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1.  A comparative analysis of genetic variation in rootstocks and scions of old olive trees – a window into the history of olive cultivation practices and past genetic variation 
BMC Plant Biology  2014;14:146.
Background
Past clonal propagation of olive trees is intimately linked to grafting. However, evidence on grafting in ancient trees is scarce, and not much is known about the source of plant material used for rootstocks. Here, the Simple Sequence Repeat (SSR) marker technique was used to study genetic diversity of rootstocks and scions in ancient olive trees from the Levant and its implications for past cultivation of olives. Leaf samples were collected from tree canopies (scions) and shoots growing from the trunk base (suckers). A total of 310 trees were sampled in 32 groves and analyzed with 14 SSR markers.
Results
In 82.7% of the trees in which both scion and suckers could be genotyped, these were genetically different, and thus suckers were interpreted to represent the rootstock of grafted trees. Genetic diversity values were much higher among suckers than among scions, and 194 and 87 multi-locus genotypes (MLGs) were found in the two sample groups, respectively. Only five private alleles were found among scions, but 125 among suckers. A frequency analysis revealed a bimodal distribution of genetic distance among MLGs, indicating the presence of somatic mutations within clones. When assuming that MLGs differing by one mutation are identical, scion and sucker MLGs were grouped in 20 and 147 multi-locus lineages (MLLs). The majority of scions (90.0%) belonged to a single common MLL, whereas 50.5% of the suckers were single-sample MLLs. However, one MLL was specific to suckers and found in 63 (22.6%) of the samples.
Conclusions
Our results provide strong evidence that the majority of olive trees in the study are grafted, that the large majority of scions belong to a single ancient cultivar containing somatic mutations, and that the widespread occurrence of one sucker genotype may imply rootstock selection. For the majority of grafted trees it seems likely that saplings were used as rootstocks; their genetic diversity probably is best explained as the result of a long history of sexual reproduction involving cultivated, feral and wild genotypes.
doi:10.1186/1471-2229-14-146
PMCID: PMC4049413  PMID: 24886387
Domestication; Grafting; Microsatellites; Olive cultivars; Propagation
2.  Genetic Structure of Wild and Cultivated Olives in the Central Mediterranean Basin 
Annals of Botany  2006;98(5):935-942.
• Background and Aims Olive cultivars and their wild relatives (oleasters) represent two botanical varieties of Olea europaea subsp. europaea (respectively europaea and sylvestris). Olive cultivars have undergone human selection and their area of diffusion overlaps that of oleasters. Populations of genuine wild olives seem restricted to isolated areas of Mediterranean forests, while most other wild-looking forms of olive may include feral forms that escaped cultivation.
• Methods The genetic structure of wild and cultivated olive tree populations was evaluated by amplified fragment length polymorphism (AFLP) markers at a microscale level in one continental and two insular Italian regions.
• Key Results The observed patterns of genetic variation were able to distinguish wild from cultivated populations and continental from insular regions. Island oleasters were highly similar to each other and were clearly distinguishable from those of continental regions. Ancient cultivated material from one island clustered with the wild plants, while the old plants from the continental region clustered with the cultivated group.
• Conclusions On the basis of these results, we can assume that olive trees have undergone a different selection/domestication process in the insular and mainland regions. The degree of differentiation between oleasters and cultivated trees on the islands suggests that all cultivars have been introduced into these regions from the outside, while the Umbrian cultivars have originated either by selection from local oleasters or by direct introduction from other regions.
doi:10.1093/aob/mcl178
PMCID: PMC2803593  PMID: 16935868
Olea europaea; AFLP; genetic diversity; population structure; wild populations
3.  Genetic Biodiversity of Italian Olives (Olea europaea) Germplasm Analyzed by SSR Markers 
The Scientific World Journal  2014;2014:296590.
The olive is an important fruit species cultivated for oil and table olives in Italy and the Mediterranean basin. The conservation of cultivated plants in ex situ collections is essential for the optimal management and use of their genetic resources. The largest ex situ olive germplasm collection consists of approximately 500 Italian olive varieties and corresponding to 85% of the total Italian olive germplasm is maintained at the Consiglio per la Ricerca e sperimentazione per l'Agricoltura, Centro di Ricerca per l'Olivicoltura e l'Industria Olearia (CRA-OLI), in Italy. In this work, eleven preselected nuclear microsatellite markers were used to assess genetic diversity, population structure, and gene flows with the aim of assembling a core collection. The dendrogram obtained utilizing the unweighted pair group method highlights the presence of homonymy and synonymy in olive tree datasets analyzed in this study. 439 different unique genotype profiles were obtained with this combination of 11 loci nSSR, representing 89.8% of the varieties analyzed. The remaining 10.2% comprises different variety pairs in which both accessions are genetically indistinguishable. Clustering analysis performed using BAPS software detected seven groups in Italian olive germplasm and gene flows were determined among identified clusters. We proposed an Italian core collection of 23 olive varieties capturing all detected alleles at microsatellites. The information collected in this study regarding the CRA-OLI ex situ collection can be used for breeding programs, for germplasm conservation, and for optimizing a strategy for the management of olive gene pools.
doi:10.1155/2014/296590
PMCID: PMC3958686  PMID: 24723801
4.  Genomic profiling of plastid DNA variation in the Mediterranean olive tree 
BMC Plant Biology  2011;11:80.
Background
Characterisation of plastid genome (or cpDNA) polymorphisms is commonly used for phylogeographic, population genetic and forensic analyses in plants, but detecting cpDNA variation is sometimes challenging, limiting the applications of such an approach. In the present study, we screened cpDNA polymorphism in the olive tree (Olea europaea L.) by sequencing the complete plastid genome of trees with a distinct cpDNA lineage. Our objective was to develop new markers for a rapid genomic profiling (by Multiplex PCRs) of cpDNA haplotypes in the Mediterranean olive tree.
Results
Eight complete cpDNA genomes of Olea were sequenced de novo. The nucleotide divergence between olive cpDNA lineages was low and not exceeding 0.07%. Based on these sequences, markers were developed for studying two single nucleotide substitutions and length polymorphism of 62 regions (with variable microsatellite motifs or other indels). They were then used to genotype the cpDNA variation in cultivated and wild Mediterranean olive trees (315 individuals). Forty polymorphic loci were detected on this sample, allowing the distinction of 22 haplotypes belonging to the three Mediterranean cpDNA lineages known as E1, E2 and E3. The discriminating power of cpDNA variation was particularly low for the cultivated olive tree with one predominating haplotype, but more diversity was detected in wild populations.
Conclusions
We propose a method for a rapid characterisation of the Mediterranean olive germplasm. The low variation in the cultivated olive tree indicated that the utility of cpDNA variation for forensic analyses is limited to rare haplotypes. In contrast, the high cpDNA variation in wild populations demonstrated that our markers may be useful for phylogeographic and populations genetic studies in O. europaea.
doi:10.1186/1471-2229-11-80
PMCID: PMC3115843  PMID: 21569271
5.  Comparative Transcriptome Analysis of Two Olive Cultivars in Response to NaCl-Stress 
PLoS ONE  2012;7(8):e42931.
Background
Olive (Olea europaea L.) cultivation is rapidly expanding and low quality saline water is often used for irrigation. The molecular basis of salt tolerance in olive, though, has not yet been investigated at a system level. In this study a comparative transcriptomics approach was used as a tool to unravel gene regulatory networks underlying salinity response in olive trees by simulating as much as possible olive growing conditions in the field. Specifically, we investigated the genotype-dependent differences in the transcriptome response of two olive cultivars, a salt-tolerant and a salt-sensitive one.
Methodology/Principal Findings
A 135-day long salinity experiment was conducted using one-year old trees exposed to NaCl stress for 90 days followed by 45 days of post-stress period during the summer. A cDNA library made of olive seedling mRNAs was sequenced and an olive microarray was constructed. Total RNA was extracted from root samples after 15, 45 and 90 days of NaCl-treatment as well as after 15 and 45 days of post-treatment period and used for microarray hybridizations. SAM analysis between the NaCl-stress and the post-stress time course resulted in the identification of 209 and 36 differentially expressed transcripts in the salt–tolerant and salt–sensitive cultivar, respectively. Hierarchical clustering revealed two major, distinct clusters for each cultivar. Despite the limited number of probe sets, transcriptional regulatory networks were constructed for both cultivars while several hierarchically-clustered interacting transcription factor regulators such as JERF and bZIP homologues were identified.
Conclusions/Significance
A systems biology approach was used and differentially expressed transcripts as well as regulatory interactions were identified. The comparison of the interactions among transcription factors in olive with those reported for Arabidopsis might indicate similarities in the response of a tree species with Arabidopsis at the transcriptional level under salinity stress.
doi:10.1371/journal.pone.0042931
PMCID: PMC3431368  PMID: 22952621
6.  Arbuscular Mycorhizal Fungi Associated with the Olive Crop across the Andalusian Landscape: Factors Driving Community Differentiation 
PLoS ONE  2014;9(5):e96397.
Background
In the last years, many olive plantations in southern Spain have been mediated by the use of self-rooted planting stocks, which have incorporated commercial AMF during the nursery period to facilitate their establishment. However, this was practised without enough knowledge on the effect of cropping practices and environment on the biodiversity of AMF in olive orchards in Spain.
Methodology/Principal Findings
Two culture-independent molecular methods were used to study the AMF communities associated with olive in a wide-region analysis in southern Spain including 96 olive locations. The use of T-RFLP and pyrosequencing analysis of rDNA sequences provided the first evidence of an effect of agronomic and climatic characteristics, and soil physicochemical properties on AMF community composition associated with olive. Thus, the factors most strongly associated to AMF distribution varied according to the technique but included among the studied agronomic characteristics the cultivar genotype and age of plantation and the irrigation regimen but not the orchard management system or presence of a cover crop to prevent soil erosion. Soil physicochemical properties and climatic characteristics most strongly associated to the AMF community composition included pH, textural components and nutrient contents of soil, and average evapotranspiration, rainfall and minimum temperature of the sampled locations. Pyrosequencing analysis revealed 33 AMF OTUs belonging to five families, with Archaeospora spp., Diversispora spp. and Paraglomus spp., being first records in olive. Interestingly, two of the most frequent OTUs included a diverse group of Claroideoglomeraceae and Glomeraceae sequences, not assigned to any known AMF species commonly used as inoculants in olive during nursery propagation.
Conclusions/Significance
Our data suggests that AMF can exert higher host specificity in olive than previously thought, which may have important implications for redirecting the olive nursery process in the future as well as to take into consideration the specific soils and environments where the mycorrhized olive trees will be established.
doi:10.1371/journal.pone.0096397
PMCID: PMC4010464  PMID: 24797669
7.  Genetic structure and core collection of the World Olive Germplasm Bank of Marrakech: towards the optimised management and use of Mediterranean olive genetic resources 
Genetica  2011;139(9):1083-1094.
The conservation of cultivated plants in ex-situ collections is essential for the optimal management and use of their genetic resources. For the olive tree, two world germplasm banks (OWGB) are presently established, in Córdoba (Spain) and Marrakech (Morocco). This latter was recently founded and includes 561 accessions from 14 Mediterranean countries. Using 12 nuclear microsatellites (SSRs) and three chloroplast DNA markers, this collection was characterised to examine the structure of the genetic diversity and propose a set of olive accessions encompassing the whole Mediterranean allelic diversity range. We identified 505 SSR profiles based on a total of 210 alleles. Based on these markers, the genetic diversity was similar to that of cultivars and wild olives which were previously characterised in another study indicating that OWGB Marrakech is representative of Mediterranean olive germplasm. Using a model-based Bayesian clustering method and principal components analysis, this OWGB was structured into three main gene pools corresponding to eastern, central and western parts of the Mediterranean Basin. We proposed 10 cores of 67 accessions capturing all detected alleles and 10 cores of 58 accessions capturing the 186 alleles observed more than once. In each of the 10 cores, a set of 40 accessions was identical, whereas the remaining accessions were different, indicating the need to include complementary criteria such as phenotypic adaptive and agronomic traits. Our study generated a molecular database for the entire OWGB Marrakech that may be used to optimise a strategy for the management of olive genetic resources and their use for subsequent genetic and genomic olive breeding.
Electronic supplementary material
The online version of this article (doi:10.1007/s10709-011-9608-7) contains supplementary material, which is available to authorized users.
doi:10.1007/s10709-011-9608-7
PMCID: PMC3247671  PMID: 21960415
Olea europaea; SSR characterisation; Chloroplast DNA markers; Model-based Bayesian clustering; Allelic diversity capture
8.  High Genetic Diversity Detected in Olives beyond the Boundaries of the Mediterranean Sea 
PLoS ONE  2014;9(4):e93146.
Background
Olive trees (Olea europaea subsp. europaea var. europaea) naturally grow in areas spanning the Mediterranean basin and towards the East, including the Middle East. In the Iranian plateau, the presence of olives has been documented since very ancient times, though the early history of the crop in this area is shrouded in uncertainty.
Methods
The varieties presently cultivated in Iran and trees of an unknown cultivation status, surviving under extreme climate and soil conditions, were sampled from different provinces and compared with a set of Mediterranean cultivars. All samples were analyzed using SSR and chloroplast markers to establish the relationships between Iranian olives and Mediterranean varieties, to shed light on the origins of Iranian olives and to verify their contribution to the development of the current global olive variation.
Results
Iranian cultivars and ecotypes, when analyzed using SSR markers, clustered separately from Mediterranean cultivars and showed a high number of private alleles, on the contrary, they shared the same single chlorotype with the most widespread varieties cultivated in the Mediterranean.
Conclusion
We hypothesized that Iranian and Mediterranean olive trees may have had a common origin from a unique center in the Near East region, possibly including the western Iranian area. The present pattern of variation may have derived from different environmental conditions, distinct levels and selection criteria, and divergent breeding opportunities found by Mediterranean and Iranian olives.These unexpected findings emphasize the importance of studying the Iranian olive germplasm as a promising but endangered source of variation.
doi:10.1371/journal.pone.0093146
PMCID: PMC3977848  PMID: 24709858
9.  SNP Discovery by Illumina-Based Transcriptome Sequencing of the Olive and the Genetic Characterization of Turkish Olive Genotypes Revealed by AFLP, SSR and SNP Markers 
PLoS ONE  2013;8(9):e73674.
Background
The olive tree (Olea europaea L.) is a diploid (2n = 2x = 46) outcrossing species mainly grown in the Mediterranean area, where it is the most important oil-producing crop. Because of its economic, cultural and ecological importance, various DNA markers have been used in the olive to characterize and elucidate homonyms, synonyms and unknown accessions. However, a comprehensive characterization and a full sequence of its transcriptome are unavailable, leading to the importance of an efficient large-scale single nucleotide polymorphism (SNP) discovery in olive. The objectives of this study were (1) to discover olive SNPs using next-generation sequencing and to identify SNP primers for cultivar identification and (2) to characterize 96 olive genotypes originating from different regions of Turkey.
Methodology/Principal Findings
Next-generation sequencing technology was used with five distinct olive genotypes and generated cDNA, producing 126,542,413 reads using an Illumina Genome Analyzer IIx. Following quality and size trimming, the high-quality reads were assembled into 22,052 contigs with an average length of 1,321 bases and 45 singletons. The SNPs were filtered and 2,987 high-quality putative SNP primers were identified. The assembled sequences and singletons were subjected to BLAST similarity searches and annotated with a Gene Ontology identifier. To identify the 96 olive genotypes, these SNP primers were applied to the genotypes in combination with amplified fragment length polymorphism (AFLP) and simple sequence repeats (SSR) markers.
Conclusions/Significance
This study marks the highest number of SNP markers discovered to date from olive genotypes using transcriptome sequencing. The developed SNP markers will provide a useful source for molecular genetic studies, such as genetic diversity and characterization, high density quantitative trait locus (QTL) analysis, association mapping and map-based gene cloning in the olive. High levels of genetic variation among Turkish olive genotypes revealed by SNPs, AFLPs and SSRs allowed us to characterize the Turkish olive genotype.
doi:10.1371/journal.pone.0073674
PMCID: PMC3772808  PMID: 24058483
10.  Genetic Diversity and Population Structure of Wild Olives from the North-western Mediterranean Assessed by SSR Markers 
Annals of Botany  2007;100(3):449-458.
Background and Aims
This study examines the pattern of genetic variability and genetic relationships of wild olive (Olea europaea subsp. europaea var. sylvestris) populations in the north-western Mediterranean. Recent bottleneck events are also assessed and an investigation is made of the underlying population structure of the wild olive populations.
Methods
The genetic variation within and between 11 wild olive populations (171 individuals) was analysed with eight microsatellite markers. Conventional and Bayesian-based analyses were applied to infer genetic structure and define the number of gene pools in wild olive populations.
Key Results
Bayesian model-based clustering identified four gene pools, which was in overall concordance with the Factorial Correspondence Analysis and Fitch–Margoliash tree. Two gene pools were predominantly found in southern Spain and Italian islands, respectively, in samples gathered from undisturbed forests of the typical Mediterranean climate. The other two gene pools were mostly detected in the north-eastern regions of Spain and in continental Italy and belong to the transition region between the temperate and Mediterranean climate zones.
Conclusions
On the basis of these results, it can be assumed that the population structure of wild olives from the north-western Mediterranean partially reflects the evolutionary history of these populations, although hybridization between true oleasters and cultivated varieties in areas of close contact between the two forms must be assumed as well. The study indicates a degree of admixture in all the populations, and suggests some caution regarding genetic differentiation at the population level, making it difficult to identify clear-cut genetic boundaries between candidate areas containing either genuinely wild or feral germplasm.
doi:10.1093/aob/mcm132
PMCID: PMC2533604  PMID: 17613587
Olea europaea; genetic variability; gene pools; microsatellites; oleasters; population structure
11.  Soil Properties and Olive Cultivar Determine the Structure and Diversity of Plant-Parasitic Nematode Communities Infesting Olive Orchards Soils in Southern Spain 
PLoS ONE  2015;10(1):e0116890.
This work has studied for the first time the structure and diversity of plant-parasitic nematodes (PPNs) infesting olive orchard soils in a wide-region in Spain that included 92 locations. It aims at determining which agronomical or environmental factors associated to the olive orchards are the main drivers of the PPNs community structure and diversity. Classical morphological and morphometric identification methods were used to determine the frequency and densities of PPNs. Thirteen families, 34 genera and 77 species of PPNs were identified. The highest diversity was found in Helicotylenchus genus, with six species previously reported in Spain and with H. oleae being a first report. Neodolichorhynchus microphasmis and Diptenchus sp., Diphtherophora sp., and Discotylenchus sp., usually considered fungal feeders, were also reported for the first time associated to olive rhizosphere. PPNs abundance ranged from 66 to 16,288 individuals/500-cm3 of soil with Helicotylenchus digonicus being the most prevalent species, followed by Filenchus sp., Merlinius brevidens and Xiphinema pachtaicum. Nematode abundance and diversity indexes were influenced by olive cultivar, and orchard and soil management practices; while olive variety and soil texture were the main factors driving PPN community composition. Soil physicochemical properties and climatic characteristics most strongly associated to the PPN community composition included pH, sand content and exchangeable K, and maximum and minimum average temperature of the sampled locations. Our data suggests that there is a high diversity of PPNs associated to olive in Southern Spain that can exert different damage to olive roots depending on the olive variety and their abundance. Further analysis to determine the resistance levels of most common olive varieties to the prevalent PPNs in Spain will help to choose the most appropriate ones for the establishment of new plantations. This choice will take into consideration the specific soils and environments where those olive varieties will be established.
doi:10.1371/journal.pone.0116890
PMCID: PMC4308072  PMID: 25625375
12.  Population genetics of Mediterranean and Saharan olives: geographic patterns of differentiation and evidence for early generations of admixture 
Annals of Botany  2013;112(7):1293-1302.
Background and Aims
The olive (Olea europaea subsp. europaea) was domesticated in the Mediterranean area but its wild relatives are distributed over three continents, from the Mediterranean basin to South Africa and south-western Asia. Recent studies suggested that this crop originated in the Levant while a secondary diversification occurred in most westward areas. A possible contribution of the Saharan subspecies (subsp. laperrinei) has been highlighted, but the data available were too limited to draw definite conclusions. Here, patterns of genetic differentiation in the Mediterranean and Saharan olives are analysed to test for recent admixture between these taxa.
Methods
Nuclear microsatellite and plastid DNA (ptDNA) data were compiled from previous studies and completed for a sample of 470 cultivars, 390 wild Mediterranean trees and 270 Saharan olives. A network was reconstructed for the ptDNA haplotypes, while a Bayesian clustering method was applied to identify the main gene pools in the data set and then simulate and test for early generations of admixture between Mediterranean and Saharan olives.
Key Results
Four lineages of ptDNA haplotypes are recognized: three from the Mediterranean basin and one from the Sahara. Only one haplotype, primarily distributed in the Sahara, is shared between laperrinei and europaea. This haplotype is detected once in ‘Dhokar’, a cultivar from the Maghreb. Nuclear microsatellites show geographic patterns of genetic differentiation in the Mediterranean olive that reflect the primary origins of cultivars in the Levant, and indicate a high genetic differentiation between europaea and laperrinei. No first-generation hybrid between europaea and laperrinei is detected, but recent, reciprocal admixture between Mediterranean and Saharan subspecies is found in a few accessions, including ‘Dhokar’.
Conclusions
This study reports for the first time admixture between Mediterranean and Saharan olives. Although its contribution remains limited, Laperrine's olive has been involved in the diversification of cultivated olives.
doi:10.1093/aob/mct196
PMCID: PMC3806528  PMID: 24013386
Admixture; domestication; Laperrine's olive; Mediterranean basin; microsatellite; Olea europaea; population genetic simulations; Sahara; secondary diversification; wild genetic resources
13.  The molecular biology of the olive fly comes of age 
BMC Genetics  2014;15(Suppl 2):S8.
Background
Olive cultivation blends with the history of the Mediterranean countries since ancient times. Even today, activities around the olive tree constitute major engagements of several people in the countryside of both sides of the Mediterranean basin. The olive fly is, beyond doubt, the most destructive pest of cultivated olives. The female fly leaves its eggs in the olive fruit. Upon emergence, the larvae feed on the olive sap, thus destroying the fruit. If untreated, practically all olives get infected. The use of chemical insecticides constitutes the principal olive fly control approach. The Sterile Insect Technique (SIT), an environmentally friendly alternative control method, had been tried in pilot field applications in the 1970's, albeit with no practical success. This was mainly attributed to the low, non-antagonistic quality of the mixed-sex released insects. Many years of experience from successful SIT applications in related species, primarily the Mediterranean fruit fly, Ceratitis capitata, demonstrated that efficient SIT protocols require the availability of fundamental genetic and molecular information.
Results
Among the primary systems whose understanding can contribute towards novel SIT approaches (or its recently developed alternative RIDL: Release of Insects carrying a Dominant Lethal) is the reproductive, since the ability to manipulate the reproductive system would directly affect the insect's fertility. In addition, the analysis of early embryonic promoters and apoptotic genes would provide tools that confer dominant early-embryonic lethality during mass-rearing. Here we report the identification of several genes involved in these systems through whole transcriptome analysis of female accessory glands (FAGs) and spermathecae, as well as male testes. Indeed, analysis of differentially expressed genes in these tissues revealed higher metabolic activity in testes than in FAGs/spermathecae. Furthermore, at least five olfactory-related genes were shown to be differentially expressed in the female and male reproductive systems analyzed. Finally, the expression profile of the embryonic serendipity-α locus and the pre-apoptotic head involution defective gene were analyzed during embryonic developmental stages.
Conclusions
Several years of molecular studies on the olive fly can now be combined with new information from whole transcriptome analyses and lead to a deep understanding of the biology of this notorious insect pest. This is a prerequisite for the development of novel embryonic lethality female sexing strains for successful SIT efforts which, combined with improved mass-reared conditions, give new hope for efficient SIT applications for the olive fly.
doi:10.1186/1471-2156-15-S2-S8
PMCID: PMC4255830  PMID: 25472866
Tephritidae; Bactrocera oleae; sry-α; hid
14.  Forensic Botany: Potential Usefulness of Microsatellite-based Genotyping of Croatian Olive (Olea europaea L.) in Forensic Casework 
Croatian medical journal  2007;48(4):556-562.
Aim
To assess genotyping with microsatellite-based markers of the olive (Olea europaea L.) for potential application of olive as legal case evidence, with regard to the degree of variability within the Croatian olive genomic pool and to the effectiveness of the chosen set of microsatellite-based markers in revealing olive divergence.
Methods
The total of 44 autochthonous Croatian olive specimens were subjected to genotyping with 16 previously described and developed microsatellite-based markers. According to previous morphological analyses, 44 specimens were classified into 30 cultivars with the exception of an additional, previously unassigned specimen.
Results
Genotyping of 44 specimens distinguished a total of 44 different genotype profiles by 16 microsatellite-based loci. Average expected heterozigosity amounted to 0.758, which points to significant diversity of Croatian olives.
Conclusion
Croatian olive genotyping showed strong varietal discrimination up to the single tree and considerable potential application of olive as evidence in investigation of crime, accident, and suicide circumstances.
PMCID: PMC2080570  PMID: 17696311
15.  Valuable Nutrients and Functional Bioactives in Different Parts of Olive (Olea europaea L.)—A Review 
The Olive tree (Olea europaea L.), a native of the Mediterranean basin and parts of Asia, is now widely cultivated in many other parts of the world for production of olive oil and table olives. Olive is a rich source of valuable nutrients and bioactives of medicinal and therapeutic interest. Olive fruit contains appreciable concentration, 1–3% of fresh pulp weight, of hydrophilic (phenolic acids, phenolic alchohols, flavonoids and secoiridoids) and lipophilic (cresols) phenolic compounds that are known to possess multiple biological activities such as antioxidant, anticarcinogenic, antiinflammatory, antimicrobial, antihypertensive, antidyslipidemic, cardiotonic, laxative, and antiplatelet. Other important compounds present in olive fruit are pectin, organic acids, and pigments. Virgin olive oil (VOO), extracted mechanically from the fruit, is also very popular for its nutritive and health-promoting potential, especially against cardiovascular disorders due to the presence of high levels of monounsaturates and other valuable minor components such as phenolics, phytosterols, tocopherols, carotenoids, chlorophyll and squalene. The cultivar, area of production, harvest time, and the processing techniques employed are some of the factors shown to influence the composition of olive fruit and olive oil. This review focuses comprehensively on the nutrients and high-value bioactives profile as well as medicinal and functional aspects of different parts of olives and its byproducts. Various factors affecting the composition of this food commodity of medicinal value are also discussed.
doi:10.3390/ijms13033291
PMCID: PMC3317714  PMID: 22489153
Mediterranean diet; high-value components; bioactives; phytochemicals; virgin olive oil; medicinal uses; therapeutic potential
16.  Olive phenolic compounds: metabolic and transcriptional profiling during fruit development 
BMC Plant Biology  2012;12:162.
Background
Olive (Olea europaea L.) fruits contain numerous secondary metabolites, primarily phenolics, terpenes and sterols, some of which are particularly interesting for their nutraceutical properties. This study will attempt to provide further insight into the profile of olive phenolic compounds during fruit development and to identify the major genetic determinants of phenolic metabolism.
Results
The concentration of the major phenolic compounds, such as oleuropein, demethyloleuropein, 3–4 DHPEA-EDA, ligstroside, tyrosol, hydroxytyrosol, verbascoside and lignans, were measured in the developing fruits of 12 olive cultivars. The content of these compounds varied significantly among the cultivars and decreased during fruit development and maturation, with some compounds showing specificity for certain cultivars. Thirty-five olive transcripts homologous to genes involved in the pathways of the main secondary metabolites were identified from the massive sequencing data of the olive fruit transcriptome or from cDNA-AFLP analysis. Their mRNA levels were determined using RT-qPCR analysis on fruits of high- and low-phenolic varieties (Coratina and Dolce d’Andria, respectively) during three different fruit developmental stages. A strong correlation was observed between phenolic compound concentrations and transcripts putatively involved in their biosynthesis, suggesting a transcriptional regulation of the corresponding pathways. OeDXS, OeGES, OeGE10H and OeADH, encoding putative 1-deoxy-D-xylulose-5-P synthase, geraniol synthase, geraniol 10-hydroxylase and arogenate dehydrogenase, respectively, were almost exclusively present at 45 days after flowering (DAF), suggesting that these compounds might play a key role in regulating secoiridoid accumulation during fruit development.
Conclusions
Metabolic and transcriptional profiling led to the identification of some major players putatively involved in biosynthesis of secondary compounds in the olive tree. Our data represent the first step towards the functional characterisation of important genes for the determination of olive fruit quality.
doi:10.1186/1471-2229-12-162
PMCID: PMC3480905  PMID: 22963618
Olea europaea; Phenolics; Secoiridoids; RT-qPCR; Transcriptome; Secondary metabolism
17.  Construction of Core Collections Suitable for Association Mapping to Optimize Use of Mediterranean Olive (Olea europaea L.) Genetic Resources 
PLoS ONE  2013;8(5):e61265.
Phenotypic characterisation of germplasm collections is a decisive step towards association mapping analyses, but it is particularly expensive and tedious for woody perennial plant species. Characterisation could be more efficient if focused on a reasonably sized subset of accessions, or so-called core collection (CC), reflecting the geographic origin and variability of the germplasm. The questions that arise concern the sample size to use and genetic parameters that should be optimized in a core collection to make it suitable for association mapping. Here we investigated these questions in olive (Olea europaea L.), a perennial fruit species. By testing different sampling methods and sizes in a worldwide olive germplasm bank (OWGB Marrakech, Morocco) containing 502 unique genotypes characterized by nuclear and plastid loci, a two-step sampling method was proposed. The Shannon-Weaver diversity index was found to be the best criterion to be maximized in the first step using the Core Hunter program. A primary core collection of 50 entries (CC50) was defined that captured more than 80% of the diversity. This latter was subsequently used as a kernel with the Mstrat program to capture the remaining diversity. 200 core collections of 94 entries (CC94) were thus built for flexibility in the choice of varieties to be studied. Most entries of both core collections (CC50 and CC94) were revealed to be unrelated due to the low kinship coefficient, whereas a genetic structure spanning the eastern and western/central Mediterranean regions was noted. Linkage disequilibrium was observed in CC94 which was mainly explained by a genetic structure effect as noted for OWGB Marrakech. Since they reflect the geographic origin and diversity of olive germplasm and are of reasonable size, both core collections will be of major interest to develop long-term association studies and thus enhance genomic selection in olive species.
doi:10.1371/journal.pone.0061265
PMCID: PMC3646834  PMID: 23667437
18.  Identification of new polymorphic regions and differentiation of cultivated olives (Olea europaea L.) through plastome sequence comparison 
BMC Plant Biology  2010;10:211.
Background
The cultivated olive (Olea europaea L.) is the most agriculturally important species of the Oleaceae family. Although many studies have been performed on plastid polymorphisms to evaluate taxonomy, phylogeny and phylogeography of Olea subspecies, only few polymorphic regions discriminating among the agronomically and economically important olive cultivars have been identified. The objective of this study was to sequence the entire plastome of olive and analyze many potential polymorphic regions to develop new inter-cultivar genetic markers.
Results
The complete plastid genome of the olive cultivar Frantoio was determined by direct sequence analysis using universal and novel PCR primers designed to amplify all overlapping regions. The chloroplast genome of the olive has an organisation and gene order that is conserved among numerous Angiosperm species and do not contain any of the inversions, gene duplications, insertions, inverted repeat expansions and gene/intron losses that have been found in the chloroplast genomes of the genera Jasminum and Menodora, from the same family as Olea.
The annotated sequence was used to evaluate the content of coding genes, the extent, and distribution of repeated and long dispersed sequences and the nucleotide composition pattern. These analyses provided essential information for structural, functional and comparative genomic studies in olive plastids. Furthermore, the alignment of the olive plastome sequence to those of other varieties and species identified 30 new organellar polymorphisms within the cultivated olive.
Conclusions
In addition to identifying mutations that may play a functional role in modifying the metabolism and adaptation of olive cultivars, the new chloroplast markers represent a valuable tool to assess the level of olive intercultivar plastome variation for use in population genetic analysis, phylogenesis, cultivar characterisation and DNA food tracking.
doi:10.1186/1471-2229-10-211
PMCID: PMC2956560  PMID: 20868482
19.  Variability of Virgin Olive Oil Phenolic Compounds in a Segregating Progeny from a Single Cross in Olea europaea L. and Sensory and Nutritional Quality Implications 
PLoS ONE  2014;9(3):e92898.
Virgin olive oil phenolic compounds are responsible for its nutritional and sensory quality. The synthesis of phenolic compounds occurs when enzymes and substrates meet as olive fruit is crushed during the industrial process to obtain the oil. The genetic variability of the major phenolic compounds of virgin olive oil was studied in a progeny of the cross of Picual x Arbequina olive cultivars (Olea europaea L.). They belong to four different groups: compounds that included tyrosol or hydroxytyrosol in their molecules, lignans, flavonoids, and phenolic acids. Data of phenolics in the oils showed that the progeny displayed a large degree of variability, widely transgressing the genitor levels. This high variability can be of interest on breeding programs. Thus, multivariate analysis allowed to identify genotypes within the progeny particularly interesting in terms of phenolic composition and deduced organoleptic and nutritional quality. The present study has demonstrated that it is possible to obtain enough degree of variability with a single cross of olive cultivars for compounds related to the nutritional and organoleptic properties of virgin olive oil.
doi:10.1371/journal.pone.0092898
PMCID: PMC3961445  PMID: 24651694
20.  A New Avenue for Classification and Prediction of Olive Cultivars Using Supervised and Unsupervised Algorithms 
PLoS ONE  2012;7(9):e44164.
Various methods have been used to identify cultivares of olive trees; herein we used different bioinformatics algorithms to propose new tools to classify 10 cultivares of olive based on RAPD and ISSR genetic markers datasets generated from PCR reactions. Five RAPD markers (OPA0a21, OPD16a, OP01a1, OPD16a1 and OPA0a8) and five ISSR markers (UBC841a4, UBC868a7, UBC841a14, U12BC807a and UBC810a13) selected as the most important markers by all attribute weighting models. K-Medoids unsupervised clustering run on SVM dataset was fully able to cluster each olive cultivar to the right classes. All trees (176) induced by decision tree models generated meaningful trees and UBC841a4 attribute clearly distinguished between foreign and domestic olive cultivars with 100% accuracy. Predictive machine learning algorithms (SVM and Naïve Bayes) were also able to predict the right class of olive cultivares with 100% accuracy. For the first time, our results showed data mining techniques can be effectively used to distinguish between plant cultivares and proposed machine learning based systems in this study can predict new olive cultivars with the best possible accuracy.
doi:10.1371/journal.pone.0044164
PMCID: PMC3434224  PMID: 22957050
21.  Validation of reference genes for gene expression analysis in olive (Olea europaea) mesocarp tissue by quantitative real-time RT-PCR 
BMC Research Notes  2014;7:304.
Background
Gene expression analysis using quantitative reverse transcription PCR (qRT-PCR) is a robust method wherein the expression levels of target genes are normalised using internal control genes, known as reference genes, to derive changes in gene expression levels. Although reference genes have recently been suggested for olive tissues, combined/independent analysis on different cultivars has not yet been tested. Therefore, an assessment of reference genes was required to validate the recent findings and select stably expressed genes across different olive cultivars.
Results
A total of eight candidate reference genes [glyceraldehyde 3-phosphate dehydrogenase (GAPDH), serine/threonine-protein phosphatase catalytic subunit (PP2A), elongation factor 1 alpha (EF1-alpha), polyubiquitin (OUB2), aquaporin tonoplast intrinsic protein (TIP2), tubulin alpha (TUBA), 60S ribosomal protein L18-3 (60S RBP L18-3) and polypyrimidine tract-binding protein homolog 3 (PTB)] were chosen based on their stability in olive tissues as well as in other plants. Expression stability was examined by qRT-PCR across 12 biological samples, representing mesocarp tissues at various developmental stages in three different olive cultivars, Barnea, Frantoio and Picual, independently and together during the 2009 season with two software programs, GeNorm and BestKeeper. Both software packages identified GAPDH, EF1-alpha and PP2A as the three most stable reference genes across the three cultivars and in the cultivar, Barnea. GAPDH, EF1-alpha and 60S RBP L18-3 were found to be most stable reference genes in the cultivar Frantoio while 60S RBP L18-3, OUB2 and PP2A were found to be most stable reference genes in the cultivar Picual.
Conclusions
The analyses of expression stability of reference genes using qRT-PCR revealed that GAPDH, EF1-alpha, PP2A, 60S RBP L18-3 and OUB2 are suitable reference genes for expression analysis in developing Olea europaea mesocarp tissues, displaying the highest level of expression stability across three different olive cultivars, Barnea, Frantoio and Picual, however the combination of the three most stable reference genes do vary amongst individual cultivars. This study will provide guidance to other researchers to select reference genes for normalization against target genes by qPCR across tissues obtained from the mesocarp region of the olive fruit in the cultivars, Barnea, Frantoio and Picual.
doi:10.1186/1756-0500-7-304
PMCID: PMC4062307  PMID: 24884716
Reference genes; qRT-PCR; Olea europaea; Gene expression
22.  Control of the olive fruit fly using genetics-enhanced sterile insect technique 
BMC Biology  2012;10:51.
Background
The olive fruit fly, Bactrocera oleae, is the major arthropod pest of commercial olive production, causing extensive damage to olive crops worldwide. Current control techniques rely on spraying of chemical insecticides. The sterile insect technique (SIT) presents an alternative, environmentally friendly and species-specific method of population control. Although SIT has been very successful against other tephritid pests, previous SIT trials on olive fly have produced disappointing results. Key problems included altered diurnal mating rhythms of the laboratory-reared insects, resulting in asynchronous mating activity between the wild and released sterile populations, and low competitiveness of the radiation-sterilised mass-reared flies. Consequently, the production of competitive, male-only release cohorts is considered an essential prerequisite for successful olive fly SIT.
Results
We developed a set of conditional female-lethal strains of olive fly (named Release of Insects carrying a Dominant Lethal; RIDL®), providing highly penetrant female-specific lethality, dominant fluorescent marking, and genetic sterility. We found that males of the lead strain, OX3097D-Bol, 1) are strongly sexually competitive with wild olive flies, 2) display synchronous mating activity with wild females, and 3) induce appropriate refractoriness to wild female re-mating. Furthermore, we showed, through a large proof-of-principle experiment, that weekly releases of OX3097D-Bol males into stable populations of caged wild-type olive fly could cause rapid population collapse and eventual eradication.
Conclusions
The observed mating characteristics strongly suggest that an approach based on the release of OX3097D-Bol males will overcome the key difficulties encountered in previous olive fly SIT attempts. Although field confirmation is required, the proof-of-principle suppression and elimination of caged wild-type olive fly populations through OX3097D-Bol male releases provides evidence for the female-specific RIDL approach as a viable method of olive fly control. We conclude that the promising characteristics of OX3097D-Bol may finally enable effective SIT-based control of the olive fly.
doi:10.1186/1741-7007-10-51
PMCID: PMC3398856  PMID: 22713628
olive fly; Bactrocera oleae; sterile insect technique; SIT; release of insects carrying a dominant lethal; RIDL; autocidal control; insect transgenics
23.  Genetic analysis and molecular characterization of Chinese sesame (Sesamum indicum L.) cultivars using Insertion-Deletion (InDel) and Simple Sequence Repeat (SSR) markers 
BMC Genetics  2014;15:35.
Background
Sesame is an important and ancient oil crop in tropical and subtropical areas. China is one of the most important sesame producing countries with many germplasm accessions and excellent cultivars. Domestication and modern plant breeding have presumably narrowed the genetic basis of cultivated sesame. Several modern sesame cultivars were bred with a limited number of landrace cultivars in their pedigree. The genetic variation was subsequently reduced by genetic drift and selection. Characterization of genetic diversity of these cultivars by molecular markers is of great value to assist parental line selection and breeding strategy design.
Results
Three hundred and forty nine simple sequence repeat (SSR) and 79 insertion-deletion (InDel) markers were developed from cDNA library and reduced-representation sequencing of a sesame cultivar Zhongzhi 14, respectively. Combined with previously published SSR markers, 88 polymorphic markers were used to assess the genetic diversity, phylogenetic relationships, population structure, and allele distribution among 130 Chinese sesame accessions including 82 cultivars, 44 landraces and 4 wild germplasm accessions. A total of 325 alleles were detected, with the average gene diversity of 0.432. Model-based structure analysis revealed the presence of five subgroups belonging to two main groups, which were consistent with the results from principal coordinate analysis (PCA), phylogenetic clustering and analysis of molecular variance (AMOVA). Several missing or unique alleles were identified from particular types, subgroups or families, even though they share one or both parental/progenitor lines.
Conclusions
This report presented a by far most comprehensive characterization of the molecular and genetic diversity of sesame cultivars in China. InDels are more polymorphic than SSRs, but their ability for deciphering genetic diversity compared to the later. Improved sesame cultivars have narrower genetic basis than landraces, reflecting the effect of genetic drift or selection during breeding processes. Comparative analysis of allele distribution revealed genetic divergence between improved cultivars and landraces, as well as between cultivars released in different years. These results will be useful for assessing cultivars and for marker-assisted breeding in sesame.
doi:10.1186/1471-2156-15-35
PMCID: PMC4234512  PMID: 24641723
Sesame; InDel; Microsatellites; Genetic diversity; Population structure; Allele distribution
24.  QTL Mapping of Flowering and Fruiting Traits in Olive 
PLoS ONE  2013;8(5):e62831.
One of the challenge fruit growers are facing is to balance between tree production and vegetative growth from year to year. To investigate the existence of genetic determinism for reproductive behaviour in olive tree, we studied an olive segregating population derived from a cross between ‘Olivière’ and ‘Arbequina’ cultivars. Our strategy was based on (i) an annual assessment of individual trees yield, and (ii) a decomposition of adult growth units at the crown periphery into quantitative variables related to both flowering and fruiting process in relation to their growth and branching. Genetic models, including the year, genotype effects and their interactions, were built with variance function and correlation structure of residuals when necessary. Among the progeny, trees were either ‘ON’ or ‘OFF’ for a given year and patterns of regular vs. irregular bearing were revealed. Genotype effect was significant on yield but not for flowering traits at growth unit (GU) scale, whereas the interaction between genotype and year was significant for both traits. A strong genetic effect was found for all fruiting traits without interaction with the year. Based on the new constructed genetic map, QTLs with small effects were detected, revealing multigenic control of the studied traits. Many were associated to alleles from ‘Arbequina’. Genetic correlations were found between Yield and Fruit set at GU scale suggesting a common genetic control, even though QTL co-localisations were in spe`cific years only. Most QTL were associated to flowering traits in specific years, even though reproductive traits at GU scale did not capture the bearing status of the trees in a given year. Results were also interpreted with respect to ontogenetic changes of growth and branching, and an alternative sampling strategy was proposed for capturing tree fruiting behaviour. Regular bearing progenies were identified and could constitute innovative material for selection programs.
doi:10.1371/journal.pone.0062831
PMCID: PMC3656886  PMID: 23690957
25.  Polyploidy in the Olive Complex (Olea europaea): Evidence from Flow Cytometry and Nuclear Microsatellite Analyses 
Annals of Botany  2007;101(1):25-30.
Background
Phylogenetic and phylogeographic investigations have been previously performed to study the evolution of the olive tree complex (Olea europaea). A particularly high genomic diversity has been found in north-west Africa. However, to date no exhaustive study has been addressed to infer putative polyploidization events and their evolutionary significance in the diversification of the olive tree and its relatives.
Methods
Representatives of the six olive subspecies were investigated using (a) flow cytometry to estimate genome content, and (b) six highly variable nuclear microsatellites to assess the presence of multiple alleles at co-dominant loci. In addition, nine individuals from a controlled cross between two individuals of O. europaea subsp. maroccana were characterized with microsatellites to check for chromosome inheritance.
Key Results
Based on flow cytometry and genetic analyses, strong evidence for polyploidy was obtained in subspp. cerasiformis (tetraploid) and maroccana (hexaploid), whereas the other subspecies appeared to be diploids. Agreement between flow cytometry and genetic analyses gives an alternative approach to chromosome counting to determine ploidy level of trees. Lastly, abnormalities in chromosomes inheritance leading to aneuploid formation were revealed using microsatellite analyses in the offspring from the controlled cross in subsp. maroccana.
Conclusions
This study constitutes the first report for multiple polyploidy in olive tree relatives. Formation of tetraploids and hexaploids may have played a major role in the diversification of the olive complex in north-west Africa. The fact that polyploidy is found in narrow endemic subspecies from Madeira (subsp. cerasiformis) and the Agadir Mountains (subsp. maroccana) suggests that polyploidization has been favoured to overcome inbreeding depression. Lastly, based on previous phylogenetic analyses, we hypothesize that subsp. cerasiformis resulted from hybridization between ancestors of subspp. guanchica and europaea.
doi:10.1093/aob/mcm275
PMCID: PMC2701839  PMID: 18024415
Flow cytometry; hexaploidy; High Atlas; Macaronesia; Olea europaea; olive; SSR; tetraploidy

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