Olive genetic diversity assessed using amplified fragment length polymorphisms

 Amplified fragment length polymorphism (AFLP) analysis was used to study the genetic variation within and among populations of genus Olea. A group of genotypes, all of them cultivated varieties of a single species, Olea europaea, was compared with wild olives and with a group of individuals belonging to different Olea species. Five primer combinations were used which produced about 290 polymorphic bands. The data obtained were elaborated with the Nei’s genetic similarity coefficient, applying different clustering methods and the Principal Coordinate Analysis. Cultivars, wild olives and North-West African species formed groups clustering together at a similarity level of 0.56, while the Olea species from East Africa and Asia grouped separately. Species from the Indian Ocean and Australia showed the highest diversity. We hypothesize that cultivars and wild plants are different forms of the same O. europaea species. The Olea from East Africa and Asia may be assigned to a different species, while the role of O. laperrini as well as that of O. maroccana as an intermediary form is confirmed. Received: 30 April 1998 / Accepted: 13 August 1998     

Centennial olive trees as a reservoir of genetic diversity

Background and Aims

Genetic characterization and phylogenetic analysis of the oldest trees could be a powerful tool both for germplasm collection and for understanding the earliest origins of clonally propagated fruit crops. The olive tree (Olea europaea L.) is a suitable model to study the origin of cultivars due to its long lifespan, resulting in the existence of both centennial and millennial trees across the Mediterranean Basin.

Methods

The genetic identity and diversity as well as the phylogenetic relationships among the oldest wild and cultivated olives of southern Spain were evaluated by analysing simple sequence repeat markers. Samples from both the canopy and the roots of each tree were analysed to distinguish which trees were self-rooted and which were grafted. The ancient olives were also put into chronological order to infer the antiquity of traditional olive cultivars.

Key Results

Only 9·6 % out of 104 a priori cultivated ancient genotypes matched current olive cultivars. The percentage of unidentified genotypes was higher among the oldest olives, which could be because they belong to ancient unknown cultivars or because of possible intra-cultivar variability. Comparing the observed patterns of genetic variation made it possible to distinguish which trees were grafted onto putative wild olives.

Conclusions

This study of ancient olives has been fruitful both for germplasm collection and for enlarging our knowledge about olive domestication. The findings suggest that grafting pre-existing wild olives with olive cultivars was linked to the beginnings of olive growing. Additionally, the low number of genotypes identified in current cultivars points out that the ancient olives from southern Spain constitute a priceless reservoir of genetic diversity.     

<Emphasis Type="Italic">Olea europaea</Emphasis> (Oleaceae) phylogeography based on chloroplast DNA polymorphism

Chloroplast DNA diversity in the olive (Olea europaea L.) complex was studied using PCR-RFLP and microsatellite markers. Fifteen chlorotypes were distinguished. We constructed a cpDNA phylogenetic tree in which five clades were recognised and located in distinct geographic areas: clade A in Central and Southern Africa, clade C in Asia, clade M in North-West Africa, clade E1 in the Mediterranean Basin and Sahara, and clade E2 in West Mediterranea. Cultivated olive clustered with Mediterranean and Saharan wild forms (clades E1 and E2). Strong genetic differentiation for cpDNA markers was observed between eastern and western Mediterranean olives, suggesting that these areas have represented different glacial refugia. Humans most likely spread one eastern chlorotype, preponderant in cultivars, across the western Mediterranean Basin. Its presence in O. e. subsp. laperrinei from the Sahara suggests a possible Mediterranean olive origin in an African population, which may have overlapped in the Southern Mediterranean during the Quaternary.     

Transferability of olive microsatellite loci across the genus <Emphasis Type="Italic">Olea</Emphasis>

The transferability of microsatellite markers developed for olive cultivars (Olea europaea L.) has been tested and confirmed in the Olea complex. Thirty two genotypes, belonging to different taxa of the genus Olea, have been analyzed with four olive SSRs. Positive amplifications at all loci were obtained in 13 taxa (at least one accession per species). Sixty seven different alleles have been detected at the four loci analyzed. Polymorphic products have been observed at the inter- and intra-species level. Some SSR loci have shown multiple amplification products in some species. The high number of unique alleles has allowed the unambiguous discrimination of most accessions. Similarity coefficients and relationships among the Olea taxa have been calculated based on SSR amplification results. The reliability of SSRs as markers for intra-species variability evaluation has been confirmed while their use to explore relationships at the inter-species level is discussed, being dependent on the locus analyzed.Communicated by H.F. Linskens     

Embryogenesis and regeneration of green plantlets from oat (Avena sativa L.) leaf-base segments: influence of nitrogen balance, sugar and auxin

The nitrogen composition and sugar and auxin concentrations of callus induction medium were optimized in order to improve the regeneration of green plants from two elite oat cultivars, Aslak and Veli. For both cultivars, the production of green plantlets was doubled by optimization. However, the results obtained also clearly demonstrated that cultivars of the same species may differ drastically in their requirements for essential media components. Veli clearly required higher total amounts of nitrogen (67.8 mM) than Aslak (44.9 mM) but less maltose and 2,4-dichlorophenoxyacetic acid (28 g l^-1 and 0.6 mg l^-1) than Aslak (38 g l^-1 and 2 mg l^-1). This result indicates that the optimal production of green plantlets through embryogenesis requires that media be optimized for each cultivar separately.     

Enhancement of microspore culture efficiency of recalcitrant barley genotypes

The culture response of isolated microspores of seven recalcitrant cultivars of barley has been largely improved by identifying an appropriate pretreatment and utilizing ovary co-cultivation. After comparison of three pretreatment media, medium B was shown to be most efficient for inducing microspore embryogenesis, while 0.3 M mannitol frequently used for the responsive cv. Igri was found to be ineffective for recalcitrant genotypes. A further significant improvement of embryogenesis was achieved by using ovary co-culture, which resulted in an overall 2.1-fold increase in embryo formation and 2.4-fold increase in green plant regeneration from all cultivars compared with the control. Optimal co-culture conditions were identified as 5 ovaries/ml medium kept over 20 days in induction culture. Microspore plating densities in cultures with and without co-culture were found to be optimal at 4᎒⁴/ml and 8-12᎒⁴/ml, respectively. The most effective and reproducible method for culturing microspores of recalcitrant genotypes appeared to be the combination of medium B pretreatment with ovary co-culture. By using this procedure, the genotypic difference in microspore embryogenesis could be reduced. It was found that medium B mainly enhanced percent live embryogenic microspores, and ovary co-culture subsequently improved cell division and embryogenic development. The method described here is important for the application of the microspore culture technique to barley breeding and biotechnology.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated production of transgenic plants of<Emphasis Type="Italic"> Muscari armeniacum</Emphasis> Leichtl. ex Bak.

A system for the production of transgenic plants has been developed for the Liliaceous ornamental plant Muscari armeniacum Leichtl. ex Bak via Agrobacterium-mediated transformation of embryogenic cultures. Leaf-derived embryogenic cultures were co-cultivated with each of three A. tumefaciens strains, all of which harbored the binary vector carrying the neomycin phosphotransferase II (nptII), hygromycin phosphotransferase (hpt) and intron-containing #-glucuronidase (gus-intron) genes in the T-DNA region. Following co-cultivation, the embryogenic cultures were cultured on a medium containing 500 mg l^-1 cefotaxime for 1 week followed by a medium containing 75 mg l^-1 hygromycin in addition to cefotaxime. After 4-5 weeks, several hygromycin-resistant (Hyg^r) cell clusters were produced from the co-cultivated embryogenic cultures. The highest efficiency of production of Hyg^r cell clusters was obtained when embryogenic cultures were inoculated with A. tumefaciens EHA101/pIG121Hm in the presence of 100 µM acetosyringone (AS) and 0.1% (v/v) of a surfactant (Tween20) followed by co-cultivation in the presence of 100 µM AS. Hyg^r embryogenic cultures developed into complete plants via somatic embryogenesis, and most of them were verified to be transgenic by GUS histochemical assay and polymerase chain reaction analysis. Southern blot analysis revealed the integration of one to five copies of the transgene into the genome of transgenic plants, but most of them had one or two copies.     

Establishing the genetic relationships between the wild and cultivated olives using a nuclear intron from nitrate reductase (nia-i3)

In Oleaceae the most outstanding biological issue is to clarify the taxonomic relationships of cultivated and wild olives. To establish the genetic relationships between the wild (Olea europaea subsp. europaea var. sylvestris (Mill.) Lehr.), the cultivated olive (Olea europaea subsp. europaea var. europaea), and other taxa of the genus Olea (Olea europaea subsp. cuspidata (Wall. ex G. Don) Cif., Olea europaea subsp. cerasiformis G. Kunkel & Sunding, Olea paniculata R. Br.) and other Oleaceae (represented by Ligustrum vulgaris) we carried out the amplification by polymerase chain reaction (PCR) and the sequencing of the third nuclear intron of the nitrate reductase gene (nia-i3). Sequence analyses showed the presence of two different functional variants of the intron (nia1 and nia2) in the Oleaceae, in addition to a shorter non-functional one. Notably, while the shortest and the nia1 variants were present in all the taxa analysed, the nia2 variant was present only in the wild and the cultivated olive. These data confirm the close phylogenetic relationship between wild and cultivated olives and suggest that this gene could be duplicated in these two taxa after its divergence from the remaining Oleaceae. The presence of a target for AflII enzyme in nia2 and its absence in nia1 variant enables easy distinction by PCR-RFLP between, on the one hand, wild and cultivated olive, and on the other the remaining subspecies of the Olea europaea L. complex (O. e. subsp. cuspidata and O. e. subsp. cerasiformis) as well as other Oleaceae (O. paniculata, L. vulgaris L.). Additionally, nia1 sequences provide useful information about phylogeny of the wild and cultivated olives inside the genus Olea.     

The complex history of the olive tree: from Late Quaternary diversification of Mediterranean lineages to primary domestication in the northern Levant

The location and timing of domestication of the olive tree, a key crop in Early Mediterranean societies, remain hotly debated. Here, we unravel the history of wild olives (oleasters), and then infer the primary origins of the domesticated olive. Phylogeography and Bayesian molecular dating analyses based on plastid genome profiling of 1263 oleasters and 534 cultivated genotypes reveal three main lineages of pre-Quaternary origin. Regional hotspots of plastid diversity, species distribution modelling and macrofossils support the existence of three long-term refugia; namely the Near East (including Cyprus), the Aegean area and the Strait of Gibraltar. These ancestral wild gene pools have provided the essential foundations for cultivated olive breeding. Comparison of the geographical pattern of plastid diversity between wild and cultivated olives indicates the cradle of first domestication in the northern Levant followed by dispersals across the Mediterranean basin in parallel with the expansion of civilizations and human exchanges in this part of the world.     

Genetic structure of wild and cultivated olives in the central Mediterranean basin

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.     

Pollen Ole e 1 content variations in olive cultivars of different Portugal regions

Olive is recognized as a crop with great impact in agricultural, socioeconomic, environmental and public health sectors. The last is becoming more important during recent years as consequence of the increase of the pollen allergy in south Europe prompted by the widespread Olea pollen allergic reactions. The aim of the study was to quantify, for the first time, the variations of the Ole e 1 allergen amount in Olea pollen grains from four cultivars in three regions of Portugal. How weather parameters can affect the allergen production was also assessed. The study was conducted in three olive producer areas of Portugal from 2010 to 2013, Santarém (Central), Elvas (Southeast) and Mirandela (Trás-os-Montes region, Northeast). Mature pollen of four different cultivars (Cobrançosa, Arbequina, Picual and Verdeal) was collected during the olive flowering season. Ole e 1 was quantified using specific 2-site antibody ELISA. Pollen of the olive groves at the boundary Olea bioclimatic distribution in the Mirandela registered the higher allergen content for all varieties in each study year. Arbequina was the variety that showed the lower Ole e 1 allergen concentration, whereas the higher content was registered for Cobrançosa. The main meteorological parameters that influenced the allergen Ole e 1 concentration in the pollen grains were the rainfall and temperatures related variables. The knowledge of the allergenicity in different olive cultivars is an important tool in the selection of the most adequate for planting as ornamental crop and to adjust the pollen extracts used for diagnosis or even immunotherapy.

     

Mapping of a thermo-sensitive earliness <Emphasis Type="Italic">per se</Emphasis> gene on <Emphasis Type="Italic">Triticum monococcum</Emphasis> chromosome 1A<Superscript>m</Superscript>

An earliness per se gene, designated Eps-A^m1, was mapped in diploid wheat in F₂ and single-seed descent mapping populations from the cross between cultivated (DV92) and wild (G3116) Triticum monococcum accessions. A QTL with a peak on RFLP loci Xcdo393 and Xwg241, the most distal markers on the long arm of chromosome 1A^m, explained 47% of the variation in heading date (LOD score 8.3). Progeny tests for the two F_2:3 families with critical recombination events between Xcdo393 and Xwg241 showed that the gene was distal to Xcdo393 and linked to Xwg241. Progeny tests and replicated experiments with line #3 suggested that Eps-A^m1 was distal to Xwg241. This gene showed a large effect on heading date in the controlled environment experiments, and a smaller, but significant, effect under natural conditions. Eps-A^m1 showed significant epistatic interactions with photoperiod and vernalization treatments, suggesting that the different classes of genes affecting heading date interact as part of a complex network that controls the timing of flowering induction. Besides its interactions with other genes affecting heading date, Eps-A^m1 showed a significant interaction with temperature. The effect of temperature was larger in plants carrying the DV92 allele for late flowering than in those carrying the G3116 allele for early flowering. Average differences in heading date between the experiments performed at 16 °C and 23 °C were approximately 11 days (P < 0.001) for the lines carrying the Eps-A^m1 allele for early flowering but approximately 50 days (P < 0.0001) for the lines carrying the allele for late flowering. The large differences in heading time (average 80 days) observed between plants carrying the G3116 and DV92 alleles when grown at 16 °C, suggest that it would be possible to produce very detailed maps for this gene to facilitate its future positional cloning.     

Cytochrome oxidase sensitivity to carbon monoxide in leaves of various tall and dwarf wheat cultivars and their crosses

In order to investigate the possible role of Rht genes in the regulation of the redox condition of cytochrome a₃ (cytochrome c oxidase) during steady-state respiration, wheat cultivars belonging to one of two groups - NP 710, NP 846 and NP 875 belonging to the tall group and Olesons dwarf, HD 1982 and HD 2122 of the dwarf group - and the reciprocal crosses between the varieties of these two groups were examined for carbon monoxide (CO) sensitivity in terms of the inhibition of mitochondrial electron transport. Leaves of young wheat seedlings were used. Differences in the redox state of cytochrome a₃ were monitored using the in vivo aerobic assay of nitrate reduction after a 1-min exposure to CO. Dwarf cultivars possessing Rht genes responded marginally (᜖%) to CO inhibition, whereas the response of tall cultivars to CO was higher (51-70%). Since CO forms a complex only with reduced cytochrome a₃, the results indicate differences in the redox state of cytochrome a₃ during in situ respiration of leaves from tall and dwarf plants that are likely to be controlled by cytoplasmic factors.     

Effect of Crossbreeding on the Chemical Composition and Biological Characteristics of Tunisian New Olive Progenies

Olive fruit characteristics (weight, pulp/stone ratio, and oil and moisture content) and the iodine value (IV) of 31 new olive progenies (Olea europaea L.) were determined. To evaluate the effect of the genetic variability on these parameters, the new olive progenies, obtained through cross‐pollination between Tunisian and Mediterranean olive cultivars, were planted in a selected grove guaranteeing the homogeneity of the pedologic and climatic conditions. A strong genetic effect and significant differences between genotypes were obtained for the IV and the fruit characteristics evaluated. Discriminant analysis was used to classify the new progenies as distinct from each other, based on their IV, and their pulp and stone weight. An almost full discrimination of the olives from different genotypes was only achieved when the fruit characteristics (pulp and stone weight) and the IV data were analyzed together.     

Intergeneric hybridization between Erucastrum canariense and Brassica rapa. Genetic relatedness between E(C) and A genomes

An intergeneric hybrid between a wild species, Erucastrum canariense (2n = 18; E^CE^C), and a cultivated oilseed brassica species, Brassica rapa (2n = 20; AA), was synthesized through ovary culture in White's basal medium supplemented with casein hydrolysate. Morphological, cytological and DNA-based analysis helped to establish the hybrid nature of the derived plants. Hybrid plants were morphologically intermediate between the two parents and were completely male, as well as female sterile. Cytological analysis revealed the occurrence of 19 I in about 38% of the PMCs investigated. However 1-8 bivalents/PMC were also observed, indicating a significant level of homology between the two genomes. Normal chromosome pairing and pollen fertility was restored following colchiploidy. The intergeneric amphiploid developed during the investigation can be used as a bridging species for the transfer of desirable genes from E^C to cultivated genomes (especially A and C), and for resistance to Alternaria blight and mustard aphid. Under field conditions, the E. canariense intergeneric hybrid and the amphiploid appeared to be moderately resistant to Alternaria blight and also harboured a significantly lower population of mustard aphid than the cultivated B. rapa.     

Mapping of DNA markers to arms and sub-arm regions of Nicotiana sylvestris chromosomes using aberrant alien addition lines

Seven monosomic addition plants, each containing the full complement of Nicotiana plumbaginifolia (2n = 20, genome constitution PP) and an aberrant chromosome of Nicotiana sylvestris (2n = 24, SS), were produced from backcrosses of hyperdiploid derivatives of the sesquidiploid hybrid PPS to N. plumbaginifolia. The N. sylvestris chromosomes in these plants were characterized by karyotype analysis, Southern hybridization with DNA markers previously localized on N. sylvestris chromosomes and a 269-bp fragment from the 3' end of 25S rDNA, and fluorescence in situ hybridization using 25S rDNA, 5S rDNA and telomere repeats (TTTAGGG)_n as probes. The N. sylvestris chromosomes in these plants were identified to be telocentrics 6S, 7S and 8S, and deletions 7S, 10, 12S and 12L, respectively. The successful identification of aberrant chromosomes in these lines enabled us to assign DNA markers to arms and sub-arm regions of N. sylvestris chromosomes. All aberrant chromosomes in the addition lines could be transmitted through mitosis and meiosis. The potential applications of the addition lines in high-resolution physical mapping, the isolation of N. sylvestris chromosomes by flow cytometry, and an understanding of the chromosomal distribution of 45S rDNA in N. sylvestris are discussed.     

14C transfer between the spring ephemeral Erythronium americanum and sugar maple saplings via arbuscular mycorrhizal fungi in natural stands

We investigated in the field the carbon (C) transfer between sugar maple (Acer saccharum) saplings and the spring ephemeral Erythronium americanum via the mycelium of arbuscular mycorrhizal (AM) fungi. Sugar maple saplings and E. americanum plants were planted together in pots placed in the ground of a maple forest in 1999. Ectomycorrhizal yellow birches (Betula alleghaniensis) were added as control plants. In spring 2000, during leaf expansion of sugar maple saplings, the leaves of E. americanum were labelled with ¹⁴CO₂. Seven days after labelling, radioactivity was detected in leaves, stem and roots of sugar maples. Specific radioactivity in sugar maples was 13-fold higher than in yellow birches revealing the occurrence of a direct transfer of ¹⁴C between the AM plants. The quantity of ¹⁴C transferred to sugar maple saplings was negatively correlated with the percentage of ¹⁴C allocated to the storage organ of E. americanum. A second labelling was performed in autumn 2000 on sugar maple leaves during annual growth of E. americanum roots. Radioactivity was detected in 7 of 22 E. americanum root systems and absent in yellow birches. These results suggest that AM fungi connecting different understorey species can act as reciprocal C transfer bridges between plant species in relation with the phenology of the plants involved.     

Optimization of in vitro micropropagation and regeneration for Populus × interamericana and Populus × euramericana hybrids (P. deltoides, P. trichocarpa, and P. nigra)

A rapid and efficient micropropagation method has been established for six European poplar cultivars of economic interest - four Populus 2 interamericana and two Populus 2 euramericana. Using a three-step procedure, we were able to regenerate plantlets from callus and acclimate them within 4 months. In the first step, callogenesis was induced when explants were cultured for 25 days on culture medium supplemented with 10 µM !-naphthaleneacetic acid and 5 µM N⁶(2-isopentenyl)adenine. Bud regeneration followed by shoot elongation was then obtained from callus tissue by combining the cytokinin-like compound thidiazuron with the surfactant Pluronic F-68 at concentrations adjusted for each cultivar. The usefulness of this procedure in the area of genetic engineering is discussed.     

Joint aerobic biodegradation of wastewater from table olive manufacturing industries and urban wastewater

Wastewater generated in the elaboration of table olives has been treated using activated sludge from a municipal wastewater plant after adequate acclimation. To avoid bactericide properties of some chemical structures present in this type of effluents, synthetic urban wastewater has been used to dilute the original wastewater. The main parameters affecting efficiency of biological processes have been studied. Thus, initial biomass concentration, temperature up to 303 K (upper working temperature limit = 313 K) and initial substrate concentration exerted a positive influence on COD degradation rate. The optimum pH was found to be around 7, experiencing a slight inhibition on cell activity at pH 4. Under the experimental conditions investigated other parameters like polyphenol content, absorbance at 254 nm and total organic carbon were also reduced to some extent. Only nitrates amount was increased after the biological process took place. A kinetic model based on Monod equation was proposed and applied to experimental results. The maximum specific growth rate was calculated by means of the aforementioned kinetic model. The value of this parameter as a function of temperature was fitted to an Arrhenius expression, w_max = 9.43 2 10¹⁰ exp(72021/RT) h^у (R in J mol^у K^у283 K < T < 303 K, pH , 7-10).