A transformação plastidial oferece uma série de vantagens em relação à transformação nuclear, como: altos níveis de expressão de proteínas, capacidade de expressar múltiplos transgenes em operons e contenção gênica pela ausência de transmissão pelo pólen. Devido ao alto número de cópias do genoma plastidial por cloroplasto e ao alto número de cloroplastos por células vegetais, são necessários ciclos de regeneração sob condições seletivas para obter transformantes homoplásmicos. A análise de homoplasmia é realizada pela metodologia de Southern blot ou pelo teste de herança do transgene pela germinação de sementes em meio seletivo. O Southern blot é trabalhoso, demorado e para maior sensibilidade envolve o uso de radioisótopos, enquanto o teste de germinação é realizado somente após a produção de sementes necessitando de um ciclo de reprodução da planta. Assim, o objetivo deste trabalho foi desenvolver um método rápido, sensível e eficaz para determinar o grau de homoplasmia de plantas transplastômicas, baseado na técnica de PCR em tempo real. Folhas de fumo foram transformadas com vetores compostos pelos genes 9 dessaturase (pMR1), 15 dessaturase (pMR3), -3 elongase (pMR5) e 12/3 dessaturase (pMR10), todos contendo o gene de seleção aadA. No total, 44 plantas foram obtidas, sendo 21 plantas positivas para a inserção do transgene. O grau de homoplasmia foi determinado pela proporção entre o número de cópias do transgene e o número de cópias do gene endógeno. Inicialmente, misturas de DNA de plantas transplastômicas homoplásmicas (pMR1 e pMR3) com DNA de planta tipo selvagem foram preparadas para simular diferentes graus de homoplasmia. DNA da planta transplastômica ou do plasmídeo foi diluído em série para construção das curvaspadrão, com a quantidade dos genes sendo estimada por meio da plotagem nessas curvas. Os índices de homoplasmia detectados na PCR em tempo real foram compatíveis com os resultados do teste de germinação com valores abaixo de 1 para plantas heteroplásmicas, 1 para a planta homoplásmica e 0 para as plantas sem a inserção do transgene. Os resultados das análises de amostras coletadas após o primeiro ciclo de regeneração mostraram que 13 das 21 plantas já se apresentavam em estado homoplásmico não sendo necessários mais ciclos de regeneração. A PCR em tempo real mostrou ser um método eficiente para análise do grau de homoplasmia de plantas transplastômicas. / Plastid transformation offers several advantages in relation to nuclear transformation, such as high-level of protein expression, the feasibility of expressing multiple transgenes in operons and gene containment through the lack of pollen transmission. Due to the high copy number of plastidial genome in chloroplasts and the high number of chloroplasts per plant cells, regeneration cycles under selective conditions are necessary to obtain homoplasmic transformants. Homoplasmy analysis is performed by Southern blot methodology or transgene inheritance test through seed germination in selective medium. Southern blot is laborious, time consuming and for more sensitivity it would require the use of radioisotopes, while germination test can be performed only after seed production which require a plant reproduction cycle. The objective of this study was to develop a fast, sensitive and effective method to determine the homoplasmy degree of transplastomic plants, based on real-time PCR. Tobacco leaves were transformed with vectors containing the 9 desaturase (pMR1), 15 desaturase (pMR3), -3 elongase (pMR5) and 12/3 desaturase (pMR10) each one with the aadA selection gene. In total, 44 plants were obtained, of which 21 were positive for the insertion of the transgene. The homoplasmy degree was determined by the proportion between the number of transgene copies and the number of endogenous gene copies. Initially, mixtures of homoplastomic plants DNA (pMR1 and pMR3) with wild-type plant DNA were prepared to simulate different degrees of homoplasmy. Transplastomic plant DNA or plasmid DNA was diluted to construct the standard curves and the gene amount was detected by plotting in this curves. The homoplasmy rate detected in real-time PCR were consistent with the results of germination test with values below 1 for heteroplasmic plants, 1 for homoplasmic plants and 0 for plants without the transgene insertion. The results obtained from the samples collected after the first regeneration cycle showed that 13 of the 21 plants were already in a homoplasmic state and did not require more cycles of regeneration. The real-time PCR proved to be an effective method for analyzing the homoplasmy degree of transplastomic plants.

A transformação plastidial oferece uma série de vantagens em relação à transformação nuclear, como: altos níveis de expressão de proteínas, capacidade de expressar múltiplos transgenes em operons e contenção gênica pela ausência de transmissão pelo pólen. Devido ao alto número de cópias do genoma plastidial por cloroplasto e ao alto número de cloroplastos por células vegetais, são necessários ciclos de regeneração sob condições seletivas para obter transformantes homoplásmicos. A análise de homoplasmia é realizada pela metodologia de Southern blot ou pelo teste de herança do transgene pela germinação de sementes em meio seletivo. O Southern blot é trabalhoso, demorado e para maior sensibilidade envolve o uso de radioisótopos, enquanto o teste de germinação é realizado somente após a produção de sementes necessitando de um ciclo de reprodução da planta. Assim, o objetivo deste trabalho foi desenvolver um método rápido, sensível e eficaz para determinar o grau de homoplasmia de plantas transplastômicas, baseado na técnica de PCR em tempo real. Folhas de fumo foram transformadas com vetores compostos pelos genes 9 dessaturase (pMR1), 15 dessaturase (pMR3), -3 elongase (pMR5) e 12/3 dessaturase (pMR10), todos contendo o gene de seleção aadA. No total, 44 plantas foram obtidas, sendo 21 plantas positivas para a inserção do transgene. O grau de homoplasmia foi determinado pela proporção entre o número de cópias do transgene e o número de cópias do gene endógeno. Inicialmente, misturas de DNA de plantas transplastômicas homoplásmicas (pMR1 e pMR3) com DNA de planta tipo selvagem foram preparadas para simular diferentes graus de homoplasmia. DNA da planta transplastômica ou do plasmídeo foi diluído em série para construção das curvaspadrão, com a quantidade dos genes sendo estimada por meio da plotagem nessas curvas. Os índices de homoplasmia detectados na PCR em tempo real foram compatíveis com os resultados do teste de germinação com valores abaixo de 1 para plantas heteroplásmicas, 1 para a planta homoplásmica e 0 para as plantas sem a inserção do transgene. Os resultados das análises de amostras coletadas após o primeiro ciclo de regeneração mostraram que 13 das 21 plantas já se apresentavam em estado homoplásmico não sendo necessários mais ciclos de regeneração. A PCR em tempo real mostrou ser um método eficiente para análise do grau de homoplasmia de plantas transplastômicas. / Plastid transformation offers several advantages in relation to nuclear transformation, such as high-level of protein expression, the feasibility of expressing multiple transgenes in operons and gene containment through the lack of pollen transmission. Due to the high copy number of plastidial genome in chloroplasts and the high number of chloroplasts per plant cells, regeneration cycles under selective conditions are necessary to obtain homoplasmic transformants. Homoplasmy analysis is performed by Southern blot methodology or transgene inheritance test through seed germination in selective medium. Southern blot is laborious, time consuming and for more sensitivity it would require the use of radioisotopes, while germination test can be performed only after seed production which require a plant reproduction cycle. The objective of this study was to develop a fast, sensitive and effective method to determine the homoplasmy degree of transplastomic plants, based on real-time PCR. Tobacco leaves were transformed with vectors containing the 9 desaturase (pMR1), 15 desaturase (pMR3), -3 elongase (pMR5) and 12/3 desaturase (pMR10) each one with the aadA selection gene. In total, 44 plants were obtained, of which 21 were positive for the insertion of the transgene. The homoplasmy degree was determined by the proportion between the number of transgene copies and the number of endogenous gene copies. Initially, mixtures of homoplastomic plants DNA (pMR1 and pMR3) with wild-type plant DNA were prepared to simulate different degrees of homoplasmy. Transplastomic plant DNA or plasmid DNA was diluted to construct the standard curves and the gene amount was detected by plotting in this curves. The homoplasmy rate detected in real-time PCR were consistent with the results of germination test with values below 1 for heteroplasmic plants, 1 for homoplasmic plants and 0 for plants without the transgene insertion. The results obtained from the samples collected after the first regeneration cycle showed that 13 of the 21 plants were already in a homoplasmic state and did not require more cycles of regeneration. The real-time PCR proved to be an effective method for analyzing the homoplasmy degree of transplastomic plants.

Les champignons mycorhiziens à arbuscules (CMA) sont des organismes microscopiques du sol qui jouent un rôle crucial dans les écosystèmes naturels et que l’on retrouve dans tous les habitats de la planète. Ils vivent en relation symbiotique avec la vaste majorité des plantes terrestres. Ils sont des biotrophes obligatoires, c'est-à-dire qu'ils ne peuvent croître qu'en présence d'une plante hôte. Cette symbiose permet entre autres à la plante d'acquérir des nutriments supplémentaires, en particulier du phosphore et du nitrate. Malgré le fait que cette symbiose apporte des services importants aux écosystèmes, la richesse des espèces, la structure des communautés, ainsi que la diversité fonctionnelle des CMA sont mal connues et l'approfondissement des connaissances dans ces domaines dépend d’outils de diagnostic moléculaire. Cependant, la présence de polymorphisme nucléaire intra-isolat combiné à un manque de données génomiques dans différents groupes phylogénétique de ces champignons complique le développement de marqueurs moléculaires et la détermination de l'affiliation évolutive à hauts niveaux de résolution (c.a.d. entre espèces génétiquement similaires et/ou isolats de la même espèce). . Pour ces raisons, il semble une bonne alternative d’utiliser un système génétique différent en ciblant le génome mitochondrial, qui a été démontré homogène au sein d'un même isolat de CMA. Cependant, étant donné le mode de vie particulier de ces organismes, une meilleure compréhension des processus évolutifs mitochondriaux est nécessaire afin de valoriser l'utilisation de tels marqueurs dans des études de diversité et en génétique des populations. En ce sens, mon projet de doctorat consistait à investiguerétudier: i) les vecteurs de divergences inter-isolats et -espèces génétiquement rapprochéesphylogénétiquement apparentées, ii) la plasticité des génomes mitochondriaux, iii) l'héritabilité mitochondriale et les mécanismes potentiels de ségrégation, ainsi que iv) la diversité mitochondriale intra-isolat in situ. À l'aide de la génomique mitochondriale comparative, en utilisant le séquençage nouvelle génération, on a démontré la présence de variation génétique substantielle inter-isolats et -espèces, engendrées par l'invasion d'éléments mobiles dans les génomes mitochondriaux des CMA, donnant lieu à une évolution moléculaire rapide des régions intergéniques. Cette variation permettait de développer des marqueurs spécifiques à des isolats de la même espèce. Ensuite, à l'aide d'une approche analytique par réseaux de gènes sur des éléments mobiles, on a été en mesure de démontrer des évènements de recombinaisons homologues entre des haplotypes mitochondriaux distincts, menant à des réarrangements génomiques. Cela a permis d'ouvrir les perspectives sur la dynamique mitochondriale et l'hétéroplasmie dans un même isolatsuggère une coexistence de différents haplotypes mitochondriaux dans les populations naturelles et que les cultures monosporales pourraient induirent une sous-estimation de la diversité allélique mitochondriale. Cette apparente contradiction avec l'homogénéité mitochondriale intra-isolat généralement observée, a amené à investiguer étudier les échanges génétiques à l'aide de croisements d'isolats génétiquement distincts. Malgré l'observation de quelques spores filles hétéroplasmiques, l'homoplasmie était le statut par défaut dans toutes les cultures monosporales, avec un biais en faveur de l'un des haplotypes parentaux. Ces résultats suggèrent que la ségrégation opère durant la formation de la spore et/ou le développement de la coloniedu mycélium. De plus, ils supportent la présence d'une machinerie protéique de ségrégation mitochondriale chez les CMAAMF, où l'ensemble des gènes impliqués dans ce mécanisme ont été retrouvé et sont orthologues aux autres champignons. Finalement, on est revenue aux sources avecon a étudié le polymorphisme mitochondrial intra-isolat à l'aide d'une approche conventionnelle de PCR en utilisant une Taq polymérase de haute fidélité, suivie de clonage et de séquençage Sanger, sur deux isolats de R. irregularis. Cela a permis l'observation d'hétéroplasmie in situ, ainsi que la co-expression de variantes de variantes de protéines'ARNm dans une souche in vitro. Les résultats suggèrent que d'autres études basées sur le séquençage nouvelle génération aurait potentiellement ignorée cette variation, offrant ainsi plusieurs nouveaux arguments permettant de considérer les CMA comme des organismes possédant une population de génomes mitochondriaux et nucléaires distincts. / The association between arbuscular mycorrhizal fungi (AMF) and plant roots is one of the most widespread symbioses involving plants, and thus has an important role in terrestrial ecosystems. In exchange for carbohydrates, AMF improve plant fitness by enhancing mineral nutrient uptake, especially in particular phosphate and nitrate. Although this symbiosisDespite the fact that these symbioses contribute provides to important services toin ecosystems, the species richness, community structure and functional diversity of AMF is not well understood due to a lack of reliable molecular tools. The intra-isolate genetic polymorphism of nuclear DNA observed in AMF, combined with a lack of genomic data in a broad range of phylogenetic groups, has made it difficult to develop molecular markers and to determine evolutionary relatedness at high levels of resolution (i.e. between genetically-similar species and/or isolates). For these reasons, it seems a good alternative to use a different genetic system by targeting the mitochondrial genome, which have been shown to be homogeneous within AMF isolates. However, given the peculiar lifestyle of these organisms, a better understanding of the mitochondrial evolutionary processes and dynamics were is necessary in order to validate the usefulness of such markers in diversity and population genetics studies. In that regard, the objectives of my PhD project were to investigate: i) the divergence between closely related species and isolates, ii) mitochondrial genomes plasticity, iii) mitochondrial heritability and potential segregation mechanisms and iv) in situ mitochondrial intra-isolate allelic diversity. With Using comparative mitochondrial genomics using and next generation sequencing (NGS) sequencing, we found substantial sequence variation in intergenic regions caused by the invasion of mobile genetic elements. This variation gives risecontributes to rapid mitochondrial genome evolution among closely related isolates and species, which makes it possible to design reliable intra- and inter-specific markers. Also, an extensive gene similarity network-based approach allowed us to provide strong evidence of inter-haplotype recombination in AMF, leading to a reshuffled mitochondrial genome. These findings suggest the coexistence of distinct mtDNA haplotypes in natural populations and raise questions as to whether AMF single spore cultivations artificially underestimates mitochondrial genetic diversity in natural population.. This apparent contradiction with the intra-isolate mtDNA homogeneity usually observed in these fungi, led to the investigation of mitochondrial heritability in the spore progeny resulting from crossed-cultures. Although an heteroplasmic state was observed in some daughter spores, we found that homoplasmy was the dominant state in all monosporal cultures, with an apparent bias towards one of the parental haplotypes. These results strongly support the presence of a putative mitochondrial segregation proteic machinery in AMF, whose complete set of genes were orthologous with those found in other fungi. Our findings suggest that segregation takes place either during spore formation or colony mycelium development. Finally, we performed a conventional PCR based approach with a high fidelity Taq polymerase, followed by downstream cloning and Sanger sequencing using the model organism Rhizophagus irregularis. We found in situ heteroplasmy along with substantial intra-isolate allelic variation within the mtDNA that persists in the transcriptome. Our study also suggest that genetic variation in Glomeromycota is higher than meets the eye and might be critically underestimated in most NGS based-AMF studies both in nuclei and mitochondria.