Disentangling the link between leaf photosynthesis and turgor in fruit growth.

Despite the importance of understanding plant growth, the mechanisms underlying how plant and fruit growth declines during drought remain poorly understood. Specifically, it remains unresolved whether carbon or water factors are responsible for limiting growth as drought progresses. We examine questions regarding the relative importance of water and carbon to fruit growth depending on the water deficit level and the fruit growth stage by measuring fruit diameter, leaf photosynthesis, and a proxy of cell turgor in olive (Olea europaea). Flow cytometry was also applied to determine the fruit cell division stage. We found that photosynthesis and turgor were related to fruit growth; specifically, the relative importance of photosynthesis was higher during periods of more intense cell division, while turgor had higher relative importance in periods where cell division comes close to ceasing and fruit growth is dependent mainly on cell expansion. This pattern was found regardless of the water deficit level, although turgor and growth ceased at more similar values of leaf water potential than photosynthesis. Cell division occurred even when fruit growth seemed to stop under water deficit conditions, which likely helped fruits to grow disproportionately when trees were hydrated again, compensating for periods with low turgor. As a result, the final fruit size was not severely penalized. We conclude that carbon and water processes are able to explain fruit growth, with importance placed on the combination of cell division and expansion. However, the major limitation to growth is turgor, which adds evidence to the sink limitation hypothesis.

Olive Varieties under UV-B Stress Show Distinct Responses in Terms of Antioxidant Machinery and Isoform/Activity of RubisCO.

In recent decades, atmospheric pollution led to a progressive reduction of the ozone layer with a consequent increase in UV-B radiation. Despite the high adaptation of olive trees to the Mediterranean environment, the progressive increase of UV-B radiation is a risk factor for olive tree cultivation. It is therefore necessary to understand how high levels of UV-B radiation affect olive plants and to identify olive varieties which are better adapted. In this study we analyzed two Italian olive varieties subjected to chronic UV-B stress. We focused on the effects of UV-B radiation on RubisCO, in terms of quantity, enzymatic activity and isoform composition. In addition, we also analyzed changes in the activity of antioxidant enzymes (SOD, CAT, GPox) to get a comprehensive picture of the antioxidant system. We also evaluated the effects of UV-B on the enzyme sucrose synthase. The overall damage at biochemical level was also assessed by analyzing changes in Hsp70, a protein triggered under stress conditions. The results of this work indicate that the varieties (Giarraffa and Olivastra Seggianese) differ significantly in the use of specific antioxidant defense systems, as well as in the activity and isoform composition of RubisCO. Combined with a different use of sucrose synthase, the overall picture shows that Giarraffa optimized the use of GPox and opted for a targeted choice of RubisCO isoforms, in addition to managing the content of sucrose synthase, thereby saving energy during critical stress points.

Phylogenomics of the olive tree (Olea europaea) reveals the relative contribution of ancient allo- and autopolyploidization events.

BACKGROUND: Polyploidization is one of the major evolutionary processes that shape eukaryotic genomes, being particularly common in plants. Polyploids can arise through direct genome doubling within a species (autopolyploidization) or through the merging of genomes from distinct species after hybridization (allopolyploidization). The relative contribution of both mechanisms in plant evolution is debated. Here we used phylogenomics to dissect the tempo and mode of duplications in the genome of the olive tree (Olea europaea), one of the first domesticated Mediterranean fruit trees. RESULTS: Our results depict a complex scenario involving at least three past polyploidization events, of which two-at the bases of the family Oleaceae and the tribe Oleeae, respectively-are likely to be the result of ancient allopolyploidization. A more recent polyploidization involves specifically the olive tree and relatives. CONCLUSION: Our results show the power of phylogenomics to distinguish between allo- and auto polyploidization events and clarify the contributions of duplications in the evolutionary history of the olive tree.

OeFAD8, OeLIP and OeOSM expression and activity in cold-acclimation of Olea europaea, a perennial dicot without winter-dormancy.

MAIN CONCLUSION: Cold-acclimation genes in woody dicots without winter-dormancy, e.g., olive-tree, need investigation. Positive relationships between OeFAD8, OeOSM , and OeLIP19 and olive-tree cold-acclimation exist, and couple with increased lipid unsaturation and cutinisation. Olive-tree is a woody species with no winter-dormancy and low frost-tolerance. However, cold-tolerant genotypes were empirically selected, highlighting that cold-acclimation might be acquired. Proteins needed for olive-tree cold-acclimation are unknown, even if roles for osmotin (OeOSM) as leaf cryoprotectant, and seed lipid-transfer protein for endosperm cutinisation under cold, were demonstrated. In other species, FAD8, coding a desaturase producing α-linolenic acid, is activated by temperature-lowering, concomitantly with bZIP-LIP19 genes. The research was focussed on finding OeLIP19 gene(s) in olive-tree genome, and analyze it/their expression, and that of OeFAD8 and OeOSM, in drupes and leaves under different cold-conditions/developmental stages/genotypes, in comparison with changes in unsaturated lipids and cell wall cutinisation. Cold-induced cytosolic calcium transients always occurred in leaves/drupes of some genotypes, e.g., Moraiolo, but ceased in others, e.g., Canino, at specific drupe stages/cold-treatments, suggesting cold-acclimation acquisition only in the latter genotypes. Canino and Moraiolo were selected for further analyses. Cold-acclimation in Canino was confirmed by an electrolyte leakage from leaf/drupe membranes highly reduced in comparison with Moraiolo. Strong increases in fruit-epicarp/leaf-epidermis cutinisation characterized cold-acclimated Canino, and positively coupled with OeOSM expression, and immunolocalization of the coded protein. OeFAD8 expression increased with cold-acclimation, as the production of α-linolenic acid, and related compounds. An OeLIP19 gene was isolated. Its levels changed with a trend similar to OeFAD8. All together, results sustain a positive relationship between OeFAD8, OeOSM and OeLIP19 expression in olive-tree cold-acclimation. The parallel changes in unsaturated lipids and cutinisation concur to suggest orchestrated roles of the coded proteins in the process.

New insights into the early steps of oil body mobilization during pollen germination.

In some plants, pollen grains accumulate storage lipids that serve as energy supply during germination. Here, three enzymes involved in early steps of oil body mobilization in the male gametophyte were functionally characterized for the first time. The effect of extracellular sugars on pollen performance and oil body dynamics was also analysed. Olive pollen oil bodies showed phospholipase A, lipase, and lipoxygenase activities on their surface. Enzyme activity levels increased during germination with a maximum after 3h. Removal of extracellular sugars from the germination medium did not affect pollen performance but increased enzyme activity rates and sped up oil body mobilization. Inhibitors seriously hampered pollen germination and pollen tube growth, leading to a characteristic accumulation of oil bodies in the germinative aperture. It can be concluded that storage lipids are sufficient for proper olive pollen germination. A lipase and a lipoxygenase are likely involved in oil body mobilization. Extracellular sugars may modulate their function, while a phospholipase A may promote their access to the storage lipids.

Cell and tissue dynamics of olive endocarp sclerification vary according to water availability.

Endocarp developmental timing in drupe-type fruits, involving tissue expansion and sclerification processes, is increasingly used as marker for biological studies and crop management. In spite of its wide application, however, little is known regarding how these morphogenetic processes unfold or the factors that modify it. This study evaluates endocarp expansion and sclerification of olive (Olea europaea) fruits, used as an example of drupe-type fruits, from trees growing under different water regimes: full irrigated, deficit irrigated (moderate reduction of water availability) and rainfed (severe reduction of water availability). Fruits were sampled weekly until pit hardening, and fruit and endocarp areas were evaluated in histological preparations. An image analysis process was tested and adjusted to quantify sclerified area and distribution within the endocarp. Individual stone cells differentiated independently but distribution and timing indicated the overall coordination of endocarp tissue sclerification. Increase in sclerified area was initially gradual, accelerated abruptly the week prior to the end of endocarp expansion and then continued at an intermediate rate. These results suggest that the end of the expansion period is driven by sclerification and the morphogenetic signals involved act first on sclerification rather than endocarp size. Intensification of sclerification and the end of expansive growth occurred first with lowest water supply. Moderate and severe reductions in water availability proportionately decreased endocarp expansion and prolonged the sclerification, delaying the date of physically perceived hardening but not affecting the final degree of endocarp sclerification.

Regulation of polyamine metabolism and biosynthetic gene expression during olive mature-fruit abscission.

Exogenous ethylene and some inhibitors of polyamine biosynthesis can induce mature-fruit abscission in olive, which could be associated with decreased nitric oxide production as a signaling molecule. Whether H2O2 also plays a signaling role in mature-fruit abscission is unknown. The possible involvement of H2O2 and polyamine in ethylene-induced mature-fruit abscission was examined in the abscission zone and adjacent cells of two olive cultivars. Endogenous H2O2 showed an increase in the abscission zone during mature-fruit abscission, suggesting that accumulated H2O2 may participate in abscission signaling. On the other hand, we followed the expression of two genes involved in the polyamine biosynthesis pathway during mature-fruit abscission and in response to ethylene or inhibitors of ethylene and polyamine. OeSAMDC1 and OeSPDS1 were expressed differentially within and between the abscission zones of the two cultivars. OeSAMDC1 showed slightly lower expression in association with mature-fruit abscission. Furthermore, our data show that exogenous ethylene or inhibitors of polyamine encourage the free putrescine pool and decrease the soluble-conjugated spermidine, spermine, homospermidine, and cadaverine in the olive abscission zone, while ethylene inhibition by CoCl2 increases these soluble conjugates, but does not affect free putrescine. Although the impact of these treatments on polyamine metabolism depends on the cultivar, the results confirm that the mature-fruit abscission may be accompanied by an inhibition of S-adenosyl methionine decarboxylase activity, and the promotion of putrescine synthesis in olive abscission zone, suggesting that endogenous putrescine may play a complementary role to ethylene in the normal course of mature-fruit abscission.

Identification of novel virulence genes and metabolic pathways required for full fitness of Pseudomonas savastanoi pv. savastanoi in olive (Olea europaea) knots.

Comparative genomics and functional analysis of Pseudomonas syringae and related pathogens have mainly focused on diseases of herbaceous plants; however, there is a general lack of knowledge about the virulence and pathogenicity determinants required for infection of woody plants. Here, we applied signature-tagged mutagenesis (STM) to Pseudomonas savastanoi pv. savastanoi during colonization of olive (Olea europaea) knots, with the goal of identifying the range of genes linked to growth and symptom production in its plant host. A total of 58 different genes were identified, and most mutations resulted in hypovirulence in woody olive plants. Sequence analysis of STM mutations allowed us to identify metabolic pathways required for full fitness of P. savastanoi in olive and revealed novel mechanisms involved in the virulence of this pathogen, some of which are essential for full colonization of olive knots by the pathogen and for the lysis of host cells. This first application of STM to a P. syringae-like pathogen provides confirmation of functional capabilities long believed to play a role in the survival and virulence of this group of pathogens but not adequately tested before, and unravels novel factors not correlated previously with the virulence of other plant or animal bacterial pathogens.

Peroxynitrite mediates programmed cell death both in papillar cells and in self-incompatible pollen in the olive (Olea europaea L.).

Programmed cell death (PCD) has been found to be induced after pollination both in papillar cells and in self-incompatible pollen in the olive (Olea europaea L.). Reactive oxygen species (ROS) and nitric oxide (NO) are known to be produced in the pistil and pollen during pollination but their contribution to PCD has so far remained elusive. The possible role of ROS and NO was investigated in olive pollen-pistil interaction during free and controlled pollination and it was found that bidirectional interaction appears to exist between the pollen and the stigma, which seems to regulate ROS and NO production. Biochemical evidence strongly suggesting that both O(2)(-) and NO are essential for triggering PCD in self-incompatibility processes was also obtained. It was observed for the first time that peroxynitrite, a powerful oxidizing and nitrating agent generated during a rapid reaction between O(2)(-) and NO, is produced during pollination and that this is related to an increase in protein nitration which, in turn, is strongly associated with PCD. It may be concluded that peroxynitrite mediates PCD during pollen-pistil interaction in Olea europaea L. both in self-incompatible pollen and papillar cells.

Morphological, histological and ultrastructural changes in the olive pistil during flowering.

Sexual reproduction is essential for the propagation of higher plants. From an agronomical point of view, this is a particularly key process because fertilization guarantees fruit formation in most cultivated fruit species. In the olive, however, in spite of its agricultural importance, little attention has been paid to the study of sexual reproduction. In order to investigate the cellular and molecular mechanisms that regulate pollen-pistil interactions in the olive during the progamic phase, it is essential to first have a good knowledge of the reproductive structures involved in such interactions. This study characterizes the anatomical and ultrastructural changes in the olive pistil, beginning from the young pistil developing within the bud until the time of petal loss and visible stigma senescence. We have correlated changes in the pistil with a series of defined floral developmental stages and determined that olive pistil structures cannot be considered completely mature and ready to be pollinated and fertilized until the onset of anthesis. Our results clearly show histological and ultrastructural variation during the diverse flowering events. We discuss whether the changes observed might influence or result from pollen-pistil interactions during the progamic phase.

Ripening-related cell wall modifications in olive (Olea europaea L.) fruit: A survey of nine genotypes.

The production of olive (Olea europaea L.) is very important economically in many areas of the world, and particularly in countries around the Mediterranean basin. Ripening-associated modifications in cell wall composition and structure of fruits play an important role in attributes like firmness or susceptibility to infestations, rots and mechanical damage, but limited information on these aspects is currently available for olive. In this work, cell wall metabolism was studied in fruits from nine olive cultivars ('Arbequina', 'Argudell', 'Empeltre', 'Farga', 'Manzanilla', 'Marfil', 'Morrut', 'Picual' and 'Sevillenca') picked at three maturity stages (green, turning and ripe). Yields of alcohol-insoluble residue (AIR) recovered from fruits, as well as calcium content in fruit pericarp, decreased along ripening. Cultivar-specific diversity was observed in time-course change patterns of enzyme activity, particularly for those acting on arabinosyl- and galactosyl-rich pectin side chains. Even so, fruit firmness levels were associated to higher pectin methylesterase (PME) activity and calcium contents. In turn, fruit firmness correlated inversely with ascorbate content and with α-l-arabinofuranosidase (AFase) and ß-galactosidase (ß-Gal) activities, resulting in preferential loss of neutral sugars from cell wall polymers.

A morphological analysis of fresh and brine-cured olives attacked by Bactrocera oleae using light microscopy and ESEM-EDS.

The present study investigated the morphology of fresh and brine-cured table olives (TOs) as well as the changes that occur when drupes are attacked by the fruit fly Bactrocera oleae. Morphological analyses were performed using light microscopy (LM) and environmental scanning electron microscopy coupled with energy dispersive spectroscopy (ESEM-EDS). The LM analysis was carried out with visible light to evaluate sections stained with either PAS or Azan mixtures as well as unstained sections observed at fluorescence microscopy. The results of the analyses showed that: i) Azan and PAS staining played a useful complementary role, increasing the information provided by the histological analysis. Indeed, in both fresh and brine-cured TOs, epidermal layers and mesocarpal cells were clearly revealed, including sclereid cells. The histological analysis allowed also to identifying the presence of secoiridoid-biophenols (seco-BPs) in both cell walls and vacuoles, as well as in the drupe regions that had been attacked by fruit flies, where they were found at higher concentrations; ii) in fresh and brine-cured olives, the excitation at 480 nm revealed the distribution of the fluorophores, among which the seco-BP are enclosed; iii) the ESEM-EDS analysis revealed the natural morphology of fresh olives, including the dimensions of their cell layers and the size and depth of the mechanical barriers of suberized or necrotic cells around the larva holes. In addition, the elemental composition of regions of interest of the drupe was determined in fresh and brine-cured TOs. The results highlighted the effectiveness of combined use of LM and ESEM-EDS in order to obtain a picture, as complete as possible, of the structural morphology of TOs. Such analytical combined approach can be used to support multidisciplinary studies aimed at the selection of new cultivars more resistant to fly attack.

Morphological and cytological development and starch accumulation in hermaphrodite and staminate flowers of olive (Olea europaea L.).

In olive (Olea europaea L.), the formation of functionally staminate flowers rather than fully functional hermaphrodites is one of the major factors limiting fruit set, as flowers with aborted pistils are incapable of producing fruit. Studies conducted on various angiosperm species have shown a correlation between flower abortion and starch content. Thus, it is important to know if starch content plays a role in regulating pistil development in olive and if so, what mechanism regulates starch distribution. Cyto-histological observations of staminate and hermaphrodite olive flowers show that pistil development in staminate flowers is interrupted after the differentiation of the megaspore mother cell. At that stage, starch grains were only detected in the ovary, style and stigma of the hermaphrodite flowers. No starch was observed in the pistils of the staminate flowers. This finding suggests a tight correlation between starch content and pistil development. The secondary origin of starch within the flower is indicated by low chlorophyll content in the gynoecium, undetectable Rubisco activity in the pistils of these two kinds of flowers and by the ultrastructure of the plastids observed by transmission electron microscope analysis. The plastids have few thylakoid membranes and grana and in the staminate flowers appeared very similar to proplastids. Considering differences in starch content between staminate and hermaphrodite flowers and the secondary origin of the starch, differences in pistil development in the staminate and hermaphrodite flowers could be related to differences in the sink strength of these two types of flowers.

Water deficit-induced changes in mesocarp cellular processes and the relationship between mesocarp and endocarp during olive fruit development.

A field experiment was conducted during two consecutive growing seasons to determine and quantify the growth response of the olive (Olea europaea L. cv. Leccino) fruit and of its component tissues to tree water status. Pre-dawn leaf water potential (Psi(w)) and fruit volume were measured at about weekly intervals, and fresh weight (FW) and dry weight (DW) of the fruit tissues at 15, 20 and 21 weeks after full bloom (AFB). Fruit anatomical sections were prepared at 8, 15 and 21 weeks AFB for area determinations and cell counts. Fruit volume of the well-watered trees (average Psi(w) = -0.97 MPa) increased rapidly and reached the greatest final size, that from the most stressed (average Psi(w) = -2.81 MPa) grew most slowly and were smallest. In general, equatorial transverse areas of the mesocarp increased with increasing Psi(w), and this response was more evident at 21 than at 15 weeks AFB. By 21 weeks AFB, the mesocarp of the well-watered trees reached values more than three times higher than those measured at 8 weeks AFB. The endocarp FW and DW did not increase between 15 and 21 weeks AFB. Within each sampling date the endocarp area, FW and DW responded weakly to Psi(w). The mesocarp-to-endocarp ratio (FW and DW) increased from 15 to 21 weeks AFB regardless of water status, mainly due to the mesocarp growth. In both years at 20 and 21 weeks AFB, low values of the mesocarp-to-endocarp ratio were found with Psi(w) below -2.5 MPa. Within the mesocarp, cell size was more responsive to water deficit than to cell number. At 8 weeks AFB, the number of cells in the mesocarp was unaffected by tree water deficit, whereas cell size decreased, although slightly, in fruits sampled from trees in which Psi(w) was < -3.0 MPa. At 21 weeks AFB, cell size showed a linear decrease with increasing level of water deficit, whereas the number of cells at 21 weeks AFB decreased as the Psi(w) decreased below -2.5 MPa and seemed unaffected above that range. Overall, the results clarify the complexity of the water-induced response of mesocarp and endocarp growth and cellular processes of olive fruits.

Feasible production of biomass and natural antioxidants through callus cultures in response to varying light intensities in olive (Olea europaea. L) cult. Arbosana.

Light is the most important physical factor in growth and development of plants. Light intensity is directly proportional to the growth and accumulation of natural antioxidants during in vitro cultures of various medicinal plants. The present research study was designed to determine the effect of different light intensities i.e. normal light (2000-2500 lx), diffused light (500-1000 lx) and complete dark (0 lx) on callus growth dynamics and production of natural antioxidants in olive cult. Arbosana. Highest callus induction frequency (50%) was observed in the stem explants pre-treated with silver nanoparticles suspension (AgNPs: 50 ppm) and cultured on MS media supplemented with combination of 6-Benzylaminopurine (BAP: 2 mg/l), Gibberellic acid (GA3: 1.5 mg/l) plus Naphthalene acetic acid (NAA: 0.5 mg/l). Maximum callus biomass (FW = 1414 mg/l) was recorded when the cultured explants were incubated initially for seven days in complete darkness, followed by transference to diffused light for one week and then finally placed under normal light in total fifty six days culture period. Moreover, phytochemical analysis of the callus cultures showed significantly higher activities of antioxidant enzymes i.e. SOD, POD, CAT and APx (2.45, 2.96, 2.57 and 1.67 U/mg. protein) in the callus cultures grown under dark condition as compared with other light treatments. For non-enzymatic antioxidant potential, maximum activity of TPC, TFC, PAL and DPPH (2.42 mg GAE/g, 1.50 mg QAE/g, 3.95 U/mg and 75%) were recorded in the calli raised in vitro under diffused light. This is the first report on the production of natural antioxidants in response to different light intensities in callus cultures of Olea europaea. Future studies should focus on large scale production of callus cultures in order to yield maximum biomass from this high valued plant.

Biogenesis of protein bodies during legumin accumulation in developing olive (Olea europaea L.) seed.

Much of our current knowledge about seed development and differentiation regarding reserves synthesis and accumulation come from monocot (cereals) plants. Studies in dicotyledonous seeds differentiation are limited to a few species and in oleaginous species are even scarcer despite their agronomic and economic importance. We examined the changes accompanying the differentiation of olive endosperm and cotyledon with a focus on protein bodies (PBs) biogenesis during legumin protein synthesis and accumulation, with the aim of getting insights and a better understanding of the PBs' formation process. Cotyledon and endosperm undergo differentiation during seed development, where an asynchronous time-course of protein synthesis, accumulation, and differential PB formation patterns was found in both tissues. At the end of seed maturation, a broad population of PBs, particularly in cotyledon cells, was distinguishable in terms of number per cell and morphometric and cytochemical features. Olive seed development is a tissue-dependent process characterized by differential rates of legumin accumulation and PB formation in the main tissues integrating seed. One of the main features of the impressive differentiation process is the specific formation of a broad group of PBs, particularly in cotyledon cells, which might depend on selective accumulation and packaging of proteins and specific polypeptides into PBs. The nature and availability of the major components detected in the PBs of olive seed are key parameters in order to consider the potential use of this material as a suitable source of carbon and nitrogen for animal or even human use.

Metabolic control analysis reveals an important role for diacylglycerol acyltransferase in olive but not in oil palm lipid accumulation.

We applied metabolic control analysis to the Kennedy pathway for triacylglycerol formation in tissue cultures from the important oil crops, olive (Olea europaea L.) and oil palm (Elaeis guineensis Jacq.). When microsomal fractions were incubated at 30 degrees C rather than 20 degrees C, there was an increase in triacylglycerol labelling. This increase was accompanied by a build up of diacylglycerol (DAG) radioactivity in olive but not in oil palm, suggesting that the activity of DAG acyltransferase (DAGAT) was becoming limiting in olive. We used 2-bromooctanoate as a specific inhibitor of DAGAT and showed that the enzyme had a flux control coefficient under the experimental conditions of 0.74 in olive but only 0.12 in oil palm. These data revealed important differences in the regulation of lipid biosynthesis in cultures from different plants and suggest that changes in the endogenous activity of DAGAT is unlikely to affect oil accumulation in oil palm crops.

On the use of leaf spectral indices to assess water status and photosynthetic limitations in Olea europaea L. during water-stress and recovery.

Diffusional limitations to photosynthesis, relative water content (RWC), pigment concentrations and their association with reflectance indices were studied in olive (Olea europaea) saplings subjected to water-stress and re-watering. RWC decreased sharply as drought progressed. Following rewatering, RWC gradually increased to pre-stress values. Photosynthesis (A), stomatal conductance (gs), mesophyll conductance (gm), total conductance (gt), photochemical reflectance index (PRI), water index (WI) and relative depth index (RDI) closely followed RWC. In contrast, carotenoid concentration, the carotenoid to chlorophyll ratio, water content reflectance index (WCRI) and structural independent pigment index (SIPI) showed an opposite trend to that of RWC. Photosynthesis scaled linearly with leaf conductance to CO2; however, A measured under non-photorespiratory conditions (A1%O2) was approximately two times greater than A measured at 21% [O2], indicating that photorespiration likely increased in response to drought. A1%O2 also significantly correlated with leaf conductance parameters. These relationships were apparent in saturation type curves, indicating that under non-photorespiratory conditions, CO2 conductance was not the major limitations to A. PRI was significant correlated with RWC. PRI was also very sensitive to pigment concentrations and photosynthesis, and significantly tracked all CO2 conductance parameters. WI, RDI and WCRI were all significantly correlated with RWC, and most notably to leaf transpiration. Overall, PRI correlated more closely with carotenoid concentration than SIPI; whereas WI tracked leaf transpiration more effectively than RDI and WCRI. This study clearly demonstrates that PRI and WI can be used for the fast detection of physiological traits of olive trees subjected to water-stress.

Construction of core collections suitable for association mapping to optimize use of Mediterranean olive (Olea europaea L.) genetic resources.

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.