Computational annotation of genes differentially expressed along olive fruit development

Background  

Olea europaea L. is a traditional tree crop of the Mediterranean basin with a worldwide economical high impact. Differently from other fruit tree species, little is known about the physiological and molecular basis of the olive fruit development and a few sequences of genes and gene products are available for olive in public databases. This study deals with the identification of large sets of differentially expressed genes in developing olive fruits and the subsequent computational annotation by means of different software.     

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.     

Assessment of genetic stability of two micropropagated wild olive species using flow cytometry and microsatellite markers

Micropropagated plants from two wild-olive species, Olea maderensis and O. europaea ssp. europaea var. sylvestris were screened for genetic stability. O. maderensis shoots were elongated/multiplied on OMG medium with zeatin (9.12 μM), and rooted on 1/2 OMG with NAA (3.22 μM). O. europaea var. sylvestris shoots were elongated/multiplied on OM medium with zeatin, and rooting was optimal after a hormonal shock (IBA 100 μM) followed by transfer to the same medium without growth regulators. In both species, acclimatization was successful and plants looked normal and morphologically identical to the donor field trees. Genetic variability was assessed at several stages of the micropropagation process using flow cytometry (FCM) and nuclear microsatellites (SSR). No changes in ploidy level were found among micropropagated plants, though small deviations, putatively due to the negative effects of cytosolic compounds on propidium iodide staining, between these and field plants were observed. In SSRs analyses, ten SSR markers were able to distinguish between genotypes. No mutations were found in these tested SSR loci among the donor tree and micropropagated plants, suggesting, for the tested markers, genetic uniformity throughout the process. The FCM and SSR results obtained do not exclude the occurrence of other changes in the nuclear genome but, considering the morphological stability of micropropagated plants, indicate that both protocols are suitable and efficient for large scale, true-to-type micropropagation of these two wild olive species.     

Dynamic analysis of olive trees in intensive orchards under forced vibration

The mechanical harvesting of fruit for oil production in an intensive olive tree orchard is generally accomplished by applying vibration to the tree’s trunk. This vibration is consequently transmitted to the branches, causing the fruit to detach. Although this practice is commonly used, the effects on tree behavior under forced vibration are not firmly established. Dynamic analysis was performed on 17 olive trees (Olea europaea L.) growing in an intensively-managed orchard using modal testing techniques. Modal parameter identification was focused inside the range excitation frequency used by the most commonly available trunk shakers on the market. The olive trees featuring a low morphological variability and modal parameters were obtained for a representative olive tree. The first two modes of vibration of the main tree frame were identified with damping ratios of 26.9 and 17.1% and natural frequencies of 20.2 and 37.7 Hz, respectively. A third mode of vibration of less importance was found at a higher frequency. Therefore, many local modes of vibration were detected near these natural frequencies, primarily located on secondary branches. During the testing, the olive trees behaved like a damped harmonic oscillator with predominantly mass damping in these modes.     

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.     

Bacterial and β-proteobacterial diversity in Olea europaea var. mastoidis- and O. europaea var. koroneiki-generated olive mill wastewaters: influence of cultivation and harvesting practice on bacterial community structure

Despite the fact that several approaches have been applied for the bioremediation of olive mill wastewaters, little information is available on bacteria inhabiting these agro-industrial effluents. In the present study, 16S rRNA gene clone libraries were constructed to identify bacterial diversity in olive-oil mill wastewaters generated by two olive varieties, Olea europaea var. mastoidis and O. europaea var. koroneiki. Due to chloroplast excess in wastewater produced from the processing of O. europaea var. koroneiki, a clone library using specific PCR primers for β-Proteobacteria was further constructed. The bacterial diversity in O. europaea var. mastoidis-generated olive mill wastewaters consisted mainly of members of Acetobacteriaceae, Prevotellaceae and Lactobacillaceae, while the majority of β-proteobacteria identified in O. europaea var. koroneiki-generated olive mill wastewaters were placed within the families Comamonadaceae, Oxalobacteraceae, Hydrogenophilaceae and Rhodocyclaceae. At least 17 novel phylogenetic linkages among Bacteria were identified. Olive-oil mill wastewaters microbiota appears to have originated from soil and freshwater environments, while the cultivation and harvesting practice highly influenced the bacterial community structure in olive mill wastewaters. The presence of fecal bacteria in O. europaea var. mastoidis-generated olive mill wastewaters, due to the long harvesting period, should be of concern.     

Identification of new polymorphic regions and differentiation of cultivated olives (<Emphasis Type="Italic">Olea europaea</Emphasis> L.) through plastome sequence comparison

Background  

The cultivated olive (Olea europaea L.) is the most agriculturally important species of the Oleaceae family. Although many studies have been performed on plastid polymorphisms to evaluate taxonomy, phylogeny and phylogeography of Olea subspecies, only few polymorphic regions discriminating among the agronomically and economically important olive cultivars have been identified. The objective of this study was to sequence the entire plastome of olive and analyze many potential polymorphic regions to develop new inter-cultivar genetic markers.     

Genetic variability analyses of the somatic embryogenesis induction process in Olea spp. using nuclear microsatellites

The crop species Olea europaea L. (olive tree) is of great economic importance in the Mediterranean region. Hence, many efforts have been done in the last decades to propagate this commercially valuable species by in vitro methods. On the other hand, the lesser known Olea maderensis (Lowe) Rivas Mart. & Del Arco which is a native species of the Madeira Archipelago has only been the subject of micropropagation from nodal stem cuttings. Therefore, in this work we analysed the stability of ten nuclear simple sequence repeat (SSR) markers at successive stages of the somatic embryogenesis process in two adult trees belonging to these two species from the Madeira Archipelago. For the induction of somatic embryogenesis, petiole and leaf explants were cultivated on solid Murashige and Skoog medium (MS) with 12.25 μM indole-3-butyric acid (IBA) and 4.56 μM of zeatin, in the dark. After 3 months, different callus tissues (non-embryogenic, pre-embryogenic and embryogenic) developed from leaf explants and petioles were later transferred to MS medium without growth regulators in the dark. All ten SSR markers were able to distinguish between species. However, no mutations were found at the SSR loci at any of the successive developmental stages from PEMs (pre-embryogenic masses) to somatic embryos. This genetic uniformity was observed within material derived from each genotype/species and its respective donor plant. Therefore, we conclude that the genomic integrities of both O. europaea and O. maderensis were maintained throughout the stages of the embryogenic processes in study suggesting the absence of somaclonal variation.     

UHPLC-DAD-FLD and UHPLC-HRMS/MS based metabolic profiling and characterization of different Olea europaea organs of Koroneiki and Chetoui varieties

The olive tree (Olea europaea L., Oleaceae) is one of the most important fruit trees in Mediterranean basin and has been associated with numerous biological assets. These effects have been mainly attributed to certain phenolic compounds found in fruits, olive oil and by-products of olive oil production. However, other Olea organs such as stems, roots and drupe stones have received little attention leading to limited knowledge about their phytochemical content. Thus, the main goal of the current study was the investigation of the chemical composition of diverse organs from two O. europaea varieties (i.e. Koroneiki and Chetoui) using combinations of modern analytical techniques. A fast UHPLC-DAD-FLD method was developed and applied for the profiling of different extracts of O. europaea organs as well as for the quantification of oleuropein. In addition, a dereplication strategy was developed using an Orbitrap platform (UHPLC-ESI-HRMS/MS) aiming to further characterization of the contained secondary metabolites. In total, 86 molecules were identified including compounds described for the first time in O. europaea such as coumarins. Some compounds were found to be organ specific such as nuzhenide derivatives in stone, flavonoids in leaves and oleuropein which was mainly found in Olea roots, in both varieties. Overall, it is noticeable that except olive oil, diverse organs of olive tree might comprise an alternative and valuable source of biologically active compounds.     

Scanning Electron Microscopy Analysis of Exine Patterns in Cultivars of Olive (Olea europaea L.)

Scanning electron microscope analysis of exine patterns in thepollen of nine cultivars of the olive tree (Olea europaea L.)has revealed specific differences between them. The differencesinclude variation in size, form and profile of the meshes andin the thickness of the muri. Every cultivar possesses its ownmorphological characteristics which are constant and are foundin successive years. Olea europaea L, olive, pollen, exine patterns     

Extensive gene flow blurs phylogeographic but not phylogenetic signal in Olea europaea L.

Genetic structure and evolutionary patterns of the wild olive tree (Olea europaea L.) were investigated with AFLP fingerprinting data at three geographic levels: (a) phylogenetic relationships of the six currently recognized subspecies in Eurasia and Africa; (b) lineage identification in subsp. europaea of the Mediterranean basin; and (c) phylogeography in the western Mediterranean. Two statistical approaches (Bayesian inference and analysis of molecular variance) were used to analyse the AFLP fingerprints. To determine the congruency and transferability of results across studies previous RAPD and ISSR data were analysed in a similar manner. Comparisons proved that qualitative results were mostly congruent but quantitative values differed, depending on the method of analysis. Neighbour-Joining analysis of AFLP phenotypes supported current classification of subspecies. At a Mediterranean scale no clear cut phylogeographic pattern was recovered, likely due to extensive gene flow between populations of subsp. europaea. Gene flow estimates calculated with conventional F-statistics showed that reproductive barriers separated neither populations nor lineages of O. europaea. Genetic divergence between eastern and western parts of the Mediterranean basin was observed only when geographical and population information were incorporated into the analyses through hierarchical analysis of molecular variance (AMOVA). Within the western Mediterranean, the highest genetic diversity was found in two regions: on both sides of the Strait of Gibraltar and in the Balearic archipelago. Additionally, long-lasting isolation of the northern-most populations of the Iberian Peninsula appeared to be responsible for a significant divergence.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Cellular localization of ROS and NO in olive reproductive tissues during flower development

Background  

Recent studies have shown that reactive oxygen species (ROS) and nitric oxide (NO) are involved in the signalling processes taking place during the interactions pollen-pistil in several plants. The olive tree (Olea europaea L.) is an important crop in Mediterranean countries. It is a dicotyledonous species, with a certain level of self-incompatibility, fertilisation preferentially allogamous, and with an incompatibility system of the gametophytic type not well determined yet. The purpose of the present study was to determine whether relevant ROS and NO are present in the stigmatic surface and other reproductive tissues in the olive over different key developmental stages of the reproductive process. This is a first approach to find out the putative function of these signalling molecules in the regulation of the interaction pollen-stigma.     

Monitoring of cultivar identity in micropropagated olive plants using RAPD and ISR markers

Randomly amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) markers were applied to assess the genetic stability of micropropagated olive (Olea europaea L. cv. Maurino) plants regenerated by axillary buds. Initial olive explants, isolated from one donor tree, were multiplied on Murashige and Skoog medium for 12 repeated subcultures. A total of 40 RAPD and 10 ISSR markers resulted in 301 distinct and reproducible band classes showing homogeneous RAPD and ISSR patterns. The amplification products revealed genetic stability among the micropropagated plants and between them and the donor plant. The results demonstrate the genetic stability of nine year old mature micropropagated olive plants cultured in field, and corroborated the fact that axillary multiplication is the safest mode for multiplication of true to type plants.     

Olive cultivar origin is a major cause of polymorphism for Ole e 1 pollen allergen

Background  

Pollens from different olive (Olea europaea L.) cultivars have been shown to differ significantly in their content in Ole e 1 and in their overall allergenicity. This allergen is, in addition, characterized by a high degree of polymorphism in its sequence. The purpose of this study is to evaluate the putative presence of divergences in Ole e 1 sequences from different olive cultivars.     

Coexistent Mediterranean woody species as a driving factor of Phytophthora cinnamomi infectivity and survival

The long-term conservation of Mediterranean mixed oak forests is seriously threatened by the massive mortality of Quercus suber caused by the exotic pathogen Phytophthora cinnamomi. This species frequently grows in mixed forests under natural conditions, but nothing is known about how its level of disease might be altered by the diversity and identity of coexisting neighbours varying in susceptibility to the exotic pathogen. Here we analysed the individual and combined effects of Q. suber and the main coexisting tree species (Quercus canariensis and Olea europaea subsp. europaea var. sylvestris) in mixed forests of southern Spain on the production of infective and survival spores of P. cinnamomi. Through in vitro experiments, it was demonstrated that mixtures of Q. suber and Q. canariensis highly stimulated the production of P. cinnamomi zoospores in comparison with both species in monocultures. Olea europaea did not stimulate zoospore production. Under controlled conditions, the initial and final densities of inoculum in soil planted with monocultures of O. europaea and Q. canariensis did not differ. However, inoculum densities significantly decreased along the experiment in Q. suber mixtures with O. europaea and Q. canariensis. Phytophthora cinnamomi was able to infect and cause root rot symptoms on all tree species, including O. europaea var. sylvestris. We concluded that mixed stands of Q. suber and Q. canariensis are able to stimulate P. cinnamomi infectivity and survival much more than monospecific stands, and consequently under favourable conditions for root disease development, the coexistence of Q. suber and Q. canariensis might exacerbate Mediterranean forests decline. This study also constitutes the first report of O. europaea var. sylvestris as host and inductor of P. cinnamomi sporulation under controlled conditions.     

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.     

Substantial genetic diversity in cultivated Moroccan olive despite a single major cultivar: a paradoxical situation evidenced by the use of SSR loci

To assess the genetic diversity in Moroccan cultivated olive, Olea europaea L. subsp. europaea, we performed molecular analysis of olive trees sampled in four geographic zones representing all areas of traditional olive culture. The analysis of 215 trees using 15 simple sequence repeat (SSR) loci revealed 105 alleles distributed among 60 SSR profiles. The analysis of chloroplast deoxyribonucleic acid polymorphism for these 60 olive genotypes allowed to identify four chlorotypes: 42 CE1, one CE2, nine COM1 and eight CCK. Among the 60 SSR profiles, 52 corresponded to cultivated olive trees for which neither denomination nor characterisation is available. These local olive genotypes displayed a spatial genetic structuring over the four Moroccan geographic zones (northwest, north centre, Atlas and southwest), as pairwise Fst values ranged from 0.0394 to 0.1383 and varied according to geographic distance. As single alleles detected in local olive were also observed in Moroccan oleaster populations, results suggest that plant material was mainly selected from indigenous populations. The assumption that Picholine marocaine cultivar is a multi-clonal cultivar was not supported by our data because we found a single genotype for 112 olive trees representing 31 to 93% of the olives sampled locally in the 14 different areas. Picholine marocaine and the few other named cultivars do not seem to belong to the same gene pools as the unnamed genotypes cultivated only locally. The situation is paradoxical: a substantial genetic diversity in Moroccan olive germplasm, probably resulting from much local domestication, but a single cultivar is predominant.