Apomixis is a clonal propagation method that produces offspring identical to the mother plant. With this feature, superior traits could be maintained over generations. However, our knowledge about apomixis is limited. In this study, we analyzed several apomictic Boechera embryologically to learn the details of apomixis. Meanwhile, we designed chemical treatments to successfully induce sex in apomictic plants and apomixis in sexual plants. Our experiments suggest that sex and apomixis coexist in plants and that sexual and apomictic reproduction are switchable by treating with specific chemicals.

Apomixis is an evolutionarily important phenomenon across plant lineages. The interaction of apomixis with hybridization and polyploidy can lead to complex patterns of reticulation, complicating efforts to reconstruct evolutionary history in groups where apomixis is common. Ferns, in particular, are rich in apomictic species, notably in centers of species diversity like East Asia. Eastern North America too is home to a number of apomictic species. We investigated the East Asian ferns in Polystichum sections Xiphopolystichum and Duropolystichum (Dryopteridaceae) in order to elucidate the evolutionary and biogeraphic history of seven apomictic species in the group: Polystichum tsus-simense, P. xiphophyllum, P. sinotsus-simense, P. pseudoxiphophyllum, P. mayebarae. P. rigens, and P. neolobatum. In addition, we examined the evolutionary origin of an undescribed apomictic cytotype of North American genus Phegopteris (Thelypteridaceae). The datasets comprised phylogenetic inference based on three nuclear and three plastid markers, analysis of mixed nucleotide signals from chromatograms generated from Sanger sequencing of nuclear markers, ploidy estimates based on flow cytometry data and spore length measurements, morphometric analysis of representative specimens collected in southwest China and nearby regions, and climatic niche models. By interpreting these multiple lines of evidence synthetically, we have discerned multiple highly reticulate complexes of polyploid lineages derived largely from diploid sexual progenitors. Our findings highlight the importance of understanding the role of apomictic reproduction in the context of species diversity, an understanding central to similar future inquiry into the diversity of East Asian and North American ferns.

Tesis presentada como parte de los requisitos para optar al Grado de Magíster en Ciencias Agropecuarias Mención en Producción Frutícola / La tuna es una de las especies de cactus más importante económicamente a nivel mundial, debido principalmente a la posibilidad de producirla en zonas áridas y semiáridas y los variados productos que se pueden obtener de ella. Esta es originaria del Golfo de México y el Caribe (CEZA, 2007) y desde allí se ha expandido por diversas partes del mundo. Esta especie fue introducida en España por los navegantes y desde allí se distribuyó por toda la cuenca del Mediterráneo y el resto de los continentes (Kiesling, 2012). Actualmente se encuentra en forma silvestre o cultivada en el sur de España, Francia, Grecia, Italia, Turquía, Israel y Sudáfrica, extendiéndose también dentro del mismo continente Americano desde Canadá hasta Chile (Sáenz, 2006).

The genus Sorbus is an example of a taxonomically complex group (TCG) with diversity derived from hybridisation, polyploidy and apomixis. The focus of this study was to elucidate the evolutionary relationships among nine Sorbus species including endemics of the Devon and north Somerset region of the south west UK, determine main routes of polyploid formation and investigate reproductive sustainability in order to make recommendations for Sorbus conservation. Molecular analysis showed that genetic structure patterns and genotypic diversity support the hypothesis that the study polyploids are a product of rare interspecific hybridisation, of single origins and are maintained through apomictic reproduction. PCoA, Neighbour Joining analysis and parental simulations reveal a reticulated relationship, with diversification the result of hybridisations between sexual diploid Sorbus torminalis and both tetraploid and triploid species. Hybridisation between S. torminalis and tetraploid Sorbus margaretae (subgenus Aria) have likely given rise to the study members of subgenus Tormaria through production of a triploid which has subsequently backcrossed to Sorbus torminalis to form further tetraploids. The discovery of a cryptic hybrid in subgenus Aria also suggests occasional hybridisation events among tetraploids are a possible route for further tetraploid formation These events illustrate key routes of polyploid formation, both illustrating the role of triploids in tetraploid formation via the triploid bridge and the key role in sexual diploids in diversification in Sorbus. Hand pollination experiments showed that self-incompatibility in the triploid species (Sorbus subcuneata) means reliance on congeneric pollen from sympatric tetraploid species for seed production. Reproductive sustainability in this species is severely compromised through spatial isolation from compatible congeners. Our findings are strong support for the development of conservation strategies that aim to safeguard current diversity through actions that increase reproductive sustainability and recruitment opportunities, and promote opportunities for on-going hybridisation for future diversification of Sorbus in this region.

Spore abortion rate was studied for a total of 109 samples belonging to 23 fern taxa. The resulting abortion rate was compared within several reproductive and phylogenetic groups. The method of spore abortion estimation was optimized by analyzing the amount of spores needed as well as the potential differences within frond.

La apomixis es una forma de reproducción clonal, donde no ocurre ni meiosis ni fecundación para la formación de la semilla. Existe una estrecha relación entre la apomixis y el nivel de ploidía, dado que en la mayoría de las especies que presentan el carácter los genotipos diploides son sexuales y los poliploides son apomícticos y con un grado variable de sexualidad (facultativos). Aunque se sabe que el carácter es regulado por una o más regiones del genoma, se han hallado numerosas evidencias de mecanismos epigenéticos que regulan su expresión. Conocer los factores que determinan la apomixis tendría un gran impacto en la agricultura. Eragrostis curvula (“pasto llorón”) es una gramínea forrajera originaria de Sudáfrica, que incluye genotipos con diferentes niveles de ploidía (2X hasta 8X, con X = 10), que pueden ser sexuales o apomícticos (facultativos u obligados). Nuestro grupo de trabajo realizó numerosos estudios, sobre todo en el carácter apomixis, tanto a nivel de genética estructural como funcional. El objetivo de esta tesis fue contribuir a la dilucidación de las bases genéticas y epigenéticas que regulan uno de los componentes de la apomixis (apomeiosis) en E. curvula. Se desarrolló una población de mapeo segregante para el carácter apomixis a partir del cruzamiento entre dos genotipos tetraploides, uno sexual (OTA-S) y otro apomíctico facultativo (Don Walter). La genotipificación se realizó con marcadores DArT-SNP y el fenotipado mediante citoembriología y un marcador molecular dominante. El mapa de ligamiento quedó conformado por 20 grupos de ligamiento (GL), con el posicionamiento del APO locus en el GL1, donde también se ubicaron tres marcadores SNP 100 % ligados al carácter apomeiosis. La secuencia de los SNP 100 % ligados tienen homología con proteínas anotadas en bases de datos, que podrían estar relacionadas al modo reproductivo. Se realizó un análisis de sintenia entre los GLs del mapa consenso y el genoma de referencia de la especie (cv. Victoria) que permitió asociar todos los GLs a alguno de los contigs y hallar los GLs que corresponderían potencialmente a cada uno de los genomas del alotetraploide (homeólogos). Para la validación de los marcadores se diseñaron y evaluaron cinco primers KASP que permitieron discriminar entre los alelos de los individuos sexuales y apomícticos. En el segundo capítulo se analizaron diferentes resultados obtenidos por el grupo de trabajo que evidencian la influencia de la epigenética sobre el carácter apomixis en plantas de E. curvula. Para analizar la participación de mecanismos de metilación del ADN en la regulación de la apomixis bajo condiciones de estrés hídrico, se realizó un ensayo en el cual muestras de ADN de inflorescencias fueron evaluadas mediante la “Estimación del contenido de metilación por doble corte con enzimas de restricción sensibles” (MCSeEd). En las regiones diferencialmente metiladas se lograron encontrar genes vinculados al modo reproductivo, como BABYBOOM, ubiquitinas, FBox y subtilisinas. Podría afirmarse que tanto el estrés interno como externo ejerce una gran influencia sobre el modo reproductivo de E. curvula, modificando la expresión del carácter apomixis en plantas apomícticas facultativas. / Apomixis is a type of clonal reproduction, where neither meiosis nor fertilization occurs in seed formation. There is a close relationship between apomixis and ploidy level, since in most of the apomictic species diploid genotypes are sexuals and polyploids are apomictics. Usually, apomictic plants are facultative, being some of the seeds sexuals in origin. Although it is known that this trait is regulated by one or more genomic regions, numerous evidences of epigenetic mechanisms that regulate its expression have been found. Knowing the factors that determine apomixis could have a great impact on agriculture. Eragrostis curvula, commonly known as weeping lovegrass, is a forage grass native to southern Africa, which includes genotypes with different ploidy levels (from 2X to 8X, with X = 10) and that can be sexuals or apomictics (facultative or obligate). Our work group carried out several studies on this model species, especially related with the apomixis character, both at the structural and functional genetic level. The objective of this thesis was to contribute to the elucidation of the genetic and epigenetic bases that regulate one of the components of apomixis (apomeiosis) in E. curvula. A segregating mapping population was generated for the apomixis trait from the cross between two tetraploid genotypes, one sexual (OTA-S) and the other one facultative apomictic (Don Walter). Genotyping was performed with DArT-SNP markers and phenotyping using cytoembryological techniques and a dominant molecular marker. The linkage map was made up of 20 linkage groups (LGs), with the apomeisis locus (APO locus) located on GL1, where three SNP markers 100 % linked to the APO locus were also located. All of this 100 % linked SNPs have homology with proteins, two of these annotated in databases. A synteny analysis was performed between the GLs of the consensus map and the E. curvula reference genome (cv. Victoria), which allowed all the GLs to be associated to any of the contigs and to find the GLs that would potentially correspond to each allotetraploid genome (homeologs). For the validation of the markers, five KASP primers were design and performed, allowing to discriminate between the sexual or apomictic alleles of the ndividuals of the mapping population. Furthermore, in a second chapter, several results obtained by the work group were evaluated, demonstrating an influence of epigenetic regulation on the apomixis trait in E. curvula. To analyze the involvement of DNA methylation mechanisms in the regulation of apomixis under water stress conditions, DNA samples of inflorescences under different stress conditions were evaluated, using the “Methylation content sensitive enzime double-digest restriction-site-associated DNA” (MCSeEd) method. Some of the genes identified in the differentially methylated regions were found to be related to the reproductive mode, such as BABYBOOM, ubiquitin, F-box and subtilisin genes. It could be proposed that stress, both internal and external, has a significant influence in the reproductive mode of E. curvula, modifying the expression of the trait in facultative apomicts.

As many as 70% of angiosperm species are known to contain polyploids, but many aspects of polyploid evolution are unclear in woody plants. Crataegus is a woody genus of Rosaceae comprising 140-200 species that are widely distributed in the Northern Hemisphere. Several species, particularly those in North America, are shown to contain polyploids. The overall goal of the thesis is to provide a better understanding of polyploid evolution by resolving problems from intergeneric to intraspecific levels in Crataegus using phylogenetic and population genetic approaches. Three major aspects were investigated: (1) Phylogeography of the Old and New World Crataegus; (2) Reproductive system and distribution of cytotypes of the black-fruited series Douglasianae in Pacific Northwest and; (3) Origins, population structure, and genetic diversity of diploid and polyploid species. Phylogenetic analyses of molecular data provide evidences of historical events such as trans-Beringian migrations and North Atlantic vicariance that contributed to modern distribution of Crataegus. Poor resolution and short internal branches in eastern North American species suggest genetic bottlenecks and/or rapid divergence following glaciations. In the Pacific Northwest, polyploids of series Douglasianae show a wider distribution and ecological amplitude than diploids. Parsimony tree and network analyses indicate that autotriploids and allotriploids occur in C. suksdorfii, while tetraploid C. suksdorfii are formed via the triploid bridge followed by introgression of sympatric C. douglasii. At the regional level, microsatellite data indicate a separation of the Pacific coastal diploids and triploids from the Columbia Plateau and Rocky Mountain triploids and tetraploids. High genetic differentiation among C. suksdorfii populations suggests that gene flow is limited by ploidy level differences as well as geographical distance. Within-population multilocus genotypic variation is greatest in sexual diploids, and least in apomictic triploids. Frequent gene flow via seed dispersal contributes to an appreciable level of intrapopulation diversity in apomictic tetraploids, and counterbalances the effects of apomixis and/or self-fertilization, which diminish genetic variation within and between seed families. These findings collectively clarify taxonomy and historical biogeography, provide an explicit reticulation model for polyploid formation, and shed light on evolution of natural populations in woody plants that show heterogeneous ploidy levels and reproductive systems.

As many as 70% of angiosperm species are known to contain polyploids, but many aspects of polyploid evolution are unclear in woody plants. Crataegus is a woody genus of Rosaceae comprising 140-200 species that are widely distributed in the Northern Hemisphere. Several species, particularly those in North America, are shown to contain polyploids. The overall goal of the thesis is to provide a better understanding of polyploid evolution by resolving problems from intergeneric to intraspecific levels in Crataegus using phylogenetic and population genetic approaches. Three major aspects were investigated: (1) Phylogeography of the Old and New World Crataegus; (2) Reproductive system and distribution of cytotypes of the black-fruited series Douglasianae in Pacific Northwest and; (3) Origins, population structure, and genetic diversity of diploid and polyploid species. Phylogenetic analyses of molecular data provide evidences of historical events such as trans-Beringian migrations and North Atlantic vicariance that contributed to modern distribution of Crataegus. Poor resolution and short internal branches in eastern North American species suggest genetic bottlenecks and/or rapid divergence following glaciations. In the Pacific Northwest, polyploids of series Douglasianae show a wider distribution and ecological amplitude than diploids. Parsimony tree and network analyses indicate that autotriploids and allotriploids occur in C. suksdorfii, while tetraploid C. suksdorfii are formed via the triploid bridge followed by introgression of sympatric C. douglasii. At the regional level, microsatellite data indicate a separation of the Pacific coastal diploids and triploids from the Columbia Plateau and Rocky Mountain triploids and tetraploids. High genetic differentiation among C. suksdorfii populations suggests that gene flow is limited by ploidy level differences as well as geographical distance. Within-population multilocus genotypic variation is greatest in sexual diploids, and least in apomictic triploids. Frequent gene flow via seed dispersal contributes to an appreciable level of intrapopulation diversity in apomictic tetraploids, and counterbalances the effects of apomixis and/or self-fertilization, which diminish genetic variation within and between seed families. These findings collectively clarify taxonomy and historical biogeography, provide an explicit reticulation model for polyploid formation, and shed light on evolution of natural populations in woody plants that show heterogeneous ploidy levels and reproductive systems.

The isolation and characterization of the gene(s) associated with and potentially responsible for the regulation of apomixis (asexual) and sexual reproduction in the grass species Tripsacum dactyloides is quintessential to agricultural advancement. Apomixis is the mechanism by which plants can produce seed without fertilization, where all progeny are genetically identical to the maternal parent. In natural populations of the genus Tripsacum, lower ploidy forms (i.e., diploid, 2n=36) reproduce sexually and the higher ploidy forms (triploid, 3n=54; tetraploid, 4n=72; etc.) reproduce asexually via apomixis. In order to gain a better understanding of sexual reproductive processes in plants, subtractive hybridization was performed on early and late female inflorescence gene products. This procedure allows for the recovery of gene products in the form of complimentary DNA, cDNA molecules, which correspond to messenger RNA (mRNA) present. These cDNA molecules were then used to extract unique sequence messages from the early developmental stage ovule tissue of diploid T. dactyloides. These cDNA molecules will allow for the direct isolation of the original form of the gene(s) from a large fragment library of the Tripsacum genome. Isolation and characterization of these gene(s) is of pivotal importance to our understanding of alternate modes of reproduction in Tripsacum. / Department of Biology

Manipulation de la recombinaison chez une plante cultivée, le riz.L’accroissement prévisible de la population mondiale ainsi que les conséquences du changement climatique obligent les sélectionneurs à créer de nouvelles variétés plus productives et plus résilientes. Les nouvelles combinaisons d’allèles favorables sont issues de la recombinaison génétique entre chromosomes homologues dont le siège est la prophase de première division de méiose. De récentes avancées chez la plante modèle Arabidopsis ont montré que l'inactivation de certains gènes permet de manipuler la méiose pour abolir ou au contraire augmenter très significativement la recombinaison. Les mécanismes de la méiose étant relativement bien conservés chez les eucaryotes, l’objectif de cette thèse était de transposer ces avancées chez une plante cultivée importante, le riz. Abolir la recombinaison méiotique permettrait de propager de façon clonale par grain des formules variétales hybrides F1 dont le rendement est de 20% supérieur à celui des lignées pures chez le riz mais dont les semences restent peu utilisées par les riziculteurs de subsistance. Les travaux réalisés dans une première partie de la thèse ont montré que le cumul de trois mutations Ososd1, pair1 et Osrec8, permettait d’obtenir des gamètes clonaux diploïdes mâles et femelles. Le phénotype apoméiotique obtenu, appelé MiMe (Mitosis instead of meiosis) chez Arabidopsis, peut être utilisé pour tester différentes stratégies d’induction de la parthénogenèse afin de produire des grains formant des plantes diploïdes clonales apomictiques. Une optimisation du mécanisme permettrait d'envisager l'utilisation de l'apomixie pour fixer l'hétérosis dans les semences hybrides F1. Par ailleurs, une augmentation globale ou locale de la recombinaison méiotique est recherchée car elle permettrait de diminuer la taille des populations de sélection et de réduire la taille des segments chromosomiques introduits dans les variétés élite de riz. Nous avons montré dans une seconde partie, que la mutation du gène OsRECQl4 codant pour une hélicase permet d'augmenter le taux de recombinaison d'un facteur de 3,3 fois faisant passer la taille de la carte génétique de 1670 cM à 5538 cM sans affecter la fertilité de la plante ni le déroulement de la méiose. Chez les plantes affectées dans la fonction d’une autre hélicase, OsFANCM, le taux de recombinaison a été également augmenté mais dans une moindre mesure (x 2,2). L’augmentation de la recombinaison s’opère sur l'ensemble des bras chromosomiques sauf au niveau des centromères. Ces résultats confirment ceux obtenus chez A. thaliana qui ont montré le rôle de régulateur négatif des crossing-overs (CO) des protéines RECQ4 et FANCM. La combinaison en cours de ces mutations entre elles ou avec celle affectant l’AAA-ATPase FIGL1 permet d’espérer une augmentation de la recombinaison encore supérieure. Ces résultats ouvrent la voie à l'utilisation des gènes anti-COs pour augmenter de façon globale le nombre de recombinants dans les croisements chez le riz et sans doute chez les autres céréales. Pour offrir une possibilité concrète aux sélectionneurs d'utiliser les gènes anti-CO, nous avons montré que la technologie CRISPR/cas9 permet d'éteindre l'expression de OsFANCM OsRECQl4 et OsFIGL1. / Manipulation of recombination in a crop, rice.The forecasted increase of world population as well as the consequences of global climate change oblige plant breeders to develop new varieties that are both more productive and resilient. Novel combinations of favourable alleles are generated through genetic recombination between homologous chromosomes, which occurs during the prophase of the first division of meiosis. Recent advances in the model plant Arabidopsis have demonstrated that the inactivation of some genes allows meiosis manipulation resulting in either an abolishment or in contrast, a significant enhancement of meiotic recombination. The meiosis mechanisms being relatively conserved across eucaryotes, the overall objective of this thesis was to transfer these advances to a crop of crucial importance, rice. To abolish meiotic recombination would allow the clonal propagation by seeds of F hybrids, which exhibit a 20% yield enhancement compared to that of pure lines in rice but remain rarely used in subsistence farming. In a first part, we showed that rice plants cumulating 3 mutations inactivating Ososd1, pair1 and Osrec8, formed clonal diploid male and female gametes. This apomeiotic phenotype, called MiMe (Mitosis instead of meiosis) in Arabidopsis, can serve as material to assay several strategies of parthenogenetic induction that would result in seed forming diploid clonal plants. Further optimization of the mechanisms would allow the use of apomixis to fix heterosis in hybrid seeds. Global and local enhancement of recombination is another desirable goal since it would allow a reduction in breeding population size and a downsizing of the introgressed chromosomal segments in elite plant materials. In a second part, we showed that mutation in the DNA helicase gene OsRECQl4 conducted to a 3.3 fold increase of recombination and inflated the genetic map size from de 1670 cM to 5538 cM, without altering plant fertility nor meiosis progression. Plants altered in a second DNA helicase, OsFANCM, exhibited a more modest 2.2 fold recombination enhancement. Recombination increase operated along the whole chromosome arms except at the centromere level. These results confirms the negative regulator role of RECQ4 and FANCM on crossing overs (CO), previously reported in Arabidopsis. On going combination of these mutations together with that altering the l’AAA-ATPase FIGL1 should conduct to an even higher recombination enhancement. These results pave the way to the use of anti-CO genes to enhance recombinant recovery in crosses of rice and possibly of other cereals. To provide breeders with a workable anti-CO system, we eventually showed that the CRISPR/Cas9 technology can be used to abolish OsFANCM, OsRECQl4 and OsFIGL1 expression.