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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Nieuwe Veredelingstechnieken NPBTs : Voorbeelden van perspectiefvolle toepassingen
Wiel, C.C.M. van de; Schaart, J.G. ; Lotz, L.A.P. - \ 2017
Wageningen : Wageningen University & Research - 33 p.
cultivation - plant breeding - plants - horticulture - greenhouse horticulture
Plant breeding and Intellectual Property Rights – a brief introduction : Plant Breeders’ rights and patents
Wiel, C.C.M. van de; Schaart, J.G. ; Lotz, L.A.P. - \ 2017
Wageningen : Wageningen University & Research - 9 p.
cultivation - plant breeding
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
University. Promotor(en): Richard Visser, co-promotor(en): Eric van de Weg; F. Costa. - Wageningen : Wageningen University - ISBN 9789463432054 - 177
malus domestica - apples - fruit - fruit growing - genetics - plant breeding - genome analysis - appels - fruitteelt - genetica - plantenveredeling - genoomanalyse

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

Coexistence of GMO production, labeling policies, and strategic firm interaction
Venus, Thomas Johann - \ 2017
University. Promotor(en): Justus Wesseler, co-promotor(en): Dusan Drabik; M.J. Punt. - Wageningen : Wageningen University - ISBN 9789463436670 - 148
genetically engineered organisms - food products - nutrition labeling - labelling - crops - plant breeding - germany - european union - regulations - markets - businesses - genetisch gemanipuleerde organismen - voedselproducten - etiketteren van voedingsmiddelen - etiketteren - gewassen - plantenveredeling - duitsland - europese unie - regelingen - markten - bedrijven

This dissertation analyzes the market effects of the coexistence of genetically modified organism (GMO) and conventional production, labeling policies, and strategic firm interactions through vertical product differentiation. Although we focus on GMOs, the applied frameworks can be adopted and extended to other differentiated products where similar concepts apply.

The main body of the dissertation consists of four chapters. In the first chapter, we estimate the perceived costs of legal requirements (‘coexistence measures’) for growing genetically modified (GM) Bt maize in Germany using a choice experiment. The costs of the evaluated ex-ante and ex-post coexistence measures range from zero to more than 300 euros per hectare per measure, and most of them are greater than the extra revenue the farmers in our survey expect from growing Bt maize or than the estimates in the literature. The cost estimates for temporal separation, which were the highest in our evaluation, imply that the exclusion of this measure in Germany is justified. The costliest measures that are currently applied in Germany are joint and strict liability for all damages. Our results further show that neighbors do not cause a problem and that opportunities for reducing costs through agreements with them exist. Finally, we find that farmers’ attitudes toward genetically modified crops affect the probability of adoption of Bt maize. Our results imply that strict liability will deter the cultivation of Bt maize in Germany unless liability issues can be addressed through other means, for example, through neighbor agreements.

The coexistence costs have implications for the supply of products in which GMOs are excluded from the production process (i.e., non-GM labeling). This is the topic of the second chapter. In that chapter, we discuss and illustrate the complexity of non-GM food labeling in Germany. We show how a multi-stakeholder organization that sets a voluntary private production and certification standard can combine the opposing and agreeing interests of its members. This cohesion reduces the fears of retailers of NGO pressure in the case of mislabeling. Whereas non-GM labeling in Germany started as a niche for farmer-to-consumer direct marketing and small processors, it was further driven by anti-GMO organizations. Today, retail chains label some of their store brands and are now the drivers. We also discuss how informing consumers through non-GM labeling addresses imperfect information, but at the same time, can create new information imperfections if consumers are not well informed about the labeling system itself.

Non-GM labeling, together with the EU-wide mandatory labeling of GMOs and their requirements on coexistence, have implications for the potential regulation of crops derived by new plant breeding techniques (NPBTs). In the third chapter, we analyze the market and welfare effects of regulating crops derived by NPBTs as genetically modified or conventional products. We consider the mandatory scheme for labeling GM products and a voluntary non-GM scheme for labeling livestock products derived from non-GM feed. We develop a partial equilibrium model that explicitly takes into account both the coexistence costs at the farm level and the segregation and identity preservation costs at the downstream level. By applying the model to EU rapeseed, we find that regulating NPBTs as GM (as compared to non-GM) in combination with mandatory and voluntary labeling increases prices and therefore makes producers better off. We also show that higher coexistence costs make the price increasing effect even stronger. Voluntary non-GM labeling applied to feed makes consumers in this sector overall worse off, but it benefits farmers and rapeseed oil consumers overall as long as segregation costs are low. Consumers of biodiesel and industrial products, such as lubricants produced from GM rapeseed, benefit from high segregation costs. We show that the effects of farm-level coexistence costs largely differ from the effects of downstream market segregation costs.

In the last of the four chapters, we consider the effects of market power and analyze the decision of investing in quality updating when high-quality product demand is growing. We model a decision of a duopoly that initially offers a product perceived as lower quality (e.g., GM product) to invest in an emerging high-quality (e.g., labeled non-GM) product. We investigate whether the smaller or the larger firm invests first. Either preemption or a war of attrition can result, depending on demand and cost factors. For each case, we derive the unique Nash equilibrium. We show that a firm’s timing to invest in high-quality production (e.g., implement a voluntary production standard) depends on several factors, such as the difference in firm size between competing firms and the level of vertical differentiation, growth and discount rate, demand parameters, and per-unit production costs. We show that institutions, which set private or public certification standards, can affect firms’ investment in differentiated products because the standard stringency affects the production and compliance costs as well as the level of product differentiation. Hence, through the setting of these standards, private and governmental institutions can impact the market structure as well as the growth of an emerging market. Finally, we discuss policy implications and how an adjustment of the EU-regulatory framework from a process- to a product-based system can make several issues discussed in this thesis problems of the past.

Genetic diversity of potato for nitrogen use efficiency under low input conditions in Ethiopia
Getahun, Baye Berihun - \ 2017
University. Promotor(en): Richard Visser, co-promotor(en): Gerard van der Linden. - Wageningen : Wageningen University - ISBN 9789463436595 - 219
solanum tuberosum - potatoes - genetic diversity - nitrogen - plant breeding - ethiopia - nutrient use efficiency - aardappelen - genetische diversiteit - stikstof - plantenveredeling - ethiopië - nutriëntengebruiksefficiëntie

Potato is a prime food security crop for smallholder farmers in the highland part of North western Ethiopia. In this region, nutrient availability, especially nitrogen (N) is a major constraint for crop productivity. To obtain insight in the possibility of improving potato for growth under low N input conditions in Ethiopia, we evaluated CxE diploid back cross population, modern European and Ethiopian potato cultivars and local Ethiopian cultivars for their ability to grow and produce tubers under low and high N input conditions. The experiments were conducted under rainfed and irrigation conditions. Eighty-eight Dutch cultivars and 9 Ethiopian cultivars were evaluated in three locations in North-western Ethiopia, in 2013 and in 2015. The two years represent two different growth seasons: rain-fed (June-October 2013) and irrigated cultivation (February-June 2015). Similarly 100 CxE diploid back cross potato genotypes were evaluated in both rainfed and irrigation production seasons in 2014. The Growth of the plants was monitored throughout the growth cycle using canopy cover measurements, with modelled canopy characteristics, and other agronomic traits were measured as per the description. The effect of season and location was further investigated by a GGE Biplot genotype-by-environment interaction analysis, and genetic factors determining phenotypic traits and yield were identified through QTL mapping and association mapping. Ethiopian cultivars showed a remarkable, environment-dependent difference in utilisation of the canopy for tuber production. While total photosynthetic capacity was higher in Ethiopian cultivars than in Dutch cultivars in rainfed production season at Injibara, tuber production was higher in Dutch cultivars. This low radiation use efficiency was not observed in the other rain-fed location (Debre-Tabor). A Genotype by Environment analysis using GGE biplots demonstrates that, Irrespective of the N levels and locations, rainfed production season test environments were grouped as one mega environment and irrigation production season test environments as the other mega environment, indicating most of the variation for yield and nitrogen use efficiency (NUE) in the dataset may be caused by the effect of rain-fed vs irrigation season. Further trials are needed to confirm this result. The QTL mapping with the CxE diploid population and GWAS analysis with the Dutch cultivars discovered both season-environment and N-specific QTL as well as constitutive QTLs. Overall, N availability affects Dutch and Ethiopian cultivars differentially, with strong environmental interaction on canopy and yield traits. Rainfed and irrigated seasons in Ethiopia may require different breeding programs for improved yield under varying fertilizer levels. Both constitutive and environment-specific QTLs were identified that may be targets for breeding prorgams towards improved yield under Ethiopian cultivation conditions.

Disentangling hexaploid genetics : towards DNA-informed breeding for postharvest performance in chrysanthemum
Geest, Geert van - \ 2017
University. Promotor(en): Richard Visser, co-promotor(en): Uulke van Meeteren; Paul Arens. - Wageningen : Wageningen University - ISBN 9789463436427 - 142
chrysanthemum - plant breeding - postharvest quality - hexaploidy - polyploidy - quantitative trait loci - phenotypes - linkage mapping - metabolomics - polymorphism - dna - plantenveredeling - kwaliteit na de oogst - hexaploïdie - polyploïdie - loci voor kwantitatief kenmerk - fenotypen - koppelingskartering - metabolomica - polymorfisme

DNA-informed selection can strongly improve the process of plant breeding. It requires the detection of DNA polymorphisms, calculation of genetic linkage, access to reliable phenotypes and methods to detect genetic loci associated with phenotypic traits of interest. Cultivated chrysanthemum is an outcrossing hexaploid with an unknown mode of inheritance. This complicates the development of resources and methods that enable the detection of trait loci. Postharvest performance is an essential trait in chrysanthemum, but is difficult to measure. This makes it an interesting but challenging trait to phenotype and detect associated genetic loci. In this thesis I describe the development of resources and methods to enable phenotyping for postharvest performance, genetic linkage map construction and detection of quantitative trait loci in hexaploid chrysanthemum.

Postharvest performance is a complicated trait because it is related to many different disorders that reduce quality. One of these disorders in chrysanthemum is disk floret degreening, which occurs after long storage. In chapter 2, we show that degreening can be prevented by feeding the flower heads with sucrose, suggesting carbohydrate starvation plays a role in the degreening process. To investigate the response to carbohydrate starvation of genotypes with different sensitivity to disk floret degreening, we investigated the metabolome of sugar-fed and carbohydrate-starved disk florets by 1H-NMR and HPAEC. We show that the metabolome is severely altered at carbohydrate starvation. In general, starvation results in an upregulation of amino acid and secondary metabolism. Underlying causes of genotypic differences explaining variation in disk floret degreening in the three investigated genotypes remained to be elucidated, but roles of regulation of respiration rate and camphor metabolism were posed as possible candidates.

In chapter 3, disk floret degreening was found to be the most important postharvest disorder after 3 weeks of storage among 44 white chrysanthemum cultivars. To investigate the inheritance of disk floret degreening, we crossed two genotypes with opposite phenotypic values of both disk floret degreening and carbohydrate content to obtain a population segregating for disk floret degreening. To phenotype the cultivar panel and the bi-parental population precisely and in a high throughput manner, we developed a method that quantified colour of detached capitula over time. This method was validated with visual observations of disk floret degreening during vase life tests. In a subset of the bi-parental population we measured carbohydrate content of the disk florets at harvest. The amount of total carbohydrates co-segregated with sensitivity to degreening, which shows that the difference in disk floret degreening sensitivity between the parents could be explained by their difference in carbohydrate content. However, the correlation was rather weak, indicating carbohydrate content is not the only factor playing a role.

In order to develop resources for DNA-informed breeding, one needs to be able to characterize DNA polymorphisms. In chapter 4, we describe the development of a genotyping array containing 183,000 single nucleotide polymorphisms (SNPs). These SNPs were acquired by sequencing the transcriptome of 13 chrysanthemum cultivars. By comparing the genomic dosage based on the SNP assay and the dosage as estimated by the read depth from the transcriptome sequencing data, we show that alleles are expressed conform the genomic dosage, which contradicts to what is often found in disomic polyploids. In line with this finding, we conclusively show that cultivated chrysanthemum exhibits genome-wide hexasomic inheritance, based on the segregation ratios of large numbers of different types of markers in two different populations.

Tools for genetic analysis in diploids are widely available, but these have limited use for polyploids. In chapter 5, we present a modular software package that enables genetic linkage map construction in tetraploids and hexaploids. Because of the modularity, functionality for other ploidy levels can be easily added. The software is written in the programming language R and we named it polymapR. It can generate genetic linkage maps from marker dosage scores in an F1 population, while taking the following steps: data inspection and filtering, linkage analysis, linkage group assignment and marker ordering. It is the first software package that can handle polysomic hexaploid and partial polysomic tetraploid data, and has advantages over other polyploid mapping software because of its scalability and cross-platform applicability.

With the marker dosage scores of the bi-parental F1 population from the genotyping array and the developed methods to perform linkage analysis we constructed an integrated genetic linkage map for the hexaploid bi-parental population described in chapter 3 and 4. We describe this process in chapter 6. With this integrated linkage map, we reconstructed the inheritance of parental haplotypes for each individual, and expressed this as identity-by-descent (IBD) probabilities. The phenotypic data on disk floret degreening sensitivity that was acquired as described in chapter 3, was used in addition to three other traits to detect quantitative trait loci (QTL). These QTL were detected based on the IBD probabilities of 1 centiMorgan intervals of each parental homologue. This enabled us to study genetic architecture by estimating the effects of each separate allele within a QTL on the trait. We showed that for many QTL the trait was affected by more than two alleles.

In chapter 7, the findings in this thesis are discussed in the context of breeding for heterogeneous traits, the implications of the mode of inheritance for breeding and the advantages and disadvantages of polyploidy in crop breeding. In conclusion, this thesis provides in general a significant step for DNA-informed breeding in polysomic hexaploids, and for postharvest performance in chrysanthemum in particular.

Prospects of whole-genome sequence data in animal and plant breeding
Binsbergen, Rianne van - \ 2017
University. Promotor(en): Roel Veerkamp; Fred van Eeuwijk, co-promotor(en): Mario Calus. - Wageningen : Wageningen University - ISBN 9789463431903 - 220
next generation sequencing - dna sequencing - quantitative trait loci - cattle - genomics - solanum lycopersicum - animal breeding - plant breeding - dna-sequencing - loci voor kwantitatief kenmerk - rundvee - genomica - dierveredeling - plantenveredeling

The rapid decrease in costs of DNA sequencing implies that whole-genome sequence data will be widely available in the coming few years. Whole-genome sequence data includes all base-pairs on the genome that show variation in the sequenced population. Consequently, it is assumed that the causal mutations (e.g. quantitative trait loci; QTL) are included, which allows testing a given trait directly for association with a QTL, and might lead to discovery of new QTL or higher accuracies in genomic predictions compared to currently available marker panels. The main aim of this thesis was to investigate the benefits of using whole-genome sequence data in breeding of animals and plants compared to currently available marker panels. First the potential and benefits of using whole-genome sequence data were studied in (dairy) cattle. Accuracy of genotype imputation to whole-genome sequence data was generally high, depending on the used marker panel. In contrast to the expectations, genomic prediction showed no advantage of using whole-genome sequence data compared to a high density marker panel. Thereafter, the use of whole-genome sequence data for QTL detection in tomato (S. Lycopersicum) was studied. In a recombinant inbred line (RIL) population, more QTL were found when using sequence data compared to a marker panel, while increasing marker density was not expected to provide additional power to detect QTL. Next to the RIL population, also in an association panel it was shown that, even with limited imputation accuracy, the power of a genome-wide association study can be improved by using whole-genome sequence data. For successful application of whole-genome sequence data in animals or plants, genotype imputation will remain important to obtain accurate sequence data for all individuals in a cost effective way. Sequence data will increase the power of QTL detection in RIL populations, association panels or outbred populations. Added value of whole-genome sequence data in genomic prediction will be limited, unless more information is known about the biological background of traits and functional annotations of DNA. Also statistical models that incorporate this information and that can efficiently handle large datasets have to be developed.

Unraveling the genetics of Botrytis cinerea resistance in Gerbera hybrida
Fu, Yiqian - \ 2017
University. Promotor(en): Richard Visser, co-promotor(en): Paul Arens; Jaap van Tuyl. - Wageningen : Wageningen University - ISBN 9789463431811 - 159
gerbera - plant pathogenic fungi - botrytis cinerea - disease resistance - genetic mapping - transcriptomics - quantitative trait loci - plant breeding - plantenziekteverwekkende schimmels - ziekteresistentie - genetische kartering - transcriptomica - loci voor kwantitatief kenmerk - plantenveredeling

Gerbera hybrida is one of the top five cut flowers. It is well-known to people for its variation in flower color and patterning. Gerbera breeding at the moment is done using conventional methods which are based on a phenotypic selection. This has drawbacks in breeding speed and efficiency, especially for complex traits like disease resistance. Gerbera gray mold, promoted by high humidity during the production in greenhouses or by an accumulation of condensate during transportation, is a considerable threat to the gerbera production. Gerbera gray mold is caused by Botrytis cinerea and plant resistance to B. cinerea is considered to be a polygenic trait that needs the contribution of multiple loci, and on top of that is highly affected by the environment. Conventional breeding might be inefficient for improving Botrytis resistance in gerbera.

In this study, the transcriptomes of four parents of two gerbera populations were sequenced using Illumina paired-end sequencing. Transcriptome data provides a resource for genetic dissection and an insight to explore gene functions for this ornamental crop. To identify the QTL regions leading to the phenotypic variation in Botrytis resistance, and establishing a relationship between marker genotype and phenotypic variation for marker assisted selection (MAS), genetic linkage maps were constructed with SNP markers in the two F1 segregating populations. A total of 20 QTLs were identified in the parental maps of the two populations. The number of QTLs found and the explained variance of most QTLs detected reflects the complex mechanism of Botrytis disease response. Narrowing down the QTL region and identifying the causal gene(s) underlying a QTL could maximize the effective use of MAS in breeding. Homologs of known functional genes involved in Botrytis resistance from other species were obtained in gerbera and SNP markers identified and mapped. Twenty-nine candidate genes were mapped and seven candidate genes could be mapped on both populations. Seven candidate genes were located in the vicinity of the QTLs detected. The co-localization of QTLs with CGs gives an indication that these candidate genes could probably be involved in resistance to Botrytis and provide a more precise possibility to use MAS in gerbera breeding in the future. A tobacco rattle virus (TRV) based gene silencing system which was used to inspect the function of two candidate genes. The two CGs are the homologs of the genes responsible for Botrytis resistance in tomato and both mapped in QTL regions related to Botrytis resistance in gerbera ray floret test. Silencing the two genes by VIGS, showed smaller lesion sizes upon Botrytis infection on gerbera ray florets compared with the controls.

The entire research went from the generation of four parental transcriptome data sets to development of SNP markers (Chapter 2), construction of genetic maps and to mapping QTLs for Botrytis resistance (Chapter 3). This was further on combined with candidate gene searching in other crops, querying and mapping homologous genes (Chapter 4) and characterizing the candidate genes which co-localized with QTLs (Chapter 5). The whole process not only helped us to unravel the genetics of Botrytis resistance in gerbera and develop genetic tools for gerbera improvement, but also could serve as guidance for developing marker-assisted selection for other ornamental plants from the beginning.

Genetic studies towards elucidation of drought tolerance of potato
Tessema, Biructa Bekele - \ 2017
University. Promotor(en): Richard Visser, co-promotor(en): Gerard van der Linden. - Wageningen : Wageningen University - ISBN 9789463431958 - 195
solanum tuberosum - potatoes - drought resistance - plant breeding - genetic analysis - quantitative trait loci - aardappelen - droogteresistentie - plantenveredeling - genetische analyse - loci voor kwantitatief kenmerk

Drought is a major threat to agricultural production, which makes drought tolerance a prime target for breeding approaches towards crop improvement. Drought is a complex polygenic trait and poses a challenge for drought tolerance breeding. Improving crops for drought tolerance at least requires the knowledge of the physiological mechanisms of the contributing traits and their genetic control. Thus, identification of genetic variation for drought tolerance is the first step towards drought tolerance breeding. The effect of drought stress on potato tuber yield and quality is very significant as potato is considered sensitive to water shortage. To understand the genetic factors underlying drought tolerance in potato, we performed drought stress experiments under green house and field conditions with moderate drought and severe drought stress conditions, respectively. In the field, potato genotypes were exposed to severe drought stress for two consecutive years starting from tuber initiation, which progressed to severe drought stress. In addition, we examined potato cultivars for moderate drought tolerance under greenhouse conditions where water application was reduced 50-60% from optimum amount starting from stolon formation. Morphological and physiological trait data were collected that allowed precise monitoring of the drought response of potato. Phenotypic data collected under severe drought stress conditions which includes traits like shoot and root biomass (fresh and dry), yield and chlorophyll content were used for QTL mapping while data collected under moderate drought stress conditions was used for genome wide association mapping. With QTL mapping, 60 QTLs were identified controlling those traits both under well-watered and drought stress conditions. In the drought tolerance evaluation of the potato cultivars under greenhouse conditions we identified significant marker trait associations for both above- and belowground traits. Many of the QTLs detected for drought tolerance traits were specific to either moderate or severe drought tolerance conditions. However, a few QTLs showed an overlap between these drought stress environments. This demonstrates the presence of common genomic regions controlling drought tolerance traits under moderate and severe drought stress conditions. In addition, from the two years of field drought stress experiments we selected a subset of genotypes that showed contrasting responses to drought stress. We used these genotypes to further examine the relationship between canopy development and tuber yield under severe drought stress conditions. Canopy development was measured for several time points and the data were used for curve fitting. From the curve-fit, parameters related to the different developmental phase of canopy were extracted. We observed that there is positive correlation between canopy parameters and tuber yield under drought stress conditions. The evaluation of potato for drought tolerance under field and greenhouse conditions has resulted in the identification of several QTLs that can be interesting to be used for enhancing drought tolerance in potato. Furthermore, the use of model derived parameters gave a better insight into the relationship between canopy development and tuber yield under water stress conditions.

Susceptibility genes : an additional source for improved resistance
Sun, Kaile - \ 2017
University. Promotor(en): Richard Visser, co-promotor(en): Evert Jacobsen; Yuling Bai. - Wageningen : Wageningen University - ISBN 9789463431415 - 174
solanum tuberosum - potatoes - solanum lycopersicum - tomatoes - genes - susceptibility - plant pathogenic fungi - phytophthora infestans - disease resistance - plant breeding - aardappelen - tomaten - genen - vatbaarheid - plantenziekteverwekkende schimmels - ziekteresistentie - plantenveredeling

Potato is affected by several diseases. Although, resistance can be obtained by introgression of major resistance genes from wild species, this has rarely been durable. Hence, other sources of resistance are highly needed. New research with a focus on loss of function mutations has led to the identification of disease susceptibility (S) genes in plants. The research in this thesis was aimed at the identification and characterization of potato S genes involved in the interaction with Phytophthora infestans and Botrytis cinerea. We selected 11 Arabidopsis thaliana S genes and silenced their potato orthologs by RNAi in the potato cultivar Desiree. The silencing of six genes resulted in resistance to P. infestans. Moreover, silencing of StDND1 reduced the infection of B. cinerea. Microscopic analysis showed that spore attachment and/or germination of P. infestans and B. cinerea was hampered on the leaf surface of StDND1-silenced potato plants. On StDMR1- and StDMR6-silenced potato plants, hyphal growth of P. infestans was arrested by the hypersensitive response-like cell death. Our results demonstrate that impairment of plant S genes may open a new way for breeding potatoes with resistance to pathogens like P. infestans and B. cinerea.

Hevige phytophthora-uitbraak 2016 benadrukt noodzaak resistente rassen
Lammerts Van Bueren, E. ; Hutten, R.C.B. ; Engelen, C.J.M. - \ 2016
Aardappelwereld 2016 (2016)9. - ISSN 0169-653X - p. 32 - 33.
phytophthora infestans - aardappelen - biologische landbouw - ziekteresistentie - resistentie van variëteiten - rassen (planten) - plantenveredeling - potatoes - organic farming - disease resistance - varietal resistance - varieties - plant breeding
De biologische aardappelteelt heeft het in 2016 weer zwaar te verduren gehad met de hevige uitbraak van Phytophtora infestans. Door aanhoudende regenval in juni en juli waren veel telers genoodzaakt om hun percelen met aangetaste aardappelen al vroeg in het seizoen te branden. Gecombineerd met de late pootdatum hebben velen een (te) lage opbrengst van hun vatbare rassen dit jaar. "DIt bevestigt nog maar weer eens de noodzaak van resistente rassen", zo stellen Edith Lammerts van Bueren, Ronald Hutten en Christel Engelen, van het project Bioimpuls waar de deelnemers hard aan nieuwe resistente rassen werken.
Genenweelde in oorsprongsgebieden essentieel voor toekomstige voedselzekerheid
Struik, Paul ; Hintum, Theo van - \ 2016
food security - genetic diversity - field crops - gene banks - plant breeding - genetic engineering - biodiversity - malus - varieties - kazakhstan - middle east

Theo van Hintum in tijdschrift Vork over genenbanken en oorsprongsgebieden als basis voor plantenveredeling

Behoud van genetische diversiteit is een belangrijk wapen om in te spelen op veranderingen in de leefomstandigheden van landbouwgewassen. Genenbanken vormen een onmisbare basis voor plantenveredeling, maar bieden onvoldoende waarborg voor toekomstige voedselzekerheid. Ook de lokale genenweelde in de oorsprongsgebieden moet behouden blijven, maar die wordt bedreigd door verstedelijking, verwaarlozing en klimaatverandering, constateert Michiel Löwik.

Susceptibility pays off: insights into the mlo-based powdery mildew resistance
Appiano, Michela - \ 2016
University. Promotor(en): Richard Visser, co-promotor(en): Yuling Bai; Anne-Marie Wolters. - Wageningen : Wageningen University - ISBN 9789462579484 - 265
solanum lycopersicum - tomatoes - disease resistance - susceptibility - oidium neolycopersici - genes - gene expression - genomics - molecular breeding - plant breeding - tomaten - ziekteresistentie - vatbaarheid - genen - genexpressie - genomica - moleculaire veredeling - plantenveredeling

Powdery mildew (PM) is a worldwide-occurring plant disease caused by ascomycete fungi of the order Erysiphales. A conspicuous number of plant species are susceptible to this disease, the occurrence of which is increasing due to the influence of climate change. Symptoms are easy to recognize by the powdery whitish fungal structures growing on the surface of plant organs. Severe infections cause significant losses in crops, such as tomato, cucumber and wheat, as well as in ornamentals, like rose and petunia. Accordingly, breeding crops with a robust immunity to this disease is of great economic importance.

A significant step in this direction was the discovery of mlo (mildew locus o) mutant alleles of the barley HvMlo gene, which are responsible for the non-race specific resistance to the barley PM pathogen, Blumeria graminis f.sp. hordei (Bgh). During the years, this recessively inherited resistance was observed to be durable, contrary to the short life-span of resistances conferred by dominant resistance (R-) genes used in barley breeding programs. Studies on the histological mechanisms of the mlo-based resistance showed that the PM pathogen was stopped during penetration of the cell wall by the formation of a papilla. This structure prevents the formation of the feeding structure of the pathogen, called a haustorium.

After sequencing many plant genomes, we are discovering that MLO genes are not only typical of this cereal, but are ubiquitously present in higher plant species in multiple copies per species, forming a gene family. The impairment of some members of a number of ever increasing plant species lead to broad-spectrum resistance towards their adapted PM pathogens. For example, in tomato the ol-2 gene, naturally harbored by the cherry tomato Solanum lycopersicum var. cerasiforme, represents the loss-of-function allele of the SlMLO1 gene, conferring resistance to the PM pathogen Oidium neolycopersici (On). Consequently, the use of mlo mutants represents a suitable alternative to the classical use of R-genes in breeding programs.

In Chapter 2, we describe the in silico identification of the complete tomato SlMLO gene family using the available information in the SOL genomic network database. In total, 16 tomato SlMLO members were cloned from leaf, root, flower and fruit of the susceptible tomato cv. Moneymaker to confirm the sequences retrieved from the database and to verify their actual expression in these tissues. We observed the presence of various types of splicing variants, although their possible functional meaning has not been investigated. Motif analyses of each of the translated protein sequences and phylogenetic studies highlighted, on one hand, amino acid stretches that characterize the whole MLO family, and, on the other hand, stretches conserved in MLO homologs that are phylogenetically related. Following a gene expression study upon On inoculation, we identified members of the SlMLO family that are upregulated few hours after pathogen challenge. Except SlMLO1, none of the three newly identified homologs in clade V, thus phylogenetically close to SlMLO1, are induced. Interestingly, two homologs, each found in different clades, are upregulated similarly to SlMLO1. Using an RNAi approach, we silenced the additional clade V-SlMLO homologs, namely SlMLO3, SlMLO5 and SlMLO8, to investigate their possible role in PM resistance. We observed that none of these homologs if individually silenced, leads to PM resistance. However, if SlMLO5 and SlMLO8 are silenced together with SlMLO1, a significantly higher level of resistance is achieved compared to plants carrying the ol-2 allele. The role of SlMLO3 could not be verified. We, therefore, concluded that there are three SlMLO genes in tomato unevenly contributing to the PM disease, of which SlMLO1 has a major role.

Chapter 3 focuses on the components of the tomato mlo-based resistance. In Arabidopsis, it is known that four members of the SNARE protein family, involved in membrane fusion, are involved in mlo-based resistance. In this chapter, we focused on the identification of tomato homologs of the Arabidopsis syntaxin PEN1 (AtSYP121). Among the group of syntaxins identified in tomato, two were closely related to each other and also to AtPEN1, denominated SlPEN1a and SlPEN1b. Another Arabidopsis syntaxin that shows a high level of homology with PEN1, called SYP122, was also found to group together with the newly identified SlPEN1 genes. However, the role of SYP122 in plant immunity was not shown in literature. After obtaining individual silencing RNAi constructs, we transformed the resistant ol-2 line, and we challenged the obtained transformants with the adapted PM On, and the non-adapted Bgh. Interestingly, we observed a significant On growth and an enhanced Bgh cell entry only in SlPEN1a silenced plants but not in SlPEN1b silenced ones. We performed a protein alignment of tomato and Arabidopsis functional and non-functional PEN sequences. The presence of three differently conserved non-synonymous amino-acid substitutions is hypothesised to be responsible for the specialization in plant immune function.

In Chapter 4 and Chapter 5, we build up a body of evidence pointing to the fact that the function of the MLO susceptibility genes is highly conserved between monocot and dicot plant species.

In Chapter 4 we started by identifying and functionally characterizing two new MLO genes of Solanaceous crops affected by the PM disease, tobacco (Nicotiana tabacum) and eggplant (Solanum melongena). We named them NtMLO1 and SmMLO1 in the respective species, as they are the closest homologs to tomato SlMLO1. By overexpressing these genes in the resistant ol-2 line, we obtained transgenic plants that were susceptible to the PM pathogen On. This finding demonstrates that both heterologous MLO proteins can rescue the function of the impaired ol-2 allele in tomato. In addition, we found in tobacco NtMLO1 an amino acid (Q198) of critical importance for the susceptibility function of this protein.

In Chapter 5, we used the same approach adopted in Chapter 4 to show that other MLO proteins of more distant dicot species, like pea PsMLO1, can rescue the loss-of-function of the tomato ol-2 allele. And finally, we stretched this concept also to monocot MLO proteins, using barley HvMlo. While performing these experiments, we could verify that the function of the monocot and dicot susceptibility MLO proteins does not rely on the presence of class-specific conservation. The latter can be the reason for the phylogenetic divergence, placing monocot MLO proteins in clade IV and dicot MLO proteins in clade V of the phylogenetic MLO tree. However, functional conservation might depend on crucial shared amino acids of clade IV and V MLO proteins. Therefore, we also conducted a codon-based evolutionary analysis that resulted in the identification of 130 codons under negative selection, thus strongly maintained during evolution.

In Chapter 6 we introduce the PM disease in cucumber caused by Podosphaera xanthii (Px). We cloned the candidate susceptibility gene for PM in cucumber, CsaMLO8, from susceptible and resistant genotypes. The latter was described as an advanced cucumber breeding line characterized by hypocotyl resistance. In this line, we found the presence of aberrant splicing variants of the CsaMLO8 mRNA due to the insertion in its corresponding genomic region of a Class LTR retrotransposon. Heterologous expression of the wild-type cucumber allele in the tomato ol-2 line restored its PM susceptibility, while the heterologous expression of the aberrant protein variant failed to do so. This finding confirms that the resistance of the advanced cucumber breeding line is due to the disruption of the coding region of this gene. We also showed that the expression of CsaMLO8 in the susceptible genotype is induced by Px in hypocotyl tissue, but not in cotyledon or leaf. Finally, by examination of the resequencing data of a collection of 115 cucumber accessions, we found the presence of the TE-containing allele in 31 of them among which a wild cucumber accession that might have been used in breeding programs to obtain resistance to the PM disease in cucumber.

In Chapter 7 a novel loss-of-function allele of the SlMLO1 gene is described, designated m200. This allele was found in a resistant plant (M200) from a mutagenized tomato Micro-Tom (MT) population obtained with the chemical mutagen ethyl methanesulfonate (EMS). The m200 mutation corresponds to a nucleotide transversion (T à A) which results in a premature stop codon. The length of the predicted SlMLO1 protein in the M200 plant is only 21 amino acids, thus much shorter than the predicted protein of the previously described ol-2 allele, consisting of 200 amino acids. Thanks to the development of a High-Resolution Melting (HRM) marker designed to detect the m200 mutation, we observed that this allele confers recessively inherited resistance in backcross populations of the resistant M200 plant with MT and Moneymaker. Histological study showed that the resistance of the m200 mutant is associated with papilla formation. Finally, we compared the rate of On penetration in epidermal cells of m200 plants with the one of plants carrying the ol-2 allele and the transgenic plants in which multiple SlMLO homologs were silenced, generated in Chapter 2.

Ultimately, in Chapter 8 the results of the previous chapters are discussed in the context of 1) practical applications in breeding programs aimed at introducing the mlo-based resistance in new crops, 2) possible research aimed at unraveling the function of the MLO protein and 3) the role of other SNARE proteins.

Werken aan diversiteit in tarwe en groenten : voor meer variatie op het veld, in het winkelschap en op het bord
Nuijten, Edwin ; Lammerts van Bueren, Edith - \ 2016
Driebergen : Louis Bolk Instituut (Publicatie / Louis Bolk Instituut 2016-030 LbP) - 20
kwekers - biologische landbouw - rassen (planten) - tarwe - zaden - plantenveredeling - groenten - genetische diversiteit - diversiteit - biologische plantenveredeling - growers - organic farming - varieties - wheat - seeds - plant breeding - vegetables - genetic diversity - diversity - organic plant breeding
Van 2014 tot 2016 heeft het Louis Bolk Instituut onderzoek gedaan naar de mogelijkheden van een breder assortiment in gewassen voor de teler (op het veld) en voor de consument (op het bord). Aanleiding voor het onderzoek is dat het aantal rassen dat aangepast is aan biologische teeltomstandigheden (rassen die dus zonder gebruik van kunstmest en bestrijdingsmiddelen kunnen) beperkt is en blijft. Veel veredelingsbedrijven kunnen vanwege de ontwikkelingskosten geen aparte rassen ontwikkelen voor een kleine markt. Meestal worden rassen uit het bestaande (gangbare) assortiment geselecteerd voor biologische vermeerdering. Bovendien zijn biologische telers en handelaren meegegaan in de huidige eisen voor hoge opbrengst en uniforme eindproducten. Het aanbieden van zaadvaste rassen in plaats van bijvoorbeeld hybride rassen is daarmee commercieel niet meteen vanzelfsprekend. Divers en Dichtbij Van 2014 tot 2016 heeft het Louis Bolk Instituut onderzoek gedaan naar de mogelijkheden van een breder assortiment in gewassen voor de teler (op het veld) en voor de consument (op het bord). Dit onderzoek is samen met Estafette Odin BV en de biologische dynamische telers GAOS in Swifterbant, De Groenen Hof in Esbeek en de Maatschap Dames en Heren Vos in Kraggenburg uitgevoerd. Het doel van dit project Divers en Dichtbij was de diversiteit op het veld en op het bord te vergroten. Daarmee bedoelen we niet alleen meer verschillende rassen, maar vooral andere type rassen of populaties die zelf meer genetische variatie bezitten. Dat kan door te kiezen voor zaadvaste rassen bij groentegewassen en populaties bij granen. Tot nu toe is populatieveredeling alleen toegepast bij granen en nog niet of nauwelijks bij groentegewassen (zie voor definities Box 1 op pagina 7). Dit betekent ook een keuze voor andere manieren van veredelen en selecteren. Aanleiding voor het onderzoek is dat het aantal rassen dat aangepast is aan biologische teeltomstandigheden (rassen die dus zonder gebruik van kunstmest en bestrijdingsmiddelen kunnen) beperkt is en blijft. Veel veredelingsbedrijven kunnen vanwege de ontwikkelingskosten geen aparte rassen ontwikkelen voor een kleine markt. Meestal worden rassen uit het bestaande (gangbare) assortiment geselecteerd voor biologische vermeerdering. Bovendien zijn biologische telers en handelaren meegegaan in de huidige eisen voor hoge opbrengst en uniforme eindproducten. Het aanbieden van zaadvaste rassen in plaats van bijvoorbeeld hybride rassen is daarmee commercieel niet meteen vanzelfsprekend. En toch heeft ons brede speurwerk in dit project wel degelijk een aantal interessante zaadvaste rassen opgeleverd! Want gelukkig zijn er in Europa en Amerika diverse biologische veredelaars actief in het veredelen van zaadvaste rassen en populaties. De informatie in deze brochure is bedoeld voor telers en andere ketenpartijen om meer te leren over de mogelijkheden van zaadvaste rassen bij groenten en populaties bij tarwe.
Aardappelwereld (Journal)
Lammerts van Bueren, Edith - \ 2016
Aardappelwereld (2016). - ISSN 0169-653X
potatoes - growers - plant breeding - arable farming - varieties - cooperation - organic farming
Kweekspecial 2016 - Deze productie is een uitgave van BioImpuls i.s.m. met Aardappelwereld BV.
Development and application of a 20K SNP array in potato
Vos, Peter - \ 2016
University. Promotor(en): Richard Visser; Fred van Eeuwijk, co-promotor(en): Herman van Eck. - Wageningen : Wageningen University - ISBN 9789462579569 - 166
solanum tuberosum - potatoes - genotypes - single nucleotide polymorphism - data analysis - plant breeding - linkage disequilibrium - genome analysis - tetraploidy - aardappelen - genotypen - gegevensanalyse - plantenveredeling - verstoord koppelingsevenwicht - genoomanalyse - tetraploïdie

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

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

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

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

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

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

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

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

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

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

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

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

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

Starch meets biotechnology : in planta modification of starch composition and functionalities
Xu, Xuan - \ 2016
University. Promotor(en): Richard Visser, co-promotor(en): Luisa Trindade. - Wageningen : Wageningen University - ISBN 9789462579200 - 169
starch - potato starch - potatoes - solanum tuberosum - plant biotechnology - biotechnology - genetic engineering - transgenic plants - modified starches - phosphate - arabidopsis thaliana - plant breeding - zetmeel - aardappelzetmeel - aardappelen - plantenbiotechnologie - biotechnologie - genetische modificatie - transgene planten - gemodificeerd zetmeel - fosfaat - plantenveredeling

Storage starch is an energy reservoir for plants and the major source of calories in the human diet. Starch is used in a broad range of industrial applications, as a cheap, abundant, renewable and biodegradable biopolymer. However, starch needs to be modified before it can fulfill the required properties for specific industrial applications. Genetic modification of starch, as a green technology with environmental and economic advantages, has attracted increasingly attention. Many achievements obtained from earlier studies have demonstrated the feasibility and potential of using this approach to produce starches with novel properties (Chapter 2).

The main objective of this research was to produce novel starches with enhanced functionalities through genetic modification, while gaining a better understanding of storage starch biosynthesis. A focus on potato was warranted as it represents a superior model system for storage starch biosynthesis studies and for the production of starches with novel properties. To this end, a number of enzymes from various sources have been expressed in potato tubers to modify starch phosphate content and polysaccharide structure, since these two characteristics have long been recognized as key features in starch properties.

To modify starch phosphate content and explore starch (de)phosphorylation, a human phosphatase enzyme named laforin, and modifications of it, were introduced into potato (Chapter 3). Interestingly, modified starches exhibited a significantly higher phosphate content rather than the expected lower phosphate content. Transcriptome analysis showed that the increase in phosphate content was a result of upregulation of starch phosphorylating genes, which revealed a compensatory response to the loss of phosphate content in potato starch. Furthermore, the increase of phosphate content in potato starch was reached to a threshold level. This was in line with the observations in the modified starches from overexpressed- Glucan water dikinase (GWD1) transgenic plants (Chapter 4). Furthermore, overexpression of two starch dikinases from Arabidopsis thaliana, glucan water dikinase 2 and 3 (AtGWD2 and AtGWD3), did not result in a significant increase in phosphate content of potato starch (Chapter 5). Taken together, these results indicated that phosphate content of potato starch is under strict control.

Morphological analysis of starch granules containing different levels of phosphate content confirmed the indispensible role of phosphate content in the normal formation of starch granules, since cracked granules were observed in the starches containing low phosphate content, while irregular bumpy shaped granules were observed in the tubers from plants containing high phosphate content. Interestingly, further analyses on the expression level of genes involved in starch metabolism and sugar-starch conversion suggested that starch phosphorylation might affect starch synthesis by controlling the carbon flux into starch while simultaneously modulating starch-synthesizing genes. Further studies are needed to confirm this finding (Chapter 4).

To produce starches with novel structures, an (engineered) 4, 6-α-glucanotransferase (GTFB) from Lactobacillus reuteri 121 was introduced into potato tubers (Chapter 6). The resulting starches showed severe changes in granule morphology, but not in starch fine structure. Transcriptome analysis revealed the existence of a self-repair mechanism to restore the regular packing of double helices in starch granules, which possibly resulted in the removal of novel glucose chains potentially introduced by the (engineered) GTFB.

This research successfully generated starches with various functionalities, including altered gelatinization characteristics (Chapter 3 and 4), improved freeze-thaw stability (Chapter 4) and higher digestibility (Chapter 6). The exploitation of relationships between starch characteristics and starch properties revealed that starch properties represent the outcome of the combined effect of many factors and are highly dependent on the genetic background in which the modification has been performed.

In conclusion, the research described in this thesis demonstrates the great potential of genetic modification in producing starches with novel properties. Meanwhile, these results revealed the presence of complex and exquisite molecular regulation mechanisms for starch biosynthesis in potato. In future research, these regulations need to be taken into account for the relational design of starch in planta. Certainly, a better understanding of the process of starch metabolism in storage organs would be a great step forward towards tailoring starch in an economically important crop such as potato.

Targets and tools for optimizing lignocellulosic biomass quality of miscanthus
Weijde, R.T. van der - \ 2016
University. Promotor(en): Richard Visser, co-promotor(en): Luisa Trindade; Oene Dolstra. - Wageningen : Wageningen University - ISBN 9789462578388 - 231 p.
miscanthus - bioethanol - biomass - biofuels - lignocellulose - fuel crops - plant breeding - cell walls - cell wall components - genetic diversity - genetic variation - biomass conversion - biobased economy - biomassa - biobrandstoffen - brandstofgewassen - plantenveredeling - celwanden - celwandstoffen - genetische diversiteit - genetische variatie - biomassaconversie

Miscanthus is a perennial energy grass characterized by a high productivity and resource-use efficiency, making it an ideal biomass feedstock for the production of cellulosic biofuels and a wide range of other biobased value-chains. However, the large-scale commercialization of converting biomass into cellulosic biofuel is hindered by our inability to efficiently deconstruct the plant cell wall. The plant cell wall is a complex and dynamic structure and its components are extensively cross-linked into an unyielding matrix. The production of biofuel depends on the extraction, hydrolysis and fermentation of cell wall polysaccharides, which currently requires energetically and chemically intensive processing operations that negatively affect the economic viability and sustainability of the industry. To address this challenge it is envisioned that the bioenergy feedstocks can be compositionally tailored to increase the accessibility and extractability of cell wall polysaccharides, which would allow a more efficient conversion of biomass into biofuel under milder processing conditions.

Extensive phenotypic and genetic diversity in cell wall composition and conversion efficiency was observed in different miscanthus species, including M. sinensis, M. sacchariflorus and interspecific hybrids between these two species. In multiple experiments a twofold increase in the release of fermentable sugars was observed in ‘high quality’ accessions compared to ‘low quality’ accessions. The exhaustive characterization of eight highly diverse M. sinensis genotypes revealed novel and distinct breeding targets for different bioenergy conversion routes. The key traits that contributed favourably to the conversion efficiency of biomass into biofuel were a high content of hemicellulosic polysaccharides, extensive cross-linking of hemicellulosic polysaccharides (revealed by a high content of trans-ferulic acids and a high ratio of arabinose-to-xylose), a low lignin content and extensive incorporation of para-coumaric acid into the lignin polymer.

Lignin is widely recognized as one of the key factors conveying recalcitrance against enzymatic deconstruction of the cell wall. The incorporation of para-coumaric acid into the lignin polymer is hypothesized to make lignin more easily degradable during alkaline pretreatment, one of the most widely applied processing methods that is used to pretreat biomass prior to enzymatic hydrolysis. Previous studies have shown that reducing lignin content is often implicated in reduced resistance of plants to lodging. We hypothesize that extensively cross-linked hemicellulosic polysaccharides may fulfil a similar function in supporting cell wall structural rigidity and increasing the content of hemicellulosic polysaccharides may be a way to reduce lignin content without adversely affecting cell wall rigidity. This strategy can be used to improve biomass quality for biobased applications, as hemicellulosic polysaccharides are more easily degradable during industrial processing than lignin. Furthermore, hemicellulosic polysaccharides adhere to cellulose, which negatively affects the level of cellulose crystallinity. Crystalline cellulose is harder to degrade than its more amorphous form. Therefore the reduction of cellulose crystallinity is another mechanism through which increasing the content of hemicellulosic polysaccharides positively contributes to cell wall degradability. These results provided new insights into the traits that may be targeted to improve the quality of lignocellulose feedstocks.

However, evaluation of complex biochemical traits for selection purposes is hindered by the fact that their accurate quantification is a costly, lengthy and laborious procedure. To overcome these limitations an accurate and high-throughput method was developed based on near-infrared spectroscopy. Through extensive calibration we developed accurate prediction models for a wide range of biomass quality characteristics, which may be readily implemented as a phenotyping tool for selection purposes.

Additionally, progress through breeding may substantially be improved by marker-assisted selection, which will reduce the need for the evaluation of genotype performance in multi-year field trials. To this end, a biparental M. sinensis mapping population of 186 individuals was developed and genotyped using a genotyping-by-sequencing approach. A total of 564 short-sequence markers were used to construct a new M. sinensis genetic map. Cell wall composition and conversion efficiency were observed to be highly heritable and quantitatively inherited properties. This is the first genetic study in miscanthus to map quantitative trait loci (QTLs) for biomass quality properties and is a first step towards the application of marker-assisted selection for biomass quality properties.

Through the evaluation of a diverse set of miscanthus genotypes in multiple locations we demonstrated that in addition to genotypic variation, growing conditions may have a substantial influence on cell wall composition and conversion efficiency. While further research is needed to identify which specific environmental parameters are responsible for the observed effects, these results clearly indicate that the environmental influence on biomass quality needs to be taken into account in order to match genotype, location and end-use of miscanthus as a lignocellulose feedstock. Moreover, significant genotype-by-environment interaction effects were observed for cell wall composition and conversion efficiency, indicating variation in environmental sensitivity across genotypes. Although the magnitude of the genotypic differences was small in comparison to genotype and environmental main effects, this affected the ranking of accession across environments. Stability analysis indicated some stable accessions performed relatively across diverse locations.

In addition to trialing miscanthus in diverse locations, we also evaluated miscanthus biomass quality under drought conditions for a number of reasons: 1) drought stress is linked to a differential expression of cell wall biosynthesis genes, 2) incidence of drought events is increasing due to climate change, 3) irrigation is likely to be uneconomical during the cultivation of miscanthus and 4) miscanthus has many characteristics that make it a crop with a good potential for cultivation on marginal soils, where abiotic stresses such as drought may prevail. Drought stress was shown to result in a large reduction in cell wall and cellulose content and a substantial increase in hemicellulosic polysaccharides and cellulose conversion rates. We hypothesized that the reduction in cellulose content was due to an increase in the production of osmolytes, which are well-known for their role in plant protection against drought. The results indicated that drought stress had a positive effect on the cell wall degradability of miscanthus biomass.

Overall the compendium of knowledge generated within the framework of this thesis provided insights into the variation in biomass quality properties in miscanthus, increased our understanding of the molecular, genetic and environmental factors influencing its conversion efficiency into biofuel and provided tools to exploit these factors to expand the use of miscanthus as a lignocellulose feedstock.

Adventitious root formation in Arabidopsis : underlying mechanisms and applications
Massoumi, Mehdi - \ 2016
University. Promotor(en): Richard Visser, co-promotor(en): Geert-Jan de Klerk; Frans Krens. - Wageningen : Wageningen University - ISBN 9789462578524 - 191
arabidopsis thaliana - adventitious roots - formation - plant development - quantitative traits - etiolation - auxins - explants - molecular biology - gene expression - dna methylation - rooting - ontogeny - plant breeding - adventiefwortels - formatie - plantenontwikkeling - kwantitatieve kenmerken - etiolering - auxinen - explantaten - moleculaire biologie - genexpressie - dna-methylering - beworteling - ontogenie - plantenveredeling

Adventitious root (AR) formation is indispensable in vegetative propagation and is widely used. A better understanding of the underlying mechanisms is needed to improve rooting treatments. We first established a system to study rooting in Arabidopsis, the model organism in plant biology but only occasionally used to study adventitious rooting. Inhibition of polar auxin transport reduced AR formation. The role of auxin transporter proteins (several PIN-proteins) was found to be tissue-specific. Maturation (the transition from juvenile to adult) negatively influenced AR formation. Maturation was associated with increased DNA methylation and decreased miR156 level. 5-Azacytidine, a drug that reduces DNA methylation, increased rooting. We also examined the effect of two donor plant pre-treatments, etiolation and flooding, on rooting. Both increased AR formation.

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