Дисертації з теми "Organellar genomes"

Via l'émergence de barrières à la reproduction qui isolent les populations les unes des autres, la spéciation est le processus qui conduit à la formation de nouvelles espèces. Par ailleurs, les génomes organellaires peuvent être impliqués dans ce processus, par le biais d'incompatibilités cytonucléaires. Leur mode de transmission peut également influencer l'évolution de l'isolement reproducteur (IR) entre populations. Dans ce travail de thèse, j'ai travaillé sur l'influence des génomes organellaires sur l'évolution de l'isolement reproducteur entre quatres lignées de Silene nutans et ai tenté de reconstruire le scénario évo-démographique qui a façonné leur évolution. Dans un premier temps, via l'utilisation de données génomiques et transcriptomiques, nous avons tenté d'identifier des candidats d'incompatibilités chloro-nucléaires impliquées dans l'IR entre ces lignées. Nous avons ensuite approfondi l'analyse d'un complexe candidat: le ribosome chloroplastique. Par ailleurs, l'IR semble être incomplet entre ces lignées puisque certains hybrides ont survécu. Nous avons donc testé une transmission paternelle du génome chloroplastique chez cette espèce, qui pourrait avoir sauvé certains de ces hybrides. Nous avons génotypé les hybrides survivants pour six SNP chloroplastiques et déterminé s'ils avaient hérité du génome chloroplastique paternel ou maternel. En permettant la transmission d'un génome chloroplastique moins incompatible, la fuite paternelle semble bien avoir sauvé certains de ces hybrides. Les génomes mitochondriaux pourraient également être impliqués dans l'IR, par le biais d'incompatibilités mito-nucléaires. Du fait de leur co-transmission, les génomes organellaires sont supposés être en déséquilibre de liaison étroit, présentant ainsi des schémas évolutifs similaires. Nous les avons comparés en utilisant les données génomiques des deux génomes organellaires, pour des individus des quatre lignées. Ces schémas évolutifs se sont révélés particulièrement contrastés, les gènes mitochondriaux présentant du polymorphisme partagé à l'inverse des gènes chloroplastiques contenant des substitutions fixées différemment entre lignées. Des événements de type recombinaison ont également été identifiés dans les gènes mitochondriaux. Enfin, nous avons reconstruit l'histoire évo-démographique des quatre lignées de S. nutans, en utilisant les données RNAseq et des méthodes ABC. Un scénario de spéciation allopatrique a été identifiée entre les quatre lignées, avec des temps de séparation cohérent avec les maximums glaciaires
Speciation is the process by which the emergence of reproductive barriers isolate populations from one another and ultimately lead to the formation of new species. How these reproductive barriers emerge is a core question when thinking of speciation. Organellar genomes might be involved in the speciation process, through cytonuclear incompatibilities. Their mode of transmission might also influence the pace of reproductive isolation evolution. In my PhD, I worked on how organellar genomes influence the evolution of reproductive isolation between isolated lineages of S. nutans and which evo-demographic scenario shaped their evolution. Using plastid genomic and nuclear transcriptomic data we tried, in the first chapter, to identify candidates for plastid-nuclear incompatibilities involved in RI between lineages of S. nutans. We further dug into one plastid candidate complexe, the plastid ribosome. Because RI seems to be incomplete between lineages of S. nutans as some inter-lineage hybrids survived, we tested for paternal leakage of the plastid genome. We genotyped the surviving hybrids for plastid SNPs and analyzed whether they inherited the paternal or maternal plastid genomes. By allowing the transmission of the less incompatible plastid genome, paternal leakage rescued some of the inter-lineage hybrids. The mitochondrial genome could also be involved in the RI, through mito-nuclear incompatibilities. Because of their co-transmission, organellar genomes are supposed to be in tight linkage-disequilibrium, so exhibiting similar evolutionary patterns. Using genomic data for both organellar genomes for individuals of the four lineages we compared their evolutionary patterns. They were different with mitochondrial genes exhibiting many shared polymorphisms while plastid genomes many fixed substitutions between lineages. Recombination-like events were also identified in the mitochondrial genes. Lastly, we reconstructed the evo-demographic histories of the four lineages of S. nutans, using RNAseq data and ABC methods. Allopatric speciation was identified between the four lineages, with split times consistent with the glacial maxima

Orientador: Vitor Fernandes Oliveira de Miranda
Resumo: Utricularia e Genlisea são gêneros irmãos de plantas carnívoras da família Lentibulariaceae. Possuem aproximadamente 260 espécies representadas em diversas formas de vida. Para o Brasil foram catalogadas 82 espécies, das quais 27 são consideradas endêmicas. Além de dispor das armadilhas carnívoras mais complexas entre plantas, algumas de suas espécies apresentam os menores genomas e as maiores taxas de mutações entre as angiospermas relatadas até o momento. A respeito de seus genomas organelares, os estudos são pífios. Neste contexto, há a necessidade de se investigar como são os genomas organelares, suas estruturas, seus genes e como se deu a evolução das organelas nos gêneros. Portanto este estudo teve como objetivo, a partir de sequenciamento de nova geração e montagem de genomas, estudar e comparar os genomas organelares de Utricularia e Genlisea. Neste âmbito, foram montados e sequenciados os cloroplastos das espécies Utricularia foliosa, U. reniformis, G. aurea, G. filiformis, G. pygmaea, G. repens e G. tuberosa, e o genoma mitocondrial de U. reniformis. Os resultados obtidos revelaram que possivelmente há relação entre forma de vida e presença de genes ndhs nos gêneros, em razão de que para as espécies terrestres há deleção e “pseudogenização” de genes ndhs, já as espécies aquáticas detêm todo repertório de ndhs intacto. A partir das evidências encontradas, foi possível constatar transferência horizontal de genes, inclusive de genes ndhs, em mitocôndrias.
Abstract: Utricularia and Genlisea are sister genera in the carnivorous family Lentibulariaceae. There are aprproximately 260 species representing diverse life forms. For Brasil there are 82 species, 27 considered endemic. At the moment, besides having the most complex carnivorous traps between all plants, some of its species have miniature genomes and the highest mutational rates among angiosperms. There are few studies regarding its organellar genome. In this context, it is necessary to investigate how are these organellar genomes, its structure, genes, and how evolutionary forces govern these organelles in the different genera. Therefore, the aim of this study is to study and compare the organellar genomes of Utricularia and Genlisea, using next generation sequencing and genome assembly. In this context, chloroplasts of the species Utricularia foliosa, U. reniformis, Genlisea aurea, G. filiformis, G. pygmaea, G. repens and G. tuberosa, and the mitochondrial genome of U. reniformis were assembled and sequenced. The results show that possibly there is a connection between life form and the presence of ndhs genes in the genera, since for the terrestrial species there are ndhs genes that are deleted and pseudogenization, in contrary to the aquatic species which have all intact ndhs repertoir. Concerning the evidences, it was possible to verify horizontal transfer of ndhs and other genes as there are chloroplasts genes in the mitochondria.
Doutor

Thanks to the development of next-generation sequencing (NGS) technology, whole genome data can be readily obtained from a variety of samples. Since the massive increase in available sequencing data, the development of efficient assembly algorithms has become the new bottleneck. Almost every new released tool is based on the De Brujin graph method, which focuses on assembling complete datasets with mathematical models. Although the decreasing sequencing costs made whole genome sequencing (WGS) the most straightforward and least laborious approach of gathering sequencing data, many research projects are only interested in the extranuclear genomes. Unfortunately, few of the available tools are specifically designed to efficiently retrieve these extranuclear genomes from WGS datasets. We developed a seed-and-extend algorithm that assembles organelle circular genomes from WGS data, starting from a single short seed sequence. The algorithm has been tested on several new (Gonioctena intermedia and Avicennia marina) and public (Arabidopsis thaliana and Oryza sativa) whole genome Illumina datasets and always outperformed other assemblers in assembly accuracy and contiguity. In our benchmark, NOVOPlasty assembled all genomes in less than 30 minutes with a maximum RAM memory requirement of 16 GB. NOVOPlasty is the only de novo assembler that provides a fast and straightforward manner to extract the extranuclear sequences from WGS data and generates one circular high quality contig.Heteroplasmy, the existence of multiple mitochondrial haplotypes within an individual, has been researched across different fields. Mitochondrial genome polymorphisms have been linked to multiple severe disorders and are of interest to evolutionary studies and forensic science. By utilizing ultra-deep sequencing, it is now possible to uncover previously undiscovered patterns of intra-individual polymorphism. However, it remains challenging to determine its source. Current available software can detect polymorphic sites but are not capable of determining the link between them. We therefore developed a new method to not only detect intra-individual polymorphisms within mitochondrial and chloroplast genomes, but also to look for linkage among polymorphic sites by assembling the sequence around each detected polymorphic site. Our benchmark study shows that this method can detect heteroplasmy more accurately than any method previously available and is the first tool that is able to completely or partially reconstruct the origin sequences for each intra-individual polymorphism.
Doctorat en Sciences
info:eu-repo/semantics/nonPublished

Laterale Gentransfer wurde zuerst von Schwartz und Dayhoff (1978) entdeckt, die es aber als eine Exzentrizität werteten und als solche ignorierten. Später, als mehrere DNS- und Eiweißsequenzen sequenziert und raffiniertere Phylogenien rekonstruiert wurden, hat die Rolle an Relevanz gewonnen, die der laterale (oder horizontale) Gentransfer in der evolutionären Geschichte von lebendigen Organismen gespielt hat. Außerdem existiert auch zwischen Endosymbionten und Zellkernen statt. Ich habe ein theoretisches Modell entwickelt, das den lateralen Gentransfer zwischen Endosymbionten und dem Zellkern repräsentiert. Das Modell erforscht die Bedeutung des Fehlens von Rekombination in den Organellen (Muller’s Ratchet) sowie Abweichungen von Muller’s Ratchet in Form der non-symmetrical homologous recombination in Gentransfermechanismen. Ich habe zum einen Zellkern-Inkompatibilitäten, die aus der Übertragung eines Gens resultieren, und zum anderen Zyto- und Zellkern-Inkompatibilitäten zwischen den mutierten endosymbiotischen Genomen und dem modifizierten Zellenkern untersucht. Die Ergebnisse zeigen, dass unter bestimmten Bedingungen die Existenz oder Nicht-Existenz von Rekombination die gleiche Wirkung haben können. Es zeigte sich auch, dass Rekombination, wenn sie vorkommt und wenn sie nicht symmetrisch ist, starke Auswirkungen auf die Allelenfrequenz einer Population haben kann. Es wurde auch klar, dass es eine starke Beziehung zwischen dem Zellkern und endosymbiotischen Genomen gibt, und dass das evolutionäre Schicksal des einen größtenteils von den evolutionären Kräften abhängig ist, die das andere beeinflussen. Wenn man Zellkern- und Cyto-Zellkerninkompatibilitäten in das Modell einführt, dann zeigen die Ergebnisse, dass die Inkompatibilitäten, die der laterale Gentransfer produziert hat, möglicherweise eine ähnliche Rolle im Speziationsmechanismus spielen könnten wie die Inkompatibilitäten zwischen Mitochondrien und Zellkernen in verschiedenen Nasonia-Arten.
Lateral gene transfer has played a key role in the evolution of living beings. This process was first acknowledged in 1978 by Schwartz and Dayhoff but considered a relatively infrequent eccentricity and ignored. Later on, as DNA and protein sequences accumulated and more refined phylogenies were reconstructed, the contribution of lateral (or horizontal) gene transfer to the evolutionary history of living organisms gained relevance. Besides, gene transfer is known to occur not only between independent organisms but also, and more frequently between endosymbionts including eukaryotic organelles. I developed a theoretical model to study the lateral gene transfer process between cell organelles (but extendible to other endosymbionts) and the cell nucleus. The model explores the role of the lack of recombination in the organelles (Muller''s ratchet) as well as deviations from Muller''s ratchet in the form of non-symmetrical homologous recombination in relation with the gene transfer process. Also, nuclear incompatibilities resulting from the inclusion of a transferred gene, and cyto-nuclear incompatibilities between the mutant endosymbiotic genomes and the modified nuclear genome are investigated. The results obtained show that under certain circumstances the existence recombination or its non-existence produce the same results, and that deviations from symmetry in the recombination process might have important effects on the frequency of different alleles. It is also clear that there is a strong relation between nuclear and endosymbiotic genomes, and that the evolutionary fate of one largely depends on the forces affecting the other. When nuclear and cyto-nuclear incompatibilities are introduced in the model, the results show that lateral gene transfer-induced incompatibilities could potentially play a role in the speciation process similar to the one produced by mitochondria in the Nasonia species.

Inter-organelle communication is a key feature of eukaryotic cells and has been found to be fundamental in many different cellular processes. One of the best characterized interorganelle cross talk due to membrane contact sites is that between Endoplasmic Reticulum (ER) and mitochondria. Also referred to as mitochondria associated ER-membranes (MAMs) or Mitochondria-ER contact sites (MERCs), their existence was discovered 50 years ago through electron microscopic studies, but their functional significance started to emerge only in late 90s when the role of MERCs in calcium exchange from ER to mitochondria was demonstrated. Despite the importance of these contacts sites in physiology and pathology, only few proteins have so far been identifed involved in the structural maintenace of the distance between the two organelles in mammals. Mitofusin 2 (MFN2) was the first structural tether to be identified. MFN2 has been found on both OMM and ER cytosolic face and is able to form homo and heterotypic interactions with MFN1, thus tethering the two organelles. As residual juxtaposition between the two organelles is still observed in Mfn2-/- cells, additional tethering proteins have to exist. To identify them, we set out to perform two replicates of a genome wide screening in mouse embryonic fibroblasts (MEFs). In order to perform the genome wide screening, we capitalized on the FRET based biosensor, where CFP fused with FRB domain and YFP fused with FKBP domain were targeted to ER (by a Sac 1 signaling sequence) and mitochondria (by an Akap signaling sequence) respectively (Csordas et al., 2010). We modified this probe by introducing between the cDNAs of the two fluorescent proteins a self-cleaving Tav2A peptide in order to have a single mRNA construct that allows the expression of equimolar level of the proteins. FKBP and FRB binding domain are able to heterodymerize upon addition of rapamycin, thus allowing the measurement not only of the basal level of juxtaposition between the two organelles, but also of the maximum level of contacts that can occur in a cell. We called this new construct FRET ER-mitochondria probe (FEMP). FEMP unique features allow us to discriminate between proteins whose role is keeping the two organelles closer, termed as "tethers", and proteins that keep the two organelles apart, defined as "spacers". We analyzed raw images from the screen and calculated two indexes, namely basal and maximum MERC index, mirroring the level of contacts observed at any given timepoint and the maximum possible level of contacts respectively. Following automated image analysis and statistical analysis performed on ~10,000 genes, after candidate selection we identified 205 genes as ER-mitochondria tethers (i.e., genes that once ablated increase the distance between the two organelles) and 59 genes as spacers (i.e., genes that once ablated decrease the distance between the two organelles) affecting both basal and maximum MERC index in both replicates. Moreover, we identified 625 tehters and 696 spacers affecting only the basal MERC index; 519 tethers and 67 spacers affecting only the maximum MERC indexes. Protein classes analysis of these three groups of genes by Panther predicted both already known and new protein classes that are yet to explored in terms of ER-mitochondria communication. Subcellular localization analysis to identify predicted proteins to be present in both ER and outer mitochondrial membrane (OMM) of the gene lists detailed before, revealed 13 proteins among the common tethers and spacers, 30 proteins affecting only the basal MERC index and 16 proteins affecting only the maximum MERC index localized on both organelles. One of the protein present in the last group is Leucine Rich Repeat Kinase 2 (LRRK2) and we have further characterized it as ER-mitochondria tether. Subcellular fractionation experiments showed that LRKK2 localized mostly in MAMs. As expected for a tether, levels of ER-mitochondria juxtaposition, measured with FEMP, were decrased in LRRK2-/- MEF. ER-mitochondria proximity was fully restored by reintroduction in MEF LRRK2-/- of wt protein but not of the familial PD associated mutants. In conclusion, we have developed a new method to assess the proximity between ER and mitochondria and we have utilized this technology to perform two replicates of a high content screen identifying novel structural components of the ER-mitochondria contact sites.
La comunicazione tra organelli cellulari è una caratteristica fondamentale delle cellule eucariotiche ed esercita un ruolo fondamentale in molti processi cellulari. Uno dei processi di comunicazione tra organelli cellulari tra i più caratterizzati è quello dovuto ai siti di contatto tra le membrane di mitocondri e reticolo endoplasmatico (ER). Anche noti come "Mitochondria-associated ER membranes" (MAMs) o "Mitochondria-ER contact sites" (MERCs), la loro esistenza è stata scoperta 50 anni fa tramite studi di microscopia elettronica, ma il loro significato funzionale è iniziato ad emergere solo alla fine degli anni 90 quando è stato dimosdtrato il ruolo dei MERCs nello scambio di calcio dall'ER. Nonostante l'importanza di questi siti di contatto tra organelli sia in fisiologia sia in patologia, solo poche proteine coinvolte nel mantenimento strutturale della distanza tra i due organelli sono state finora identificate nei mammiferi. Mitofusina2 (MFN2) è stato il primo "tether" strutturale ad essere identificato. E' stato rilevato che MFN2 è localizzata sia nella membrana mitocondriale esterna (OMM) sia sulla superficie citosolica dell'ER ed ' in grado di formare intrazioni omo- ed eterotipiche con MFN1, mantenendo quindi la distanza tra i due organelli. Poiché una residua giustapposizione tra i due organelli è stata osservata in cellule MFN2-/-, ulteriori proteine che esercitano questo ruolo devono esistere. Per identificarle, abbiamo stabilito un protocollo ed eseguito due repliche di uno screening genomico su larga scala in fibroblasti embrionali di topo (MEF). Per eseguire questo screening, abbiamo sfruttato un biosensore basato sulla FRET, dove la proteina fluorescente CFP fusa con il dominio funzionale FRB e la proteina fluorescente YFP fusa con il dominio funzionale FKBP vengono fatte localizzare rispettivamente all'ER (grazie alla sequenza di segnale Sac1) ed ai mitocondri (grazie alla sequenza di segnale Akap1) (Csordas G. et al., 2010). Abbiamo modificato questo costrutto inserendo tra i cDNA delle due proteine il peptide autocatalitico Tav2A per ottenere un singolo mRNA e quindi l'espressione equimolare delle due proteine. I domini funzionali FKBP e FRB sono in grado di eterodimerizzare con l'aggiunta di Rapamicina, permettendo così la misurazione non solo dei livelli di giustapposizione basale tra i due organelli, ma anche del massimo livello di contatti che possono avvenire in una cellula. Abbiamo chiamato questo nuovo costrutto FRET ER-mitochondria probe (FEMP). Le caratteristiche uniche del FEMP ci consentono didiscriminare tra le proteine il cui ruolo è quello di mantenere i due organelli vicini, chiamate "tethers", e proteine che invece tengono i due organelli più distanti, definiti "spacers". Le immagini ottenute dallo screening sono state analizzate e sono stati calcolati due indici, chiamati "basal MERC index" e "maximum MERC index", che rappresentano rispettivamente il livello di contatti osservabili in qualsiasi momento in una cellula e il massimo livello di contatti possibile. A seguito di un'analisi delle immagini automatizzata e di un'analisi statistica effettutata su ~10,000 geni, dopo un processo di selezione abbiamo identificato 205 geni come "tethers" (geni che una volta eliminati aumentano la distanza tra i due organelli) tra mitocondri e ER e 59 geni come "spacers" (geni che una volta eliminati diminuiscono la distanza tra i due organelli) che influenzano sia il basal sia il maximum MERC index in entrambe le repliche. Inoltre, sono stati identificati 625 tethers e 696 spacers che influenzano solo il basal MERC index; e 519 tethers e 67 spacers che modificano solo il maximum MERC index. Analisi delle classi di proteine presenti in questi tre gruppi tramite Panther ha rivelato sia classi di proteine il cui ruolo in questo processo era noto, sia nuove classi di proteine il cui ruolo nella comunicazione tra ER e mitocondri deve ancora essere esplorato. Analisi della localizzazione cellulare per identificare proteine localizzate sia nell'ER sia nei mitocondri delle liste di geni esposte in precedenza, ha rivelato l'esistenza di 13 proteine tra i tethers e gli spacers comuni, 30 proteine che influenzano solo il basal MERC index e 16 proteine che influenzano solo il maximum MERC index localizzate in entrambi gli organelli. Una delle proteine presente nell'ultimo gruppo è "Leucine Rich Repeat Kinase 2" (LRRK2) che abbiamo ulteriormente caratterizzato come tether tra ER e mitocondri. Esperimenti di frazionamento cellulare dimostrano che LRRK2 è localizzata principalmente nelle MAMs. Come previsto per un tether, il livello di prossimità tra ER e mitocondri, misurato tramite FEMP, sono diminuiti in MEF LRRK2-/-. La prossimità tra i due organelli è pienamente recuperata dalla reintroduzione in MEF LRRK2-/- della proteina WT, ma non dei mutanti associati alle forme di Parkinson familiare. In conclusione, abbiamo sviluppato un nuovo metodo per determinare la prossimità tra ER e mitocondri e abbiamo utilizzato questa tecnologia per eseguire due repliche di uni screening gnomico su larga scala identificando nuovi componenti strutturali dei contatti tra mitocondri e ER.

Master’s degree in Bioinformatics and Applications to the Life Sciences at the University of Trás-os-Montes and Alto Douro
The use of next-generation sequencing (NGS) technologies has been revolutionizing the study of genetics. The big amount of generated data by these technologies allows the characterization of species genomes and genomic variation between species and within the same one. The present dissertation focused on the study of single nucleotide polymorphisms (SNPs) in organelle genomes of two well-known species of Quercus: Quercus suber and Quercus ilex rotundifolia, commonly named cork oak and holm oak respectively. Chloroplasts and mitochondria are organelles present in plant cells and play a crucial role in photosynthesis and energy metabolism, respectively, among other important physiological functions. Each of these organelles has its own genome, distinct from the nuclear genome. Within the Quercus genus, the chloroplast genome sequences have been determined for over 20 species, including cork oak which was assembled by the Genosuber consortium, and only the cork oak mitochondrial genome has been determined to date. Moreover, the amount of information on genomic variation is very scarce in chloroplast genomes, or non-existent in mitochondrial genomes. Therefore, there is a clear need to increase our knowledge in this field, given the importance of these species in the ecosystem and their socio-economic impact especially in the south region of the Iberic Peninsula. The pipeline of this study involves the use of high-throughput sequencing data of 47 individuals (39 cork oaks and 8 holm oaks) using NGS techniques and tools to perform quality control, preprocessing, read mapping, variant calling and annotation. Additionally, to achieve the best performance on preprocessing and variant calling the used tools were tested using different parameters on a smaller group of individuals. As it was expected given the higher conservation of chloroplast genomes, the presented results show a higher variation on mitochondrial genomes, especially when comparing cork oak with holm oak trees. These variations suggest a different capacity in both species and some studies have been reporting that holm oak is more resistant than cork oak and these variations may be the reason for that. With this in mind, it is possible to say that holm oak trees have greater ability to withstand climate change and therefore be a good model for selection of important molecular markers.
A utilização de tecnologias de next-generation sequencing (NGS) tem vindo a revolucionar os estudos genéticos. A grande quantidade de dados gerados por estas tecnologias permite a caracterização dos genomas das espécies e a variação genómica entre espécies e dentro da mesma. A presente dissertação focou-se no estudo de polimorfismos de nucleótidos únicos (SNPs) em genomas de organelos de duas espécies bem conhecidas de Quercus: Quercus suber e Quercus ilex rotundifolia, vulgarmente designadas por sobreiro e azinheira, respetivamente. Os cloroplastos e mitocôndrias são organelos presentes nas células vegetais e desempenham um papel crucial na fotossíntese e no metabolismo energético, respetivamente, entre outras importantes funções fisiológicas. Cada um destes organelos tem o seu próprio genoma, distinto do genoma nuclear e pouco se sabe sobre eles em espécies de Quercus. No entanto, as sequências do genoma do cloroplasto foram determinadas em mais de 20 espécies, incluindo o sobreiro cujo assembly foi feito pelo consórcio Genosuber. Por outro lado, apenas o genoma mitocondrial do sobreiro foi determinado até à data. Além disso, a quantidade de informação sobre a variação genómica é muito escassa nos genomas dos cloroplastos, ou inexistente nos genomas mitocondriais. Portanto, existe uma clara necessidade de aumentar os nossos conhecimentos neste campo, dada a importância destas espécies no ecossistema e o seu impacto socioeconómico, especialmente na região sul da Península Ibérica. A estrutura deste estudo envolve a utilização de dados de sequenciação de alto rendimento de 47 indivíduos (39 sobreiros e 8 azinheiras) utilizando técnicas e ferramentas de NGS para realizar o controlo de qualidade, pré-processamento, mapeamento, determinação de variantes e anotação. Para além disso, para alcançar o melhor desempenho no pré-processamento e na determinação de variantes, as ferramentas utilizadas foram testadas utilizando diferentes parâmetros num grupo mais pequeno de indivíduos. Como era de esperar dada a maior conservação dos genomas dos cloroplastos, os resultados apresentados mostram uma maior variação nos genomas mitocondriais, especialmente quando se compara o sobreiro com a azinheira. Estas variações sugerem uma capacidade diferente em ambas as espécies e alguns estudos têm relatado que a azinheira é mais resistente do que o sobreiro e estas variações podem ser a razão para isso. Com isto em mente, é possível dizer que as azinheiras têm maior capacidade de resistir às alterações climáticas e, portanto, ser um bom modelo para a seleção de marcadores moleculares importantes.

Genomic sequence data from the three domains of life have revealed a remarkable diversity of genome architectures. The relative contributions of adaptive versus non-adaptive processes in shaping this diversity are poorly understood and hotly debated. This thesis investigates the evolution of genome architecture in the Chloroplastida (i.e., green algae and land plants), with a particular focus on the mitochondrial and plastid genomes of chlamydomonadalean algae (Chlorophyceae, Chlorophyta). Much of the work presented here describes unprecedented extremes in: i) genome compactness (i.e., the fraction of noncoding DNA in a genome), ii) genome conformation (e.g., circular vs. linear vs. linear fragmented genomes), iii) intron and repeat content; and iv) nucleotide-composition landscape (e.g., GC-rich vs. AT-rich genomes). These data are then combined with intra-population nucleotide diversity data to explore the degree to which non-adaptive forces, such as random genetic drift and mutation rate, have shaped the organelle and nuclear genomes of the Chloroplastida. The major conclusions from this dissertation are that chlamydomonadalean algae show a much greater variation in organelle genome architecture than previously thought — this group boasts some of the most unusual mitochondrial and plastid genomes from all eukaryotes — and that the majority of this variation can be explained in non-adaptive terms.

Comparativement au génome contenu dans le noyau de la cellule de plante, nos connaissances des génomes des deux organelles de cette cellule, soit le plastide et la mitochondrie, sont encore très limitées. En effet, un nombre très restreint de facteurs impliqués dans la réplication et la réparation de l’ADN de ces compartiments ont été identifiés à ce jour. Au cours de notre étude, nous avons démontré l’implication de la famille de protéines Whirly dans le maintien de la stabilité des génomes des organelles. Des plantes mutantes pour des gènes Whirly chez Arabidopsis thaliana et Zea mays montrent en effet une augmentation du nombre de molécules d’ADN réarrangées dans les plastides. Ces nouvelles molécules sont le résultat d’une forme de recombinaison illégitime nommée microhomology-mediated break-induced replication qui, en temps normal, se produit rarement dans le plastide. Chez un mutant d’Arabidopsis ne possédant plus de protéines Whirly dans les plastides, ces molécules d’ADN peuvent même être amplifiées jusqu’à cinquante fois par rapport au niveau de l’ADN sauvage et causer un phénotype de variégation. L’étude des mutants des gènes Whirly a mené à la mise au point d’un test de sensibilité à un antibiotique, la ciprofloxacine, qui cause des bris double brin spécifiquement au niveau de l’ADN des organelles. Le mutant d’Arabidopsis ne contenant plus de protéines Whirly dans les plastides est plus sensible à ce stress que la plante sauvage. L’agent chimique induit en effet une augmentation du nombre de réarrangements dans le génome du plastide. Bien qu’un autre mutant ne possédant plus de protéines Whirly dans les mitochondries ne soit pas plus sensible à la ciprofloxacine, on retrouve néanmoins plus de réarrangements dans son ADN mitochondrial que dans celui de la plante sauvage. Ces résultats suggèrent donc une implication pour les protéines Whirly dans la réparation des bris double brin de l’ADN des organelles de plantes. Notre étude de la stabilité des génomes des organelles a ensuite conduit à la famille des protéines homologues des polymérases de l’ADN de type I bactérienne. Plusieurs groupes ont en effet suggéré que ces enzymes étaient responsables de la synthèse de l’ADN dans les plastides et les mitochondries. Nous avons apporté la preuve génétique de ce lien grâce à des mutants des deux gènes PolI d’Arabidopsis, qui encodent des protéines hautement similaires. La mutation simultanée des deux gènes est létale et les simples mutants possèdent moins d’ADN dans les organelles des plantes en bas âge, confirmant leur implication dans la réplication de l’ADN. De plus, les mutants du gène PolIB, mais non ceux de PolIA, sont hypersensibles à la ciprofloxacine, suggérant une fonction dans la réparation des bris de l’ADN. En accord avec ce résultat, la mutation combinée du gène PolIB et des gènes des protéines Whirly du plastide produit des plantes avec un phénotype très sévère. En définitive, l’identification de deux nouveaux facteurs impliqués dans le métabolisme de l’ADN des organelles nous permet de proposer un modèle simple pour le maintien de ces deux génomes.
Compared to the nuclear genome, very little is known about the genomes of the two plant cytoplasmic organelles, the plastid and the mitochondria. Indeed, very few factors involved in either the replication or the repair of these genomes have been identified. Here we show the implication of the Whirly protein family in the maintenance of organellar DNA. Indeed, mutations in Whirly genes lead to DNA rearrangements in both Arabidopsis thaliana and Zea mays plastids. These rearrangements are the product of microhomology-mediated break-induced replication that rarely occurs in wild-type plants but increases in absence of Whirly proteins. In a mutant plant devoid of plastidial Whirly proteins, these new DNA molecules can be amplified up to fifty times the normal DNA level and cause a variegated phenotype. In the course of the study of the Whirly mutant plants, we developed a strategy, based on the use of the antibiotic ciprofloxacin, to induce DNA double-strand breaks specifically in plant organelles. The Arabidopsis mutant plants without Whirly proteins in the plastids are more sensitive to the antibiotic ciprofloxacin than wild-type plants. Accordingly, there is a much larger increase in the number of rearranged DNA molecules in the plastids of the mutant plants than in the control plants. Surprisingly, while the mutant plants devoid of Whirly proteins in the mitochondria do not show increased sensitivity to the drug, they do accumulate more rearrangements in their mitochondrial DNA compared to wild-type plants. These results suggest that the Whirly proteins are involved in the repair of DNA double-strand breaks in the plant organelle genomes. Our study of the plant organelle genome stability has lead us to a family of proteins homologous to the DNA polymerase I in bacteria. This family has been proposed to be responsible for most of the DNA-synthesis activity in the plant organelles. We bring genetic proof to support this hypothesis using mutants of the two PolI genes of Arabidopsis. The combined mutation of both genes is lethal and the single mutations cause a decrease in the relative DNA levels in the organelles, thus confirming the involvement of both genes in DNA replication. Interestingly, mutants of the PolIB but not PolIA gene shows increase sensitivity to ciprofloxacin suggesting a function in DNA repair. In line with these results, a cross between a PolIB mutant and the mutant of plastid Whirly genes resulted in plants with severe growth defects and numerous rearrangements in the plastid DNA. In conclusion, we have identified two factors involved in the metabolism of organelle DNA and proposed a simple model of how these genomes are maintained in the plant cell.

Le maintien de la stabilité du génome est essentiel pour la propagation de l’information génétique et pour la croissance et la survie des cellules. Tous les organismes possèdent des systèmes de prévention des dommages et des réarrangements de l’ADN et nos connaissances sur ces processus découlent principalement de l’étude des génomes bactériens et nucléaires. Comparativement peu de choses sont connues sur les systèmes de protection des génomes d’organelles. Cette étude révèle l’importance des protéines liant l’ADN simple-brin de la famille Whirly dans le maintien de la stabilité du génome des organelles de plantes. Nous rapportons que les Whirlies sont requis pour la stabilité du génome plastidique chez Arabidopsis thaliana et Zea mays. L’absence des Whirlies plastidiques favorise une accumulation de molécules rearrangées produites par recombinaison non-homologue médiée par des régions de microhomologie. Ce mécanisme est similaire au “microhomology-mediated break-induced replication” (MMBIR) retrouvé chez les bactéries, la levure et l’humain. Nous montrons également que les organelles de plantes peuvent réparer les bris double-brin en utilisant une voie semblable au MMBIR. La délétion de différents membres de la famille Whirly entraîne une accumulation importante de réarrangements dans le génome des organelles suite à l’induction de bris double-brin. Ces résultats indiquent que les Whirlies sont aussi importants pour la réparation fidèle des génomes d’organelles. En se basant sur des données biologiques et structurales, nous proposons un modèle où les Whirlies modulent la disponibilité de l’ADN simple-brin, régulant ainsi le choix des voies de réparation et permettant le maintien de la stabilité du génome des organelles. Les divers aspects de ce modèle seront testés au cours d’expériences futures ce qui mènera à une meilleure compréhension du maintien de la stabilité du génome des organelles.
Maintenance of genome stability is essential for the accurate propagation of genetic information and for cell growth and survival. Organisms have therefore developed efficient strategies to prevent DNA lesions and rearrangements. Much of the information concerning these strategies has been obtained through the study of bacterial and nuclear genomes. Comparatively little is known about how organelle genomes maintain a stable structure. This study implicates the single-stranded nucleic acid-binding proteins of the Whirly family in the maintenance of plant organelle genome stability. Here we report that the plastid-localized single-stranded DNA binding proteins of the Whirly family are required for plastid genome stability in Arabidopsis thaliana and Zea mays. Absence of plastidial Whirlies favors the accumulation of rearranged molecules that arise through a non-homologous recombination mechanism mediated by regions of microhomology. This mechanism is similar to the microhomology-mediated break-induced replication (MMBIR) described in bacteria, yeast and humans. Additionally we show that plant organelles can repair double-strand breaks using a MMBIR-like pathway. Plants lacking Whirly proteins accumulate elevated levels of microhomology-mediated DNA rearrangements upon double-strand break induction, indicating that Whirlies also contribute to the accurate repair of plant organelle genomes. Using biological and structural data, we propose a working model in which Whirlies modulate the access of repair proteins and complementary DNA to single-stranded regions, thereby regulating the choice of repair pathways and maintaining plant organelle genome stability. The various aspects of this model will be tested in future experiments which should allow a better understanding of the mechanisms underlying genome stability in plant organelles.

Les plantes doivent assurer la protection de trois génomes localisés dans le noyau, les chloroplastes et les mitochondries. Si les mécanismes assurant la réparation de l’ADN nucléaire sont relativement bien compris, il n’en va pas de même pour celui des chloroplastes et des mitochondries. Or il est important de bien comprendre ces mécanismes puisque des dommages à l’ADN non ou mal réparés peuvent entraîner des réarrangements dans les génomes. Chez les plantes, de tels réarrangements dans l’ADN mitochondrial ou dans l’ADN chloroplastique peuvent conduire à une perte de vigueur ou à un ralentissement de la croissance. Récemment, notre laboratoire a identifié une famille de protéines, les Whirly, dont les membres se localisent au niveau des mitochondries et des chloroplastes. Ces protéines forment des tétramères qui lient l’ADN monocaténaire et qui accomplissent de nombreuses fonctions associées au métabolisme de l’ADN. Chez Arabidopsis, deux de ces protéines ont été associées au maintien de la stabilité du génome du chloroplaste. On ignore cependant si ces protéines sont impliquées dans la réparation de l’ADN. Notre étude chez Arabidopsis démontre que des cassures bicaténaires de l’ADN sont prises en charge dans les mitochondries et les chloroplastes par une voie de réparation dépendant de très courtes séquences répétées (de cinq à cinquante paires de bases) d’ADN. Nous avons également montré que les protéines Whirly modulent cette voie de réparation. Plus précisément, leur rôle serait de promouvoir une réparation fidèle de l’ADN en empêchant la formation de réarrangements dans les génomes de ces organites. Pour comprendre comment les protéines Whirly sont impliquées dans ce processus, nous avons élucidé la structure cristalline d’un complexe Whirly-ADN. Nous avons ainsi pu montrer que les Whirly lient et protègent l’ADN monocaténaire sans spécificité de séquence. La liaison de l’ADN s’effectue entre les feuillets β de sous-unités contiguës du tétramère. Cette configuration maintient l’ADN sous une forme monocaténaire et empêche son appariement avec des acides nucléiques de séquence complémentaire. Ainsi, les protéines Whirly peuvent empêcher la formation de réarrangements et favoriser une réparation fidèle de l’ADN. Nous avons également montré que, lors de la liaison de très longues séquences d’ADN, les protéines Whirly peuvent s’agencer en superstructures d’hexamères de tétramères, formant ainsi des particules sphériques de douze nanomètres de diamètre. En particulier, nous avons pu démontrer l’importance d’un résidu lysine conservé chez les Whirly de plantes dans le maintien de la stabilité de ces superstructures, dans la liaison coopérative de l’ADN, ainsi que dans la réparation de l’ADN chez Arabidopsis. Globalement, notre étude amène de nouvelles connaissances quant aux mécanismes de réparation de l’ADN dans les organites de plantes ainsi que le rôle des protéines Whirly dans ce processus.
Plants must protect the integrity of three genomes located respectively in the nucleus, the chloroplasts and the mitochondria. Although DNA repair mechanisms in the nucleus are the subject of multiple studies, little attention has been paid to DNA repair mechanisms in chloroplasts and mitochondria. This is unfortunate since mutations in the chloroplast or the mitochondrial genome can lead to altered plant growth and development. Our laboratory has identified a new family of proteins, the Whirlies, whose members are located in plant mitochondria and chloroplasts. These proteins form tetramers that bind single-stranded DNA and play various roles associated with DNA metabolism. In Arabidopsis, two Whirly proteins maintain chloroplast genome stability. Whether or not these proteins are involved in DNA repair has so far not been investigated. Our studies in Arabidopsis demonstrate that DNA double-strand breaks are repaired in both mitochondria and chloroplasts through a microhomology-mediated repair pathway and indicate that Whirly proteins affect this pathway. In particular, the role of Whirly proteins would be to promote accurate repair of organelle DNA by preventing the repair of DNA double-strand breaks by the microhomology-dependant pathway. To understand how Whirly proteins mediate this function, we solved the crystal structure of Whirly-DNA complexes. These structures show that Whirly proteins bind single-stranded DNA with low sequence specificity. The DNA is maintained in an extended conformation between the β-sheets of adjacent protomers, thus preventing spurious annealing with a complementary strand. In turn, this prevents formation of DNA rearrangements and favors accurate DNA repair. We also show that upon binding long ssDNA sequences, Whirly proteins assemble into higher order structures, or hexamers of tetramers, thus forming spherical particles of twelve nanometers in diameter. We also demonstrate that a lysine residue conserved among plant Whirly proteins is important for the stability of these higher order structures as well as for cooperative binding to DNA and for DNA repair. Overall, our study elucidates some of the mechanisms of DNA repair in plant organelles as well as the roles of Whirly proteins in this process.