Luciana Baldoni

Comparative study of the discriminating capacity of RAPD, AFLP and SSR markers and of their effectiveness in establishing genetic relationships in olive

Abstract. RAPDs, AFLPs and SSRs were compared in terms of their informativeness and efficiency in a study of genetic diversity and relationships among 32 olive cultivars cultivated in Italy and Spain. SSRs presented a higher level of polymorphism and a greater information content, as assessed by the expected heterozygosity, than AFLPs and RAPDs. The lowest values of expected heterozygosity were obtained for AFLPs, which, nevertheless were the most efficient marker system due to their capacity to reveal the highest number of bands per reaction and because of the high values achieved for a considerable number of indexes. All three techniques discriminated the genotypes very effectively, but only SSRs were able to discriminate the cultivars Frantoio and Cellina. The correlation coefficients of similarity were statistically significant for all three marker systems used but were lower for the SSR data than for RAPDs and AFLPs. For all markers a high similarity in dendrogram topologies was obtained although some differences were observed. All the dendrograms, including that obtained by the combined use of all the marker data, reflect some relationships for most of the cultivars according to their geographic diffusion. AMOVA analysis detected greater genetic differentiation among cultivars within each country than it did between the two countries.

Comparative 454 pyrosequencing of transcripts from two olive genotypes during fruit development

BackgroundDespite its primary economic importance, genomic information on olive tree is still lacking. 454 pyrosequencing was used to enrich the very few sequence data currently available for the Olea europaea species and to identify genes involved in expression of fruit quality traits.ResultsFruits of Coratina, a widely cultivated variety characterized by a very high phenolic content, and Tendellone, an oleuropein-lacking natural variant, were used as starting material for monitoring the transcriptome. Four different cDNA libraries were sequenced, respectively at the beginning and at the end of drupe development. A total of 261,485 reads were obtained, for an output of about 58 Mb. Raw sequence data were processed using a four step pipeline procedure and data were stored in a relational database with a web interface.ConclusionMassively parallel sequencing of different fruit cDNA collections has provided large scale information about the structure and putative function of gene transcripts accumulated during fruit development. Comparative transcript profiling allowed the identification of differentially expressed genes with potential relevance in regulating the fruit metabolism and phenolic content during ripening.

A consensus list of microsatellite markers for olive genotyping

Cultivar identification is a primary concern for olive growers, breeders, and scientists. This study was aimed at examining the SSR markers retrieved from the literature and currently used in olive study, in order to select those most effective in characterizing the olive accessions and to make possible the comparison of data obtained by different laboratories. Olive microsatellite profiles were assessed by four independent laboratories, which analyzed 37 pre-selected SSR loci on a set of 21 cultivars. These SSR markers were initially tested for their reproducibility, power of discrimination and number of amplified loci/alleles. Independent segregation was tested for each pair of SSRs in a controlled cross and the allelic error rate was quantified. Some of them were finally selected as the most informative and reliable. Most of the alleles were sequenced and their sizes were determined. Profiles of the reference cultivars and a list of alleles with their sizes obtained by sequencing are reported. Several genetic parameters have been analysed on a larger set of cultivars allowing for a deeper characterization of the selected loci. Results of this study provide a list of recommended markers and protocols for olive genotyping as well as the allelic profile of a set of reference cultivars that would be useful for the establishment of a universal database of olive accessions.

Morphological and molecular analyses for the characterization of a group of Italian olive cultivars

A group of olive varieties, commonlycultivated in the sub-coastal area of theEmilia-Romagna region, in Italys northernolive cultivation area limit, weredescribed using both morphological andmolecular approaches. The morpho-biologicalcharacters of each accession were collectedaccording to the UPOVs descriptor list(International Union for the Protection ofNew Varieties of Plants). Biometric indexesof inflorescence, leaf, fruit, and stone,measured using the Video Image Analysis(VIA), were compared to the molecular dataobtained on the same set of cultivars usingAFLP (Amplified Fragment LengthPolymorphism) and SSR (Simple SequenceRepeat) markers. The morphological andmolecular data led to similarrepresentations of the cultivarrelationships. However, only the AFLP andSSR data were able to characterize specificolive varieties and identify erroneousdenominations and cases of synonymy.

Olive phenolic compounds: metabolic and transcriptional profiling during fruit development

BackgroundOlive (Olea europaea L.) fruits contain numerous secondary metabolites, primarily phenolics, terpenes and sterols, some of which are particularly interesting for their nutraceutical properties. This study will attempt to provide further insight into the profile of olive phenolic compounds during fruit development and to identify the major genetic determinants of phenolic metabolism.ResultsThe concentration of the major phenolic compounds, such as oleuropein, demethyloleuropein, 3–4 DHPEA-EDA, ligstroside, tyrosol, hydroxytyrosol, verbascoside and lignans, were measured in the developing fruits of 12 olive cultivars. The content of these compounds varied significantly among the cultivars and decreased during fruit development and maturation, with some compounds showing specificity for certain cultivars. Thirty-five olive transcripts homologous to genes involved in the pathways of the main secondary metabolites were identified from the massive sequencing data of the olive fruit transcriptome or from cDNA-AFLP analysis. Their mRNA levels were determined using RT-qPCR analysis on fruits of high- and low-phenolic varieties (Coratina and Dolce d’Andria, respectively) during three different fruit developmental stages. A strong correlation was observed between phenolic compound concentrations and transcripts putatively involved in their biosynthesis, suggesting a transcriptional regulation of the corresponding pathways. OeDXS, OeGES, OeGE10H and OeADH, encoding putative 1-deoxy-D-xylulose-5-P synthase, geraniol synthase, geraniol 10-hydroxylase and arogenate dehydrogenase, respectively, were almost exclusively present at 45 days after flowering (DAF), suggesting that these compounds might play a key role in regulating secoiridoid accumulation during fruit development.ConclusionsMetabolic and transcriptional profiling led to the identification of some major players putatively involved in biosynthesis of secondary compounds in the olive tree. Our data represent the first step towards the functional characterisation of important genes for the determination of olive fruit quality.

Computational annotation of genes differentially expressed along olive fruit development

BackgroundOlea 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.ResultsmRNA 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.ConclusionThe 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.

Identification of new polymorphic regions and differentiation of cultivated olives (Olea europaea L.) through plastome sequence comparison

BackgroundThe cultivated olive (Olea europaea L.) is the most agriculturally important species of the Oleaceae family. Although many studies have been performed on plastid polymorphisms to evaluate taxonomy, phylogeny and phylogeography of Olea subspecies, only few polymorphic regions discriminating among the agronomically and economically important olive cultivars have been identified. The objective of this study was to sequence the entire plastome of olive and analyze many potential polymorphic regions to develop new inter-cultivar genetic markers.ResultsThe complete plastid genome of the olive cultivar Frantoio was determined by direct sequence analysis using universal and novel PCR primers designed to amplify all overlapping regions. The chloroplast genome of the olive has an organisation and gene order that is conserved among numerous Angiosperm species and do not contain any of the inversions, gene duplications, insertions, inverted repeat expansions and gene/intron losses that have been found in the chloroplast genomes of the genera Jasminum and Menodora, from the same family as Olea.The annotated sequence was used to evaluate the content of coding genes, the extent, and distribution of repeated and long dispersed sequences and the nucleotide composition pattern. These analyses provided essential information for structural, functional and comparative genomic studies in olive plastids. Furthermore, the alignment of the olive plastome sequence to those of other varieties and species identified 30 new organellar polymorphisms within the cultivated olive.ConclusionsIn addition to identifying mutations that may play a functional role in modifying the metabolism and adaptation of olive cultivars, the new chloroplast markers represent a valuable tool to assess the level of olive intercultivar plastome variation for use in population genetic analysis, phylogenesis, cultivar characterisation and DNA food tracking.

Mutation scanning and genotyping by high-resolution DNA melting analysis in olive germplasm

The application of high-resolution melting (HRM) analysis of DNA is reported for scanning and genotyping Olea europaea germplasm. To test the sensitivity of the method, a functional gene marker, phytochrome A (phyA), was used, since this gene is correlated with important traits for the ecology of the species. We have designed a set of oligos able to produce amplicons of 307 bp to scan for the presence of single polymorphic mutations in a specific phyA fragment encompassing the chromophore attachment site (Cys323). The presence of mutations for substitution, either homozygous or heterozygous, was easily detected by melting curve analysis in a high-resolution melter. It has been established that the sensitivity of the HRM analysis can be significantly improved designing specific primers very close to the mutation sites. All SNPs found were confirmed by sequence analyses and ARMS-PCR. The method has also been confirmed to be very powerful for the visualization of microsatellite (SSR) length polymorphisms. HRM analysis has a very high reproducibility and sensitivity for detecting SNPs and SSRs, allowing olive cultivar genotyping and resulting in an informative, easy, and low-cost method able to greatly reduce the operating time.

Molecular interactions between the olive and the fruit fly Bactrocera oleae

BackgroundThe fruit fly Bactrocera oleae is the primary biotic stressor of cultivated olives, causing direct and indirect damages that significantly reduce both the yield and the quality of olive oil. To study the olive-B. oleae interaction, we conducted transcriptomic and proteomic investigations of the molecular response of the drupe. The identifications of genes and proteins involved in the fruit response were performed using a Suppression Subtractive Hybridisation technique and a combined bi-dimensional electrophoresis/nanoLC-ESI-LIT-MS/MS approach, respectively.ResultsWe identified 196 ESTs and 26 protein spots as differentially expressed in olives with larval feeding tunnels. A bioinformatic analysis of the identified non-redundant EST and protein collection indicated that different molecular processes were affected, such as stress response, phytohormone signalling, transcriptional control and primary metabolism, and that a considerable proportion of the ESTs could not be classified. The altered expression of 20 transcripts was also analysed by real-time PCR, and the most striking differences were further confirmed in the fruit of a different olive variety. We also cloned the full-length coding sequences of two genes, Oe-chitinase I and Oe-PR27, and showed that these are wound-inducible genes and activated by B. oleae punctures.ConclusionsThis study represents the first report that reveals the molecular players and signalling pathways involved in the interaction between the olive fruit and its most damaging biotic stressor. Drupe response is complex, involving genes and proteins involved in photosynthesis as well as in the production of ROS, the activation of different stress response pathways and the production of compounds involved in direct defence against phytophagous larvae. Among the latter, trypsin inhibitors should play a major role in drupe resistance reaction.

Proteome regulation during Olea europaea fruit development.

Background Widespread in the Mediterranean basin, Olea europaea trees are gaining worldwide popularity for the nutritional and cancer-protective properties of the oil, mechanically extracted from ripe fruits. Fruit development is a physiological process with remarkable impact on the modulation of the biosynthesis of compounds affecting the quality of the drupes as well as the final composition of the olive oil. Proteomics offers the possibility to dig deeper into the major changes during fruit development, including the important phase of ripening, and to classify temporal patterns of protein accumulation occurring during these complex physiological processes. Methodology/Principal Findings In this work, we started monitoring the proteome variations associated with olive fruit development by using comparative proteomics coupled to mass spectrometry. Proteins extracted from drupes at three different developmental stages were separated on 2-DE and subjected to image analysis. 247 protein spots were revealed as differentially accumulated. Proteins were identified from a total of 121 spots and discussed in relation to olive drupe metabolic changes occurring during fruit development. In order to evaluate if changes observed at the protein level were consistent with changes of mRNAs, proteomic data produced in the present work were compared with transcriptomic data elaborated during previous studies. Conclusions/Significance This study identifies a number of proteins responsible for quality traits of cv. Coratina, with particular regard to proteins associated to the metabolism of fatty acids, phenolic and aroma compounds. Proteins involved in fruit photosynthesis have been also identified and their pivotal contribution in oleogenesis has been discussed. To date, this study represents the first characterization of the olive fruit proteome during development, providing new insights into fruit metabolism and oil accumulation process.