15 research outputs found
Temporal and spatial expression of genes involved in DNA methylation during reproductive development of sexual and apomictic Eragrostis curvula
Recent reports in model plant species have highlighted a role for DNA methylation pathways in the regulation of the somatic-to-reproductive transition in the ovule, suggesting that apomixis (asexual reproduction through seeds) likely relies on RdDM downregulation. Our aim was therefore to explore this hypothesis by characterizing genes involved in DNA methylation in the apomictic grass Eragrostis curvula. We explored floral transcriptomes to identify homologs of three candidate genes, for which mutations in Arabidopsis and maize mimic apomixis (AtAGO9/ZmAGO104, AtCMT3/ZmDMT102/ZmDMT105, and AtDDM1/ZmCHR106), and compared both their spatial and temporal expression patterns during reproduction in sexual and apomictic genotypes. Quantitative expression analyses revealed contrasting expression patterns for the three genes in apomictic vs sexual plants. In situ hybridization corroborated these results for two candidates, EcAGO104 and EcDMT102, and revealed an unexpected ectopic pattern for the AGO gene during germ line differentiation in apomicts. Although our data partially support previous results obtained in sexual plant models, they suggest that rather than an RdDM breakdown in the ovule, altered localization of AtAGO9/ZmAGO104 expression is required for achieving diplospory in E. curvula. The differences in the RdDM machinery acquired during plant evolution might have promoted the emergence of the numerous apomictic paths observed in plants.Fil: Selva, Juan Pablo. 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 Biología, Bioquímica y Farmacia; ArgentinaFil: Siena, Lorena Adelina. 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: Rodrigo, Juan Manuel. 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: 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: Zappacosta, Diego Carlos. 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: Romero, José Rodolfo. 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; 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: 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; ArgentinaFil: Leblanc, O.. Universidad de Montpellier; AlemaniaFil: 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; Argentin
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
Transferencia de marcadores EST-SSR y COS desde especies modelo a Paspalum notatum y estudios de mapeo comparativo
Paspalum es un género perteneciente a la familia de las Poaceae que cuenta con
más de 300 especies. Las mismas muestran una gran variación en los niveles de ploidía
y modos de reproducción. En general, los citotipos diploides son sexuales y los
poliploides sexuales o apomícticos. Dada su complejidad, el género ha sido dividido en
subgéneros y grupos informales. Varias especies representan importantes recursos
forrajeros de las regiones tropicales y subtropicales de América. En particular, P.
notatum Flüggé es una gramínea rizomatosa perenne cuyas razas tetraploides se
reproducen casi exclusivamente por apomixis de tipo apospórica. La aposporía en P.
notatum está controlada por un locus simple dominante con segregación distorsionada.
Trabajos previos posibilitaron el desarrollo de mapas genéticos al nivel tetraploide y la
identificación de la región genómica responsable de la aposporía. A partir de los
proyectos de secuenciación masiva de genomas se han desarrollado distintos tipos de
marcadores moleculares de secuencia conocida. Los microsatélites génicos (EST-SSR)
derivan de secuencias expresadas que contienen repeticiones microsatélites (SSR)
internas. Los marcadores COS provienen de genes que contienen regiones conservadas
entre especies poco relacionadas. Estos tipos de marcadores son relativamente simples
de desarrollar y han mostrado un alto nivel de polimorfismo y transferibilidad entre
especies. Los objetivos de este trabajo de tesis fueron transferir marcadores de
secuencia conocida (EST-SSR, SSR genómicos y COS) a P. notatum y caracterizar los
grupos de ligamiento de la especie por medio de análisis comparativos. Asimismo, se
realizaron estudios para determinar la utilidad de estos marcadores en otras especies del
género y su aptitud para la realización de estudios de filogenia. Como material vegetal
se emplearon los genotipos tetraploides de P. notatum Q4188 (sexual) y Q4117
(apomíctico) y una población F1, derivada de ambos. Además fueron incluidas 33
accesiones correspondientes a 11 especies y seis grupos taxonómicos diferentes.
Marcadores EST-SSR de trigo y SSR genómicos de P. notatum fueron ensayados sobre
los genotipos Q4188 y Q4117. Paralelamente se desarrollaron marcadores COS a partir
de genes asociados a componentes de la apomixis en gramíneas. Los marcadores
obtenidos fueron localizados en los mapas de Q4188 y Q4117 utilizando los programas
de mapeo Mapmaker 3.0/exp y JoinMap 3.0. Cuarenta y cuatro y 66 nuevos marcadores
fueron integrados a los mapas de Q4188 y Q4117, respectivamente. Los marcadores
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incorporados extendieron las distancias genéticas cubiertas por ambos mapas y
posibilitaron la identificación de nuevos grupos de ligamiento. Las secuencias de varios
EST-SSR de P. notatum mostraron similitudes con los clones originales de trigo (de los
cuales derivan) y confirmaron la identificación de secuencias ortólogas entre ambas
especies. Paralelamente un análisis de mapeo in silico permitió determinar la
localización de los marcadores empleados en los genomas de arroz y maíz. Los estudios
comparativos detectaron varios segmentos cromosómicos conservados entre P. notatum,
arroz y maíz. En especial, se determinó que 12 marcadores resultaron asociados a los
grupos de ligamiento relacionados con la aposporía. Los marcadores Ksum206dd y
Ksum219bd fueron localizados a ambos lados del locus responsable del carácter. La
utilidad de los marcadores EST-SSR y SSR genómicos en otras especies del género y su
capacidad para realizar estudios de filogenia fueron analizadas. Como control externo se
utilizó trigo pan (Triticum aestivum L.) cv. Federal. Los marcadores EST-SSR
generaron 166 fragmentos con un promedio de 6,16 fragmentos polimórficos por grupo.
Los SSR genómicos produjeron 104 fragmentos totales con un promedio de 4,39
fragmentos polimórficos por grupo. Los análisis de agrupamiento permitieron
discriminar entre grupos taxonómicos y especies dentro de cada grupo. Los resultados
presentados en esta tesis demuestran la factibilidad de transferir marcadores EST-SSR y
COS a P. notatum y contribuyen al conocimiento de la estructura genómica de las razas
tetraploides de la especie. En particular, la identificación de secuencias ortólogas entre
P. notatum, arroz y maíz permitió detectar varios segmentos cromosómicos conservados
en las tres especies e iniciar estudios de mapeo comparativo. La localización de varios
marcadores en los grupos de ligamiento asociados a la aposporía permitió una mejor
caracterización de esta región genómica y la definición de un segmento del genoma de
arroz que contendría genes candidatos a controladores del carácter. Se demostró
asimismo la posibilidad de emplear estos marcadores en varias especies del género y su
utilidad para la realización de estudios de filogenia.Paspalum is a genus of the grass family with more than 300 species. Due to its
taxonomic complexity, it has been divided in several subgenera and informal groups.
Many Paspalum species consist of sexual-diploid and apomictic-polyploid cytotypes,
and several have arisen through hybridization. Moreover, some members of the genus
are important natural forages for tropical and subtropical regions of America. In
particular, tetraploid race of P. notatum (bahiagrass) reproduces by aposporous
apomixis. Apospory in the species is controlled by a single dominant locus with a
distorted segregation ratio. Previous research reported the construction of a genetic
linkage map for the species and the localization of the genomic region responsible for
apospory (ASGR). The objectives of this study were i) to transfer molecular markers of
known sequences (EST-SSR, genomic SSR and COS) to P. notatum and characterize
the genome of the species by a comparative analysis, and ii) to determine the usefulness
of these kind of markers for taxonomic studies in the genus Paspalum. As plant material
the tetraploid genotypes Q4188 (sexual) and Q4117 (apomicts) and a F1 mapping
population derived from them were used. Moreover, 33 accessions belonging to 11
species and six informal taxonomic groups were included. Hexaploid wheat (Triticum
aestivum L.) was used as external control. Sequences of both wheat EST-SSR and P.
notatum genomic SSR markers were retrieved from public databases. COS markers
were developed from genes associated with components of apospory. All markers tested
showed a high rate of transferability and a good level of polymorphism. Sequence of
several EST-SSR markers isolated from P. notatum showed similarity with the
corresponding wheat clones from which they derived, and thus, confirmed the
identification of orthologous sequences between the two species. The localization of
markers in the genetic maps of Q4188 and Q4117 extend the genetic coverage of both
maps and allowed the recognition of several chromosome segments conserved between
bahiagrass, wheat and rice. Two markers at both sides of the ASGR were identified.
Assays of markers on Paspalum accessions confirmed they capability for detecting
polymorphic loci. Moreover, markers specific of species and groups were detected.
Clustering analysis was in agreement with previous reports. Results presented in this
work proved the transferability of EST-SSR markers to Paspalum species and
contribute to the genetic characterization of the P. notatum genome. Moreover, a new
set of markers for phylogenetic analysis in the genus is presented.Fil: Apellido, Nombre. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias; ArgentinaFil: Siena, Lorena Adelina. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias; Argentin
A genetic map of tetraploid Paspalum notatum Flügge (bahiagrass) based on single-dose molecular markers
Paspalum notatum Flu¨ gge is a warmseason
forage grass with mainly diploid (2n =
20) and autotetraploid (2n = 40) representatives.
Diploid races reproduce sexually and
require crosspollination due to a self-incompatible
mating system, while autotetraploids
reproduce by aposporous apomixis. The objectives
of this work were to develop a genetic
linkage map of Paspalum notatum Flu¨gge at
the tetraploid level, identify the linkage/s
group/s associated with apomixis and carry
out a general characterization of its mode of
inheritance. A pseudo test-cross F1 family of
113 individuals segregating for the mode of
reproduction was obtained by crossing a synthetic
completely sexual tetraploid plant
(Q4188) as female parent with a natural
aposporous individual (Q4117) as pollen donor.
Map construction was based on single-dose
markers (SDAFs) segregating from both parents.
Two linkage maps (female and male)
were constructed. Within each map, homologous
groups were assembled by detecting
repulsion-phase linked SDAFs. Putative
Q4188 and Q4117 homolog groups were
identified by mapping shared single dose markers
(BSDF). The Q4188 map consisted of 263
markers distributed on 26 co-segregation
groups over a total genetic distance of 1.590.6
cM, while the Q4117 map contained 216 loci
dispersed on 39 co-segregation groups along
2.265.7 cM, giving an estimated genome coverage
of 88% and 83%, respectively. Seven and
12 putative homologous chromosomes were
detected within Q4188 and Q4117 maps,
respectively. Afterward, ten female and male
homologous chromosomes were identified by
mapping BSDFs. In the Q4117 map, a single
linkage group was associated with apospory. It
was characterized by restriction in recombination
and preferential chromosome pairing. A
BPSD marker mapping within this group
allowed the detection of the female homolog
and the putative four male groups of the set
carrying apospory.Fil: Stein, Juliana. Laboratorio de Biología Molecular, Facultad de Ciencias Agrarias, Universidad Nacional de Rosario; Argentina
Transference of wheat Expressed Sequence Tag-Simple Sequence Repits to Paspalum species and cross-species amplification of Paspalum notatum Simple Sequence Repits: Potential use in phylogenetic analysis and mapping
The genus Paspalum includes numerous species of agronomic importance. The objectives of this work were to evaluate the transferability and polymorphism of publicly available wheat EST-SSR (wEST-SSR) markers to Paspalum spp., assess the cross-species amplification of Paspalum notatum genomic-SSRs (PnSSR) within the genus, and evaluate both types of markers for phylogenetic analyses and mapping. Thirty two accessions, including 11 species were used. Moreover, 65 F1 hybrids of P. notatum were employed for mapping. Transferability ratio of wEST-SSRs was 72.72%. On average 19.25 bands per primer combination were obtained. Cross-species amplification of PnSSRs was 55.37%, with an average of 9.45 fragments per primer pair. Both types of markers differed in the amplification capacity between primers pairs and species. Clustering analysis with wEST-SSRs data discriminated accessions by species and taxonomic groups. Genomic relationships were in agreement with previous works indicating that wEST-SSRs are adequate for phylogenetic surveys in the genus. Mapping experiments showed that both, wEST-SSRs and PnSSRs mapped scattered in the genome. On hundred and two new markers were added to the existent P. notatum linkage groups. Primer pairs ksum206, ksum219 and PN03-F2 generated markers that mapped linked to apospory. Sequences of EST-SSRs experimentally mapped in P. notatum showed 46.23% to 55.27% similarity with the original wheat EST. A preliminary comparative mapping analysis was carried combining experimental and in silico mapping results.Fil: Siena, Lorena Adelina. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Laboratorio de Biología Molecular; 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. 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
Genetic and embryological evidences of apomixis at the diploid level in Paspalum rufum support recurrent auto-polyploidization in the species
Gametophytic apomixis is an asexual mode of reproduction by seeds. This trait is present in several plant families and is strongly associated with polyploidy. Paspalum rufum is a forage grass with sexual self-incompatible diploids (2n = 2x = 20) and aposporous-apomictic pseudogamous tetraploids (2n = 4x = 40). In previous work embryological observations of the diploid genotype Q3754 showed 8.8-26.8% of the ovaries having one meiotic plus an aposporous-like embryo sac, suggesting some capability for apomictic reproduction. The objective of this work was to characterize progenies derived from Q3754 to determine if aposporous sacs were functional and generated progenies via apomixis at the diploid level. Re-examination of Q3754 ovaries showed that 12.5% of them contained one sexual plus an aposporous sac confirming previous results. Progeny tests were carried out on two experimental families (H 1 and S1) employing heterozygous RAPD marker loci. Family H1 was obtained crossing Q3754 with a natural diploid genotype (Q3861) and S1 derived from the induced self-pollination of Q3754. Genetic analysis of H1 showed that all individuals derived from sexual reproduction. However, 5 out of 95 plants from S1 showed the same heterozygous state as the mother plant for 14 RAPD loci suggesting a clonal origin. Further experiments, designed to test the functionality of aposporous sacs by flow cytometric analyses, were carried out on a third family (M 1) obtained by crossing Q3754 with the tetraploid plant Q3785. Histograms of 20 M1 plants showed 15 diploids (75%), 4 triploids (20%) and 1 tetraploid (5%). Triploids and the tetraploid may have originated from functional aposporous embryo sacs. Likewise, the reconstruction of the developmental route of 40 individual seeds demonstrated that 11 of them (27.5%) derived from fertilized aposporic sacs. The results presented in this work indicate that gametophytic apomixis is effectively expressed at the diploid level in Paspalum rufum and could be the foundation of a recurrent auto-polyploidization process in the species.Fil: Siena, Lorena Adelina. Universidad Nacional de Rosario; ArgentinaFil: Sartor, Maria Esperanza. 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: 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: Quarin, Camilo Luis. 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. Universidad Nacional de Rosario; 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; Argentin
An apomixis-linked ORC3-like pseudogene is associated with silencing of its functional homolog in apomictic Paspalum simplex
Apomixis in plants consists of asexual reproduction by seeds. Here we characterized at structural and functional levels an apomixis-linked sequence of Paspalum simplex homologous to subunit 3 of the ORIGIN RECOGNITION COMPLEX (ORC3). ORC is a multiprotein complex which controls DNA replication and cell differentiation in eukaryotes. Three PsORC3 copies were identified, each one characterized by a specific expression profile. Of these, PsORC3a, specific for apomictic genotypes, is a pseudogene that was poorly and constitutively expressed in all developmental stages of apomictic flowers, whereas PsORC3b, the putative functional gene in sexual flowers, showed a precise time-related regulation. Sense transcripts of PsORC3 were expressed in the female cell lineage of both apomictic and sexual reproductive phenotypes, and in aposporous initials. Although strong expression was detected in sexual early endosperm, no expression was present in the apomictic endosperm. Antisense PsORC3 transcripts were revealed exclusively in apomictic germ cell lineages. Defective orc3 mutants of rice and Arabidopsis showed normal female gametophytes although the embryo and endosperm were arrested at early phases of development. We hypothesize that PsORC3a is associated with the down-regulation of its functional homolog and with the development of apomictic endosperm which deviates from the canonical 2(maternal):1(paternal) genome ratio.Fil: Siena, Lorena Adelina. 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: 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: Calderini, Ornella. Istituto di Bioscienze e Biorisorse; ItaliaFil: Paolocci, Francesco. Istituto di Bioscienze e Biorisorse; ItaliaFil: Cáceres, Maria E.. Istituto di Bioscienze e Biorisorse; ItaliaFil: Kaushal, Pankaj. Istituto di Bioscienze e Biorisorse; ItaliaFil: Grisan, Simone. Istituto di Bioscienze e Biorisorse; ItaliaFil: 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; ArgentinaFil: Pupilli, Fulvio. Istituto di Bioscienze e Biorisorse; Itali
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
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