Background: Olea europaea L. is a traditional tree crop of the Mediterranean basin with a worldwide economical high impact. Differently from other fruit tree species, little is known about the physiological and molecular basis of the olive fruit development and a few sequences of genes and gene products are available for olive in public databases. This study deals with the identification of large sets of differentially expressed genes in developing olive fruits and the subsequent computational annotation by means of different software. Results: mRNA from fruits of the cv. Leccino sampled at three different stages [i.e., initial fruit set (stage 1), completed pit hardening (stage 2) and veraison (stage 3)] was used for the identification of differentially expressed genes putatively involved in main processes along fruit development. Four subtractive hybridization libraries were constructed: forward and reverse between stage 1 and 2 (libraries A and B), and 2 and 3 (libraries C and D). All sequenced clones (1,132 in total) were analyzed through BlastX against non-redundant NCBI databases and about 60% of them showed similarity to known proteins. A total of 89 out of 642 differentially expressed unique sequences was further investigated by Real-Time PCR, showing a validation of the SSH results as high as 69%. Library-specific cDNA repertories were annotated according to the three main vocabularies of the gene ontology (GO): cellular component, biological process and molecular function. BlastX analysis, GO terms mapping and annotation analysis were performed using the Blast2GO software, a research tool designed with the main purpose of enabling GO based data mining on sequence sets for which no GO annotation is yet available. Bioinformatic analysis pointed out a significantly different distribution of the annotated sequences for each GO category, when comparing the three fruit developmental stages. The olive fruit-specific transcriptome dataset was used to query all known KEGG (Kyoto Encyclopaedia of Genes and Genomes) metabolic pathways for characterizing and positioning retrieved EST records. The integration of the olive sequence datasets within the MapMan platform for microarray analysis allowed the identification of specific biosynthetic pathways useful for the definition of key functional categories in time course analyses for gene groups. Conclusion: The bioinformatic annotation of all gene sequences was useful to shed light on metabolic pathways and transcriptional aspects related to carbohydrates, fatty acids, secondary metabolites, transcription factors and hormones as well as response to biotic and abiotic stresses throughout olive drupe development. These results represent a first step toward both functional genomics and systems biology research for understanding the gene functions and regulatory networks in olive fruit growth and ripening.

Jatropha curcas L. (2n = 2x = 22) is becoming a popular non-food oleaginous crop in several developed countries due to its proposed value in the biopharmaceutical industry. Despite the potentials of its oil-rich seeds as a renewable source of biodiesel and an interest in large-scale cultivation, relatively little is known with respect to plant reproduction strategies and population dynamics. Here, genomic DNA markers and FCSS analyses were performed to gain insights into ploidy variation and heterozygosity levels of multiple accessions, and genomic relationships among commercial varieties of Jatropha grown in different geographical areas. The determination of ploidy and the differentiation of either pseudogamous or autonomous apomixis from sexuality were based on the seed DNA contents of embryo and endosperm. The presence of only a high 2C embryo peak and a smaller 3C endosperm peak (ratio 2:3) is consistent with an obligate sexual reproductive system. Because of the lack of either 4C or 5C endosperm DNA estimates, the occurrence of gametophytic apomixis seems unlikely in this species but adventitious embryony cannot be ruled out. The investigation of genetic variation within and between cultivated populations was carried out using dominant RAPD and Inter-SSR markers, and codominant SSR markers. Nei’s genetic diversity, corresponding to the expected heterozygosity, was equal to H_e = 0.3491 and the fixation index as low as F_st = 0.2042. The main finding is that seeds commercialized worldwide include a few closely related genotypes, which are not representative of the original Mexican gene pool, revealing high degrees of homozygosity for single varieties and very low genetic diversity between varieties.

St. John’s wort (Hypericum perforatum L.) is a medicinal plant that produces important metabolites with antidepressant and anticancer activities. Recently gained biological information has shown that this species is also an attractive model system for the study of a naturally occurring form of asexual reproduction called apomixis, which allows cloning plants through seeds. In aposporic gametogenesis, one or multiple somatic cells belonging to the ovule nucellus change their fate by dividing mitotically and developing functionally unreduced embryo sacs by mimicking sexual gametogenesis. Although the introduction of apomixis into agronomically important crops could have revolutionary implications for plant breeding, the genetic control of this mechanism of seed formation is still not well understood for most of the model species investigated so far. We used Roche 454 technology to sequence the entire H. perforatum flower transcriptome of whole flower buds and single flower verticils collected from obligately sexual and unrelated highly or facultatively apomictic genotypes, which enabled us to identify RNAs that are likely exclusive to flower organs (i.e., sepals, petals, stamens and carpels) or reproductive strategies (i.e., sexual vs. apomictic). Here we sequenced and annotated the flower transcriptome of H. perforatum with particular reference to reproductive organs and processes. In particular, in our study we characterized approximately 37,000 transcripts found expressed in male and/or female reproductive organs, including tissues or cells of sexual and apomictic flower buds. Ontological annotation was applied to identify major biological processes and molecular functions involved in flower development and plant reproduction. Starting from this dataset, we were able to recover and annotate a large number of transcripts related to meiosis, gametophyte/gamete formation, and embryogenesis, as well as genes that are exclusively or preferentially expressed in sexual or apomictic libraries. Real-Time RT-qPCR assays on pistils and anthers collected at different developmental stages from accessions showing alternative modes of reproduction were used to identify potential genes that are related to plant reproduction sensu lato in H. perforatum. Our approach of sequencing flowers from two fully obligate sexual genotypes and two unrelated highly apomictic genotypes, in addition to different flower parts dissected from a facultatively apomictic accession, enabled us to analyze the complexity of the flower transcriptome according to its main reproductive organs as well as for alternative reproductive behaviors. Both annotation and expression data provided original results supporting the hypothesis that apomixis in H. perforatum relies upon spatial or temporal mis-expression of genes acting during female sexual reproduction. The present analyses aim to pave the way toward a better understanding of the molecular basis of flower development and plant reproduction, by identifying genes or RNAs that may differentiate or regulate the sexual and apomictic reproductive pathways in H. perforatum.

Unlike sexual reproduction, apomixis encompasses a number of reproductive strategies, which permit maternal genome inheritance without genetic recombination and syngamy. The key biological features of apomixis are the circumvention of meiosis (i.e., apomeiosis), the differentiation of unreduced embryo sacs and egg cells, and their autonomous development in functional embryos through parthenogenesis, and the formation of viable endosperm either via fertilization-independent means or following fertilization with a sperm cell. Despite the importance of apomixis for breeding of crop plants and although much research has been conducted to study this process, the genetic control of apomixis is still not well understood. Hypericum perforatum is becoming an attractive model system for the study of aposporous apomixis. Here we report results from a global gene expression analysis of H. perforatum pistils collected from sexual and aposporous plant accessions for the purpose of identifying genes, biological processes and molecular functions associated with the aposporous apomixis pathway. Across two developmental stages corresponding to the expression of aposporous apomeiosis and parthenogenesis in ovules, a total of 224 and 973 unigenes were found to be significantly up- and down-regulated with a fold change ≥ 2 in at least one comparison, respectively. Differentially expressed genes were enriched for multiple gene ontology (GO) terms, including cell cycle, DNA metabolic process, and single-organism cellular process. For molecular functions, the highest scores were recorded for GO terms associated with DNA binding, DNA (cytosine-5-)-methyltransferase activity and heterocyclic compound binding. As deregulation of single components of the sexual developmental pathway is believed to be a trigger of the apomictic reproductive program, all genes involved in sporogenesis, gametogenesis and response to hormonal stimuli were analysed in great detail. Overall, our data suggest that phenotypic expression of apospory is concomitant with the modulation of key genes involved in the sexual reproductive pathway. Furthermore, based on gene annotation and co-expression, we underline a putative role of hormones and key actors playing in the RNA dependent DNA methylation pathway in regulating the developmental changes occurring during aposporous apomixis in H. perforatum.

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 identified 396 DEGs and 1834 transcripts displaying phenotype-specific expression. Furthermore, the sequencing and assembly of the genome allowed the annotation of a 50kb sequence portion located upstream the HAPPY locus and to address the extent to which single transcripts were assembled in multiple variants . 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 expressiom 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 a subset of genes encoding for potentially interacting proteins involved in pre-mRNA splicing. Furthermore, 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.