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The release of new olive cultivars with an increased squalene content in their virgin olive oil is considered an important target in olive breeding programs. In this work, the variability of the squalene content in a core collection of 36 olive cultivars was first studied, revealing two olive cultivars, 'Dokkar' and 'Klon-14', with extremely low and high squalene contents in their oils, respectively. Next, four cDNA sequences encoding squalene synthases (SQS) were cloned from olive. Sequence analysis and functional expression in bacteria confirmed that they encode squalene synthases. Transcriptional analysis in distinct olive tissues and cultivars indicated that expression levels of these four SQS genes are spatially and temporally regulated in a cultivar-dependent manner and pointed to OeSQS2 as the gene mainly involved in squalene biosynthesis in olive mesocarp and, therefore, in the olive oil. In addition, the biosynthesis of squalene appears to be transcriptionally regulated in water-stressed olive mesocarp.

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.

The fruit size of a cultivated olive tree is consistently larger than its corresponding wild relatives because fruit size is one of the main traits associated with olive tree domestication. Additionally, large fruit size is one of the main objectives of modern olive breeding programs. However, as the long juvenile period is one main hindrance in classic breeding approaches, obtaining genetic markers associated with this trait is a highly desirable tool. For this reason, GWAS analysis of both genetic markers and the genes associated with fruit size determination, measured as fruit weight, was herein carried out in 50 genotypes, of which 40 corresponded to cultivated and 10 to wild olive trees. As a result, 113 genetic markers were identified, which showed a very high statistically significant correlation with fruit weight variability, p < 10−10. These genetic markers corresponded to 39 clusters of genes in linkage disequilibrium. The analysis of a segregating progeny of the cross of “Frantoio” and “Picual” cultivars allowed us to confirm 10 of the 18 analyzed clusters. The annotation of the genes in each cluster and the expression pattern of the samples taken throughout fruit development by RNAseq enabled us to suggest that some studied genes are involved in olive fruit weight determination.

In Spain, several local studies have highlighted the likely presence of unknown olive cultivars distinct from the approximately 260 ones previously described in the literature. Furthermore, recent advancements in identification techniques have significantly enhanced in terms of efficacy and precision. This scenario motivated a new nationwide prospecting effort aimed at recovering and characterizing new cultivated germplasm using high-throughput molecular markers. In the present study, the use of 96 EST-SNP markers allowed the identification of a considerable amount of new material (173 new genotypes) coming from areas with low intensification of production in different regions of Spain. As a result, the number of distinct national genotypes documented in the World Olive Germplasm Bank of IFAPA, Córdoba (WOGBC-ESP046) increased to 427. Likewise, 65 and 24 new synonymy and homonymy cases were identified, respectively. This rise in the number of different national cultivars allowed to deepen the knowledge about the underlying genetic structure. The great genetic variability of Spanish germplasm was confirmed, and a new hot spot of diversity was identified in the northern regions of La Rioja and Aragon. Analysis of the genetic structure showed a clear separation between the germplasm of southern and northern-northeastern Spain and indicated a significantly higher level of admixture in the latter. Given the expansion of modern olive cultivation with only a few cultivars, this cryptic germplasm is in great danger of disappearing. This underlines the fact that maintaining as many cultivars as possible will increase the genetic variability of the olive gene pool to meet the future challenges of olive cultivation.

Olive, the emblematic Mediterranean fruit crop, owns a great varietal diversity, which is maintained in ex situ field collections, such as the World Olive Germplasm Bank of Córdoba (WOGBC), Spain. Accurate identification of WOGBC, one of the world's largest collections, is essential for efficient management and use of olive germplasm. The present study is the first report of the use of a core set of 96 EST-SNP markers for the fingerprinting of 1273 accessions from 29 countries, including both field and new acquired accessions. The EST-SNP fingerprinting made possible the accurate identification of 668 different genotypes, including 148 detected among the new acquired accessions. Despite the overall high genetic diversity found at WOGBC, the EST-SNPs also revealed the presence of remarkable redundant germplasm mostly represented by synonymy cases within and between countries. This finding, together with the presence of homonymy cases, may reflect a continuous interchange of olive cultivars, as well as a common and general approach for their naming. The structure analysis revealed a certain geographic clustering of the analysed germplasm. The EST-SNP panel under study provides a powerful and accurate genotyping tool, allowing for the foundation of a common strategy for efficient safeguarding and management of olive genetic resources.

Understanding microbial associations through co-occurrence network analysis is a very effective tool to detect patterns of copresence and mutual exclusion among members of the plant-associated microbiome. By using 16S and ITS rRNA metagenomics equencing, we studied the associations of bacterial and fungal communities present in xylem sap of plantlets and adult olive trees of 'Picual', 'Arbequina', 'Arbosana', 'Frantoio' and 'Hojiblanca' olive varieties. Results based on the analysis of amplicon sequence variant and network assembly analysis showed distinct microbial association behaviors according to olive plant age and genotype. Thus, Pseudomonas, Bosea and Meiothermus showed the highest positive associations in the xylem of adult trees whereas Azospirillium, Rhizobium and Brevundimonas showed the greatest positive interactions in olive plantlets. Furthermore, MCODE method identified clusters of highly interconnected regions for each olive genotype, where Alternaria and Phaeococcomyces genera were unique to ‘Arbequina’; Rhizobium, Bradyrhizobium and Brevibacillus were exclusive to ‘Arbosana’; Gardnerella and Rathayibacter to ‘Frantoio’; Acinetobacter and Micrococcus to ‘Hojiblanca’ and Arthrobacter, Lawsonella, Massilia, Sphingopyxis, Thermaerobacter and Variovorax to ‘Picual’. Interestingly, Pseudomonas and Anoxybacillus were positioned at the network cores obtained when data were analyzed by plant age and plant genotype, respectively. Network association analysis has revealed new knowledge regarding specific microbial associations occurring among olive genotypes at different plant age, offering the identification of keystone xylem-inhabiting microbial species that may result determinant for olive growth and health.

Los microorganismos endófitos representan una estrategia innovadora para el manejo de las enfermedades vasculares en el olivo actuando como una defensa natural innata para hacer frente a la infección por patógenos que colonizan el xilema como son Verticillium dahliae y Xylella fastidiosa. Sin embargo, pocos estudios han caracterizado el efecto de la variedad y la edad de la planta en la estructura y diversidad microbiana del xilema del olivo. Por lo tanto, este trabajo se ha centrado en la caracterización mediante un enfoque metagenómico de las comunidades bacterianas y fúngicas presentes en el fluido xilemático de plantones jóvenes y olivos adultos de las variedades de olivo ‘Picual’, ‘Arbequina’, ‘Arbosana’, ‘Frantoio’ y ‘Hojiblanca’. Se han identificado un total de 440 ASVs bacterianos y 96 ASVs fúngicos que variaron en función de la edad de la planta alcanzando una diversidad (alfa) microbiana mayor en plantas adultas que en los plantones jóvenes y presentando una estructura microbiana (beta-diversidad) diferente en función de la edad, pero no de la variedad. Por otra parte, las variedades ‘Arbosana’ y ‘Hojiblanca’ mostraron una mayor riqueza bacteriana y fúngica, respectivamente. Globalmente, Meiothermus (24.61%), Anoxybacillus (18.21%) y Paenibacillus (13.15%) fueron los géneros bacterianos más representativos mientras que Aureobasidium fue el hongo más abundante (50.51%), seguido de Plectosphaerella (6.94%) y Malassezia (6.38%). Estos resultados contribuyen a mejorar la caracterización y el conocimiento de la variabilidad del microbioma xilemático de olivo, generando una información muy útil para optimizar el manejo de las enfermedades vasculares del olivo mediante el uso de sus propios microrganismos endofíticos como agentes control biológico.

[Background] Olive orchards are threatened by a wide range of pathogens. Of these, Verticillium dahliae has been in the spotlight for its high incidence, the difficulty to control it and the few cultivars that has increased tolerance to the pathogen. Disease resistance not only depends on detection of pathogen invasion and induction of responses by the plant, but also on barriers to avoid the invasion and active resistance mechanisms constitutively expressed in the absence of the pathogen. In a previous work we found that two healthy non-infected plants from cultivars that differ in V. dahliae resistance such as ‘Frantoio’ (resistant) and ‘Picual’ (susceptible) had a different root morphology and gene expression pattern. In this work, we have addressed the issue of basal differences in the roots between Resistant and Susceptible cultivars., [Results] The gene expression pattern of roots from 29 olive cultivars with different degree of resistance/susceptibility to V. dahliae was analyzed by RNA-Seq. However, only the Highly Resistant and Extremely Susceptible cultivars showed significant differences in gene expression among various groups of cultivars. A set of 421 genes showing an inverse differential expression level between the Highly Resistant to Extremely Susceptible cultivars was found and analyzed. The main differences involved higher expression of a series of transcription factors and genes involved in processes of molecules importation to nucleus, plant defense genes and lower expression of root growth and development genes in Highly Resistant cultivars, while a reverse pattern in Moderately Susceptible and more pronounced in Extremely Susceptible cultivars were observed., [Conclusion] According to the different gene expression patterns, it seems that the roots of the Extremely Susceptible cultivars focus more on growth and development, while some other functions, such as defense against pathogens, have a higher expression level in roots of Highly Resistant cultivars. Therefore, it seems that there are constitutive differences in the roots between Resistant and Susceptible cultivars, and that susceptible roots seem to provide a more suitable environment for the pathogen than the resistant ones.

Fatty acid composition of olive oil has an important effect on the oil quality to such an extent that oils with a high oleic and low linoleic acid contents are preferable from a nutritional and technological point of view. In the present work, we have first studied the diversity of the fatty acid composition in a set of eighty-nine olive cultivars from the Worldwide Olive Germplasm Bank of IFAPA Cordoba (WOGBC-IFAPA), and in a core collection (Core-36), which includes 28 olive cultivars from the previously mentioned set. Our results indicate that oleic and linoleic acid contents displayed the highest degree of variability of the different fatty acids present in the olive oil of the 89 cultivars under study. In addition, the independent study of the Core-36 revealed two olive cultivars, Klon-14 and Abou Kanani, with extremely low and high linoleic acid contents, respectively. Subsequently, these two cultivars were used to investigate the specific contribution of different fatty acid desaturases to the linoleic acid content of mesocarp tissue during olive fruit development and ripening. Fatty acid desaturase gene expression levels, together with lipid analysis, suggest that not only OeFAD2-2 and OeFAD2-5 but also the different specificities of extraplastidial acyltransferase enzymes are responsible for the variability of the oleic/linoleic acid ratio in olive cultivars. All this information allows for an advancement in the knowledge of the linoleic acid biosynthesis in different olive cultivars, which can impact olive breeding programs to improve olive oil quality.

The characterization of tree canopy traits represents a key step to identify potential cultivars adaptable to a specific growing system. Developing methodologies to rapidly evaluate canopy traits allows to classify cultivars in germplasm banks and facilitates selections in breeding programs. In the present work, the ability of low-cost unmanned aerial vehicle (UAV) was compared with on-ground traditional methodology for measuring canopy traits (namely crown diameters, tree height and crown volume) in trees of the Worldwide Olive Germplasm Bank of Córdoba. The evaluated trees belonged to a wide range of cultivars and were planted in three different dates. A wide variability was found for the four parameters measured with both on ground and UAV methodologies. Besides, a significant linear correlation was observed at tree and cultivar level, with relative errors that ranged from 4.44 to 13.9 % for axial parameters or from 17.0–29.0% for volume, depending on the planting date considered. ANOVA and Tukey's results showed a high agreement between both methodologies in identifying cultivars with similar vigour values. When comparing means over time, the UAV-based data allowed a slightly better detection of significant differences than on-ground data. The results provided by the present research indicated that the use of UAV is a reliable methodology to study tree canopy traits in olive germplasm banks, supporting breeders to the identification and definition of the genetics behind crop variability. The evaluation here performed indicates that there is a wide variability on canopy traits on the cultivated olive germplasm that could be exploited to select potential cultivars for different growing systems.

Aim of study: Crop phenology is a critical component in the identification of impacts of climate change. Then, the assessment of germplasm collections provides relevant information for cultivar selection and breeding related to phenology, being the base for identifying adaptation strategies to climate change.Area of study: The World Olive Germplasm Bank located at IFAPA Centre “Alameda del Obispo” (WOGB-IFAPA) in Cordoba (Southern Spain) was considered for the study.Material and methods: Data gathered for nine years on flowering and ripening time of olive cultivars from WOGB-IFAPA were evaluated. Thus, full flowering date (FFD) for 148 cultivars and ripening date (RD) for 86 cultivars, coming from 14 olive growing countries, were considered for characterization of olive phenology and for calibration/validation of phenological models.Main results: The characterization of WOGB-IFAPA has allowed the identification of cultivars with extreme early (‘Borriolenca’) and late (‘Ulliri i Kuq’) flowering as well as the ones with extreme early (‘Mavreya’) and late (‘Gerboui’) ripening dates. However, the very limited inter-cultivar variability, especially for FFD, resulted in a non-optimal simulation models performance. Thus, for FFD and RD the root mean square error was around 6 and 24 days, respectively. The limited inter-cultivar variability was associated to the low average temperatures registered during winter at WOGB-IFAPA generating an early accumulation of the chilling requirements, thus homogenizing FFD of all the analyzed cultivars.Research highlights: The identification of cultivars with early FFD and late RD provides useful information for breeding programs and climate change studies for identifying adaptation strategies.

Aim of study: Crop phenology is a critical component in the identification of impacts of climate change. Then, the assessment of germplasm collections provides relevant information for cultivar selection and breeding related to phenology, being the base for identifying adaptation strategies to climate change.Area of study: The World Olive Germplasm Bank located at IFAPA Centre “Alameda del Obispo” (WOGB-IFAPA) in Cordoba (Southern Spain) was considered for the study.Material and methods: Data gathered for nine years on flowering and ripening time of olive cultivars from WOGB-IFAPA were evaluated. Thus, full flowering date (FFD) for 148 cultivars and ripening date (RD) for 86 cultivars, coming from 14 olive growing countries, were considered for characterization of olive phenology and for calibration/validation of phenological models.Main results: The characterization of WOGB-IFAPA has allowed the identification of cultivars with extreme early (‘Borriolenca’) and late (‘Ulliri i Kuq’) flowering as well as the ones with extreme early (‘Mavreya’) and late (‘Gerboui’) ripening dates. However, the very limited inter-cultivar variability, especially for FFD, resulted in a non-optimal simulation models performance. Thus, for FFD and RD the root mean square error was around 6 and 24 days, respectively. The limited inter-cultivar variability was associated to the low average temperatures registered during winter at WOGB-IFAPA generating an early accumulation of the chilling requirements, thus homogenizing FFD of all the analyzed cultivars.Research highlights: The identification of cultivars with early FFD and late RD provides useful information for breeding programs and climate change studies for identifying adaptation strategies.

Olive breeding has traditionally focused on the agronomic traits but the increasing number of scientific proofs supporting the positive impact of virgin olive oil (VOO) consumption on human health has made the functional quality of this product to be considered as an olive breeding target, particularly regarding the phenolic fraction of VOO. The presence of phenolic compounds in VOO is based mainly on the content of phenolic glucosides initially present in the olive fruit tissues and on the activity of hydrolytic and oxidative enzymes acting on these glucosides during the VOO extraction process. Thus, data on olive fruit phenolic composition would be of interest in olive breeding programs with the aim of obtaining new cultivars with improved functional quality. Taking into account that the main phenolics in olive fruits are essentially similar to those found in olive leaves, the objective of the present work was to carry out a comparative study of the phenolic fraction of fruits and leaves in selected cultivars characterized by having different contents of phenolic compounds in the oils to find out if the early selection of olive seedlings could be possible through an analysis of the phenolic compounds of their leaves. Results of the study showed that there is no correlation between the level of the fruit and leaf phenolics for the different olive cultivars. In addition, no significant correlation was found between the content of tyrosol and hydroxytyrosol derivatives in the leaves of the different olive cultivars over time as it does in the case of the fruits during the ripening process. These preliminary data pointed out that although the biosynthesis of the major olive phenolics is carried out through similar biochemical pathways, they seem to be regulated differently in the different organs of the plant.

Wild subspecies of Olea europaea constitute a source of genetic variability with huge potential for olive breeding to face global changes in Mediterranean-climate regions. We intend to identify wild olive genotypes with optimal adaptability to different environmental conditions to serve as a source of rootstocks and resistance genes for olive breeding. The SILVOLIVE collection includes 146 wild genotypes representative of the six O. europaea subspecies and early-generations hybrids. These genotypes came either from olive germplasm collections or from direct prospection in Spain, continental Africa and the Macaronesian archipelago. The collection was genotyped with plastid and nuclear markers, confirming the origin of the genotypes and their high genetic variability. Morphological and architectural parameters were quantified in 103 genotypes allowing the identification of three major groups of correlative traits including vigor, branching habits and the belowground-to-aboveground ratio. The occurrence of strong phenotypic variability in these traits within the germplasm collection has been shown. Furthermore, wild olive relatives are of great significance to be used as rootstocks for olive cultivation. Thus, as a proof of concept, different wild genotypes used as rootstocks were shown to regulate vigor parameters of the grafted cultivar “Picual” scion, which could improve the productivity of high-density hedgerow orchards.

The control of the olive tree vigor is a critical factor to ensure adequate production and longevity of high-density olive cultivation. To deal with this problem, we intend to take advantage of the genetic variability present in the wild subspecies of Olea europaea to identify genotypes of reduced vigor with optimal adaptability to diverse soils and adverse environmental conditions. In this work, early vigor traits have been characterized in different wild olive subspecies that could potentially be used as rootstocks to control the vigor of grafted cultivars. Significant differences between genotypes were obtained for all vigor traits and high values of broad sense heritability were obtained for most of them, indicating strong genetic effect and therefore good possibilities for selection. Compared to ‘Arbequina’ and ‘Picual’ cultivars, some of the wild genotypes evaluated showed a clearly lower vigor at the end of the experimental period. These genotypes could be tested as dwarfing rootstocks for high-density olive plantations.

Up to date, the bacterial and fungal microbial communities from the olive (Olea europaeaL.) root systems have not been simultaneously studied. In this work, we show that microbial communities from the olive root endosphere are less diverse than those from the rhizosphere. But more relevant was to unveil that olive belowground communities are mainly shaped by the genotype of the cultivar when growing under the same environmental, pedological and agronomic conditions. Furthermore, Actinophytocola, Streptomyces and Pseudonocardia are the most abundant bacterial genera in the olive root endosphere, Actinophytocola being the most prevalent genus by far. In contrast, Gp6, Gp4, Rhizobium and Sphingomonas are the main genera in the olive rhizosphere. Canalisporium, Aspergillus, Minimelanolocus and Macrophominaare the main fungal genera present in the olive root system. Interestingly enough, a high proportion of so far unclassified fungal sequences at class level were detected in the rhizosphere. From the belowground microbial profiles here reported, it can be concluded that the genus Actinophytocolamay play an important role in olive adaptation to environmental stresses. Moreover, the huge unknown fungal diversity suggests that there are still some fungi with important ecological and biotechnological implications that have yet to be discovered.

Olive (Olea europaea L.) is one of the most important fruit crops in the Mediterranean Basin, because it occupies significant acreage in these countries and often has important cultural heritage and landscape value. This crop can be infected by several Meloidogyne species (M. javanica, M. arenaria, and M. incognita, among others), and only a few cultivars with some level of resistance to these nematodes have been found. Innovations in intensive olive growing using high planting densities, irrigation, and substantial amounts of fertilizers could increase the nematode population to further damaging levels. To further understand the interactions involved between olive and pathogenic nematodes and in the hope of finding solutions to the agricultural risks, this research aimed to determine the reaction of important olive cultivars in Spain and wild olives to M. javanica infection, including genotypes of the same and other O. europaea subspecies. All olive cultivars tested were good hosts for M. javanica, but high levels of nematode reproduction found in three cultivars (Gordal Sevillana, Hojiblanca, and Manzanilla de Sevilla) were substantially different. In the wild accessions, O. europaea subsp. cerasiformis (genotype W147) and O. europaea subsp. europaea var. sylvestris (genotype W224) were resistant to M. javanica at different levels, with strong resistance in W147 (reproduction factor [Rf] = 0.0003) and moderate resistance in W224 (Rf = 0.79). The defense reaction of W147 to M. javanica showed a strong increase of phenolic compounds but no hypersensitive reaction.

The bacterial and fungal communities from the olive (Olea europaea L.) root systems have not yet been simultaneously studied. We show in this work that microbial communities from the olive root endosphere are less diverse than those from the rhizosphere. But more relevant was to unveil that olive belowground communities are mainly shaped by the genotype of the cultivar when growing under the same environmental, pedological and agronomic conditions. Furthermore, Actinophytocola, Streptomyces and Pseudonocardia are the most abundant bacterial genera in the olive root endosphere, Actinophytocola being the most prevalent genus by far. In contrast, Gp6, Gp4, Rhizobium and Sphingomonas are the main genera in the olive rhizosphere. Canalisporium, Aspergillus, Minimelanolocus and Macrophomina are the main fungal genera present in the olive root system. Interestingly enough, a large number of as yet unclassified fungal sequences (class level) were detected in the rhizosphere. From the belowground microbial profiles here reported, it can be concluded that the genus Actinophytocola may play an important role in olive adaptation to environmental stresses. Moreover, the huge unknown fungal diversity here uncovered suggests that fungi with important ecological function and biotechnological potential are yet to be identified.

Virgin olive oil (VOO) is the main source of lipids in the Mediterranean diet and one of the main contributors to its proven protection against diseases associated with chronic inflammation states. This oil is rich in antioxidant compounds such as tocopherols, which together constitute the vitamin E stock of the oil. The purpose of the present work was to conduct a study on the diversity of the contents of vitamin E in the olive species (Olea europaea L.), and to know how the season climatic conditions and the degree of fruit ripening stage influences the final content of this vitamin in VOO. Data showed that the content of vitamin E in VOO is highly dependent on the olive cultivar, displaying a wide variability (89–1410 mg tocopherol/kg oil) in the olive species, and that is also dependent, to a lesser extent, on the crop year climate and the stage of fruit ripening. In addition, the suitability of cultivar crosses for breeding programs to obtain new cultivars with improved vitamin E content in VOO has been assessed. Our findings demonstrated that a single cross of olive cultivars may provide sufficient variability to be used in the selection of new cultivars.

Virgin olive oil is characterized by its unique aroma, which is synthesized when olive fruits are crushed during the industrial process used for oil production. The genetic variability of the major volatile compounds comprising the oil aroma was studied in a representative sample of olive cultivars from the World Olive Germplasm Collection (IFAPA, Cordoba, Spain). The analytical data demonstrated that a high degree of variability for the content of volatile compounds is found in the olive species and that most of the volatile compounds found in the oils were synthetized by the enzymes included in the so-called lipoxygenase pathway. The use of multivariate analysis to identify cultivars is particularly interesting in terms of volatile composition and deduced organoleptic quality. It can be used for identification of old olive cultivars that give rise to oils with a high organoleptic quality and in parent selection for olive breeding programs.

Virgin olive oil is unique among plant oils for its high levels of oleic acid, and the presence of a wide range of minor components, which are responsible for both its health-promoting properties and characteristic aroma, and only produced when olives are crushed during the industrial process used for oil production. The genetic variability of the major volatile compounds comprising the oil aroma was studied in a representative sample of olive cultivars from the World Olive Germplasm Collection (IFAPA, Cordoba, Spain), by means of the headspace solid-phase microextraction/gas chromatography–mass spectrometry–flame ionization detection (HS-SPME/GC-MS-FID). The analytical data demonstrated that a high variability is found for the content of volatile compounds in olive species, and that most of the volatile compounds found in the oils were synthesized by the enzymes included in the so-called lipoxygenase pathway. Multivariate analysis allowed the identification of cultivars that are particularly interesting, in terms of volatile composition and presumed organoleptic quality, which can be used both to identify old olive cultivars that give rise to oils with a high organoleptic quality, and in parent selection for olive breeding programs.

The wide olive genetic patrimony has revealed high variability for most of the agronomic and oil quality traits of interest in olive growing. Few studies, however, have addressed the interaction of this variability with the environment, a subject of particular interest considering the natural high instability of the Mediterranean climate and the challenge of the predicted climate change. The current work presents results on the interaction between genotype and environment from multi-environment trials of olive cultivars and breeding selections, planted in different edaphoclimatic conditions of Andalusia, Southern Peninsular Spain and Canary Islands. For most of the agronomic and oil quality characters evaluated (flowering phenology, flower quality, pattern of oil accumulation, fatty acid composition and phenol content and composition), significant genotype and environment effects have been observed. For example, olive cultivars grown in Tenerife under much milder winter temperatures than in the Iberian Peninsula showed substantially earlierflowering and oil accumulation. Only in the case of flowering phenology was no significant genotype effect found. Furthermore, a strong genotype x environment effect was highly consistent in all characters considered. Regarding resistance to disease, such as Verticillium wilt, the variability of results from both natural and artificial inoculations also tends to indicate a considerable environmental effect and the need for careful testing of disease evolution. All this information strongly suggests the necessity of comparative trials of olive cultivars for both adequate choice of cultivar and final selection in breeding programs.

Inventorying, characterising and conserving on-farm ancient olive trees is a priority for safeguarding their genetic, natural and agricultural value and for protecting ancient genotypes threatened with extinction. In the “Taula del Sénia” (M-TdS) area (northeast Iberian Peninsula) a highly important cultural landscape has been preserved, in which the olive groves play an outstanding social and economic role: the ancient olive trees, sustained by many local farmers, constitute a living heritage and provide a clear example of High Nature Value (HNV). A total of 4526 ancient productive olive trees, with a trunk circumference (PBH) larger than 3.5 m, were inventoried and their spatial localization and biometric measurements were collected. 41 olive trees have shown the highest category in monumentality (PBH >8.1 m). The outstanding trees might be 634–1082 years old. The endocarp morphology of a representative sample of the most ancient trees from this settlement resulted in 14 different profiles. The ancient trees genotyped, through eight simple sequence repeat (SSR) markers, revealed 43 SSR profiles. The use of SSR enabled us to verify that most of the trees (98%) belong to the local cv. ‘Farga’, a male sterile variety with a rare chlorotype, only a few trees corresponded with other local varieties, ‘Morrut’, ‘Canetera’ and ‘Sevillenca’, and ten hitherto unidentified genotypes were distinguished, some with chloroplast lineages different from the ‘Farga’ type. The M-TdS area holds a unique living and exploitable heritage with the highest concentration of ancient olive trees worldwide. On-farm conservation of this germplasm by the community of local growers is enabling preservation of this important source of genetic variation, potentially holding traits of resilience and adaptation to adverse soil and climatic conditions, demonstrated by the survival of these trees over the centuries. Farmers have undertaken initiatives to valorize the olive oil deriving from these M-Td

There is an increasing need for diversification of olive (Olea europaea L.) cultivars, mainly because of the new intensive industry and the current trend of establishing olive productions in new areas outside the Mediterranean basin. Olive breeding has traditionally been focused on the improvement of agronomic traits. However, the nutritional quality of virgin olive oil (VOO) is lately being considered as a breeding target because of the growing weight of scientific evidence supporting the positive impact of VOO on human health. Increasing VOO consumption, through improvement of its nutritional and sensory properties, will in turn have a positive effect on both consumer health and olive-producing areas. VOO metabolites responsible for its nutritional and sensory quality include components such as phenolics and volatile compounds, which are synthesized during the oil extraction process by endogenous enzymes. Here, we aimed to investigate olive diversity regarding the key VOO quality metabolites of olive accessions cultivated around the world. For this purpose, oils from 97 olive cultivars representative of the diversity found at the World Olive Germplasm Collection (CAP-UCO-IFAPA), located at IFAPA Centre “Alameda del Obispo” Cordoba, Spain, were obtained and the phenolic and volatile fractions were further analyzed. The component profiles were compared with those of a similar subset of seedlings arisen from the cross of two widely cultivated olive cultivars having contrasting oil compositions ('Arbequina' and 'Picual'). Results showed that a single cross provides enough variability to produce new olive cultivars with improved quality. The set of cultivars from the collection displayed, in general, higher levels of variability than the progeny cross for the main groups of volatile compounds. However, the latter displayed a wider variability than the set of cultivars under study for most of the main classes of phenolic compounds in the oil, except the lignans.

Ethanol is a substrate for the chemical synthesis of fatty acid ethyl esters (FAEE) during storage of virgin olive oil whose contents are officially regulated. Given the impact that the ethanol content might have on the olive oil commercialization, the level of this metabolite has been studied in an array of olive genotypes representing the diversity available in olive (Olea europaea L.). Substantial levels of ethanol have been found in the oils of all genotypes under study. Moreover, increasing levels of alcohol dehydrogenase activity have been found during olive fruit ripening in good correspondence with the accumulation of ethanol in advanced stages of fruit maturation. The results suggest that ethanol has a ubiquitous character in the fruits of Olea europaea and, therefore, in all the oils obtained from them. Besides, their concentration seems to depend on the cultivar, ripening stage and climatology, not discarding the influence of the growing conditions. Data suggest that the application of olive oil regulation for FAEE levels should consider the presence of basal levels of ethanol in the oils, which are quite high in many cultivars.

A wide genetic diversity has been reported for wild olives, which could be particularly interesting for the introgression of some agronomic traits and resistance to biotic and abiotic stresses in breeding programs. However, the introgression of some beneficial wild traits may be paralleled by negative effects on some other important agronomic and quality traits. From the quality point of view, virgin olive oil (VOO) from olive cultivars is highly appreciated for its fatty acid composition (high monounsaturated oleic acid content) and the presence of several minor components. However, the composition of VOO from wild origin and its comparison with VOO from olive cultivars has been scarcely studied. In this work, the variability for fruit characters (fruit weight and oil content, OC), fatty acid composition, and minor quality components (squalene, sterols and tocopherols content and composition) was studied in a set of plant materials involving three different origins: wild genotypes (n = 32), cultivars (n = 62) and genotypes belonging to cultivar × wild progenies (n = 62). As expected, values for fruit size and OC in wild olives were lower than those obtained in cultivated materials, with intermediate values for cultivar × wild progenies. Wild olives showed a remarkably higher C16:0 percentage and tocopherol content in comparison to the cultivars. Contrarily, lower C18:1 percentage, squalene and sterol content were found in the wild genotypes, while no clear differences were found among the different plant materials regarding composition of the tocopherol and phytosterol fractions. Some common highly significant correlations among components of the same chemical family were found in all groups of plant materials. However, some other correlations were specific for one of the groups. The results of the study suggested that the use of wild germplasm in olive breeding programs will not have a negative impact on fatty acid composition, tocopherol content, and tocopherol a

Different scientific evidences suggest that the long term dietary consumption of virgin olive oil (VOO) seems to be related to an attenuation of the inflammatory response and reduction of the associated risk of chronic inflammatory disease states. VOO phenolic compounds are claimed to be the main responsible for these positive health benefits. They are mainly synthetized from phenolic glucosides present in the olive fruit by the action of glucosidases occurring when they come together once the olive fruit is crushed during olive oil extraction. The genetic variability of the major phenolic compounds was studied in a representative sample of olive cultivars (Olea europaea L.) from the World Olive Germplasm Collection established at IFAPA Centre “ Alameda del Obispo in Cordoba, Spain. The most abundant phenolic components found in VOO are the secoiridoid derivatives resulting from the enzymatic hydrolysis of oleuropein, ligstroside and demethyloleuropein present in the fruit, which showed to be on average their main phenolic glucosides. The mean content of phenolic compounds in the oils was 494.51 µg/g oil, displaying a variability range of 63.74-1432.04 µg/g oil. The mean content of phenolic compounds in the fruits was 12384.23 µg/g fruit with a range of 3754.13-30696.39 µg/g fruit. Total phenolic compounds in the fruits and the oils were significantly correlated (r = 0.66). Thus, the composition and biochemical status of the olive fruit seems to be the most important variables determining the synthesis of the VOO phenolic compounds during the oil extraction process. On the other hand, the content of oleuropein and derivatives in the oils and fruits showed a correlation coefficient (r = 0.64) lower than that observed for ligstroside and derivatives (r = 0.73). These findings might be related to the higher oxidation rates of the former due to the action of oxidative enzymes during the oil extraction process as a consequence of the orthodiphenolic structure they posses

Phenolics are claimed to be one of the main responsible for the health benefits associated to the long term consumption of virgin olive oil (VOO). The variability of the phenolic composition was studied in an olive cultivar core collection which holds an extensive genetic diversity. The most abundant VOO phenolics were the secoiridoid derivatives resulting from the hydrolysis of oleuropein, ligstroside and demethyloleuropein present in the fruit, which showed to be on average their main phenolic glucosides. Total phenolic compounds in the fruits and the oils were, in general, significantly and positively strong correlated (r = 0.82) with p < 0.001. Thus, the fruit phenolic content seems to be the main factor determining the synthesis of VOO phenolics during oil extraction. Data on phenolic composition would be of interest for the selection of optimal parents in olive breeding programs with the aim of obtaining new cultivars with improved functional quality.

Virgin olive oil (VOO) is the only food product requiring official sensory analysis to be classified in commercial categories, in which the evaluation of the aroma plays a very important role. The selection of parents, with the aim of obtaining new cultivars with improved oil aroma, is of paramount importance in olive breeding programs. We have assessed the volatile fraction by headspace-solid-phase microextraction/gas chromatography-mass spectrometry-flame ionization detection (HS-SPME/GC-MS-FID) and the deduced aroma properties of VOO from a core set of olive cultivars (Core-36) which possesses most of the genetic diversity found in the World Olive Germplasm Collection (IFAPA Alameda del Obispo) located in Cordoba, Spain. The VOO volatile fractions of Core-36 cultivars display a high level of variability. It is mostly made of compounds produced from polyunsaturated fatty acids through the lipoxygenase pathway, which confirms to be a general characteristic of the olive species (Olea europaea L.). The main group of volatile compounds in the oils was six straight-chain carbon compounds derived from linolenic acid, some of them being the main contributors to the aroma of the olive oils according to their odor activity values (OAV). The high level of variability found for the volatile fraction of the oils from Core-36 and, therefore, for the aroma odor notes, suggest that this core set may be a very useful tool for the choice of optimal parents in olive breeding programs in order to raise new cultivars with improved VOO aroma.

Fatty acids are the main components of the olive oil and their composition has a critical influence on the oil quality. However, oil quality evaluation has not been frequently included in the selection of new bred cultivars. This can be due to the difficulties in analyzing oil quality in large set of genotypes and also to the long juvenile period of olive seedlings. Therefore, the identification of molecular markers associated to olive oil quality traits could facilitate their selection in breeding programs of this species. In the present work, the identification of the first QTLs for fatty acids on olive oil is reported. They have been located in a linkage map of a ‘Picual’ × ‘Arbequina’ progeny of the olive breeding program of Córdoba. Correlations among fatty acids are in agreement with previous reports of breeding progenies. QTLs found for oleic and linoleic acids explained 41.1 and 69.7% of the total variability, respectively, and were co-localized in the same linkage groups. In the same region, QTLs for monounsaturated, polyunsaturated and oleic/linoleic ratio were also identified. In other linkage groups, three QTLs for linolenic and one for palmitoleic acid were also located explaining 15.0–28.0% of the total variability. These results could be useful to increase the efficiency of breeding programs aimed at selecting new cultivars with high oleic acid content, and, therefore, with enhanced nutritional properties and oxidative stability of the olive oil.

The use of dwarfing rootstocks represents an optimum mechanism to control the vigour of fruit tree species. Exceptionally, olive trees are grown on their own roots and due to excessive vigour, only a very limited number of cultivars have been successfully adapted to high-density orchards. Through prospection and phenotyping of different wild subspecies, a wide range of genetic variability of the species Olea europaea is being explored to identify genotypes that can be used as rootstocks to optimize intensive olive cultivation (e.g. low vigour, low susceptibility to Verticillium dahliae and high tolerance to abiotic stress). Here, early vigour parameters were studied in genotypes belonging to the subsp. europaea (var. sylvestris), guanchica, cuspidata and crosses of cuspidata x sylvestris. Homogeneous plants were obtained from in-vitro germinated seedlings and further micropropagation. After ex-vitro acclimatization and soil planting, plant height, diameter of the main axis at different heights and length of the longest shoot were measured eight months later. Significant correlations were obtained among the different vigour parameters and high values of broad sense heritability were obtained for all of them. A wide variability in vigour parameters was observed with some clear differences regarding the origin of the plant materials. Thus, the lowest vigour was obtained in sylvestris genotypes from Jaen (Spain) and Marrakech (Morocco) provenances, while the highest vigour was obtained in sylvestris genotypes from Cadiz (Spain) and genotypes from crosses cuspidata x sylvestris. However, a wide variability was obtained in all cases, particularly in genotypes from guanchica origin. Future works will be carried out in order to determine the relationship among vigour parameters measured at different plant age and to define the most efficient strategies for selection at early stages of breeding programs. Funding source: RECUPERA-2020 and contract agreement between IFAPA and

Virgin olive oil (VOO) represents the primary dietary lipid source in the Mediterranean diet and has been linked to its positive health benefits. VOO metabolites responsible for its nutritional and organoleptic quality include certain components that are transferred almost unchanged from fruit to oil (fatty acids and tocopherols) and other compounds that are synthesized during the oil extraction process by endogenous enzymes from precursors already present in the fruit (volatile and phenolic compounds). The health - promoting characteristics of VOO seems to be related to the high level of oleic acid, an adequate balance of saturated/unsaturated fatty acids, the presence of tocopherols and, specially, the contents of phenolic compounds. Meanwhile, volatile compounds are responsible for its aroma, whereas phenolic compounds are also responsible for the bitter and pungent taste notes of this oil. The final content of these metabolites related to VOO quality is controlled by the levels of expression/activity of the corresponding genes/enzymes involved in their biosynthesis which are cultivar dependent. Given that oil quality is one of the main objectives of the breeding programs aiming at obtaining new olive cultivars, knowledge of the content of these key components for the VOO quality might be very useful for the selection. Moreover, the combination of biochemical and molecular studies would allow to identify the most important genes/enzymes in relation to the oil quality and to know their regulatory mechanisms, so that this knowledge may be used in the molecular marker assisted selection of olive clones with improved oil quality features. For this purpose, the natural variability of the olive species was assessed in a collection of cultivars from the World Olive Germplasm Bank (WOGB) located at Córdoba, and the progeny of a cross of Picual x Arbequina olive cultivars. Analytical data showed that the cultivar collection displayed, in general, a higher level of variabi

Verticillium wilt (VW) resistance was evaluated in genotypes from olive crosses including resistant cultivars as parents. Thirty-eight genotypes from three crosses were evaluated: ‘Changlot Real’ × ‘Dolce Agogia’ (16), ‘Frantoio’ × ‘Arbosana’ (13) and ‘Koroneiki’ × ‘Empeltre’ (9). These genotypes were previously selected for several agronomic traits from wider initial progenies populations. Several disease severity and plant colonization parameters were evaluated in inoculation experiments under controlled conditions by dipping roots cutting in a conidial suspension of a highly virulent defoliating isolate of Verticillium dahliae. Significant differences among the evaluated genotypes, including parents and selections from crosses, were obtained for all the disease parameters assessed. A wide variability in disease parameters was observed in the three cross combinations tested. Genotypes with lower relative susceptible index values than both parents were found in the three progenies tested and 10 out of 38 genotypes (26 %) were finally classified as resistant. The level of resistance of these genotypes will be confirmed in future studies under field conditions.

Development of methodologies for early selection is one of the most important goals of olive breeding programs at present. In this context, the identification of molecular markers associated with beneficial alleles could allow the development of marker-assisted selection (MAS) strategies in olive breeding programs. Fruit-related and plant vigor traits, which are of key importance for olive selection and breeding, were analyzed during two seasons in a progeny derived from the cross ‘Picual’ × ‘Arbequina.’ Quantitative trait loci (QTL) analyses were performed using MapQTL 4.0. A total of 22 putative QTLs were identified in the map of ‘Arbequina.’ QTLs clustered in linkage groups (LG) 1, 10 and 17. QTLs for oil-related traits located in LG 1 and 10 explained around 20–30 % of the phenotypic variability depending on the season and the trait. QTL for moisture-related traits were detected in LG 1, 10 and 17, and QTLs for the ratio pulp to stone were identified in LG 10 and 17 explaining around 15–20 %. Interaction between QTLs for the same trait was investigated. The significance of these results was discussed considering the co-localization of QTLs and Pearson correlations among traits. Five additional QTLs were detected in the map of ‘Picual.’ Four of them clustered in LG 17 indicating the presence of a QTL for fruit weight explaining around 12.7–15.2 % of the variability. Additionally, a QTL for trunk diameter was detected in LG 14 explaining 16 % of the variation. These results represent an important step toward the application of MAS in olive breeding programs.

DArT (Diversity Array Technology) markers assessed in a large set of olive cultivars have been used to evaluate their utility for the management of the World Olive Germplasm Bank (WOGB-Córdoba) of IFAPA Centre >Alameda del Obispo> Córdoba (Spain). The coefficient of similarity of Kulczynsky (K1), which calculates the ratio between common markers relative to the number of unique markers (range 0 to ∞), was used for such purpose. Low K1 values (lower than 3.61) were obtained for the majority of the pair of cultivars (99.80% of the total). Low and similar ranges of K1 values were also found in both a seedling progeny of 'Picual'. ×. 'Arbequina' and a set of wild olives included in the study. These results may suggest that most of the WOGB-Córdoba cultivars studied probably were derived from outcrossing events. As expected, low similarity values were found among different cultivars with the same naming (homonyms). On the contrary, a total of 105 pairs (0.20%) showed K1 values above 14.58, implying thus low genetic differentiation. These high K1 values suggest the presence of potential redundant germplasm in the collection which likely includes synonyms, accumulation of somatic mutations, redundancies from prospecting surveys, and planting errors. Thus, the genetic relationships revealed by DArT markers among cultivars are of great interest for management of olive collections and could be useful to increase the performance of future breeding programs by optimizing the choice of parents in combination with phenotypic information. Finally, the transferability of DArT markers to wild olives has been confirmed. © 2013 Elsevier Ltd.

Molecular markers (SSR, SNP and DArT) and agronomical traits have been used in the world's largest olive (Olea europaea L.) germplasm collection (IFAPA, Centre Alameda del Obispo, Cordoba, Spain) to study the patterns of genetic diversity and underlying genetic structure among 361 olive accessions. In addition the marker data were used to construct a set of core collections by means of two different algorithms (MSTRAT and PowerCore) based on M (maximization) strategy. Our results confirm that the germplasm collection is a useful source of genetically diverse material. We also found that geographical origin is an important factor structuring genetic diversity in olive. Subsets of 18, 27, 36, 45 and 68 olive accessions, representing respectively 5%, 7. 5%, 10%, 12. 5% and 19% of the whole germplasm collection, were selected based on the information obtained by all the data set as well as each marker type considered individually. According to our results, the core collections that represent between 19% and 10% of the total collection size could be considered as optimal to retain the bulk of the genetic diversity found in this collection. Due to its high efficiency at capturing all the alleles/traits states found in the whole collection, the core size of 68 accessions could be of special interest for genetic conservation applications in olive. The high average genetic distance and diversity and the almost equal representation of accessions from different geographical regions indicate that the core size of 36 accessions, could be the working collection for olive breeders. © 2011 Springer-Verlag.

The present study reports the setting up of Diversity Arrays Technology (DArT) markers in olive (Olea europaea L.). Two genomic representations were generated using the PstI/. TaqI combination. A first one was aimed to cover the variability available at the World Olive Germplasm Bank (WOGB) and it was based on DNA from 87 olive cultivars carefully selected from the WOGB to represent the olive variability. The second one was obtained from DNA of the parents of a mapping population, 'Picual' and 'Arbequina', and was used to increase the number of markers segregating within them. A total of 2031 and 1630 markers were used for diversity and mapping analyses, respectively to test the utility of DArT markers in olive. A set of 62 cultivars was genotyped with olive-specific DArT markers. A dendrogram was constructed confirming the relationships among olive cultivars reported in previous works. Besides, DArT markers permitted the unambiguous discrimination of all 62 genotypes confirming thus their utility for identification studies, an important issue for management of germplasm collections. DArT markers also allowed the construction of a genetic map of olive using the population derived from the cross 'Picual'. ×. 'Arbequina' and the pseudo-testcross mapping strategy. This map will be used as a framework map to add new markers derived from olive sequencing originating from the project OLEAGEN. In conclusion, olive-specific DArT markers will enhance identification and genetic studies in olive given their high throughput and low cost per data. © 2011 Elsevier B.V.

The genetic diversity of wild olive trees sampled from eight different sites from two provinces of Andalusia (Cadiz and Jaen), Southern Spain, was evaluated by means of agro-morphological traits and SSR markers. The agro-morphological traits showed a high variation between genotypes and significant correlation coefficients were obtained among the values recorded in two consecutive years, 2007/08 and 2008/09 (r= 0.59-0.78). Traits such as fruit symmetry (position A), stone shape, fruit flesh/stone ratio and olive oil content on both wet and dry matter, showed the highest values of diversity and were very useful in genotype discrimination. As expected, average values obtained in wild olives for fruit size and oil content were lower than the previously reported in cultivated material. However, it is noticeable that wild trees with fruit weights (1.3. g) and olive oil percentage in dry matter (33.8%) comparable to the values found in some olive cultivars, were also found. With both DNA-based and agro-morphological descriptors, higher levels of variability were found within each site than between sites. Genetic variation observed among the wild olive germplasm at the DNA level was higher than the agro-morphological traits, indicating the efficiency of SSR markers for detecting genetic diversity among wild olive genotypes and their relationships. The lack of consistency between the relationship studies performed with molecular and morphological markers could indicate that each marker system measures different aspects of the wild olive genetic variability. Molecular data obtained by SSR markers together with morphological and agronomical characterization of olive trees confirmed the high diversity within the wild populations and their potential use for olive breeding. © 2011 Elsevier B.V.