Molecular characterization of olive (Olea europaea L.) Sicilian cultivars using SSR markers

Olive (Olea europaea L.) is one of the economically most important fruit crops for the Mediterranean area, with production being mainly destined to oil extraction. In Sicily, olive has been cultivated since ancient times and its germplasm is characterized by a wide genetic diversity that could be related to its domestication and spread in ancient times, and to some reproductive biological peculiarities as self-incompatibility. This analysis was conducted on 65 genotypes with the purpose of characterizing a large collection of Sicilian accessions (47 genotypes) and to compare them with varieties coming from Southern Italy and from the most important countries of the Mediterranean basin. With this aim we used 8 simple sequence repeat (SSR) markers, which detected a total of 74 alleles and identified an average of 19.5 genotypes in the population investigated. A larger variability than expected was found in the analyzed genotypes, some synonymies already reported in literature were confirmed, but also some cultivars considered as identical were discriminated such as in the case of Castriciana, Ogliarola messinese and Passalunara. The whole study revealed a wide intraspecific variability within the gene pool examined, independently from the geographical origin.     

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.     

SNP-based markers for discriminating olive (Olea europaea L.) cultivars.

A set of 11 polymorphic markers (1 cleaved amplified polymorphic sequence (CAPS), 2 sequence-characterized amplified regions (SCARs), and 8 single-nucleotide polymorphism (SNP)-derived markers) was obtained for olive cultivar identification by comparing DNA sequences from different accessions. Marker development was more efficient, using sequences from the database rather than cloning arbitrary DNA fragments. Analyses of the sequences of 3 genes from 11 diverse cultivars revealed an SNP frequency of 1 per 190 base pairs in exons and 1 per 149 base pairs in introns. Most mutations were silent or had little perceptible effect on the polypeptide encoded. The higher incidence of transversions (55%) suggests that methylation is not the major driving force for DNA base changes. Evidence of linkage disequilibrium in 2 pairs of markers has been detected. The set of predominantly SNP-based markers was used to genotype 65 olive samples obtained from Europe and Australia, and was able clearly to discriminate 77% of the cultivars. Samples, putatively of the same cultivar but derived from different sources, were revealed as identical, demonstrating the utility of these markers as tools for resolving nomenclature issues. Genotyping data were used for constructing a dendrogram by UPGMA cluster analysis using the simple matching similarity coefficient. Relationships between cultivars are discussed in relation to the route of olive's spread.     

Phenological growth stages of olive trees (Olea europaea)

This paper describes the phenological growth stages of olive trees using the BBCH (Biologische Bundesanstalt, Bundessortenamt, Chemische Industrie) scale. Eight principal growth stages for bud, leaf and shoot development, inflorescence emergence, flowering, fruit development, fruit maturity and senescence and 32 secondary growth stages are described. Advantages of the BBCH scale over other methods are discussed.     

A molecular linkage map of olive (Olea europaea L) based on RAPD, microsatellite, and SCAR markers.

An integrated molecular linkage map of olive (Olea europaea L.) was constructed based on randomly amplified polymorphic DNA (RAPD), sequence characterized amplified region (SCAR), and microsatellite markers using the pseudo-testcross strategy. A mapping population of 104 individuals was generated from an F1 full-sib family of a cross between 'Frantoio' and 'Kalamata'. The hybridity of the mapping population was confirmed by genetic similarity and nonmetric multidimensional scaling. Twenty-three linkage groups were mapped for 'Kalamata', covering 759 cM of the genome with 89 loci and an average distance between loci of 11.5 cM. Twenty-seven linkage groups were mapped for 'Frantoio', covering 798 cM of the genome with 92 loci and an average distance between loci of 12.3 cM. For the integrated map, 15 linkage groups covered 879 cM of the genome with 101 loci and an average distance between loci of 10.2 cM. The size of the genomic DNA was estimated to be around 3000 cM. A sequence characterized amplified region marker linked to olive peacock disease resistance was mapped to linkage group 2 of the integrated map. These maps will be the starting point for studies on the structure, evolution, and function of the olive genome. When the mapping progeny pass through their juvenile phase and assume their adult characters, mapping morphological markers and identification of quantitative trait loci for adaptive traits will be the primary targets.     

Lipoxygenase pathway in olive callus cultures (Olea europaea)

Stimulation of the lipoxygenase pathway in olive fruit initiates a cascade of reactions that begins with the regio- and stereospecific di-oxygenation of polyunsaturated fatty acids containing a cis, cis-1,4 pentadiene moiety. Later products of the pathway include volatiles that influence the organoleptic properties of harvested olive oil. In this study, we have investigated lipoxygenase activity in olive callus cultures, and found that there is evidence of several isoforms of the enzyme with different pH optima and substrate specificities. Endogenous lipoxygenase activity was detected throughout the growth cycle of olive callus, particularly during the log phase of growth, suggesting that olive lipoxygenases are intimately involved in growth. The most prominent lipoxygenase activity in tissue cultures was found to be soluble but significant activities were detected in the plastid fraction. In addition, hydroperoxide lyase (HPL) activity was measured in the calli; both 13- and 9-HPL activities were found which were particulate.     

Morphology and discrimination features of pollen from Italian olive cultivars (Olea europaea L.)

Pollen morphology of 14 cultivars of Olea europaea subsp. europaea var. europaea was analysed in order to discriminate main pollen types. The cultivars were selected from the most spread and early flowering crops grown in Italy. Morphometric parameters were observed on acetolysed pollen by means of light microscopy and scanning electron microscopy. Polar axis (P), equatorial diameter (E), P/E ratio, maximum distance between colpi in mesocolpium, distance between the apices of two colpi, exine thickness, maximum length of lumina in mesocolpium and in apocolpium, and exine reticulum thickness in mesocolpium have been measured. According to P and E, the 14 olive cultivars of this study can be divided into the three groups of small (P: 21.75 µm, E: 22.55 µm; ‘Manna’ and ‘Tonda di Cagliari’), large (P: 25.1 µm, E: 26.1 µm; ‘Pescarese’ and ‘Rotondella di Sanza’) and medium size (P: 23.49 µm, E: 24.54 µm, ‘Carolea’, ‘Grossa di Cassano’, ‘Giarraffa’, ‘Nocellara messinese’, ‘Nocellara del Belice’, ‘Santagatese’, ‘Intosso’, ‘Maiatica di Ferrandina’, ‘Nostrale di Fiano Romano’, ‘Santa Caterina’). Maximum length of lumina and exine thickness are useful parameters for further distinction of olive pollen groups, since these parameters are able to provide a specific pollen profile for each cultivar.     

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     

Polyploidy in the olive complex (Olea europaea): evidence from flow cytometry and nuclear microsatellite analyses

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.     

Multiple origins for Mediterranean olive (Olea europaea L. ssp. europaea) based upon mitochondrial DNA polymorphisms

A study of nuclear and cytoplasmic genetic diversity of cultivated olive, oleaster and other taxa belonging to the complex O. europaea was performed. Nuclear DNA polymorphism (RAPDs) in oleaster displays a gradient between the east and west of the Mediterranean Basin. In cultivars, the gradient is less visible owing to their diffusion and selection. Furthermore, three mitotypes (ME1, MOM and MCK) were detected in both cultivated olive and oleaster. A fourth mitotype, ME2, was unique to some cultivars. The preponderant mitotype, ME1, marks the Near Eastern origin of olive in oleaster. In the west of the Mediterranean, another mitotype, MOM, was found in most oleaster. and a few cultivars. The third, MCK, was found in a few oleaster from the west and in cultivars originating in Kabylie and Languedoc. We argue that MCK marks an ancestral Mediterranean population. The mitotypes mark independent cultivated olive origins which were not detected with DNA nuclear diversity.     

Forecasting olive (Olea europaea) crop production by monitoring airborne pollen

Forecasting harvests of olives destined for the production of olive oil can be based on counts of airborne olive pollen, and meteorological and agronomic observations. This study was carried out during six consecutive years (1990–1995) in the Campiña Alta (an olive-producing region in the province of Córdoba, south-west Spain). Olive pollen totals are the annual sum of the concentrations recorded for the periods that the filters of a Cour trap were exposed. The meteorological data are the values of accumulated rainfall between 1 September and the following 15 April (a date prior to the beginning of olive flowering). The agronomic data are the forecast and actual productions for the province of Córdoba, supplied by the Board of Agriculture of the Andalusian government, and the actual production of the Campiña Alta, supplied at the end of harvest by private olive-growing co-operatives. The data were combined, and four mathematical equations were obtained to forecast the crop 6 months in advance, with varying degrees of reliability. The reliability was very high for an appropriate agricultural area. The most accurate equation isY=?1.90×10⁴+2.35X+53.94 (which forecasts the production of the Campiña Alta), whereY is the olive production (MT),X the olive pollen count,Z the rainfall prior to flowering, anda, b andc are constants. The least accurate equation is that relating olive pollen concentrations with olive production in the province of Córdoba.     

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.     

Investigations of genetic variation between olive (Olea europaea L.) cultivars using arbitrarily primed polymerase chain reaction (AP-PCR)

Characterization and selection of olive clones for the production of olive oil is essential in Turkey because of its profitable exploitation. AP-PCR (Arbitrarily-Primed PCR) is a technique that can distinguish the genetic relationship among plant species and other organisms. In this study, AP-PCR approach was used in order to determine the genetic relationship of different six olive clones. The purity of DNA is one of the most important factors affecting the product of the AP-PCR method. In this respect, modified genomic DNA isolation procedure from Oleae europaea clones was developed so that this procedure can be used to obtain plant genomic DNA from diverse aromatic plants, which produce essential oils and secondary metabolites. By following the optimized AP-PCR amplification protocol, unique DNA fingerprint profiles for each olive clone were produced. AP-PCR-generated unique DNA fingerprint profiles can be used in the identification, distribution and diversity of various olive cultivars.     

The age of monumental olive trees (Olea europaea) in northeastern Spain

Trees can reach ages that in some cases amount to thousands of years. In the Mediterranean region, olive trees (Olea europaea) have traditionally been considered a particularly long-lived species. The main objective of this study was to assess the age of large olive trees considered to be millenarian and classified as monumental trees in northeastern Spain. We extracted cores of 14 individuals and obtained 8 sections of trees which had already been cut in the area where the largest olive trees in the northeastern Iberian Peninsula are found. The age of the sampled olive trees was assessed by counting the number of annual growth rings. Tree rings did not cross-date well, neither within nor between individuals, but boundaries between likely annual rings were clearly distinct. We found a linear relationship between DBH and tree age (in years) (Age = 2.11 × diameter(cm) + 88.93, R² = 0.80), which was used to estimate the age of unsampled olive trees. The maximum estimated age (627 ± 110 years) is among the greatest ages reported for olive trees around the world (700 years) and among the oldest trees in Mediterranean ecosystems.     

Low soil temperatures induce water deficits in olive (Olea europaea) trees

Olive trees are often subjected to low temperatures during winter. To quantify the effects of low temperatures on the water relations of olive trees, we studied the responses to low soil temperatures on winter days of variable evaporative demand (ET₀) in 1-year-old potted olive (Oleo europaea L. cv. Picual) trees in 1996 and 1997. Low night (2.5 and 5.2°C) but ambient day soil temperatures (above 10°C) did not affect stomatal conductance (g_s), leaf (Ψ_leaf) and stem (Ψ_stem) water potentials. Soil temperature levels inducing water stress in olive trees were determined for winter days with ET₀ typical for southern Spain (ET₀= 1.5 ± 0.3 mm day^?1). Leaf and stem water potential decreased and root hydraulic resistance (r_root) increased when trees were exposed to night and day soil temperatures below 10°C. Stomatal conductance was not affected at soil temperatures between 6.4 and 10°C, but decreased at temperatures below 6.4°C. The soil temperature levels affecting the water uptake of olive trees remained relatively constant over the range of ET₀ of 1-2 mm day^?1 during winter and early spring months. However, the soil temperature influencing gs appeared to be more variable and was affected by ET₀. Olive tree recovery from low soil temperature stress depended on stress duration and severity and interacted with ET₀. Recovery of ψ started already during the stress period, probably induced by stomatal closure and high r_root, thus allowing tree rehydration overnight. Root hydraulic resistance contributed the major part of whole-tree hydraulic resistance in response to cold stress, accounting for 76 and 89% at 6.4 and 4.6°C, respectively; which indicates that r_root is the primary control of the water status in olive trees under low temperatures.     

Effects of drought stress on phenolic accumulation in greenhouse-grown olive trees (Olea europaea)

The present study evaluates the effects of severe drought stress on the content of phenolic compounds in olive leaves, namely hydroxytyrosol, tyrosol, p-hydroxybenzoic acid, catechin, luteolin 7-O-rutinoside, luteolin 7-O-glucoside, apigenin 7-O-glucoside, quercetin, apigenin, pinoresinol, oleuropein and verbascoside in greenhouse-grown plantlets. The results showed that oleuropein, verbascoside, luteolin 7-O-glucoside and apigenin 7-O-glucoside were the most important phenolic compound of stressed olive plants and can represent up to 84% of the total amount of the identified phenolic compounds. Application of drought stress caused a significant increase in the level of oleuropein (87%), verbascoside (78%), luteolin 7-O-glucoside (72%) and apigenin 7-O-glucoside (85%), when compared to the control. The elevated values of these phenolic compounds can help controlling the water status of olive plants and avoiding serious oxidative damage induced by water deficit stress. To our knowledge, this is the first report to show the boost in the concentrations of verbascoside, luteolin 7-O-glucoside and apigenin 7-O-glucoside in the leaves of olive trees after water deficit stress.     

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     

LTR retrotransposon dynamics in the evolution of the olive (Olea europaea) genome

Improved knowledge of genome composition, especially of its repetitive component, generates important information for both theoretical and applied research. The olive repetitive component is made up of two main classes of sequences: tandem repeats and retrotransposons (REs). In this study, we provide characterization of a sample of 254 unique full-length long terminal repeat (LTR) REs. In the sample, Ty1-Copia elements were more numerous than Ty3-Gypsy elements. Mapping a large set of Illumina whole-genome shotgun reads onto the identified retroelement set revealed that Gypsy elements are more redundant than Copia elements. The insertion time of intact retroelements was estimated based on sister LTR’s divergence. Although some elements inserted relatively recently, the mean insertion age of the isolated retroelements is around 18 million yrs. Gypsy and Copia retroelements showed different waves of transposition, with Gypsy elements especially active between 10 and 25 million yrs ago and nearly inactive in the last 7 million yrs. The occurrence of numerous solo-LTRs related to isolated full-length retroelements was ascertained for two Gypsy elements and one Copia element. Overall, the results reported in this study show that RE activity (both retrotransposition and DNA loss) has impacted the olive genome structure in more ancient times than in other angiosperms.     

Ethylene evolution from various developing organs of olive (Olea europaea) after excision

Ethylene evolution from leaves, stems, inflorescences and fruits of the olive plant ( Olea europaea L.) cv. Manzanillo was studied at various stages of their development. Mature non-growing organs, particularly leaves, have a constant, low, and uniform rate of ethylene evolution. Ethylene evolution from detached mature olive leaves was constant during the first 12 h after excision. Leaves on shoots maintained in vitro kept a constant rate of ethylene evolution for at least the first 5–6 days. Leaf injury significantly increased ethylene evolution. Ethylene evolution from injured and non-injured control leaves could be markedly inhibited aminoethoxyvinylglycine (AVG) applied to the leaves or fed to the shoot. The use of excised olive shoots and leaves as an in vitro model system for studies of induced metabolic processes such as abscission and developing water stress was suggested.