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.

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.

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.

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.

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.

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.

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.

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.

Plasticity of fruit and oil traits in olive among different environments.

Olive is a long-living perennial species with a wide geographical distribution, showing a large genetic and phenotypic variation in its growing area. There is an urgent need to uncover how olive phenotypic traits and plasticity can change regardless of the genetic background. A two-year study was conducted, based on the analysis of fruit and oil traits of 113 cultivars from five germplasm collections established in Mediterranean Basin countries and Argentina. Fruit and oil traits plasticity, broad-sense heritability and genotype by environment interaction were estimated. From variance and heritability analyses, it was shown that fruit fresh weight was mainly under genetic control, whereas oleic/(palmitic + linoleic) acids ratio was regulated by the environment and genotype by environment interaction had the major effect on oil content. Among the studied cultivars, different level of stability was observed, which allowed ranking the cultivars based on their plasticity for oil traits. High thermal amplitude, the difference of low and high year values of temperature, negatively affected the oil content and the oleic acid percentage. Information derived from this work will help to direct the selection of cultivars with the highest global fitness averaged over the environments rather than the highest fitness in each environment separately.

Behavior of Four Olive Cultivars During Salt Stress.

Olive is considered as a moderately salt tolerant plant, however, tolerance to salt appears to be cultivar-dependent and genotypic responses have not been extensively investigated. In this work, saline stress was induced in four olive cultivars: Arbequina, Koroneiki, Royal de Cazorla and Fadak 86. The plants were grown in 2.5 l pots containing 60% peat and 40% of pumice mixture for 240 days and were irrigated three times a week with half-strength Hoagland solution containing 0, 100 and 200 mM NaCl. The effects of salt stress on growth, physiological and biochemical parameters were determined after 180, 210, and 240 days of treatment. Saline stress response was evaluated in leaves by measuring the activity of GSH and CAT enzymatic activity, as well as proline levels, gas exchanges, leaves relative water content and chlorophyll content, and proline content. All the studied cultivars showed a decrease in Net Photosynthesis, leaves chlorophyll content and plant growth (mainly leaves dry weight) and an increase in the activity of GSH and CAT. In addition, the reduction of proline content in leaf tissues, induced an alteration of osmotic regulation. Among the studied cultivars Royal and Koroneiki better counteracting the effects of saline stress thanks to a higher activity of two antioxidant enzymes.

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.

Genetic Mapping of the Incompatibility Locus in Olive and Development of a Linked Sequence-Tagged Site Marker.

The genetic control of self-incompatibility (SI) has been recently disclosed in olive. Inter-varietal crossing confirmed the presence of only two incompatibility groups (G1 and G2), suggesting a simple Mendelian inheritance of the trait. A double digest restriction associated DNA (ddRAD) sequencing of a biparental population segregating for incompatibility groups has been performed and high-density linkage maps were constructed in order to map the SI locus and identify gene candidates and linked markers. The progeny consisted of a full-sib family of 229 individuals derived from the cross 'Leccino' (G1) × 'Dolce Agogia' (G2) varieties, segregating 1:1 (G1:G2), in accordance with a diallelic self-incompatibility (DSI) model. A total of 16,743 single nucleotide polymorphisms was identified, 7,006 in the female parent 'Leccino' and 9,737 in the male parent 'Dolce Agogia.' Each parental map consisted of 23 linkage groups and showed an unusual large size (5,680 cM in 'Leccino' and 3,538 cM in 'Dolce Agogia'). Recombination was decreased across all linkage groups in pollen mother cells of 'Dolce Agogia,' the parent with higher heterozygosity, compared to megaspore mother cells of 'Leccino,' in a context of a species that showed exceptionally high recombination rates. A subset of 109 adult plants was assigned to either incompatibility group by a stigma test and the diallelic self-incompatibility (DSI) locus was mapped to an interval of 5.4 cM on linkage group 18. This region spanned a size of approximately 300 Kb in the olive genome assembly. We developed a sequence-tagged site marker in the DSI locus and identified five haplotypes in 57 cultivars with known incompatibility group assignment. A combination of two single-nucleotide polymorphisms (SNPs) was sufficient to predict G1 or G2 phenotypes in olive cultivars, enabling early marker-assisted selection of compatible genotypes and allowing for a rapid screening of inter-compatibility among cultivars in order to guarantee effective fertilization and increase olive production. The construction of high-density linkage maps has led to the development of the first functional marker in olive and provided positional candidate genes in the SI locus.

Usefulness of a New Large Set of High Throughput EST-SNP Markers as a Tool for Olive Germplasm Collection Management.

Germplasm collections are basic tools for conservation, characterization, and efficient use of olive genetic resources. The identification of the olive cultivars maintained in the collections is an important ongoing task which has been performed by both, morphological and molecular markers. In the present study, based on the sequencing results of previous genomic projects, a new set of 1,043 EST-SNP markers has been identified. In order to evaluate its discrimination capacity and utility in diversity studies, this set of markers was used in a representative number of accessions from 20 different olive growing countries and maintained at the World Olive Germplasm Collection of IFAPA Centre 'Alameda del Obispo' (Córdoba, Spain), one of the world's largest olive germplasm bank. Thus, the cultivated material included: cultivars belonging to previously defined core collections by means of SSR markers and agronomical traits, well known homonymy cases, possible redundancies previously identified in the collection, and recently introduced accessions. Marker stability was tested in repeated analyses of a selected number of accessions, as well as in different trees and accessions belonging to the same cultivar. In addition, 15 genotypes from a cross 'Picual' × 'Arbequina' cultivars from the IFAPA olive breeding program and a set of 89 wild genotypes were also included in the study. Our results indicate that, despite their relatively wide variability, the new set of EST-SNPs displayed lower levels of genetic diversity than SSRs in the set of olive core collections tested. However, the EST-SNP markers displayed consistent and reliable results from different plant material sources and plant propagation events. The EST-SNPs revealed a clear cut off between inter- and intra-cultivar variation in olive. Besides, they were able to reliably discriminate among different accessions, to detect possible homonymy cases as well as efficiently ascertain the presence of redundant germplasm in the collection. Additionally, these markers were highly transferable to the wild genotypes. These results, together with the low genotyping error rates and the easy and fully automated procedure used to get the genotyping data, validate the new set of EST-SNPs as possible markers of choice for olive cultivar identification.

High Levels of Variation Within Gene Sequences of Olea europaea L.

Gene sequence variation in cultivated olive (Olea europaea L. subsp. europaea var. europaea), the most important oil tree crop of the Mediterranean basin, has been poorly evaluated up to now. A deep sequence analysis of fragments of four genes, OeACP1, OeACP2, OeLUS and OeSUT1, in 90 cultivars, revealed a wide range of polymorphisms along all recognized allele forms and unexpected allele frequencies and genotype combinations. High linkage values among most polymorphisms were recorded within each gene fragment. The great sequence variability corresponded to a low number of alleles and, surprisingly, to a small fraction of genotype combinations. The distribution, frequency, and combination of the different alleles at each locus is possibly due to natural and human pressures, such as selection, ancestrality, or fitness. Phylogenetic analyses of allele sequences showed distant and complex patterns of relationships among cultivated olives, intermixed with other related forms, highlighting an evolutionary connection between olive cultivars and the O. europaea subspecies cuspidata and cerasiformis. This study demonstrates how a detailed and complete sequence analysis of a few gene portions and a thorough genotyping on a representative set of cultivars can clarify important issues related to sequence polymorphisms, reconstructing the phylogeny of alleles, as well as the genotype combinations. The identification of regions representing blocks of recombination could reveal polymorphisms that represent putatively functional markers. Indeed, specific mutations found on the analyzed OeACP1 and OeACP2 fragments seem to be correlated to the fruit weight.