Sturgeons (Chondrostei: Acipenseriformes) display markedly disjunct distributions with a wide occurrence in the northern hemisphere. Their unique benthic specializations, conserved morphology, evolutionary age, the variation in their basic diadromous life history, and the large public interest due to their near extinction or critically endangered status make sturgeons and paddlefishes one of the most interesting group of vertebrates. In addition to that, ploidy diversity of Acipenseriformes possessing three ploidy groups having ~ 120 chromosomes, ~ 240 ? 270 chromosomes and ~ 360 chromosomes provides unique model for investigation of evolutionary processes which were going through the genome duplication events. Sturgeons are also notoriously known for their strong propensity to interspecific and intergeneric hybridization which can result in hybrids with various ploidy levels. All these facts make sturgeon genetics and cytogenetics a thriving but also complicated area for research. In the present work, the role of genome duplication and functional reduction evens in evolution of sturgeon species as well as sturgeons? ploidy levels and ploidy relationships among Acipenseriformes were investigated using molecular markers. In addition to that, clarification of origin of abnormal ploidy levels and observation of segregation pattern of microsatellite alleles in the course of hybridization of polyploid sturgeon species were included into this study. With regard to the all considerations and observations provided by this study we concluded that evolution of sturgeon species is still widely dynamic and ongoing process which might goes through the allopolyplodization as well as autopolyplidization events.

Eight of fifteen species in genus Anthoxanthum (Poaceae) can be found in Europe. Five of them are perennials forming A. odoratum complex, the remaining three are annual, more or less mediterranean taxa (A. aristatum, A. ovatum and A. gracile). Within the A. odoratum s. l. complex the following taxa are distinguished: widely spread A. odoratum s. str. (4x; 2n = 20), arcto-alpine A. alpinum (2x a 4x; 2n = 10 and 20), Madeiran endemic species A. maderense (2x; 2n = 10), endemic species of Balkan mountains A. pauciflorum (2x; 2n = 10) and the Iberian peninsula endemic A. amarum (?x; 2n ~ 90). The aim of this thesis is to clearify the unknown evolutionary relationships between the taxa, between the annuals and perennials, diploids and polyploids. The following questions should be answered in this study: 1) What is the origin and distribution of the rediscovered diploid perennial taxon and what is its relationship to the other members of the group; 2) What is the distribution pattern of the perennial taxa of the genus Anthoxanthum in Europe and what is their haplotype differenciation (overall distribution of the taxa and haplotypes and the existence of their sympatric occurence); 3) What evolutionary ties exist among the species and what is the origin of allotetraploid taxon A. odoratum s.str. The...

A vast range of factors shape the genetic structure of plant populations. In this thesis, I focus on two of them. The first factor, polyploidization, is a process of chromosome set multiplication through whole-genome duplication within a single species (autopolyploids) or hybridization of two different species (allopolyploids). It rapidly brings changes into genomes, allowing species to occupy distinct niches, adapt to new habitats, colonize them, or adapt to changing environment in their native range. But it comes at a certain cost - difficulties in mitosis and meiosis, changes in cellular architecture. Furthermore, after cytotype establishment, new individuals have to deal with cytotype exclusion effect, competition with parental individuals and higher nutrition requirements. In this thesis, I present the effect of polyploidization on populations of three species- Arabidopsis arenosa, Aster amellus and Festuca rubra. The second factor changing the population genetic structure presented in this thesis is the fragmentation of populations. Population fragmentation can be caused by natural or anthropogenic activities and often leads to overall reduction in population size and reduced connectivity among fragments. Restricted gene flow may threaten long-term population survival due to inbreeding...

Made available in DSpace on 2016-12-23T14:37:31Z (GMT). No. of bitstreams: 1 Stefanie Cristina Oliveira.pdf: 1100238 bytes, checksum: e4200efbe53a66bdaa4fc82fbfac51ed (MD5) Previous issue date: 2012-07-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O uso de bioenergia tem sido apontado como uma estratégia para diminuição da utilização de combustíveis fósseis. Fundamentado nesse fato, muitos países, incluindo o Brasil, aumentaram os esforços para financiar pesquisas com espécies vegetais produtoras de biocombustíveis. Dentre essas espécies, Jatropha curcas L. destaca-se pelo alto potencial para produção de óleo. Visando à propagação clonal e massal de acessos elite de J. curcas, técnicas de cultura de tecidos têm sido aplicadas. Entretanto, diferentes autores frisaram a necessidade de ampliação de estudos envolvendo ferramentas da cultura de tecidos vegetais, dentre elas a poliploidização in vitro. A indução de poliploides in vitro é uma importante ferramenta no melhoramento genético de plantas, por possibilitar a obtenção de maior variabilidade de materiais com possíveis incrementos de características agronômicas desejáveis. Nesse sentindo, um dos objetivos deste trabalho foi estabelecer um protocolo de indução in vitro de plântulas poliploides da variedade / Gonçalo/ (2C = 0.85 pg, 2n = 2x = 22 cromossomos). Os resultados de citometria de fluxo evidenciaram que tetraploides e mixoploides foram obtidos a partir de ápices caulinares tratados com diferentes concentrações de colchicina e tempos de exposição. Fundamentado em três parâmetros (número de plântulas sobreviventes, tetraploides e mixoploides), o tratamento envolvendo 0,5 mM de colchicina a um pulso de 96 horas foi considerado o mais adequado. Com base nesse resultado, um segundo experimento de poliploidização foi realizado somente com a dosagem de 0,5 mM com pulsos de 96, 120, 144 e 168 horas. Os resultados confirmaram que a concentração de 0,5 mM por 96 horas é ideal para indução de tetraploides de J. curcas / Gonçalo/ . Plântulas mixoploides também foram encontradas nos tratamentos com0,5 mM de colchicina aos pulsos de 96, 120 e 144 horas. Visto a relevância da propagação clonal e massal de acessos elite de J. curcas, inclusive das plântulas poliploides obtidas no primeiro estudo, o presente trabalho também teve como objetivo induzir a calogênese in vitro a partir de explantes foliares. Os resultados mostraram que a combinação ácido 2,4-diclorofenoxiacético (2,4-D)/cinetina (KN) promoveu maior número de explantes com calos friáveis quando comparado à combinação ácido indol-3-butírico (AIB)/KN. Dessa forma, o procedimento envolvendo a combinação auxina/citocinina é fundamental para gerar calos friáveis, os quais representam a base para estabelecimento de um sistema embriogênico in vitro de acessos elite. Pela primeira vez, um sistema de cultura de tecidos foi adaptado para indução, propagação e recuperação de tetraploides e mixoploides de J. curcas / Gonçalo/ . Além disso, os dados gerados contribuem para criação e ampliação de bancos de germoplasma in vitro da espécie / The use of bioenergy has been suggested as a strategy for reducing the utilization of fossil fuels. Thereafter, many countries, including Brazil, have increased efforts to fund the research of plant species for biofuel production. Among these species, Jatropha curcas L. stands out mainly due to its high potential for oil production. Thus, tissue culture techniques have been applied aiming at the mass and clonal propagation of J. curcas elite lines. However, different authors have emphasized the necessity of expanding studies that involve plant tissue culture tools such as in vitro polyploidization. In this sense, the present study sought to establish a protocol for in vitro induction of polyploid seedlings from shoot tips of the variety Gonzalo (2C = 0.85 pg, 2n = 2x = 22 chromosomes). Flow cytometry revealed mixoploids and tetraploids obtained from shoot tips treated with different colchicine concentrations and exposure times. Based on three criteria (survival rate of the explants, and numbers of tetraploid and mixoploid plantlets), the treatment with 0.5 mM colchicine in a 96 hour pulse was statistically considered the most appropriate. Based on these data, a second polyploidization experiment was carried out, using a dosage of 0.5 mM colchicine in pulses of 96, 120, 144 and 168 hours. The results confirmed that the concentration of 0.5 mM for 96 hours was the ideal treatment for J. curcas Gonçalo tetraploid induction. Mixoploid seedlings were also detected in the treatments with 0.5 mM colchicine in pulses of 96, 120 and 144 hours. Considering the relevance of mass and clonal multiplication of elite J. curcas accessions, and the polyploid seedlings obtained in the first investigation, this study further aimed to establish in vitro callogenesis from leaf explants. The results showed that the combination of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (Kn) provided greater number of explants with friable calli in relation to the combination indol-3-butyric acid (IBA) and Kn. Thus, 2,4-D/Kn was the treatment of choice to generate friable calli, which are essential for somatic embryogenesis. Ultimately, a tissue culture system was adapted for induction, propagation and recovery of in vitro tetraploids from the J. curcas variety Gonçalo, as well as other inbred lines. Besides providing a new protocol for polyploidy generation, this study also contributed to the germplasm of J. curcas and the achievement of genetic variability through obtainment of tetraploid and mixoploid seedlings

This thesis aimed on comparison of environmental requirements and morphological and biological traits of diploid and triploid cytotypes of wetland plant flowering rush (Butomus umbellatus), occurring in Eastern Slovak Lowland in unique mixed populations, and to develop primers for variable microsatellite loci suitable for further genetic studies. In total, 72 populations of Butomus umbellatus were found. The analysis of relative genome size by the flow cytometry (FCM) technique revealed 34 exclusively triploid cytotype populations, 8 exclusively diploid populations and 30 mixed populations of Butomus umbellatus. In mixed populations, the numbers of diploid and triploid individuals are quite equivalent and the plants create combined compact communities. Analyses of environmental characteristics of sites with specific cytotypes (PCA, RDA, ANOVA) revealed no difference in site requirements of diploid and triploid Butomus umbellatus plants. Morphometric analyses (PCA, CDA) of traits of 36 diploid and 54 triploid plants confirmed differing morphology of both cytotypes occurring in the nature. Subsequent replanting of these plants into pots and cultivation further increased these differences. The ratio of width and length of internal perianthium is the most reliable parameter to distinguish diploid and...

Contact zones of cytotypes harbour a unique opportunity to study the dynamics and evolution of mixed-ploidy systems. Butomus umbellatus (flowering rush) is one of a few species in which diploid and triploid cytotype is present in nature. The first known mixed-ploidy populations of B. umbellatus were discovered in south-eastern Slovakia during the previous studies. Common occurrence of the species in an area with substantially natural dynamics of wetland ecosystems represents a unique model system for unbiased comparisons of traits and genetic diversity between diploid and triploid individuals along with their dispersal capabilities on a landscape level. The main aim of of this thesis is to uncover the truth behind evolutionary processes that are taking place in the contact zone of diploid and triplioid cytotype of B. umbellatus in south-eastern Slovakia. Using flow cytometry and molecular methods (microsatellites) we will test whether this is a primary or secondary contact zone and assess the level of genetic variability between individuals and populations of both cytotypes. Using the analysis od microsatellite loci we discovered that the contact zone of cytotype is of both primary and secondary character and gene flow between cytotypes was also detected. These results were supported by high production and...

This thesis presents a novel approach to reconstructing ancestral genomes of a number of descendant species related by a phylogeny. Traditional methods face challenges due to cycles of whole genome doubling followed by fractionation in plant lineages. In response, the thesis proposes a new approach that first accumulates a large number of candidate gene adjacencies specific to each ancestor in a phylogeny. A subset of these which to produces long ancestral contigs are chosen through maximum weight matching. The strategy results in more complete reconstructions than existing methods, and a number of quality measures are deployed to assess the results. The thesis also presents a new computational technique for estimating the ancestral monoploid number of chromosomes, involving a "g-mer" analysis to resolve a bias due to long contigs and gap statistics to estimate the number. The method is applied to a set of phylogenetically related descendant species, and the monoploid number is found to be 9 for all rosid and asterid orders. Additionally, the thesis demonstrates that this result is not an artifact of the method, by deriving a monoploid number of approximately 20 for the metazoan ancestor. The reconstructed ancestral genomes are functionally annotated and visualized through painting ancestral projections on descendant genomes and highlighting syntenic ancestor-descendant relationships. The proposed method is applied to genomes drawn from a broad range of plant orders. The Raccroche pipeline reconstructs ancestral gene orders and chromosomal contents of the ancestral genomes at all internal vertices of a phylogenetic tree, and constructs chromosomes by counting the frequencies of ancestral contig co-occurrence on the extant genomes, clustering these for each ancestor, and ordering them. Overall, this thesis presents a significant contribution to the field of ancestral genome reconstruction, offering a new approach that produces more complete reconstructions and provides valuable insights into the evolutionary process giving rise to the gene content and order of extant genomes.

The presented study tries to reveal the evolutionary history of polyploid complex Galium pusillum aiming on a rare czech endemic species G. sudeticum, an ideal model to study allopatric speciation in the context of Central European (post)glacial development. The species G. sudeticum grows in Krkonose Mts. and on serpentines in western Bohemia (Slavkovský les) more than 200 kilometers away. Our morphological and molecular data suggest a clearly different history of its two isolated areas. The populations in Krkonose Mts. are probable relics of mountainous species G. anisophyllon which was there on its northern border of occurence and hybridized with lowland species G. valdepilosum during postglacial vegetations shifts. This fact is well supported by intermediate position of Krkonose Mts. populations in both morphological and molecular AFLP analyses and by sharing the same chloroplast haplotype with geographically close lowland populations. On the other hand the serpentine lowland populations in western Bohemia are both morphologicaly and genetically indistinguishable from G. valdepilosum. Distinct genetic lineage of few populations belonging to G. valdepilosum (incl. one traditionaly referred as G. sudeticum) was found in western Bohemia and Bavaria, located on relic stands (calcareous and serpentine...

Dans l’histoire évolutive des végétaux, la polyploïdisation a été un phénomène récurrent qui a façonné les génomes, aurait contribué à l’avènement de grandes étapes évolutives et aurait favorisé la survie de nombreuses lignées lors de crises écologiques majeures. Le principal mécanisme d’apparition d’espèces polyploïdes est la polyploïdisation sexuelle, qui implique la formation de gamètes 2n résultant de modifications de la division méiotique. Récemment plusieurs mutants produisant des taux élevés de gamètes 2n ont été identifiés chez A. thaliana. Chez cette espèce, la perte de fonction du gène AtPS1 conduit à la mise en place de fuseaux parallèles en méiose II et celle du gène AtCYCA1;2/TAM à l’omission de la seconde division méiotique. L’objectif de cette thèse a été de déterminer des facteurs et mécanismes responsables de la formation de gamètes 2n, en utilisant le rosier comme modèle végétal. Ces travaux ont permis : (i) d’identifier un facteur abiotique, la température élevée, comme inducteur de production de forts taux de gamètes 2n, (ii) de montrer que la fenêtre de sensibilité à ce facteur est restreinte à la méiose et (iii) de révéler que ces gamètes 2n produits sont principalement issus de la mise en place de fuseaux parallèles en méiose II. Afin de déterminer les mécanismes moléculaires à l’origine de leur formation, deux gènes candidats, RhPS1 et RhCYCA1 ont été identifiés chez Rosa. L’analyse de leur expression a révélé : (i) en condition non inductible, leur forte expression dans les étamines au stade méiose et (ii) la répression rapide de leurs niveaux de transcrits en condition d’induction de gamètes 2n. La fonction méiotique du gène RhPS1 a été validée par complémentation du mutant atps1-1 d’A. thaliana et par l’obtention d’une lignée rosier transgénique p35S::ARNi-RhPS1. Compte tenu de ces résultats, l’étude de la polyploïdisation et de ses mécanismes peut désormais être replacée dans le contexte actuel de changement climatique. / In the evolutionary history of plants, polyploidization has been a recurring phenomenon that has shaped the genomes, might have contributed to the occurrence of major evolutionary step and might have facilitated the survival of many plant families during major ecological crises. The main mechanism of polyploidization is sexual polyploidization, which involves the formation of 2n gametes resulting from meiotic division changes. Recently, mutants highly producing 2n gametes have been isolated in A. thaliana. Loss of AtPS1 gene function leads to parallel spindles orientation in meiosis II and loss of AtCYCA1;2/TAM gene function leads to the omission of the second meiotic division. The aim of this PhD project was to identify factors and mechanisms responsible for the 2n gametes formation, using Rosa as a model. This work permitted to: (i) discover an abiotic factor, high temperature, that can induce a high production of 2n gametes, (ii) show that the sensitivity window to this factor is narrow and restricted to meiosis and (iii) reveal that 2n gamete production in inductive condition, results from parallel spindle orientation in meiosis II. To determine molecular mechanisms responsible for their formation, two candidate genes, RhPS1 and RhCYCA1 were identified in Rosa. Analysis of their expression revealed: (i) their high expression level in stamens at meiosis stage in non-inductive condition and (ii) the rapid repression of their transcript levels under inductive condition. Meiotic gene function of RhPS1 was validated by complementation of atps1-1 mutant and by generating a rose transgenic line p35S:: RNAi-RhPS1. According to these results, polyploidization and its mechanisms can now be replaced in the context of the current climate.

Les angiospermes (ou plantes à fleurs) regroupent environ 350 000 espèces ayant divergé il y a 150 à 200 millions d’années en deux familles botaniques principales, les monocotylédones (les orchidées, les palmiers, les bananiers, les joncs, les graminées...) et les eudicotylédones (les Brassicaceae, les Rosaceae, les légumineuses...) représentant respectivement 20% et 75% des plantes à fleurs. Les angiospermes font l’objet de nombreux travaux de recherche, en particulier en génomique depuis 2000 avec le séquençage du premier génome de plantes (Arabidopsis thaliana) qui a précédé le décryptage des génomes d’un nombre important d’autres espèces modèles et/ou d’intérêt agronomique (environ 100 aujourd’hui). L’accès croissant à la séquence des génomes de plantes a permis de mettre à jour une importante diversité structurale de leur génome, en termes de taille physique, de nombre de chromosomes, de nombre de gènes et de richesse en éléments transposables. Les forces évolutives ayant permis une telle diversité structurale des génomes au cours de l’évolution sont au cœur des travaux de cette thèse. La paléogénomique se propose d’étudier à travers la reconstruction de génomes ancestraux, comment ces espèces ont divergé à partir d’ancêtres communs et quels mécanismes ont contribué à une telle plasticité de structure génomique. Dans cet objectif, les travaux de cette thèse ont mis en œuvre des méthodes basées sur la génomique comparée permettant l’étude de l’évolution structurale des génomes via la reconstruction des génomes ancestraux fondateurs des espèces modernes. Ainsi, un génome ancestral des angiospermes a été reconstruit constitué de 5 chromosomes et porteur de 6707 gènes ordonnés sur ceux-ci, permettant d’intégrer dans un même modèle les monocotylédones et les eudicotylédones et élucider leur histoire évolutive, notamment pour les espèces d’intérêt agronomique majeur telles que les céréales, les rosids et les Brassicaceae. L’inférence de ces génomes ancestraux des plantes modernes a permis l’identification et l’étude de l’impact des évènements de polyploïdie (doublement génomique), ubiquitaires chez les plantes. Nous avons montré que les génomes tendent à revenir à une structure diploïde suite à un évènement de polyploïdie. Cette diploïdisation structurale se fait au niveau caryotypique (par le biais de réarrangements chromosomiques impliquant la perte des centromères et télomères ancestraux) mais aussi géniques (par le biais de pertes de gènes ancestraux en double copies). Il a été montré que cette perte se faisait préférentiellement sur un des sous-génomes post-polyploïdie, menant au phénomène de « dominance des sous-génomes ». Ces biais de plasticité structurale (on parle de compartimentation de la plasticité) se font différentiellement entre les espèces, les chromosomes, les compartiments chromosomiques mais aussi les types de gènes, aboutissant à la diversité structurale observée entre les génomes modernes de plantes. Ces travaux qui rentrent dans le cadre de la recherche fondamentale ont également un fort aspect appliqué à travers la recherche translationnelle en ayant permis de créer des passerelles entre les différentes espèces travaillées en agriculture. Le passage d’une espèce à une autre via les génomes ancestraux fondateurs reconstruits permet notamment le transfert de connaissances des gènes ou de régions d’intérêt des espèces modèles aux espèces cultivées. Les travaux de thèse, par la reconstruction d’ancêtres, permettent une comparaison de haute-résolution des génomes de plantes et in fine l’étude de leur plasticité acquise au cours de l’évolution, et revêtent donc à la fois un aspect fondamental (pour comprendre l’évolution des espèces) mais aussi appliqué (pour l’amélioration des espèces d’intérêt agronomique à partir des modèles). / Angiosperms (or flowering plants) consist in approximatively 350 000 species that have diverged 150 to 200 million years ago in two main families, monocots (orchids, palm trees, banana, bulrushes, grasses...) and dicots (Brassicaceae, Rosaceae, legumes...) representing respectively 20% and 75% of flowering plants. Angiosperms are the subject of intense researches, in particular in genomics since 2000 with the sequence release of the first plant genome (Arabidopsis thaliana) preceding a large number of genomes of plant models and/or species of agronomical interest (around 100 today). Increasing access to plant genome sequences has allowed the identification of their structural diversity, in terms of genome size, number of chromosomes and genes as well as transposable element content. The evolutionary forces that have shaped such structural genomic divergence are at the center of this thesis. Our paleogenomics approach will investigate, through ancestral genome reconstructions, how modern species have diverged from common ancestors and which mechanisms have contributed to such present-day genome plasticity. In this thesis, we have developed methods based on comparative genomics to study plant genome evolution and reconstruct ancestral genomes, extinct progenitors of the modern angiosperm species. An ancestral angiosperm genome has been reconstructed made of 5 chromosomes and 6707 ordered genes allowing the integration in the same model of monocots and eudicots and finally elucidating evolutionary trajectories for species of major agricultural interest such as cereals, rosids and Brassicaceae. The reconstructed paleohistory of modern flowering plants enabled the identification as well as the investigation of the impact of polyploidy events (WGD, whole genome duplications), ubiquitous in plants, as a major driver of the observed structural plasticity of angiosperms. We established that genomes tend to return to a diploid status following a polyploidy event. This structural diploidization is performed at the karyotypic level through chromosomal rearrangements (involving ancestral centromeres and telomeres losses) as well as the gene level (through ancestral duplicates loss). It has been shown that this diploidization is preferentially done on one of the post-polyploidy subgenome, leading to the "sub-genome dominance" phenomenon. This structural plasticity bias (also referenced as plasticity partitioning) is acting differentially between species, chromosomes, chromosomal compartments, gene types, resulting in the structural diversity observed between the present-day plant genomes. This thesis is clearly within the scope of fundamental researches but also has a strong applied objective through translational research in creating bridges between species of major relevance for agriculture. The comparison of one species to another through the reconstructed ancestral genomes allows transferring knowledge gained on genes or any region of interest from model species to crops. Paleogenomics, in reconstructing ancestral genome and unveiling the forces driving modern plant genome plasticity, is therefore of fundamental (toward understanding species evolution) but also applied (toward improving orphan species from knowledge gained in models) objectives.