Search

Late blight caused by the oomycete Phytophthora infestans, remains the most devastating disease for potato. Wild Solanum species provide a valuable resource of resistance (R) genes. Yet, for educated R gene deployment, molecular knowledge of the corresponding avirulence (Avr) gene is essential. AVR2 is an RXLR effector of P. infestans with an important role in late blight development. AVR2 is recognized by the R2 family from wild Mexican Solanum species and by the dissimilar Rpi-mcq1 family from Peru. Herewith, we present the first example of two sequence unrelated R proteins that recognize the same effector. Interestingly, the recognition of AVR2 by Rpi-mcq1 and R2 displays an overlapping, but not identical specificity. Disease tests with a wide panel of P. infestans isolates on transgenic R2 and Rpi-mcq1 potatoes show different patterns of disease resistance. Studies of the AVR2 family revealed that this is a highly diverse effector family that is differentially expressed in P. infestans isolates. We found new AVR2 alleles and we hypothesize that the AVR2 family is co-evolving with two different R gene families. R2-mediated AVR2 recognition requires the interaction with the BSU-like 1 (BSL1) protein, whereas the Rpi-mcq1-mediated recognition involves a different signaling pathway. These studies contribute to understanding the R-AVR interaction, which is instrumental to improve R gene-based breeding and deployment against late blight in potato.

This thesis focused on two important fruit quality aspects, fruit shelf life and fruit flavour by utilisation of two sources of genetic variation: (1) segregating populations based on cultivated tomato germplasm and (2) a diverse tomato core collection that consist of wild relatives, land races and old cultivars. In chapter 2 and 3 of this thesis we have evaluated a tomato core collection consisting of 122 landraces, heirloom varieties and wild accessions for variation in several plant growth and fruit quality-related traits. This collection provided a good resource of superior alleles to incorporate in future breeding programs. 66 of the 88 cultivated accessions of this collection have been sequenced and explored for the presence of known mutations or sequence variations in key genes underlying important domestication and agronomic traits. In chapter 3, the 88 cultivated accessions of this collection plus two additional modern genotypes have been characterised in more detail for post-harvest shelf-life and thus we have now uncovered in that material novel sources for fruit shelf-life improvement. in chapter 4 and 5 of this thesis, we have investigated two metabolite QTLs involved in flavour of tomato by using a segregating population based on cultivated tomato genotypes. In chapter 4 we have fine mapped a major QTL for a volatile organic compound (2-phenyethanol) which is highly associated with consumer liking and flavour intensity (Knudsen et al., 1993; Tieman et al., 2007; Tieman et al., 2017). In chapter 5 we have identified and fine mapped the genomic region controlling the amino acid glutamate, which is a flavour enhancer and key factor conferring umami taste in tomato (Yilmaz, 2000). These results facilitate the molecular breeding of cultivars with improved flavour and also lead to a better understanding of the biosynthesis pathway underlying these two metabolites.

One of the major global challenges of today is to meet the food demands of an ever increasing population (food demand will increase by 50% in 2030). One approach to address this challenge is to breed new crop varieties that yield more even under unfavorable conditions e.g. have improved tolerance to drought and/or resistance to pathogens. However, designing a breeding program is a laborious and time consuming effort that often lacks the capacity to generate new cultivars quickly in response to the required traits. Recent advances in biotechnology and genomics data science have the potential to accelerate and precise breeding programs greatly. As large-scale genomic data sets for crop species are available in multiple independent data sources and scientific literature, this thesis provides innovative technologies that use natural language processing (NLP) and semantic web technologies to address challenges of integrating genomic data for improving plant breeding. Firstly, in this research study, we developed a supervised Natural language processing (NLP) model with the help of IBM Watson, to extract knowledge networks containing genotypic-phenotypic associations of potato tuber flesh color from the scientific literature. Secondly, a table mining tool called QTLTableMiner++ (QTM) was developed which enables knowledge discovery of novel genomic regions (such as QTL regions), which positively or negatively affect the traits of interest. The objective of both above mentioned, NLP techniques was to extract information which is implicitly described in the literature and is not available in structured resources, like databases. Thirdly, with the help of semantic web technology, a linked-data platform called Solanaceae linked data platform(pbg-ld) was developed, to semantically integrates geno- and pheno-typic data of Solanaceae species. This platform combines both unstructured data from scientific literature and structured data from publicly available biological databases u

One to two per cent of the population has Coeliac Disease (CD), an immune reaction to gluten. Wheat grains contain gluten, a mixture of glutenin and gliadin proteins, which build a network that gives wheat bread its unique properties and quality. Most gliadins and part of the glutenins contain immunogenic epitopes, which are the actual trigger of the immune reaction. A gluten-free diet, excluding wheat, barley and rye, is currently the only remedy for coeliac patients. This diet is not easy to adhere to, partly because wheat gluten is added to many processed food products for their viscoelastic properties. In addition, gluten-free products typically require the inclusion of numerous additives to adjust their texture and taste, resulting in products that are often less healthy than gluten-based equivalents, and more expensive. Thus there is a need to develop healthier food products for coeliac patients. One can now use CRISPR/Cas to remove all gluten genes, which would produce a gluten-free wheat which is interesting for many people who want to eat gluten-free, but it would have an inferior baking quality. In this PhD thesis, Aurélie Jouanin describes an alternative use of gene editing with CRISPR/Cas9 to precisely modify gliadin genes and strip them of immunogenic epitopes, to develop wheat with safe gluten. As a proof of principle she generated wheat plants in which some gliadin genes were modified or removed. These edited wheat plants are not yet safe for CD patients, as there is a large number of gluten genes present in wheat and not all gluten genes have been targeted. She has therefore also developed high-throughput methods to determine which genes have been modified and which remain to be edited in future steps towards a safe wheat variety. The regulation of gene editing as Genetic Modification (GM) in Europe is currently a hot topic. She discusses the inconsistency of the European regulation of gene editing in plants by displaying the similarities of mutation

Two of the most limiting diseases in cucumber production are downy mildew (DM), caused by the oomycete Pseudoperonospora cubensis, and powdery mildew (PM), caused by the fungi Podosphaera xanthii and Golovinomyces orontii. Whereas the pathogens causing DM and PM are not related, they share a similar obligate biotrophic lifestyle. In my PhD thesis we aimed at identifying genes involved in susceptibility and resistance to these diseases, in order to better understand the interactions between cucumber and mildew pathogens, and contribute to breeding disease resistant cucumbers. One focus point in my research was the concept of susceptibility (S) genes. Loss-of-function of S-genes lead to resistance, which is more durable than classical race-specific resistance (R) genes. A second pillar of my PhD research was to identify causal genes for QTL contributing to disease resistance, which are frequently used in cucumber breeding. Regarding PM, the most important source of resistance is “hypocotyl resistance”, which causes partial resistance characterized by PM-free hypocotyls and stems, and can lead to full resistance when combined with other sources of resistance. One of the most famous examples of an S gene is MLO, loss-of-function alleles of which provide durable PM resistance in barley already for several decades. After the cloning of the causal gene it was found that MLO genes occur in all sequenced plant genomes to date, and that in several plant species MLO homologues in phylogenetic clades IV and V were found to be S genes for PM. In the first two chapters of my thesis I investigated MLO genes in cucumber, leading to the identification and functional characterization of a loss-of-function allele of the clade V CsaMLO8 gene as causal for hypocotyl resistance in cucumber (Chapter 2). Furthermore, we functionally characterized the other clade V MLO genes in cucumber, CsaMLO1 and CsaMLO11, and found that both are functional S-genes too, although they are rather weakly ex

Plant pathogens can have great social and economic impact, and are a continuous threat to food security. This is clearly the case for Synchytrium endobioticum, the species causing potato wart disease. S. endobioticum is an obligate biotrophic fungus of the phylum Chytridiomycota (chytrids), which is a basal lineage in the fungal kingdom. The lack of chemical control agents, the impact of the pathogen (complete loss of tuber yields from infected field have been reported), and the production of resting spores that remain viable and infectious in infested soils for decades, led to a quarantine status for S. endobioticum in most countries world-wide. Potato wart disease has been reported from all continents where potato is cultivated, and strict phytosanitary control measures are enforced to prevent the introduction and spread of the pathogen. The use of resistant potato varieties has proven successful in achieving these goals. In chapter 1, over a century of potato wart research is reviewed to place our current knowledge in historic perspective. Observations from light microscopy and electron microscopy studies of S. endobioticum performed in the twentieth century are combined with recent molecular studies. Based on our current knowledge on molecular plant-pathogen interactions, a model is presented to describe the interaction between S. endobioticum and its host in particular with respect to plant resistance. Chapter 2 describes the independent sequencing, assembly and functionally annotation of two S. endobioticum genomes. A comparative genomics approach, in which knowledge acquired from other fungal taxa is exploited, is used to gain insights into genomic features underlying the obligate biotrophic and pathogenic lifestyle of the pathogen. Our study underlines the high diversity in chytrids compared to the well-studied Ascomycota and Basidiomycota, and reflects biological differences between the phyla. Moreover, it highlights the surprising commonalities between pla

Potato (Solanum tuberosum L.) can reproduce sexually through flowering and asexually through tuberization. While tuberization has been thoroughly studied, little research has been done on potato flowering. Flower bud development in the strictly short-day tuberizing S. tuberosum group Andigena is impaired under short-day conditions. This impaired development may indicate that tuberization negatively influences flowering. Here, we determine how tuberization affects flower bud development. To find out whether the absence of tubers improves flowering, we prevented tuberization by: (i) grafting potato scions onto wild potato rootstocks, which were unable to form tubers; (ii) removing stolons, the underground structures on which tubers form; and (iii) using plants that were silenced in the tuberization signal StSP6A. Additionally, transgenic plants with increased StSP6A expression were used to determine if flower bud development was impaired. The absence of a tuber sink alone did not accelerate flower bud development, nor did it allow more plants to reach anthesis (open flowering stage) or have more open flowers. Interestingly, reducing StSP6A expression improved flower bud development, and increasing expression impaired it. Our results show that flower bud development in potato is repressed by the tuberization signal StSP6A, and not by competition with the underground tuber sink.

Clavibacter michiganensis subsp. michiganensis (Cmm) is a quarantine organism in Europe and in many other countries. It is one of the most severe bacterial pathogens affecting tomato. Screening tomato plants for their resistance levelto Cmm requires a large amount of space under quarantine conditions and is therefore costly. This project developed a new inoculation protocol on in vitro tomato plants to facilitate a more economic and higher throughput disease screening. A new method using the PathoScreen system was tested to localize green fluorescent protein-tagged Cmm in planta and to quantify the pathogen based on the percentage of corrected GFP (cGFP%). The system was sensitive in detecting the GFP-tagged Cmm in the shoots, but in the roots a high autofluorescence masked detection and thus sensitivity of the assay. The in vitro protocol was tested on several wild relatives of tomato, which were previously screened in a greenhouse assay. The correlation between wilt symptoms in vitro and wilt symptoms in the greenhouse was overall moderate (r = 0.6462). The protocol worked well in differentiating the two parents that were used in the mapping studies. This study shows that the in vitro protocol can be efficiently used for resistance breeding in many tomato genotypes.

Potato plants form tuberous storage organs on underground modified stems called stolons. Tubers are rich in starch, proteins, and other important nutrients, making potato one of the most important staple food crops. The timing of tuber development in wild potato is regulated by day length through a mechanism that is closely related to floral transition [1, 2]. Tuberization is also known to be regulated by the availability of assimilates, in particular sucrose, the transported form of sugar, required for starch synthesis. During the onset of tuber development, the mode of sucrose unloading switches from apoplastic to symplastic [3]. Here, we show that this switch may be mediated by the interaction between the tuberization-specific FT homolog StSP6A and the sucrose efflux transporter StSWEET11 [4]. The binding of StSP6A to StSWEET11 blocked the leakage of sucrose to the apoplast, and is therefore likely to promote symplastic sucrose transport. The direct physical interaction between StSWEET11 and StSP6A proteins represents a link between the sugar and photoperiodic pathways for the regulation of potato tuber formation. Our data suggest that a previously undiscovered function for the FT family of proteins extends their role as mobile signals to mediators of source-sink partitioning, opening the possibility for modifying source-sink interactions.

Synchytrium endobioticum is an obligate biotrophic fungus of the phylum Chytridiomycota. It causes potato wart disease, has a world-wide quarantine status and is included on the HHS and USDA Select Agent list. S. endobioticum isolates are grouped in pathotypes based on their ability to evade host-resistance in a set of differential potato varieties. So far, thirty-nine pathotypes are reported. A single dominant gene (Sen1) governs pathotype 1 resistance and we anticipated that the underlying molecular model would involve a pathogen effector (AvrSen1) that is recognized by the host. The S. endobioticum specific secretome of fourteen isolates representing six different pathotypes was screened for effectors specifically present in pathotype 1(D1) isolates but absent in others. We identified a single AvrSen1 candidate. Expression of this candidate in potato Sen1 plants showed a specific hypersensitive response, which co-segregated with the Sen1 resistance in potato populations. No HR was obtained with truncated genes found in pathotypes that evaded recognition by Sen1. These findings established that our candidate gene was indeed Avrsen1. The S. endobioticum AvrSen1 is a single copy gene and encodes a 376 amino acid protein without predicted function or functional domains, and is the first effector gene identified in Chytridiomycota, an extremely diverse yet underrepresented basal lineage of fungi.

Background: A quantitative trait locus (QTL) is a genomic region that correlates with a phenotype. Most of the experimental info

Potato (Solanum tuberosum) is the most important non-cereal crop for human consumption and its starch and fibers are also used to produce several industrial products. Potato suffers from many pests and diseases. The most threatening and re-emerging disease of potato worldwide is late blight, which is caused by the notorious oomycete Phytophthora infestans (Chapter 1). This pathogen is a hemi-biotrophic organism that secretes a huge arsenal of apoplastic and host-translocated cytoplasmic effectors in order to colonize the host. Interestingly, wild potato plants have evolved receptors that recognize some of those effectors and trigger defense responses. These wild plants are a major source of resistance genes that can be transferred to the cultivated potato. Resistant cultivars obtained by breeding are highly desirable because the costs for chemical control of the disease are very high and there is also a need to reduce their use in order to preserve the environment. Moreover, the pathogen has developed resistance to some of those chemicals. Receptors that recognize cytoplasmic effectors often belong to the nucleotide-binding leucine-rich repeat (NLR) family of resistance genes, (R genes), and they have been used for several years in resistance breeding. Despite the effectivity of NLRs in providing resistance, so far, most single NLR genes introduced into cultivated potato have been defeated by P. infestans rather quickly. To effectively control the pathogen in the long term, resistance gene stacking approaches in combination with new layers of defense have to be considered. Like NLRs, cell surface-residing receptors, or pattern recognition receptors (PRRs) trigger defense responses and contribute to basal or non-host resistance against pathogens. They do so by recognizing apoplastic effectors or microbe-associated molecular patterns (MAMPs). Usually PRR-triggered resistance is quantitative and not as robust as NLR-based resistance, however it is believed to be more d

Miscanthus is a perennial grass with a C4 photosynthetic pathway and characterized by high productivity and resource use efficiency, which makes it favorable for bioenergy production. In order to avoid competition with food crops, Miscanthus will need be grown on underutilized marginal lands like saline soils. Planting Miscanthus in marginal areas and maintaining economically viable yields and quality depend on suitable genotypes, and evaluation of the performance of Miscanthus germplasm under salt stress and identifying salt tolerant genotypes for cultivation on saline soils or as breeding parents to improve salt tolerance are essential for sustainable bioenergy production. Therefore, several screening systems and experimental approaches were applied to identify genetic variation for salt tolerance, elucidate the salt tolerance mechanisms underlying this variation in Miscanthus. The result showed that genotypes with high yield and quality under salt stress are recommended as potential genotypes for breeding or direct production on marginal lands.

How to feed the world has become a huge challenge with the increasing world population. Potato, the most important non-cereal crop, is widely consumed as staple food in the Western world, and recently it has proposed to be also promoted as a staple food in developing countries like China. However, the global production of potato has greatly been hampered by a disastrous disease, called potato late blight. This disease is caused by the oomycete pathogen Phytophthora infestans, which triggered the great Irish famine in the 1840s. Besides chemical control, resistance (R) genes from wild relatives of potato have been introduced into modern cultivars by traditional breeding as well as by transgenic technology (GMO). However, most R genes that belong to the nucleotide-binding domain and leucine-rich repeat containing (NLR) class, which are generally rapidly defeated by the fast-evolving P. infestans population in the field. On the other hand, another form of disease resistance that constitutes the first layer of plant defense in the apoplastic space, has not received enough attention by breeders. This apoplastic defense is mediated by the plant surface immune receptors. These receptors can recognize microbe- or pathogen-associated molecular patterns (MAMPs/PAMPs) or apoplastic effectors of pathogens and trigger a defense response. MAMP/PAMP-triggered immunity (MTI/PTI) is believed to be more wide spectrum and more durable than the typical effector triggered immunity (ETI) that is mediated by cytoplasmic NLR receptors. In this thesis, I studied various apoplastic effectors from P. infestans and their receptors in the host, with the ultimate aim to achieve a broader and more durable resistance to late blight. In chapter 1, I summarized the history of potato late blight resistance breeding, the current knowledge of the plant immune system, particularly the plant surface immune receptors and the effector biology in the age of genome sequencing. To understand the surface immun

Lily (genus Lilium, family Liliaceae) is one of the most important ornamental crops for the production of flower bulbs and cut flowers. Conventional breeding might be inefficient for introducing variation in flower colours in L. longiflorum due to hybrid sterility and deficiency of genetic resources in related, crossable species. An alternative is provided by the technology of genetic modification, but lily is among the monocot species that is recalcitrant to genetic transformation by A. tumefaciens and thus exhibits a low transformation efficiency (TE). There is a need to enhance TE in lily. Because lily transformation is proven to be difficult and time consuming, we developed and optimized a simple agroinfiltration method in order to monitor transient gene expression of anthocyanin structural genes and regulatory genes in lily tepals. In addition, we determined molecular mechanisms regulating flower colour in lilies by an expression analysis of anthocyanin biosynthesis-related genes during multiple flower developmental stages. As a result, DFR was found to be the main bottleneck for not having colour in L. longiflorum cv. ‘Lincoln’ and the Oriental hybrid cv. ‘Rialto’. A follow up of this research should focus on a careful selection and characterization of a functional and suitable DFR gene copy from other lilies or another crop in order to produce novel flower colour in both cultivars.

The quarantine pathogen, Clavibacter michiganensis subsp. michiganensis (Cmm), is one of the most important bacterial pathogens affecting tomatoes. The quarantine status and the devastating disease symptoms caused by Cmm are problematic for many parties. Even though resistance/tolerance breeding is one of the most important and successful ways to manage diseases, other paths can also be walked to manage disease symptoms. We developed a new and an efficient way to screen big tomato populations in vitro and used it to fine map the QTL on chromosome 7. The region has been reduced to 211 Kb which resulted in 15 annotated genes. Furthermore, several susceptibility (S) genes were used to test effectiveness against Cmm and we found WAT1 confers higher tolerance. Lastly, alternative approaches were applied to reduce Cmm disease symptoms by applying grafting and utilizing biocontrol agents, respectively. From this method, we found reduced wilting symptoms only when the pathogen enters from the roots. In conclusion, we developed a new protocol that could ease disease screening, fine mapped the QTL on chromosome 7 to a smaller region, found a potential new tolerance against Cmm by manipulating the WAT1 gene and tested two alternative approaches to minimize wilting symptom.

Many of our most important crop species are polyploid – an unusual phenomenon whereby each chromosome is present in multiple copies (more than the usual two copies). The most common such arrangement is tetraploidy, where each chromosome is present four times. Plant species can tolerate this condition quite well (the same cannot be said of animals or humans). In fact, polyploidy can confer certain advantages such as larger fruits and flowers, seedless fruits (useful for fruit growers) or improved tolerance to environmental stresses. However, carrying multiple copies of each chromosome complicates things, particularly when crop breeders would like to use DNA information to help inform selection decisions. This PhD project looked at how DNA information of polyploids should be best analysed, developing methods and new software tools to achieve this. We analysed DNA information from polyploid crops such as potato, rose and chrysanthemum, yielding many novel insights and important results.

The world’s third most important food crop, potato, suffers yield reductions during drought stress. Drought scenarios are predicted to become more frequent and severe due to climate change. Therefore, we investigated the feasibility of improving drought tolerance in potato using a set of 103 European potato cultivars. We employed a multidisciplinary approach in our study including the phenotyping of multi-location and multi-year field trials, modelling of potato canopy growth and tuber size distribution, association mapping of tuber size distribution parameters with a 14K SNP array, magnetic resonance imaging of xylem and phloem conduits, microscopic study of stem cross-sections and transcriptomic analyses. We observed that late foliage maturity and a delayed attainment of maximum canopy cover favoured tuber yield during drought conditions. We found a marker-trait association of a region on chromosome three with marketable tuber size and spread of tuber size distribution under drought. Our findings indicate that water and assimilate transport are critical for carbon partitioning to tubers during drought. We observed that increased xylem density per unit stem area and reduction of xylem vessel diameter under drought contributed to tolerance. An integration of these findings in potato breeding programs will facilitate the improvement of drought tolerance in potato.

The durability and effectiveness of nonhost resistance suggests promising practical applications for crop breeding, relying upon elucidation of key aspects of this type of resistance. Inheritance and mapping studies are valuable for identifying genes that are determinants of specificity of the host or nonhost status. To overcome the problems associated with inheritance studies involving two different plant species, it is possible to study the genetics of resistance in plants that display natural variation in the degree of resistance to a non-adapted pathogen, the so-called “near-nonhosts”. Barley can be considered a near nonhost to non-adapted powdery mildews of cereals and grasses (Blumeria graminis ff. spp.), because a few accessions are somewhat susceptible at seedling stage. In the present thesis, the main goal was to investigate the genetic factors responsible for the specificity of the (non)host status in barley to B. graminis ff. spp. We also investigated the causes of an unknown condition affecting the grain quality in three mapping populations having the barley line SusPtrit as a parent.

The leafy head is the harvested organ of Chinese cabbage and is composed of a large number of tightly wrapped heading leaves surrounding the shoot apex. For both farmers and consumers, the Chinese cabbage leafy head trait has clear advantages, related to yield, stress resistance and storability. The leafy head is an obvious doemstication trait, also found in cababges (B. oleracea ssp. Capitata), lettuce (Lactuca sativa L.) and radicchio of Treviso (Cychorium intibus L.). The formation of the leafy head is genetically determined, however affected by both internal and external factors. The developmental and genetic mechanisms underlying leafy head formation are however poorly understood. In recent years, scientists started to study this domestication trait, which resulted in identification of domestication signals and QTL for leafy head formation. This thesis link morphological observations (plant, tissue and cell level) to genetic regulation (by population genetic analysis, trancriptome analysis) and gene functional experiments to explain how leafy heads form in Chinese cabbage. The relationship between variation in rosette-leaves and leafy heads by phenotyping 152 Chinese cabbage accessions was determined and QTL explaining heading and heading related traits were identified. Genome wide transcriptome analysis and the morphological differences at cellular level between heading and non-heading B. rapa pak choi and Chinese cabbage were studied to help us explain how Chinese cabbage heads form. Furthermore, the function of leafy head formation associated gene BrARF3.1 was studied. Taken together, research described in this thesis links the morphological observations on leaf and leafy head development to molecular genetic results, both at the population and cellular levels.

Downy mildew, caused by the oomycete Bremia lactucae, is a devastating disease in lettuce (Lactuca sativa) cultivation, leading to high yield losses. An effective method of downy mildew control is the deployment of resistant lettuce cultivars. Over 50 monogenic, dominant resistance (R) genes have been deployed in lettuce breeding. R genes provide high levels of resistance, but are continually rendered ineffective by the occurrence of pathogenic strains with new virulence characteristics. This demonstrates the presence of an arms race between plant and pathogen. R genes may be an effective but short-term solution to control downy mildew in lettuce, as long as sufficient resistance sources can continuously provide novel R genes. However, the continuous introgression of new R genes in elite breeding material demands much time of breeders and is not a durable solution. Until now, most resistance genes have been derived from the primary gene pool of cultivated lettuce. The wild lettuce species L. saligna is a member of the secondary gene pool, completely resistant to all B. lactucae isolates and considered as a nonhost for downy mildew. Despite its potential attractiveness as a resistance source, L. saligna has hardly been exploited for lettuce breeding. In chapter 2, we explored the potential of screening for R genes in L. saligna by use of pathogen candidate effectors. Effectors are pathogenic proteins to aid infection of specific plant species. We transiently expressed 16 candidate effectors from B. lactucae in a diverse Lactuca germplasm set (n=150). Accessions that react with a hypersensitive response (HR) are supposed to carry an R gene that recognizes the effector as an avirulence factor. Two candidate effectors (BLN08 and BLR31) induced an HR in L. saligna accessions. BLN08 triggered an HR in 55% of the accessions, but in segregating populations responsiveness did not co-segregate with resistance to Bl:24, the B. lactucae race from which the effectors were cloned

Durum wheat (Triticum durum) is among the most important food crops of the Mediterranean Basin, encompassing regions in Southern Europe and North Africa, as well as the Northern Great Plains of the U.S. and parts of Southern Asia. Particularly in North Africa it is the prime economical and dietary crop for small holder farmers in marginal areas and has greatly contributed to the existing genetic diversity of bread wheat. Despite its standing as a staple crop, primarily in North Africa and Southern Europe, the overall vulnerability of durum wheat germplasm to fungal diseases is well known and frequently reported. Among those is septoria tritici blotch (STB), the major foliar disease of wheat in Europe that is caused by Zymoseptoria tritici. However, the scientific community has limited attention for this crop and mostly focused on bread wheat. Therefore, very little is known about the genetic basis of the resistance to STB in tetraploid wheat. In bread wheat, 21 Stb major genes and manifold quantitative trait loci (QTLs) have been identified and intensively deployed in breeding programs, whereas Stb genes are neither recognized nor mapped in the largely under- investigated tetraploid wheats. One of the reasons is the reported dichotomy of the pathogenicity of Z. tritici isolates towards bread and durum wheat. This has been an additional hurdle hampering breeding for resistance in durum wheat because well characterized Z. tritici isolates that are pathogenic on bread wheat cannot be used in durum wheat phenotyping assays (and vice versa). On top of that, specificity in either of these wheat systems has been questioned for a long time and therefore hindered effective breeding strategies. Thus, deciphering the genetics of the wheat-Z. tritici interaction, specifically for tetraploid wheats, greatly contributes to enhancing our understanding of this important pathosystem and thereby to more effective breeding strategies in this important cereal staple crop. Chapter 1 is

The identification of haplotypes in tetraploid potato allows to improve genetic studies and facilitate marker-assisted selection. For many years, only bi-allelic molecular markers were used for application in genetic studies and they undoubtedly improved our understanding of the inheritance of important agronomical traits. However, these undertakings are complicated by the lack of knowledge about linkage between these SNPs and thus their underlying haplotype structure. The inability of geneticists to achieve haplotype reconstruction was mainly due to complications of the higher ploidy level of cultivated potato (2x = 4x = 48), as a single potato variety contains four copies of each chromosome (tetraploid). In this thesis, methods are described that allow haplotype reconstruction in tetraploid potato, either from sequencing data of a single variety or by use of SNP information over multiple varieties. We employed these methods on genotypic data of potato varieties and used the reconstructed haplotypes to detect which alleles influence traits such as plant maturity, tuber shape and flesh color. The starting point of this thesis was a genetic study of the inheritance of potato tuber shape and eye depth. In Chapter 2 we identified a strong marker-trait association for tuber shape on potato chromosome 10 (Ro locus), that co-localises with a major effect QTL for eye depth. Subsequent fine mapping in a diploid full-sib potato population (C × E) refined the associated region of 3.1 Mb to a small region of 280 Kb. In this region, a repeat cluster of peroxidase genes is located. In Chapter 3 we started with the development of methods for haplotype reconstruction. We introduced a novel method to use short-read DNA sequencing data to reconstruct haplotypes. A previous study genotyped ~800 potato genes in 83 tetraploid varieties using Illumina short reads. This information was used as input for our haplotype reconstruction pipeline and allowed us to generate haplotype blocks of

Late blight, caused by the oomycete pathogen Phytophthora infestans, is the most devastating disease in potato. For sustainable management of this economically important disease, resistance breeding relies on the availability of resistance (R) genes. Such R genes against P. infestans have evolved in wild tuber-bearing Solanum species from North, Central and South America, upon co-evolution with cognate avirulence (Avr) genes. Here, we report how effectoromics screens with Avr2 of P. infestans revealed defense responses in diverse Solanum species that are native to Mexico and Peru. We found that the response to AVR2 in the Mexican Solanum species is mediated by R genes of the R2 family that resides on a major late blight locus on chromosome IV. In contrast, the response to AVR2 in Peruvian Solanum species is mediated by Rpi-mcq1, which resides on chromosome IX and does not belong to the R2 family. The data indicate that AVR2 recognition has evolved independently on two genetic loci in Mexican and Peruvian Solanum species, respectively. Detached leaf tests on potato cultivar ‘Désirée’ transformed with R genes from either the R2 or the Rpi-mcq1 locus revealed an overlapping, but distinct resistance profile to a panel of 18 diverse P. infestans isolates. The achieved insights in the molecular R – Avr gene interaction can lead to more educated exploitation of R genes and maximize the potential of generating more broad-spectrum, and potentially more durable control of the late blight disease in potato.

Background Recent advances in ~omics technologies such as transcriptomics, metabolomics and proteomics along with genotypic profiling have permitted the genetic dissection of complex traits such as quality traits in non-model species. To get more insight into the genetic factors underlying variation in quality traits related to carbohydrate and starch metabolism and cold sweetening, we determined the protein content and composition in potato tubers using 2D–gel electrophoresis in a diploid potato mapping population. Upon analyzing we made sure that the proteins from the patatin family were excluded to ensure a better representation of the other proteins. Results We subsequently performed pQTL analyses for all other proteins with a sufficient representation in the population and established a relationship between proteins and 26 potato tuber quality traits (e.g. flesh colour, enzymatic discoloration) by co-localization on the genetic map and a direct correlation study of protein abundances and phenotypic traits. Over 1643 unique protein spots were detected in total over the two harvests. We were able to map pQTLs for over 300 different protein spots some of which co-localized with traits such as starch content and cold sweetening. pQTLs were observed on every chromosome although not evenly distributed over the chromosomes. The largest number of pQTLs was found for chromosome 8 and the lowest for chromosome number 10. For some 20 protein spots multiple QTLs were observed. Conclusions From this analysis, hotspot areas for protein QTLs were identified on chromosomes three, five, eight and nine. The hotspot on chromosome 3 coincided with a QTL previously identified for total protein content and had more than 23 pQTLs in the region from 70 to 80 cM. Some of the co-localizing protein spots associated with some of the most interesting tuber quality traits were identified, albeit far less than we had anticipated at the onset of the experiments.

Crambe abyssinica (crambe) is a new industrial oil crop that can grow on saline soil and tolerates salty water irrigation. Genetically engineered crambe in which the seed‐oil composition is manipulated for more erucic acid and less polyunsaturated fatty acid (PUFA) would be highly beneficial to industry. In this research, lysophosphatidic acid acyltransferase 2 RNA interference (CaLPAT2‐RNAi) was introduced into the crambe genome to manipulate its oil composition. The result showed in comparison with wild type, CaLPAT2‐RNAi could significantly reduce linoleic and linolenic acid content, simultaneously increasing erucic acid content. Systematic metabolism engineering was then carried out to further study CaLPAT2‐RNAi, combined with the overexpression of Brassica napus fatty acid elongase (BnFAE), Limnanthes douglasii LPAT (LdLPAT), and RNAi of endogenous fatty acid desaturase 2 (CaFAD2‐RNAi). Oil composition analysis on the tranformants' seeds showed that (a) with CaFAD2‐RNAi, PUFA content could be dramatically decreased, in comparison with BnFAE + LdLPAT + CaFAD2‐RNAi, and BnFAE + LdLPAT + CaFAD2‐RNAi + CaLPAT2‐RNAi seeds showed lower linolenic acid content; (b) BnFAE + LdLPAT + CaFAD2‐RNAi + CaLPAT2‐RNAi could increase the erucic acid content in crambe seed oil from less than 66.6% to 71.6%, whereas the highest erucic acid content of BnFAE + LdLPAT + CaFAD2‐RNAi was 79.2%; (c) although the four‐gene combination could not increase the erucic acid content of seed oil to a higher level than the others, it led to increased carbon resource deposited into C22:1 and C18:1 moieties and lower PUFA. Summarily, the present research indicates that suppression of LPAT2 is a new, promising strategy for seed‐oil biosynthesis pathway engineering, which would increase the value of crambe oil.

Polyploid organisms carry more than two copies of each chromosome, a condition rarely tolerated in animals but which occurs relatively frequently in the plant kingdom. One of the principal challenges faced by polyploid organisms is to evolve stable meiotic mechanisms to faithfully transmit genetic information to the next generation upon which the study of inheritance is based. In this review we look at the tools available to the research community to better understand polyploid inheritance, many of which have only recently been developed. Most of these tools are intended for experimental populations (rather than natural populations), facilitating genomics-assisted crop improvement and plant breeding. This is hardly surprising given that a large proportion of domesticatedplant species are polyploid. We focus on three main areas: (1) polyploid genotyping; (2) genetic and physical mapping; and (3) quantitative trait analysis and genomic selection. We also briefly review some miscellaneous topics such as the mode of inheritance and the availability of polyploid simulation software. The current polyploid analytic toolbox includes software for assigning marker genotypes (and in particular, estimating the dosage of marker alleles in the heterozygous condition), establishing chromosome-scale linkage phase among marker alleles, constructing (short-range) haplotypes, generating linkage maps, performing genome-wide association studies (GWAS) and quantitative trait locus (QTL) analyses, and simulating polyploid populations. These tools can alsohelp elucidate the mode of inheritance (disomic, polysomic or a mixture of bothas in segmental allopolyploids) or reveal whether double reduction and multivalent chromosomal pairing occur. An increasing number of polyploids (or associated diploids) are being sequenced, leading to publicly available reference genome assemblies. Much work remains in order to keep pace with developments in genomic technologies. However, such technologies als

Most regulations worldwide stipulate that a new genetically modified (GM) crop event has to be compared to its closest non-GM counterpart as a corner stone of the pre-market risk assessment. To this end the GM crop and its comparator should be grown in field trials for a phenotypic comparison as well as for subsequent detailed analysis of the composition of the two crop varieties. A more in-depth globally harmonised approach for the conduct of these field trials is lacking. Only a few countries have formulated detailed protocols for the set-up of GM field trials. In some countries, commercial non-GM reference varieties need to be included in a field study to compile reliable data that indicate the range of natural variation for the compounds tested at the specific location. Detailed analysis of pre-market assessment reports have so far not shown the added value of including these reference varieties in the field trials. In all cases where specific values were found to be outside of the range of the reference varieties, it proved possible to draw conclusions on the part of the pre-market risk assessment that relates to the compositional analysis, on the basis of already available compositional data. With the increasing quality of several databases on compositional data of a growing number of crop species, it seems unlikely that reference varieties will become more important on future occasions. It was furthermore investigated whether this part of the risk assessment can be related to field trial requirements for variety registration with the explicit intention of reducing the data burden on producers of new GM plant varieties. Field trials for variety registration so far include an assessment of phenotypic characteristics that do not cover safety aspects, with the exception of establishment of the glycoalkaloid content in potatoes in the Netherlands and Sweden. It may, however, under certain conditions be relatively easy to exchange data from compositional measuremen

Usually, mapping studies in potato are performed with segregating populations from crosses between highly heterozygous diploid or tetraploid parents. These studies are hampered by a high level of genetic background noise due to the numerous segregating alleles, with a maximum of eight per locus. In the present study, we aimed to increase the mapping efficiency by using progenies from diploid inbred populations in which at most two alleles segregate. Selfed progenies were generated from a cross between S. tuberosum (D2; a highly heterozygous diploid) and S. chacoense (DS; a homozygous diploid clone) containing the self-incompatibility overcoming S locus inhibitor (Sli-gene). The Sli-gene enables self-pollination and the generation of selfed progenies. One F2 population was used to map several quality traits, such as tuber shape, flesh and skin color. Quantitative trait loci were identified for almost all traits under investigation. The identified loci partially coincided with known mapped loci and partially identified new loci. Nine F3 populations were used to validate the QTLs and monitor the overall increase in the homozygosity level.

Contains fulltext : 175484.pdf (publisher's version ) (Open Access)

Contains fulltext : 175479.pdf (publisher's version ) (Open Access)

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 frui

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 af

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 tes

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.

Tomato production is threatened by climate change. High temperatures lead to a decrease of fruit set which correlates with a decrease of pollen fertility. The low viability of tomato pollen under heat stress was previously shown to be associated with alterations in specific metabolites. In this thesis, we used untargeted metabolomics approaches to broaden the identification of metabolites affected by heat stress. We assessed the suitability of pollen isolation methods for metabolomics analysis and considered the pitfalls for our further analysis. We explored the developmental metabolomes of pollen and anthers of different tomato genotypes under control and high temperature conditions and identified that microsporogenesis is a critical developmental stage for the production of mature and fertile pollen grain under heat stress. Several metabolites were putatively associated with tolerance to high temperature such as specific flavonoids, polyamines and alkaloids. These metabolites can be further used as markers in breeding programs to develop new genotypes tolerant to high temperatures.

The button mushroom, Agaricus bisporus var. bisporus, is one of the most cultivated mushrooms worldwide. Even though wild isolates of this variety have a broad genetic variation, the traditional and present-day hybrids only have a very narrow genetic base. The button mushroom has a typical meiotic recombination landscape (MRL) in which crossover (CO) events are predominantly restricted to the extreme ends of the chromosomes. This has been one of the main obstacles for mushroom breeders in improving or generating new mushroom hybrids due to a considerable linkage drag. A wild variety of A. bisporus, i.e., burnettii appeared to have CO spread more evenly across the genome. The existence of two extremely different MRLs in two compatible A. bisporus varieties offers an excellent opportunity to study the genetic basis for positioning CO in meiosis. The main objective of the research presented in this thesis initially was to examine meiosis of the var. burnettii in more detail and subsequently to identify genomic regions revealing the difference in MRL of the two A. bisporus varieties. The availability of genome sequences in the bisporus variety has produced many more informative markers such as SNP. We aimed to de novo sequence one of the haplotypes of a heterokaryotic strain of the burnettii variety using the PacBio sequencing technique and resequencing the other haplotype using Illumina HiSeq. In parallel to this, we used Genotyping by Sequencing (GBS) to construct the first linkage map of the burnettii variety, showing a more or less even distribution of COs across the genome. The constructed linkage map has also proved to be a useful tool for de novo assembly of the burnettii variety genome sequence. In addition, we performed comparative genome sequence studies between the burnettii variety and the previously sequenced genomes of two of the bisporus variety homokaryons, indicating high levels of collinearity between all three genomes. The only chromosomal rearrangeme

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 bot

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 drou

Candidate effectors from lettuce downy mildew (Bremia lactucae) enable high-throughput germplasm screening for the presence of resistance (R) genes. The nonhost species Lactuca saligna comprises a source of B. lactucae R genes that has hardly been exploited in lettuce breeding. Its cross-compatibility with the host species L. sativa enables the study of inheritance of nonhost resistance (NHR). We performed transient expression of candidate RXLR effector genes from B. lactucae in a diverse Lactuca germplasm set. Responses to two candidate effectors (BLR31 and BLN08) were genetically mapped and tested for co-segregation with disease resistance. BLN08 induced a hypersensitive response (HR) in 55% of the L. saligna accessions, but responsiveness did not co-segregate with resistance to Bl:24. BLR31 triggered an HR in 5% of the L. saligna accessions, and revealed a novel R gene providing complete B. lactucae race Bl:24 resistance. Resistant hybrid plants that were BLR31 nonresponsive indicated other unlinked R genes and/or nonhost QTLs. We have identified a candidate avirulence effector of B. lactucae (BLR31) and its cognate R gene in L. saligna. Concurrently, our results suggest that R genes are not required for NHR of L. saligna.

Plants are exposed to combinations of various biotic and abiotic stresses, but stress responses are usually investigated for single stresses only. Here, we investigated the genetic architecture underlying plant responses to 11 single stresses and several of their combinations by phenotyping 350 Arabidopsis thaliana accessions. A set of 214 000 single nucleotide polymorphisms (SNPs) was screened for marker-trait associations in genome-wide association (GWA) analyses using tailored multi-trait mixed models. Stress responses that share phytohormonal signaling pathways also share genetic architecture underlying these responses. After removing the effects of general robustness, for the 30 most significant SNPs, average quantitative trait locus (QTL) effect sizes were larger for dual stresses than for single stresses. Plants appear to deploy broad-spectrum defensive mechanisms influencing multiple traits in response to combined stresses. Association analyses identified QTLs with contrasting and with similar responses to biotic vs abiotic stresses, and below-ground vs above-ground stresses. Our approach allowed for an unprecedented comprehensive genetic analysis of how plants deal with a wide spectrum of stress conditions.

Whole Genome Shotgun (WGS) sequences of plant species often contain an abundance of reads that are derived from the chloroplast genome. Up to now these reads have generally been identified and assembled into chloroplast genomes based on homology to chloroplasts from related species. This re-sequencing approach may select against structural differences between the genomes especially in non-model species for which no close relatives have been sequenced before. The alternative approach is to de novo assemble the chloroplast genome from total genomic DNA sequences. In this study, we used k-mer frequency tables to identify and extract the chloroplast reads from the WGS reads and assemble these using a highly integrated and automated custom pipeline. Our strategy includes steps aimed at optimizing assemblies and filling gaps which are left due to coverage variation in the WGS dataset. We have successfully de novo assembled three complete chloroplast genomes from plant species with a range of nuclear genome sizes to demonstrate the universality of our approach: Solanum lycopersicum (0.9 Gb), Aegilops tauschii (4 Gb) and Paphiopedilum henryanum (25 Gb). We also highlight the need to optimize the choice of k and the amount of data used. This new and cost-effective method for de novo short read assembly will facilitate the study of complete chloroplast genomes with more accurate analyses and inferences, especially in non-model plant genomes.

BackgroundBotrytis cinerea, a necrotrophic pathogenic fungus, attacks many crops including potato and tomato. Major genes for complete resistance to B. cinerea are not known in plants, but a few quantitative trait loci have been described in tomato. Loss of function of particular susceptibility (S) genes appears to provide a new source of resistance to B. cinerea in Arabidopsis.ResultsIn this study, orthologs of Arabidopsis S genes (DND1, DMR6, DMR1 and PMR4) were silenced by RNAi in potato and tomato (only for DND1). DND1 well-silenced potato and tomato plants showed significantly reduced diameters of B. cinerea lesions as compared to control plants, at all-time points analysed. Reduced lesion diameter was also observed on leaves of DMR6 silenced potato plants but only at 3 days post inoculation (dpi). The DMR1 and PMR4 silenced potato transformants were as susceptible as the control cv Desiree. Microscopic analysis was performed to observe B. cinerea infection progress in DND1 well-silenced potato and tomato leaves. A significantly lower number of B. cinerea conidia remained attached to the leaf surface of DND1 well-silenced potato and tomato plants and the hyphal growth of germlings was hampered.ConclusionsThis is the first report of a cytological investigation of Botrytis development on DND1-silenced crop plants. Silencing of DND1 led to reduced susceptibility to Botrytis, which was associated with impediment of conidial germination and attachment as well as hyphal growth. Our results provide new insights regarding the use of S genes in resistance breeding.