Study of the genetic relationships and evolutionary histories of pupfish populations (Cyprinodontidae: Cyprinodon spp.) from the remnant aquatic habitats of Death Valley was approached by exploring the genetic structure and divergence within and among populations using mitochondrial and nuclear DNA markers. The findings of these studies illustrate the influences of population size and isolation time in the divergence of small, fragmented populations largely via genetic drift. The information revealed in this study has implications for assessing priorities in the conservation of the unique evolutionary heritage among populations of the Death Valley pupfishes. A new retrotransposable element family was identified and characterized. This family of genetic elements was uncovered during a search of the pupfish genome for transposable elements to be used as molecular markers for population analyses. The description of this element family, named "Swimmer 1" (SW1), provides new insights into the evolution of long interspersed nuclear elements (LINEs) in vertebrates. Therefore, a full characterization of the SW1 element family was undertaken in the Japanese medaka (Oryzias latipes) as well as in the pupfish genome. The Japanese medaka is a model organism widely used for genetic and developmental biology studies. / Ph. D.

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.

Les éléments transposables (ET) sont une source majeure de variation génétique, ce qui leur confère un rôle essentiel dans l’évolution des génomes. Certes présents dans tous les génomes analysés à ce jour, leurs proportions sont fortement variables entre espèces et aussi entre populations, suggérant une relation unique entre génome hôte et ET. Grâce à un système modèle composé de populations naturelles de deux espèces proches (Drosophila melanogaster et D. simulans) avec des quantités différentes en ET, nous avons pu comparer les relations génome hôte/ET. Nous nous sommes particulièrement interessés à l’élément helena qui, chez D. simulans, montre une activité faible, malgré un nombre de copies élevé.Cette activité moindre est associée à de nombreuses délétions internes des copies, suggérant un mécanisme de régulation d’ET par des délétions de l’ADN. Un autre système de régulation de l’activité des ET utilise le contrôle épigénétique, ce qui permet le maintien des copies d’ET dans le génome mais un blocage de leur activité. Le remodelage de la chromatine est un système épigénétique bien décrit chez la drosophile. Les régions chromatiniennes des génomes sont associées à différents types de modifications d’histone. Nous avons mis en évidence, dans des populations de D. melanogaster et D. simulans, une variation conséquente de modifications d’histones de type hétérochromatique, H3K27me3 et H3K9me2, associées àdes copies de différents ET. De plus, nous avons décrit des populations chez D. simulans dites déréprimées, chez lesquelles certains éléments sont surexprimés et présentent des localisations probablement hétéchromatiques. Les ET sont donc contrôlés par le génome hôte par des délétions internes et probablement par un système épigénétique variable. De plus, dans certaines populations, des copies peuvent échapper à ce contrôle et envahir le génome. Les ET sont donc des grands créateurs de variabilité génétique mais permettent aussi une territorialisation chromatinienne du génome car ils portent des modifications épigénétiques précises et sont capables de les étendre à leurs environnements génomiques. Ceci leur confére la fonction "d'épigénétique mobile". / Transposable elements (TEs) are one major force of genome evolution thanks to theirability to create genetic variation. TEs are ubiquitous and their proportion is variable between species and also populations, suggesting that a tight relationship exists between genomes and TEs. The model system composed of the natural populations of the twin sisters Drosophila melanogaster and D. simulans is interesting to compare host/TE relationship, since both species harbour different amounts of TE copies. The helena element is nearly silenced in D.simulans natural populations despite a very high copy number. Such repression is associated to abundant internally deleted copies suggesting a regulatory mechanism of TEs based on DNA deletion. Another pathway of TE regulation is through epigenetics where the host genome is able to keep intact the DNA sequences of TEs and still silence their activities.Chromatin remodelling is well known in drosophila and specific histone modifications can be associated to specific chromatin domains. We observed an important variation on H3K27me3and H3K9me2, two heterochromatic marks, on TE copies in D. melanogaster and D. simulans natural populations. Also, we show that derepressed lines of D. simulans exist for specific elements, have high TE transcription rates and are highly associated to non constitutive heterochromatic marks. TEs are therefore controlled by the host genome through DNA deletion and a possible chromatin remodelling mechanism. Not only genetic variability is enhanced by TEs but also epigenetic variability, allowing the host genome to be partitioned into chromatin domains. TEs are therefore mandatory to gene network regulation through their ability of “jumping epigenetics”.

Les éléments transposable (Ets) ont un rôle important dans l’évolution, puisque ce sont des séquences d’ADN qui ont la capacité de se déplacer dans le génome hôte. Nous cherchons à comprendre l’activité dans les croisements entre la Drosophile mojavensis et D. arizonae. La thèse est divisée en quatre chapitres, le première présente une analyse détaillée d'un rétrotransposon non-LTR appelé I , connu pour causer dysgénésie hybride D. melanogaster. Les analyses phylogénétiques réalisés, ont montré que les séquences I chez D. mojavensis et nourri par ceux d'autres espèces de Drosophila appartiennent, à des familles différentes d’ET. Les analyses d'expression par RTQ-PCR, a montré que cet élément est une activité de transcription dans les ovaires et les testicules des deux espèces et leurs hybrides, et ont une grande expression dans les testicules mais pas dans les ovaires des hybrides, qui pourraient être associés avec le mâle hybride phénotype de stérilité. Dans le deuxième chapitre sont présentées les analyses de TE exprimés dans les ovaires des deux souches parentales et leurs hybrides par l'ARN-Seq. Les résultats montrent des espèces spécifiques expression de TE chez les parents et les hybrides et d'une manière sans précédent, ET sont généralement réglementés en ce qui concerne les hybrides avec leurs parents, bien que certains d'entre eux sont surexprimés. Dans le troisième chapitre sont présentés les résultats de l'expression de quatre rétrotransposons (Helena, I, Copia et Osvaldo) quantifiés par RTQ -PCR; et enfin, dans le dernier chapitre, nous avons présenté des estimations de la taille du génome (C-valeur) dans les deux espèces parentales et hybrides réciproques. Dans l'ensemble, cette thèse révèle un scénario d'expression de TE spécifiques D. mojavensis et D. arizonae, et de sa réglementation dans les hybrides de rares exceptions près, qui peut nous aider à comprendre la complexité de la dynamique et de l'action de ces éléments mobiles dans la spéciation procédé de différentes espèces / The transposable elements (TEs) have an important role in evolution, since they are DNA sequences that have the ability to move into the host genome. We seek to understand the activity of the TEs in crosses between Drosophila mojavensis and D. arizonae. The thesis is divided into four chapters. The first presents a detailed analysis of an non- LTR retrotransposon called I, known to cause hybrid dysgenesis in D. melanogaster. Putatively active sequences similar to the I element were identified and characterized in the genome of D. mojavensis. The performed phylogenetic analyzes showed that the I sequences in D. mojavensis and those harbored by other Drosophila species belong to different I families. Expression analyses by RTq-PCR showed that this element is transcriptionally active in ovaries and testes of both species and their hybrids, and have high expression in the testes, but not in the hybrids ovaries, which could be associated with the male hybrid sterility phenotype. In the second chapter are presented analyses of expressed TEs in the ovaries of two parental strains and their hybrids by RNA-Seq. The results show species-specific expression of TEs in the parents and hybrids; and, in an unprecedented manner, that TEs are generally regulated in hybrids regarding with their parents, although some of them are overexpressed. In the third chapter are presented results of expression of four retrotransposons (Helena , I, Copia and Osvaldo) quantified by RTq-PCR; and finally, in the last chapter, we presented estimates of the genome size ( C - value), in both parental species and reciprocal hybrids. Overall, this thesis reveal a scenario of expression of specific TEs in D. mojavensis and D. arizonae, and its regulation in hybrids with rare exceptions, which can help us to understand the complexity of the dynamics and action of these mobile elements in the speciation process of different species

Elementos transponíveis (TEs) são sequências de DNA que possuem a capacidade de transposição no genoma hospedeiro. O principal objetivo deste trabalho reside na investigação em torno de possíveis contribuições de TEs nos genomas das algas C. reinhardtii e V. carteri, mais especificamente, na arquitetura dos genes ortólogos nestas espécies. Neste contexto, análises em sílico em larga escala foram realizadas, buscando-se identificar associações entre TEs e os genes ortólogos. Os resultados indicaram que os genes em C. reinhardtii tendem a acumular mais cópias de TEs em relação aos seus ortólogos em V. carteri. C. reinhardtii apresentou maior densidade de cópias de TEs para as regiões flanqueadora 5´ , flanqueadora 3´ e intrônica quando comparada a V. carteri; o inverso foi verificado quando analisada a densidade de TEs nas regiões codificantes. Análises para apurar a distribuição dos elementos em regiões intergênicas e intragênicas foram estabelecidas, nas quais a frequência observada dos elementos foi comparada à frequência esperada segundo a distribuição randômica de TEs no genoma, simulada computacionalmente. Foram constatadas regiões em que a presença dos elementos encontra-se significativamente abaixo do esperado, a exemplo de intervalos adjacentes ao início e ao término dos genes, o que provavelmente reflete a seleção negativa de eventos de integração nestas delimitações, em virtude dos efeitos deletérios associados à disrupção de estruturas de regulação da expressão gênica. De forma geral, nas regiões flanqueadoras 5´ e 3´, foi identificada a tendência de elevação da frequência padronizada de TEs à medida que a classe de distância avaliada se distancia do início e do término do gene, respectivamente. A baixa representatividade dos elementos também foi constatada em regiões intragênicas. O estudo da distribuição de TEs nos íntrons dos genes ortólogos indicou a preservação destas regiões quanto à fixação de TEs, sendo a representatividade abaixo do esperado mais evidente em intervalos adjacentes ao éxon, o que minimiza a chance de ruptura no padrão de splicing dos genes. Em sequências codificantes, a escassez de TEs - esperada devido ao provável efeito deletério destes eventos para a função do gene - foi constatada nos ortólogos das duas espécies. No entanto, inovações decorrentes da integração dos elementos em regiões codificantes podem resultar em efeitos evolutivos positivos, embora estes eventos sejam raros. Nas espécies analisadas foram identificados dois casos, de especial interesse, em que um domínio da sequência peptídica encontra-se localizado em região derivada de TE: o primeiro refere-se ao gene Cre06.g262800, em C. reinhardtii, no qual foi identificado o domínio PHD-finger associadao ao elemento Gypsy-5-LTR_CR; o segundo remete ao gene Vocar20001092m.g, em V. carteri, no qual o domínio zinc knuckle foi reconhecido em região derivada do elemento Gypsy3-LTR_VC. Estes genes constituem exemplos da contribuição de TEs na evolução de sequências codificadoras nas espécies C. reinhardtii e V. carteri, corroborando a hipótese de que os TEs podem contribuir na evolução da arquitetura dos genes, apesar do efeito disruptivo inerente à integração dos mesmos em regiões gênicas. / Transposable elements (TEs) are DNA sequences able to transpose in the host genome. The aim of this study resides in the investigation of TEs contributions in the algae C. reinhardtii and V. carteri genomes, more specifically in the architecture of orthologous genes in these species. In this context, large scale in silico analysis were performed to identify associations between TEs and orthologous genes. The results indicated that genes in the C. reinhardtii specie tend to accumulate more TEs copies than orthologous genes in V. carteri. C. reinhardtii showed higher density of TEs copies in the 5´ flanking, 3´ flanking and intronic regions when compared to V. carteri; the opposite was observed in coding regions. Investigation of the elements distribution in the intergenic and intragenic regions was performed, in which the observed TE frequency was compared to expected TE frequency from the simulated random distribution of the elements in the genome. It was verified regions where TE frequency was significantly lower than expected, as in gene boundaries adjacencies, probably reflecting a negative selection of the TE integration events in these delimitations due to deleterious effects associated with disruption of gene regulatory structures. In general terms, it was observed an increasing standardized frequency in the 5´ and 3´ flanking regions as the distance from gene start and gene end, respectively, increases. TEs underrepresentation was also verified in the intragenic regions. The study of TEs distribution in the introns of orthologous genes revealed the preservation of these structures in relation to TEs fixation, with a stronger underrepresentation near exon, which minimizes the chance of gene splicing pattern disruption. In the coding sequences, the TEs scarcity - expected due the likely deleterious effects to gene function - was verified in the orthologous of both species. However, in rare instances, innovations mediated by TEs integration in the coding regions can lead to positive evolutionary effects. In the species analyzed two instances of particular interest were observed, in which the domain of peptide sequence is located in the region derived from TE. The first one refers to the Cre06.g262800 gene, in the C. reinhardtii specie, which has a PHD-finger domain associated with Gypsy-5-LTR_CR element. The second one refers to the Vocar20001092m.g gene, in V. carteri, in which the zinc knuckle was recognized in region derived from Gypsy3-LTR_VC element. These genes are examples of TEs contributions in the evolution of coding sequences in the C. reinhardtii and V. carteri species, corroborating the hypothesis that TEs can contribute to the evolution of gene architecture, despite the inherent disruptive effect in their integration in the gene regions.

Les éléments transposables (ET) et leur reliques sont des composants majeurs des génomes eucaryotes. Ils sont potentiellement hautement mutagéniques car leur prolifération peut engendrer des réarrangements chromosomiques, des interruptions de gènes ou affecter l’expression génique par interférence transcriptionnelle. Néanmoins, peu d’ET sont généralement mobiles dans les génomes grâce à l’action de mécanismes qui restreignent leur activité comme la méthylation de l’ADN chez les mammifères et les plantes. De fait, chez Arabidopsis thaliana, une perte sévère de méthylation de l’ADN causée par une mutation dans le gène codant la protéine remodeleuse de chromatine DDM1 (DECREASE IN DNA METHYLATION 1) engendre l’accumulation massive de transcrits correspondants à des séquences d’ET. En revanche, peu d’ET semblent être mobilisés suite à cette réactivation transcriptionnelle. Nous proposons ici de déterminer (1) l’étendue de la mobilisation des ET suite à la perte de méthylation de l’ADN, (2) la distribution des nouvelles insertions d’ET le long du génome d’Arabidopsis et (3) les conséquences des nouvelles insertions d’ET sur l’expression des gènes situés à proximité. Dans ce but, nous avons séquencé le génome d’une cinquantaine d’epiRIL (epigenetic Recombinant Inbred Lines) dérivées d’un croisement entre une plante sauvage et un mutant ddm1. Suite au croisement retour de la F1 avec une plante sauvage et sélection des individus F2 homozygotes pour l’allèle sauvage DDM1, les epiRIL ont été propagées au travers de 6 autofécondations successives. Les epiRIL permettent donc l’étude détaillée des évènements de transpositions juste après qu’ils aient eu lieu. Pour identifier les évènements de transpositions dans ces lignées nous avons mis au point TE-tracker, un programme basé sur les données issues du séquençage Illumina de banques maite-pair. Par cette approche, nous avons montré que les ET mobiles dans ddm1 et les epiRIL appartiennent à seulement une quinzaine environ des >300 familles identifiées dans le génome d’Arabidopsis. Qui plus est, on observe des variations importantes de fréquences et dynamiques de transpositions entre les différentes familles d’ET ce qui suggère l’existence de mécanismes additionnels contrôlant la transposition. Les analyses moléculaires réalisées sur un sous-ensemble des ET mobilisés appartenant à différentes familles ont notamment montré que ces différences sont dues en grande partie aux différentes modalités d’établissement du contrôle épigénétique sur les ET nouvellement insérés. D’autre part, nos analyses indiquent que la distribution des nouvelles insertions d’ET diffère grandement de celle des copies résidentes. Ce résultat suggère donc que la suraccumulation des séquences d’ET dans les régions péricentromériques du génome d’Arabidopsis n’est pas due à un ciblage spécifique des insertions dans ces régions, mais est plutôt la conséquence de leur élimination des bras chromosomiques. Enfin, nous avons cherché à déterminer dans quelle mesure la méthylation de l’ADN associée aux séquences répétées a un impact sur l’expression des gènes situés à proximité en étudiant des mutants affectés dans les différentes voies de la méthylation de l’ADN. Par des analyses phénotypiques et moléculaires nous avons montré que, même si la plupart des gènes d’Arabidopsis n’est pas affectée par l’état de méthylation des séquences répétées situées à proximité, deux voies de la méthylation de l’ADN agissent ensemble pour maintenir l’expression normale d’un petit nombre de gènes ayant des effets pléiotropes situés proximité de séquences répétées. La méthylation de l’ADN agit donc comme un double système de contrôle pour assurer l’expression normale d’un petit nombre de gènes clefs localisés a proximité de séquences répétées. / Transposable elements (TEs) and their relics are major components of eukaryotic genomes. TEs are potentially highly mutagenic as their proliferation can cause chromosomal rearrangements, disrupt genes or affect gene expression through transcriptional interference. However, few TEs are usually mobile within genomes at any one time thanks to potent mechanisms that restrain their activity, such as DNA methylation in mammals and plants. Thus, in the flowering plant Arabidopsis, severe loss of DNA methylation caused by mutations in the chromatin remodeler gene DDM1 triggers massive accumulation of transcripts corresponding to TEs. Yet, comparatively few TEs appear to be mobilized as a result. Here, we set out to determine (1) the extent to which DNA methylation prevents TE mobilization, (2) where do TEs insert following their reactivation and (3) what are the consequences of new TE insertions on the expression of neighboring genes. To this ends, we have sequenced the genome of over 50 epigenetic Recombinant Inbred Lines (epiRILs) that were derived from a cross between a wild type and an isogenic ddm1 mutant line. After backcrossing of the F1 and selection of the progeny homozygous for wild-type DDM1, the epiRILs were propagated through six rounds of selfing. The epiRILs therefore permit a detailed assessment of transposition events soon after they have occurred. In order to identify TE mobilization in the epiRILs we developed TE-tracker, a pipeline based on Illumina sequencing of mate pairs libraries. Using this approach, we could show that although both retroelements and DNA transposons are mobilized in ddm1 and the epiRILs, mobile TEs belong to only a dozen or so of the >300 TE families identified in the Arabidopsis genome. Furthermore the rate and dynamics of transposition vary dramatically between TE families, suggesting the existence of additional mechanisms controlling transposition. Molecular analysis performed on a subset of those mobile TEs belonging to distinct families show that these differences are greatly due to different modality of establishment of epigenetic control over newly inserted TEs. In addition, our analysis indicates that the distribution of new TE insertions differs dramatically from the one of resident copies. These findings provide compelling evidence that over accumulation of TE sequences in the pericentromeric regions of the Arabidopsis genome is not due to specific targeting of TEs, but rather to their elimination from chromosome arms. Finally, we assessed the extent to which repeat-associated DNA methylation impacts the expression of neighboring genes by studying mutants affected in different methylation pathways. Phenotypic and molecular analyses reveal that, even though most Arabidopsis genes are not detectably sensitive to the methylation status of neighboring repeats, two DNA methylation pathways act together to maintain the normal expression of only a very small number of genes near repeats and that these genes tend to have pleiotropic effects. Then, DNA methylation acts as a double-lock system to ensure the normal expression of a small number of key genes located near repeats.

Leptosphaeria maculans ‘brassicae’ (Lmb) est un champignon filamenteux de la classe des Dothideomycètes faisant partie du complexe d’espèces Leptosphaeria maculans-Leptosphaeria biglobosa composé d’agents pathogènes des crucifères. Lmb est particulièrement adapté au colza (Brassica napus) et provoque la maladie qui lui est la plus dommageable : la nécrose du collet. Dans le but de mieux comprendre et contrôler cette maladie, l’équipe d’accueil a initié un projet de génomique visant à identifier de façon systématique les gènes impliqués dans le pouvoir pathogène. Les premières données génomiques montraient deux aspects très importants et potentiellement spécifiques de Lmb : (i) tous les gènes d'avirulence caractérisés expérimentalement étaient localisés dans de grandes régions riches en bases AT et composées d'éléments transposables (ET), (ii) ces régions riches en AT préfiguraient une structure génomique particulière, qui, si elle se généralisait à l'ensemble du génome, aurait été totalement inédite chez un micro-organisme eucaryote. La première partie de cette thèse présente la description du génome de Lmb en se focalisant sur sa structure en isochores, résultant d’une invasion du génome par des ET qui ont ensuite été inactivés par un mécanisme de défense spécifique aux champignons ascomycètes, le RIP (Repeat-Induced Point mutation). Puis, l’impact potentiel de cette structure sur la diversification et l’évolution des protéines jouant un rôle clé lors de l’interaction agent pathogène-plante a été évalué, mettant ainsi en avant l’existence d’un génome à « deux vitesses ». Afin de mieux comprendre le rôle potentiel joué par les ET au niveau des capacités d’adaptation de Lmb au colza, une étude de génomique comparative et évolutive de cinq membres du complexe d’espèces a été réalisée. Ce travail montre que Lmb est la seule espèce du complexe dont le génome a été envahi par les ET, et que ces derniers sont impliqués dans (i) des réarrangements intrachromosomiques potentiellement liés à la spéciation entre Lmb et l’espèce la plus proche, (ii) la présence de gènes espèce-spécifiques et (iii) des déplacements dans des régions génomiques très dynamiques de gènes codant des effecteurs. Les travaux constituant cette thèse participent à la généralisation du concept selon lequel un lien fort existe chez les champignons filamenteux phytopathogènes entre ET et gènes impliqués dans la pathogenèse ou l’adaptation à l’hôte. / Leptosphaeria maculans ‘brassicae’ (Lmb) is a filamentous ascomycete from class Dothideomycetes. It belongs to the Leptosphaeria maculans-Leptosphaeria biglobosa species complex which comprises pathogens of crucifers. Lmb is specifically adapted to oilseed rape (Brassica napus) and is responsible for the most damaging disease of this crop: “stem canker”. In order to better understand and control the disease, the host team initiated a genomic project aiming at systematically identify genes involved in pathogenicity, analyse genome plasticity and evaluate their incidence on adaptability to host. Preliminary genome data firstly showed that all characterized avirulence genes were localized in large AT-rich regions, mainly composed of Transposable Elements (TEs). In addition, these AT-rich regions were the first hints that the Lmb genome may present a very unusual structure compared to other microorganisms. The first part of this thesis describes the Lmb genome with a special focus on its isochore structure, which is the result of a massive TE invasion of the genome followed by an inactivation of TEs by an ascomycete-specific defense mechanism called RIP (Repeat-Induced Point mutation). The potential impacts of this genome structure on diversification and evolution of proteins involved in the plant-pathogen interaction were assessed and highlighted the existence of a “two speed” genome. To better understand how TEs are involved in adaptation of Lmb towards oilseed rape, a comparative and evolutionary genomic analysis of five members of the species complex was conducted. This study shows that Lmb is the only species of the complex with genome invaded by TEs at such an extent, and that TEs are involved in (i) intrachromosomal rearrangements putatively related to the speciation event between Lmb and its closest relative species, (ii) the presence of species-specific genes, (iii) translocations of effector genes into highly dynamic genomic regions. Our data contribute to the generalization of the “two speed” genome concept in filamentous phytopathogens postulating that highly plastic regions of the genome are enriched in genes involved in niche adaptation and that a strong link exists between TEs and genes involved in pathogenesis or host adaptation.

Transposable Elements (TEs) or jumping genes are the DNA sequences that have an intrinsic capability to move within a host genome from one genomic location to another. Studies show that the presence of a TE within or adjacent to a functional gene may alter its expression. TEs can also cause an increase in the rate of mutation and can even promote gross genetic arrangements. Thus, the proper classification of the identified jumping genes is important to understand their genetic and evolutionary effects. While computational methods have been developed that perform either binary classification or multi-label classification of TEs, few studies have focused on their hierarchical classification. The existing methods have limited accuracy in classifying TEs. In this study, we examine the performance of a variety of machine learning (ML) methods and propose a robust augmented Stacking-based ML method, ClassifyTE, for the hierarchical classification of TEs with high accuracy.

Present in almost all living organisms, cytochrome P450s form one of the biggest enzyme superfamilies. They are versatile biocatalysts, capable of performing a range of biochemical reactions and are involved in a wide spectrum of biological functions. The vinegar fly, Drosophila melanogaster, has 85 P450s in its sequenced genome. Six of these have been found to catalyse the synthesis of the important insect molting hormone, 20-hydroxyecdysone and a handful have been implicated in insecticide resistance. The other P450s remained largely uncharacterised. / In the first half of this thesis, the expression patterns of P450s in the D. melanogaster genome were characterised by in situ hybridisation at the third instar larval stage. Most P450s have defined expression patterns at this stage of development. A majority of P450s are expressed in the midgut, Malpighian tubules and fat body, tissues that are involved in the metabolism of xenobiotics. Other P450s are expressed in specific tissues, such as the prothoracic glands, the salivary glands and the gonads, where they might have roles in development or reproduction. In particular, Cyp6g2 is expressed in the corpus allatum (CA), where it could play a role in juvenile hormone synthesis. An RNAi lethality screen using lines that were available from the Vienna Drosophila RNAi Centre identified a number of P450s which are essential for development and viability. / In the second half of the thesis, the transcriptional regulation of a P450 involved in insecticide resistance, Cyp6g1, was investigated. Cyp6g1 was regulated by two discrete cis-regulatory modules/enhancers, one controlling expression in the Malpighian tubules and one controlling expression in the midgut and fat body. Phenobarbital induction of Cyp6g1 is tissue-specific and is mediated by a fragment in the 5’ regulatory region that interacts with both enhancers. Characterisation of the long terminal repeat (LTR) of the Accord transposable element in the 5’ region of Cyp6g1, present in insecticide resistant populations, shows that the Accord LTR contains cis-regulatory elements which increase expression of Cyp6g1 in the fat body, midgut and Malpighian tubules, and contribute to insecticide resistance in these populations. / This study shows that the diverse tissue distribution of different P450s in D. melanogaster is related to the diverse biological functions of the enzymes encoded. This is exemplified by the detailed examination of the regulation of the insecticide resistance-conferring P450, Cyp6g1. Its expression pattern reflects its detoxification function in the fly. The role of transposable element insertions in changing gene expression patterns and contributing to selectable variation in genomes is also demonstrated through the Cyp6g1 study.

ABSTRACT Chromatin insulators are required for proper temporal and spatial expression of genes in metazoans. Insulators are thought to play an important role in the regulation of gene expression through the formation of higher-order chromatin structures. One of the best characterized insulators is the Drosophila gypsy insulator, which is located in the gypsy retrovirus. Several proteins are required for gypsy insulator function, including Su(Hw), Mod(mdg4), and CP190. In addition to the gypsy insulator, these proteins are located throughout the genome at sites which are thought to correspond to endogenous insulators. Analysis of the distribution of insulator proteins across a region of chromosome 2R in Drosophila polytene chromosomes shows that Su(Hw) is found in three structures differentially associated with insulator proteins: bands, interbands and domains of coexpressed genes. Bands are formed by condensation of chromatin within genes containing one or more Su(Hw) binding sites, while Su(Hw) sites in interbands appear to form structures normally associated with open chromatin. Bands characterized by the lack of CP190 and BEAF-32 insulator proteins are formed by clusters of coexpressed genes, and these bands correlate with the distribution of specific chromatin marks. Conservation of the band interband pattern, as well as the distribution of insulator proteins in nurse cells, suggests that this organization may represent the basic organization of interphasic chromosomes. We also show that, in addition to the gypsy insulator, sequence analysis predicts the presence of Su(Hw) binding sites within a number of transposable elements. Su(Hw) binds to predicted sites within gtwin and jockey, which possesses enhancer-blocking activity. Su(Hw) affects the tissue-specific expression of transposable elements, although this effect is unrelated to the presence of Su(Hw) binding sites within the element or control of the elements via the piRNA pathway. Additionally, the effect of Su(Hw) on transposable element expression often differs from that of Mod(mdg4). Taken together, these results suggest that insulator proteins associate specifically with, and may help to define, various levels of chromatin organization on polytene chromosomes. Also, gypsy insulator proteins may influence the expression of transposable elements in a way that does not depend on Su(Hw) binding sites within the elements themselves.