Development and characterization of microsatellite loci from Olea europaea

This study reports 19 simple sequence repeat loci developed from a genomic library of the olive tree (Olea europaea L.), of which 12 revealed to be polymorphic and informative, ranging from two to 14 alleles.     

Cytoplasmic male sterility in the olive (Olea europaea L.)

The olive tree is usually hermaphrodite but self-incompatible. In the Western Mediterranean some cultivars are totally male-sterile. Three different male-sterile phenotypes have been recognised. To infer the genetic basis of male sterility we studied its inheritance and cytoplasmic diversity in wild (oleaster) and cultivated Mediterranean olive. In the cross Olivière×Arbequina, the male-sterile trait was maternally inherited and affected all progenies. We also checked that both chloroplast and mitochondrial DNAs are maternally inherited. RFLP studies on chloroplast and mitochondrial DNAs revealed several cytotypes: two chlorotypes and four mitotypes in cultivars and oleaster (wild or feral Mediterranean olive). Furthermore, a total linkage desequilibrium between the CCK chlorotype and the MCK mitotype in cultivars and oleaster from different regions supports the fact that paternal leakage of organelles was not observed. The male sterility (ms 2) displayed by Olivière, plus six other cultivars and three oleaster was strictly associated with the CCK chlorotype and the MCK mitotype. These facts suggest that Olivière carries cytoplasmic male sterility. Male-fertile and male-sterile oleasters carrying this cytotype showed the presence of restorer alleles. This CMS might be due to a distant cross between olive taxa. The two other male-sterile phenotypes displayed by Lucques (ms 1) and Tanche (ms 3) were associated with the ME1 mitotype but we have not demonstrated CMS. Received: 26 July 1999 / Accepted: 27 August 1999     

Novel Secoiridoid Glucosides in Olea europaea Leaves Suffering from Boron Deficiency

From the methanol extract of boron deficient Olea europaea leaves, two secoiridoid glycosides, not detected in leaf extracts of untreated plants, 6′-E-p-coumaroyl-secologanoside and 6′-O-[(2E)-2,6-dimethyl-8-hydroxy-2-octenoyloxy]-secologanoside, were isolated together with three known secoiridoid glycosides, oleuropein, oleoside dimethyl ester, and secologanoside. The structures of the isolated compounds were established by means of NMR and MS spectral analyses. The above novel secoiridoids were synthesized by the plant as a physiological response to nutrient stress.     

Chloroplast-DNA variation in cultivated and wild olive (Olea europaea L.)

Polymorphism in the lengths of restriction fragments of the whole cpDNA molecule was studied in cultivated olive and in oleaster (wild olive) over the whole Mediterranean Basin. Seventy two olive cultivars, 89 very old trees cultivated locally, and 101 oleasters were scored for ten endonucleases. Moreover, maternal inheritance of cpDNA in olive was shown by analysing the progeny of a controlled cross between two parents which differed in their cpDNA haplotypes. In the whole species, three site- and three length-mutations were observed, corresponding to five distinct chlorotypes. The same chlorotype (I) was predominant in both oleasters and cultivated olive trees, confirming that these are closely related maternally. Three other chlorotypes (II, III and IV) were observed exclusively in oleaster material and were restricted either to isolated forest populations or to a few individuals growing in mixture with olive trees possessing the majority chlorotype. An additional chlorotype (V) was characterised by three mutations located in distinct parts the cpDNA molecule but which were never observed to occur separately. This chlorotype, more widely distributed than the other three, in both cultivated and wild olive, and occurring even in distant populations, was observed exclusively in male-sterile trees showing the same specific pollen anomaly. However, in the present study, no evidence was provided for a direct relationship between the occurrence of the cpDNA mutations and male sterility. It is suggested that the large geographic distribution of chlorotype V may be related to the high fruit production usually observed on male-sterile trees. These may be very attractive for birds which are fond of olive fruit and spread the stones efficiently. Probably for the same reason, people preserved male-sterile oleasters for long periods and, in several places, used male-sterile cultivars over large areas. Received: 25 November 1998 / Accepted: 19 December 1998     

Paternity tests support a diallelic self‐incompatibility system in a wild olive (Olea europaea subsp. laperrinei,Oleaceae)

Self‐incompatibility (SI) is the main mechanism that favors outcrossing in plants. By limiting compatible matings, SI interferes in fruit production and breeding of new cultivars. In the Oleeae tribe (Oleaceae), an unusual diallelic SI system (DSI) has been proposed for three distantly related species including the olive (Olea europaea), but empirical evidence has remained controversial for this latter. The olive domestication is a complex process with multiple origins. As a consequence, the mixing of S‐alleles from two distinct taxa, the possible artificial selection of self‐compatible mutants and the large phenological variation of blooming may constitute obstacles for deciphering SI in olive. Here, we investigate cross‐genotype compatibilities in the Saharan wild olive (O. e. subsp. laperrinei). As this taxon was geographically isolated for thousands of years, SI should not be affected by human selection. A population of 37 mature individuals maintained in a collection was investigated. Several embryos per mother were genotyped with microsatellites in order to identify compatible fathers that contributed to fertilization. While the pollination was limited by distance inside the collection, our results strongly support the DSI hypothesis, and all individuals were assigned to two incompatibility groups (G1 and G2). No self‐fertilization was observed in our conditions. In contrast, crosses between full or half siblings were frequent (ca. 45%), which is likely due to a nonrandom assortment of related trees in the collection. Finally, implications of our results for orchard management and the conservation of olive genetic resources are discussed.     

Ultrastructural aspects of the cytoplasmic origin and accumulation of oil in olive fruit (Olea europaea)

The development of oil bodies and oil droplets in fruits of olive was examined at the ultrastructural level. Both oil bodies that form in young fruits and oil droplets that develop with fruit maturation are cytoplasmic bodies. The formation of the small oil bodies occurs in localized regions of the cytoplasm. These bodies are closely associated and fuse together, forming a small oil droplet that protruded against and indented the vacuolar membrane. As the fruit matures, new oil bodies appear to form and fuse with the oil droplet, resulting in the formation of a single large oil droplet of about 30 μm in diameter in most mature mesocarp cells. The cytoplasmic region where the oil bodies formed had a granulate, ultrastructural appearance, and cytoplasmic components such as membranes and ribosomes were noticeably absent in these regions. The granulate material coated the oil bodies and oil droplets, and appeared as a thin, compressed band between the round inner surface of the droplets and the indented tonoplast. We suggest that this granulate material is involved in the synthesis of the oil and, with enlargement of the oil bodies, this coat becomes thinner in regions where they are closely associated, resulting in zones where confluence of the oil occurs.     

Identification of simple sequence repeats (SSRs) in olive ( Olea europaea L.)

A small insert genomic library of Olea europaea L., highly enriched in (GA/CT)n repeats, was obtained using the procedure of Kandpal et al. (1994). The sequencing of 103 clones randomly extracted from this library allowed the identification of 56 unique genomic inserts containing simple sequence repeat regions made by at least three single repeats. A sample of 20 primer pairs out of the 42 available were tested for functionality using the six olive varieties whose DNA served for library construction. All primer pairs succeeded in amplifying at least one product from the six DNA samples, and ten pairs detecting more than one allele were used for the genetic characterisation of a panel of 20 olive accessions belonging to 16 distinct varieties. A total of 57 alleles were detected among the 20 genotypes at the ten polymorphic SSR loci. The remaining primer pair allowed the amplification of a single SSR allele for all accessions plus a longer fragment for some genotypes. Considering the simple sequence repeat polymorphism, 5.7 alleles were scored on average for each of the ten SSR loci. A genetic dissimilarity matrix, based on the proportion of shared alleles among all the pair-wise combinations of genotypes, was constructed and used to disentangle the genetic relationships among varieties by means of the UPGMA clustering algorithm. Graphical representation of the results showed the presence of two distinct clusters of varieties. The first cluster grouped the varieties cultivated on the Ionian Sea coasts. The second cluster showed two subdivisions: the first sub-cluster agglomerated the varieties from some inland areas of Calabria; the second grouped the remaining varieties from Basilicata and Apulia cultivated in nearby areas. Results of cluster analysis showed a significant relationship between the multilocus genetic similarities and the geographic origin of the cultivars. Received: 2 February 2001 / Accepted: 1 June 2001     

Genetic relationships in the olive (Olea europaea L.) reflect multilocal selection of cultivars

One hundred and two olive RAPD profiles were sampled from all around the Mediterranean Basin. Twenty four clusters of RAPD profiles were shown in the dendrogram based on the Ward’s minimum variance algorithm using chi-square distances. Factorial discriminant analyses showed that RAPD profiles were correlated with the use of the fruits and the country or region of origin of the cultivars. This suggests that cultivar selection has occurred in different genetic pools and in different areas. Mitochondrial DNA RFLP analyses were also performed. These mitotypes supported the conclusion also that multilocal olive selection has occurred. This prediction for the use of cultivars will help olive growers to choose new foreign cultivars for testing them before an eventual introduction if they are well adapted to local conditions. Received: 10 April 2000 / Accepted: 15 May 2000     

Abnormalities induced by agricultural pesticides in the microsporogenesis of olive tree (Olea europaea L.) cultivars

The study evaluated the microsporogenesis of olive trees subjected to different agricultural pesticide applications during flowering. Inflorescences of cultivars Arbequina and MGS GRAP541 were subjected to agricultural pesticides: mineral oil, neem oil, dimethoate and deltamethrin. The floral buds were fixed in Carnoy for the microsporogenesis analysis and in Karnovsky for scanning electron microscopy. The slides were prepared by squash technique and staining with propionic carmine. The pollen viability was determined by Alexander’s stain and in vitro germination. Results show that the quantification of abnormalities in meiosis in the two cultivars caused significant effect among the treatments, being that all differed statistically from the control group. Both methods showed a higher percentage of viable pollens in the control treatment and lower percentage of viability with the agricultural pesticides. The method of pollen viability by staining presented the highest averages of viable pollens, but when compared together, both methods presented a strongly related positive linear correlation. It was concluded that the used chemical products increased the percentage of chromosomal abnormalities during microsporogenesis, which interfered in the pollen viability of the two analyzed cultivars. The product deltamethrin caused the strongest effect on meiosis and on pollen viability.     

A set of primers for length and nucleotide‐substitution polymorphism in chloroplastic DNA of Olea europaea L. (Oleaceae)

Chloroplastic DNA (cpDNA) variation at five microsatellite motifs, two insertion‐deletion sites, and eight nucleotide substitution sites was investigated in the Olea europaea complex. Primers were designed for flanking regions of these sites to amplify short cpDNA regions. They provided polymorphism when polymerase chain reaction (PCR) products from a representative sample of 128 O. europaea individuals were either resolved by size into polyacrylamide gels (length polymorphism) or digested with restriction enzymes (nucleotide‐substitution polymorphism). These polymorphisms serve to distinguish most of the cytoplasmic haplotypes previously recognized. Potential application of these markers in O. europaea includes phylogeography, conservation and germplasm identification, even when using poorly preserved material from herbarium specimens or forensic and archaeological materials.     

Stomatal and photosynthetic responses of olive (Olea europaea L.) leaves to water deficits

The leaf gas exchange of mature olive trees (Olea europaea L.) was characterized over a wide range of water deficits in the field during 1998, in Cordoba, Spain. Leaf photosynthesis (A) and stomatal conductance (g_l) responded diurnally and seasonally to variations in tree water status and evaporative demand. In the absence of water stress, A and g_l were generally high during autumn and low in days of high vapour pressure deficits (VPD). Leaf A varied between 0 and 2 µmol m^?2 s^?1 under severe water deficits that lowered the stem water potential (Ψ_x) to ?8·0 MPa, but recovered rapidly following rehydration. Transpiration efficiency (TE) was curvilinearly related to VPD and not influenced by water deficits except in cases of severe water stress, where low TE values were observed at Ψ_x below ?4 MPa. Three models of leaf conductance were calibrated and validated with the experimental data; two were based on the model proposed by Leuning (L) and the other was derived from the widely used Jarvis (J) model. The L models performed better than the J model in two validation tests. The scatter of the predictions and the limited accuracy of all three models suggest that, in addition to the physiological and environmental variables considered, there are additional endogenous factors influencing the g_l of olive leaves.     

A possible role for flowering locus T‐encoding genes in interpreting environmental and internal cues affecting olive (Olea europaea L.) flower induction

Olive (Olea europaea L.) inflorescences, formed in lateral buds, flower in spring. However, there is some debate regarding time of flower induction and inflorescence initiation. Olive juvenility and seasonality of flowering were altered by overexpressing genes encoding flowering locus T (FT). OeFT1 and OeFT2 caused early flowering under short days when expressed in Arabidopsis. Expression of OeFT1/2 in olive leaves and OeFT2 in buds increased in winter, while initiation of inflorescences occurred i n late winter. Trees exposed to an artificial warm winter expressed low levels of OeFT1/2 in leaves and did not flower. Olive flower induction thus seems to be mediated by an increase in FT levels in response to cold winters. Olive flowering is dependent on additional internal factors. It was severely reduced in trees that carried a heavy fruit load the previous season (harvested in November) and in trees without fruit to which cold temperatures were artificially applied in summer. Expression analysis suggested that these internal factors work either by reducing the increase in OeFT1/2 expression or through putative flowering repressors such as TFL1. With expected warmer winters, future consumption of olive oil, as part of a healthy Mediterranean diet, should benefit from better understanding these factors.     

Changes in non-structural carbohydrates in olive (Olea europaea) leaves during root zone salinity stress

Self-rooted olive ( Olea europaea L.) plants were grown in hydroponics at various NaCl concentrations (from 0 to 200m M ) for 28 to 32 days followed by 28 to 30 days of relief from salinity over two growing seasons. Olive leaves accumulated both glucose and mannitol during the period of salinity stress. The concentrations of fructose, myo -inositol, galactose, galactinol, sucrose, raffinose, and stachyose were not significantly affected by salinity. Starch content was decreased by salinity. The mannitol/glucose and mannitol/soluble carbohydrates ratios increased as the external NaCl concentration was increased, but returned to the control levels during the relief period. The increase in mannitol or glucose molar concentrations, expressed on a leaf tissue water basis, was partially due to a reduction in leaf tissue water content under salinity stress. However, an increase in mannitol concentration was also observed when expressed on a dry weight basis. The accumulation of mannitol in leaf tissue preceded any reduction in leaf area rate or net assimilation rate. The increase in leaf mannitol or glucose concentration was positively correlated with the increasing level of salinity at the root zone, but not with the accumulation of Na⁺ in the shoot. The role of mannitol. a potential osmoregulator in leaf mesophyll during salinity stress, is discussed in relation to the complex carbohydrate composition of olive leaves.     

Somatic embryogenesis and plant regeneration in olive (Olea europaea L.)

Leaf discs from olive (Olea europaea L.) grown in vitro and immature zygotic embryos collected at 50, 75, 90 and 105 days after full bloom were tested for their somatic embryogenic capacity. The embryos were grown in half-strength MS medium and half-strength OM medium with BAP combinated with either 2,4-D or NAA. Incubation was either in an initial dark period followed by 16h daylight or in 16h daylight throughout. Somatic embryogenesis, approx. 40%, mostly directly from the embryos, was observed only in 75-day-old embryos in medium containing low cytokinin and auxin concentrations. Differentiation was inhibited by 2,4-D whereas NAA did not. In leaf discs and younger and older zygotic embryos, only callus and root formation was observed. Somatic embryos were germinated and then potted-up to soil.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - NAA naphtaleneacetic acid     

Phenotypic plasticity and integration across the canopy of Olea europaea subsp. guanchica (Oleaceae) in populations with different wind exposures

Woody plants, as sessile and long-lived organisms, are expected to have effective mechanisms for dealing with recurrent environmental stresses. In the present study, we hypothesized that phenotypic plasticity (the ability to express alternative phenotypes) and integration (covariation among functionally related traits) are elicited in plants under stressful wind speed conditions. We investigated the within-crown variation of nine vegetative traits of a tree species (Olea europaea subsp. guanchica) in six populations that represented a gradient of wind speed exposures. Wind-exposed twigs in outer-canopy layers had smaller leaves; thinner, lighter, and shorter internodes; and a larger internode cross-sectional area to leaf area ratio. Comparison between field and greenhouse trials revealed that field differences among populations were mediated by phenotypic plasticity. Outer-canopy twigs expressed plastic responses in populations exposed to high wind speeds, whereas inner-canopy twigs displayed high phenotypic convergence among populations. In addition, phenotypic integration increased with wind exposure (outer canopy > inner canopy > greenhouse) and was consequently affected by canopy openness. We conclude that exposure to wind above a certain speed threshold in this woody species elicits a plastic response that is associated with increased integration among traits and involves mechanical and hydraulic rearrangements in more exposed parts of the trees.     

The role of staminate flowers in the breeding system of Olea europaea (Oleaceae): an andromonoecious, wind-pollinated taxon

BACKGROUND AND AIMS: Andromonoecy, as a breeding system, has generated a considerable body of theory in terms of sexual selection, but extended records comparing the performance of pollen grains from staminate versus hermaphrodite flowers are still sparse. The objective in this study was to elucidate the role of staminate flowers in the andromonoecious breeding system of olive (Olea europaea). METHODS: To determine the meaning of staminate flowers, an evaluation was made of resource allocation to, and phenology of, staminate and hermaphrodite flowers in the cultivar 'Mission', and a comparison was made of the male function between both kinds of flowers. KEY RESULTS: Dry weight of hermaphrodite flowers was 19 % greater than dry weight of staminate flowers arising in comparable positions of the panicle. This difference was mainly due to pistil and petal weight; there were no significant differences in stamen weight. There were no significant differences between staminate and hermaphrodite flowers in either amount of pollen per anther, or pollen quality as determined by pollen viability, germinability or ability to fertilize other flowers. There was no significant link between gender and time of anthesis. However, position of the flower within the panicle correlated with time of anthesis and gender. Flowers at the apex and at primary pedicels tended to be hermaphrodite and open earlier, whereas flowers arising in secondary pedicels were mainly staminate and were commonly the last to reach anthesis. CONCLUSIONS: It is proposed that the main advantage provided by production of staminate flowers in olive is to enhance male fitness by increasing pollen output at the whole plant level, although a relict function of attracting pollinators cannot be completely discarded.     

Historical biogeography of olive domestication (Olea europaea L.) as revealed by geometrical morphometry applied to biological and archaeological material

Aim This study intends to improve our understanding of historical biogeography of olive domestication in the Mediterranean Basin, particularly in the north-western area. Location Investigations were performed simultaneously on olive stones from extant wild populations, extant cultivated varieties from various Mediterranean countries, and archaeological assemblages of Spanish, French and Italian settlements. Methods A combination of morphometrics (traditional and geometrical) allowed us to study both the size and shape of endocarp structure. Concerning shape, a size-standardized method coupled with fitted polynomial regression analysis was performed. Results We found morphological criteria for discriminating between wild and cultivated olive cultivars, and established patterns of morphological variation of olive material according to the geographical origin (for extant material) and to the age of the olive forms (for archaeological material). Levels of morphological convergences and divergences between wild olive populations and cultivated varieties are presented as evidence. Main conclusions Morphological changes of endocarps of olive under domestication at both geographical and chronological scales provide new criteria for the identification of olive cultivars. They allow to determine the origins of cultivated forms created and/or introduced in the north-western Mediterranean regions and to understand how human migrations affected the rest of the Western Mediterranean regions. A model of diffusion of olive cultivation is proposed. It shows evidence of an indigenous origin of the domestication process, which is currently recognized in the north-western area since the Bronze Age.     

Behavior of storage lipids during development and germination of olive ( Olea europaea L.) pollen

Summary.  The presence of abundant oil bodies in the mature olive pollen grain has led us to focus on the behavior of these lipid bodies during pollen development and in vitro pollen germination. The appearance, increase, and accumulation of lipid bodies have been determined by following the sequential development of the pollen grain. Semithin slices of anthers and pollen grains were stained with Sudan Black B in order to identify neutral lipids. Ultrastructural studies were also carried out. Our results show a notable increase in lipid bodies between the young-pollen-grain stage and the mature-pollen-grain stage. Substantial polarization of lipid bodies was observed after 1 or 2 h of pollen incubation in germination medium. During pollen tube growth, the lipid bodies are located near the germinative aperture after 3 h of incubation, as well as inside the pollen tube, thus suggesting that the lipid bodies move from the pollen grain to the pollen tube. After 7 h of germination the presence of lipid bodies inside the pollen tube is no longer substantial. Our results support the idea that lipid bodies are involved in pollen germination, stigma penetration, and pollen tube growth. These results are discussed in connection with their implications for the pollen germination process. Received June 4, 2002; accepted October 29, 2002; published online April 8, 2003 RID="*" ID="*" Correspondence and reprints: Departamento de Bioquímica, Biología Celular y Molecular de Plantas, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Profesor Albareda 1, 18008 Granada, Spain.