The increasing availability of molecular tools is facilitating marker-assisted selection (MAS) in plant improvement programs. The objectives of this research were to: 1) populate the framework buffelgrass genome map with additional molecular markers, 2) develop polymerase chain reaction (PCR)-based markers from selected, informative restriction fragment length polymorphism (RFLP) markers on the buffelgrass genome map, and 3) increase marker resolution near the locus conferring apomixis (PApo1). Buffelgrass [Pennisetum ciliare (L.) Link syn. Cenchrus ciliaris L.] (2n=4x=36), a highly polymorphic, apomictic, perennial forage grass, is well-suited for genetic linkage analyses. One hundred and seventy one probes from an apomictic, spikelet-specific, complementary deoxyribonucleic acid (cDNA) library and 70 expressed sequence tag simple sequence repeats (EST-SSRs) from apomictic pistil cDNAs were evaluated and added to the framework buffelgrass genome map. The improved linkage map contains 851 markers from 11 grass species and covers approximately 80-85% of the buffelgrass genome. Two RFLPs from the buffelgrass genome map were converted to PCR-based markers for both the identification of hybrids and quantification of sexual versus apomictic reproduction. A gel-free, high-throughput technique was developed to analyze these markers directly in 96-well plates. Five additional markers were placed onto the buffelgrass linkage group with the PApo1 apomixis locus through comparative mapping of candidate orthologs from the sorghum genome map and bulked-segregant analysis of amplified-fragment-lengthpolymorphisms (BSA-AFLP). Increasing the mapping population size did not increase map resolution in the PApo1 region. Association mapping revealed that the recombination suppression near PApo1 is moderate and would complicate comparative map-based cloning efforts of the orthologous region in sorghum.

Apomixis holds vast potential for improving agriculture worldwide. It would make plant breeding faster, allow for fixation of hybrid genotypes, and help genetically isolate transgenic crops. However, efforts to introduce apomixis into agriculturally important crops, through either plant breeding or molecular genetics, have failed to produce any new apomictic varieties. This could possibly be remedied if researchers better understood the genetics of apomixis, including the underlying genes, their regulation, and the cellular pathways they control. My work increased our knowledge of these processes by using a mixture of novel methods and underutilized resources. In this study I pursued a two-pronged approach that involved both traditional and reverse molecular genetic techniques. I analyzed the Semigamy mutation from Pima Cotton (Gossypium barbadense), which produces large numbers of haploid and chimeric offspring. Although interesting and potentially useful, little is known about this gene, its expression, regulation, and localization, or the cellular processes in which it is involved. This knowledge advanced our understanding of both this part of apomixis and plant reproduction in general. I was able to determine that the Semigamy mutation is recessive to the wild-type and is incompletely expressed even in the homozygous state. Second, I attempted to recreate the various steps of apomixis in the model species Arabidopsis thaliana using the host of tools made available by its fully sequenced genome. Mutants in various organisms, including microbes, plants, and animals, have phenotypes resembling various components of apomixis, and the sequences of most of the genes involved are available in public databases. I identified homologous Arabidopsis genes by comparing these sequences against the entire Arabidopsis genome. Mutants carrying altered versions of these genes were then studied and characterized using various techniques to see if they had any effect on plant reproduction. This method had not been used to study apomixis before this study and revealed several new reproductive Arabidopsis mutations, specifically those in genes homologous to the S. cerevisiae ste12 and rec8 genes. These results will advance the study of apomixis and potentially enable researchers to one day apply it to agriculturally important crop species.

Apomixis holds vast potential for improving agriculture worldwide. It would make plant breeding faster, allow for fixation of hybrid genotypes, and help genetically isolate transgenic crops. However, efforts to introduce apomixis into agriculturally important crops, through either plant breeding or molecular genetics, have failed to produce any new apomictic varieties. This could possibly be remedied if researchers better understood the genetics of apomixis, including the underlying genes, their regulation, and the cellular pathways they control. My work increased our knowledge of these processes by using a mixture of novel methods and underutilized resources. In this study I pursued a two-pronged approach that involved both traditional and reverse molecular genetic techniques. I analyzed the Semigamy mutation from Pima Cotton (Gossypium barbadense), which produces large numbers of haploid and chimeric offspring. Although interesting and potentially useful, little is known about this gene, its expression, regulation, and localization, or the cellular processes in which it is involved. This knowledge advanced our understanding of both this part of apomixis and plant reproduction in general. I was able to determine that the Semigamy mutation is recessive to the wild-type and is incompletely expressed even in the homozygous state. Second, I attempted to recreate the various steps of apomixis in the model species Arabidopsis thaliana using the host of tools made available by its fully sequenced genome. Mutants in various organisms, including microbes, plants, and animals, have phenotypes resembling various components of apomixis, and the sequences of most of the genes involved are available in public databases. I identified homologous Arabidopsis genes by comparing these sequences against the entire Arabidopsis genome. Mutants carrying altered versions of these genes were then studied and characterized using various techniques to see if they had any effect on plant reproduction. This method had not been used to study apomixis before this study and revealed several new reproductive Arabidopsis mutations, specifically those in genes homologous to the S. cerevisiae ste12 and rec8 genes. These results will advance the study of apomixis and potentially enable researchers to one day apply it to agriculturally important crop species.

Heidelberg, Univ., 2008.

Plants that are heterozygous for genetic markers but do not produce segregating progeny may be suspected of carrying a mutation conditioning apomixis. Seed stocks in which heterozygous plants could be identified phenotypically were treated with a chemical mutagen. These seed stocks were heterozygous for recessive genetic markers, and/or heterozygous for a chromosome translocation. Spikes from heterozygous M1 plants were harvested and seeded in bulk. Spikes from heterozygous M2 plants were harvested and planted in M3 rows. The M3 rows were observed for the absence of segregating progeny and/or were observed cytologically for the presence of a heterozygous translocation. M3 rows not segregating for the genetic markers were crossed onto plants homozygous for the genetic markers. The F1 progenies were observed for an expected ratio of 1 normal: 1 recessive plant. All nonsegregating lines were found to be non-heterozygous. These lines most likely occurred due to seed and pollen contamination or were the result of crossing over between genetic markers. Fertile M2 plants were harvested from the treated heterozygous translocation seed stock. Normally, barley plants heterozygous for a translocation will produce semisterile spikes. Plants that would normally be semisterile but are fertile could be carrying a mutation conditioning apomixis. Progeny of the fertile M2 plants were examined cytologically for the presence of the heterozygous translocation. All selected lines contained the normal seven pairs of chromosomes and were the result of seed or pollen contamination. Seed stocks which could eliminate the problem of contamination in future experiments were developed and discussed. Haploviable mutants closely linked with the male sterile locus, msg2, were isolated in these seed stocks. Haploviable mutants are recognized by upset genetic ratios of alleles linked with the mutant. Selfed progenies of plants carrying a haploviable mutation contained fertile and male sterile plants in about a 1:1 ratio. Mostly male sterile progenies were obtained when plants heterozygous for the haploviable mutant and the male sterile allele were crossed onto male sterile plants. Four lines containing haploviable mutants were evaluated for their usefulness in producing all male sterile lines for hybrid barley production.

"April 20th 2003." / Includes bibliographical references (leaves 202-220) / xii, 220 leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Agricultural Science, 2003

Apomixis is an intriguing and agronomically valuable asexual reproductive pathway resulting in seeds that give rise to plants that are identical in genotype to the female parent. Apomixis is absent in agriculturally important seed crops and our work has focused on the analysis of apomixis in the daisy-like genus Hieracium which contains sexual and apomictic species. Prior studies have shown that apomixis in Hieracium is controlled by two dominant loci. The LOA controls the avoidance of meiosis during female gametophyte (embryo sac) formation and the LOP locus is required for fertilization independent embryo formation during seed initiation. The genes conferring apomixis are unknown. In this study we focused on the events of autonomous seed initiation. Cytological examination of apomictic mutants that have lost LOA or LOP and analysis of their progeny enabled us to characterize developmental aspects associated with the function of these loci. Upon removal of LOA meiosis occurs normally and LOP segregates with a 1:1 ratio in the progeny, characterizing maternal gametophytic control. We also show that autonomous embryo formation segregates with autonomous endosperm formation, suggesting that these two loci are closely linked. However, upon meiotic division, embryo lethal components arise and embryo development in apomeiosis mutants was generally defective and seed set was low. Similarly, upon removal of LOP, apomixis initiation occurs normally and unreduced embryo sacs can only form seeds if pollinated. Autonomous seed initiation is actively repressed in the sexual model plant Arabidopsis by the action of a chromatin remodelling complex encoded by the FERTILIZATION INDEPENDENT SEED (FIS)-class genes. These genes are homologues of the Drosophila PcG complex that also repress gene expression throughout Drosophila development. Mutations in the FIS-class genes lead to elements of apomixis, such as autonomous endosperm, and in one particular mutant, autonomous egg cell development. Given the similarity in apomictic and FISclass gene mutant phenotype we isolated three homologues from sexual and apomictic Hieracium plants: FERTILIZATION INDEPENDENT ENDOSPERM (FIE), MULTICOPY SUPPRESSOR OF IRA1 (MSI1) AND RETINOBLASTOMA (RBR). FIS-class genes from sexual and apomicit Hieracium and examined their expression, interaction and function during seed initiation. The isolated Hieracium FIS-class genes were highly conserved in sexual and apomictic plants in terms of gene sequence and temporal and spatial expression pattern. Analysis of protein interactions by yeast-two hybrid showed that the HFIE gene from sexual and apomictic plants does not interact with other complex members in the same manner found in Arabidopsis. Protein modelling uncovered structural differences between the Arabidopsis and Hieracium FIE proteins. RNAi- mediated down-regulation of HFIE in sexual Hieracium did not lead to autonomous seed initiation indicating HFIE was not part of a repressive complex. Down-regulation of HFIE in sexual and apomictic plants revealed the gene was essential for embryo growth and viability. Therefore, FIS-complex genes interact differently in Arabidopsis and Hieracium and have different developmental roles. In summary, the results presented here suggest that the FIS-genes are not mutated in apomictic Hieracium plants, but they interact differently relative to the Arabidopsis counterparts and play a fundamental role in embryogenesis. Thus, engineering autonomous seed into crops will not depend on mutating these genes but rather in uncovering the molecular signal that triggers apomictic development. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1337073 / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2008

Thesis (doctoral)--Swedish University of Agricultural Sciences, 2000. / Includes bibliographical references.

Eragrostis curvula es una gramínea estudiada por su tolerancia al estrés por sequía y por tener la capacidad de reproducirse por apomixis, la cual es definida como un mecanismo que permite la clonación natural por medio de semillas resultando la descendencia genéticamente idéntica a la planta madre. E. curvula tiene un número básico de cromosomas de 10 y contiene genotipos con distintos niveles de ploidias. Una de las características la especie es que los genotipos diploides son siempre sexuales mientras que los poliploides son generalmente apomícticos aunque existen tetraploides sexuales. Desde hace décadas la apomixis ha sido estudiada con el fin de identificar las bases moleculares y genéticas para ser transferidas a otros cultivos de importancia económica. Las técnicas de secuenciación de ADN son una herramienta sumamente útil para descubrir genes y regiones asociadas a distintos caracteres biológicos, así como para realizar análisis filogenéticos y de sintenia entre especies. En el trabajo de esta tesis se propuso obtener secuencias genómicas de E. curvula utilizando tecnologías de última generación con el fin de identificar los genes relacionados a la apomixis y otros genes de interés como los de la tolerancia al estrés por sequía y la calidad forrajera. El primer genoma de la especie secuenciado fue el genotipo diploide sexual Victoria que fue originado del tetraploide apomíctico Tanganyika INTA. Las plataformas utilizadas para su secuenciación fueron PacBio, Chicago y Hi-C y fue ensamblado a través del software FALCON con el que se obtuvo un N50 de ~43 mega pares de bases (Mb) y 1.143 contigs. Los genes anotados fueron 56.469 los cuales fueron utilizados para determinar la historia evolutiva de la especie. Además se caracterizaron las secuencias de los genes de las vías de la lignina relacionados con la calidad forrajera y los genes de la familia WRKY, vinculados a la tolerancia al estrés por sequía. Por otro lado se encontraron genes de E. curvula ortólogos a los expresados en etapas específicas de la vía sexual de Oryza sativa. Los genomas tetraploides apomícticos secuenciados fueron el del cultivar Tanganyika INTA obtenido mediante la plataforma Illumina y el de Don Walter combinando las tecnologías Chromium 10X y Oxford Nanopore. El ensamblado de Tanganyika INTA con el software Masurca resultó en un N50 de 4.715 pb y 293.300 contigs, mientras que en el genoma de Don Walter ensamblado con Supernova+DBG2OLC se obtuvo un N50 de 224.390 pb y 7.542 contigs. Por medio de un análisis comparativo se pudo identificar la región vinculada la apomixis en todos los genomas en la cual se encontraron genes que podrían tener un rol activo en la regulación del modo reproductivo de acuerdo con la literatura. A través de esta tesis se pudieron secuenciar, ensamblar y anotar tres genomas de E. curvula lo cual representa un avance significativo en la caracterización de la especie. La identificación de regiones asociadas a la apomixis y sus posibilidades de expresión y regulación permitirá, en una instancia futura, manipular el carácter e intentar la introgresión del mismo en especies sexuales de interés agrícola por medio de ingeniería genética. / Eragrostis curvula is forage grass studied for its tolerance to draught stress and because its characteristic reproductive pathway, the apomixis. Apomixis is an asexual way of reproduction by seeds in which the progeny result genetically identical to the mother plant. The basic number of chromosomes of E. curvula is 10 and has different ploidy levels being the diploids always sexual while the polyploids are mainly apomictic despite there are fully sexual tetraploids. Apomixis has been studied in order to identify its molecular and genetic basis to transfer this trait to economically import crops. The DNA sequencing is a powerful tool to detect genes and/or regions associated to different traits and disclose the evolutionary history of the species. In this Thesis the latest generation technologies have been used to sequencing and assembly the E. curvula genome to identify the genes related to apomixis and other interesting traits like draught tolerance and forage quality. The first sequenced genome of the species was the diploid sexual genotype Victoria originated from the apomictic tetraploid Tanganyika INTA. The technologies used for the sequencing were a combination of PacBio, Chicago and Hi-C and the assembler with best performance was FALCON. The final N50 obtained here was ~43 Mb and the number of contigs was 1,143. The 56,469 genes annotated in this genome were used to unravel the evolutionary history of E. curvula. Furthermore we identify genes in the lignin pathway associated to the forage quality and genes of the WRKY family which are related to al multiple abiotic stress like drought stress. The genes related to the sexual pathway were evaluated through a comparative analysis with specific development stages in Oryza sativa. The tetraploids genomes of E. curvula sequenced here were derived from Tanganyika INTA, sequenced with Illumina, and Don Walter using a combination of Chromium 10x and Oxford Nanopore. The Tanganyika INTA assembly was performed with Masurca software obtaining an N50 of 4,715 bp and 293,000 contigs, while Don Walter was assembled with Supernova+DBG2OLC rendering an N50 of 224,390 bp and 7,542 contigs. Interestingly a region linked to apomixis was found through a comparative analysis between the genomes of E. curvula that could have an active role in the regulation of the reproductive pathway based on the literature. In this Thesis were sequenced, assembled and annotated three genomes of E. curvula representing a significant step forward in terms of characterisation of the species. The identification of the regions linked to apomixis, its expression and regulation could allow handling the trait and, in future instances, to introduce apomixis to sexual economically important crops.

El estrés ejerce sus efectos sobre el organismo, no sólo a través de las vías de respuesta fisiológica, sino también a través de vías genéticas y epigenéticas. Muchos autores sostienen que el estrés es un factor clave para explicar el papel evolutivo del sexo en los organismos de modo reproductivo facultativo. Por lo tanto, es esperable que los mismos se crucen más frecuentemente bajo situaciones de estrés, facilitando la adaptación a ambientes cambiantes. Eragrostis curvula es un complejo polimórfico donde la mayoría de sus miembros se reproducen por apomixis, reproducción asexual a través de semillas, que conduce a la generación de progenies genéticamente idénticas a la planta madre. El objetivo de esta tesis fue determinar el efecto de diferentes factores que generan estreses genómicos sobre la expresión de la apomixis en pasto llorón, como el estrés hídrico, el cultivo in vitro, la poliploidización y la hibridación intraespecífica. Para ello, se analizaron sacos embrionarios de plantas de distintos genotipos de pasto llorón luego de haber sido sometidas a dichas condiciones. Además, se realizaron pruebas de progenie con marcadores moleculares y se analizó el nivel de metilación de los genomas luego de los diferentes tratamientos. Antes de evaluar el efecto de situaciones estresantes sobre el modo reproductivo fue necesario analizar la estabilidad del carácter a lo largo del periodo de floración, hallándose que el cv. Tanganyika INTA presenta una sexualidad promedio menor al 2 % en condiciones de invernáculo. Plantas del cv. Tanganyika INTA bajo condiciones de estrés por déficit hídrico mostraron un 14 % de sacos sexuales, indicando un incremento de expresión de la sexualidad respecto de las plantas control. Luego de un periodo de rehidratación mostraron una sexualidad residual del 5,3 %, indicando una disminución significativa de la misma, siendo, sin embargo, significativamente diferente del control. Se realizaron pruebas de progenie con marcadores RAPDs y se detectaron polimorfismos solamente en las plantas sometidas a estrés hídrico, aunque en una proporción menor a la esperada de acuerdo a los análisis citoembriológicos, indicando que no todos los sacos embrionarios sexuales producidos dan lugar a plantas viables. El cv. Tanganyika USDA no evidenció en ningún momento la capacidad de generar sacos sexuales, clasificándoselo como apomíctico obligado. El genotipo facultativo UNST1131 presentó una proporción de procesos sexuales del 18,5 % bajo condiciones de estrés hídrico. Sin embargo este valor no fue significativamente distinto al control (10 %), debido principalmente a la gran variabilidad observada. Para evaluar efectos mediados por ABA se realizaron ensayos de aplicación foliar en plantas del cv. Tanganyika INTA normalmente regadas. El análisis citoembriológico no permitió detectar diferencias significativas en el número de sacos sexuales en relación con el control, descartándose momentáneamente a la concentración de esta hormona como mediadora del efecto generado por estrés. También se analizó el efecto del cultivo in vitro realizando diferentes ensayos en los que se varió el tipo de explanto, el medio de cultivo y la duración de los mismos. Se evaluaron los cultivares Tanganyika USDA y Tanganyika INTA. Solo uno de los ensayos (exposición a medios selectivos) generó un efecto significativo sobre el número de sacos sexuales producidos, que incrementaron, demostrando una cierta plasticidad del carácter en situaciones de estrés. Estos resultados indicarían que situaciones de estrés previas y durante la etapa de floración podrían estar regulando la expresión de la sexualidad en genotipos apomícticos facultativos de pasto llorón. Es evidente que en los cvs. Tanganyika USDA, Tanganyika INTA y UNS1131 expresan diferentes niveles de apomixis/sexualidad, desde cero a 33%. Por otro lado, este estudio permitió demostrar que no solo las condiciones externas promueven estreses genómicos que alteran la expresión de la apomixis en pasto llorón. Condiciones como la poliploidización y la hibridación intraespecífica también afectan al modo reproductivo. El análisis de la expresión de la apomixis a través del tiempo en dos híbridos (105 y 60) obtenidos a partir de la cruza entre materiales contrastantes para el modo reproductivo mostró diferencias en la expresión del carácter. El primero (105) mostró un comportamiento errático en las proporciones de procesos apomíctico/sexuales, con estabilización del carácter tendiente a una relación 1:1 mientras que el otro, (60) presentó solo sacos sexuales en las dos primeras floraciones y una pequeña proporción de sacos apomícticos en la tercera. Los resultados obtenidos en las pruebas de progenie mostraron correlación con los observados por estudios citoembriológicos. Dada la relación entre la apomixis y el ambiente, podría especularse que los genes involucrados en el modo reproductivo son controlados por factores epigenéticos como la metilación del ADN. Se realizaron análisis comparativos sobre los genomas (AFLP) y epigenomas (MSAP) de varios grupos de plantas con el fin de establecer el efecto de estreses genómicos sobre el genoma y epigenoma. Los grupos de plantas comparadas fueron: 1) tratadas por estrés hídrico y plantas control del cv. Tanganyika INTA y 2) plantas híbridas del primer año de análisis y luego de tres años. En el primer caso, no se hallaron cambios genéticos en el tiempo en las plantas sometidas a estrés hídrico y control, mientras que las comparaciones con marcadores MSAP permitieron evidenciar un gran número de marcadores polimórficos debidos a procesos de metilación y desmetilación del ADN entre los dos tiempos de muestreo. Si bien no existe una diferencia estadística entre la suma de cambios ocurridos en el tiempo entre el grupo de plantas control y tratadas, se observó un alto grado de correlación (R2= 0,7949) entre los porcentajes de cambios ocurridos en el tiempo y los porcentajes de sexualidad calculados a través del análisis citoembriológico para cada individuo. En el híbrido 60 no se hallaron cambios genéticos en el tiempo, pero si se hallaron en el híbrido 105. En el análisis de los perfiles epigenéticos, el híbrido 60 mostró una mayor proporción de procesos de metilación, mientras que el 105 reveló una tendencia mayoritaria hacia la desmetilación del ADN. Esto podría atribuirse a una mayor inestabilidad genómica, que podría asociarse a un comportamiento más errático del modo reproductivo. La secuenciación de una muestra de los polimorfismos obtenidos por AFLP y MSAP mostró una alta proporción (55 %) de secuencias sin homología con regiones conocidas del genoma, pudiendo tratarse de metilaciones y desmetilaciones en regiones no codificantes o en secuencias del genoma aún no informadas. Las secuencias que mostraron homología con secuencias previamente anotadas fueron principalmente elementos transponibles. Los resultados aquí presentados y los previos obtenidos por el grupo de trabajo nos permiten hipotetizar que existe una región determinante de la apomixis, que estaría ausente en las plantas tetraploides sexuales. La misma podría contener regiones repetitivas y con redundancia génica que estarían silenciando la sexualidad en plantas apomícticas. Existen numerosas evidencias sobre el control mediado por pequeños ARNs en el silenciamiento de regiones repetitivas y elementos transponibles. Ante determinadas circunstancias, como condiciones de estrés prolongado, en esta región podría estar ocurriendo un efecto de des-represión epigenética, permitiendo un aumento en la proporción de procesos sexuales. Si bien aún resta trabajo experimental para comprobar esta hipótesis, los procesos de metilación y desmetilación observados y los elementos transponibles secuenciados serían evidencias preliminares a considerar. / Stress affects the organisms generating physiological, genetic and epigenetic responses. Many authors argue that stress is a key factor to explain the evolutionary role of sex in organisms with facultative reproductive mode. Therefore, it is expected that facultative organisms will cross more frequently under stress, facilitating the adaptation to changing environments. Eragrostis curvula constitutes a polymorphic complex where most of its members reproduce by apomixis, asexual reproduction through seeds, which leads to the generation of progenies genetically identical to the mother plant. The aim of this work was to study the effect of different factors that generate genomic stress on the expression of apomixis in weeping lovegrass, such as water stress, in vitro culture, polyploidization and intraspecific hybridization. For this purpose, embryo sacs of different weeping lovegrass genotypes were observed after being the plants subjected to these stress conditions. Moreover, progeny tests were conducted by using molecular markers and the genome methylation status after different treatments was also assessed. Before evaluating the effect of the mentioned stressors on the reproductive mode it was necessary to analyze the stability of the trait throughout the flowering period, finding that plants of the cv. Tanganyika INTA growing in the greenhouse exhibited an average of sexuality less than 2 %. Plants of this cultivar subjected to water stress showed an average of 14% of sexual embryo sacs, indicating an increased expression of sexuality compared to control plants. After a rehydration period these plants showed an average of sexuality of 5.3 %, indicating a significant reduction in the number of sexual embryo sacs, being, however, significantly different from control plants. Progeny tests were performed with RAPDs markers detecting polymorphisms only in plants subjected to water stress, although in a less ratio than the expected according to the cyto-embryological analysis, indicating that not all the sexual embryo sacs produced develop into viable plants. Plants of the cv. Tanganyika USDA did not show at any time the ability to generate sexual embryo sacs and being classified as full apomictic one. The facultative genotype UNST1131 showed an expression of sexual processes of 18.5 % under water stress conditions, however this value was not significantly different to the control (10%), mainly due to the large variability exhibited by this material. To assess effects mediated by ABA, foliar application were performed on normally watered cv. Tanganyika INTA plants. The cyto-embryologic analysis was unable to detect significant differences in the number of sexual embryo sacs compared to the control, dismissing for the moment the hormone concentration as a mediator of the effect generated by stress. The effect of in vitro culture was also analyzed performing different tests in which the explant type, culture media and time under culture were changed. Tanganyika USDA and Tanganyika INTA cultivars were evaluated. In only one of the trials (exposure to selective media) a significant effect on the number of sexual embryo sacs was observed, which increased, showing a certain plasticity of the trait when the plants were growing under stress conditions. These results indicate that stress situations before and during the flowering stage might be regulating the expression of sexuality in facultative apomictic genotypes of weeping lovegrass. It is evident that Tanganyika USDA, Tanganyika INTA and UNS1131 cultivars express different levels of apomixis/sexuality ratios, from zero to 33%. This study also demonstrated that not only external conditions promote genomic stresses that alter the expression of apomixis in weeping lovegrass. Conditions such as intraspecific hybridization and polyploidization also affect the reproductive mode. The analysis of the expression of apomixis over time in two hybrids (#105 and #60) obtained from crosses between sexual X apomictic materials showed differences in the expression of the trait. The first one (#105) showed an erratic behavior in the ratio of apomictic/sexual processes, ending after two years with a ratio of 1/1, while the other (#60), only showed sexual embryo sacs on the two first blooms and a small proportion of apomictic embryo sacs during the third one. The results of progeny tests correlated very well with the cytoembryological observations. Given the close relationship between apomixis and environment, it might be speculated that the genes involved in the control of the reproductive mode could be subjected to epigenetic regulation, involving DNA methylation in some way. Comparative analyses at genomic (AFLP) and epigenetic (MSAP) levels were performed on different groups of plants in order to analyze the effect of genomic stresses. The groups compared were: 1) plants under water stress and normally watered plants (control) belonging to cv. Tanganyika INTA; 2) recently hybridized plants (first blooming) and the same plants in the third blooming period. In the first case (water stressed vs. control plants), no genetic changes over time were observed while comparisons with MSAP showed a large number of polymorphic markers due to processes of DNA methylations and de-methylations, showing a high correlation level (R2 = 0.7949) between the percentages of changes over time and the number of sexual embryo sacs. In the hybrid #60 no genetic changes were detected as it was in the #105. However, the analysis of epigenetics profiles showed a higher proportion of methylation processes in the hybrid #60 than in #105, while DNA demethylation levels were in the opposite direction. This could be associated with a higher genomic instability in #105 than in #60 that could be correlated with an increased erratic behavior of the reproductive mode. The sequencing of a sample of the polymorphic AFLP and MSAP bands showed a high proportion (55 %) of sequences without homology to previously annotated sequences, being probably noncoding regions or unknown genes. The other sequences were mainly transposable elements. The results presented here and those previously reported by our group allow us to hypothesize that there is a critical region conditioning apomixis, which would be absent in sexual tetraploid plants. This region could contain repetitive elements and gene redundancy that might be silencing sexuality in apomictic plants. Under certain circumstances, such as long stress periods, the control could be relaxed, allowing an increase in the proportion of sexual processes.