Specific expression of apomixis-linked alleles revealed by comparative transcriptomic analysis of sexual and apomictic Paspalum simplex Morong flowers

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Ovule Gene Expression Analysis in Sexual and Aposporous Apomictic Hypericum perforatum L. (Hypericaceae) Accessions

Hypericum perforatum L. (2n = 4x = 32) is an attractive model system for the study of aposporous apomixis. The earliest phenotypic features of aposporous apomixis in this species are the mitotic formation of unreduced embryo sacs from a somatic cell of the ovule nucellus and the avoidance of meiosis. In this research we addressed gene expression variation in sexual and apomictic plants, by focusing on the ovule nucellus, which is the cellular domain primarily involved into the differentiation of meiocyte precursors and aposporous embryo sacs, at a pre-meiotic developmental stage. Gene expression analyses performed by RNAseq identified 396 differentially expressed genes and 1834 transcripts displaying phenotype-specific expression. Furthermore, the sequencing and assembly of the genome from a diploid sexual accession allowed the annotation of a 50 kb sequence portion located upstream the HAPPY locus and to address the extent to which single transcripts were assembled in multiple variants and their co-expression levels. About one third of identified DEGs and phenotype-specific transcripts were associated to transcript variants with alternative expression patterns. Additionally, considering DEGs and phenotype-specific transcript, the co-expression level was estimated in about two transcripts per locus. Our gene expression study shows massive differences in the expression of several genes encoding for transposable elements. Transcriptional differences in the ovule nucellus and pistil terminal developmental stages were also found for subset of genes encoding for potentially interacting proteins involved in pre-mRNA splicing. Furthermore, the sexual and aposporous ovule transcriptomes were characterized by differential expression in genes operating in RNA silencing, RNA-mediated DNA methylation (RdDM) and histone and chromatin modifications. These findings are consistent with a role of these processes in regulating cell fate determination in the ovule, as indicated by forward genetic studies in sexual model species. The association between aposporous apomixis, pre-mRNA splicing and DNA methylation mediated by sRNAs, which is supported by expression data and by the enrichment in GO terms related to these processes, is consistent with the massive differential expression of multiple transposon-related sequences observed in ovules collected from both sexual and aposporous apomictic accessions. Overall, our data suggest that phenotypic expression of aposporous apomixis is concomitant with the modulation of key genes involved in the two interconnected processes: RNA splicing and RNA-directed DNA methylation

Structure, target-specificity and expression of PN_LNC_N13, a long non-coding RNA differentially expressed in apomictic and sexual Paspalum notatum

Key message: ncRNA PN_LNC_N13 shows contrasting expression in reproductive organs of sexual and apomictic Paspalum notatum genotypes. Abstract: 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.Fil: Ochogavía, Ana 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: Galla, Giulio. Università di Padova; ItaliaFil: Seijo, José 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; ArgentinaFil: González, Ana María. 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: Bellucci, Michele. Consiglio Nazionale delle Ricerche; ItaliaFil: Pupilli, Fulvio. Consiglio Nazionale delle Ricerche; ItaliaFil: Barcaccia, Gianni. Università di Padova; ItaliaFil: Albertini, Emidio. Università di Perugia; ItaliaFil: Pessino, Silvina Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentin

Structure, target-specificity and expression of PN_LNC_N13, a long non-coding RNA differentially expressed in apomictic and sexual Paspalum notatum.

The 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

Variation for polyphenol oxidase activity in stems of Medicago species

Polyphenol oxidase (PPO) activity was detected in stems of both glandular-haired and glabrous wild Medicago species. Two PPO isoforms appeared in each Medicago species, the heavier isoform ranged from 120 to 180 kDa and the lighter one from 65 to 75 kDa. The substrate affinity for 4-methyl catechol was quite low (Km around 0.5 mM) in all the species examined. The PPO specific activity present in the stem epidermis of insect-resistant Medicago spp. was remarkably higher than that measured in the same tissue of susceptible alfalfa, thus suggesting a possible role of PPO in resistance against biotic stresses. (© Inra/Elsevier, Paris.)L'activité de la polyphénoloxydase (PPO) dans des tiges d'espèces de Medicago. L'activité de la polyphénoloxydase a été détectée dans des tiges d'espèces spontanées de Medicago, aussi bien du type à poils glandulaires que du type glabre. Deux isoformes de PPO sont apparues dans chacune des espèces de Medicago, la plus lourde allant de 120 à 180 kDa et la plus légère de 65 à 75 kDa. L'affinité du substrat pour le 4-méthylcatéchol est restée sur des valeurs plutôt basses (Km aux alentours de 0,5 mM) chez toutes les espèces examinées. L'activité spécifique de la PPO relevée dans l'épiderme de la tige des espèces résistantes aux insectes est apparue comme nettement plus forte que celle mesurée dans le même tissu chez la luzerne sensible, ce qui suggère que la PPO pourrait jouer un rôle dans la résistance aux stress biotiques. (© Inra/Elsevier, Paris.

Optimal population size for RFLP-assisted cultivar identification in alfalfa (Medicago sativa L.)

The ability of RFLP markers to distinguish between 2 heterogeneous alfalfa ecotypes, "Vogherese" and "Maremmana", was correlated with the number of plants sampled in either single-plant or bulk analyses. Independent subsample populations of 20 and 50 plants were compared for variance component partitioning and band frequencies in 20-, 50- and 100-plant subpopulations of Maremmana. Homogeneity within 3 independent bulks of the same size as given above was taken as a measure of bulk optimal size. A minimum of 50 plants is required for both single-plant and bulk analyses; however, for a large majority of bands analysed, no significant differences were detected for their frequency among populations of 20, 50 and 100 plants, with the exception of 6 bands out of 48. One ecotype-specific marker was found through bulk analysis. Results are discussed in relation to the improvement of RFLP methodology for cultivar identification in alfalfa.Taille de population optimale pour l'identification des cultivars par RFLP chez la luzerne (Medicago sativa L.). La capacité des marqueurs RFLP de distinguer entre deux écotypes hétérogènes de luzerne, "Vogherese " et "Maremmana ", a été comparée avec le nombre de plantes composant un échantillon utilisé aussi bien pour l'analyse sur des plantes individuelles que sur des plantes en mélange ("bulk "). La partition des composantes de la variance a été comparée dans des sous-échantillons de populations indépendantes de 20 et 50 plantes, et les fréquences de bandes ont été aussi comparées dans des sous-populations de Maremmana constituées par 20, 50 et 100 plantes. Le degré d'homogénéité révelé à l'intérieur de 3 "bulks " indépendants, de tailles identiques à celles qui viennent d'être mentionnées, a été pris comme mesure de la taille optimale du "bulk ". Un minimum de 50 plantes est nécessaire aussi bien pour l'analyse sur plantes individuelles qu'en "bulk ", alors que pour la grande majorité des bandes analysée, aucune différence significative de leur fréquence n'a été relevée entre les populations de 20, 50 et 100 plantes, à l'exception de 6 bandes sur 48. Un marqueur spécifique d'un écotype a été trouvé au moyen de l'analyse "bulk ". Les résultats sont discutés dans l'optique d'une amélioration de la méthodologie RFLP servant à l'identification des cultivars chez la luzerne

The chromosome segment related to apomixis in Paspalum simplex is homeologous to the telomeric region of the long arm of rice chromosome 12

Apomixis is a form of asexual reproduction that in plants leads to the production of seed progeny that are exact copies of the mother individual. A mapping population generated by backcrossing a sexual with an apomictic genotype of Paspalum simplex, both at the tetraploid level, was used to find markers co-segregating with apomixis. Genetic analysis showed that apomixis is under the control of a single dominant allele assuming a random chromatid assortment. Five rice markers, mapped in the telomeric region of the long arm of rice chromosome 12, showed tight linkage with apomixis. Genetic and molecular data strongly indicate that the potentiality to express apomixis in P. simplex is given by a relatively large chromosome segment that is inherited as a single genetic unit.Fil: Pupilli, Fulvio. Istituto di Ricerche sul Miglioramento Genetico delle Piante Foraggere; ItaliaFil: Labombarda, Paola. Istituto di Ricerche sul Miglioramento Genetico delle Piante Foraggere; ItaliaFil: Cáceres, María Emilia. Istituto di Ricerche sul Miglioramento Genetico delle Piante Foraggere; ItaliaFil: Arcioni, Sergio. Istituto di Ricerche sul Miglioramento Genetico delle Piante Foraggere; ItaliaFil: 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; Argentin

How to become an apomixis model: The multifaceted case of Paspalum

In the past decades, the grasses of the Paspalum genus have emerged as a versatile model allowing evolutionary, genetic, molecular, and developmental studies on apomixis as well as successful breeding applications. The rise of such an archetypal system progressed through integrative phases, which were essential to draw conclusions based on solid standards. Here, we review the steps adopted in Paspalum to establish the current body of knowledge on apomixis and provide model breeding programs for other agronomically important apomictic crops. In particular, we discuss the need for previous detailed cytoembryological and cytogenetic germplasm characterization; the establishment of sexual and apomictic materials of identical ploidy level; the development of segregating populations useful for inheritance analysis, positional mapping, and epigenetic control studies; the development of omics data resources; the identification of key molecular pathways via comparative gene expression studies; the accurate molecular characterization of genomic loci governing apomixis; the in-depth functional analysis of selected candidate genes in apomictic and model species; the successful building of a sexual/apomictic combined breeding scheme.Fil: 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: Pupilli, Fulvio. Consiglio Nazionale delle Ricerche; ItaliaFil: Acuña, Carlos Alberto. 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: Leblanc, Olivier. No especifíca;Fil: 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; Argentin