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Silvina Claudia Pessino - One of the best experts on this subject based on the ideXlab platform.

  • Small RNA-seq reveals novel regulatory components for apomixis in Paspalum notatum.
    BMC genomics, 2019
    Co-Authors: Juan Pablo A. Ortiz, Lorena Adelina Siena, Maricel Podio, Olivier Leblanc, Cristian Oscar Rohr, Mauricio Javier Grisolía, Carolina Colono, Celeste Antonela Azzaro, Silvina Claudia Pessino
    Abstract:

    Apomixis is considered an evolutionary deviation of the sexual reproductive pathway leading to the generation of clonal maternal progenies by seeds. Recent evidence from model and non-model species suggested that this trait could be modulated by epigenetic mechanisms involving small RNAs (sRNAs). Here we profiled floral sRNAs originated from apomictic and sexual Paspalum notatum genotypes in order to identify molecular pathways under epigenetic control that might be involved in the transition from sexuality to agamospermy. The mining of genes participating in sRNA-directed pathways from floral Paspalum transcriptomic resources showed these routes are functional during reproductive development, with several members differentially expressed in apomictic and sexual plants. Triplicate floral sRNA libraries derived from apomictic and a sexual genotypes were characterized by using high-throughput sequencing technology. EdgeR was apply to compare the number of sRNA reads between sexual and apomictic libraries that map over all Paspalum floral transcripts. A total of 1525 transcripts showed differential sRNA representation, including genes related to meiosis, plant hormone signaling, biomolecules transport, transcription control and cell cycle. Survey for miRNA precursors on transcriptome and genome references allowed the discovery of 124 entities, including 40 conserved and 8 novel ones. Fifty-six clusters were differentially represented in apomictic and sexual plants. All differentially expressed miRNAs were up-regulated in apomictic libraries but miR2275, which showed different family members with opposed representation. Examination of predicted miRNAs targets detected 374 potential candidates. Considering sRNA, miRNAs and target surveys together, 14 genes previously described as related with auxin metabolism, transport and signaling were detected, including AMINO ACID/AUXIN PERMEASE 15, IAA-AMIDO SYNTHETASE GH3–8, IAA30, miR160, miR167, miR164, miR319, ARF2, ARF8, ARF10, ARF12, AFB2, PROLIFERATING CELL FACTOR 6 and NITRATE TRANSPORTER 1.1. This work provides a comprehensive survey of the sRNA differential representation in flowers of sexual and apomictic Paspalum notatum plants. An integration of the small RNA profiling data presented here and previous transcriptomic information suggests that sRNA-mediated regulation of auxin pathways is pivotal in promoting apomixis. These results will underlie future functional characterization of the molecular components mediating the switch from sexuality to apomixis.

  • The vesicle trafficking regulator PN_SCD1 is demethylated and overexpressed in florets of apomictic Paspalum notatum genotypes.
    Scientific reports, 2018
    Co-Authors: Marika Bocchini, Silvina Claudia Pessino, Juan Pablo A. Ortiz, Giulio Galla, Michele Bellucci, Fulvio Pupilli, Gianni Barcaccia, Emidio Albertini
    Abstract:

    Apomixis (asexual reproduction through seeds) is considered a deviation of the sexual reproductive pathway leading to the development of clonal progenies genetically identical to the mother plant. Here we used the Methylation-Sensitive Amplification Polymorphism (MSAP) technique to characterize cytosine methylation patterns occurring in florets of sexual and aposporous Paspalum notatum genotypes, in order to identify epigenetically-controlled genes putatively involved in apomixis development. From twelve polymorphic MSAP-derived sequences, one (PN_6.6, later renamed PN_SCD1) was selected due to its relevant annotation and differential representation in apomictic and sexual floral transcriptome libraries. PN_SCD1 encodes the DENN domain/WD repeat-containing protein SCD1, which interacts with RAB GTPases- and/or MAPKs to promote specialized cell division, functions in clathrin-mediated membrane transport and acts as potential substrate receptor of CUL4 E3 ubiquitin ligases. Quantitative RT-PCR and comparative RNAseq analyses of laser microdissected nucellar cells confirmed PN_SCD1 upregulation in florets of apomictic plants and revealed that overexpression takes place just before the onset of apospory initials. Moreover, we found that several SCD1 molecular partners are expressed in P. notatum florets and upregulated in apomictic plants. Our results disclosed a specific vesicle trafficking molecular pathway epigenetically modulated during apomixis.

  • Structure, target-specificity and expression of PN_LNC_N13 , a long non-coding RNA differentially expressed in apomictic and sexual Paspalum notatum
    Plant Molecular Biology, 2017
    Co-Authors: Ana Ochogavía, José Guillermo Seijo, Ana María González, Giulio Galla, Michele Bellucci, Fulvio Pupilli, Gianni Barcaccia, Emidio Albertini, Silvina Claudia Pessino
    Abstract:

    ncRNA PN_LNC_N13 shows contrasting expression in reproductive organs of sexual and apomictic Paspalum notatum genotypes. Apomictic plants set genetically maternal seeds whose embryos derive by parthenogenesis from unreduced egg cells, giving rise to clonal offspring. Several Paspalum notatum apomixis related genes were identified in prior work by comparative transcriptome analyses. Here, one of these candidates (namely N13) was characterized. N13 belongs to a Paspalum gene family including 30–60 members, of which at least eight are expressed at moderate levels in florets. The sequences of these genes show no functional ORFs, but include segments of different protein coding genes. Particularly, N13 shows partial identity to maize gene BT068773 (RESPONSE REGULATOR 6). Secondary structure predictions as well as mature miRNA and target cleavage detection suggested that N13 is not a miRNA precursor. Moreover, N13 family members produce abundant 24-nucleotide small RNAs along extensive parts of their sequences. Surveys in the GREENC and CANTATA databases indicated similarity with plant long non-coding RNAs (lncRNAs) involved in splicing regulation; consequently, N13 was renamed as PN_LNC_N13. The Paspalum BT068773 predicted ortholog (N13TAR) originates floral transcript variants shorter than the canonical maize isoform and with possible structural differences between the apomictic and sexual types. PN_LNC_N13 is expressed only in apomictic plants and displays quantitative representation variation across reproductive developmental stages. However, PN_LNC_N13-like homologs and/or its derived sRNAs showed overall a higher representation in ovules of sexual plants at late premeiosis. Our results suggest the existence of a whole family of N13-like lncRNAs possibly involved in splicing regulation, with some members characterized by differential activity across reproductive types.

  • A reference floral transcriptome of sexual and apomictic Paspalum notatum.
    BMC genomics, 2017
    Co-Authors: Juan Pablo A. Ortiz, Santiago Revale, Lorena Adelina Siena, Maricel Podio, Luciana Delgado, Juliana Stein, Olivier Leblanc, Silvina Claudia Pessino
    Abstract:

    Paspalum notatum Flugge is a subtropical grass native to South America, which includes sexual diploid and apomictic polyploid biotypes. In the past decade, a number of apomixis-associated genes were discovered in this species through genetic mapping and differential expression surveys. However, the scarce information on Paspalum sequences available in public databanks limited annotations and functional predictions for these candidates. We used a long-read 454/Roche FLX+ sequencing strategy to produce robust reference transcriptome datasets from florets of sexual and apomictic Paspalum notatum genotypes and delivered a list of transcripts showing differential representation in both reproductive types. Raw data originated from floral samples collected from premeiosis to anthesis was assembled in three libraries: i) sexual (SEX), ii) apomictic (APO) and iii) global (SEX + APO). A group of physically-supported Paspalum mRNA and EST sequences matched with high level of confidence to both sexual and apomictic libraries. A preliminary trial allowed discovery of the whole set of putative alleles/paralogs corresponding to 23 previously identified apomixis-associated candidate genes. Moreover, a list of 3,732 transcripts and several co-expression and protein –protein interaction networks associated with apomixis were identified. The use of the 454/Roche FLX+ transcriptome database will allow the detailed characterization of floral alleles/paralogs of apomixis candidate genes identified in prior and future work. Moreover, it was used to reveal additional candidate genes differentially represented in apomictic and sexual flowers. Gene ontology (GO) analyses of this set of transcripts indicated that the main molecular pathways altered in the apomictic genotype correspond to specific biological processes, like biotic and abiotic stress responses, growth, development, cell death and senescence. This data collection will be of interest to the plant reproduction research community and, particularly, to Paspalum breeding projects.

  • Development of a modified transformation platform for apomixis candidate genes research in Paspalum notatum (bahiagrass)
    In Vitro Cellular & Developmental Biology - Plant, 2014
    Co-Authors: Micaela Mancini, Silvina Claudia Pessino, Juan Pablo A. Ortiz, Lorena Adelina Siena, Maricel Podio, Nadia Woitovich, Hugo R. Permingeat, Silvina Andrea Felitti
    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.

Juan Pablo A. Ortiz - One of the best experts on this subject based on the ideXlab platform.

  • Small RNA-seq reveals novel regulatory components for apomixis in Paspalum notatum.
    BMC genomics, 2019
    Co-Authors: Juan Pablo A. Ortiz, Lorena Adelina Siena, Maricel Podio, Olivier Leblanc, Cristian Oscar Rohr, Mauricio Javier Grisolía, Carolina Colono, Celeste Antonela Azzaro, Silvina Claudia Pessino
    Abstract:

    Apomixis is considered an evolutionary deviation of the sexual reproductive pathway leading to the generation of clonal maternal progenies by seeds. Recent evidence from model and non-model species suggested that this trait could be modulated by epigenetic mechanisms involving small RNAs (sRNAs). Here we profiled floral sRNAs originated from apomictic and sexual Paspalum notatum genotypes in order to identify molecular pathways under epigenetic control that might be involved in the transition from sexuality to agamospermy. The mining of genes participating in sRNA-directed pathways from floral Paspalum transcriptomic resources showed these routes are functional during reproductive development, with several members differentially expressed in apomictic and sexual plants. Triplicate floral sRNA libraries derived from apomictic and a sexual genotypes were characterized by using high-throughput sequencing technology. EdgeR was apply to compare the number of sRNA reads between sexual and apomictic libraries that map over all Paspalum floral transcripts. A total of 1525 transcripts showed differential sRNA representation, including genes related to meiosis, plant hormone signaling, biomolecules transport, transcription control and cell cycle. Survey for miRNA precursors on transcriptome and genome references allowed the discovery of 124 entities, including 40 conserved and 8 novel ones. Fifty-six clusters were differentially represented in apomictic and sexual plants. All differentially expressed miRNAs were up-regulated in apomictic libraries but miR2275, which showed different family members with opposed representation. Examination of predicted miRNAs targets detected 374 potential candidates. Considering sRNA, miRNAs and target surveys together, 14 genes previously described as related with auxin metabolism, transport and signaling were detected, including AMINO ACID/AUXIN PERMEASE 15, IAA-AMIDO SYNTHETASE GH3–8, IAA30, miR160, miR167, miR164, miR319, ARF2, ARF8, ARF10, ARF12, AFB2, PROLIFERATING CELL FACTOR 6 and NITRATE TRANSPORTER 1.1. This work provides a comprehensive survey of the sRNA differential representation in flowers of sexual and apomictic Paspalum notatum plants. An integration of the small RNA profiling data presented here and previous transcriptomic information suggests that sRNA-mediated regulation of auxin pathways is pivotal in promoting apomixis. These results will underlie future functional characterization of the molecular components mediating the switch from sexuality to apomixis.

  • The vesicle trafficking regulator PN_SCD1 is demethylated and overexpressed in florets of apomictic Paspalum notatum genotypes.
    Scientific reports, 2018
    Co-Authors: Marika Bocchini, Silvina Claudia Pessino, Juan Pablo A. Ortiz, Giulio Galla, Michele Bellucci, Fulvio Pupilli, Gianni Barcaccia, Emidio Albertini
    Abstract:

    Apomixis (asexual reproduction through seeds) is considered a deviation of the sexual reproductive pathway leading to the development of clonal progenies genetically identical to the mother plant. Here we used the Methylation-Sensitive Amplification Polymorphism (MSAP) technique to characterize cytosine methylation patterns occurring in florets of sexual and aposporous Paspalum notatum genotypes, in order to identify epigenetically-controlled genes putatively involved in apomixis development. From twelve polymorphic MSAP-derived sequences, one (PN_6.6, later renamed PN_SCD1) was selected due to its relevant annotation and differential representation in apomictic and sexual floral transcriptome libraries. PN_SCD1 encodes the DENN domain/WD repeat-containing protein SCD1, which interacts with RAB GTPases- and/or MAPKs to promote specialized cell division, functions in clathrin-mediated membrane transport and acts as potential substrate receptor of CUL4 E3 ubiquitin ligases. Quantitative RT-PCR and comparative RNAseq analyses of laser microdissected nucellar cells confirmed PN_SCD1 upregulation in florets of apomictic plants and revealed that overexpression takes place just before the onset of apospory initials. Moreover, we found that several SCD1 molecular partners are expressed in P. notatum florets and upregulated in apomictic plants. Our results disclosed a specific vesicle trafficking molecular pathway epigenetically modulated during apomixis.

  • A reference floral transcriptome of sexual and apomictic Paspalum notatum.
    BMC genomics, 2017
    Co-Authors: Juan Pablo A. Ortiz, Santiago Revale, Lorena Adelina Siena, Maricel Podio, Luciana Delgado, Juliana Stein, Olivier Leblanc, Silvina Claudia Pessino
    Abstract:

    Paspalum notatum Flugge is a subtropical grass native to South America, which includes sexual diploid and apomictic polyploid biotypes. In the past decade, a number of apomixis-associated genes were discovered in this species through genetic mapping and differential expression surveys. However, the scarce information on Paspalum sequences available in public databanks limited annotations and functional predictions for these candidates. We used a long-read 454/Roche FLX+ sequencing strategy to produce robust reference transcriptome datasets from florets of sexual and apomictic Paspalum notatum genotypes and delivered a list of transcripts showing differential representation in both reproductive types. Raw data originated from floral samples collected from premeiosis to anthesis was assembled in three libraries: i) sexual (SEX), ii) apomictic (APO) and iii) global (SEX + APO). A group of physically-supported Paspalum mRNA and EST sequences matched with high level of confidence to both sexual and apomictic libraries. A preliminary trial allowed discovery of the whole set of putative alleles/paralogs corresponding to 23 previously identified apomixis-associated candidate genes. Moreover, a list of 3,732 transcripts and several co-expression and protein –protein interaction networks associated with apomixis were identified. The use of the 454/Roche FLX+ transcriptome database will allow the detailed characterization of floral alleles/paralogs of apomixis candidate genes identified in prior and future work. Moreover, it was used to reveal additional candidate genes differentially represented in apomictic and sexual flowers. Gene ontology (GO) analyses of this set of transcripts indicated that the main molecular pathways altered in the apomictic genotype correspond to specific biological processes, like biotic and abiotic stress responses, growth, development, cell death and senescence. This data collection will be of interest to the plant reproduction research community and, particularly, to Paspalum breeding projects.

  • Transcriptome analysis of seed development in apomictic Paspalum notatum
    Annals of Applied Biology, 2015
    Co-Authors: Silvina Andrea Felitti, Juan Pablo A. Ortiz, Carlos Alberto Acuña, Camilo L. Quarin
    Abstract:

    The seed developmental process involves various tissues with several ploidy levels and different genetic origins. Therefore, its characterisation at the transcriptome level is certainly a challenge. The hypothesis of endosperm balance number (EBN) postulates that each species has an effective number that is important for normal endosperm and seed development to occur. Understanding endosperm formation in apomictic plants is crucial for the perspective of transferring apomixis to sexual species of agronomic interest. Since sexual tetraploid Paspalum plants fit the EBN premise, the EBN insensitivity observed in apomictic plants might be a requirement for the spread of pseudogamous apomixis. Crosses using several cytotypes of Paspalum notatum were made in order to induce the development of seeds with different maternal/paternal genomic ratios in the endosperm. A transcriptome characterisation of ovaries 3 h after pollination was performed using cDNA-AFLP methodology. Forty-six of the 100 differentially expressed transcript-derived fragments (DETDFs) were specifically found in crosses in which apomictic plants were used as the female parent and presented a predicted m : p ratio in the endosperm that was different to the 2:1 requirement of the EBN. Moreover, 12 of the DETDFs presented identity with proteins that were differentially expressed in response to changes in the levels of extracellular ATP (eATP) in Arabidopsis cell suspension cultures. eATP is an important molecular switch in plants that tightly controls organellar energy metabolism and activates gene expression controlling specific growth and developmental programmes. The results suggest that eATP-mediated signalling could be involved in the regulation of endosperm development.

  • Development of a modified transformation platform for apomixis candidate genes research in Paspalum notatum (bahiagrass)
    In Vitro Cellular & Developmental Biology - Plant, 2014
    Co-Authors: Micaela Mancini, Silvina Claudia Pessino, Juan Pablo A. Ortiz, Lorena Adelina Siena, Maricel Podio, Nadia Woitovich, Hugo R. Permingeat, Silvina Andrea Felitti
    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.

Maricel Podio - One of the best experts on this subject based on the ideXlab platform.

  • Small RNA-seq reveals novel regulatory components for apomixis in Paspalum notatum.
    BMC genomics, 2019
    Co-Authors: Juan Pablo A. Ortiz, Lorena Adelina Siena, Maricel Podio, Olivier Leblanc, Cristian Oscar Rohr, Mauricio Javier Grisolía, Carolina Colono, Celeste Antonela Azzaro, Silvina Claudia Pessino
    Abstract:

    Apomixis is considered an evolutionary deviation of the sexual reproductive pathway leading to the generation of clonal maternal progenies by seeds. Recent evidence from model and non-model species suggested that this trait could be modulated by epigenetic mechanisms involving small RNAs (sRNAs). Here we profiled floral sRNAs originated from apomictic and sexual Paspalum notatum genotypes in order to identify molecular pathways under epigenetic control that might be involved in the transition from sexuality to agamospermy. The mining of genes participating in sRNA-directed pathways from floral Paspalum transcriptomic resources showed these routes are functional during reproductive development, with several members differentially expressed in apomictic and sexual plants. Triplicate floral sRNA libraries derived from apomictic and a sexual genotypes were characterized by using high-throughput sequencing technology. EdgeR was apply to compare the number of sRNA reads between sexual and apomictic libraries that map over all Paspalum floral transcripts. A total of 1525 transcripts showed differential sRNA representation, including genes related to meiosis, plant hormone signaling, biomolecules transport, transcription control and cell cycle. Survey for miRNA precursors on transcriptome and genome references allowed the discovery of 124 entities, including 40 conserved and 8 novel ones. Fifty-six clusters were differentially represented in apomictic and sexual plants. All differentially expressed miRNAs were up-regulated in apomictic libraries but miR2275, which showed different family members with opposed representation. Examination of predicted miRNAs targets detected 374 potential candidates. Considering sRNA, miRNAs and target surveys together, 14 genes previously described as related with auxin metabolism, transport and signaling were detected, including AMINO ACID/AUXIN PERMEASE 15, IAA-AMIDO SYNTHETASE GH3–8, IAA30, miR160, miR167, miR164, miR319, ARF2, ARF8, ARF10, ARF12, AFB2, PROLIFERATING CELL FACTOR 6 and NITRATE TRANSPORTER 1.1. This work provides a comprehensive survey of the sRNA differential representation in flowers of sexual and apomictic Paspalum notatum plants. An integration of the small RNA profiling data presented here and previous transcriptomic information suggests that sRNA-mediated regulation of auxin pathways is pivotal in promoting apomixis. These results will underlie future functional characterization of the molecular components mediating the switch from sexuality to apomixis.

  • A reference floral transcriptome of sexual and apomictic Paspalum notatum.
    BMC genomics, 2017
    Co-Authors: Juan Pablo A. Ortiz, Santiago Revale, Lorena Adelina Siena, Maricel Podio, Luciana Delgado, Juliana Stein, Olivier Leblanc, Silvina Claudia Pessino
    Abstract:

    Paspalum notatum Flugge is a subtropical grass native to South America, which includes sexual diploid and apomictic polyploid biotypes. In the past decade, a number of apomixis-associated genes were discovered in this species through genetic mapping and differential expression surveys. However, the scarce information on Paspalum sequences available in public databanks limited annotations and functional predictions for these candidates. We used a long-read 454/Roche FLX+ sequencing strategy to produce robust reference transcriptome datasets from florets of sexual and apomictic Paspalum notatum genotypes and delivered a list of transcripts showing differential representation in both reproductive types. Raw data originated from floral samples collected from premeiosis to anthesis was assembled in three libraries: i) sexual (SEX), ii) apomictic (APO) and iii) global (SEX + APO). A group of physically-supported Paspalum mRNA and EST sequences matched with high level of confidence to both sexual and apomictic libraries. A preliminary trial allowed discovery of the whole set of putative alleles/paralogs corresponding to 23 previously identified apomixis-associated candidate genes. Moreover, a list of 3,732 transcripts and several co-expression and protein –protein interaction networks associated with apomixis were identified. The use of the 454/Roche FLX+ transcriptome database will allow the detailed characterization of floral alleles/paralogs of apomixis candidate genes identified in prior and future work. Moreover, it was used to reveal additional candidate genes differentially represented in apomictic and sexual flowers. Gene ontology (GO) analyses of this set of transcripts indicated that the main molecular pathways altered in the apomictic genotype correspond to specific biological processes, like biotic and abiotic stress responses, growth, development, cell death and senescence. This data collection will be of interest to the plant reproduction research community and, particularly, to Paspalum breeding projects.

  • Development of a modified transformation platform for apomixis candidate genes research in Paspalum notatum (bahiagrass)
    In Vitro Cellular & Developmental Biology - Plant, 2014
    Co-Authors: Micaela Mancini, Silvina Claudia Pessino, Juan Pablo A. Ortiz, Lorena Adelina Siena, Maricel Podio, Nadia Woitovich, Hugo R. Permingeat, Silvina Andrea Felitti
    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.

  • Characterization and expression analysis of SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) genes in sexual and apomictic Paspalum notatum.
    Plant Molecular Biology, 2013
    Co-Authors: Maricel Podio, Silvina Claudia Pessino, Lorena Adelina Siena, Luciana Delgado, José Guillermo Seijo, Ana María González, Silvina Andrea Felitti, Micaela Mancini, Juan Pablo A. Ortiz
    Abstract:

    The SOMATIC EMBRYOGENESIS RECEPTOR-LIKE 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.

  • Sequence characterization, in silico mapping and cytosine methylation analysis of markers linked to apospory in Paspalum notatum
    Genetics and molecular biology, 2012
    Co-Authors: Maricel Podio, Silvina Claudia Pessino, Camilo L. Quarin, Lorena Adelina Siena, Juliana Stein, Silvina Andrea Felitti, María Pía Rodríguez, Erci Javier Martínez, Juan Pablo A. Ortiz
    Abstract:

    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 regions 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 molecular 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 mapping in silico in the apospory-syntenic regions of rice and maize. Approximately 5 kb corresponding to different markers 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.

Camilo L. Quarin - One of the best experts on this subject based on the ideXlab platform.

  • Transcriptome analysis of seed development in apomictic Paspalum notatum
    Annals of Applied Biology, 2015
    Co-Authors: Silvina Andrea Felitti, Juan Pablo A. Ortiz, Carlos Alberto Acuña, Camilo L. Quarin
    Abstract:

    The seed developmental process involves various tissues with several ploidy levels and different genetic origins. Therefore, its characterisation at the transcriptome level is certainly a challenge. The hypothesis of endosperm balance number (EBN) postulates that each species has an effective number that is important for normal endosperm and seed development to occur. Understanding endosperm formation in apomictic plants is crucial for the perspective of transferring apomixis to sexual species of agronomic interest. Since sexual tetraploid Paspalum plants fit the EBN premise, the EBN insensitivity observed in apomictic plants might be a requirement for the spread of pseudogamous apomixis. Crosses using several cytotypes of Paspalum notatum were made in order to induce the development of seeds with different maternal/paternal genomic ratios in the endosperm. A transcriptome characterisation of ovaries 3 h after pollination was performed using cDNA-AFLP methodology. Forty-six of the 100 differentially expressed transcript-derived fragments (DETDFs) were specifically found in crosses in which apomictic plants were used as the female parent and presented a predicted m : p ratio in the endosperm that was different to the 2:1 requirement of the EBN. Moreover, 12 of the DETDFs presented identity with proteins that were differentially expressed in response to changes in the levels of extracellular ATP (eATP) in Arabidopsis cell suspension cultures. eATP is an important molecular switch in plants that tightly controls organellar energy metabolism and activates gene expression controlling specific growth and developmental programmes. The results suggest that eATP-mediated signalling could be involved in the regulation of endosperm development.

  • Sequence characterization, in silico mapping and cytosine methylation analysis of markers linked to apospory in Paspalum notatum
    Genetics and molecular biology, 2012
    Co-Authors: Maricel Podio, Silvina Claudia Pessino, Camilo L. Quarin, Lorena Adelina Siena, Juliana Stein, Silvina Andrea Felitti, María Pía Rodríguez, Erci Javier Martínez, Juan Pablo A. Ortiz
    Abstract:

    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 regions 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 molecular 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 mapping in silico in the apospory-syntenic regions of rice and maize. Approximately 5 kb corresponding to different markers 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.

  • Frequencies and variation in cytosine methylation patterns in diploid and tetraploid cytotypes of Paspalum notatum
    Biologia plantarum, 2012
    Co-Authors: M. P. Rodriguez, Camilo L. Quarin, Gerardo Domingo Lucio Cervigni, Juan Pablo A. Ortiz
    Abstract:

    Paspalum notatum Flugge is a grass species organized as an agamic complex. The objective of the current research was to survey the frequencies and variation of cytosine methylation at CCGG sequences in diploid and tetraploid genotypes, and to determine the occurrence of methylation changes associated with tetraploidization by using methylation-sensitive amplification polymorphism (MSAP) markers. No differences were found in the average proportions of methylated CCGG sites between cytotypes, but methylation patterns were significantly more variable in tetraploids. In both groups of plants, epigenetic and non-epigenetic variation correlated significantly when compared by Mantel tests. The evaluation of 159 common MSAP markers showed that 18.86 % of them differed in their methylation status in the different ploidies. Dendrogram analysis, reflecting epigenetic distances, showed that the four diploids and one experimentally-obtained sexually-reproducing tetraploid, grouped together. MSAP analysis performed on a diploid plant and its autotetraploid derivative showed that new epialleles emerged after tetraploidization. Sequencing of several MASP markers showed homologies with low copy genes, non-coding sequences and transposon/retrotransposon elements.

  • Residual sexuality and its seasonal variation in natural apomictic Paspalum notatum accessions
    Biologia plantarum, 2011
    Co-Authors: R. N. Rebozzio, Camilo L. Quarin, Maria Esperanza Sartor, Francisco Espinoza
    Abstract:

    Traditionally, tetraploid Paspalum notatum was considered an obligate or a facultative apomict according to cytoembryological analyses. The degree of facultativeness was usually determined by the relative amount of mature ovules bearing aposporous or meiotic (sexual) embryo sacs, or both together. We established, through progeny tests conducted with the aid of AFLP markers, the degree of residual sexuality expressed in four selected biotypes. The results showed it to be substantially and significantly lower than predicted by previous embryological analyses for the same biotypes. Moreover, the lowest expression of residual sexuality was coincident with maximum flowering period. Seed development in facultative apomictic P. notatum shows a definite bias against meiotic embryo sacs.

  • A genetic map of tetraploid Paspalum notatum Flügge (bahiagrass) based on single-dose molecular markers
    Molecular Breeding, 2007
    Co-Authors: Juliana Stein, Silvina Claudia Pessino, Camilo L. Quarin, Eric Javier Martínez, Lorena Adelina Siena, María Pía Rodríguez, Juan Pablo A. Ortiz
    Abstract:

    Paspalum notatum Flugge is a warm-season 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 Flugge 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.

José Guillermo Seijo - One of the best experts on this subject based on the ideXlab platform.

  • Structure, target-specificity and expression of PN_LNC_N13, a long non-coding RNA differentially expressed in apomictic and sexual Paspalum notatum.
    Plant molecular biology, 2017
    Co-Authors: Ana Ochogavía, José Guillermo Seijo, Ana María González, Giulio Galla, Michele Bellucci, Fulvio Pupilli, Gianni Barcaccia, Emidio Albertini, Silvina Pessino
    Abstract:

    KEY MESSAGE: ncRNA PN_LNC_N13 shows contrasting expression in reproductive organs of sexual and apomictic Paspalum notatum genotypes. Apomictic plants set genetically maternal seeds whose embryos derive by parthenogenesis from unreduced egg cells, giving rise to clonal offspring. Several Paspalum notatum apomixis related genes were identified in prior work by comparative transcriptome analyses. Here, one of these candidates (namely N13) was characterized. N13 belongs to a Paspalum gene family including 30-60 members, of which at least eight are expressed at moderate levels in florets. The sequences of these genes show no functional ORFs, but include segments of different protein coding genes. Particularly, N13 shows partial identity to maize gene BT068773 (RESPONSE REGULATOR 6). Secondary structure predictions as well as mature miRNA and target cleavage detection suggested that N13 is not a miRNA precursor. Moreover, N13 family members produce abundant 24-nucleotide small RNAs along extensive parts of their sequences. Surveys in the GREENC and CANTATA databases indicated similarity with plant long non-coding RNAs (lncRNAs) involved in splicing regulation; consequently, N13 was renamed as PN_LNC_N13. The Paspalum BT068773 predicted ortholog (N13TAR) originates floral transcript variants shorter than the canonical maize isoform and with possible structural differences between the apomictic and sexual types. PN_LNC_N13 is expressed only in apomictic plants and displays quantitative representation variation across reproductive developmental stages. However, PN_LNC_N13-like homologs and/or its derived sRNAs showed overall a higher representation in ovules of sexual plants at late premeiosis. Our results suggest the existence of a whole family of N13-like lncRNAs possibly involved in splicing regulation, with some members characterized by differential activity across reproductive types.

  • Structure, target-specificity and expression of PN_LNC_N13 , a long non-coding RNA differentially expressed in apomictic and sexual Paspalum notatum
    Plant Molecular Biology, 2017
    Co-Authors: Ana Ochogavía, José Guillermo Seijo, Ana María González, Giulio Galla, Michele Bellucci, Fulvio Pupilli, Gianni Barcaccia, Emidio Albertini, Silvina Claudia Pessino
    Abstract:

    ncRNA PN_LNC_N13 shows contrasting expression in reproductive organs of sexual and apomictic Paspalum notatum genotypes. Apomictic plants set genetically maternal seeds whose embryos derive by parthenogenesis from unreduced egg cells, giving rise to clonal offspring. Several Paspalum notatum apomixis related genes were identified in prior work by comparative transcriptome analyses. Here, one of these candidates (namely N13) was characterized. N13 belongs to a Paspalum gene family including 30–60 members, of which at least eight are expressed at moderate levels in florets. The sequences of these genes show no functional ORFs, but include segments of different protein coding genes. Particularly, N13 shows partial identity to maize gene BT068773 (RESPONSE REGULATOR 6). Secondary structure predictions as well as mature miRNA and target cleavage detection suggested that N13 is not a miRNA precursor. Moreover, N13 family members produce abundant 24-nucleotide small RNAs along extensive parts of their sequences. Surveys in the GREENC and CANTATA databases indicated similarity with plant long non-coding RNAs (lncRNAs) involved in splicing regulation; consequently, N13 was renamed as PN_LNC_N13. The Paspalum BT068773 predicted ortholog (N13TAR) originates floral transcript variants shorter than the canonical maize isoform and with possible structural differences between the apomictic and sexual types. PN_LNC_N13 is expressed only in apomictic plants and displays quantitative representation variation across reproductive developmental stages. However, PN_LNC_N13-like homologs and/or its derived sRNAs showed overall a higher representation in ovules of sexual plants at late premeiosis. Our results suggest the existence of a whole family of N13-like lncRNAs possibly involved in splicing regulation, with some members characterized by differential activity across reproductive types.

  • Characterization and expression analysis of SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) genes in sexual and apomictic Paspalum notatum.
    Plant Molecular Biology, 2013
    Co-Authors: Maricel Podio, Silvina Claudia Pessino, Lorena Adelina Siena, Luciana Delgado, José Guillermo Seijo, Ana María González, Silvina Andrea Felitti, Micaela Mancini, Juan Pablo A. Ortiz
    Abstract:

    The SOMATIC EMBRYOGENESIS RECEPTOR-LIKE 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.

  • Expression of lorelei-like genes in aposporous and sexual Paspalum notatum plants
    Plant Molecular Biology, 2011
    Co-Authors: Silvina Andrea Felitti, Juan Pablo Amelio Ortiz, Maricel Podio, José Guillermo Seijo, Ana María González, Natalia Verónica Laspina, Lorena Siena, Silvina Claudia Pessino
    Abstract:

    Gametophytic apomictic plants form non-reduced embryo sacs that generate clonal embryos by parthenogenesis, in the absence of both meiosis and egg-cell fertilization. Here we report the sequence and expression analysis of a lorelei -like Paspalum notatum gene, n20gap - 1 , which encodes a GPI-anchored protein previously associated with apomixis in this species. Phylogeny trees showed that n20gap - 1 was evolutionary related to the Arabidopsis thaliana lorelei genes At4g26466 and At5g56170. The lorelei At4g26466 disruption was shown to be detrimental to sperm cell release in arabidopsis. RFLP (Restriction Fragment Length Polymorphism) analysis revealed the occurrence of several homologous sequences in the Paspalum notatum genome, exhibiting polymorphisms genetically linked to apomixis. Real-time PCR showed that lorelei -family genes present a minor activity peak at pre-meiosis and a major one at anthesis. The apomictic genotype analyzed showed a significantly increased activity at pre-meiosis, post-meiosis and anthesis with respect to a sexual genotype. In situ hybridization assays revealed expression in integuments, nucellus and the egg-cell apparatus. Several n20gap - 1 alleles differing mainly at the 3′ UTR sequence were identified. Allele-specific real-time PCR experiments showed that allele 28 was significantly induced in reproductive tissues of the apomictic genotype with respect to the sexual genotype at anthesis. Our results indicate that P. notatum lorelei -like genes are differentially expressed in representative sexual (Q4188) and apomictic (Q4117) genotypes, and might play a role in the final stages of the apomixis developmental cascade. However, the association of n20gap - 1 expression with the trait should be confirmed in significant number of sexual and apomictic genotypes.

  • Characterization of retrotransposon sequences expressed in inflorescences of apomictic and sexual Paspalum notatum plants.
    Sexual plant reproduction, 2011
    Co-Authors: Ana Ochogavía, Juan Pablo A. Ortiz, Maricel Podio, José Guillermo Seijo, Ana María González, Erica Duarte Silveira, Ana Luiza Machado Lacerda, Vera Tavares De Campos Carneiro, Silvina Claudia Pessino
    Abstract:

    Apomixis, an asexual mode of reproduction through seeds, holds much promise for agricultural advances. However, the molecular mechanisms underlying this trait are still poorly understood. We previously isolated several transcripts representing novel sequences differentially expressed in reproductive tissues of sexual and apomictic plants. Here, we report the characterization of two of these unknown RNA transcripts (experimental codes N17 and N22). Since original fragments showed no significant homologies to sequences at databases, preliminary genomic PCR experiments were carried out to discard possible contaminations. RACE extension on flanking regions provided longer sequences for the candidates and additional related transcripts, which revealed similarity to LTR retrotransposons carrying short transduplicated segments of protein-coding genes. Interestingly, some transduplicated segments corresponded to genes previously associated with apomictic development. Gene copy number estimations revealed a moderate representation of the elements in the genome, with significantly increased numbers in a sexual genotype with respect to an apomictic one. Genetic mapping of N17 showed that a copy of this particular element was located onto Paspalum notatum linkage group F3c, at a central non-recombinant region resembling a centromere. Expression analysis showed an increased activity of N17 and N22 sense strands in ovules of the sexual genotypes. A retrotransposon-specific differential display analysis aimed at detecting related sequences allowed the identification of a complex family, with the majority of its members represented in the sexual genotype. Our results suggest that these elements could be participating in regulatory pathways related to apomixis and sexuality.