Meta-analysis of transcriptome reveals key genes relating to oil quality in olive.

BACKGROUND: Olive oil contains monounsaturated oleic acid up to 83% and phenolic compounds, making it an excellent source of fat. Due to its economic importance, the quantity and quality of olive oil should be improved in parallel with international standards. In this study, we analyzed the raw RNA-seq data with a meta-analysis approach to identify important genes and their metabolic pathways involved in olive oil quality. RESULTS: A deep search of RNA-seq published data shed light on thirty-nine experiments associated with the olive transcriptome, four of these proved to be ideal for meta-analysis. Meta-analysis confirmed the genes identified in previous studies and released new genes, which were not identified before. According to the IDR index, the meta-analysis had good power to identify new differentially expressed genes. The key genes were investigated in the metabolic pathways and were grouped into four classes based on the biosynthetic cycle of fatty acids and factors that affect oil quality. Galactose metabolism, glycolysis pathway, pyruvate metabolism, fatty acid biosynthesis, glycerolipid metabolism, and terpenoid backbone biosynthesis were the main pathways in olive oil quality. In galactose metabolism, raffinose is a suitable source of carbon along with other available sources for carbon in fruit development. The results showed that the biosynthesis of acetyl-CoA in glycolysis and pyruvate metabolism is a stable pathway to begin the biosynthesis of fatty acids. Key genes in oleic acid production as an indicator of oil quality and critical genes that played an important role in production of triacylglycerols were identified in different developmental stages. In the minor compound, the terpenoid backbone biosynthesis was investigated and important enzymes were identified as an interconnected network that produces important precursors for the synthesis of a monoterpene, diterpene, triterpene, tetraterpene, and sesquiterpene biosynthesis. CONCLUSIONS: The results of the current investigation can produce functional data related to the quality of olive oil and would be a useful step in reducing the time of cultivar screening by developing gene specific markers in olive breeding programs, releasing also new genes that could be applied in the genome editing approach.

Genealogical tracing of Olea europaea species and pedigree relationships of var. europaea using chloroplast and nuclear markers.

BACKGROUND: Olive is one of the most cultivated species in the Mediterranean Basin and beyond. Despite being extensively studied for its commercial relevance, the origin of cultivated olive and the history of its domestication remain open questions. Here, we present a genealogical and kinship relationships analysis by mean of chloroplast and nuclear markers of different genera, subgenus, species, subspecies, ecotypes, cultivated, ancient and wild types, which constitutes one of the most inclusive research to date on the diversity within Olea europaea species. A complete survey of the variability across the nuclear and plastid genomes of different genotypes was studied through single nucleotide polymorphisms, indels (insertions and deletions), and length variation. RESULTS: Fifty-six different chlorotypes were identified among the Oleaceae family including Olea europaea, other species and genera. The chloroplast genome evolution, within Olea europaea subspecies, probably started from subsp. cuspidata, which likely represents the ancestor of all the other subspecies and therefore of wild types and cultivars. Our study allows us to hypothesize that, inside the subspecies europaea containing cultivars and the wild types, the ancestral selection from var. sylvestris occurred both in the eastern side of the Mediterranean and in the central-western part of Basin. Moreover, it was elucidated the origin of several cultivars, which depends on the introduction of eastern cultivars, belonging to the lineage E1, followed by crossing and replacement of the autochthonous olive germplasm of central-western Mediterranean Basin. In fact, our study highlighted that two main 'founders' gave the origin to more than 60% of analyzed olive cultivars. Other secondary founders, which strongly contributed to give origin to the actual olive cultivar diversity, were already detected. CONCLUSIONS: The application of comparative genomics not only paves the way for a better understanding of the phylogenetic relationships within the Olea europaea species but also provides original insights into other elusive evolutionary processes, such as chloroplast inheritance and parentage inside olive cultivars, opening new scenarios for further research such as the association studies and breeding programs.

OeBAS and CYP716C67 catalyze the biosynthesis of health-beneficial triterpenoids in olive (Olea europaea) fruits.

The bioactive properties of olive (Olea europaea) fruits and olive oil are largely attributed to terpenoid compounds, including diverse triterpenoids such as oleanolic, maslinic and ursolic acids, erythrodiol, and uvaol. They have applications in the agri-food, cosmetics, and pharmaceutical industries. Some key steps involved in the biosynthesis of these compounds are still unknown. Genome mining, biochemical analysis, and trait association studies have been used to identify major gene candidates controlling triterpenoid content of olive fruits. Here, we identify and functionally characterize an oxidosqualene cyclase (OeBAS) required for the production of the major triterpene scaffold ß-amyrin, the precursor of erythrodiol, oleanolic and maslinic acids, and a cytochrome P450 (CYP716C67) that mediates 2α oxidation of the oleanane- and ursane-type triterpene scaffolds to produce maslinic and corosolic acids, respectively. To confirm the enzymatic functions of the entire pathway, we have reconstituted the olive biosynthetic pathway for oleanane- and ursane-type triterpenoids in the heterologous host, Nicotiana benthamiana. Finally, we have identified genetic markers associated with oleanolic and maslinic acid fruit content on the chromosomes carrying the OeBAS and CYP716C67 genes. Our results shed light on the biosynthesis of olive triterpenoids and provide new gene targets for germplasm screening and breeding for high triterpenoid content.

Volatile Olfactory Profiles of Umbrian Extra Virgin Olive Oils and Their Discrimination through MOX Chemical Sensors.

Extra virgin olive oil (EVOO) is the best vegetable oil worldwide but, at the same time, is one of the product victims of fraud in the agri-food sector, and the differences about quality within the extra-virgin olive oil category are often missed. Several scientific techniques were applied in order to guarantee the authenticity and quality of this EVOO. In the present study, the volatile compounds (VOCs) by gas chromatography-mass spectrometry with solid-phase micro-extraction detection (GC-MS SPME), organoleptic analysis by the official Slow Food panel and the detection by a Small Sensor System (S3) were applied. Ten EVOOs from Umbria, a central Italian region, were selected from the 2021 Slow Food Italian extra virgin olive oil official guide, which includes hundreds of high-quality olive oils. The results demonstrated the possibility to discriminate the ten EVOOs, even if they belong to the same Italian region, by all three techniques. The result of GC-MS SPME detection was comparable at the discrimination level to the organoleptic test with few exceptions, while the S3 was able to better separate some EVOOs, which were not discriminated perfectly by the other two methods. The correlation analysis performed among and between the three methodologies allowed us to identify 388 strong associations with a p value less than 0.05. This study has highlighted how much the mix of VOCs was different even among few and localized EVOOs. The correlation with the sensor detection, which is faster and chipper compared to the other two techniques, elucidated the similarities and discrepancies between the applied methods.

Characterization of Differentially Expressed Genes under Salt Stress in Olive.

Climate change, currently taking place worldwide and also in the Mediterranean area, is leading to a reduction in water availability and to groundwater salinization. Olive represents one of the most efficient tree crops to face these scenarios, thanks to its natural ability to tolerate moderate salinity and drought. In the present work, four olive cultivars (Koroneiki, Picual, Royal de Cazorla and Fadak86) were exposed to high salt stress conditions (200 mM of NaCl) in greenhouse, in order to evaluate their tolerance level and to identify key genes involved in salt stress response. Molecular and physiological parameters, as well as plant growth and leaves' ions Na+ and K+ content were measured. Results of the physiological measurements showed Royal de Cazorla as the most tolerant cultivar, and Fadak86 and Picual as the most susceptible ones. Ten candidate genes were analyzed and their complete genomic, CDS and protein sequences were identified. The expression analysis of their transcripts through reverse transcriptase quantitative PCR (RT-qPCR) demonstrated that only OeNHX7, OeP5CS, OeRD19A and OePetD were upregulated in tolerant cultivars, thus suggesting their key role in the activation of a salt tolerance mechanism.

The eastern part of the Fertile Crescent concealed an unexpected route of olive (Olea europaea L.) differentiation.

Background and Aims: Olive is considered a native plant of the eastern side of the Mediterranean basin, from where it should have spread westward along the Mediterranean shores, while little is known about its diffusion in the eastern direction. Methods: Genetic diversity levels and population genetic structure of a wide set of olive ecotypes and varieties collected from several provinces of Iran, representing a high percentage of the entire olive resources present in the area, was screened with 49 chloroplast and ten nuclear simple sequence repeat markers, and coupled with archaeo-botanical and historical data on Mediterranean olive varieties. Approximate Bayesian Computation was applied to define the demographic history of olives including Iranian germplasm, and species distribution modelling was performed to understand the impact of the Late Quaternary on olive distribution. Key Results: The results of the present study demonstrated that: (1) the climatic conditions of the last glacial maximum had an important role on the actual olive distribution, (2) all Iranian olive samples had the same maternal inheritance as Mediterranean cultivars, and (3) the nuclear gene flow from the Mediterranean basin to the Iranian plateau was almost absent, as well as the contribution of subspecies cuspidata to the diversity of Iranian olives. Conclusions: Based on this evidence, a new scenario for the origin and distribution of this important fruit crop has been traced. The evaluation of olive trees growing in the eastern part of the Levant highlighted a new perspective on the spread and distribution of olive, suggesting two routes of olive differentiation, one westward, spreading along the Mediterranean basin, and another moving towards the east and reaching the Iranian plateau before its domestication.

Overexpression of the olive acyl carrier protein gene (OeACP1) produces alterations in fatty acid composition of tobacco leaves.

Taking into account that fatty acid (FA) biosynthesis plays a crucial role in lipid accumulation in olive (Olea europaea L.) mesocarp, we investigated the effect of olive acyl carrier protein (ACP) on FA composition by overexpressing an olive ACP cDNA in tobacco plants. The OeACP1.1A cDNA was inserted in the nucleus or in the chloroplast DNA of different tobacco plants, resulting in extensive transcription of the transgenes. The transplastomic plants accumulated lower olive ACP levels in comparison to nuclear-transformed plants. Moreover, the phenotype of the former plants was characterized by pale green/white cotyledons with abnormal chloroplasts, delayed germination and reduced growth. We suggest that the transplastomic phenotype was likely caused by inefficient olive ACP mRNA translation in chloroplast stroma. Conversely, total lipids from leaves of nuclear transformants expressing high olive ACP levels showed a significant increase in oleic acid (18:1) and linolenic acid (18:3), and a concomitant significant reduction of hexadecadienoic acid (16:2) and hexadecatrienoic acid (16:3). This implies that in leaves of tobacco transformants, as likely in the mesocarp of olive fruit, olive ACP not only plays a general role in FA synthesis, but seems to be specifically involved in chain length regulation forwarding the elongation to C18 FAs and the subsequent desaturation to 18:1 and 18:3.

Polymorphisms in the AOX2 gene are associated with the rooting ability of olive cuttings.

KEY MESSAGE: Different rooting ability candidate genes were tested on an olive cross progeny. Our results demonstrated that only the AOX2 gene was strongly induced. OeAOX2 was fully characterised and correlated to phenotypical traits. The formation of adventitious roots is a key step in the vegetative propagation of trees crop species, and this ability is under strict genetic control. While numerous studies have been carried out to identify genes controlling adventitious root formation, only a few loci have been characterised. In this work, candidate genes that were putatively involved in rooting ability were identified in olive (Olea europaea L.) by similarity with orthologs identified in other plant species. The mRNA levels of these genes were analysed by real-time PCR during root induction in high- (HR) and low-rooting (LR) individuals. Interestingly, alternative oxidase 2 (AOX2), which was previously reported to be a functional marker for rooting in olive cuttings, showed a strong induction in HR individuals. From the OeAOX2 full-length gene, alleles and effective polymorphisms were distinguished and analysed in the cross progeny, which were segregated based on rooting. The results revealed a possible correlation between two single nucleotide polymorphisms of OeAOX2 gene and rooting ability.

The homomorphic self-incompatibility system in Oleaceae is controlled by a hemizygous genomic region expressing a gibberellin pathway gene.

In flowering plants, outcrossing is commonly ensured by self-incompatibility (SI) systems. These can be homomorphic (typically with many different allelic specificities) or can accompany flower heteromorphism (mostly with just two specificities and corresponding floral types). The SI system of the Oleaceae family is unusual, with the long-term maintenance of only two specificities but often without flower morphology differences. To elucidate the genomic architecture and molecular basis of this SI system, we obtained chromosome-scale genome assemblies of Phillyrea angustifolia individuals and related them to a genetic map. The S-locus region proved to have a segregating 543-kb indel unique to one specificity, suggesting a hemizygous region, as observed in all distylous systems so far studied at the genomic level. Only one of the predicted genes in this indel region is found in the olive tree, Olea europaea, genome, also within a segregating indel. We describe complete association between the presence/absence of this gene and the SI types determined for individuals of seven distantly related Oleaceae species. This gene is predicted to be involved in catabolism of the gibberellic acid (GA) hormone, and experimental manipulation of GA levels in developing buds modified the male and female SI responses of the two specificities in different ways. Our results provide a unique example of a homomorphic SI system, where a single conserved gibberellin-related gene in a hemizygous indel underlies the long-term maintenance of two groups of reproductive compatibility.

Cold stress resilience of Iranian olive genetic resources: evidence from autochthonous genotypes diversity.

Olive (Olea europaea L.) is one of the most cultivated tree species in Iran. This plant is characterized by its tolerance to drought, salt, and heat stresses while being vulnerable to frost. During the last decade, periods of frost have occurred several times in Golestan Province, in the northeast of Iran, which caused severe damage to olive groves. This study aimed to evaluate and individuate autochthonous Iranian olive varieties with regard to frost tolerance and good agronomic performance. For this purpose, 218 frost-tolerant olive trees were selected from 150,000 adult olive trees (15-25 years old), following the last harsh autumn of 2016. The selected trees were reassessed at different intervals, i.e., 1, 4, and 7 months after the cold stress in field conditions. Using 19 morpho-agronomic traits, 45 individual trees with relatively stable frost-tolerance were reevaluated and selected for this research. Ten highly discriminating microsatellite markers were used for the genetic profiling of the 45 selected olive trees, and, ultimately, five genotypes with the highest tolerance among 45 selected ones were placed in a cold room at freezing temperatures for image analyses of cold damage. The results of morpho-agronomic analyses evidenced no bark splitting or symptoms of leaf drop in the 45 cold-tolerant olives (CTOs). The oil content of the cold-tolerant trees comprised almost 40% of the fruit dry weight, highlighting the potential of these varieties for oil production. Moreover, through molecular characterization, 36 unique molecular profiles were individuated among the 45 analyzed CTOs that were genetically more similar to the Mediterranean olive cultivars than the Iranian ones. The present study demonstrated the high potential of local olive varieties, which would be promising and more suitable than commercial olive varieties, with regard to the establishment of olive groves under cold climate conditions. This could be a valuable genetic resource for future breeding activities to face climate changes.

The Ancient Olive Trees (Olea europaea L.) of the Maltese Islands: A Rich and Unexplored Patrimony to Enhance Oliviculture.

A prospecting campaign in the Maltese Islands has ensured the survival of several ancient olive trees (Olea europaea L.), genetically distant from known cultivars. Most of these plants were abandoned or partially cultivated. A two-year evaluation of fruit characteristics and compositions was performed on samples collected from the main representatives of these indigenous genotypes. Analyses were carried out using Gas Chromatography, High-Performance Liquid Chromatography and Near Infrared Spectrometry. Among the fruit samples, a wide range of variations was observed. Some of the genotypes showed fruit traits suitable for table olive production. This is the case of samples with a pulp/pit ratio higher than four, such as 1Wardija, 1Caritas, 1Plattini, 1Bingemma Malta and 3Loretu, whilst 1Bidni, 1Mellieha, 2Qnotta, 3Loretu, 1Bingemma Malta and 1Caritas were suitable for dual purpose. The total phenol content ranged from 6.3 (1Wardija) to 117.9 (2Mtarfa) g/kg of fresh pulp. The average percentage of MUFA was quite low for most of the varieties. These genotypes, which presumably originated in the Maltese Islands and are well adapted to the local pedo-climatic conditions, are being propagated for the following evaluation of their bio-agronomical performance (production, suitability to intensive cultivation, environmental sustainability, product quality, etc.). The purpose is to select, among these local genotypes, the most outstanding varieties, in terms of phenolic and FA profile and agronomical potential, to spread into cultivation, thereby contributing to an increase in the quality of the local table and olive oil production, strongly linked to the territory.

Olive phenolic compounds: metabolic and transcriptional profiling during fruit development.

BACKGROUND: Olive (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. RESULTS: The 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. CONCLUSIONS: Metabolic 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.

Bioactive Compound Profiling of Olive Fruit: The Contribution of Genotype.

The health, therapeutic, and organoleptic characteristics of olive oil depend on functional bioactive compounds, such as phenols, tocopherols, squalene, and sterols. Genotype plays a key role in the diversity and concentration of secondary compounds peculiar to olive. In this study, the most important bioactive compounds of olive fruit were studied in numerous international olive cultivars during two consecutive seasons. A large variability was measured for each studied metabolite in all 61 olive cultivars. Total phenol content varied on a scale of 1-10 (3831-39,252 mg kg-1) in the studied cultivars. Squalene values fluctuated over an even wider range (1-15), with values of 274 to 4351 mg kg-1. Total sterols ranged from 119 to 969 mg kg-1, and total tocopherols varied from 135 to 579 mg kg-1 in fruit pulp. In the present study, the linkage among the most important quality traits highlighted the scarcity of cultivars with high content of at least three traits together. This work provided sound information on the fruit metabolite profile of a wide range of cultivars, which will facilitate the studies on the genomic regulation of plant metabolites and development of new olive genotypes through genomics-assisted breeding.

Correction: Transcript Analysis and Regulative Events during Flower Development in Olive (Olea europaea L.).

[This corrects the article DOI: 10.1371/journal.pone.0152943.].

How Temperatures May Affect the Synthesis of Fatty Acids during Olive Fruit Ripening: Genes at Work in the Field.

A major concern for olive cultivation in many extra-Mediterranean regions is the adaptation of recently introduced cultivars to environmental conditions different from those prevailing in the original area, such as the Mediterranean basin. Some of these cultivars can easily adapt their physiological and biochemical parameters in new agro-environments, whereas others show unbalanced values of oleic acid content. The objective of this study was to evaluate the effects of the thermal regime during oil synthesis on the expression of fatty acid desaturase genes and on the unsaturated fatty acid contents at the field level. Two cultivars (Arbequina and Coratina) were included in the analysis over a wide latitudinal gradient in Argentina. The results suggest that the thermal regime exerts a regulatory effect at the transcriptional level on both OeSAD2 and OeFAD2-2 genes and that this regulation is cultivar-dependent. It was also observed that the accumulated thermal time affects gene expression and the contents of oleic and linoleic acids in cv. Arbequina more than in Coratina. The fatty acid composition of cv. Arbequina is more influenced by the temperature regime than Coratina, suggesting its greater plasticity. Overall, findings from this study may drive future strategies for olive spreading towards areas with different or extreme thermal regimes serve as guidance for the evaluation olive varietal patrimony.

Evolution of Extra Virgin Olive Oil Quality under Different Storage Conditions.

The extent and conditions of storage may affect the stability and quality of extra virgin olive oil (EVOO). This study aimed at evaluating the effects of different storage conditions (ambient, 4 °C and -18 °C temperatures, and argon headspace) on three EVOOs (low, medium, and high phenols) over 18 and 36 months, analyzing the main metabolites at six time points. The results showed that low temperatures are able to maintain all three EVOOs within the legal limits established by the current EU regulations for most compounds up to 36 months. Oleocanthal, squalene, and total phenols were affected by storage temperatures more than other compounds and degradation of squalene and α-tocopherol was inhibited only by low temperatures. The best temperature for 3-year conservation was 4 °C, but -18 °C represented the optimum temperature to preserve the organoleptic properties. The present study provided new insights that should guide EVOO manufacturers and traders to apply the most efficient storage methods to maintain the characteristics of the freshly extracted oils for a long conservation time.

Diallelic self-incompatibility is the main determinant of fertilization patterns in olive orchards.

Self-incompatibility (SI) in flowering plants potentially represents a major obstacle for sexual reproduction, especially when the number of S-alleles is low. The situation is extreme in the commercially important olive tree, where in vitro pollination assays suggested the existence of a diallelic SI (DSI) system involving only two groups (G1 and G2). Varieties belonging to the same SI group cannot fertilize each other, such that successful fruit production is predicted to require pollination between varieties of different groups. To test this prediction, we explored the extent to which the DSI system determines fertilization patterns under field conditions. One hundred and seventeen olive cultivars were first genotyped using 10 highly polymorphic dinucleotide Simple Sequence Repeat (SSR) markers to ascertain varietal identity. Cultivars were then phenotyped through controlled pollination tests to assign each of them to one of the two SI groups. We then collected and genotyped 1440 open pollinated embryos from five different orchards constituted of seven local cultivars with known group of incompatibility groups. Embryos genotype information were used: (i) to assign embryos to the most likely pollen donor genotype in the neighbourhood using paternity analysis, and (ii) to compare the composition of the pollen cloud genetic among recipient trees in the five sites. The paternity analysis showed that the DSI system is the main determinant of fertilization success under field open pollination conditions: G1 cultivars sired seeds exclusively on G2 cultivars, and reciprocally. No self-fertilization events were observed. Our results demonstrate that DSI is a potent force determining pollination success among varieties within olive orchards used for production. They have the potential to improve management practices by guiding the selection of compatible varieties to avoid planting orchards containing sets of varieties with strongly unbalanced SI groups, as these would lead to suboptimal olive production.

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

BACKGROUND: The 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. RESULTS: The 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. CONCLUSIONS: In 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.

EST-SNP Study of Olea europaea L. Uncovers Functional Polymorphisms between Cultivated and Wild Olives.

BACKGROUND: The species Olea europaea includes cultivated varieties (subsp. europaea var. europaea), wild plants (subsp. europaea var. sylvestris), and five other subspecies spread over almost all continents. Single nucleotide polymorphisms in the expressed sequence tag able to underline intra-species differentiation are not yet identified, beyond a few plastidial markers. METHODS: In the present work, more than 1000 transcript-specific SNP markers obtained by the genotyping of 260 individuals were studied. These genotypes included cultivated, oleasters, and samples of subspecies guanchica, and were analyzed in silico, in order to identify polymorphisms on key genes distinguishing different Olea europaea forms. RESULTS: Phylogeny inference and principal coordinate analysis allowed to detect two distinct clusters, clearly separating wilds and guanchica samples from cultivated olives, meanwhile the structure analysis made possible to differentiate these three groups. Sequences carrying the polymorphisms that distinguished wild and cultivated olives were analyzed and annotated, allowing to identify 124 candidate genes that have a functional role in flower development, stress response, or involvement in important metabolic pathways. Signatures of selection that occurred during olive domestication, were detected and reported. CONCLUSION: This deep EST-SNP analysis provided important information on the genetic and genomic diversity of the olive complex, opening new opportunities to detect gene polymorphisms with potential functional and evolutionary roles, and to apply them in genomics-assisted breeding, highlighting the importance of olive germplasm conservation.

Physiological, epigenetic and genetic regulation in some olive cultivars under salt stress.

Cultivated olive, a typical fruit crop species of the semi-arid regions, could successfully face the new scenarios driven by the climate change through the selection of tolerant varieties to salt and drought stresses. In the present work, multidisciplinary approaches, including physiological, epigenetic and genetic studies, have been applied to clarify the salt tolerance mechanisms in olive. Four varieties (Koroneiki, Royal de Cazorla, Arbequina and Picual) and a related form (O. europaea subsp. cuspidata) were grown in a hydroponic system under different salt concentrations from zero to 200 mM. In order to verify the plant response under salt stress, photosynthesis, gas exchange and relative water content were measured at different time points, whereas chlorophyll and leaf concentration of Na+, K+ and Ca2+ ions, were quantified at 43 and 60 days after treatment, when stress symptoms became prominent. Methylation sensitive amplification polymorphism (MSAP) technique was used to assess the effects of salt stress on plant DNA methylation. Several fragments resulted differentially methylated among genotypes, treatments and time points. Real time quantitative PCR (RT-qPCR) analysis revealed significant expression changes related to plant response to salinity. Four genes (OePIP1.1, OePetD, OePI4Kg4 and OeXyla) were identified, as well as multiple retrotransposon elements usually targeted by methylation under stress conditions.