Cellular networks are required for a variety of processes in complex organisms. Caenorhabditis elegans is a useful model to gain insight into the gene regulatory networks that assemble cellular networks. Mutations in a variety of genes can affect the sinusoidal locomotive pattern of C. elegans. We isolated the mutant jd1500 from a standard genetic screen looking for mutants in C. elegans that exhibit asymmetric locomotive patterns. The two aims of this study were to: 1) identify the gene and characterize its role in the gene regulatory network and 2) characterize the cells affected by the mutation. We reasoned that jd1500 likely disrupts the proper balance between dorsal and ventral body wall muscle contractions. By using three-point genetic mapping, we predicted the locus of jd1500 between -9.42 and -11.73 centimorgans of the X chromosome. Our results implicate the embryonic, cholinergic DB motor neurons as likely cellular targets of the mutation.

The study of aflatoxin in Aspergillus spp. has garnered the attention of many researchers due to aflatoxin's carcinogenic properties and frequency as a food and feed contaminant. Significant progress has been made by utilizing the model organism Aspergillus nidulans to characterize the regulation of sterigmatocystin (ST), the penultimate precursor of aflatoxin. A previous forward genetic screen identified 23 A. nidulans mutants involved in regulating ST production. Six mutants were characterized from this screen using classical mapping (five mutations in mcsA) and complementation with a cosmid library (one mutation in laeA). The remaining mutants were backcrossed and sequenced using Illumina and Ion Torrent sequencing platforms. All but one mutant contained one or more sequence variants in predicted open reading frames. Deletion of these genes resulted in identification of mutant alleles responsible for the loss of ST production in 12 of the 17 remaining mutants. Eight of these mutations were in genes already known to affect ST synthesis (laeA, mcsA, fluG, and stcA), while the remaining four mutations (in laeB, sntB, and hamI) were in previously uncharacterized genes not known to be involved in ST production. Deletion of laeB, sntB, and hamI in A. flavus results in loss of aflatoxin production, confirming that these regulators are conserved in the aflatoxigenic aspergilli. This report highlights the multifaceted regulatory mechanisms governing secondary metabolism in Aspergillus. Additionally, these data contribute to the increasing number of studies showing that forward genetic screens of fungi coupled with whole-genome resequencing is a robust and cost-effective technique. IMPORTANCE In a postgenomic world, reverse genetic approaches have displaced their forward genetic counterparts. The techniques used in forward genetics to identify loci of interest were typically very cumbersome and time-consuming, relying on Mendelian traits in model organisms. The current work was pursued not only to identify alleles involved in regulation of secondary metabolism but also to demonstrate a return to forward genetics to track phenotypes and to discover genetic pathways that could not be predicted through a reverse genetics approach. While identification of mutant alleles from whole-genome sequencing has been done before, here we illustrate the possibility of coupling this strategy with a genetic screen to identify multiple alleles of interest. Sequencing of classically derived mutants revealed several uncharacterized genes, which represent novel pathways to regulate and control the biosynthesis of sterigmatocystin and of aflatoxin, a societally and medically important mycotoxin.

Salt stress is a global problem that limits agricultural production. The early responses to salinity, independent of toxic shoot-ion accumulation, are still largely unknown. Here, optimised salt treatment and high-throughput phenotyping protocols were developed and used to examine the natural variation in the early responses to salt stress of 191 Arabidopsis thaliana accessions. Common and novel traits of plants grown under salt treatment were captured through time using RGB and chlorophyll fluorescence imaging. Phenotypic data was combined with the Arabidopsis 10M SNP markers for genome-wide association studies to identify genetic components underlying the early responses to salt stress. The most promising candidate loci were selected based on association strength, allele frequency and number of traits associating to the same locus. In silico analysis highlighted interesting allelic variations across the identified loci, and by phenotypically characterising the candidate gene mutants under salt stress, the associations were experimentally validated. This work comprises a detailed study of the natural variation in the early responses to salt stress, which can give insights into the mechanisms contributing to salinity tolerance and provide the fundaments for crop improvements under saline conditions across the globe.

Strawberry and potato are both important crop species in the world providing various nutritional values. The cultivated strawberry, Fragaria ananassa, is a fruit crop with a complex genome (2n=8x=56) whereas the diploid woodland strawberry, Fragaria vesca, has a smaller genome (2n=2x=14, 240 Mb) and lots of other qualities that make it a good model for genetic and genomic study, such as high yield of seeds and efficient transformation. Potato (Solanum tuberosum, 2n=4x=48) is an important vegetable crop in the world and is highly heterozygous. The successful sequencing of the homozygous doubled monoploid clone of potato provides good insight into the study of important genes in this species in improving the pest resistance and improving yield. One approach to characterize gene function in a model system is having large populations of T-DNA insertional or transposon tagged mutants. The idea of using AcDs construct to create transposon tagged mutant populations has also been applied in many species. Here we transformed two species, Fragaria vesca and a monoploid potato, Solanum phureja 1-3-516, which is the progenitor of the sequenced doubled monoploid clone, with the same AcDs construct, Ac-DsATag-Bar_gosGFP, to generate mutant collection, compare the marker gene performance and transposition efficiency, as well as characterizing phenotypic mutants with genes of interest. Transposants were found to reinsert to unlinked sites from the launch pad site in the strawberry genome, whereas in potato transposants tended to locate locally from the launch pad position when using the same construct. One transposon based activation tagging strawberry mutant, with its insertion in the promoter region of gene of interest in strawberry from the Ac-DsATag-Bar_gosGFP population was studied. In a segregating T2 population, expression level of the candidate gene, epidermis-specific secreted glycoprotein EP1 precursor, was 670 fold higher in petioles of homozygotes than in wild type plants, suggesting the function of this gene involved in maintaining mechanical strength of petioles. Since the often-used transposase gene was cloned from the monocot species maize, the efficiency of obtaining germinal transposants was many times lower than expected in order to saturate the genome for diploid species. In order to improve the chance of getting unique transposants, we attempted to codon optimize the transposase gene, as well as switching to microspore specific promoters that had been well characterized to control timing of expression of the transposase gene. Transposants were found in both T0 primary regenerates and anther culture derived potatoes using both the pAcDs-AtSCP and pAcDs-AmDEFH125 constructs. Sequencing of the empty donor site revealed that excision occurred in different cells during anther culture. A strawberry mutant with sugar transport deficiency due to T-DNA insertion near a sucrose transporter-2 gene showing stunted phenotype with increased level of anthocyanin was also characterized. The concentrations of sucrose, glucose, and fructose were significantly greater in source leaves of the mutant than wild type plants, suggesting these compounds might be substrates of this gene in transporting to sink leaves and roots. / Ph. D.

To identify novel and essential components of the plant membrane trafficking mechanisms, Arabidopsis membrane trafficking mutants from fluorescent protein-based forward genetic screens were characterized. First, four novel glutathione synthase (GSH2) mutant alleles featured swollen endoplasmic reticulum (ER)-derived bodies that accumulated a soluble secretory marker. Consistent with the role of GSH2 in glutathione biosynthesis, the loss-of-function mutant alleles exhibited gamma-glutamylcysteine (γ-EC) hyperaccumulation and glutathione deficiency. The aberrant ER morphology was ascribed to the γ-EC accumulation. Redox-sensitive fluorescent protein revealed that gsh2 seedlings maintained a reduced cytoplasm at steady state but were more sensitive to oxidative challenge. Second, Mut 21 was a conditional mutant that accumulated a secretory marker in the alkalized apoplast at restrictive temperature (31˚C). The mutant was identified as carrying a mutant allele of tuftelin-interacting protein 11 (TFIP11), which has been implicated in regulating redifferentiation and cell proliferation through a cytokinin signalling pathway. Hence, it was postulated that the changes in response to cytokinin affect auxin-mediated acidification of the apoplast. Third, Mut 43 was a conditional mutant that accumulated a soluble secretory marker in the ER and unidentified punctate structures at restrictive temperature, and exhibited perturbations in ER export of a soluble protein marker. Moreover, the mutant showed severe growth defects and abnormal radial root swelling in the apical elongation zone. A mutation identification method through deep-sequencing of the wild-type siblings in outcrossed heterozygous mutant families was developed and tested in Mut 43. At the time when this thesis was prepared, bioinformatic analysis has assigned Mut 43 to the bottom arm of chromosome two and predicted a 300kb mapping interval based on the observed bias in single nucleotide polymorphism ratios. This work demonstrates the feasibility of using forward genetics to study plant-specific aspects of membrane trafficking mechanisms and incorporates new technology to streamline the process of gene identification.

Lat est clé dans le développement des cellules T. En l'absence de Lat, le développement des cellules T au niveau du thymus est complètement bloqué au stade DN3. Le développement des lymphocytes T est très diminué dans des souris knockin LatY136F dans lesquelles la tyrosine présente en position 136 de la protéine Lat est remplacée par une phénylalanine. De manière paradoxale, le petit nombre de cellules T qui émergent dans la périphérie des souris LatY136F donnent naissance à un syndrome lymphoprolifératif Th2 sévère. Ce projet vise à identifier les facteurs génétiques qui contribuent au développement d'un tel syndrome lymphoprolifératif. A la suite d'un crible génétique basée sur une mutagenèse à l'ENU (N-éthyl-N-nitrosurea), des lignées de souris mutantes LatY136F présentant une pathologie atténuée ou exacerbée ont été fixées et les mutations induites chimiquement ont été cartographiées et identifiées. En particulier, nous avons obtenu une lignée mutante appelée Basilic dépourvue du syndrome lymphoprolifératif. Le clonage positionnel assisté par les techniques de séquençage à haut-débit de l'ADN ont permis l'identification d'un gène appelé, Rltpr ou Lrrc16c, et ne possédant aucune fonction immunologique connue. Nous avons montré que Rltpr constitue un élément clé de la voie de signalisation opérée par la molécule de costimulation CD28. Les résultats principaux obtenus au cours de la thèse sont présentés sous forme d'un article publié. Un chapitre de perspective est consacré ensuite aux améliorations que nous pourrions apporter au type d'approche que nous avons réalisé. Enfin une dernière rubrique traite des outils permettant la manipulation du génome de la souris. / Lat is essential for proper T cell development. In the absence of Lat, intra thymic T cell development is completely blocked at an early stage known as the DN3 stage. LatY136F mice in which the tyrosine found at position 136 of Lat was replaced by a phenylalanine, also showed an impaired sequence of intrathymic T cell development. Paradoxically, the small number of improperly selected T cells that reach the periphery of LatY136F mice expand in an uncontrolled manner and trigger a severe Th2 lymphoproliferative disorder. The present thesis project aimed at identifying genetic factors contributing to the development of such a lymphoproliferative disorder. Using a sensitized ENU mutagenesis screen, we identified and characterized mutations that dampened or exacerbated the LatY136F pathology. A mutation that is called Basilic and that acts in a T cell intrinsic manner and completely prevented the LatY136F lymphoproliferative disorder was characterized in a comprehensive manner. Positional cloning assisted by next generation DNA sequencing demonstrated that Basilic corresponds to a mutation in a gene called Rltpr or Lrrc16c, the function of which was previously completely unknown in the frame of the immune system. Further functional studies performed during this project showed that Rltpr corresponded to a “missing link” involved in the signaling cascade that mediates the function of the co-stimulatory molecule CD28. Consistent with that view, nullozygous Cd28 mutant mice were found to be a phenocopy of the Basilic mice and as such prevented lymphoproliferative disorder in LatY136F mice. The Basilic mutation also resulted in a defect in regulatory T cell development.

<p>  </p> <p>Forward genetics discovery of the molecular basis of induced mutants has fundamentally contributed to our understanding of basic biological processes such as metabolism, cell dynamics, growth, and development. Advances in Next-Generation Sequencing (NGS) technologies enabled rapid genome sequencing but also come with limitations such as sequencing errors, dependence on reference genome accuracy, and alignment errors. By incorporating pedigree information to help correct for some errors I optimized variant calling and filtering strategies to respond to experimental design. This led to the identification of multiple causative alleles, the detection of pedigree errors, and an ability to explore the mutational spectrum of multiple mutagens in Arabidopsis. Similar to the problems in forward genetic discovery of mutant alleles, variation in genomes complicates the analysis of gene expression affected by natural variation. The plant hypersensitive response (HR) is a highly localized and rapid form of programmed cell death that plants use to contain biotrophic pathogens. Substantial natural variation exists in the mechanisms that trigger and control HR, yet a complete understanding of the molecular mechanisms modulating HR is lacking. I explored the gene expression consequences of the plant HR in maize using a semi-dominant mutant encoding a constitutively active HR-inducing Nucleotide Binding Site Leucine Rich Repeat protein, <em>Rp1-D21,</em> derived from the receptor responsible for perceiving certain strains of the common rust <em>Puccinia sorghi</em>. Differentially expressed genes (DEG) in response to <em>Rp1-D21</em> were identified in different genetic backgrounds and hybrids that exhibit divergent enhancing (NC350) or suppressing (H95, B73) effects on the visual manifestations of HR. To enable this analysis, I created anonymized reference genomes for each comparison, so that the reference genome induced less bias in the mapping steps. Comprehensive identification of DEG corroborated the visual phenotypes and provided the identities of genes influential in plant hypersensitive response for further studies. The locations of expression quantitative trait loci (eQTL) that determined the differential response of NC350 and B73 were identified using 198 F1 families generated by crossing B73 x NC350 RIL population and <em>Rp1-D21</em>/+ in H95. This identified 3514 eQTL controlling the variability in differential expression between mutant versus wild-type. <em>Trans-</em>eQTL were dramatically arranged in the genome and identified 17 hotspots with more than 200 genes influenced by each locus. A single locus significantly affected expression variation in 5700 genes, 5396 (94.7%) of which were DGE. An allele specific expression analysis of NC350 x H95 and B73 x H95 F1 hybrids with and without <em>Rp1-D21</em> identified <em>cis-</em>eQTL and ASE at a subset of these genes. Bias in the confirmation of eQTL by ASE was still present despite the anonymized reference genomes indicating that additional efforts to improve signal processing in these experiments is needed.</p>

Mosquitoes belonging to the genera Anopheles transmit malaria parasites, attributing the highest mortality of any vector-borne disease worldwide. Mosquitoes belonging to the genera Aedes transmit arboviruses including dengue, which has become the most important vector-borne virus due to a drastic surge in disease incidence. The scope of the studies in this dissertation is broad, with investigations bringing together elements of classical genetics, recent advances in sequencing and genome-editing technologies, and the use of modern forward genetics approaches. Chapter 2 of this dissertation explores the use of the Oxford Nanopore Sequencing Technology for the first time in mosquitoes. This new technology provides long reads that were used to piece together the AabS3 chromosomal assembly for Anopheles albimanus. The utility of this genomic resource is demonstrated by the discovery of novel telomeric repeats at the ends of the chromosomes that could have important implications in mosquito biology and control. Chapter 3 describes a forward genetics strategy called 'Marker-Assisted Mapping' (MAM) that enables high-resolution mapping of the causal gene locus of a mutant phenotype. The principle and effectiveness of MAM is first demonstrated by mapping a known transgene insertion. MAM is then used to identify cardinal as a candidate causal gene for the spontaneous red-eye (re) mutation. Genetic crosses between the re mutant and cardinal knocking out individuals generated using CRISPR/Cas9 confirmed that cardinal indeed is the causal gene for re mutation. Chapter 4 explores three innovative strategies for mosquito sex separation by exploiting several sex-linked marker lines. We show that by linking a transgenic marker to the male-determining locus (M locus), or by linking the male-determining Nix gene to a marker, males can be precisely separated from females. We also produce a two-marker transgenic line that allows for both non-transgenic male separation and for efficient line maintenance. Finally, we discuss further applications of the resources generated and future directions stemming from these findings. Altogether, the studies described in this dissertation contribute to the overall goal of understanding mosquito biology and of controlling mosquito-borne infectious diseases. / Doctor of Philosophy / Female mosquitoes bite and transmit deadly pathogens including the malaria parasite, and viruses such as dengue, Zika, and West Nile. Control programs that attempt to limit the spread of these deadly diseases rely on the control of mosquitoes themselves. These mosquito control methods have relied heavily on indoor and outdoor insecticidal spraying. However, the efficacy of these methods has been jeopardized by the increasing prevalence of insecticide resistance. Thus, it is necessary to implement other methods for effective mosquito control. Genetic control strategies such as the Sterile Insect Technique (SIT) and Wolbachia-based Incompatible Insect Technique (IIT) are excellent solutions to overcome the limitations of current control strategies. As female mosquitoes bite and transmit disease-causing pathogens, only males are released, which necessitate the separation of the non-biting males from females before release. The aim of this work was to use recent technological advancements to better understand the genome and basic genetics of vector mosquito species, and to identify possible approaches to improve current sex separation practices. To develop a deep understanding of mosquito biology and genetics, it is crucial that a high-quality and accurate genome assembly is available. However, many mosquito genome assemblies remain fragmented. To address this limitation, we used recent advances in sequencing technologies to produce a high-quality genome assembly for the New World malaria mosquito, Anopheles albimanus. These sequencing and assembly efforts led to the discovery of novel telomere sequences at the ends of chromosomes, which could have implications for mosquito control. Forward genetics, which identifies the gene(s) responsible for a given phenotype, has been hindered by the low recombination rate in the yellow and dengue fever mosquito, Aedes aegypti. We develop a Marker-Assisted Mapping (MAM) strategy to address this problem. We first demonstrate this method by mapping the known insertion of a transgene. MAM is then used to identify cardinal as a candidate causal gene for the spontaneous red-eye (re) mutation. MAM identification of the Cardinal gene was then verified by knocking out Cardinal, which represents the first successful gene mapping in Aedes aegypti using forward genetics. The MAM strategy has broad implications as it could enable the discovery of genes involved in important traits such as insecticide resistance. To improve sex separation methods, we took advantage of several sex-linked transgenic lines to develop three novel strategies. First, we demonstrate that screening for a genetic marker that is tightly linked to the male-determining locus (M locus) is an effective approach to reduce female contamination. Second, we demonstrate that instead of linking a marker to the M locus, we can link the male-determining factor, Nix, to a genetic marker. When a Nix transgene is located adjacent to the red-eye locus with extremely tight linkage, the red-eye phenotype becomes a faithful marker for separation of males and females. Finally, we developed a two-marker genetic sexing strain that produces non-transgenic males that could be used for release, and transgenic marked males and females for efficient line maintenance.

La taille du fruit et la morphologie des tissus du fruit sont des caractères clés définissant la qualité finale du fruit. Parmi la grande diversité de fruits observée dans la nature, la domestication et la sélection ont entrainé d’importantes modifications de la taille et de la morphologie des tissus du fruit. Jusqu'à présent, seuls quelques régulateurs génétiques ont été identifiés, et les mécanismes cellulaires et moléculaires par lesquels la morphologie des tissus du fruit est définie restent imprécis. Dans ce contexte, l'objectif de ma thèse est d'identifier et de caractériser de nouveaux régulateurs impliqués dans la morphologie des tissus du fruit. Pour cela, j'ai utilisé une collection de mutants EMS de tomate comme source de diversité génétique et phénotypique et j'ai sélectionné deux mutants présentant des tendances opposées et extrêmes d'épaisseur du péricarpe. Grace à une stratégie de cartographie par séquençage, j’ai identifié une région génétique du chromosome 10, associée au phénotype péricarpe épais. J'ai également étudié le rôle de la Guanylate Binding Protein (GBP) à l’origine du phénotype péricarpe fin chez la tomate. La GBP est une grosse GTP binding protein qui n’a jamais été caractérisée chez les plantes. Afin d'approfondir l’étude de cette protéine, j'ai étudié en parallèle son rôle dans les modèles tomate et Arabidopsis" thaliana. J'ai démontré que les deux protéines homologues sont localisées dans le noyau. La mutation de la GBP chez la tomate induit de fortes altérations de la division et de l'expansion cellulaire à l'intérieur du péricarpe ainsi qu'une altération de la croissance des racines latérales chez la tomate et Arabidopsis, une caractéristique classiquement retrouvée chez les mutants altérés dans la mitose. Cette étude suggère que le GBP joue un rôle dans le contrôle précis des divisions cellulaires dans le péricarpe de tomate. / Fruit size and morphology are key characters defining the final fruit quality. Among the large fruit diversity observed in the nature, human domestication and selection has induced changes in fruit size and tissue morphology. Only a few genetic regulators have been identified so far, thus cellular and molecular mechanisms by which fruit tissue morphology is defined remain incomplete. In this context, the aim of my thesis is to identify and characterize new regulators of fruit tissue morphology. For this purpose, I used a collection of tomato EMS mutants as a source of genetic and phenotypic diversity. I selected two mutants presenting opposite trends of pericarp thickness. Through a mapping-by-sequencing strategy, I identified a genetic region on chromosome 10, associated with an extreme thick pericarp phenotype. I also investigated the role of the Guanylate Binding Protein (GBP) at the origin of a thin pericarp phenotype. The GBP is a large GTP binding protein that was never characterized in plants so far. In order to go deeper into its functional characterization in plants, I studied in parallel the role of the protein in tomato and Arabidopsis "thaliana" models. I showed that both homolog proteins are localized at the nucleus. Mutation of GBP in tomato induced strong alterations in cell division and cell expansion inside the pericarp and altered lateral root growth in tomato and Arabidopsis, a classical feature for mutants impaired in mitosis. This study suggests a role for the GBP in the fine control of cell division in the tomato pericarp.

Quinoa has been utilized for millennia in the Andes region of South America as a nutritious and hardy food crop. In recent years interest in quinoa has grown as need increases for an alternative to traditional cereal crops that can tolerate marginal environments while offering superior nutrition. Growers outside the Andes have experienced several complications adopting quinoa, including undesirable secondary metabolites, poor yield, lodging, and height inconsistency. Unfortunately, access to native ecotypes for crop improvement is limited, and desirable traits are difficult to introduce into available quinoa cultivars because of its allotetraploid genome and tendency to self-pollinate. A genome-wide survey of induced mutations in 244 sequenced M2 families was created from a bank of EMS-treated quinoa seeds and assembled into a library of mutant lineages with predicted variants and their effects on genes to assist in identifying agronomically valuable mutations in target genes as a supplement to crop improvement efforts. Using this library, eight families containing mutations in genes associated with reduced height "GAI1, GA20OX, GID1, and L " were identified. Several individuals exhibited a shorter than average phenotype; however, because each family contains thousands of EMS-induced mutations, the causative mutation of the reduced height phenotype in each family could not be definitively identified. In one family, absence of the GAI1 mutant allele, but the presence of a mutant CKX3 allele, provided a correlation between a mutation and the short phenotype. Genotyping each generation would be required for a targeted mutant allele to be tracked through selection.