Innocenzo Muzzalupo

A consensus list of microsatellite markers for olive genotyping

Cultivar identification is a primary concern for olive growers, breeders, and scientists. This study was aimed at examining the SSR markers retrieved from the literature and currently used in olive study, in order to select those most effective in characterizing the olive accessions and to make possible the comparison of data obtained by different laboratories. Olive microsatellite profiles were assessed by four independent laboratories, which analyzed 37 pre-selected SSR loci on a set of 21 cultivars. These SSR markers were initially tested for their reproducibility, power of discrimination and number of amplified loci/alleles. Independent segregation was tested for each pair of SSRs in a controlled cross and the allelic error rate was quantified. Some of them were finally selected as the most informative and reliable. Most of the alleles were sequenced and their sizes were determined. Profiles of the reference cultivars and a list of alleles with their sizes obtained by sequencing are reported. Several genetic parameters have been analysed on a larger set of cultivars allowing for a deeper characterization of the selected loci. Results of this study provide a list of recommended markers and protocols for olive genotyping as well as the allelic profile of a set of reference cultivars that would be useful for the establishment of a universal database of olive accessions.

ANALYSIS BY HPLC-MS/MS OF BIOPHENOLIC COMPONENTS IN OLIVES AND OILS

The aim of this work was to analyze by HPLC-MS-MS natural biophenolic compounds in complex natural samples like olives and olive oils. Eleven benzoic and cinnamic acids, vanillin and other eight biophenolic compounds were studied by liquid chromatography-atmospheric pressure ionization mass spectrometry/mass spectrometry with a Turbo Ion source in the negative mode. To confirm the specific presence, with great sensitivity, of such compounds in olive samples, in brine samples and in extravirgin olive oil the fragmentation of precursor ions into the product species acquisition was used in Multiple Reaction Monitoring (MRM) mode.

Microsatellite markers for identification of a group of italian olive accessions

Cultivar characterization for fruit trees certification requires fast, efficient and reliable techniques. Microsatellite markers (SSR) were used in the molecular characterization of 23 genotypes of Olea europaea subsp europaea. The DNA from the olive cultivars was analyzed using nine pre-selected SSR primers (GAPU59, GAPU71A, GAPU71B, GAPU103A, UDO99-01, UDO99-12, UDO99-28 and UDO99-39) and revealed 29 alleles, which allowed each genotype to be identified. In the dendrogram, the nine primers allowed the 23 olive genotypes to be grouped into subgroups corresponding to the same cultivar denominations. SSR markers proved to be efficient and reliable for the molecular characterization of Italian olive cultivars.

Transcript Levels of CHL P Gene, Antioxidants and Chlorophylls Contents in Olive (Olea europaea L.) Pericarps: A Comparative Study on Eleven Olive Cultivars Harvested in Two Ripening Stages

The effects of ripening stage on the antioxidant content in olive pericarps were evaluated in eleven olive genotypes grown in the same bioagronomic conditions in Southern Italy. We examined the transcript levels of geranylgeranyl reductase (CHL P) gene and the content of tocopherols, phenolic compounds and chlorophylls in the pericarps. The examined genotypes showed an increase of CHL P transcripts during pericarps ripening. Significant differences were reported in the antioxidant proportions in the same cultivars at different pericarp ripening stage. We show an inverse correlation between phenols and tocopherols content. In particular, during the ripening phase, tocopherols increased rapidly in olive pericarps while phenolic compounds and chlorophyll levels declined significantly. The significant amounts of these antioxidants confirm the nutritional and medicinal value of olive drupes and its products (table olives and olive oil). We suggest, for the first time, a link between CHL P transcript levels and tocopherols content during the ripening of olive pericarps. Besides, we revealed that this trend of CHL P transcript levels during pericarps ripening is independent from the olive genotypes.

Molecular Characterization of Italian Olive Cultivars by Microsatellite Markers.

Olive (Olea europaea L.) is a very important oil-producing crop in the Mediterranean area. Despite the long cultivation history and the great social-economic interest of the olive tree, its germplasm is today poorly characterized on the whole. Indeed, elaiographical and biometrical studies are surprisingly insufficient to well address its management and preservation. In addition, reliable molecular standardized methods in order to elucidate the potential occurrence of homonyms (one denomination for several genotypes) or synonyms (one genotype with several denominations) are needed to eliminate ambiguities in variety identification. Genetic diversity studies using microsatellite analysis were carried out in a set of 100 Italian olive (Olea europaea L.) cultivars (14 cvs from Abruzzo region, 9 cvs from Basilicata, 7 cvs from Calabria, 2 cvs from Campania, 4 cvs from Lazio, 1 cv from Liguria, 12 cvs from Molise, 12 cvs from Apulia, 29 cvs from Sicilia and 10 cvs from Toscana region). Samples of olive leaves were harvested from plants growing in the olive germplasm collection of the Consiglio per la Ricerca e Sperimentazione in Agricoltura (C.R.A.) – Istituto Sperimentale per l’Olivicoltura (ISOl) of Rende (CS), Italy. All microsatellites were polymorphic. A total of 52 alleles over nine loci were observed, ranging from 2.0 at UDO01 to 11.0 alleles at UDO39, with a mean of 5.8 alleles per locus. The expected heterozygosity ranged from 0.39 at UDO01 to 0.79 at UDO39 and the discrimination power varied from 0.40 at UDO01 to 0.89 GAPU103A. The results of alleles identification were then used to create a qualitative data matrix of presence (1) and absence (0) that was processed using NTSYS-PC software. Pairwise similarities between cultivars were calculated using Dice coefficient for qualitative data. The resulting similarity matrix was used to construct a dendrogram by means of the UPGMA (unweighted pairgroup method with arithmetical averages) algorithm. The following three presumable synonyms were detected: 1‘Filare’ and ‘Grossa di Venafro’, 2‘Giarraffa’ and ‘Pizzo di Corvo’ 3‘Frantoio’ and ‘Ogliarola barese’. Therefore, synonyms characterisation is very important to avoid genotype redundancy in order to maximise genetic diversity in Italian olive germplasm collection. This study showed that the use of molecular markers like SSRs is very useful to build a data base available for variety analysis and for olive germplasm collection management.

Direct DNA amplification from virgin olive oil for traceability and authenticity

The olive tree is one of the most common fruit species cultivated for oil and table olives in Italy and, particularly, in the Mediterranean area. DNA fingerprinting methods include different markers; however, our work is based on the identification of olive oil cultivars by using simple sequence repeats analysis. As previously reported in the literature, this proposed method shows good capability to amplify, for example, DNA from wine and table grape varieties. In our paper, we suggest an easy methodology, which allows direct amplification of DNA and bypassing of the extraction of DNA using an engineered DNA polymerase, KAPA3G Plant DNA polymerase, improved to tolerate plant PCR inhibitors. This new procedure is more efficient, faster and cheaper than traditional methods of DNA extraction and amplification and leads to more accurate results. This innovative protocol, without the addition of chemical solutions, has provided good results and has permitted traceability of virgin olive oils.

Chemical and Sensory Analysis of Some Egyptian Virgin Olive Oils

In the paper here presented, virgin olive oils produced in the year crop 2010/2011 in two different areas of Egypt, Siwa oasis and Giza, were characterised by their chemical-physical parameters. Also, the analysis of volatile compounds by SPME/GC/MS was carried out and the results compared with those provided by the panel test for the same samples. SPME/GC/MS analysis revealed that most of the volatile compounds determined in the virgin olive oil samples under investigation, contribute to characterise the sensory notes related to the rancid perceptions, oily and fatty persistency justifying and hence the negative attributes noticed by the sensory evaluation. Sensory and SPME/GC/MS analysis of olive oils samples here investigated reveal that most of the volatile compounds are characterised by sensory notes related to the rancid perceptions.

Deposition on mica and scanning force microscopy imaging of DNA molecules whose original B structure is retained

The supercoiling of topologically constrained DNA molecules is altered by modifications of their original B structure. Contact mode, scanning force microscope (SFM) imaging requires a fairly tight binding of the DNA molecules to a flat surface, generally mica, and is often accomplished using divalent or trivalent cations and dehydrating the sample thoroughly immediately upon deposition, following procedures known to cause at least a partial B‐DNA to A‐DNA transition. We report a modification of the DNA deposition method presented by Koller et al. [Biopolymers 13, 995 (1974)] that is suitable to SFM imaging of DNA and allows complete control of the conditions of deposition (ionic strength, temperature). We have measured the contour length of linear DNA molecules in SFM images of depositions prepared with the reported method. Our data show that the B structure is preserved.

De Novo Transcriptome Sequencing of Olea europaea L. to Identify Genes Involved in the Development of the Pollen Tube

In olive (Olea europaea L.), the processes controlling self-incompatibility are still unclear and the molecular basis underlying this process are still not fully characterized. In order to determine compatibility relationships, using next-generation sequencing techniques and a de novo transcriptome assembly strategy, we show that pollen tubes from different olive plants, grown in vitro in a medium containing its own pistil and in combination pollen/pistil from self-sterile and self-fertile cultivars, have a distinct gene expression profile and many of the differentially expressed sequences between the samples fall within gene families involved in the development of the pollen tube, such as lipase, carboxylesterase, pectinesterase, pectin methylesterase, and callose synthase. Moreover, different genes involved in signal transduction, transcription, and growth are overrepresented. The analysis also allowed us to identify members in actin and actin depolymerization factor and fibrin gene family and member of the Ca2+ binding gene family related to the development and polarization of pollen apical tip. The whole transcriptomic analysis, through the identification of the differentially expressed transcripts set and an extended functional annotation analysis, will lead to a better understanding of the mechanisms of pollen germination and pollen tube growth in the olive.

Olive Oil Traceability

The market for imported, premium priced foods has increased dramatically over recent years, as consumers become ever more aware of products originating from around the world. There are many food products that are of superior quality (taste, texture, fragrance etc.) because of the locale in which they are cultivated. Environmental conditions, such as local climate and soil characteristics, combine to yield crops that exhibit specific traits. Clearly, higher quality products demands higher market prices, therefore unscrupulous traders may attempt to increase profits by deliberately mislabelling foods, or by increasing the volume of a good quality batch through adulteration with sub-standard produce.