Control genético del tamaño floral en Antirrhinum majus

[SPA] El objetivo principal del presente trabajo de tesis es entender los mecanismos genéticos del tamaño floral en Antirrhinum majus. Para ello se ha llevado a cabo un estudio genético de mutantes de A. majus que afectan al tamaño floral. En primer lugar se evaluó la estabilidad de los parámetros en estudio frente a condiciones de hacinamiento y reducción artificial del área foliar en plantas silvestre (165E). Estos resultados demostraron que los órganos laterales del cuerpo tienden a reducir la masa total frente al hacinamiento. Las hojas disminuyen de forma preferencial en tamaño, manteniendo el número. De forma opuesta, se reduce la producción de flores mientras el tamaño floral permanece prácticamente imperturbable. Estos resultados indican que el tamaño floral es un carácter estable frente a las condiciones de hacinamiento y a la reducción del área foliar. Se seleccionaron mutantes de A. majus en genes que afectan el tamaño y las proporciones florales: COMPACTA (CO), GRANDIFLORA (GRAF) Y FORMOSA (FO). En primer lugar se realizaron análisis de segregación en poblaciones F2 y se observó que los mutantes co y fo son alelos recesivos mientras el mutante Graf es un alelo dominante. El mutante co presenta reducción en el tamaño de todos los órganos de las plantas incluyendo hojas y flores. Afecta de forma específica los sépalos, pétalos y gineceos reduciendo su tamaño y provocando variaciones en la expansión y/o la división celular dependiendo de los diferentes órganos o verticilos de la flor. Graf, que presente un efecto prácticamente exclusivo en el tamaño de la flor, parece provocar cambios opuestos a co incrementando el tamaño de todos los órganos florales. Provoca un aumento en la expansión celular de la superficie de los pétalos y en la división en los órganos sexuales. El análisis de los dobles mutantes Graf co sugiere una posible participación de ambos genes en una misma ruta de control del tamaño de los pétalos. Los dobles mutantes entre co y un gen de identidad de clase B (que también reduce el tamaño de los órganos florales) muestran sinergismo entre ambos genes en cuanto al tamaño y la identidad del pétalo. Estos resultados sugieren que ambos genes participan en rutas paralelas en el control del tamaño e identidad de los órganos del segundo verticilo. El mutante fo, afecta exclusivamente el tamaño de la flor con órganos florales más grandes en los tres verticilos externos. Produce un incremento en el número de células en todos los órganos provocando un aumento de los niveles de expresión de AmAINTEGUMENTA en los meristemos florales así como un aumento de la expresión de genes relacionados con la actividad del ciclo celular en pétalos. También reduce la expansión celular de pétalos y gineceos causando una mayor expresión del gen AmBigPetal. El análisis de los dobles mutantes con un gen de identidad de clase C muestra que la acción diferencial de fo en cuanto al aumento en el tamaño de los órganos no parece ser específica de la identidad de los órganos. En el doble mutante también se observa sinergismo en cuanto al aumento del tamaño producido por fo lo que sugiere una posible participación de ambos genes en control del tamaño de los pétalos por vías redundantes. La presencia de mutantes pleiotrópicos afectando el tamaño de todos los órganos laterales de las plantas y de genes con efectos específicos en el tamaño floral indican que existen al menos dos grupos de genes que controlan el tamaño floral, uno que controla tanto el crecimiento vegetativo como reproductivo y otro específico de la flor.[ENG] The main objective of this thesis is to uncover the genetic control of floral size in Antirrhinum majus. We have done a characterization of A. majus mutants. First step was the evaluation of the stability of floral size against crowding conditions and artificial foliar reduction, in wild type 165E plants. These results demonstrated that plant lateral organs tend to reduce total mass upon crowding. Leaf number proofed a stable character, while leaf size decreased. In contrary, flower number was reduced while floral size was stable. Results indicate that floral size is a stable character both under crowding and foliar reduction. The following genes of A. majus whose mutants affect floral size and proportions were selected: COMPACTS (CO), GRANDIFLORA (GRAF) Y FORMOSA (FO). A segregation analysis in F2 populations revealed that co and fo mutants are recessive alleles while Graf mutant is a dominant allele. The mutant co shows a reduction in the size of all plant lateral organs including flowers and leaves. In the flowers we observed a size reduction of sepals, petals and carpels either due to changes in cell expansion and/or cell division, depending on the floral organ identity or the floral whorl. Graf, which almost exclusive effect flower size, shows a phenotype opposite to co with an increase in size of all floral organs. Changes are due to an increment on cell expansion in petals and cell division in sexual organs. Analysis of the double mutant, Graf co, suggests that both genes act in the same route controlling petal size. Double mutants between co and a class B floral identity gene (which also reduces floral organ size) show a synergistic effect between both genes concerning organ size and petal identity. This suggests that the two genes could participate in organ size control and petal identity in parallel routes. The mutant fo affect exclusively flowers showing bigger floral organs in the three outer whorls. Increases cell number in all floral organs. Expression level of AINTEGUMENTA is elevated in floral meristems and those of cell cycle related genes in mutant petals. Cell expansion is reduced in petals and carpels and this coincides with an up regulation of BigPetal gene. Double mutant analysis between fo and a class C flower identity gene indicates that fo effect is not specific of organ identity. The fo double mutant also shows synergism with the class C identity gene suggesting a putative participation of both genes in redundant routes in the control of petal size. The presences of pleiotropic mutants affecting all plant lateral organs, and genes that disturb only flower size indicate the presence of at least two gene groups, one controlling vegetative and reproductive growth and another flower specific one.Universidad Politécnica de CartagenaPrograma de doctorado en Tecnología Agraria y Alimentari

Analysis of variation for apomictic reproduction in diploid Paspalum rufum

Background and Aims: The diploid cytotype of Paspalum rufum (Poaceae) reproduces sexually and is self-sterile; however, recurrent autopolyploidization through 2n + n fertilization and the ability for reproduction via apomixis have been documented in one genotype of the species. The objectives of this work were to analyse the variation in the functionality of apomixis components in diploid genotypes of P. rufum and to identify individuals with contrasting reproductive behaviours. Methods: Samples of five individuals from each of three natural populations of P. rufum (designated R2, R5 and R6) were used. Seeds were obtained after open pollination, selfing, conspecific interploidy crosses and interspecific interploidy self-pollination induction. The reproductive behaviour of each plant was determined by using the flow cytometric seed screen (FCSS) method. Embryo sacs were cleared using a series of ethanol and methyl salicylate solutions and observed microscopically. Key Results: In open pollination, all genotypes formed seeds by sexual means and no evidence of apomeiotic reproduction was detected. However, in conspecific interploidy crosses and interspecific interploidy self-pollination induction, variations in the reproductive pathways were observed. While all plants from populations R2 and R6 formed seeds exclusively by sexual means, three genotypes from the R5 population developed seeds from both meiotic and aposporous embryo sacs, and one of them (R5#49) through the complete apomictic pathway (apospory + parthenogenesis + pseudogamy). Cytoembryological observations revealed the presence of both meiotic and aposporous embryo sacs in all the genotypes analysed, suggesting that parthenogenesis could be uncoupled from apospory in some genotypes. Conclusions: The results presented demonstrate the existence of variation in the functionality of apomixis components in natural diploid genotypes of P. rufum and have identified individuals with contrasting reproductive behaviours. Genotypes identified here can be crossed to generate segregating populations in order to study apomixis determinants at the diploid level. Moreover, analysis of their expression patterns, quantification of their transcript levels and an understanding of their regulation mechanisms could help to design new strategies for recreating apomixis in a diploid genome environment.Fil: Delgado Benarroch, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Nordeste. Instituto de Botánica del Nordeste (i); Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Laboratorio de Biología Molecular; ArgentinaFil: Galdeano, Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Nordeste. Instituto de Botánica del Nordeste (i); ArgentinaFil: Sartor, Maria Esperanza. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Nordeste. Instituto de Botánica del Nordeste (i); ArgentinaFil: Quarin, Camilo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Nordeste. Instituto de Botánica del Nordeste (i); ArgentinaFil: Espinoza, Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Nordeste. Instituto de Botánica del Nordeste (i); ArgentinaFil: Ortiz, Juan Pablo Amelio. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Laboratorio de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Nordeste. Instituto de Botánica del Nordeste (i); Argentin

A molecular recombination map of Antirrhinum majus

<p>Abstract</p> <p>Background</p> <p>Genetic recombination maps provide important frameworks for comparative genomics, identifying gene functions, assembling genome sequences and for breeding. The molecular recombination map currently available for the model eudicot <it>Antirrhinum majus </it>is the result of a cross with <it>Antirrhinum molle</it>, limiting its usefulness within <it>A. majus</it>.</p> <p>Results</p> <p>We created a molecular linkage map of <it>A</it>. <it>majus </it>based on segregation of markers in the F2 population of two inbred lab strains of <it>A. majus</it>. The resulting map consisted of over 300 markers in eight linkage groups, which could be aligned with a classical recombination map and the <it>A. majus </it>karyotype. The distribution of recombination frequencies and distorted transmission of parental alleles differed from those of a previous inter-species hybrid. The differences varied in magnitude and direction between chromosomes, suggesting that they had multiple causes. The map, which covered an estimated of 95% of the genome with an average interval of 2 cM, was used to analyze the distribution of a newly discovered family of MITE transposons and tested for its utility in positioning seven mutations that affect aspects of plant size.</p> <p>Conclusions</p> <p>The current map has an estimated interval of 1.28 Mb between markers. It shows a lower level of transmission ratio distortion and a longer length than the previous inter-species map, making it potentially more useful. The molecular recombination map further indicates that the <it>IDLE </it>MITE transposons are distributed throughout the genome and are relatively stable. The map proved effective in mapping classical morphological mutations of <it>A. majus</it>.</p

Characterization and expression analysis of Somatic Embryogenesis Receptor Kinase (SERK) genes in sexual and apomictic Paspalum notatum

The Somatic Embryogenesis Receptor Kinase (SERK) gene plays a fundamental role in somatic embryogenesis of angiosperms, and is associated with apomixis in Poa pratensis. The objective of this work was to isolate, characterize and analyze the expression patterns of SERK genes in apomictic and sexual genotypes of Paspalum notatum. A conserved 200-bp gene fragment was amplified from genomic DNA with heterologous primers, and used to initiate a chromosomal walking strategy for cloning the complete sequence. This procedure allowed the isolation of two members of the P. notatum SERK family; PnSERK1, which is similar to PpSERK1, and PnSERK2, which is similar to ZmSERK2 and AtSERK1. Phylogenetic analyses indicated that PnSERK1 and PnSERK2 represent paralogous sequences. Southern-blot hybridization indicated the presence of at least three copies of SERK genes in the species. qRT-PCR analyses revealed that PnSERK2 was expressed at significantly higher levels than PnSERK1 in roots, leaves, reproductive tissues and embryogenic calli.Moreover, in situ hybridization experiments revealed that PnSERK2 displayed a spatially and chronologically altered expression pattern in reproductive organs of the apomictic genotype with respect to the sexual one. PnSERK2 is expressed in nucellar cells of the apomictic genotype at meiosis, but only in the megaspore mother cell in the sexual genotype. Therefore, apomixis onset in P. notatum seems to be correlated with the expression of PnSERK2 in nucellar tissue.Fil: Podio, Maricel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Nordeste. Instituto de Botánica del Nordeste (i); Argentina; Universidad Nacional de Rosario. Facultad de Cs.agrarias. Laboratorio de Biologia Molecular; Argentina;Fil: Felitti, Silvina Andrea. Universidad Nacional de Rosario. Facultad de Cs.agrarias. Laboratorio de Biologia Molecular; Argentina;Fil: Siena, Lorena Adeli. Universidad Nacional de Rosario. Facultad de Cs.agrarias. Laboratorio de Biologia Molecular; Argentina;Fil: Delgado Benarroch, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Nordeste. Instituto de Botánica del Nordeste (i); Argentina; Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias; Argentina;Fil: Mancini, Micaela. Universidad Nacional de Rosario. Facultad de Cs.agrarias. Laboratorio de Biologia Molecular; Argentina;Fil: Seijo, Jose Guillermo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Nordeste. Instituto de Botánica del Nordeste (i); Argentina;Fil: Gonzalez, Ana María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Nordeste. Instituto de Botánica del Nordeste (i); Argentina;Fil: Pessino, Silvina Claudia. Universidad Nacional de Rosario. Facultad de Cs.agrarias. Laboratorio de Biologia Molecular; Argentina;Fil: Ortiz, Juan Pablo Amelio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Nordeste. Instituto de Botánica del Nordeste (i); Argentina; Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias; Argentina; Universidad Nacional de Rosario. Facultad de Cs.agrarias. Laboratorio de Biologia Molecular; Argentina

Formation and Shaping of the Antirrhinum Flower through Modulation of the CUP Boundary Gene

Boundary domain genes, expressed within or around organ primordia, play a key role in the formation, shaping, and subdivision of planar plant organs, such as leaves. However, the role of boundary genes in formation of more elaborate 3D structures, which also derive from organ primordia, remains unclear. Here we analyze the role of the boundary domain gene CUPULIFORMIS (CUP) in formation of the ornate Antirrhinum flower shape. We show that CUP expression becomes cleared from boundary subdomains between petal primordia, most likely contributing to formation of congenitally fused petals (sympetally) and modulation of growth at sinuses. At later stages, CUP is activated by dorsoventral genes in an intermediary region of the corolla. In contrast to its role at organ boundaries, intermediary CUP activity leads to growth promotion rather than repression and formation of the palate, lip, and characteristic folds of the closed Antirrhinum flower. Intermediary expression of CUP homologs is also observed in related sympetalous species, Linaria and Mimulus, suggesting that changes in boundary gene activity have played a key role in the development and evolution of diverse 3D plant shapes

pcrEfficiency: a Web tool for PCR amplification efficiency prediction

<p>Abstract</p> <p>Background</p> <p>Relative calculation of differential gene expression in quantitative PCR reactions requires comparison between amplification experiments that include reference genes and genes under study. Ignoring the differences between their efficiencies may lead to miscalculation of gene expression even with the same starting amount of template. Although there are several tools performing PCR primer design, there is no tool available that predicts PCR efficiency for a given amplicon and primer pair.</p> <p>Results</p> <p>We have used a statistical approach based on 90 primer pair combinations amplifying templates from bacteria, yeast, plants and humans, ranging in size between 74 and 907 bp to identify the parameters that affect PCR efficiency. We developed a generalized additive model fitting the data and constructed an open source Web interface that allows the obtention of oligonucleotides optimized for PCR with predicted amplification efficiencies starting from a given sequence.</p> <p>Conclusions</p> <p>pcrEfficiency provides an easy-to-use web interface allowing the prediction of PCR efficiencies prior to web lab experiments thus easing quantitative real-time PCR set-up. A web-based service as well the source code are provided freely at <url>http://srvgen.upct.es/efficiency.html</url> under the GPL v2 license.</p

Monoaminergic and histaminergic strategies and treatments in brain diseases

The monoaminergic systems are the target of several drugs for the treatment of mood, motor and cognitive disorders as well as neurological conditions. In most cases, advances have occurred through serendipity, except for Parkinson's disease where the pathophysiology led almost immediately to the introduction of dopamine restoring agents. Extensive neuropharmacological studies first showed that the primary target of antipsychotics, antidepressants, and anxiolytic drugs were specific components of the monoaminergic systems. Later, some dramatic side effects associated with older medicines were shown to disappear with new chemical compounds targeting the origin of the therapeutic benefit more specifically. The increased knowledge regarding the function and interaction of the monoaminergic systems in the brain resulting from in vivo neurochemical and neurophysiological studies indicated new monoaminergic targets that could achieve the efficacy of the older medicines with fewer side-effects. Yet, this accumulated knowledge regarding monoamines did not produce valuable strategies for diseases where no monoaminergic drug has been shown to be effective. Here, we emphasize the new therapeutic and monoaminergic-based strategies for the treatment of psychiatric diseases. We will consider three main groups of diseases, based on the evidence of monoamines involvement (schizophrenia, depression, obesity), the identification of monoamines in the diseases processes (Parkinson's disease, addiction) and the prospect of the involvement of monoaminergic mechanisms (epilepsy, Alzheimer's disease, stroke). In most cases, the clinically available monoaminergic drugs induce widespread modifications of amine tone or excitability through neurobiological networks and exemplify the overlap between therapeutic approaches to psychiatric and neurological conditions. More recent developments that have resulted in improved drug specificity and responses will be discussed in this review.peer-reviewe

Heterochronic reproductive developmental processes between diploid and tetraploid cytotypes of Paspalum rufum

BACKGROUND AND AIMS: Apomixis is an asexual reproductive mode via seeds that generate maternal clonal progenies. Although apomixis in grasses is mainly expressed at the polyploid level, some natural diploid genotypes of Paspalum rufum produce aposporous embryo sacs in relatively high proportions and are even able to complete apomixis under specific conditions. However, despite the potential for apomixis, sexuality prevails in diploids, and apomixis expression is repressed for an as yet undetermind reason. Apomixis is thought to derive from a deregulation of one or a few components of the sexual pathway that could be triggered by polyploidy and/or hybridization. The objectives of this work were to characterize and compare the reproductive development and the timing of apospory initial (AI) emergence between diploid genotypes with potential for apomixis and facultative apomictic tetraploid cytotypes of P. rufum. METHODS: Reproductive characterization was performed by cytoembryological observations of cleared ovaries and anthers during all reproductive development steps and by quantitative evaluation of the ovule growth parameters. KEY RESULTS: Cytoembryological observations showed that in diploids, both female and male reproductive development is equally synchronized, but in tetraploids, megasporogenesis and early megagametogenesis are delayed with respect to microsporogenesis and early microgametogenesis. This delay was also seen when ovary growth was taken as a reference parameter. The analysis of the onset of AIs revealed that they emerge during different developmental periods depending on the ploidy level. In diploids, the AIs appeared along with the tetrad (or triad) of female meiocytes, but in tetraploids they appeared earlier, at the time of the megaspore mother cell. In both cytotypes, AIs can be seen even during megagametogenesis. CONCLUSIONS: Overall observations reveal that female sexual reproductive development is delayed in tetraploids as compared with diploid genotypes, mainly at meiosis. In tetraploids, AIs appear at earlier sexual developmental stages than in diploids, and they accumulate up to the end of megasporogenesis. The longer extension of megasporogenesis in tetraploids could favour AI emergence and also apomixis success.Fil: Soliman, Mariano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Investigaciones en Ciencias Agrarias de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Instituto de Investigaciones en Ciencias Agrarias de Rosario; ArgentinaFil: Espinoza, Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Botánica del Nordeste. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias. Instituto de Botánica del Nordeste; ArgentinaFil: Ortiz, Juan Pablo Amelio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Investigaciones en Ciencias Agrarias de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Instituto de Investigaciones en Ciencias Agrarias de Rosario; ArgentinaFil: Delgado Benarroch, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Investigaciones en Ciencias Agrarias de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Instituto de Investigaciones en Ciencias Agrarias de Rosario; Argentin

Quantitative levels of Deficiens and Globosa during late petal development show a complex transcriptional network topology of B function

The transcriptional network topology of B function in Antirrhinum, required for petal and stamen development, is thought to rely on initial activation of transcription of DEFICIENS (DEF) and GLOBOSA (GLO), followed by a positive autoregulatory loop maintaining gene expression levels. Here, we show that the mutant compacta (co), whose vegetative growth and petal size are affected, plays a role in B function. Late events in petal morphogenesis such as development of conical cell area and scent emissions were reduced in co and def nicotianoides (def nic), and absent in co def nic double mutants, suggesting a role for CO in petal identity. Expression of DEF was down-regulated in co but surprisingly GLO was not affected. We investigated the levels of DEF and GLO at late stages of petal development in the co, def nic and glo-1 mutants, and established a reliable transformation protocol that yielded RNAi-DEF lines. We show that the threshold levels of DEF or GLO required to obtain petal tissue are approximately 11% of wild-type. The relationship between DEF and GLO transcripts is not equal or constant and changes during development. Furthermore, down-regulation of DEF or GLO does not cause parallel down-regulation of the partner. Our results demonstrate that, at late stages of petal development, the B function transcriptional network topology is not based on positive autoregulation, and has additional components of transcriptional maintenance. Our results suggest changes in network topology that may allow changes in protein complexes that would explain the fact that not all petal traitsappear early in development.Fil: Manchado Rojo, María. Universidad Politécnica de Cartagena; EspañaFil: Delgado Benarroch, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Botánica del Nordeste. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias. Instituto de Botánica del Nordeste; Argentina. Universidad Politécnica de Cartagena; EspañaFil: Roca, María José. Universidad Politécnica de Cartagena; EspañaFil: Weiss, Julia. Universidad Politécnica de Cartagena; EspañaFil: Marcos, Egea Cortines. Universidad Politécnica de Cartagena; Españ