Plant gene families organized as linked clusters are capable of evolving by a process of unequal crossing-over. This results in the formation of chimeric genes that may impart a novel function. However, the frequency and functional consequences of these unequal cross-over events are poorly characterized. Plant disease resistance genes (R genes) genes are frequently organized as gene clusters. In this study, I constructed an elaborately designed reconfigurable synthetic RPP1 (for resistance to Paranospora parasitica) gene cluster (synthRPP1) to model R gene evolution by meiotic recombination. This experimental design utilizes gain-of-luciferase phenotype (luc+) to identify and isolate recombinant R genes and uses two alternatively marked alleles to distinguish and measure different types of meiotic recombination (intra- vs. inter-chromosomal). Two putative single copy transgenic plants containing the synthRPP1 gene cluster were generated. These synthRPP1 gene clusters were reconfigured in vivo by two kinds of site-specific recombination systems (CRE/Lox, FLP/FRT) to generate two alternative versions of the synthRPP1 gene clusters in vivo. These lines, as well as others being developed, will be used in future genetic crosses to identify and characterize plants expressing chimeric RPP1 genes. My second area of research was to use a previously developed synthetic RBCSB gene cluster (synthRBCSB) gene cluster to investigate the relative frequency of meiotic unequal crossing over between paralogous genes located on either homologous chromosomes (homozygous lines) or sister chromatids (hemizygous lines). In contrast to published somatic recombination frequencies using a different reporter gene system, no statistically significant difference of meiotic unequal crossing over was observed between homo- and hemi-zygous synthRBCSB lines. This result suggests that meiotic unequal crossing-over between paralogs located on homologous chromosomes occurs at about the same frequency as paralogs located on sister chromatids. To investigate the rate of somatic recombination in synthRBCSB lines, a QRT-PCR method was developed to estimate the frequency of somatic recombination. Preliminary results suggest that the somatic recombination frequency was about 10,000 fold higher than meiotic recombination in the same generation. Moreover, two of five cloned chimeric genes that formed by somatic recombination indicated a different distribution of resolution sites than those observed in meiotic recombination. This finding suggests there are significant differences in both the frequency and character of somatic versus meiotic unequal crossing-over between paralogous genes in Arabidopsis. / Ph. D.

Retrogenes are a group of functional genes produced by gene retroduplication events during evolution. It has been observed that many retrogenes have formed since the evolutionary divergence of Anopheles mosquitoes from the Aedes lineage as a result of developing heteromorphic sex chromosomes. It has been further observed that these retroduplications predominately occur from parent genes on the heteromorphic X chromosome to autosomes and have a predisposition to have enriched expression in testis. In order to investigate the nature of this male-biased expression in testis, we utilized bioinformatic techniques to identify retrotransposition events and assign them relative ages based on evolutionary branches of divergence. This list of parent genes and retrogenes were then analyzed and a total of twenty-five gene pairs were selected for further examination. Available gene expression data in the form of RNA-seq and DNA microarray were used in tandem with gene annotation data to computationally investigate gene pairs in An. coluzzii. These pairs were further investigated experimentally by means of RT-PCR conducted on dissected head, thorax, abdomen, and reproductive organs in both male and female Anopheles coluzzii Mopti strain. Testis and male accessory glands (MAGs) were also investigated by this method in An. coluzzii. Available expression data support previously observed testis enriched expression of retrogenes and provides evidence for the predominate expression of retrogenes occurring in postmeiotic cells suggesting retrogene involvement in sperm development. Experimental evidence revealed a small group of five retrogenes which exhibit the expected male-biased expression in male testis with little to no expression in female ovaries, although a shared expression in the heads of both sexes was observed. Of the five retrogenes, four carry out energy related functions involving mitochondria, suggesting contribution to energy requirements of developing sperm. Testis and MAG experiments in An. coluzzii revealed a predisposition for retrogenes to be expressed in testis while parent genes tended to have higher expression in MAGs, and this phenomenon is partially supported by DNA microarray expression data. Overall, these results suggest further investigation of retrogenes in An. coluzzii may reveal unique functions in male mosquito fertility that are exploitable in genetic approaches to mosquito control. / Master of Science in Life Sciences / Malaria is a potentially deadly disease which effects thousands of people every year. Malaria around the world is spread by multiple species of mosquitoes in a genus called Anopheles. Controlling the populations of these disease spreading mosquitoes is essential to preventing the spread of malaria. Current insecticide-based approaches used to stop mosquitoes are becoming less effective overtime as mosquitoes become resistant. A potential way to develop new techniques for mosquito control is through research involving mosquito reproductive genetics. Understanding the genes involved in how mosquitoes reproduce could improve future techniques designed to reduce or prevent mosquitos from reproducing. This research focuses on a group of genes called retrogenes which have formed over the evolution of these mosquitoes via the duplication from a separate parent gene. In mosquitoes these retrogenes are understood to be involved in male reproduction. The retrogenes involved in male mosquito reproduction could have important functions in male sexual reproduction and sterility. These important genes could be manipulated to interrupt whatever important functions these genes have in reproduction. In this research we first computationally identified retrogenes and their parent genes and categorized them by age. We then utilized available annotation and expression data to analyze the potential significance of retrogenes to male fertility and found that multiple retrogenes tended to be expressed during sperm development. Lastly, we conducted gene expression experiments using dissected head, thorax, abdomen, and reproductive organs in both male and female Anopheles mosquitoes. Results revealed unique patterns of expression that suggest male specific roles of five retrogenes in testes and head expression in both males and females suggesting a possible role in mating behavior. These results provide evidence that retrogenes do have functional roles in male fertility specifically related to the maturation and development of sperm.

No description available.

La méiose est la division cellulaire qui aboutit à la production de gamètes haploïdes. Lors de la méiose, un unique évènement de réplication est suivi de deux divisions afin de réduire la ploïdie. Lors de ces deux divisions, la cohésion entre chromatides sœurs est éliminée de façon séquentielle pour permettre la succession de deux ségrégations de chromosomes équilibrées. La progression du ‘’cycle méiotique’’ est contrôlée par des régulateurs communs à la mitose et à la méiose mais également par des mécanismes nécessitant des protéines spécifiques à la méiose. L’objectif de de mon travail de thèse était de décrypter les mécanismes moléculaires permettant l’enchainement de deux divisions équilibrées pour la production de gamètes haploïdes. Nous avons pu montrer que la protéine OSD1 inhibait l’APC/C pour permettre la progression méiotique. Nous avons également mis en évidence un réseau fonctionnel, comprenant OSD1, CYCA1;2/TAM et TDM, indispensable à trois étapes clés de la progression méiotique chez Arabidopsis ; la transition prophase-méiose I, la transition méiose I-méiose II et la sortie de méiose. Ces travaux ont également permis de caractériser chez Arabidopsis les deux paralogues de Shugoshin, qui sont des protéines conservées et impliquées dans la protection de la cohésion centromérique. Nous avons également identifié Patronus comme un nouveau protecteur de la cohésion centromérique en méiose. Les résultats obtenus suggèrent que Patronus est un régulateur de l’APC/C qui permet d’empêcher l’élimination de la cohésion centromérique en interkinèse méiotique. / Meiosis is a specialized type of cell division that generates haploid gametes. At meiosis, two divisions follow a single DNA replication event leading to ploidy halving. A stepwise sister chromatids cohesion release also occurs to allow the two successive balanced rounds of chromosome segregation. In addition to general cell-cycle regulators, meiosis requires specific proteins. The aim of this thesis was to understand the molecular mechanisms leading to two successive balanced chromosome segregations. We show that OSD1 promotes meiotic progression through APC/C inhibition and we identified a functional network between OSD1, CYCA1;2/TAM and TDM in Arabidopsis. This functional network controls three key steps of meiotic progression; the prophase-meiosis I transition, the meiosis I-meiosis II transition and the meiosis exit. In addition, we characterized the two Arabidopsis thaliana Shugoshin paralogs, which are conserved proteins involved in sister chromatid cohesion protection. We also identified Patronus, an uncharacterized protein, as a novel protector of meiotic centromeric cohesion. We suggest that Patronus is a novel APC/C regulator that prevents cohesins release during meiotic interkinesis. This work identified two APC/C regulators essential for meiosis in Arabidopsis thaliana.

A cana-de-açúcar constitui uma das principais fontes de alimento e de energia renovável no mundo. Entre os fatores bióticos que reduzem sua produtividade, destaca-se o carvão, doença causada pelo fungo biotrófico Sporisorium scitamineum, cujos danos à cultura podem ultrapassar 80%. No entanto, poucos estudos foram desenvolvidos sobre a genética desse patógeno. Assim, o presente trabalho estudou a dinâmica de marcas RFLP associados à região subtelomérica (tel-RFLP) e marcas GFP em três populações do patógeno (a, b e c) obtidas em ciclos sucessivos da doença. A população a inicialmente foi formada pela inoculação com teliósporos F1 oriundos do isolado 39 produzido em trabalho anterior (REIS, 2012). As populações b e c foram produzidas inicialmente por cruzamentos controlados. Estes envolveram como parentais esporídios haploides de tipos de reação sexual compatíveis e perfis tel-RFLP conhecidos. Na população b os esporídios parentais (18A e 39B) foram isolados de teliósporos distintos, apresentaram perfis tel-RFLP mais contrastantes e simularam fecundação cruzada. Na população c, os esporídios parentais 39Agfp e 39Bgfp apresentaram perfis tel- RFLP mais semelhantes. Esses mutantes foram obtidos pela inserção heteróloga do gene gfp em esporídios obtidos de um único teliósporo simulando autofecundação (reação sexual intra-tétrade). A comparação entre os perfis de tel-RFLP dos esporídios selvagens e mutantes GFP detectou a inserção do gene gfp possivelmente na região subtelomérica do individuo 39Agfp (parental). A cada ciclo da doença teliósporos selvagens e mutantes GFP foram obtidos, sendo isolados dois esporídios para os cruzamentos controlados que levaram à produção do ciclo seguinte de doença. O uso de cruzamentos controlados possibilitou a produção de teliósporos idênticos geneticamente, permitindo inferir sobre eventos meióticos ocorrentes numa mesma progênie. A fenotipagem sexual dos esporídios que tiveram os perfis de tel-RFLP analisados reduziu o viés advindo do desvio da segregação esperada de 1:1 entre os tipos sexuais de esporídios pertencentes à mesma divisão meiótica. Além disso, auxiliou na detecção de recombinantes, visto que os loci de reação sexual segregam na meiose I. A análise das marcas tel-RFLP das progênies mutantes GFP e selvagens, obtida respectivamente em 1 e 2 ciclos da doença, evidenciou eventos de recombinação na segregação de cromossomos homólogos e eventos de recombinação homóloga. Mesmo apresentando número elevado, as marcas tel-RFLP foram capazes de rastrear indivíduos pertencentes a uma mesma população, o que poderá auxiliar em futuras avaliações epidemiológicas do carvão da cana-de-açúcar. Por fim, a expressão do gene gfp em células de pró-basídios mutantes possibilitou a detecção de recombinação homóloga associada a essa marca e a observação inédita de tétrades lineares de quatro células para essa espécie. Observou-se elevado potencial de utilização do gene gfp em estudos genéticos aplicados a esse patossistema. A recombinação sugerida na região subtelomérica mostrou frequência de recombinação ainda não descrita para essa espécie a ser estudada posteriormente. / Sugarcane is a major source of food and renewable energy in the world. Among the biotic factors that reduce its productivity we highlight the sugarcane smut disease, caused by the biotrophic fungus Sporisorium scitamineum, whose damages to the crop can overpass 80%. However, few genetic studies have targeted this pathogen. Thus, the present work studied the dynamics of RFLP marks associated to the subtelomeric region (tel-RFLP) and GFP marks of three populations of the pathogen (a, b and c) obtained from consecutive disease cycles. Population a was initially formed with inoculation using teliospores F1 from the whip 39 obtained in previous work (REIS, 2012). Populations b and c were initially produced by controlled crosses of sexual compatible haploid sporidia with known tel-RFLP profiles as parents. In population b the parent sporidia (18A and 39B) were isolated from distinct teliospores, had more different tel-RFLP profiles (7 polymorphic marks) and simulated outcrossing. In population c, the parental sporidia 39Agfp and 39Bgfp had more similar tel-RFLP profiles. These mutants were obtained by heterologous insertion of gfp gene in sporidia obtained from single teliospore, simulating selfing (intra-tetrad mating type). The comparison between wild-type and GFP mutant sporidia tel-RFLP profiles had detected the gfp gene insertion in the subtelomeric region of the individual 39Agfp (parent). For each disease cycle wild-type and GFP mutant teliospores were obtained and two sporidia were isolated to perform controlled crosses that lead to the next disease cycle. The use of controlled crosses resulted in the production of genetically identical teliospores, which allowed inferring about same progeny meiotic occurring events. The sexual phenotyping of the sporidia that had their tel-RFLP analyzed reduced the bias resulted from deviation of expected 1:1 ratio segregation among the mating types of same meiotic division progeny. Besides, it helped in the recombinants detection, due to mating type loci segregation in meiosis I. The analysis of wild-type and GFP mutant progenies tel- RFLP marks, obtained for 2 and 1 disease cycles, has pointed out events of homologous chromosomes recombination and crossing-over events. Despite of the high amount of marks obtained, the tel-RFLP marks allowed the tracking of individuals from the same population, which can benefit future sugarcane smut disease epidemiologic evaluations. Moreover, the gfp gene expression of mutant probasidium cells led to the detection of crossing-over involving the GFP mark and the first observation of linear four-cells tetrads to this species. A high potential use of gfp gene in genetic studies applied to this pathosystem was observed. The recombination suggested for the subtelomeric region showed a recombination frequency haven\'t described for this species that needs to be further studied.

Hybrid male sterility (HMS) is typically the first mechanism fortifying reproductive isolation resulting from genomic incompatibilities. Three interspecies feline breeds derived from domestic cat crosses to wild cat species (Asian leopard cat and African serval) manifest HMS through several generations of backcrossing before eventually regaining fertility. This work utilized 199 hybrid individuals with varying fertilities in a genome wide association study (GWAS) comprising 63,000 genome wide SNPs. Leveraging these results with whole-testis transcriptome sequencing and quantitative real-time PCR data facilitated the comparison of transcripts in sterile and fertile hybrids. This dissertation describes four loci with highly significant and fifty with moderately significant association to sterility within each individual hybrid domestic breed and combinations of breeds. These associations help identify epistatic targets for hybrid incompatibility contributing to sterility. Comparative QTL mapping between pairs of species provides a framework to describe the accumulation of clade-specific reproductive isolating loci. Detailed exploration of gene misregulation between domestic and hybrid individuals, as well as between littermate hybrids of varying fertilities outlines a pattern of expression consistent with a meiotic sex-chromosome inactivation failure in early generations and apoptotic failure in later hybrid generations. Combining comparative genomic association and transcriptomic characterization among hybrid felids of varying divergence, new insight is gained into the mechanisms of mammalian reproductive isolation.

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No. of bitstreams: 1 nunes_gb_me_jabo.pdf: 1276204 bytes, checksum: 3aba64a51e863966cadeab46cfa01561 (MD5) Previous issue date: 2018-02-28 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Este estudo foi desenvolvido com o objetivo de avaliar o efeito do precursor do peptídeo natriurético tipo C (NPPC) durante o cultivo de pré-maturação in vitro (pré-MIV) de oócitos bovinos sobre: 1) a progressão da maturação nuclear; 2) a expressão gênica das células do cumulus e 3) a aquisição da competência do oócito para o desenvolvimento embrionário in vitro. Os complexos cumulus-oócito (CCOs) foram pré-MIV com 100 nM NPPC por 8 horas (grupo NPPC) e, ao final do período, foram lavados para completa remoção do NPPC e submetidos à MIV por 22h. Após 8h de pré-MIV e após 8h de pré-MIV seguidas de 22h de MIV (duração total do cultivo = 30h) foram avaliadas a progressão da maturação nuclear e a expressão relativa de mRNA nas células do cumulus. O grupo controle (C) foi maturado na ausência de NPPC por até 30h, e as mesmas avaliações anteriores foram realizadas imediatamente após a remoção do ambiente folicular (C0), após 8h de cultivo (C8), após 22h de cultivo (C22) e após 30h de cultivo (C30). Em outro experimento, cujos tratamentos foram idênticos aos supramencionados, os oócitos foram fecundados ao término da MIV e foi avaliado o desenvolvimento embrionário até a fase de blastocistos. Após 8h de cultivo de pré-MIV, a análise da progressão da meiose demonstrou que o grupo C0 apresentou 58,9% de estruturas com configuração de GV1, enquanto que o grupo C8 apresentou apenas 13,9% de estruturas nesta configuração (P<0,05). A proporção de GV1 no grupo NPPC foi semelhante a ambos estes grupos (22,8%; P>0,05). Por outro lado, o grupo C8 apresentou maior taxa de GV3 em relação ao C0 (53,1% vs. 3,62%; P<0,05), sem diferenças com o grupo NPPC (38,7%; P>0,05). Ao final do cultivo de MIV, não foi observada diferença entre os grupos (C22 vs. NPPC vs. C30) com relação à maturação nuclear, todavia, houve maior taxa de oócitos degenerados no C30 em comparação com C22 (11,4% vs. 2,8%; P<0,05). A análise da expressão relativa de genes das células do cumulus após 8h de pré-MIV com NPPC evidenciou aumento (P<0,05) na expressão de genes relacionados à expansão destas células (GREM1, PTGS2/COX2, PTX3, TNFAIP6 e VCAN), à maturação oocitária (BDNF, EGFR, NOS3, PDE5A, PRKCD e STAT3) e ao desenvolvimento embrionário (IGF1R, KRT8 e LUM). Ao final do cultivo de MIV, observou-se no grupo NPPC que o gene PTX3, relacionado à expansão das células do cumulus, além dos genes AREG e BDNF, relacionados à maturação oocitária, e o gene LUM, relacionado ao desenvolvimento embrionário estavam mais expressos em comparação com o grupo C30 (P<0,05). Com relação ao desenvolvimento embrionário, os grupos experimentais não apresentaram diferença (P>0,05) quanto à taxa de clivagem (média de 73,22%). Embora o grupo NPPC não tenha diferido (P>0,05) de C22 e C30 quanto à taxa de blastocistos, houve diferença entre C22 e C30 (69,3 vs. 37,4; P<0,05). Todavia, não houve diferença entre os grupos com relação ao número de células totais (blastômeros) e apoptóticas (P>0,05). Em conclusão, o cultivo de pré-MIV de oócitos bovinos por 8h com 100nM NPPC não bloqueou a retomada da meiose, mas a progressão da meiose ocorreu de forma mais lenta e impediu o envelhecimento e degeneração dos oócitos. O cultivo de oócitos por tempo prolongado (30h) na ausência de NPPC foi prejudicial para o desenvolvimento embrionário, mas o tratamento com NPPC (8h pré-MIV+22h MIV = duração total de 30h) reverteu parcialmente este índice. / The aim of this study was to evaluate the effect of the C-type natriuretic peptide precursor (NPPC) during pre in vitro maturation culture (pre-IVM) of bovine oocytes on: 1) nuclear maturation progress; 2) gene expression in cumulus cells and 3) acquisition of competence for in vitro embryo development. Cumulus oocyte complexes (COCs) were pre-IVM with 100 nM NPPC for 8 hours (NPPC group) and then were washed for the complete removal of NPPC and submitted to IVM for 22h. After 8h pre-IVM followed by 22h IVM (total culture time = 30h) oocytes were evaluated for nuclear maturation progress and cumulus cells for relative mRNA expression. Control group (C) was IVM in the absence of NPPC for up to 30h and the same evaluations were made immediately after follicle removal (C0) and after 8h (C8), 22h (C22) and after 30h of culture (C30). In another experiment with the same treatment, the oocytes were fertilized at the end of IVM and embryo development to the blastocyst stage was evaluated. After 8 hours of pre-IVM culture, meiosis progression analysis showed 58.9% of oocytes in GV1 configuration in C0, while C8 had only 13.9% (P<0.05). The GV1 rates in NPPC did not differ from any group (22.8%; P>0.05). On the other hand, C8 showed higher rates of GV3 in comparison with C0 (53.1% vs. 3.62%; p<0.05) and no differences compared to NPPC (38.7%; P<0.05). At the end of IVM culture, no differences between groups (C22 vs. NPPC vs. C30) were observed in nuclear maturation, however, higher rates of degenerated oocytes were observed in C30 in comparison with C22 (11.4% vs. 2.8%; P<0.05). The relative gene expression analysis in cumulus cells after 8h pre-IVM with NPPC showed an up-regulation in genes related to cumulus cells expansion (GREM1, PTGS2/COX2, PTX3, TNFAIP6 and VCAN), oocyte maturation (BDNF, EGFR, NOS3, PDE5A, PRKCD e STAT3) and embryo development (IGF1R, KRT8 and LUM). At the end of IVM culture, the cumulus cells expansion related gene, PTX3, the oocyte maturation genes, AREG and BDNF, and the embryo development gene, LUM, were up-regulated in NPPC in comparison with C30 (p<0.05). Regarding embryo development, the cleavage rates did not differ in experimental groups (mean around 73,22%). Besides, blastocyst rates did not differ between NPPC (P>0.05) and the other groups, but there was a difference between C22 and C30 (69.3 vs. 37.4%; P<0.05). There was no difference between the groups in total cell number (blastomeres) and apoptotic cells (P<0.05). In conclusion, the pre-IVM culture of bovine oocytes for 8h with 100nM NPPC did not block meiosis resumption, but meiosis progression occurred more slowly and prevented aging and degeneration of the oocytes. The prolonged time of oocyte culture (30h) in the absence of NPPC was detrimental to embryo development, but NPPC treatment (8h pre-IVM + 22h IVM = total duration of 30h) partially reversed this effect.

A cana-de-açúcar constitui uma das principais fontes de alimento e de energia renovável no mundo. Entre os fatores bióticos que reduzem sua produtividade, destaca-se o carvão, doença causada pelo fungo biotrófico Sporisorium scitamineum, cujos danos à cultura podem ultrapassar 80%. No entanto, poucos estudos foram desenvolvidos sobre a genética desse patógeno. Assim, o presente trabalho estudou a dinâmica de marcas RFLP associados à região subtelomérica (tel-RFLP) e marcas GFP em três populações do patógeno (a, b e c) obtidas em ciclos sucessivos da doença. A população a inicialmente foi formada pela inoculação com teliósporos F1 oriundos do isolado 39 produzido em trabalho anterior (REIS, 2012). As populações b e c foram produzidas inicialmente por cruzamentos controlados. Estes envolveram como parentais esporídios haploides de tipos de reação sexual compatíveis e perfis tel-RFLP conhecidos. Na população b os esporídios parentais (18A e 39B) foram isolados de teliósporos distintos, apresentaram perfis tel-RFLP mais contrastantes e simularam fecundação cruzada. Na população c, os esporídios parentais 39Agfp e 39Bgfp apresentaram perfis tel- RFLP mais semelhantes. Esses mutantes foram obtidos pela inserção heteróloga do gene gfp em esporídios obtidos de um único teliósporo simulando autofecundação (reação sexual intra-tétrade). A comparação entre os perfis de tel-RFLP dos esporídios selvagens e mutantes GFP detectou a inserção do gene gfp possivelmente na região subtelomérica do individuo 39Agfp (parental). A cada ciclo da doença teliósporos selvagens e mutantes GFP foram obtidos, sendo isolados dois esporídios para os cruzamentos controlados que levaram à produção do ciclo seguinte de doença. O uso de cruzamentos controlados possibilitou a produção de teliósporos idênticos geneticamente, permitindo inferir sobre eventos meióticos ocorrentes numa mesma progênie. A fenotipagem sexual dos esporídios que tiveram os perfis de tel-RFLP analisados reduziu o viés advindo do desvio da segregação esperada de 1:1 entre os tipos sexuais de esporídios pertencentes à mesma divisão meiótica. Além disso, auxiliou na detecção de recombinantes, visto que os loci de reação sexual segregam na meiose I. A análise das marcas tel-RFLP das progênies mutantes GFP e selvagens, obtida respectivamente em 1 e 2 ciclos da doença, evidenciou eventos de recombinação na segregação de cromossomos homólogos e eventos de recombinação homóloga. Mesmo apresentando número elevado, as marcas tel-RFLP foram capazes de rastrear indivíduos pertencentes a uma mesma população, o que poderá auxiliar em futuras avaliações epidemiológicas do carvão da cana-de-açúcar. Por fim, a expressão do gene gfp em células de pró-basídios mutantes possibilitou a detecção de recombinação homóloga associada a essa marca e a observação inédita de tétrades lineares de quatro células para essa espécie. Observou-se elevado potencial de utilização do gene gfp em estudos genéticos aplicados a esse patossistema. A recombinação sugerida na região subtelomérica mostrou frequência de recombinação ainda não descrita para essa espécie a ser estudada posteriormente. / Sugarcane is a major source of food and renewable energy in the world. Among the biotic factors that reduce its productivity we highlight the sugarcane smut disease, caused by the biotrophic fungus Sporisorium scitamineum, whose damages to the crop can overpass 80%. However, few genetic studies have targeted this pathogen. Thus, the present work studied the dynamics of RFLP marks associated to the subtelomeric region (tel-RFLP) and GFP marks of three populations of the pathogen (a, b and c) obtained from consecutive disease cycles. Population a was initially formed with inoculation using teliospores F1 from the whip 39 obtained in previous work (REIS, 2012). Populations b and c were initially produced by controlled crosses of sexual compatible haploid sporidia with known tel-RFLP profiles as parents. In population b the parent sporidia (18A and 39B) were isolated from distinct teliospores, had more different tel-RFLP profiles (7 polymorphic marks) and simulated outcrossing. In population c, the parental sporidia 39Agfp and 39Bgfp had more similar tel-RFLP profiles. These mutants were obtained by heterologous insertion of gfp gene in sporidia obtained from single teliospore, simulating selfing (intra-tetrad mating type). The comparison between wild-type and GFP mutant sporidia tel-RFLP profiles had detected the gfp gene insertion in the subtelomeric region of the individual 39Agfp (parent). For each disease cycle wild-type and GFP mutant teliospores were obtained and two sporidia were isolated to perform controlled crosses that lead to the next disease cycle. The use of controlled crosses resulted in the production of genetically identical teliospores, which allowed inferring about same progeny meiotic occurring events. The sexual phenotyping of the sporidia that had their tel-RFLP analyzed reduced the bias resulted from deviation of expected 1:1 ratio segregation among the mating types of same meiotic division progeny. Besides, it helped in the recombinants detection, due to mating type loci segregation in meiosis I. The analysis of wild-type and GFP mutant progenies tel- RFLP marks, obtained for 2 and 1 disease cycles, has pointed out events of homologous chromosomes recombination and crossing-over events. Despite of the high amount of marks obtained, the tel-RFLP marks allowed the tracking of individuals from the same population, which can benefit future sugarcane smut disease epidemiologic evaluations. Moreover, the gfp gene expression of mutant probasidium cells led to the detection of crossing-over involving the GFP mark and the first observation of linear four-cells tetrads to this species. A high potential use of gfp gene in genetic studies applied to this pathosystem was observed. The recombination suggested for the subtelomeric region showed a recombination frequency haven\'t described for this species that needs to be further studied.

A genome is shaped by many different forces. Recombination can for instance both create and maintain genetic diversity, but the need to locally reduce recombination rates will also leave specific signatures. Genetic elements can act selfishly and spreading at the expense of the rest of the genome can leave marks of their activity, as can mechanisms that suppresses them, in a phenomenon known as genomic conflict. In this thesis, I have studied the forces driving genome evolution, using modern genome sequencing techniques and with a special focus on a class of selfish genetic elements known as Spore killers found in the fungus Neurospora. First, we show novel findings on large-scale suppression of recombination by non-structural means in the N. tetrasperma genomes. In contrary, in the genomic region harbouring the spore killer elements Sk-2 and Sk-3 of N. intermedia, a dense set of inversions that are interspersed with transposable elements have accumulated. The inversions are unique for each killer type, showing that they have a long separated evolutionary history and likely have established themselves independently. For the Sk-2 haplotype, where we have polymorphism data, we see signs of relaxed selection, which is consistent with the hypothesis that recombination suppression reduces the efficacy of selection in this region. These results show the strong effects the divergent selective forces of genomic conflicts can have on chromosome architecture. Furthermore, we investigate the hypothesis that spore killing can drive reproductive isolation, by comparing the fertility of crosses between N. metzenbergii and either killer or non-killer N. intermedia strains. We show that crosses with spore killer strains have lower fertility, which cannot be explained by the killing itself, but is potentially caused by an incompatibility gene captured in the non-recombining region. Finally, we identified the genetic element responsible for causing spore killing in the Sk-1 spore killer strains found in N. sitophila. Unlike the Sk-2 and Sk-3 elements, Sk-1 is not connected to a large, non-recombining region, but is caused by a single locus, and we also find indications that this locus was introgressed from N. hispaniola.

L’agriculture moderne doit assurer notre sécurité alimentaire dans le contexte d’un changement climatique qui entraîne une baisse des rendements. Une meilleure compréhension des facteurs contrôlant la recombinaison méiotique ouvrirait la voie à la modification du nombre et de la répartition des crossing-overs, ce qui permettrait une localisation plus précise des facteurs génétiques contrôlant les caractères d’intérêt agronomique et faciliterait le pyramidage d’allèles favorables au sein d’un même génotype élite. Pendant ma thèse, j’ai développé une méthode de mesure à haut débit de la recombinaison chez la levure Saccharomyces cerevisiae pour étudier la diversité de la recombinaison et de l’interférence au sein d’une collection de 24 souches représentatives de la diversité de l’espèce ainsi qu’au sein d’un dispositif di-allèle à cinq parents. Les résultats montrent un nombre moyen de crossing-overs par méiose compris entre 24 et 61, ce qui est plus élevé que chez la majorité des autres espèces. Plus particulièrement, les profils de recombinaison diffèrent entre souches, atteignant un écart d’un facteur 9 dans certaines régions. Les souches originaires d’habitats peu stables n’ont cependant pas un niveau de recombinaison plus élevé que les souches originaires d’environnements stables. En outre, la plupart des souches montrent de l’interférence dont la force est corrélée positivement avec le niveau de recombinaison. L’étude de la relation entre niveau de recombinaison et similarité de séquence entre homologues, à différentes échelles locales ou globales, indique que la recombinaison est contrôlée à la fois par des éléments cis et des facteurs trans. Par ailleurs, l’hétérozygotie chez les hybrides a un effet négatif sur le niveau de recombinaison, mais les homozygotes ont aussi un niveau de recombinaison réduit par un effet de dépression de consanguinité. Ce travail permettra maintenant d’étudier la réponse de la recombinaison à la sélection et de détecter les QTL de nombre de crossing-overs, afin d’identifier des gènes qui contrôlent la recombinaison. / Modern agriculture must ensure food security in a context of climate change that will lower yields. A better understanding of the factors controlling meiotic recombination could pave the way to modifying the number and distribution of crossing-over, which would allow a more precise localization of genetic factors controlling agronomic traits, and facilitate gene pyramiding in selection programs. During my thesis, I developed a method for high-throughput measurement of recombination rates in the yeast Saccharomyces cerevisiae. This allowed me to study the diversity of recombination and interference in a collection of 24 strains representing most of the diversity of the species, as well as within a five-parent di-allele design. The results show an average number of crossovers per meiosis ranging between 24 and 61, higher than in the majority of other species. Furthermore, recombination patterns differ between strains, and ratios of local recombination rates show 9-fold differences in some regions. Strains from unstable habitats, however, do not have a higher level of recombination than those from stable environments. In addition, most strains show interference whose strength is positively correlated with the level of recombination. The study of the relationship between recombination rate and sequence similarity between homologs at different scales (from local to global) indicates that recombination is controlled by both cis elements and trans factors. Lastly, heterozygosity in hybrids has a negative effect on crossing-over, but homozygotes also have a reduced level of recombination due to inbreeding depression. This work will now be used to study the response of recombination to selection and to detect QTL of crossover number in order to identify genes controlling recombination.