Development of a modified transformation platform for apomixis candidate genes research in Paspalum notatum (bahiagrass)

Abstract The aim of this work was to improve existing transformation protocols and to transform specific genotypes of Paspalum notatum (bahiagrass) for functional analyses of candidate genes involved in reproduction. Three different explants were assayed for in vitro plant regeneration: mature seeds, mature embryos, and shoot meristems. Plant regeneration was achieved with all explant types, but mature seeds produced the optimal rate (78.0%) and were easiest to manipulate. A method based on serial re-induction of calli from meristems of the regenerated lines was also developed, which could be useful in plant breeding strategies pursuing somaclonal variation. Transient transformation experiments were performed on calli obtained from mature seeds using a compressed helium gene gun. Transient transformation constructs included anthocyanin-synthesis genes cloned under the CAMV 35S promoter and an enhanced green fluorescent protein gene (egfp) driven by the rice actin1 (act1) promoter. Selection curves for ammonium glufosinate were developed in order to determine the optimal selective pressure for stable transformation (1.0 mg/L). Stable co-transformation experiments were carried out with two different constructs containing: (1) the reporter egfp gene cloned under the rice act1 promoter and (2) the selector bar gene driven by the ubiquitin promoter. A total of 27 (64.2%) transgenic plants out of 42 resistant plants analyzed were obtained. The presence of the transgenes in regenerated plants was confirmed by polymerase chain reaction and DNA gel blot analysis. Gene expression was demonstrated by eGFP fluorescence detection and in vivo assays for ammonium glufosinate tolerance. This platform is being used to generate transgenic plants of P. notatum to analyze the function of apomixis-associated candidate genes.Fil: Mancini, Micaela. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Laboratorio de Biología Molecular; ArgentinaFil: Woitovich Valetti, Nadia. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Laboratorio de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Cientifico Tecnológico Rosario; ArgentinaFil: Permingeat, Hugo Raúl. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Laboratorio de Biología Molecular; ArgentinaFil: Podio, Maricel. 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); ArgentinaFil: Siena, Lorena Adelina. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Laboratorio de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Cientifico Tecnológico Rosario; 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); ArgentinaFil: Pessino, Silvina Claudia. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Laboratorio de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Cientifico Tecnológico Rosario; ArgentinaFil: Felitti, Silvina Andrea. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Laboratorio de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Cientifico Tecnológico Rosario; Argentin

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

First insight into divergence, representation and chromosome distribution of reverse transcriptase fragments from L1 retrotransposons in peanut and wild relative species

Peanut is an allotetraploid (2n = 2x = 40, AABB) of recent origin. Arachis duranensis and A. ipaënsis, the most probable diploid ancestors of the cultigen, and several other wild diploid species with different genomes (A, B, D, F and K) are used in peanut breeding programs. However, the genomic relationships and the evolutionary pathways of genome differentiation of these species are poorly understood. We performed a sequence-based phylogenetic analysis of the L1 reverse transcriptase and estimated its representation and chromosome distribution in species of five genomes and three karyotype groups with the aim of contributing to the knowledge of the genomic structure and evolution of peanut and wild diploid relatives. All the isolated rt fragments were found to belong to plant L1 lineage and were named ALI. The best supported phylogenetic groups were not concordant with the genomes or karyotype groups. The copy number of ALI sequences was higher than the expected one for plants and directly related to genome size. FISH experiments revealed that ALI is mainly located on the euchromatin of interstitial and distal regions of most chromosome arms. Divergence of ALI sequences would have occurred before the differentiation of the genomes and karyotype groups of Arachis. The representation and chromosome distribution of ALI in peanut was almost additive of those of the parental species suggesting that the spontaneous hybridization of the two parental species of peanut followed by chromosome doubling would not have induced a significant burst of ALI transposition.Fil: Samoluk, Sergio Sebastián. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias; Argentina. 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: Robledo, Germán. 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 Nacional del Nordeste. Facultad de Cs.exactas Naturales y Agrimensura. Departamento de Agrimensura; ArgentinaFil: Podio, Maricel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Laboratorio de Biología Molecular; ArgentinaFil: Chalup, Laura María Isabel. 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 Nacional del Nordeste. Facultad de Ciencias Agrarias; ArgentinaFil: Ortiz, Juan Pablo Amelio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias; ArgentinaFil: Pessino, Silvina Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias; ArgentinaFil: Seijo, Jose Guillermo. 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 Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura; Argentin

Iudex Litem Suam Facere. Estudi des de l'Antiga Roma fins al nostre temps

En aquest el nostre treball ens endinsem en un món del Dret Romà poc tractat en els nostres manuals i en les classes magistrals. Es tracta de la figura dels quasidelictes de l'Antiga Roma, una institució a cavall del Dret Civil i el Dret Penal. En concret el nostre treball se centrarà en un dels quatre quasidelictes de les Res Cottidianare el Iudex Litem Suam Facere, quan el jutge es fa seu el litigi. Estudiarem el tracte des de llavors fins el nostre temps amb aportacions actuals

CYTOGENETIC ANALYSIS IN APOMICTIC AND SEXUAL PASPALUM NOTATUM AND MOLECULAR CHARACTERIZATION OF SEQUENCES ASSOCIATED WITH APOSPORY

Paspalum notatum Flügge es una gramínea rizomatosa perenne ampliamente difundida en las regiones tropicales y subtropicales de Sudamérica. Las razas tetraploides naturales de esta especie se reproducen casi exclusivamente por apomixis de tipo apospórica. La aposporía (un componente de la apomixis apospórica) en P. notatum es controlada por un locus único (denominado ACL) que presenta segregación distorsionada y supresión de la recombinación. La presencia de inversiones u otras alteraciones cromosómicas fueron propuestas para explicar el tipo de herencia observado para la aposporía. La determinación de la estructura cromosómica que contiene al ACL puede aportar conocimiento sobre la transmisión del carácter a la progenie y contribuir a la identificación de secuencias codificantes asociadas con su expresión. Por otro lado, varios marcadores moleculares completamente ligados a la aposporía y transcriptos de ARNm diferencialmente expresados en inflorescencias de plantas apomícticas y sexuales fueron identificados en P. notatum y otras especies de gramíneas. Sin embargo, hasta el momento solo unos pocos de ellos han sido estudiados en detalle a nivel genético y funcional. El objetivo general de este trabajo de Tesis fue profundizar los estudios tendientes a determinar las características citogenéticas y moleculares del locus responsable de la aposporía en P. notatum y caracterizar genes cuya expresión fue asociadas a la apomixis en gramíneas así como secuencias genómicas localizadas específicamente en el locus de la aposporía. En el Capítulo I se describen los estudios citogenéticos realizados en meiocitos de plantas apomícticas y sexuales de P. notatum. Como material vegetal fueron incluidas cinco accesiones apomícticas naturales, tres individuos sexuales obtenidos experimentalmente y 16 híbridos F1 clasificados por el modo de reproducción. Las observaciones citogenéticas revelaron que tanto las plantas apomícticas como las sexuales presentaron algunas anormalidades meióticas como, cromosomas rezagados, puentes de cromatina y micronúcleos. Sin embargo, un análisis cuantitativo de éstas reveló diferencias significativas entre las accesiones apomícticas y sexuales. Asimismo, el análisis de los híbridos confirmó las diferencias observadas en los progenitores con diferente modo de reproducción. Un estudio de FISH reveló que el ACL se encuentra en hemicigosis, muy próximo a regiones ricas en heterocromatina. Los resultados presentados en este Capítulo demostraron que las plantas apomícticas presentan un mayor número de anormalidades meióticas en la anafase I que las sexuales y que estas anormalidades se transmiten a la descendencia asociadas con el modo de reproducción. Asimismo fue posible determinar que el locus responsable de la aposporía se transmite solo a uno de los productos de la meiosis I. Esta estructura cromosómica podría explicar la distorsión de la segregación y la supresión de la recombinación asociada a la transmisión de la apomixis en la especie. En el Capítulo II se describe la caracterización molecular de los genes SERK y EXS en P. notatum. El gen SERK (somatic embryogenesis receptor like kinase) fue asociado a la apomixis en Poa pratensis. A partir del apilamiento de secuencias conocidas fue posible aislar dos secuencias genómicas de P. notatum con alta similitud a genes SERK: PnSERK1 (1750 pb) y PnSERK2 (2050pb). Al menos 3 copias de PnSERK están presentes en el genoma de P. notatum. Estudios de expresión por PCR en tiempo real (qRT-PCR) en diferentes estadios del desarrollo reproductivo del genotipo Q4188 (sexual) y Q4117 (apomíctico) mostraron que PnSERK2 se expresa a niveles superiores de PnSERK1 y que muestra diferencias significativas en los estadios de premeiosis, postmeiosis y antesis entre ambos genotipos. Experimentos de hibridación in situ de tejidos, mostraron una expresión contrastante entre el genotipo sexual y el apomíctico. Los resultados obtenidos indican que PnSERK2 presenta una expresión diferencial entre plantas apomícticas y sexuales y podría formar parte de la cascada de genes asociados a la expresión de la apomixis en P. notatum. Se analizó también el gen EXS. Una secuencia codificante para el dominio EXS fue identificada en un clon de BAC conteniendo marcadores moleculares completamente ligados a la aposporía en P. simplex. Mediante experimentos similares a los descriptos anteriormente se aisló un fragmento de 2298 pb con alta similitud al gen EXS de arroz y maíz de los genotipos Q4188 y Q4117. El análisis del número de copias reveló que existen entre 2 y 4 secuencias homólogas en el genoma de P. notatum. Estudios de expresión por qRT-PCR no mostraron diferencias significativas entre los genotipos sexuales y apomícticos en distintos estadios del desarrollo reproductivo. Sin embargo, estudios de hibridación in situ de tejido mostraron expresión de EXS en tejido ovárico del genotipo sexual mientras que solo se observó expresión en células que rodean a los sacos embrionarios en el genotipo apomíctico. El análisis de mapeo in silico, indicó que PnEXS se localiza en regiones que ya han sido reportadas como asociadas a la aposporía. Estos resultados indican que PnEXS podría estar diferencialmente regulado en plantas apomícticas y sexuales. En el Capítulo III se presentan los estudios realizados en la caracterización de secuencias específicas del ACL en P. notatum y el mapeo de transcriptos de ARNm asociados con el modo de reproducción. Varias de las secuencias correspondientes al ACL mostraron similitud con elementos repetitivos, proteínas hipotéticas, proteínas ribosomales y dominios codificantes. El mapeo de seis transcriptos expresados diferencialmente entre plantas apomícticas y sexuales mostró que N54 resultó ligado en repulsión al ACL. Los resultados obtenidos en esta Tesis permitieron detectar la presencia de un rearreglo cromosomal que se trasmite a la progenie asociado a la aposporía. Además, se logró localizar el ACL en un único cromosoma de P. notatum y determinar su condición hemicigota. Paralelamente, se pudo demostrar que PnSERK2 se encuentra funcionalmente asociado a la aposporía y que PnEXS estaría regulado diferencialmente en plantas apomícticas y sexuales. El análisis de marcadores 100% ligados al carácter puso en evidencia la presencia de secuencias codificantes que podrían estar asociadas a la aposporía.Paspalum notatum Flügge is a perennial rhizomatous grass species distributed in tropical and subtropical regions of South America. Natural tetraploid cytotypes reproduce by obligated aposporous apomixis. Apospory (a component of apomictic reproduction) in P. notatum is controlled by a single locus (ACL) with distorted segregation ratio and suppression of recombination. The presence of an inversion or other type of chromosomal alteration was proposed to explain the observed segregation fashion of the trait. Disclosing the chromosomal structure of the ACL can contribute to the understanding of the transmission of apospory and the identification of sequences associated with its expression. On the other hand, several molecular markers fully linked to the ACL as well as mRNA transcripts differentially expressed in inflorescences of apomictic and sexual plants were identified in P. notatum and other species of grasses. However, only a few of them have been studied in detail at the genetic and functional levels so far. The objective of this work was to determine the cytogenetic characteristic of apomictic and sexual tetraploid P. notatum and to characterize genes whose expression was associated with apomixis in grasses, as well as, a group of genomic sequences mapping at ACL in the species. Chapter I describes the studies carried out in meiocytes of apomictic and sexual plants of P. notatum. Five natural apomictic accessions, three completely sexual experimentallyobtained tetraploid individuals and 16 F1 hybrids classified by the mode of reproduction were used as plant material. The cytogenetic observations revealed that both sexual and apomictic plants showed meiotic abnormalities as, lagging chromosomes, chromatin bridges and micronuclei. However, a quantitative analysis of these configurations at anaphase I revealed significant differences between apomictic and sexual individuals. The analysis of hybrids confirmed the differences between accessions with different reproductive mode. FISH analysis showed that the ACL is hemizygous and it is placed close to a region rich in heterochromatin. These results indicated that apomictic plants showed a greater number of meiotic abnormalities in anaphase I than sexual ones and that abnormalities were transmitted to the offspring associated with the mode of reproduction. It was also possible to determine that the ACL is transmitted only to one of the products of meiosis I. This chromosomal structure could explain the segregation distortion and the suppression of recombination associated with transmission of apospory in the species. Chapter II describes the molecular characterization of genes SERK and EXS in P. notatum. The SERK (somatic embryogenesis receptor like kinase) gene was associated with apomixis in Poa pratensis. Two sequence PnSERK1 (1750 BP) and PnSERK2 (2050pb) were isolated. At least 3 copies of PnSERK were present in the P. notatum. Expression analysis by real time PCR (qRT-PCR) at different stages of the reproductive development of Q4188 (sexual) and Q4117 (apomictic) genotypes showed that PnSERK2 is expressed at higher levels than PnSERK1 and showed significant differences between genotypes at premeiosis, postmeiosis and anthesis. In situ hybridization with PnSERK2, showed a contrasting expression between the apomictic and sexual plants. Results presented in this work indicate that PnSERK2 participate in the cascade of genes associated with the expression of apomixis in P. notatum. On the other hand, a coding sequence showing an EXS domain was identified in a BAC clone containing molecular makers completely linked apospory in P. simplex. A fragment of 2298 pb highly similar to the EXS domain of rice and maize was isolated from genotypes Q4188 and Q4117. Copy number analysis revealed that there are between 2 and 4 homologous sequences in the P. notatum genome. Expression analysis by qRT-PCR at different stages of the reproductive development showed no significant differences among genotypes. However, in situ tissue hybridization showed expression of EXS in ovaries of the sexual genotype, while EXS was expressed only in the surrounding cells of the embryo sac in the apomictic genotype. In silico mapping, indicated that PnEXS is located in regions that have associated previously with apospory. These results indicate that EXS could be under a different expression control in sexual and apomictic plants. Chapter III presents studies focused on the characterization of specific sequences mapping at the ACL in P. notatum and the localization of mRNA transcripts associated with the mode of reproduction. Several sequences of the ACL showed similarity with repetitive elements, hypothetical proteins, ribosomal proteins and coding domains. Mapping assays of six transcripts differentially expressed between apomictic and sexual plants showed that clone N54 was linked in repulsion to the ACL. This can be a good candidate for further analysis. The results presented in this Thesis showed the presence of a chromosomal rearrangement associated with apospory that it is transmitted to the progeny. In addition, it was determined that ACL of P. notatum is placed in a hemizygous region. Analysis of PnSERK2 and PnEXS indicated that both genes are associated with apomixis. Moreover, analysis of markers 100% linked to the character revealed the presence of coding sequence in this region.Fil: Apellido, Nombre. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias; ArgentinaPodio, Maricel. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias; Argentin

Disclosing the Molecular Link between Apospory and Diplospory

Gametophytic apomixis forms seeds with clonal embryos identical to the mother plant through two different mechanisms, namely apospory and diplospory, both of them involving the formation of nonreduced megagametophytes within the ovule. These nonreduced embryo sacs are originated from companion nucellar cells (apospory) or the megaspore mother cell, after absence/failure of meiosis (diplospory). In both paths the nonreduced egg cells produce embryos through parthenogenesis. Our hypothesis here was that, at least for some candidate genes, apomeiosis and parthenogenesis associate, respectively, with contrasting or identical representation patterns in aposporous vs. diplosporous differential mRNA expression comparisons. The rationale supporting this proposition is that in aposporous and diplosporous plants apomeiosis might involve some common genes under an opposite spatio-temporal regulation pattern (OE transcripts), while parthenogenesis should be identically activated in both reproductive types (IE candidates). We compared two sets of Roche/454 sequences differentially expressed in reproductive organs of sexual vs. apomictic plants of Paspalum notatum (aposporous) and Eragrostis curvula (diplosporous) from premeiosis to anthesis, to identify OE and IE transcripts. Then, the selected sequences were investigated in TruSeq/Hiseq Illumina RNA floral libraries constructed at different reproductive stages. We found 89 opposite expression transcripts exclusively represented at premeiosis and 66 equal expression transcripts occurring at anthesis only. These candidate genes were used to produce String interaction networks with others previously related with apomeiosis and parthenogenesis, and their location is being mapped within the Paspalum and Eragrostis genomic ACL (apomixis controlling loci). Functional analysis of selected sequences is at work.Fil: Garbus, Ingrid. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Centro de Recursos Naturales Renovables de la Zona Semiárida. Universidad Nacional del Sur. Centro de Recursos Naturales Renovables de la Zona Semiárida; Argentina. Universidad Nacional del Sur. Departamento de Ciencias de la Salud; ArgentinaFil: Podio, Maricel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; ArgentinaFil: Echenique, Carmen Viviana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Centro de Recursos Naturales Renovables de la Zona Semiárida. Universidad Nacional del Sur. Centro de Recursos Naturales Renovables de la Zona Semiárida; Argentina. Universidad Nacional del Sur. Departamento de Agronomía; ArgentinaFil: Pessino, Silvina Claudia. 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; Argentina7th Series of Seminars on Advances in Apomixis ResearchArgentinaMechanisms of Apomictic Developmen

The Auxin-Response Repressor IAA30 Is Down-Regulated in Reproductive Tissues of Apomictic Paspalum notatum

International audienceThe capacity for apomixis in Paspalum notatum is controlled by a single-dominant genomic region, which shows strong synteny to a portion of rice chromosome 12 long arm. The locus LOC_Os12g40890, encoding the Auxin/Indole-3-Acetic Acid (Aux/IAA) family member OsIAA30, is located in this rice genomic segment. The objectives of this work were to identify transcripts coding for Aux/IAA proteins expressed in reproductive tissues of P. notatum, detect the OsIAA30 putative ortholog and analyze its temporal and spatial expression pattern in reproductive organs of sexual and apomictic plants. Thirty-three transcripts coding for AUX/IAA proteins were identified. Predicted protein alignment and phylogenetic analysis detected a highly similar sequence to OsIAA30 (named as PnIAA30) present in both sexual and apomictic samples. The expression assays of PnIAA30 showed a significant down-regulation in apomictic spikelets compared to sexual ones at the stages of anthesis and post-anthesis, representation levels negatively correlated with apospory expressivity and different localizations in sexual and apomictic ovules. Several PnIAA30 predicted interactors also appeared differentially regulated in the sexual and apomictic floral transcriptomes. Our results showed that an auxin-response repressor similar to OsIAA30 is down-regulated in apomictic spikelets of P. notatum and suggests a contrasting regulation of auxin signaling during sexual and asexual seed formation

Sequence characterization, in silico mapping and cytosine methylation analysis of markers linked to apospory in Paspalum notatum

In previous studies we reported the identification of several AFLP, RAPD and RFLP molecular markers linked to apospory in Paspalum notatum . The objective of this work was to sequence these markers, obtain their flanking re -gions by chromosome walking and perform an in silico mapping analysis in rice and maize. The methylation status of two apospory-related sequences was also assessed using methylation-sensitive RFLP experiments. Fourteen mo -lecular markers were analyzed and several protein-coding sequences were identified. Copy number estimates and RFLP linkage analysis showed that the sequence PnMAI3 displayed 2-4 copies per genome and linkage to apospory. Extension of this marker by chromosome walking revealed an additional protein-coding sequence map- ping in silico in the apospory-syntenic regions of rice and maize. Approximately 5 kb corresponding to different mark- ers were characterized through the global sequencing procedure. A more refined analysis based on sequence information indicated synteny with segments of chromosomes 2 and 12 of rice and chromosomes 3 and 5 of maize. Two loci associated with apomixis locus were tested in methylation-sensitive RFLP experiments using genomic DNA extracted from leaves. Although both target sequences were methylated no methylation polymorphisms associated with the mode of reproduction were detected.Laboratorio de Biología Molecular, Facultad de Ciencias Agrarias, Universidad Nacional de Rosario, Zavalla, Santa Fe, Argentin

Evaluation of meiotic abnormalities and pollen viability in aposporous and sexual tetraploid Paspalum notatum (Poaceae)

We analyzed anaphase I configurations and pollen viability in aposporous and sexual tetraploid (2n = 4x = 40) cytotypes of Paspalum notatum. Five natural aposporous accessions and three experimentally obtained sexual individuals were used. In addition, 16 (8aposporous and 8 sexual) F1 hybrids, previously classified by their mode of reproduction, were analyzed. Cytogenetic observations revealed normal and abnormal anaphase I configurations in both aposporous and sexual genotypes. Anaphase I abnormalities were mainly laggard chromosomes, chromatin bridges, and micronuclei. On average, 44.36 % of aposporous meiocytes and 29.66 % of sexual ones showed abnormal anaphase I configurations. The total numbers of normal and abnormal anaphase I were highly significantly different between aposporous and sexual strains. The pollen viability test indicated that aposporous individuals had significantly more non-viable pollen than sexual ones; a positive correlation (r = 0.71; r2 = 0.50) between the variables was detected. Analysis of aposporous and sexual hybrids confirmed differences in the numbers of normal and abnormal anaphase I patterns in the aposporous and sexual parents. However, similar proportions of viable pollen were produced by both groups of hybrids. In this case, the variables were not correlated (r = 0.23; r2 = 0.05). Data from this study indicated that aposporous strains had a genetic rearrangement affecting meiosis that was absent in the experimentally obtained sexual individuals and that it was transmitted to the progeny. The possible association between meiotic abnormalities and the inheritance of apospory is discussed.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 Ciencias Agrarias. Laboratorio de Biología Molecular; ArgentinaFil: Siena, Lorena Adelina. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Laboratorio de Biología Molecular; ArgentinaFil: Hojsgaard, Diego Hernan. Universitat of Gottingen; AlemaniaFil: Stein, Juliana. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Laboratorio de Biología Molecular; ArgentinaFil: Quarin, Camilo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Nordeste. Instituto de Botánica del Nordeste (i); ArgentinaFil: Ortiz, Juan Pablo Amelio. 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; Argentin

Spotting the Targets of the Apospory Controller TGS1 in Paspalum notatum

International audienceSexuality and apomixis are interconnected plant reproductive routes possibly behaving as polyphenic traits under the influence of the environment. In the subtropical grass Paspalum notatum, one of the controllers of apospory, a main component of gametophytic apomixis reproduction, is TRIMETHYLGUANOSINE SYNTHASE 1 (TGS1), a multifunctional gene previously associated with RNA cleavage regulation (including mRNA splicing as well as rRNA and miRNA processing), transcriptional modulation and the establishment of heterochromatin. In particular, the downregulation of TGS1 induces a sexuality decline and the emergence of aposporous-like embryo sacs. The present work was aimed at identifying TGS1 target RNAs expressed during reproductive development of Paspalum notatum. First, we mined available RNA databases originated from spikelets of sexual and apomictic plants, which naturally display a contrasting TGS1 representation, to identify differentially expressed mRNA splice variants and miRNAs. Then, the role of TGS1 in the generation of these particular molecules was investigated in antisense tgs1 sexual lines. We found that CHLOROPHYLL A-B BINDING PROTEIN 1B-21 (LHC Ib-21, a component of the chloroplast light harvesting complex), QUI-GON JINN (QGJ, encoding a MAP3K previously associated with apomixis) and miR2275 (a meiotic 24-nt phasi-RNAs producer) are directly or indirectly targeted by TGS1. Our results point to a coordinated control exercised by signal transduction and siRNA machineries to induce the transition from sexuality to apomixis