Les interactions mito-nucléaire sont au centre des fonctions de la mitochondrie, telles que la production d’énergie. Or, peu d’informations sont connues sur comment le génome mitochondrial peut influencer la réponse nucléaire. Les cybrides (hybrides cytoplasmiques) peuvent présenter des modifications phénotypiques (adaptatives ou non) dûe à ces interactions. Dans cette étude, les différents mitotypes (haplotype mitochondriale) de l'espèce Chrosomus eos furent utilisés comme modèle afin d’étudier plus en profondeur les interactions entre les différents génomes d’une cellule. Ce complexe présente en plus du type sauvage, deux types de cybrides, dont les mitochondries proviennent de deux refuges glaciaires différents : Mississippien et Atlantique. Cette étude fut effectuée sur des individus en sympatrie afin de prendre en compte l’influence environnementale. Des populations en allopatrie furent également mesurés séparément afin de complémenter les études précédentes. Afin de répondre à ces objectifs, plusieurs approches furent employées, adressant ainsi divers niveaux d’intégration : la méthylation, la transcription, la protéomique et l’activité enzymatique. Des différences entre les divers mitotypes furent observées, mais de magnitude inférieure à l’influence environnementale. Ce résultat suggère donc que les différences associées au mitotype en sympatrie affecte leur répartition et leur habileté à coloniser différents types d’environnements, créant ainsi la similarité intra-mitotype inter-lac observé dans les études précédentes. Pour adresser l’analyse transcriptomique, une référence a été produite. Cette référence offre beaucoup d’informations pour des applications futures. / The mito-nuclear interactions are at the center of the mitochondrial functions, such as energy production. Despite their importance, little is known about how the influence of the mitochondrial genome on the nuclear genes expressions. The cybrids (cytoplasmic hybrids) can present phenotype modification (adaptive or not) caused by the mito-nuclear interactions. In this study, different mitotype of Cybrids of the Chrosomus eos species were used as model to further study the interaction between a cell’s genomes. This complex of species has two types of cybrids, the mitochondria of which originates from two different glacial refugia : Atlantic and Mississippian. This set up of this study was a sympatric lake, to minimise the environmental factor. Allopatric population were also separately analysed to supplement past studies. To accomplish these goals, multiple methods were used, assessing also multiple levels of gene expression: Methylation, transcription, protein and enzymatic activity. Differences between mitotypes were observed, but of lesser magnitude then the environmental factor. Thus, this result suggest that the differences associated to the sympatric mitotypes drive a deference in environment colonization, which would create the inter-lake intra-mitotype similarity observed in past studies. To assess the transcription of mitotype, a transcriptomic reference was produced. This reference offers a lot of information and future applications.

Lignocellulosic plants are the most abundant source of terrestrial biomass and are one of the potential sources of renewable energy that can replace the use of fossil fuels. For a country such as Sweden, where the forest industry accounts for 10% of the total export, there would be large economical benefits associated with increased biomass yield. The availability of research on wood development conducted in conifer tree species, which represent the majority of the forestry in Sweden, is limited and the majority of research has been conducted in model angiosperm species such as Arabidopsis thaliana. However, the large evolutionary distance between angiosperms and gymnosperms limits the possibility to identify orthologous genes and regulatory pathways by comparing sequence similarity alone. At such large evolutionary distances, the identification of gene similarity is, in most cases, not sufficient and additional information is required for functional annotation. In this thesis, two high-spatial resolution datasets profiling wood development were processed; one from the angiosperm tree Populus tremula and the other from the conifer species Picea abies. These datasets were each published together with a web resource including tools for the exploration of gene expression, co-expression and functional enrichment of gene sets. One developed resource allows interactive, comparative co-expression analysis between species to identify conserved and diverged co-expression modules. These tools make it possible to identifying conserved regulatory modules that can focus downstream research and provide biologists with a resource to identify regulatory genes for targeted trait improvement. / Lignocellulosa är den vanligast förekommande källan till markburen biomassa och är en av de förnybara energikällor som potentiellt kan ersätta användningen av fossila bränslen. För ett land som Sverige, där skogsindustrin som står för 10 \% av den totala exporten, skulle därför en ökad produktion av biomassa kunna ge stora ekonomiska fördelar. Forskningen på barrträd, som utgör majoriteten av svensk skog är begränsad och den huvudsakliga forskningen som har bedrivits på växter, har skett i modell organismer tillhörande gruppen gömfröiga växter som till exempel i Arabidopsis thaliana. Det evolutionära avståndet mellan gömfröiga (blommor och träd) och nakenfröiga (gran och tall) begränsar dock möjligheten att identifiera regulatoriska system mellan dessa grupper. Vid sådana stora evolutionära avstånd krävs det mer än att bara identifiera en gen i en modellorganism utan ytterligare information krävs som till exempel genuttrycksdata. I denna avhandling har två högupplösta experiment som profilerar vedens utveckling undersökts; ett från gömfröiga träd Populus tremula och det andra från nakenföriga träd (barrträd) Picea abies. Datat som behandlats har publicerats tillsammans med webbsidor med flera olika verktyg för att bland annat visa genuttryck, se korrelationer av genuttryck och test för anrikning av funktionella gener i en grupp. En resurs som utvecklats tillåter interaktiva jämförelser av korrelationer mellan arter för att kunna identifiera moduler (grupper av gener) som bevaras eller skilts åt mellan arter över tid. Identifieringen av sådana bevarade moduler kan hjälpa att fokusera framtida forskning samt ge biologer en möjlighet att identifiera regulatoriska gener för en riktad förbättring av egenskaper hos träd.

Les impacts environnementaux dues à l'extraction minière sont considérables. C'est l'action des microorganismes, en utilisant leur métabolisme du soufre sur les déchets miniers, qui engendre les plus grands défis. Jusqu'à présent, peu de recherches ont été effectués sur les microorganismes environnementaux pour la compréhension globale de l'action du métabolisme du soufre dans une optique de prévention et de rémédiation des impacts environnementaux de l'extraction minière. Dans cette étude, nous avons étudié une bactérie environnementale, Acidithiobacillus thiooxidans, dans le but de comprendre le métabolisme du soufre selon le milieu de culture et le niveau d'acidité du milieu. Nous avons utilisé la transcriptomique à haut débit, RNA-seq, en association avec des techniques de biogéochimie et de microscopie à électrons pour déterminer l'expression des gènes codants les enzymes du métabolisme du soufre. Nous avons trouvé que l'expression des gènes des enzymes du métabolisme du soufre chez ce microorganisme sont dépendantes du milieu, de la phase de croissance et du niveau d'acidité présent dans le milieu. De plus, les analyses biogéochimiques montrent la présence de composés de soufre réduits et d'acide sulfurique dans le milieu. Finalement, une analyse par microscopie électronique révèle que la bactérie emmagasine des réserves de soufre dans son cytoplasme. Ces résultats permettent une meilleure compréhension de son métabolisme et nous rapprochent de la possibilité de développer une technique de prédiction des réactions ayant le potentiel de causer des impacts environnementaux dus à l'extraction minière. / The environmental impact of mining extraction is important. The action of microorganisms using their sulfur metabolism to metabolise compounds in mining waste contributes to reactions that may impact water quality and the environment. Few studies have been conducted on environmental microorganisms to advance the global comprehension of their sulfur metabolism in an attempt to study their impact on the environment. In this study, we cultivate an environmental bacterium, Acidithiobacillus thiooxidans, in an attempt to understand its sulfur metabolism in different growth media and at different levels of acidity. We used high-throughput RNA sequencing in association with sulfur biogeochemistry and electron microscopy to determine the expression of the genes encoding sulfur metabolism enzymes. The expression of genes encoding sulfur metabolism enzymes was media and pH-dependent. Also, the biogeochemical analysis showed the presence of reduced sulfur intermediates and of sulfuric acid in the medium. Finally, an electron microscopic analysis revealed that the bacteria stock sulfur in the cytoplasm. These results resulted in a better comprehension of its sulfur metabolism and it opens the possibility to predict reactions in mining operations that have impact on the environment.

Odorantes são detectados por centenas de receptores olfatórios (ORs) que pertencem à superfamília dos receptores acoplados à proteína G. Estes receptores são expressos nos neurônios sensoriais olfatórios localizados na cavidade nasal. Cada neurônio sensorial olfatório expressa um único alelo de gene OR de uma grande família de genes OR. Este padrão característico da expressão de genes OR resulta na formação de um mapa olfatório espacial no bulbo olfatório, que é necessário para a discriminação de odorantes pelo sistema olfatório. Os mecanismos envolvidos nesta regulação ainda não são bem conhecidos. O DNA genômico em neurônios olfatórios é coberto com marcas repressivas de metilação de histonas, indicando que a regulação da estrutura da cromatina deve desempenhar um papel importante na regulação da expressão de genes OR. Trabalhos anteriores demonstraram que RNAs não codificadores (ncRNAs) estão envolvidos na deposição de marcas de histonas em determinados genes. No entanto, os ncRNAs expressos no epitélio olfatório ainda não são conhecidos. Neste trabalho, identificamos e catalogamos o repertório completo de ncRNAs anotados, incluindo os miRNAs, expressos no epitélio olfatório de camundongos recémnascidos e adultos. Muitos destes, apesar de já anotados como ncRNAs, ainda não foram descritos na literatura como expressos no MOE. Identificamos ao todo 1161 miRNAs e 295 lincRNAs expressos no epitélio olfatório, e pudemos verificar como os níveis de expressão destes RNAs variam durante o desenvolvimento. A partir deste repertório, selecionamos lincRNAs que são preferencialmente expressos no epitélio olfatório quando comparados a outros tecidos de camundongo. Dez destes lincRNAs foram selecionados para validação utilizando-se RT-PCR. Cinco lincRNAs foram validados e analisados quanto à sua expressão em diferentes tecidos. Nosso trabalho estabelece uma plataforma de dados que permitirá o estudo do papel desempenhado por ncRNAs no epitélio olfatório. Além disto, os nossos resultados mostram que a abordagem utilizada permite a identificação de novos lincRNAs que apresentam expressão restrita ou preferencial no epitélio olfatório, e que, portanto, devem apresentar uma função relevante para o olfato. / Odorants are detected by hundreds of odorant receptors (ORs) which belong to the superfamily of G protein-coupled receptors. These receptors are expressed in the olfactory sensory neurons of the nose. Each olfactory sensory neuron expresses one single OR gene allele from a large family of OR genes. This characteristic pattern of OR gene expression results in the formation of a spatial olfactory map in the olfactory bulb, which is required for odorant discrimination by the olfactory system. The mechanisms involved in this regulation are unknown. OR genomic DNA in olfactory neurons is covered with repressive histone methylation marks, indicating that the chromatin structure should play an important role in the regulation of OR gene expression. Previous studies suggest that noncoding RNAs (ncRNAs) are involved in the deposition of histone marks in certain genes. However, the ncRNAs expressed in the olfactory epithelium are completely unknown. In this work, we used RNA-seq to identify and catalogue the complete repertoire of ncRNAs, including miRNAs, expressed in the olfactory epithelium from newborn and adult mice. In this way, we were able to identify 1161 miRNAs and 295 lincRNAs and analyze how their levels of expression varies during development. Out of these repertoire, we selected lincRNAs that are preferentially expressed in the olfactory epithelium when compared to other mouse tissues. Ten out of these lincRNAs were selected for validation by using RTPCR, and five of them could be validated and further analyzed. Our work establishes a data platform which will enable the study of the role played by ncRNAs in the olfactory epithelium. In addition, our results show that our approach can be successfully used to identify ncRNAs that are restrictedly or preferentially expressed in the olfactory epithelium, and which therefore must be relevant for olfaction.

A utilização de toxinas Cry de Bacillus thuringiensis (Bt) no controle de lepidópteros-praga, principalmente em áreas onde a estratégia de refúgio não é regulamentada, facilita a evolução da resistência em populações de pragas-alvo. Há três relatos de resistência à campo para Spodoptera frugiperda (J.E. Smith), dois para a toxina Cry1F e um para Cry1Ab. No Brasil, ocorrem populações resistentes à Cry1F e Cry1Ab. Esse trabalho foi voltado à identificação do mecanismo de resistência de uma população de S. frugiperda à toxina Cry1F, baseando-se nas hipóteses existentes para explicar o modo de ação de toxinas Cry. Uma dessas hipóteses é baseada na formação de poros na membrana do epitélio intestinal, enquanto a outra na transdução de sinal intracelular e ativação do processo de morte celular. Para a identificação do mecanismo de resistência de S. frugiperda à toxina Cry1F, o receptor caderina de linhagens suscetível (SUS) e resistente (RES) foi caracterizado, bem como realizado estudos de expressão gênica diferencial comparativa pela análise do transcritoma dessas linhagens. Estudos de expressão gênica diferencial comparativa também foram realizados pela análise do transcritoma do intestino de lagartas de linhagens SUS e resistente isogênica (RESiso), para a identificação do mecanismo molecular de resistência à toxina Cry1F. A caracterização do transcrito do gene caderina das linhagens suscetível, resistente e resistente isogênica revelou diferenças na composição de aminoácidos da proteína caderina predita entre as linhagens suscetível e resistente à toxina Cry1F nos domínios de repetição CR5, CR6 e CR10 e no domínio C-terminal. Também foi verificado que das mutações encontradas na linhagem RES, apenas as mutações da região C-terminal foram fixadas na linhagem RESiso. A análise comparativa do transcritoma de linhagens SUS e RES indicou a maior expressão de genes relacionados à metabolização de xenobióticos, como as monoxigenases do citocromo P450, glutationa-S-transferases e carboxilcolinesterases, em lagartas resistentes, mas não foram encontradas diferenças na expressão de receptores Cry, como aminopeptidase N e fosfatase alcalina. Porém, caderina foi superexpressa e o transportador ABCg5 teve expressão reduzida na linhagem RES. O ABCg5 foi indicado como o provável mecanismo de resistência dessa linhagem à toxina Cry1F, juntamente com o aumento da capacidade de detoxificação relatada. A análise comparativa do transcritoma de linhagens SUS e RESiso produziu resultados semelhantes à análise anterior quanto ao padrão de expressão de enzimas de detoxificação, mas nesse caso foi observada redução da transcrição de caderina na linhagem RESiso em relação à SUS. A análise da linhagem isogênica também indicou alteração na expressão de transportadores ABC na linhagem RESiso; porém, para o transportador ABCb1. A análise comparativa do transcritoma de linhagens SUS e RESiso corroborou a participação do sistema de detoxificação e acrescentou a redução na expressão do receptor caderina como mecanismo de resistência dessa população à toxina Cry1F, assim como a de transportadores ABC, apesar do transportador ABCg5 não ter sido identificado nessa análise comparativa. / The broad use of Cry toxins from Bacillus thuringiensis (Bt) to control lepidopteran pests, particularly where refuge strategies are not legalized or implemented, has facilitated the evolution of resistance of pest populations. There are three records of resistance of Spodoptera frugiperda (J.E Smith) in field condition to Bt toxins so far, two of them to Cry1F and one to Cry1Ab toxins. In Brazil, field-evolved resistance of S. frugiperda has been recorded for both toxins. Thus, we aimed to identify the mechanisms associated to the resistance of S. frugiperda to Cry1F toxin based on the two concurrent hypotheses on the mode of action of Cry toxins. One of such hypotheses is based on the potential of Cry toxins to form pores in the membrane of the gut epithelium, while the other is based on the production of an intracellular transduction signal and the activation of the process of cell death. To identify the resistance mechanisms of S. frugiperda to Cry1F toxin, we characterized the transcript of the cadherin receptor of susceptible (SUS) and resistant (RES) strains of S. frugiperda to search for mutations and performed a comparative analysis of the transcriptome from SUS and RES strains. We also analyzed the transcriptome from the gut of SUS and isogenic resistant strains (RESiso) in order to identify the molecular mechanisms associated to the resistance of S. frugiperda to Cry1F. The characterization of cadherin receptor in SUS, Res and REsiso strains showed differences in the amino acid composition of the repeated domains CR5, CR6 and CR10 and in the C-terminal domain. Only mutations occurring on C-terminal of the RES strain were maintained in the RESiso strain. The comparative transcriptome between SUS and RES strains indicated a higher expression of genes related to the detoxification process, such as cytochrome P450s, glutathione-S-transferases and carboxylcholinesterases in the RES strain, while no differences in the expression of Cry receptors, such aminopeptidade N and alkaline phosphatase, were observed. However, transcriptomic analysis indicated up-regulation of cadherin and down-regulation of the ABCg5 transporter was down-regulated in the RES strain. We propose that ABCg5 is one of the mechanisms involved in S. frugiperda resistance to Cry1F, together with the increased detoxification activity observed. Analysis of the gut transcriptome from SUS and RESiso yielded similar results regarding the differential expression of detoxifying enzymes, but on this case cadherin was down-regulated in RESiso as compared to the SUS strain. Down-regulation of ABC transporters in the RESiso strain was also observed, but for the ABCb1 transporter. Analyses of the transcriptome of SUS and RESiso strains also indicated the resistance of S. frugiperda to Cry1F is related to an increased transcription of detoxifying enzymes and a reduced transcription of the cadherin receptor. Our data also demonstrates the resistance is due to the existence of adequate constitutive levels of transcription of genes that respond to the intoxication with Cry1F.

La métamorphose des amphibiens est le processus rapide et irréversible par lequel un têtard aquatique se transforme en une grenouille respirant à la surface. Cette transition écologique, réminiscente de la période périnatale chez les mammifères, s'accompagne de changements spectaculaires (régime alimentaire, organes locomoteurs, système respiratoire...). Ces modifications morphologiques et physiologiques nécessitent la réponse concertée à un signal hormonal, les hormones thyroïdiennes (HT), de différents tissus vers des destin parfois opposés : apoptose (dans la queue), prolifération (dans les pattes), et remodelage (dans les intestins et le système nerveux central). Toutefois, la synchronisation de la réponse des différents tissus fait appel à d'autres signaux hormonaux, et notamment les glucocorticoïdes (GC). Ces derniers sont également les médiateurs principaux de la réponse au stress. Les processus endocriniens de la métamorphose et la réponse au stress sont fortement couplés. Les GC peuvent ainsi jouer le rôle d'interface permettant l'intégration de signaux environnementaux au niveau de réseaux de régulation. Dans le cadre de mon doctorat, j'ai analysé les transcriptomes des bourgeons de membres postérieurs et de l'épiderme caudal de têtards de Xenopus tropicalis traités ponctuellement avec des HT et / ou des GC. La comparaison de ces deux tissus a permis de caractériser la diversité des profils d'expression des gènes cibles des HT et des GC.Il en ressort plusieurs résultats majeurs. Tout d'abord, la diversité des profils d'interaction entre ces deux voies est limitée, et la majorité des types de profils sont communs aux deux tissus. Indépendamment du tissu, certains profils sont caractéristiques de fonctions biologiques spécifiques comme le remodelage de la matrice extracellulaire et le système immunitaire. Les gènes impliqués dans ces fonctions communes aux deux tissus sont cependant différents. Enfin, plusieurs facteurs impliqués dans la méthylation de l'ADN sont régulés par les deux hormones. / Amphibian metamorphosis is the rapid and irreversible process during which an aquatic tadpole transforms into an air breathing adult frog. This ecological transition, reminiscent of the mammalian perinatal period, comes with spectacular changes (diet, locmotor organs, respiratory system...). These morphological and physiological modifications necessitate the properly timed response to a single hormonal signal, the thyroid hormones (TH), in various tissues to lead them to sometimes opposite fates : apoptosis (in the tail), cell prolifération and differenciation (in the limbs) and remodeling (in the intestine and the central nervous system).However, TH do not act alone. In particular, glucocorticoids (GC) play important roles during this process. They also are the main mediator of the stress response. Endocrine processes of the metamorphosis and the stress response are deeply intertwined. GC can thus act as an interface to integrate environmental inputs into regulatory networks.During my doctorate, I analyzed the possible transcriptional crosstalks between TH and GC in two larval tissues : the tailfin (TF) and the hindlimb buds (HLB). Comparing these two tissues allowed me to caracterize the diversity of TH and GC target gene expression profiles. This resulted in several major results. First, the diversity of the profiles of crosstalk between these two pathways is limited, and the majority of the types of profiles is common to both tissues. Next, independently ofthe tissues, some profiles are caracteristic of spécific biological functions such as extracellular matrix remodeling and the immune system. Yet, the genes involved in these shared functions are different between the TF and the HLB. Finally, several factors involved in DNA methylation are subject to a crosstalk between the two hormones.

O trigo (Triticum aestivum L.) é uma importante cultura no Brasil. Poucas cultivares são recomendadas para produção do tipo sequeiro no Bioma Cerrado onde a escassez de água limita o rendimento de grãos. Aqui reportamos uma análise de transcriptoma do MGS1 Aliança (cultivar de trigo adaptada ao Cerrado) sob estresse de seca. Um grupo de 4.422 transcritos diferencialmente expressos foi encontrado em raízes e folhas. O número de transcritos reprimidos em raiz (1.102) foi menor que os transcritos induzidos (1.706), enquanto o oposto ocorreu em folhas (1,017 induzidos e 647 reprimidos). O número de transcritos comuns entre ambos órgaõs foi 1.249, enquanto 2.124 foram específicos para raíz e 1.049 específicos para folhas. Análises de RT-qPCR de 35 transcritos selecionados ao acaso revelou uma correlação de 0,78 com os dados de transcriptoma. Os transcritos diferencialmente expressos foram distribuídos por todos os cromossomos e componentes do genoma. O número de transcritos no genoma B foi maior do que nos genomas A e D. Ainda, um grande número de transcritos relacionados à seca foi mapeado nos cromossomos 3B, 5B e 2B. Quando consideramos ambos órgãos, 116 diferentes rotas metabólicas foram alteradas. Uma rota em comum, entre as três mais alteradas em ambos órgãos, foi o metabolismo do amido e da sacarose. A comparação de transcritos derivados de raiz e de folha permite a identificação de transcritos importantes relacionados à respota ao estresse de seca em cada um destes órgãos. Os dados obtidos, também, abrem caminho para o desenvolvimento de futuros marcadores e seleção de genes candidatos ligados à característica. Estes resultados são úteis para o entendimento de rotas metabólicas envolvidas na tolerância à seca em trigo. A informação gerada será usada, a mais longo prazo, para propósitos de transgenia. Para isto, a metodologia de duplo-haploides é desejável e uma primeira investigação sobre a eficiência de protocolo se mostrou necessária. Micrósporos são células gaméticas com capacidade de dar origem a uma nova planta via embriogênese in vitro. Plantas duplo-haploides geradas pela cultura de micrósporos isolados são completamente homozigotas e representam uma importante ferramenta para estudos genéticos e melhoramento de plantas O processo androgenético é desencadeado por diferentes pré-tratamentos de estresse, os quais são empregados para mudar os micrósporos da rota gametofítica para a rota esporofítica. Embora a cultura de micrósporos isolados tenha inúmeras vantagens, importantes limitações tem impedido sua apliação em larga escala. Diferenças genotípicas na resposta androgenética e na formação de plantas albinas ainda constituem desafios. Embora o albinismo seja principalmente uma característica genética, pré-tratamentos e meios de cultura apropriados podem evitar este fenômeno até certo ponto. A resposta androgenética de cinco genótipos de trigo brasileiro foi avaliada no presente estudo. Dois pré-tratamentos foram testados: frio (4°C) e ácido 2-hidroxinicotinico (100 mg/L). O frio foi melhor que o pré-tratamento químico, produzindo mais plantas verdes em quatro de cinco genótipos. Somente dois genótipos brasileiros tratados com ácido 2-hidroxinicotinico produziram plantas, e um deles apenas uma única planta albina. Nossos reultados mostram, também, que o meio semilíquido (contendo 10% de Ficoll) promoveu uma maior resposta androgenética que o meio líquido, aumentando o número de embriões e plantas regeneradas. / Wheat (Triticum aestivum L.) is an important crop cultivated in Brazil. Few cultivars are recommended for rainfed production in the Cerrado Biome where water scarcity limits grain yield. Here we report a transcriptome analysis of MGS1 Aliança (a wheat cultivar adapted to the Cerrado) under drought stress. A set of 4,422 differentially expressed transcripts was found in roots and leaves. The number of down-regulated transcripts in roots (1,102) was lower than the up-regulated transcripts (1,706), while the opposite occurred in leaves (1,017 induced and 647 repressed). The number of common transcripts between the two tissues was 1,249, while 2,124 were specific to roots and 1,049 specific to leaves. Quantitative RT-PCR analysis of 35 randomly selected transcripts revealed a 0.78 correlation with the transcriptome data. The differentially expressed transcripts were distributed across all chromosomes and component genomes. The number of transcripts on the B genome was greater than on the A and D genomes. Additionally, a greater number of drought related transcripts was mapped on chromosomes 3B, 5B and 5D. When considering both tissues, 116 different metabolic pathways were changed. One common pathway, among the top three changed pathways in both tissues, was starch and sucrose metabolism. The comparison of root- and leaf-derived transcripts allows the identification of important transcripts related to water stress response in each of these tissues. It also paves the way for future marker development and selection of candidate genes linked to that trait. These results are useful for understanding the metabolic pathways involved in wheat drought response. The information generated will be used for transgenic wheat purposes. For this the doubled-haploid method is desirable and an investigation about the protocol eficiency is needed. Microspores are gametic cells with capacity to give rise to a new plant via in vitro embryogenesis. Doubled haploid plants generated by isolated microspore culture are completely homozygous and represent an important tool for plant genetics and breeding research. This process is triggered by different stress pretreatments, which are employed to switch microspores from gametophytic to a sporophytic pathway. Although isolated microspore culture has innumerous advantages, important limitations have prevented its application on a large scale. Genotypic differences in androgenic response and the formation of albino plants remain great challenges. Although albinism is a major genetic characteristic, appropriated pretreatments and culture medium can avoid this phenomenon to some extent. The androgenic response of five Brazilian wheat genotypes was evaluated in the present study. Two pretreatments were tested: cold (4°C) and 2-hydroxynicotinic acid (100 mg/L). Cold was better than chemical pretreatment, producing more green plants in four out of five genotypes. Only two Brazilian genotypes treated with 2-hydroxynicotinic acid produced plants, and one of them produced a single albino plant. Our results also show that semi-liquid medium (containing 10% Ficoll) promoted a higher androgenic response than did liquid medium, increasing the number of embryos and regenerated plants.

Transcriptomics and gene expression profiling enables the elucidation of the genetic response of an organism to various environmental cues. Transcriptomics enables the deciphering of differences between two closely related organisms to the same environment and in contrast, enables the elucidation of genetic responses of the same organism to different environmental cues. Two major methods are utilized for the study of transcriptomes, high-throughput sequencing and microarray analysis. High-throughput sequencing technologies such as the Illumina platform are relatively new and protocols must be developed for the analyses of transcriptomes (RNA-sequencing). A RNA-seq protocol was developed and refined for the Illumina sequencing platform. This protocol was then utilized for the de novo sequencing of the steelhead salmon transcriptome. Hatchery steelhead exhibit a reduced fitness compared to wild steelhead that has been shown to be genetically based. Consequently, the steelhead transcriptome was assembled, annotated, and used to identify gene expression differences between hatchery and wild fish. We uncovered many differentially expressed genes involved in metabolic processes and growth and development. This work has created a better understanding of the genetic differences between hatchery and wild steelhead salmon. Brachypodium distachyon is a monocot grass important as a model for cereal crops and potential biofuels feedstocks. To better understand the genetic response of this plant to different environmental cues, a comprehensive assessment of the transcriptomic response was conducted under a variety of conditions including diurnal/circadian light/dark/temperature environments and different abiotic stress conditions. Using a whole-genome tiling DNA microarray, we identified that the majority of transcripts in Brachypodium exhibit a daily rhythm in their abundance that is conserved between rice and Brachypodium. We also identified numerous cis-regulatory elements dictating these rhythmic expression patterns. We also identified the genetic response to abiotic stresses such as salinity, drought, cold, heat, and high light. We uncovered a core set of genes which responds to all stresses, indicating a core stress response. A large number of transcription factors were uncovered as potential nodes for regulating the abiotic stress response in Brachypodium. Moreover, promoter elements that drive specific responses to discrete abiotic stresses were uncovered. Altogether, the transcriptome analyses in this work furthers our understandings of how particular organisms respond to environmental cues and better elucidates the relationship between genes and the environment. / Graduation date: 2012 / Access restricted to the OSU Community at author's request from Oct. 5, 2011 - April 5, 2012.

This thesis is about the use of massive DNA sequencing to investigate the transcriptome. During recent decades, several studies have made it clear that the transcriptome comprises a more complex set of biochemical machinery than was previously believed. The majority of the genome can be expressed as transcripts; and overlapping and antisense transcription is widespread. New technologies for the interroga- tion of nucleic acids have made it possible to investigate such cellular phenomena in much greater detail than ever before. For each application, special requirements need to be met. The work presented in this thesis focuses on the transcrip- tome and the development of technology for its analysis. In paper I, we report our development of an automated approach for sample preparation. The procedure was benchmarked against a publicly available reference data set, and we note that our approach outperformed similar manual procedures in terms of reproducibility. In the work reported in papers II-IV, we used different massive sequencing technologies to investigate the transcriptome. In paper II we describe a concatemerization approach that increased throughput by 65% using 454 sequencing,and we identify classes of transcripts not previously described in Populus. Papers III and IV both report studies based on SOLiD sequencing. In the former, we investigated transcripts and proteins for 13% of the human gene and detected a massive overlap for the upper 50% transcriptional levels. In the work described in paper IV, we investigated transcription in non-genic regions of the genome and detected expression from a high number of previ- ously unknown loci. / QC 20100723

Understanding the genetic basis of biological processes is a fundamental component of modern ecology and evolutionary biology studies. With the recent advent of next generation sequencing (NGS) technologies, it is now possible to perform large genome and transcriptome projects for ecologically important non-model species. In this thesis, I focused on the development and application of genomic resources of two non-model bird species, the black grouse (Tetrao tetrix) and the great snipe (Gallinago media). Using the chicken genome as a reference, I developed a reference guided NGS pipeline to assemble the complete draft genome of black grouse. The draft genome has a good coverage of the main 29 chromosomes of the chicken genome. The genome was used to develop a vast number of genetic markers. Comparing this genome with that of other species, I identified the genomic regions which were important for the lineage specific evolution of black grouse. I also sequenced and characterised the spleen transcriptome of the black grouse. I identified and validated a large number of gene-based microsatellite markers from the transcriptome and identified and confirmed the expression of immune related genes. Using a similar RNA-Seq approach, I also sequenced the blood transcriptomes of 14 great snipe males with different mating success. I identified genes and single nucleotide polymorphisms (SNPs) which might be related to male mating success in this species, both in terms of gene expression levels and genetic variation structure. For the immunologically important major histocompatibility complex (MHC) gene region of black grouse, I constructed a fosmid library and used it to sequence the complete core MHC region of this species. This resource allowed me to perform a comprehensive comparative genomics analysis of the galliform MHC, by which I found that some genes in this region were affected by selective forces. I was also able to develop a single locus genotyping protocol for the duplicated MHC BLB (class IIB) genes and found that the two black grouse BLB loci followed different evolutionary trajectories. This thesis set an example of developing genomic resources in non-model species and applying them in addressing questions relevant to ecology and evolutionary biology.