Orientadores: Anete Pereira de Souza, Antonio Augusto Franco Garcia / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-19T06:27:16Z (GMT). No. of bitstreams: 1 Marconi_ThiagoGibbin_D.pdf: 16468359 bytes, checksum: 01e191ac164eff1d269733328f5be31e (MD5) Previous issue date: 2011 / Resumo: A utilização dos marcadores moleculares em estudos de mapeamento genético e de QTLs (Quantitative Trait Loci) tem proporcionado um importante progresso no conhecimento da genética e da estrutura genômica da cana-de-açúcar. O projeto de sequenciamento de ESTs (Expressed Sequence Tags) do programa Genoma da FAPESP (SUCEST) identificou aproximadamente 43 mil clusters que representam os genes de cana-de-açúcar. Sabe-se que os ESTs apresentam grande potencial para serem utilizados no desenvolvimento de marcadores genético-moleculares. Tendo em vista os avanços possíveis no melhoramento genético da cana-de-açúcar com a construção de um mapa genético funcional a partir de ESTs de interesse, este trabalho teve como objetivos o mapeamento genético em uma população F1 de cana-de-açúcar utilizando marcadores moleculares do tipo EST-SSRs (Expressed Sequence Tags - Simple Sequence Repeats) e SNP (Single Nucleotide Polymorphism), desenvolvidos a partir de seqüências ESTs homólogas a genes de interesse. Os SNPs desenvolvidos e mapeados demonstraram novos tipos de segregações possíveis de serem incorporadas ao mapeamento genético em cana-de-açúcar, representando avanços para a análise genética de poliplóides e possibilitando a saturação do mapa genético com marcadores completamente informativos. Os marcadores moleculares EST-SSRs e SNPs desenvolvidos e integrados ao mapa genético da cana-de-açúcar aumentaram sua resolução e também as possibilidades de mapeamento dos QTLs com maior precisão / Abstract: The use of molecular markers in genetic mapping studies and QTL (Quantitative Trait Loci) has provided an important advance in knowledge of genetics and genomic structure of sugarcane. The sequencing project of ESTs (Expressed Sequence Tags) form FAPESP's Genome Program (SUCEST) identified approximately 43 000 clusters representing the sugarcane genes. It is known that the ESTs have great potential for use in the development of genetic molecular markers. Given the possible advances in genetic breeding of sugarcane with the construction of a functional genetic map from ESTs of interest, the aim of this study was the construction of a genetic map in a F1 population of sugarcane using molecular markers EST-SSR (Expressed Sequence Tags - Simple Sequence Repeats) and SNP (Single Nucleotide Polymorphism) derived from ESTs sequences homologous to genes of interest. The developed and mapped SNPs demonstrated new types of segregation ratio that could be incorporated in the genetic mapping of sugarcane, representing advances for the genetic analysis of polyploid and allowing the saturation of the genetic map with fully informative markers. The EST-SSR markers and SNPs developed and integrated into the genetic map of sugarcane increased the resolution, coverage of the genome and also the possibilities of mapping QTLs with greater precision / Doutorado / Genetica Vegetal e Melhoramento / Doutor em Genetica e Biologia Molecular

Genome duplication plays a significant role in plant evolution. Formation of new polyploids is generally considered to be rare. Nevertheless, under natural conditions mixed-ploidy populations occur in relatively large numbers. Only the observations in the cytotype contact zone can identify all the factors affecting the stability or instability of the population. Number of research focusing on study of cytotype coexistence in natural mixed-ploidy populations is still low. As a model system for the study of mechanisms governing cytotype coexistence was chosen the Tripleurospermum inodorum. Research focused on natural mixed-ploidy populations and also on planted mixed-ploidy populations. Permanent plots were located in south, west, northwest and north Bohemia. Field observations were supplemented by cultivation experiments carried out in the greenhouse. Several phenomena were discovered at the level of whole populations. Cytotype distribution in the plot was random. The spatial structure of natural populations was quickly changing even within a single season. Even between single plot evaluations the cytotype ratios varied. Three percent of all plants were triploid hybrids. Most often detected cytotype in soil seed bank was diploid cytotype. The study of population dynamics shows, that tetraploids are...

La recombinaison méiotique via les Crossing-Overs (COs) est le principal mécanisme permettant le brassage de la diversité génétique. Cependant, le nombre et la position des COs entre paires de chromosomes homologues sont strictement régulés, limitant la séparation des loci en sélection variétale. Dans le cas du colza B. napus, l’utilisation d’allotriploïdes (AAC, 2n=3x=29), issus du croisement entre le colza (AACC, 2n=4x=38) et l’un de ses progéniteurs B. rapa (AA, 2n=2x=20), permet d’augmenter considérablement le nombre de COs entre chromosomes homologues A. L’objectif de cette étude était de déterminer les conséquences d’une telle variation sur la distribution des COs le long des chromosomes ainsi que d’identifier des facteurs régulant ce phénomène. Suite à la production et à la caractérisation cytogénétiques d’hybrides F1 présentant différents caryotypes, la recombinaison homologue a été évaluée par des analyses génétiques via des marqueurs SNPs physiquement ancrés sur l’ensemble duNous avons montré que l’addition du génome C chez les allotriploïdes conduit toujours à (1) la formation de COs surnuméraires, dont le nombre varie fonction des méioses mâle/femelle et du fond génétique, (2) une modification des profils de recombinaison, notamment au voisinage des centromères, et (3) une réduction de l’intensité d’interférence. De plus, nous avons révélé que le contrôle génétique de ces variations est imputé à des chromosomes C spécifiques et aurait divergé dans un contexte polyploïde. Nous avons donc identifié un levier permettant d’optimiser le brassage de la diversité gén / Meiotic recombination via crossovers (COs) is the main mechanism responsible for mixing genetic diversity. However, the number and position of COs between the pairs of homologous chromosomes are strictly regulated, limiting the loci separation in plant breeding. In the case of the rapeseed B. napus, the use of allotriploids (AAC, 2n=3x=29), resulting from the cross between rapeseed (AACC, 2n=4x=38) and one of its progenitors B. rapa (AA, 2n=2x=20), allows a substantial increase of the number of COs between homologous A chromosomes. The objective of this study was to determine the consequences of such a variation on the distribution of COs along the chromosomes and to identify factors regulating this phenomenon. Following the production and cytogenetic characterization of F1 hybrids with different karyotypes, homologous recombination was assessed by genetic analyzes via SNPs markers physically anchored on the whole A genome.We showed that the additional C genome in allotriploids always leads to (1) the formation of extra COs, for which the number depends on the male/female meiosis and the genetic background, (2) the modification of the recombination landscapes, especially in the vicinity of centromeres, and (3) the decrease of CO interference. In addition, we revealed that the genetic control of these variations is assigned to specific C chromosomes and could have evolved in a polyploid context. We have therefore identified a way to optimize the shuffling of genetic diversity in rapeseed breeding.

The co-occurrence of polyploidy and clonal reproduction among plant species has long been recognized, but the evolutionary mechanisms underlying the association are unknown. Here, I investigate whether polyploidy increases the magnitude of clonality, either directly or indirectly, by comparing the extent and spatial structure of clones between diploid and tetraploid Chamerion angustifolium in a greenhouse environment and natural populations. In the greenhouse, tetraploid plants allocated 90.4% more dry mass to root buds, the primary mechanism of clonal reproduction, than diploids. Per unit root mass, tetraploids produced 44% fewer root buds and the average position of the root buds along the root was 47% closer to the stem than in diploids. In natural populations, the magnitude of clonality in tetraploid C. angustifolium was similar or less than in diploids. However, clones were spatially aggregated in all diploid populations but only in two of five tetraploid populations. Average clone patch diameter, however, was not significantly different between diploids (3.9 m) and tetraploids (2.5 m). These data do not support the hypothesis that clonality increases as a result of genome duplication. Rather, it is possible that clonality is linked to genome duplication because clonal diploids are predisposed for polyploid formation and establishment. / National Science and Engineering Research Council, Canada Research Chair Program, Canadian Foundation for Innovation

Polyploidization (whole-genome duplication) is widely considered one of the most important evolutionary forces driving the diversification of flowering plants. Polyploids tend to originate recurrently and many plant species retain individuals of two or more different ploidy levels in certain parts of their distributional range of even within their populations. The main aim of this thesis was to address the understudied aspects of polyploid speciation by employing new, convenient methods and/or studying plant model systems with unique features. Difference in monoploid genome size of Tripleurospermum inodorum (Asteraceae) cytotypes provided a unique opportunity for addressing the rate of spontaneous polyploidization in natural populations by enabling the easy distinction of neopolyploid mutants from long-established polyploids in routine flow-cytometric analyses. Repeated ploidy screening in mixed-ploidy populations of annual T. inodorum have been, to our knowledge, the very first attempt to document temporal changes in cytotype composition in situ. In spite of considerable between- year oscillations in cytotype frequencies, both diploids and tetraploids usually persisted locally for several consecutive years. The common incidence of such ploidy mixtures along with a partial fertility of triploid...

[EN] Polyploidy is very common in higher plants and is one of the main forces driving evolution, as it leads to changes that may favor plant adaptation. In citrus, polyploid breeding has provided great advantage, enabling the development of new tetraploid (4x) rootstocks and triploid seedless varieties. Rootstock breeding programs aim to combine the characters of interest in a single genotype to provide the required tolerances to biotic and abiotic constraints. In Spain, the presence of citrus tristeza virus (CTV) and the abundance of calcareous and saline soils limit the number of suitable citrus rootstocks. Citrus breeding has been traditionally carried out by sexual hybridization but this method has limitations which are mainly imposed by their complex reproductive biology and their high heterozygosity. To overcome these restrictions, some breeding programs working at tetraploid level have been initiated to facilitate the development of new citrus rootstock ideotypes. The spontaneous appearance of autotetraploid plants in seedlings of apomictic citrus genotypes brings a relatively simple breeding method, offering the opportunity of evaluating their properties and selecting them as new rootstocks. The impact of genome duplication on citrus is variable between species and remains poorly described. To assess the usefulness of 4x citrus rootstocks it is required to evaluate the effect of genome duplication on stress physiology, tree vigor and agronomical behavior. Citrus protoplast fusion allows the somatic hybridization between sexually incompatible species, leading to combine the whole genomes of parents in a single genotype and adding complementary dominant characters, regardless of parental heterozygosity. The purpose of this thesis was to assess the potential interest of the 4x versions of the main rootstocks used in Spanish citrus industry, which are citrange Carrizo (Citrus sinensis (L.) Osb. × Poncirus trifoliata (L.) Raf.) (CC) and C. macrophylla W. (CM) and describe the anatomical, physiological and molecular determinants of the differences found. Additionally, the characterization of two allotetraploid somatic hybrids obtained by protoplast fusion from these parents was also performed, as a preliminary step for their possible use as rootstocks. Results show that 4xCC has a reducing effect on the size of orange trees without changing the productive efficiency or the fruit quality, effects that might contribute to increase the productivity and facilitate the orchard management. In both rootstocks, genome duplication modified the mineral composition in the leaf of the grafted variety and enhanced the salinity tolerance of seedlings. Boron uptake in excess conditions was reduced in 4xCC when compared to 2x, while this property was not observed in 4xCM. The salt and boron exclusion abilities are associated to lower transpiration rates, reduced root hydraulic conductivity and root anatomical changes observed in 4x roots. However, the ability of 4x CC and CM to cope with iron chlorosis is not different from the 2x genotypes. These 4x rootstocks might therefore provide smaller citrus tress more tolerant to certain stresses, while maintaining the general behavior of the diploid genotype. Furthermore, somatic hybrids differ from each other in their genome composition, indicating that losses and rearrangements of the genetic material occurred during the fusion process. Both inherited the tolerance to stem pitting caused by CTV from CC, are tolerant to iron chlorosis like CM and have a higher tolerance to salinity than CC. Thus, these hybrids have a great potential for Mediterranean citrus industry. The provided knowledge on the effects of genome duplication and somatic hybridization on the anatomy, physiology, agronomic performance and molecular biology of citrus rootstocks will be key for developing breeding programs that aim to address current and future needs of citrus industry. / [ES] La poliploidía es una característica muy común en plantas superiores y actúa como motor evolutivo promoviendo su adaptación. En cítricos, la mejora genética a nivel poliploide agiliza el desarrollo de portainjertos tetraploides (4x) y permite obtener variedades sin semilla triploides. El portainjerto es un elemento fundamental en la citricultura para proporcionar al cultivo la tolerancia a estreses bióticos y abióticos. En España, el alto contenido en caliza y sales de la mayoría de los suelos citrícolas y el virus de la tristeza de los cítricos (CTV) limitan en el número de portainjertos adecuados para el cultivo de cítricos. El desarrollo de nuevos portainjertos tiene como objetivo reunir en un genotipo las características de interés. La hibridación sexual ha sido el método tradicionalmente empleado, aunque en cítricos plantea inconvenientes debido a su compleja biología reproductiva y a su alta heterocigosidad. La autotetraploidía, que aparece espontáneamente en los cítricos apomícticos, posibilita la selección y evaluación de las variantes 4x de cada portainjerto. El efecto de la tetraploidía es variable entre las diferentes especies de cítricos y apenas ha sido descrito. Conocer su influencia sobre la fisiología y morfología de los cítricos permitiría valorar su utilidad agronómica. La alotetraploidía, obtenida mediante fusión de protoplastos, permite hibridar especies sexualmente incompatibles y reunir los genomas completos de los parentales, acumulando sus caracteres dominantes complementarios con independencia de la heterozigosidad. El propósito de ésta tesis ha sido evaluar las variantes 4x de los portainjertos más utilizados en España, citrange Carrizo (Citrus sinensis (L.) Osb. × Poncirus trifoliata (L.) Raf.) (CC) y C. macrophylla (CM) para valorar su utilidad agronómica y describir sus propiedades. Además se han caracterizado dos híbridos alotetraploides obtenidos mediante fusión de protoplastos de CM y CC para valorar su posible utilización como portainjertos para la citricultura mediterránea. La tetraploidía en CC y CM modifica la composición mineral foliar de la variedad injertada y mejora la tolerancia de éstas plantas a la salinidad, mientras que la capacidad de hacer frente a la clorosis férrica no difiere entre ploidías en éstos portainjertos. El portainjerto CC4x ejerce un efecto reductor del tamaño de copa en árboles de naranjo sin modificar su eficiencia productiva ni la calidad del fruto y reduce la absorción y transporte de boro a la variedad injertada en condiciones de exceso, mejorando su tolerancia. La mayor capacidad de exclusión de sales y boro en las plantas 4x se relaciona con su menor transpiración. En CC interviene también en este comportamiento la menor conductancia hidráulica de las raíces 4x inducida por sus modificaciones anatómicas. Los híbridos alotetraploides obtenidos mediante fusión de protoplastos de CC y CM difieren en la composición de su genoma, indicando que se han producido pérdidas puntuales y reorganizaciones durante el proceso de fusión. Ambos reúnen los genomas nucleares parentales, han heredado de CC la tolerancia a las acanaladuras en la madera causadas por CTV, son similares a CM en su tolerancia a la clorosis férrica y manifiestan mayor tolerancia a la salinidad que CC. Por ello, reúnen características de gran interés para la citricultura mediterránea. La evaluación y selección de portainjertos 4x permitiría obtener árboles más pequeños y más tolerantes a ciertos estreses que mantengan el comportamiento general ya conocido de los diploides originales. La reducción del tamaño de copa contribuiría a mejorar el rendimiento productivo y a facilitar el manejo de las plantaciones citrícolas. El conocimiento generado sobre los efectos de la tetraploidización y de la hibridación somática en la anatomía, fisiología, comportamiento agronómico y genética de los portainjertos de cítricos ser / [CAT] La poliploïdia és molt comuna en plantes superiors i es un dels principals motors evolutius per promoure canvis que afavoreixen la adaptació. En cítrics, la millora genètica a nivell poliploide ha contribuït agilitzant el desenvolupament de nous portaempelts tetraploides (4x) i de varietats sense llavors triploides. En Espanya, el virus de la tristesa dels cítrics (CTV) i l'alt contingut salí i calcari de la majoria dels sols limiten el numere de portaempelts adequats per al cultiu. La millora de portaempelts de cítrics té l'objectiu de reunir el major numere de característiques d'interés en un únic cultivar. La millora genètica tradicional de cítrics, duta a terme mitjançant hibridació sexual, planteja inconvenients deguts a la seua complexa biologia reproductiva i a l'alta heterocigositat. Per aixó s'han iniciat programes de millora de portaempelts a nivell 4x, amb l'objectiu d'agilitzar el seu desenvolupament. La autotetraploïdia, que apareix de manera espontània en especies apomíctiques de cítrics, permet seleccionar les variants 4x dels portaempelts, com un mètode de millora relativament senzill. L'efecte de la tetraploïdia és variable entre les espècies de cítrics i la seua incidència a penes havia sigut descrita. Conéixer la seua repercussió en la tolerància a estressos i el vigor de l'arbre permetria avaluar la utilitat de les versions 4x de cada portaempelt. D'altra banda, l'al¿lotetraploïdia, obtinguda mitjançant la fusió de protoplastos, possibilita l'obtenció d'híbrids d'espècies sexualment incompatibles i reunir en una sola hibridació els genomes complets de les parentals d'interés, permetent l'addició de caràcters dominants complementaris amb independència de l'heterozigositat dels parentals. La finalitat d'esta tesi ha sigut avaluar les propietats d'interés agronòmic de les variants 4x dels portaempelts més utilitzats en la citricultura española, citrange Carrizo (Citrus sinensis (L.) Osb. × Poncirus trifoliata (L.) Raf.) (CC) i C. macrophylla (CM), per valorar la seua utilitat i descriure els determinants de les diferències trobades. A més a més, s'ha pretés realitzar una caracterització molecular i fisiològica de dos híbrids al¿lotetraploides obtinguts de la fusió de protoplastos de CM i CC, com un pas previ per a la seua possible utilització com a portaempelts. El portaempelt CC4x exerceix un efecte reductor de la grandària de copa en arbres de taronger sense modificar l'eficiència productiva ni la qualitat del fruit. Este caràcter milloraría el rendiment productiu i a facilitaría el maneig de les plantacions citricoles. En els dos portaempelts estudiats, la tetraploïdia modifica la composició mineral foliar de la varietat empeltada i proporciona major tolerancia a la salinitat. En CC, la tetraploïdia redueix l'absorció del boro en excés, metre que esta propietat no s'ha observat en CM4x. La menor absorció del boro i l'exclusió del clorur es troben associades a la menor transpiració, a la reduïda conductivitat hidràulica i a les característiques anatòmiques de les arrels 4x. No obstant això, la tolerancia a la clorosi fèrrica no difereix entre ploidíes en estos portaempelts. La selecció de portaempelts 4x permetria obtenir arbres més menuts i més tolerants a certs estressos que mantenen el comportament ja conegut, de la variant diploide. Per la seua banda, els híbrids somàtics difereixen en la composició del genoma, indicant que s'han produït reorganitzacions i pèrdues puntuals durant el procés de fusió. Els dos han heretat de CC la tolerància a les estries en la fusta provocades per CTV, son tolerants a la clorosi fèrrica com CM i toleren la salinitat millor que CC. Per tot, reuneixen un conjunt de característiques de gran interés potencial per a la citricultura mediterrània. El coneixement generat en esta tesi serà de gran utilitat en els programes de millora basades en necessitats actuals i futures. / Ruiz Valdés, M. (2016). Identificación de caracteres fisiológicos y moleculares para la tolerancia a estreses abióticos en portainjertos de cítricos tetraploides [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/73725 / TESIS