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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    Persistent organic pollutants : aberrant DNA methylation underlying potential health effects
    Dungen, M.W. van den - \ 2016
    Wageningen University. Promotor(en): Tinka Murk; Ellen Kampman, co-promotor(en): Wilma Steegenga; Dieuwertje van Gils-Kok. - Wageningen : Wageningen University - ISBN 9789462577893 - 207
    persistent organic pollutants - dna methylation - molecular genetics - epigenetics - health hazards - toxic substances - endocrine disruptors - eels - fish consumption - toxicology - persistente organische verontreinigende stoffen - dna-methylering - moleculaire genetica - epigenetica - gezondheidsgevaren - toxische stoffen - hormoonverstoorders - palingen - visconsumptie - toxicologie

    Wild caught fish, especially marine fish, can contain high levels of persistent organic pollutants (POPs). In the Netherlands, especially eel from the main rivers have high POP levels. This led to a ban in 2011 on eel fishing due to health concerns. Many of the marine POPs have been related to adverse health effects such as endocrine disruption, neurodevelopmental problems, immune suppression and cancer. Although some mechanisms of action of POPs are clear, like dioxins binding to the aryl hydrocarbon receptor and OH-PCBs binding to thyroid transport proteins, not all adverse health effects can be explained by these mechanisms of action. Epigenetic phenomena, such as DNA methylation, have been proposed as a possible molecular mechanism underlying adverse health effects. DNA methylation is a heritable modification, which refers to the addition of a methyl group to cytosine in a CpG dinucleotide. Observational studies have indeed shown that POPs can affect global DNA methylation, although results are inconsistent. Some animal studies as well as in vitro experiments suggest that POPs can affect gene-specific DNA methylation, however, the biological significance and relevance for humans is not clear. Therefore, this thesis aimed to 1) study the accumulation of POPs in men consuming eel from high-polluted areas 2) elucidate whether seafood-related POPs can induce aberrant DNA methylation and 3) to determine whether DNA methylation is related to functional endpoints and gene expression in vitro.

    For this purpose eight POPs that are abundantly present in seafood were chosen, namely 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), polychlorobiphenyl (PCB) 126 and 153, perfluorooctanesulfonic acid (PFOS), hexabromocyclododecane (HBCD), 2,2′,4,4′- tetrabromodiphenyl ether (BDE-47), tributyltin (TBT), and methylmercury (MeHg). Chapter 2 describes the in vitro effects of these POPs and mixtures thereof in H295R adrenocortical carcinoma cells. Relative responses for 13 steroid hormones and 7 genes involved in the steroidogenic pathway, and CYP1A1, were analysed. PFOS induced the most pronounced effects on steroid hormone levels by significantly affecting 9 out of 13 hormone levels measured, with the largest increases found for 17β-estradiol, corticosterone, and cortisol. Furthermore, TCDD, both PCBs, and TBT significantly altered steroidogenesis. Increased steroid hormone levels were accompanied by related increased gene expression levels. The differently expressed genes were MC2R, CYP11B1, CYP11B2, and CYP19A1 and changes in gene expression levels were more sensitive than changes in hormone levels. The POP mixtures tested showed mostly additive effects, especially for DHEA and 17β-estradiol levels. This study shows that some seafood POPs are capable of altering steroidogenesis in H295R cells at concentrations that mixtures might reach in human blood, suggesting that adverse health effects cannot be excluded. DNA methylation was not measured in this study due to the short exposure time, which was expected not to be sufficient for long-term epigenetic marks. Therefore, in chapters 3A and 3B a differentiation experiment was performed enabling long-term exposure to POPs. Human mesenchymal stem cells (hMSCs) were differentiated into mature adipocytes over a time-course of 10 days. The transcriptional regulatory cascade involved in adipocyte differentiation has been extensively studied, however the mechanisms driving the transcription are poorly understood. In chapter 3A we therefore first explored the involvement of DNA methylation in transcriptional regulation during adipocyte differentiation. Genome-wide changes in DNA methylation were measured as well as the expression of adipogenic genes. The majority of these genes showed significant expression changes during the differentiation process. There were, however, only a couple of these differentially expressed genes that were differentially methylated. Genome-wide DNA methylation changes were most often located in intergenic regions, and underrepresented close to the transcription start site. This suggested that changes in DNA methylation are not the underlying mechanism regulating gene expression during adipocyte differentiation. Nevertheless, we explored DNA methylation differences after continuous exposure to POPs to investigate whether this could be an underlying mechanism by which POPs affect adipocyte differentiation. TCDD and PFOS decreased lipid accumulation, while TBT increased lipid accumulation. TCDD and TBT induced opposite gene expression profiles, whereas after PFOS exposure gene expression remained relatively stable. Genome-wide DNA methylation analysis showed that all three POPs affected DNA methylation patterns in adipogenic and other genes, but without concomitant gene expression changes. Differential methylation was again predominantly detected in intergenic regions, where the biological relevance of alterations in DNA methylation is unclear. This study demonstrated that POPs, at environmentally relevant levels, are able to induce differential DNA methylation in differentiating adipocytes. However, the biological relevance of this aberrant DNA methylation remains unclear.

    The in vitro results showed a proof of principle that POPs could be capable of altering DNA methylation. To this date, no human studies were performed investigating the relationship between POP levels and genome-wide DNA methylation. In order to investigate this, we first measured POP levels in eel consumers from the high-polluted areas (areas with a ban on eel fishing) and compared these levels to men consuming eel from low-polluted areas or aquaculture (chapter 4). We aimed to investigate the accumulation of these POPs and determine whether the predictions made in an earlier risk assessment were valid. This was indeed the case as levels of dioxins and dioxin-like compounds were on average 2.5 times higher in men consuming eel from high-polluted areas. Furthermore, PCBs with their hydroxylated metabolites, and perfluoroalkyl substances (PFASs) were, up to ten times, higher in these consumers. Especially the high levels of dioxins and dioxin-like compounds as well as the OH-PCBs are expected to be of health concern. We continued this research in chapter 5 by associating all the measured POPs to clinical parameters related to e.g. thyroid hormones and liver enzymes, but found no relationship. Subsequently, we investigated the association between dioxins and dioxin-like compounds, the sum of seven indicator PCBs, and PFOS with genome-wide DNA methylation. We detected a number of differentially methylated regions (DMRs) related to genes involved in carcinogenesis (e.g. BRCA1, MAGEE2, HOXA5), the immune system (e.g. RNF39, HLA-DQB1), in retinol homeostasis (DHRS4L2), or in metabolism (CYP1A1). In contrast to the in vitro data, most significant effects were detected in CpG islands and were annotated close to the promoter region. This suggests that the differential methylation might be related to differential expression and possibly induce adverse health effects. The hypermethylation of some of these gene related to cancer could be an explanation of the carcinogenic effects that are observed with POP exposure.

    Based on the results of this thesis we can conclude that the consumption of eel from high-polluted areas lead to accumulation of POPs above safe levels and that POP levels are associated with gene-specific DNA methylation in vitro as well as in environmentally exposed men. More research, however, is needed to fully elucidate the biological implications of this aberrant DNA methylation. A first step can be to measure histone modifications, as these two epigenetic marks together are likely better in predicting gene expression. The second step can be to investigate the potential health effects related to these epigenetic marks and to determine whether there is a causal relationship. Although at this point there is a lack of knowledge with regard to health effects caused by DNA methylation, the consumption of eel from these high-polluted areas is ill- advised, because adverse health effects cannot be excluded based on our results and can even be expected based on literature.

    Using genetic transformation to produce enhanced levels of erucic acid and novel wax esters in Crambe abyssinica
    Qi, W. - \ 2014
    Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Robert van Loo; Frans Krens. - Wageningen : Wageningen University - ISBN 9789462570368 - 117
    genetische modificatie - moleculaire veredeling - moleculaire genetica - crambe abyssinica - olieleverende planten - industriële gewassen - biobased economy - erucazuur - esters - genetic engineering - molecular breeding - molecular genetics - crambe abyssinica - oil plants - industrial crops - biobased economy - erucic acid - esters
    Crambe abyssinica (crambe) is een oliehoudend gewas dat grote potentie heeft als industrieel uitgangsmateriaal. In het onderzoek beschreven in dit proefschrift werd een solide transformatieprotocol ontwikkeld voor crambe. Om tegemoet te komen aan de eisen voor introductie in het milieu die de EU stelt aan GM gewassen en om de kans op acceptatie door het publiek te vergroten is het 'merker-vrije' genetische vector systeem dat door Wageningen UR Plant Breeding is ontwikkeld in crambe toegepast en uitgetest. Verschillende transformatie experimenten zijn uitgevoerd om het gehalte erucazuur in de zaadolie van crambe te verhogen. Ten slotte is GM crambe gebruikt als productieplatform voor hoogwaardige, industriële wasesters van het type jojoba wasesters.
    Vergelijkende genoomanalyse geeft inzicht in de evolutie en biologie van pathogene oömyceten
    Seidl, M.F. ; Govers, F. - \ 2013
    Gewasbescherming 44 (2013)4. - ISSN 0166-6495 - p. 109 - 112.
    genomica - oömyceten - pathogenen - biologie - evolutie - moleculaire genetica - plantenziekteverwekkende schimmels - bio-informatica - plantenziekten - phytophthora infestans - genomics - oomycetes - pathogens - biology - evolution - molecular genetics - plant pathogenic fungi - bioinformatics - plant diseases - phytophthora infestans
    Hoewel oömyceten nog maar kortgeleden het genomica-tijdperk zijn binnengetreden hebben de nieuwe ‘-omics’-technieken al geleid tot een overvloed aan kwantitatieve data. Vergelijkende en geïntegreerde genomica is cruciaal om deze schatkist met data te ontsluiten. In het proefschrift ‘Exploring Evolution and Biology of Oomycetes: Integrative and Comparative Genomics’ zijn met succes de eerste stappen gezet om deze data te gebruiken om zodoende de evolutie en biologie van oömyceten verder te ontrafelen en dit heeft reeds geleid tot waardevole nieuwe inzichten.
    Phylogenetic relationships within major nematode clades based on multiple molecular markers
    Rybarczyk-Mydlowska, K. - \ 2013
    Wageningen University. Promotor(en): Jaap Bakker, co-promotor(en): Hans Helder; Geert Smant. - [S.l. : s.n. - ISBN 9789461736529 - 125
    vrijlevende nematoden - dorylaimidae - mononchidae - aphelenchidae - fylogenetica - moleculaire merkers - moleculaire genetica - free living nematodes - dorylaimidae - mononchidae - aphelenchidae - phylogenetics - molecular markers - molecular genetics

    Nematodes are probably the most abundant Metazoans on our planet. They are present in densities of millions of individuals per square meter in soil and sediments. However, these inconspicuous animals are hardly known to the general public as most individuals are colorless and smaller than 1 mm. If people are aware of nematodes, it is because of the damage they inflict on humans and animals such as elephantiasis (Wucheria bancrofti) and ascariasis (in humans Ascaris lubricoides; in pigs Ascaris suum), or on plants such as potato (potato cyst nematode) and tomato (root-knot nematodes). To a far lesser extent it is known that majority of nematodes are key players in the soil food web, and as such they can be used as indicators for the biological condition of the environment they live in.

    This PhD thesis focuses on terrestrial nematodes belonging to four orders: Dorylaimida, Mononchida, Aphelenchida and Tylenchida, which represent animals of ecological and economical relevance. Members of families belonging to the first two are highly sensitive to the environmental disturbances, and are informative as biological indicators. The order Aphelenchida harbors numerous facultative plant-parasitic species. In the absence of a host plant, most of them are able to feed on fungi as an alternative food source. This is in contrast to the distal representatives of the order Tylenchida that are invariably obligate parasites of higher plants.

    Stress-sensitive nematode orders Dorylaimida and Mononchida have a high potential for soil health assessment. The SSU rDNA-based analysis of these two orders resulted in two highly distinct phylogenies. Relationships among the Mononchida, an order dominated by carnivorous nematodes, were to some extent in accordance with the classical nematode systematics. It is noted that the families Mylonchulidae, Mononchidae and Anatonchidae are not monophyletic. Nevertheless, it was possible to design family-specific primers for rDNA-based molecular detection. On the contrary, resolution of the SSU rDNA tree of the Dorylaimida was extremely poor, except for the plant-parasitic family Longidoridae and the mainly predaceous family Nygolaimidae. Analysis of a 1,000 bp fragment of the 5’ region of large subunit (LSU) rDNA resulted in an improved resolution. Twelve subclades were distinguished and this topology was only in slight agreement with the classical systematics of the suborder Dorylaimina. The poor resolution generated by SSU rDNA sequence analysis within this species-rich suborder is remarkable; it has not been observed in any other suborder in the phylum Nematoda. Possibly, Dorylaimina diversification is the result of rapid speciation events.

    A plant-parasitic lifestyle apparently accelerates the rate of change of rDNA genes. This was not only true for the obligate plant-parasitic Longidoridae, but also for the facultative plant-parasitic Aphelenchoididae. Most members of the genus Aphelenchoides are fungivores, but a few of them feed on higher plants as well. As they feed on aboveground parts of higher plants they are usually called ‘foliar nematodes’. Species such as Aphelenchoides besseyi, A. fragariae and A. ritzemabosi parasitize on ornamental plants in greenhouses and nurseries, and some field crops such as rice or strawberry. Moreover, A. subtenuis causes serious damage infecting flower bulbs. Identification of foliar nematode species, and the distinction between plant- parasitic species and other, mostly harmless, fungal feeding representatives of the genus Aphelenchoides is hampered by the scarcity of informative morphological characters and lack of well-established systematics. Based on nearly full-length SSU rDNA sequences, a phylogenetic tree was generated, where the four target species appeared as distinct, well-supported, monophyletic groups. The presence of species-specific DNA motifs made it possible to design PCR primers for the detection and quantification of the foliar nematode species in complex DNA backgrounds such as plant material and soil samples.

    The order Tylenchida is dominated by obligatory plant-parasitic nematode taxa and includes economically high-impact species such as the lesion (Pratylenchus spp.), root-knot (Meloidgyne spp.) and cyst (Heterodera, Globodera) nematodes. The sequence diversification of cellulases, a non-neutral, plant pathogenicity-related genes, was investigated. Unlike by far most other animals, nematodes do not depend on endosymbionts for the production of cell wall-degrading enzymes. Among a repertoire of these proteins, glycoside hydrolase family 5 (GHF5) cellulases are best studied. It is hypothesized that they were acquired by one or multiple horizontal gene transfer (HGT) events. Moreover, the nature of the donor - hypothesized to be a plant-parasitic soil bacterium - and the recipient, possibly a bacterivorous nematode is fully unclear. Using a range of primers, partial GHF5 cellulase sequences spanning the core catalytic domain were amplified and sequenced from basal Meloidogyne, and a range of Pratylenchus and Hirschmanniella species. Phylogenetic analysis of more than 100 partial GHF5 cellulase sequences resulted in a division of the enzymes’ catalytic domains into three types (A, B, C). Type B was numerically dominant, and notably the P. thornei cellulase was positioned sister to all type B root-knot nematode cellulases. Moreover, the overall topology of the catalytic domain B-type showed remarkable resemblance with trees based on rDNA sequences. This analysis suggests that most likely the cellulases were passed on by ancestors of a family nowadays known as the Pratylenchidae, and root-knot and cyst nematodes did not acquire these genes directly by lateral genes transfer.

    To further elucidate the relationship between a part of the family Pratylenchidae (namely the subfamilies Pratylenchinae and Hirschmanniellinae) and the Meloidogynidae, two neutral (= pathogenicity-unrelated) genes were taken into consideration: SSU rDNA and a part of the largest subunit of the RNA polymerase II gene (rpb1). Both morphological and molecular data seem to point at root-knot nematodes being a subclade branching from the migratory endoparasites Pratylenchidae. Extension of the SSU rDNA data set - more sequences from a broader range of species – did not result in a well- resolved relationship between the Pratylenchidae and the Meloidogynidae. A switch to another gene that was previously exploited to investigate relationships within the genus Meloidogyne, a fragment of the largest subunit of RNA polymerase II sequences, did not provide us with more robust information about the evolutionary transition between lesion and root-knot nematodes. The genus Pratylenchus comprises more than 100 species. It is referred to as a stenomorphic genus since only a few subtle characteristics are used for species identification. In the current study, only a subset of these species was taken to consideration; predominantly species that are relatively well-characterized as pathogens in agro-ecosystems. It was postulated that the Pratylenchus species closest to the basal root-knot nematodes should be sought among the less well-known and agronomically less relevant lesion nematode species.

    'Intraspecific pathogen variation' Verslag KNPV/Plantum/EPS-eendagsconferentie : Wageningen, 22 januari 2013
    Folkertsma, R.T. ; Goverse, A. ; Posthuma, E. ; Gilijamse, T. - \ 2013
    Gewasbescherming 44 (2013)2. - ISSN 0166-6495 - p. 32 - 34.
    plantenziekteverwekkers - genetische variatie - genotypische variatie - dna-sequencing - moleculaire technieken - moleculaire genetica - plantenziekten - conferenties - plant pathogens - genetic variation - genetic variance - dna sequencing - molecular techniques - molecular genetics - plant diseases - conferences
    Dinsdag 22 januari 2013 werd in Wageningen een eendagsconferentie gehouden getiteld ‘Intraspecific pathogen variation - implications and opportunities’. Deze conferentie werd georganiseerd naar aanleiding van discussies over het werken met intraspecifieke variatie voor diagnostiek en veredeling binnen de Nematodenwerkgroep van de KNPV en de Isolaten-beheerwerkgroep van Plantum. Het doel van de bijeenkomst was a.) onderzoekers uit de private en de publieke sector samenbrengen om recente ontwikkelingen te bespreken in fundamentele en toegepaste aspecten van het werken met intraspecifieke variatie, en b.) het stimuleren van uitwisselen van ideeën binnen en tussen beide groepen, voor mogelijke vervolginitiatieven.
    De tomatenkaart is klaar, wat nu?
    Finkers, H.J. ; Visser, R.G.F. - \ 2013
    Kennis Online 10 (2013)mei. - p. 3 - 5.
    moleculaire genetica - plantengenetica - dna-sequencing - tomaten - rassen (planten) - plantenveredeling - genetische bronnen van plantensoorten - genomen - molecular genetics - plant genetics - dna sequencing - tomatoes - varieties - plant breeding - plant genetic resources - genomes
    In 2012 publiceerde Nature de genomische sequentie van de tomaat. Maar daarmee is het werk niet af, zegt Richard Finkers. Hij bepaalde de basenvolgorde van nog eens 150 verwanten van de modeltomaat, om plantenveredelaars in staat te stellen op zoek te gaan naar nieuwe genen in oude rassen.
    'Ui is een puzzel met 150 duizend stukjes'
    Smulders, M.J.M. - \ 2013
    Kennis Online 10 (2013)mei. - p. 12 - 12.
    moleculaire genetica - dna-sequencing - genomen - plantenveredeling - onderzoek - molecular genetics - dna sequencing - genomes - plant breeding - research
    Om zeer grote genomen in kaart te kunnen brengen, moeten eerst fundamentele vragen worden opgelost. Zonder hulp van de overheid komen die antwoord er niet, zegt René Smulders. Terwijl grondige sequencing veredeling aantoonbaar versnelt.
    Imaging genetics of seed performance
    Joosen, R.V.L. - \ 2013
    Wageningen University. Promotor(en): Linus van der Plas, co-promotor(en): Henk Hilhorst; Wilco Ligterink. - S.l. : s.n. - ISBN 9789461734976 - 196
    zaden - zaadkieming - kiemkracht - genetica - kiemrust - genomica - arabidopsis thaliana - moleculaire genetica - moleculaire biologie - loci voor kwantitatief kenmerk - seeds - seed germination - germinability - genetics - seed dormancy - genomics - arabidopsis thaliana - molecular genetics - molecular biology - quantitative trait loci

    The Netherlands has a long history of plant breeding which has resulted in a leading position in the world with respect to the sales of vegetable seeds. Nowadays high-tech methods are used for crop-production which demands high standards for the quality of the starting materials. While breeding has mainly focused on crop yield and disease resistance in the past, it now becomes equally important to create seeds that rapidly and uniformly germinate under a wide range of production environments. A better understanding of the molecular processes that are underlying seed quality is a crucial first step to enable targeted breeding. In this thesis we describe the results of new methods that were used to map the genetics of seed germination.

    For this research we have used the leading plant science model species Arabidopsis thaliana which has a short generation time and a fully sequenced genome. Further, the large scientific community working on this model species is providing a wealth of resources ranging from large collections of worldwide accessions, genetic mapping populations, mutants and knowledge about gene, protein and metabolite action. A disadvantage of using Arabidopsis is the small size of the seeds, which requires evaluation of the germination of individual seeds with the use of magnifying glasses. This problem has been solved by using image analysis to create an automated procedure to obtain detailed information for parameters such as rate, uniformity and maximum germination. This procedure, called ‘the Germinator’, is described in Chapter 2 and has been enthusiastically adopted by the seed community.

    Plants cannot walk away from the environment at which the seed is dispersed. To survive and to enable reproduction, plants adapt to the prevailing environment which results in considerable genetic variation. This ‘natural variation’ is a great resource to study the mechanisms of adaptation. In Chapter 3 we have used two distinct Arabidopsis accessions, one originating from Germany (Bayreuth) and the other from high altitude in the Pamiro-Alay Mountains in Tadjikistan (Shahdara). In contrast to the Bayreuth accession, the Shahdara accession is well adapted to survive harsh conditions and is known to be stress tolerant to a range of environments. A genetic mapping (recombinant inbred line; RIL) population, consisting of 165 lines, that was derived from these two accessions is therefore particularly suitable to locate the genomic regions with genetic differences that influence seed germination. Such genomic regions are commonly referred to as quantitative trait loci (QTL). With help of the Germinator system we were able to evaluate germination of this RIL population under many different conditions. This resulted in a description of the ‘genetic landscape of seed performance’ in which we identified many QTLs for Arabidopsis seed germination.

    QTL regions are often large and identification of the causal gene requires intensive follow up research. We therefore aimed for a high throughput analysis using modern ‘omics’ techniques to analyze differences in metabolite levels and gene expression between the lines. A method to classify and visualize the vast amount of data derived from such an approach is described in Chapter 4. The so called genetical ‘omics’ experiments are expensive and therefore often force researchers to limit their study to a single developmental stage or environment only. A novel generalized setup overcomes this limitation and was tested for metabolite level changes in Chapter 5. This setup offers a unique reduction of experimental load with minimal effect on statistical power and is of great potential in the field of system genetics. Four different developmental stages of seed germination were tested in the RIL population. This approach resulted in a large dataset for which efficient analytical procedures were lacking. Thus, Chapter 5 also includes a description of a newly developed statistical procedure to analyze this type of data. The same approach and material were used in Chapter 6 to evaluate the genetics of genome wide gene expression.

    Another approach to zoom in on the molecular mechanisms underlying seed performance is described in Chapter 7. Here, the genetic diversity was maximized by using 360 different Arabidopsis accessions which had been subjected to ultra-high density genotyping. In potential, such a genome wide association (GWA) study can provide high resolution mapping of genetic variation resulting in only a few candidate genes per association for the phenotype under study. Although we were able to replicate experiments over two years with a high level of heritability, no significant associations were found. This emphasizes the need to critically review the power of such an approach for traits that are expected to be determined by many small effect loci.

    Finally, closing in on the molecular mechanisms underlying the seed traits that we studied might be possible by a full integration of the datasets that were described in the different chapters. Two examples that show the potential and the complexity of such integration are described in the General Discussion (Chapter 8). Research focused on seed quality does not end here but has gained an impulse by the described new methods and hypotheses to continue on both the fundamental and applied level in the coming years.

    Next Generation Plant Breeding
    Goud, J.C. - \ 2012
    Gewasbescherming 43 (2012)6. - ISSN 0166-6495 - p. 188 - 190.
    plantenveredeling - plantenveredelingsmethoden - innovaties - plantengenetica - moleculaire genetica - conferenties - selectiemethoden - resistentieveredeling - plant breeding - plant breeding methods - innovations - plant genetics - molecular genetics - conferences - selection methods - resistance breeding
    Van 11-14 november 2012 vond in de Reehorst de conferentie ‘Next Generation Plant Breeding’ plaats. Tijdens deze bijeenkomst kwamen de grote uitdagingen van de toekomstige plantenveredeling aan de orde: de opkomst van nieuwe sequencing-technieken, de bijbehorende enorme hoeveelheid gegevens die geproduceerd wordt, inzicht in de genetica van de plant, de bijbehorende statistische methoden, geautomatiseerde waarneming van planten, en veel meer. Een impressie.
    Hoogleraar en aio’s gaan geheimen bol ontrafelen”.
    Immink, G.H. - \ 2012
    BloembollenVisie 2012 (2012)259. - ISSN 1571-5558 - p. 20 - 21.
    bloembollen - moleculaire genetica - plantenfysiologie - landbouwkundig onderzoek - plantenveredeling - ornamental bulbs - molecular genetics - plant physiology - agricultural research - plant breeding
    Sinds 1 november is het team van hoogleraar Richard Immink compleet. Samen met twee assistenten-in-opleiding gaat hij de komende vijf jaar na of de jeugdfase van vooral de tulp is te verkorten en de vermeerdering van tulpen is te versnellen. Daarnaast staat de genetische kant van een aantal processen centraal, zoals winterrust en bloeitijdstip.
    Rationalization of a genebank cucumber collection with SSR markers
    Dooijeweert, W. van; Treuren, R. van - \ 2012
    genenbanken - komkommers - cucumis sativus - simple sequence repeats (ssr) - moleculaire genetica - genetische merkers - gene banks - cucumbers - cucumis sativus - simple sequence repeats - molecular genetics - genetic markers
    The cucumber (Cucumis sativus) collection of the Centre for Genetic Resources, the Netherlands (CGN) consists of 937 accessions. The collection mainly includes old cultivars but also contains landraces and the crop wild relative C. sativus var. hardwickii. Passport data were updated in 2002, and used to rationalize the collection. Recently, the main part of the collection was screened for microsatellite (SSR) variation.
    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 - herpesviridae - european eels - 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.
    Met hagel op aardappels schieten
    Rietman, H. - \ 2012
    Gewasbescherming 43 (2012)3. - ISSN 0166-6495 - p. 83 - 85.
    resistentieveredeling - ziekteresistentie - phytophthora infestans - aardappelen - genetisch bepaalde resistentie - genen - genlokalisatie - moleculaire genetica - resistance breeding - disease resistance - phytophthora infestans - potatoes - genetic resistance - genes - gene location - molecular genetics
    Op 20 juni 2011 promoveerde Hendrik Rietman aan Wageningen University (WUR) op zijn proefschrift getiteld: “Putting the Phytophthora infestans genome sequence at work; multiple novel avirulence and potato resistance gene candidates revealed”. Zijn promotor, Prof. Dr. Richard G. F. Visser, en co-promotor, Dr. Ir. Vivianne G. A. A. Vleeshouwers, zijn beiden verbonden aan de leerstoelgroep Plantenveredeling van de WUR, waar ook het onderzoek werd uitgevoerd. Tevens is er samengewerkt met de leerstoelgroep Fytopathologie (WUR), het Sainsbury Lab en het James Hutton Instituut (beiden gevestigd in het Verenigd Koninkrijk). Financiering was afkomstig van het ‘Parapluplan Phytophthora’ en WUR Plantenveredeling.
    Genetic analysis of symbiosome formation
    Ovchinnikova, E. - \ 2012
    Wageningen University. Promotor(en): Ton Bisseling; I.A. Tikhonovich, co-promotor(en): Erik Limpens. - S.l. : s.n. - ISBN 9789461733610 - 147
    rhizobium - fabaceae - endosymbiose - wortelknolletjes - genregulatie - moleculaire genetica - moleculaire biologie - plant-microbe interacties - rhizobium - fabaceae - endosymbiosis - root nodules - gene regulation - molecular genetics - molecular biology - plant-microbe interactions

    Endosymbiotic interactions form a fundament of life as we know it and are characterized by the formation of new specialized membrane compartments, in which the microbes are hosted inside living plant cells. A striking example is the symbiosis between legumes and nitrogen-fixing Rhizobium bacteria (rhizobia), which represents the most important source of biologically fixed nitrogen. The accommodation of rhizobia as novel nitrogen-fixing organelles, called symbiosomes, inside the cells of a novel organ, the root nodule, forms the heart of this ecologically and agriculturally important symbiosis. Understanding how these organelles are made will be keystone to exploit this symbiosis for sustainable agriculture in the future. In this thesis, we undertook a genetic approach to identity key components that control symbiosome formation especially in the genetically well-characterized garden pea (Pisum sativum) system. At the start of this thesis, the most extensive and morphologically best-characterized collection of mutants impaired in symbiosome formation was, and currently still is, available in pea. However, the cloning of the corresponding genes is severely hampered by its large genome size and recalcitrance to genetic transformation. Therefore, we used the model legume Medicago truncatula (Medicago) as reference genome to clone pea symbiosome mutants via a synteny-based cloning approach. We focused especially on three mutants in pea, named sym33, sym41 and sym31, which are affected most early in symbiosome formation: namely blocked in the release of bacteria from cell wall bound infection threads inside root nodule cells (sym33 and sym41) or induction of the subsequent differentiation of the symbiosomes (sym31).
    In Chapter 1, a general introduction is given on the process of symbiosome formation in legume root nodules. In this introduction, we focus on mechanisms by which these new nitrogen-fixing organelles are formed and address some of the recent insights, most of which were obtained after the start of this thesis, into plant components that control this process, which have been obtained from genetic studies in pea and the model legumes Medicago and Lotus japonicus (Lotus).
    Pea is part of the Papillionoid legume subfamily and closely related to the model legume Medicago. It has been shown that there is extensive synteny between the pea and Medicago genomes, which offers an efficient strategy to clone pea gene using Medicago as intergenomic cloning vehicle. In Chapter 2, we outline this synteny-based cloning approach and the molecular tools that we created to clone the pea genes required for symbiosome formation. In addition, we describe an efficient method to obtain transgenic roots via Agrobacterium rhizogenes mediated root transformation in pea that facilitates the functional analysis of pea genes in root endosymbioses.
    In Chapter 3, we report the cloning of the pea Sym33 and Medicago SYM1 genes those mutants are most strongly impaired in their ability to form symbiosomes, i.e. the release of rhizobia from the cell wall bound infection threads. Both pea Sym33 and Medicago SYM1 encode the interacting protein of DMI3, IPD3. IPD3 was shown to interact with DMI3/CCaMK, a calcium- and calmodulin-dependent kinase that is an essential component of the common symbiotic signalling pathway for both rhizobial symbiosis and arbuscular mycorrhiza. Our data reveal a novel, key role for IPD3 in symbiosome formation and development. Further, we show that MtIPD3 is required for the expression of a nodule-specific remorin MtSYMREM1, which is required for proper infection thread growth and essential for symbiosome formation.
    In Chapter 4, we report the synteny-based cloning of the pea sym41 mutant that is also impaired in the release of the bacteria from the infection threads. We show that Sym41 represents a weak allele of the common symbiotic signalling gene PsSym19/MtDMI2, a leucine-rich repeat domain containing receptor kinase that is essential for both rhizobial and mycorrhizal endosymbioses. Sym41 contains a splice-site mutation in intron 9, by which the formation of a functional transcript is reduced by ~90%. The implication of Sym19/DMI2 together with the identified role of Sym33/IPD3 in symbiosome formation (Chapter 3) strongly indicate that rhizobia have co-opted the signalling pathway from the ancient arbuscular mycorrhiza to be hosted as new organelles inside root nodule cells.
    In Chapter 5, we describe the synteny-based mapping of pea sym31, a mutant impaired in symbiosome differentiation. By making use of the synteny with Medicago, we fine mapped the Sym31 gene to a region of ~2.5 cM, which corresponds to a <450 kb region in Medicago. In this syntenic region, one gene MtN3.1, a putative sugar transporter stands out as prime candidate to control symbiosome differentiation. We describe and discuss our efforts to determine the role of this gene in symbiosome differentiation in pea and Medicago.
    In Chapter 6, we summarize and discuss our current insight into symbiosome formation and its relation to the arbuscular mycorrhiza and we give a perspective on the future of cloning the pea genes required for endosymbioses.

    A molecular cytogenetic analysis of chromosome behavior in Lilium hybrids
    Xie, S.L. - \ 2012
    Wageningen University. Promotor(en): Richard Visser; Jaap van Tuyl, co-promotor(en): Paul Arens. - S.l. : s.n. - ISBN 9789461732248 - 115
    lilium - cytogenetica - moleculaire genetica - genetische analyse - hybriden - chromosomen - recombinatie - soortkruising - meiose - lilium - cytogenetics - molecular genetics - genetic analysis - hybrids - chromosomes - recombination - interspecific hybridization - meiosis

    Lily (Lilium) has become one of the top bulbous crops for the cut flower industry in the past two decades. The genus Lilium comprises of approximately 80 species, which have been classified into seven sections. Each section possesses distinctive phenotypic characters, such as flower color, flower shape and resistances to diseases and pests. Crosses between species in the same section are relatively easy and the resulting hybrids are in general fertile, while interspecific crosses between species from different sections are rather difficult and the resulting hybrids are in general sterile. As a result, different hybrid groups have been bred in the 20th century. Within these different hybrid groups, Longiflorum (L), Asiatic (A) and Oriental (O), which are derived from the section Leucolirion, Sinomartagon and Archelirion respectively, are of commercial importance and hence, are the most widely cultivated lilies worldwide.

    Lily hybrids provide an ideal model for molecular cytogenetic research. With the development of techniques of overcoming pre- and post- crossing barriers of interspecific crosses, as well as the application of asexual and sexual polyploidization to restore the fertility of F1 lily hybrids, combining of desirable traits from different hybrid groups has become feasible. As a result, interspecific hybridization and polyploidization have been widely used in the breeding of new cultivars of lily. These cultivars, as well as other breeding materials from interspecific crosses, facilitate the application of molecular cytogenetic analysis due to three reasons: 1) the chromosomes of lily are large enough for cytological observations; 2) genomes of different hybrid groups are homoeologous; and 3) these homoeologous genomes can be simultaneously distinguished by DNA in situ hybridization. Using these lily hybrids combined with genomic in situ hybridization (GISH) and florescence in situ hybridization (FISH), the interaction of homoeologous genomes can be studied though meiotic observation of the F1 hybrids. Meanwhile, chromosome sequential variation with relevance to crossover and chromosome rearrangements can also be observed.

    For this purpose, interspecific crosses between the Lilium longiflorum cultivar ‘White Fox’ and the Asiatic cultivar ‘Connecticut King’ were made, and some of these F1 hybrids, which show a relatively high fertility with the production of unreduced gametes, were backcrossed with an Asiatic cultivar . The meiosis of the interspecific hybrids, as well as the sexual polyploidized progenies, were analysed by GISH and FISH. In addition, one population of sexual polyploidized AOA hybrids was also analysed for the genome composition. Results showed that there was no evidence that lily allopolyploids possess any noticeable chromosome rearrangements. The equal segregation of reciprocal and non-reciprocal recombinant product showed that the intergenomic recombination in the sexual polyploidized progenies was indeed from a natural process-chiasmata formation and crossovers and hence, should not be considered as translocations as was suggested in literature for intergenomic recombination. This conclusion was further confirmed by meiotic observation of the interspecific F1 hybrids.

    Detailed meiotic observations were carried out in interspecific hybrids between Longiflorum × Asiatic groups of lilies (Lilium) which were used as parents to generate sexual polyploids with intergenomic recombination. Bivalents involving two homoeologous chromosomes, as well as unpaired univalents were the main configurations at metaphase I. However, in two genotypes, multivalents and bivalents both involving non-homologous pairing of two Asiatic chromosomes were observed. This indicated the presence of a duplication which was common to two non-homologous chromosomes in the hybrids. It is deduced that there was a reciprocal translocation in the Asiatic parent cv. ‘Connecticut King’ and these two genotypes resulted from duplication-deficiency gametes. Results from Anaphase I showed that chiasma formation involving non-sister chromatids gave rise to two strand single, two strand double, three strand double, four strand double and multiple exchanges. It is also noticeable that there was a high frequency of multiple crossovers in the genotypes with duplication, indicating a reduced crossover interference in multivalents. Beside the normal crossovers, also chromosome bridges at anaphase I of meiosis were observed. GISH and FISH painting showed that these bridges involve not only non-sister chromatids but also sister-chromatids. The bridges, without any differentiation along their length, were always accompanied by fragments with a variable size. These results indicated that the bridges, together with the accompanying fragments, were derived from U-type exchanges. Other than homologous recombination (HR), nonhomologous end joining (NHEJ) probably led to the production of bridges when repairing the double strand breaks (DSBs) during meiosis.

    Progenies from unilateral polyploidization of crosses between LA hybrids and Asiatic cultivars were predominant triploids. However, three exceptional plants, which possessed 35 normal chromosomes and a small aberrant chromosome instead of the expected normal number of 36, were observed. In all three cases the small aberrant chromosomes were isochromosomes which had obviously originated during the first backcross generation, and the length of the arms of these aberrant chromosomes were always related with the length of the short arm of the missing chromosome. Furthermore, one of these three chromosomes possessed 45S rDNA hybridization sites in the proximal positions, which resembles the short arm of the missing chromosome (chromosome 4 of L genome). Combined with the results of chromosome breakage during meiosis, centric breakage and fusion is a putative mechanism of the production of these isochromosomes. Meanwhile, two small, supernumerary or B chromosomes were detected as extra chromosomes in a tetraploid plant derived from chromosome doubling of an intersectional hybrid (2n=2x=24) between a cultivar of the Longiflorum (L) and the Trumpet (T) group. When this tetraploid LLTT hybrid was crossed with a triploid LLO hybrid (O=Oriental), the B chromosome was transmitted to 73.4% of the progenies. Based on GISH and FISH characterization it was shown that the B chromosome found consisted of two identical arms, with 5S rDNA hybridizing to the majority of it, which were flanked by normal telomeres, suggesting that this is an isochromosome.

    The results of current investigations are of practical implication for a number of reasons. Firstly, the behavior of homoeologous chromosomes during meiotic processes in lily hybrids was studied in detail, and it can be used to explain the profound genetic changes in the early generations during hybrid speciation. Secondly, some problems that go unnoticed in genetic mapping can be predicted and well explained by the occurrence of chromosome rearrangements in the parents which are used to produce the segregation population and thirdly, the discovery of U-type exchanges during meiosis and de novo isochromosomes in the backcross progenies supplies an alternative mechanism for the origin of B chromosomes.

    Waarom maakt aardappelplant knollen?
    Bachem, C.W.B. - \ 2012
    Kennis Online 9 (2012)mei. - p. 8 - 8.
    aardappelen - genomica - moleculaire genetica - genomen - genen - genexpressie - potatoes - genomics - molecular genetics - genomes - genes - gene expression
    Nu het aardappelgenoom in kaart is gebracht, kan het werk eigenlijk pas goed beginnen. ‘We kunnen doelgerichter zoeken naar genen die betrokken zijn bij resistenties en kwaliteit.’ En naar waarom de aardappel knollen vormt en zijn zusje de tomaat niet.
    Een kaart van het gigantische leliegenoom en de genetische hutspot van prei
    Arens, P. ; Scholten, O.E. - \ 2012
    Kennis Online 9 (2012)april. - p. 10 - 11.
    moleculaire genetica - dna-sequencing - genomen - lilium - allium porrum - chromosoomkaarten - molecular genetics - dna sequencing - genomes - lilium - allium porrum - chromosome maps
    Van allerlei organismen is de complete DNA-volgorde bekend. Lelieveredelaars moeten het echter nog doen met een kaart van niks. Dat komt door de onwaarschijnlijke omvang van het genoom van de bloem. Een lelie heeft tien keer meer DNA dan een mens. Niet alleen de lelie, ook prei laat zich lastig in kaart brengen. Dat komt niet alleen door de omvang van het DNA, maar vooral door de organisatie ervan. Waar mensen en veel planten van elk chromosoom twee kopieën hebben, heeft prei er vier.
    Genetical metabolomics in apples (Malus x domestica Borkh)
    Khan, S.A. - \ 2012
    Wageningen University. Promotor(en): Evert Jacobsen, co-promotor(en): Henk Schouten. - S.l. : s.n. - ISBN 9789461731371 - 184
    appels - malus - plantenveredeling - moleculaire veredeling - metabolomica - moleculaire genetica - genetische modificatie - apples - malus - plant breeding - molecular breeding - metabolomics - molecular genetics - genetic engineering

    The aim of this thesis was finding genes that control the production of potentially health beneficial metabolites in apple fruits. The approach was genetic mapping of secondary metabolites such as phenolic compounds in an F1 progeny, leading to the detection of genetic loci that controlled these metabolites. At these genetic loci candidate genes were identified, using the whole genome sequence of apple, and it was investigated whether the expression of these candidate genes in the F1 progeny correlated with the metabolite levels.

    The cultivated apple (Malus x domestica Borkh) is among the most diverse and ubiquitously cultivated fruit species. It belongs to the family of Rosaceae which includes many commercial fruit species such as pear, strawberry, cherry, peach, apricot, almond, black cherry, and crab apple. Apple has a haploid chromosome number of 17. It is a self-incompatible and highly heterozygous crop. The breeding is further hampered by the long juvenile period which makes breeding in this crop a very slow process.

    The saying “An apple a day keeps the doctor away” has encouraged many researchers to search for the “magic” ingredients found in apple. Due to the beneficial role of apple phenolics, it is also called as a “new agrochemical crop”. Apple possesses many health beneficial properties for human beings as it is a rich source of phenolic compounds.It has been associated with reducing the risks of certain diseases such as cancers, particularly prostate, liver, colon, and lung cancers, cardiovascular diseases, coronary heart diseases, asthma, type-2 diabetes, thrombotic stroke, and ischemic heart disease.

    The second chapter of this thesis describes the construction of genetic linkage maps of the parents of a segregating population derived from the cross between the cultivars ‘Prima’ and ‘Fiesta’. For this purpose the already available linkage maps, as described in this chapter, were made denser by inclusion of 240 Diversity Array Technology (DArT) markers. Thus the total number of markers for ‘Prima’ and ‘Fiesta’ integrated map reached to 820. DArT-markers are hybridization based dominant DNA-markers. DArT provides a high-throughput whole genome genotyping platform for the detection and scoring of hundreds of polymorphic loci without any need for prior sequence information. This is the first report on DArT in horticultural trees. Genetic mapping of DArT markers in two mapping populations and their integration with other marker types showed that DArT is a powerful high throughput method for obtaining accurate and reproducible marker data, at low cost per data point. This method appears to be suitable for aligning the genetic maps of different segregating populations. Sequencing of the marker clones showed that they are significantly enriched for low copy, gene rich regions.

    Chapter 3 describes metabolic diversity of Malus. Wild germplasm was compared to advanced breeding selections and to the segregating F1 population from the cross between the cultivars ‘Prima’ and ‘Fiesta’. The metabolic profiles were analyzed by means of liquid chromatography-mass spectrometry (LC-MS). LC-MS is an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography with the mass analysis capabilities of mass spectrometry. This resulted in the detection of 418 putative metabolites in the peel and 254 in the flesh. Fruits from 23 wild species, eight advanced selections and the segregating F1 population were analyzed. The data were subjected to Principle Components Analysis (PCA). Variance analysis of the first PC showed that genetic variation accounted for 96.6 % in peel and 97.4 % in flesh of the total metabolic variation. Technical variation accounted for 1.4 % and 0.8%, while environmental variation accounted for 2.0% and 1.8% in peel and flesh respectively. The genetic variation between wild genotypes was very large, compared to the advanced selections and the F1 progeny. Only 8 % of the genetic variation of the first principle component was captured by the advanced selections. This indicates strong genetic erosion during breeding. This genetic erosion was mainly caused by reduction of the levels of several flavonoids including catechin, epicatechin and procyanidins. PCA of the F1 progeny of the ‘Prima’ x ‘Fiesta’ cross showed a clear 3:1 Mendelian segregation of metabolites. These metabolites were 4.2 fold less in both peel and flesh in progeny that had inherited the recessive alleles of a gene at the top of Linkage Group16 (LG16) from the heterozygous parents.

    We found a separate group of 11 metabolites in peel and 12 in flesh. These metabolites were putatively identified as glycosylated forms of b-glycols: R-octane-1, 3-diol and its unsaturated form R-5-(Z)-octene-1, 3-diol which have a potential role in controlling infection by microorganisms and influence the aroma of some ciders. The levels of these metabolites were up to 50 fold more abundant in some progeny compared to both parents. Genetic mapping showed that this strong increase was caused by one locus at the top of LG8, in progeny that had inherited only the recessive alleles of that locus from the heterozygous parents. This research illustrates not only the strong genetic erosion in apple breeding regarding metabolic diversity, and strong reduction of flavonoids in some progeny, but also shows that inbreeding can lead to a strong increase of metabolites that were present at much lower levels in both parents and advanced selections. This loss and gain of metabolites was especially observed in case of accumulation of recessive alleles during inbreeding.

    The genetic factors controlling metabolite composition were studied in more detail in Chapter 4. We investigated the genetic factors of the quantitative variation of these potentially beneficial compounds (Chapter 3, 4), by combining the genetic maps (Chapter 2) with the LC-MS data for thesegregating F1 population from the cross ‘Prima’ x ‘Fiesta’. This resulted into metabolite quantitative trait loci (mQTLs). When using the software MetaNetwork, 669 significant mQTLs were detected: 488 in the peel and 181 mQTLs in the flesh. Four linkage groups (LGs) i.e. LG1, LG8, LG13 and LG16 were found to contain mQTL hotspots, mainly regulating metabolites that belong to the phenylpropanoid pathway. These include various metabolites i.e. sinapate hexoside, coumaroyl hexoside, phloridzin, quinic acids, phenolic esters, kaempferol glycosides, quercetin glycosides, cyanidin pnetoside, flavan-3-ols (catechin, epicatechin), and procyanidins. The genetics of annotated metabolites was studied in more detail using MapQTL®. It was found that quercetin conjugates had mQTLs on LG1 and LG13. The most important mQTL hotspot with the largest number of metabolites was, however, detected at the top of LG16: mQTLs for 32 peel-related and 17 flesh-related phenolic compounds. The metabolites that mapped in the mQTL hotspot on LG16 all belong to the phenylpropanoid pathway of secondary metabolites. These compounds showed a monogenic Mendelian inheritance in a 3:1 segregation ratio. Procyanidins dimer II was used as a representative of the numerous compounds that mapped at the LG16 mQTL hotspot. By means of graphical genotyping of this monogenic trait, a genetic window could be made in which the gene that caused the mQTL hotspot should reside. We located structural genes involved in the phenolic biosynthetic pathway, using the genetic map together with the published whole genome sequence of apple. The structural gene leucoanthocyanidin reductase (MdLAR1) was detected in the mQTL hotspot window on LG16, as were seven transcription factor genes. To our knowledge, this is the first time that a QTL analysis was performed on such a high number of metabolites in an outbreeding plant species.

    The expression of the candidate genes found in the mQTL window on LG16 was studied and discussed in Chapter 5. qPCR was used for this purpose and it was found that the expression of only the structural gene MdLAR1 was strongly positively correlated with the metabolite procyanidin dimer II content. Neither the expression profiles of other structural genes of the phenylpropanoid pathway, the transcription factor genes at the mQTL hotspot, nor of transcription factor genes outside the mQTLs hotspot, showed any significant correlation with the procyanidin dimmer II content that mapped at the mQTL hotpot. This indicates that MdLAR1 was the gene, which caused this mQTL hotspot (Chapter 5). The progeny that had inherited one or two copies of the dominant alleles (Mm, MM) showed on the average a 4.4 and 11.8 fold higher expression level of MdLAR1 respectively, compared to the progeny that had inherited the recessive alleles only (mm). This led to a 4.0 fold increase of procyanidin dimer II level at the ripe stage.

    Strikingly, at the mQTL hotspot at the top of LG16, there is also a locus that controls acidity of the ripe fruits. However, the dominant alleles for acidity appeared to be in repulsion to the dominant alleles for high metabolite levels (Chapter 6). This shows that acidity is controlled by another gene than the metabolite levels. The combination of the genetic position based on the whole apple genome sequence, annotation of potential genes, and expression profiling indicated that the malic acid transporter gene MdALMT2 was responsible for the clear differences in malic acid content and pH in mature apple fruits of the segregating F1 population. The genetic inheritance of at least one dominant allele (MaMa/Mama) of this gene sufficed for a three-fold increase of the malic acid concentration and a reduction of the pH from 4 to 3 in ripe apples, compared to the presence of only the lower expressed recessive allele (mama). This malic acid transporter gene is located at the top of LG16.Malic acid is the predominant organic acid associated with the pH in apple fruits. It is synthesized in the cytoplasm and transported into the cell vacuole. The concentration of malic acid in the cell vacuole determines the pH of the cell. pH is very important for the overall taste of many fruits, including apple, and has profound effects on the organoleptic quality of apples. The pH of mature apples was genetically mapped on LG16 in the segregating population from the cross ‘Prima’ x ‘Fiesta’. To our knowledge, this is the first time that the genetic segregation of the pH in apple is assigned to a specific gene. Further, this gene has not been reported yet in conjunction to pH of apples or other fruits. After cloning of the MdALMT2 gene, it can be used for, proof of principle, influencing the acidic of existing varieties either by silencing this gene in more acidic cultivars or by inserting this gene into the low acidic cultivars. Another step would be to develop an allele specific molecular marker for selection (Marker Assisted Selection) of the acidity of fruits already at seedling stage, five years before the trees carry fruits.

    In another study, a dominantly mutated allele of the transcription factor gene MdMYB10,including its upstream promoter, coding region and terminator sequence, was introduced by transformation into apple, strawberry and potato plants. The dominantly inherited mutant allele of MdMYB10 from apple induces anthocyanin production throughout the plant, also at the early stage after transformation. The aim was to determine whether MdMYB10 could be used as a visible selectable marker for plant transformation as an alternative to chemically selectable markers, such as kanamycin resistance. After transformation, the color of calli, shoots and well-growing plants were evaluated. Red and green shoots were harvested from apple explants and examined for the presence of the MdMYB10 gene by PCR analysis. Red shoots of apple explants always contained the MdMYB10 gene but not all MdMYB10 containing shoots were red. Strawberry plants transformed with the MdMYB10 gene showed anthocyanin accumulation in leaves and roots. No visible accumulation of anthocyanin could be observed in potato plants grown in vitro, even the ones carrying the MdMYB10 gene. However, acid methanol extracts of potato shoots or roots carrying the MdMYB10 gene contained up to four times higher anthocyanin content than control plants. Therefore, anthocyanin production as a result of the dominant MdMYB1010 gene can be used as a selectable marker for apple, strawberry and potato transformation, replacing kanamycin resistance gene such as nptII. We reported this MdMYB10 as a cisgenic selectable marker gene for apple transformation (Chapter 7). The results from all experimental chapters have been discussed in a broader sense in the general discussion (Chapter 8). The future prospectives and potential challenges in the genetical metabolomics are also highlighted. The approaches we developed in the current thesis could be used not only for developing potentially a more healthy and improved apple but can also be applied for the genetical metabolomics studies in other important crops.

    Breeding programs for indigenous chicken in Ethiopia : analysis of diversity in production systems and chicken populations
    Dana, N.M. - \ 2011
    Wageningen University. Promotor(en): Johan van Arendonk, co-promotor(en): Liesbeth van der Waaij; T. Dessie. - [S.l. : S.n. - ISBN 9789085858720 - 149
    kippen - veredelingsprogramma's - genetische verbetering - prestatiekenmerken - dorpen - ecotypen - karakterisering - moleculaire genetica - genetische diversiteit - genetische parameters - agrarische productiesystemen - ethiopië - fowls - breeding programmes - genetic improvement - performance traits - villages - ecotypes - characterization - molecular genetics - genetic diversity - genetic parameters - agricultural production systems - ethiopia
    The aim of this research was to generate information required to establish a sustainable breeding program for improving the productivity of locally adapted chickens to enhance the livelihood of rural farmers in Ethiopia. The first step was to characterize village poultry production environments and farmers’ objectives for keeping chickens, and to identify factors affecting the choice of genetic stock used in villages. This was achieved by carrying out a questionnaire survey and a participatory group discussion with village farmers in different geographic regions of Ethiopia. The low input nature of village environments, the prevalence of disease and predators, and other factors such as the use of chickens both as sources of eggs and meat, and income determined the choice of chicken breed used by farmers, and thus, should be considered carefully before initiating new breeding programs. The highest importance attached to adaptation traits and the existence of particular preferences for chickens of certain plumage colours and comb shapes were also found to have effects on developing new breeds for village systems.
    The next part of the thesis focused on identifying important and unique gene pools in local populations. This was achieved by characterizing the local chicken ecotypes both morphologically and molecular genetically. This way the genetic difference between the local populations and the level of genetic diversity within the populations was determined. Attributes important in breeding for tropical conditions such as the pea comb gene, and the naked neck gene have been identified. It was also revealed that the variability found within a single population could explain most of the genetic diversity (97%) in Ethiopian chicken populations. The result of this work is important both from conservation and utilization perspective and assists in maintaining indigenous genetic diversity for current and future generations.
    Finally, the pedigreed Horro population that was kept on station was used for estimating genetic parameters for the production traits, monthly and cumulative part period egg numbers and growth to 16 weeks of age. Because the pedigreed population was established only recently, data of only 2 generations were available for estimating these genetic parameters. The results are promising but inaccurate due to insufficient amount of data. They would need to be re-estimated when more generations have been produced and thus more data has been generated.

    Selecting Sole: breeding programs for natural - mating populations
    Blonk, R.J.W. - \ 2010
    Wageningen University. Promotor(en): Johan van Arendonk, co-promotor(en): Hans Komen. - [S.l. : S.n. - ISBN 9789085857129 - 174
    solea - tong (vis) - veredelingsprogramma's - natuurlijke paring - inteelt - heritability - fokwaarde - moleculaire genetica - stamboom - solea - dover soles - breeding programmes - natural mating - inbreeding - heritability - breeding value - molecular genetics - pedigree
    The aim of this thesis was to design a breeding program for increased productivity of farmed common sole, Solea solea, 1) using natural mating in groups to obtain offspring and 2) using present farm infrastructures as much as possible. Parental allocation with DNA marker data on offspring from natural mating parents showed that parental contributions were highly skewed. This indicates that few animals contribute to majority of the offspring. Levels of coancestry in offspring populations were high (2-4%) showing that selection methods need to restrict rates of inbreeding in future generations.
    Genetic variances of body weight and body length in common sole were measured at harvest and it was shown that genetic improvement of these traits is possible. It was pointed out that selection on growth of common sole needs to be accompanied by selection for shape to compensate for undesired correlated responses in shape. Further, it was demonstrated that in populations with skewed contributions, use of marker data can be more efficient once breeding values are estimated with continuous molecular relatedness rather than with a reconstructed pedigree. To decrease costs of breeding programs, selection of parents may be from production populations directly. Results indicated that estimation of genetic parameters is affected by husbandry practices as grading, but that effects are predictable. To optimise breeding programs with natural mating of parents, 2-stage selection schemes with optimal contribution selection from mass selected and genotyped fractions were compared with mass selection schemes. Using 2-stage selection schemes, rates of inbreeding can be restricted with a smaller nucleus than with mass selection schemes. However, response is lower as well. The different findings in this thesis are put into context of a breeding program for common sole and discussed in relation to profitability, benefits of natural mating and correlated responses to selection for growth.

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