwbare karakter. Helaas is de populatie klein. Dan is een goed doordacht fokbeleid nodig voor het behoud van genetische diversiteit en een gezonde populatie
    High-throughput open source computational methods for genetics and genomics
    Prins, J.C.P. - \ 2015
    Wageningen University. Promotor(en): Jaap Bakker; R.C. Jansen, co-promotor(en): Geert Smant. - Wageningen : Wageningen University - ISBN 9789462574595 - 136
    plantenparasitaire nematoden - dna-sequencing - next generation sequencing - verwerkingscapaciteit - computational science - genetica - genomica - plant parasitic nematodes - dna sequencing - next generation sequencing - throughput - computational science - genetics - genomics

    Biology is increasingly data driven by virtue of the development of high-throughput technologies, such as DNA and RNA sequencing. Computational biology and bioinformatics are scientific disciplines that cross-over between the disciplines of biology, informatics and statistics; which is clearly reflected in this thesis. Bioinformaticians often contribute crucial insights and novelty to scientific research because they are central to data analysis and contribute concrete algorithms and software solutions. In addition, bioinformaticians have an important role to play when it comes to organising data and software and making it accessible to others. In this thesis, in addition to contributing to biological questions, I discuss issues around accessing and sharing data, with the challenges of handling large data, input/output (IO) bottlenecks and effective use of multi-core computations.

    By creating software solutions together with molecular biologists, I contributed and published insights in biological processes in nematodes and plants. I published software solutions that made it easier for others to analyse data, which impacts the wider research community. I created solutions that made it easier for others to publish software solutions by themselves. The introduction of computing and the internet makes it possible to share ideas and computational methods. I am convinced it is a good idea to publish software solutions as `free and open source' software (FOSS) in the public domain so that we can continue to build on the work of others.

    Chapter 2 presents a computational method for identifying gene families in a sequenced genome that may be involved in pathogenicity, i.e., those genes that code for proteins that interact with molecules of an infected host. Such nematode proteins are known to contain highly variable DNA sections that code for the biochemical properties of an interaction site. By applying phylogenetic analysis through maximum likelihood (PAML) and comparison of homologues sequences in other organisms with comparable and different life styles, we discovered 77 unique candidate sequence families in the plant pathogen Meloidogyne incognita that deserve further investigation in the laboratory.

    Chapter 3 presents GenEST, a computational method for predicting which fragments captured by the cDNA-AFLP high-throughput technology matched known expressed sequence tags (ESTs). The cDNA-AFLP biochemical process was calculated in silico and fragments matching the fragment lengths as given by cDNA-AFLP were matched. Through this technique novel effectors from the nematode Globodera rostochiensis, putatively involved in pathogenicity, were identified and partly confirmed in the laboratory.

    Chapter 4 presents GenFrag, a computational method that expands on GenEST for predicting which fragments captured by cDNA-AFLP matched fragments of a fully sequenced genome with its known spliced gene variants. Through this in silico technique genes were identified in the plant Arabidopsis thaliana putatively involved in maternal genomic imprinting and partly confirmed in the laboratory.

    Chapter 5 presents multiple QTL mapping (MQM), a high-throughput computational method for predicting what sections of a genome correlate with, for example, gene expression. The study of finding such eQTL is challenging, not least because many of them are potentially false positives. The MQM parallelized algorithm is embedded in the R/qtl software package which makes it widely available to researchers. The impact thereof means that it is widely cited by studies on model organisms, such as mouse, rat, the nematode Caenorhabditis elegans and the plant A. thaliana.

    Chapter 6 presents a theoretical framework in the form of a review for identifying plant-resistance genes (R-genes) that combines the lessons learnt in the previous chapters. Plants lack an adaptive immune system and therefore, next to having physical defences, use R-genes to code for proteins that recognise molecules and proteins from invading pathogens, with an example on A. thaliana. These R-genes can be viewed as the counterparts of effectors identified in Chapter 3 and Chapter 4. By introducing the concept of a prior the chapter discusses eQTL or broader xQTL techniques as presented in the Chapter 5 to narrow down on gene candidates involved in plant defence.

    Chapter 7 and Chapter 8 present FOSS bioinformatics tools, and modules that make use the Ruby programming language. BioRuby (Chapter 7) has components for sequence analysis, pathway analysis, protein modelling and phylogenetic analysis; it supports widely used data formats and provides access to databases, external programs and public web services. All Ruby software created in the context of this thesis was contributed initially to the main BioRuby project, e.g. the PAML parser of Chapter 2, and later as individual Biogems (Chapter 8), e.g. the bio-blastxmlparser, bio-alignment, bigbio and bio-rdf biogems for Chapter 2, and three Genfrag related biogems for Chapter 4. Over 16 modules were contributed by the author as Ruby FOSS projects and are listed on the website. Because of the open nature of the BioRuby project, both BioRuby and BioGem software modules are increasingly used and cited in biomedical research, not only in genomics, but also in phylogenetics and prediction of protein structural complexes and data integration.

    Chapter 9 presents sambamba, a software tool for scaling up next generation sequencing (NGS) alignment processing through the use of multiple cores on a computer. Sambamba is a replacement for samtools, a commonly used software tool for working with aligned output from sequencers. Sambamba makes use of multi-core processing and is written in the D programming language. Not only does sambamba outperform samtools, but it already comes with an improved deduplication routine and other facilities, such as easy filtering of data. The Sambamba software is now used in the large sequencing centres around the world.

    Chapter 10 `Big Data, but are we ready?' gives a response to a publication on using cloud computing for large data processing. The chapter discusses computational bottlenecks and proves prescient because the number of citations of this paper increases every year.

    Chapter 11 `Towards effective software solutions for big biology' discusses the need for a change of strategy with regard to bioinformatics software development in the biomedical sciences to realise big biology software projects. This includes improved scientific career tracks for bioinformaticians and dedicated funding for big data software development.

    Chapter 12 discusses the computational methods and software solutions presented in this thesis, painting a picture of further challenges in bioinformatics computational solutions for the elucidation of biological processes. The chapter starts with a discussion on the merits and shortcomings of each individual software solution presented in this thesis, followed by a perspective on next generation sequencing, data integration and future research in software solutions.

    Linkage disequilibrium and genomic selection in pigs
    Veroneze, R. - \ 2015
    Wageningen University. Promotor(en): Johan van Arendonk; S.E.F. Guimarães, co-promotor(en): John Bastiaansen. - Wageningen : Wageningen University - ISBN 9789462574151 - 142
    varkens - verstoord koppelingsevenwicht - loci voor kwantitatief kenmerk - genomica - populaties - kruising - inteeltlijnen - fokwaarde - selectief fokken - genetica - pigs - linkage disequilibrium - quantitative trait loci - genomics - populations - crossbreds - inbred lines - breeding value - selective breeding - genetics

    Securing a sufficiently large set of genotypes and phenotypes can be a limiting factor when implementing genomic selection. This limitation may be overcome by combining data from multiple populations or by using information of crossbred animals. The research described in this thesis characterized linkage disequilibrium (LD) patterns in different pig populations and evaluated whether the consistency of LD between populations allows us to make predictions about the performance of genomic selection when multiple populations are included in the prediction and/or validation datasets.

    In chapter 2 I evaluated the persistence of LD and patterns of LD decay of pure and crossbred pig populations using real data that was representative of the crossbreeding structure of pig production. The persistence of phase between the crosses and their parental populations was high, indicating that similar marker effects might be expected across these populations. Across the purebred populations the persistence of phase was low therefore higher density panels should be used to have the same marker-QTL associations across these populations.

    In chapter 3, the well-known nonlinear model developed by Sved (1971) was compared against a an alternative, loess regression, to describe LD decay. The loess regression model was found to be less influenced by the lack of residual normality, independence and homogeneity of variance than the nonlinear regression model. The loess regression model resulted in more reliable LD predictions and can be used to formally compare the LD decay curves between populations.

    Chapter 4 showed the utility of different reference sets (across- and multi-population) for the prediction of genomic breeding values, as well as the potential of using crossbred performance in genomic prediction. None of the accuracies obtained using across-population, or multi-population genomic prediction, nor the accuracies obtained using crossbred data, followed the expectations based on LD that was described in chapter 2. I showed that across-population prediction accuracy was negligible even when the populations had common breeds in their genetic background. The variable accuracies of multi-population prediction and moderate accuracy of prediction of crossbred performance appeared to be a result of the differences in genetic architecture between pure populations and between purebred and crossbred animals.

    In chapter 5, a methodology that uses information from genome wide association analyses in the genomic predictions was developed and evaluated. The aim in chapter 5 was to let the genomic prediction model use information from the genetic architecture in single- and multi-population genomic prediction. I showed that using weights based on GWAS results from a combined population did result in higher accuracies of GBLUP in single- as well as in multi-population predictions.

    In chapter 6 I placed my results in a broader context. I discussed about the theoretical and practical aspects of linkage disequilibrium in breeding and in the estimation of effective population size. I also discussed the application of genomic selection in a small population and in practical pig breeding, including the prospects of using whole genome sequence for genomic prediction.

    On the evolution of allorecognition and somatic fusion in ascomycete filamentous fungi
    Bastiaans, E. - \ 2015
    Wageningen University. Promotor(en): Bas Zwaan, co-promotor(en): Duur Aanen; Fons Debets. - Wageningen : Wageningen University - ISBN 9789462572973 - 128
    ascomycota - schimmels - genetica - moleculaire herkenning - celgroei - evolutie - ascomycota - fungi - genetics - molecular recognition - cell growth - evolution

    Cooperation -behaviour that benefits other individuals- can be beneficial at the level of the group. For example, a large group is better protected against predators than a small group, and a group of individuals dividing certain tasks may be more efficient than a group of individuals that perform all tasks separately. A cooperating group can thus reach a higher reproduction than a group of non-cooperating individuals. However, even though cooperation increases fitness at the level of the group, it is difficult to explain the evolution of cooperation: within a cooperative group, non-cooperative individuals, which still do profit from other, cooperating, individuals will have more resources to spend on reproduction, and thus are predicted to have a higher fitness. Natural selection will thus select these cheaters until there are no cooperating individuals left.

    Hamilton’s kin-selection theory predicts that stable cooperation can evolve when cooperation is directed with a higher probability towards genetically related individuals (kin) than towards unrelated individuals. In his formulized condition (rB - C > 0), cooperation is stable when the cost of helping (expressed as the number of offspring not produced because of the cooperative allele) is lower than the benefit (the number of additional offspring as a consequence of the cooperative allele), multiplied by the average relatedness of the individuals that receive the help. Kin-selection theory is not the only theory that can explain cooperation, but is generally accepted as an important factor in many forms of cooperation. One mechanism to direct help preferentially towards kin is population viscosity. Little dispersal results in progeny and ancestors staying close together in a group. If individuals are more motile, active kin discrimination becomes important. In order to direct cooperation preferentially towards kin, many organisms have developed specialized genetic kin recognition mechanisms, based on one or more polymorphic recognition loci. These organisms only cooperate with individuals that partially or fully match their own recognition genes. Crozier made a model based on marine invertebrates that form colonies. When colonies are close to each other they cooperatively fuse with neighbouring colonies when they are clonally related, or actively compete when they are less related. The decision to cooperate is based on genetic allorecognition. Crozier’s model predicted that if fusion increases fitness, common alleles will be favoured since individuals with common recognition alleles will fuse more often. This will lead to selection of the most frequent recognition alleles until recognition polymorphism disappears completely. Thus, there is a cost of allorecognition that may reduce the genetic variation upon which allorecognition crucially relies, a prediction now known as ‘Crozier’s paradox’. An important hypothesis that can solve this paradox is to incorporate the effect of cheating. Cheating will lead to a cost to the group of cooperating individuals and therefore can impose selection pressure to maintain allorecognition. Another hypothesis is that allorecognition diversity may be selected for another function.

    Multicellularity is an extreme example of cooperation: the cells of an individual usually show division of labour, and altruism is strong because only a fraction of the cells reach the germline. A multicellular individual thus essentially is a cooperating group of cells, and evolution acts at different levels. The multicellular individual gets selected based on its fitness compared to the multicellular individuals it competes with, while at the same time the cells within the multicellular individual are under natural selection. This can lead to a potential conflict, where cheating cells evolve that have a higher fitness relative to other cells within the individual, but at the same time reduce the fitness of the multicellular individual. Theory predicts that a high relatedness among the cells of an individual reduces the opportunities for such cheating cells. Consistent with this hypothesis, there are some important mechanisms, which maintain high relatedness observed in multicellular organisms. One mechanism is regular single-celled bottlenecks in the lifecycle such as spores or seeds or zygotes from which multicellular individuals develop by mitotic division. Another important mechanism to maintain the high relatedness after the single-celled bottleneck is allorecognition to prevent fusion with non-self cells. Allorecognition is found in most multicellular organisms, but seems most relevant for organisms in which fusion between individuals or aggregation of cells is a notable part of their lifecycle.

    In this thesis, I have used filamentous ascomycete fungi as a model for the evolution of stable multicellularity and allorecognition. The fungi have regular single-celled bottlenecks in the form of spore formation, from which they develop by clonal division of the nuclei to form a tubular network known as the fungal mycelium or colony. An interesting aspect of fungal growth is that the mycelium is not clearly divided into cell compartments, with the result that cytoplasm and nuclei can freely move through parts of the colony. This implies that organelles (nuclei, mitochondria), and not cells, are the main potential selective unit below the individual. Another important feature of fungi is that fungal colonies can fuse. Whether neighbouring colonies fuse or reject each other is determined by a highly polymorphic genetic allorecognition mechanism.

    In this thesis, I have used these fungi to address the theoretical problem identified by Crozier, the evolutionary stability of genetic kin recognition. We first tested whether fusion between colonies indeed is beneficial compared to allorecognition, and whether this can lead to erosion of allorecognition diversity (chapter 2). We used the ascomycete fungus Neurospora crassa, a well-established model species for genetic research, of which numerous strains are available of different allotype. We found that cultures grown from a single allotype have a higher spore production than cultures grown from a mixture of different allotypes. This shows that fusion is beneficial relative to allorecognition. We determined the precise causes of this relative cost of allorecognition, by using a fusion mutant that partially mimics the effect of allorecognition. Colonies remain separated from each other, similar to colonies separated due to allorecognition. However, in contrast to confrontations between colonies with a different allotype, in which part of the mycelium is sacrificed in a cell death reaction, in confrontations between different colonies of the fusion mutant, there is no active rejection. This comparison showed that the benefit of fusion is due not only to absence of mutual antagonism, which occurs upon allorecognition, but also to an increase in colony size per se. We then experimentally demonstrated that the benefit of fusion selects against allorecognition diversity, as predicted by Crozier. We show that there is a positive correlation between the frequency of an allotype and its competitive fitness, thus showing that positive frequency dependent selection acts on allotype diversity, thus leading to erosion of allotype diversity.

    In the remaining part of the thesis, I have used different ascomycete fungus models to test various hypotheses to explain the evolutionary stability of allorecognition. One hypothesis considers allorecognition as a means to protect against cheating genotypes, genotypes that have a competitive advantage in combination with a wildtype genotype, but that reduce total reproductive output (chapter 3). According to recent theoretical models that simulate the evolution of allorecognition in combination with the possibility of somatic cheating, high allorecognition diversity can evolve in combination with low frequencies of cheating. The main condition is that cheating can evolve from cooperative genotypes. In order to test the hypothesis that cheating is a realistic threat to multicellular growth in fungi, we used an experimental evolution approach with N. crassa that maximised the potential for cheating genotypes by selecting under low relatedness, conditions: a high inoculation density, complete mixing at each transfer and in the absence of allorecognition. Within less than 300 generations, all eight replicate lines we evolved under these conditions significantly decreased their average asexual spore production. This yield reduction was caused by genotypes that matched the criteria for cheating: they had increased competitive fitness relative to a cooperative ancestral type, but spore production was significantly decreased when grown in mono culture or together with a cooperative type. A parallel control experiment, in which relatedness was kept high within the colony by using a fusion mutant, did not result in a reduction in asexual spore yield, showing that maintaining high relatedness provides efficient protection against cheating. From these results we can conclude that cheating can evolve quickly from cooperative genotypes, but that cheating only is selected when relatedness is low. This explains that cheating genotypes are generally not picked up from nature, since relatedness will usually be higher under natural conditions. First, the extremely high density used in our experiments is unlikely to occur in nature, so that there is more clonal outgrowth relative to fusion. Second, the high diversity of allorecognition alleles observed in nature will increase the average relatedness among the nuclei of a single individual. At the same time, the threat of cheating creates selection pressure to maintain allorecognition.

    A different hypothesis, specific to fungi, is the possibility that allorecognition provides protection against cytoplasmic cheaters (chapter 4). Usually, mitochondria are restricted in their movement by cell compartments, so that there is selection at the level below that of the cells. In fungi, mitochondria can move through the mycelium similar to the nuclei. For this reason, mitochondria can be selected within a fungal colony similar to the way nuclei can be selected within a fungal colony. We studied the evolutionary dynamics of mutant mitochondria that cause senescence in Neurospora intermedia, a species closely related to N. crassa. The mitochondria mutate under the influence of a natural occurring mitochondrial plasmid that acts as a mutagen. The mutated mitochondria have a selective advantage within the fungal colony, which allows them to increase in frequency at the cost of colony fitness. Once the mutated mitochondria reach a high frequency, the colony dies. Therefore, these mitochondrial mutants are typical cheaters, which increase their own relative fitness at the cost of the colony. We performed evolution experiments where we varied relatedness by varying fusion and bottleneck size. We show that reduction of the bottleneck size reduces the predictability of selection of mutant mitochondria. Then, we show that evolution with a fusion mutant effectively selects against mutant mitochondria and prevents senescence of the cultures. In a following experiment we then show that allorecognition can prevent or delay senescence in a similar way as what happens in cultures with a fusion mutant. These experiments confirmed that cheating mitochondrial genotypes provide a realistic threat to fungal multicellularity and that allorecognition can help keeping these mutants at a low frequency.

    Although the selective pressure by cheating appears to be sufficient to maintain the allorecognition diversity observed in fungi, it does not exclude the hypothesis that allorecognition diversity can also be the result of selection for another function. In chapter 5, I describe the highly polymorphic ­het-c locus in Podospora anserina. The het-c locus determines allorecognition together with two unlinked loci termed het-d and het-e. Each het-c allele is incompatible with a specific subset of the het-d and het-e alleles. We found that the het-c allorecognition gene is under diversifying selection and more polymorphic than most other fungal allorecognition genes. Several aspects hint to a possible function in pathogen recognition for the het-c, het-d and het-e allorecognition system, such as its high variability and structural and sequence homologies to plant defence genes. Therefore, we argue that diversity in these genes may be selected for both maintaining allorecognition and pathogen recognition. The characteristics of these genes seem an exception and have not been found for other fungal allorecognition genes. The functioning of these genes in pathogen recognition and defence remains to be demonstrated. So although these results are interesting, cheating remains the most probable solution to explain the evolution of allorecognition diversity.

    The results described in this thesis emphasize the influence of somatic cheating on the evolution of allorecognition in fungi. Fungi are economically and medically very important for society. Therefore, the results described in this thesis are very useful since they give new insight in how high relatedness can keep fungal growth stable if this is desired and how cheating might be useful to use against undesired fungal growth. Finally, I discuss that cheating is a risk in most multicellular organisms and that allorecognition is very important to prevent such cheating genotypes from spreading between individuals.

    Study of natural variation for Zn deficiency tolerance in Arabidopsis thaliana
    Campos, A.C.A.L. - \ 2015
    Wageningen University. Promotor(en): Maarten Koornneef, co-promotor(en): Mark Aarts. - Wageningen : Wageningen University - ISBN 9789462572515 - 232
    arabidopsis thaliana - voedingsstoffentekorten - sporenelementtekorten - zink - genetische variatie - tolerantie - variatie - genetica - arabidopsis thaliana - nutrient deficiencies - trace element deficiencies - zinc - genetic variation - tolerance - variation - genetics

    English summary

    Zinc is an important structural component and co-factor of proteins in all living organisms. The model plant species for genetic and molecular studies, Arabidopsis thaliana, expresses more than 2,000 proteins with one or more Zn binding domains. Low Zn availability in arable soils is a widespread problem around the world which results in agricultural losses and the production of grains with low Zn content. The long-term consumption of low-Zn-content food items leads to severe health problems in humans as a result of severe or mild dietary Zn deficiency. Hence the importance of studying Zn homeostasis in plants and mechanisms involved in Zn deficiency tolerance aiming to enhance Zn concentration in plants edible parts and to develop varieties with a higher tolerance to Zn deficiency.

    Plants are sessile organisms which trough evolution have developed specific traits in order to adapt to certain environmental conditions in their surroundings. As a result some plant genotypes are more tolerant to Zn deficiency and when exposed to low Zn conditions are able to perform better than others. To investigate the physiological mechanisms involved in Zn deficiency tolerance I examined natural variation present in a set of twenty diverse Arabidopsis thaliana accessions. In chapter 2, differences in shoot biomass production, Zn usage index (ZnUI), ionome (concentration of elements) and expression level of six key Zn deficiency responsive genes were studied. Accessions did not show large natural variation for shoot Zn concentration under Zn deficiency, while the decreases in shoot biomass and ZnUI were more variable. The conclusion from this is that accessions differ for the minimum Zn concentration required for growth which is associated with differences in Zn deficiency tolerance. We also found that the gene expression levels of three Zn transmembrane transporters (IRT3, ZIP3 and 4) in shoot were positively correlated with ZnUI and shoot biomass, but negatively correlated with shoot Zn concentration. This implies that a higher tolerance to Zn deficiency in A. thaliana is associated with an increased Zn translocation from root to shoot under low Zn. Furthermore, I used a logistic regression model to demonstrate that differences in the shoot ionome can be used as a biomarker to identify the plant Zn physiological state. Based on the changes in the concentrations of some elements in each of the Zn deficiency treatments it was possible to predict the Zn physiological state of the plants similarly to when Zn concentration is used alone.

    The adaptive response to Zn deficiency involves physiological changes in shoots, but also in roots which play a key role in the acquisition of nutrients. In chapter 3 I used the same twenty A. thaliana accessions as described in chapter 2 to identify root system architecture traits and changes in the root ionome involved in a higher tolerance to Zn deficiency in plants. Similar to shoots, all accessions showed a strong reduction in root Zn concentration under Zn deficiency, whereas changes in other root system architecture traits were more variable between the accessions. These analyses showed that differences between the accessions in root system architecture traits and minimum Zn concentration required for growth are important for Zn deficiency tolerance. The Zn deficiency treatment also affects the formation of lateral roots and thus root system architecture. It was therefore not surprising that the Zn deficiency treatment induced changes in the concentrations of other elements which were correlated with changes in root traits.

    Plants respond to different concentrations of Zn supply by changing the expression levels of genes involved in the Zn homeostasis network. This is important for the control of the Zn concentration and sequestration in plant cells, tissues and organs and involves the uptake, accumulation, transport and redistribution of Zn within the plant. Based on the work described in chapter 2, three A. thaliana accessions were selected with contrasting tolerance to Zn deficiency, and used for a whole genome transcription profiling analysis using RNA sequencing. Chapter 4 describes the identification of sets of general and core genes used by A. thaliana in its response to Zn deficiency. The purpose of using three accessions was to complement previous studies, which used only one accession, and identify new candidate genes involved in the general response to Zn deficiency in A. thaliana. General transcriptional changes were observed in the regulation of carbohydrate metabolism, glucosinolate biosynthesis and the circadian clock. As the transcriptional changes were recorded at two time points, it was also possible to distinguish early and late responses to Zn deficiency. The early response to Zn deficiency was stronger in roots with the induction of several Zn homeostasis genes and repression of Fe uptake genes. The late response to Zn deficiency comprised of the strong induction of several Zn uptake, transport and remobilization genes in both roots and shoots. These analysis confirmed several genes previously identified in Col-0 to have a general role in the Zn deficiency response, but it also led to the identification of new candidate genes, such as defensins and defensin-like genes, as very promising new actors in the A. thaliana Zn deficiency homeostasis network.

    Chapter 5 describes the A. thaliana accession-specific Zn deficiency responsive transcript profiles, comparing Tsu-0, Pa-2 and Col-0, with the aim to identify biological processes involved in the observed differences in Zn deficiency tolerance between these three accessions. Tsu-0 displayed a high tolerance to Zn deficiency in shoot, Col-0 (reference accession) showed a high tolerance to Zn deficiency in both root and shoot, whereas Pa-2 root and shoot were more sensitive to Zn deficiency. Some of the accession-specific Zn deficiency responsive transcripts were involved in similar biological processes, such as defence response, programmed cell death and carbohydrates and glucosinolates metabolism. The differential regulation of these processes between the three accessions may reflect their differences in Zn deficiency tolerance. Among the Col-0 specific transcripts were several genes encoding proteins kinases which may play a role in a more specific separation of the abiotic and biotic stress responses in this accession and possibly involved in its higher tolerance to Zn deficiency in both shoots and roots. Tsu-0 specifically changes the expression of a set of shoot transcripts encoding ethylene responsive transcription factors which are involved in the regulation of shoot growth and plant tolerance to abiotic and biotic stresses, corresponding well with the observed shoot Zn deficiency tolerance. Accession Pa-2 down-regulated transcripts involved in cell wall organization in roots which correlates with its high sensitivity to Zn deficiency in this organ. Finally, the accessions specific response to Zn deficiency also resulted in the differential regulation of transcripts encoding transposases which may reflect large scale chromatin reorganization or demethylation in response to the stress condition.

    The main findings of the research described in this thesis and their implications are described in the General Discussion (chapter 6). By investigating the response to Zn deficiency in a diverse set of A. thaliana accessions both at the physiological and transcriptional level important mechanisms involved in Zn deficiency tolerance were identified. Furthermore, several key candidate genes among the accessions general and accession-specific Zn deficiency responsive transcripts were identified. The further functional characterization of these genes is expected to reveal important new steps in the regulation of Zn homeostasis and Zn deficiency tolerance in A. thaliana.

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