Sónia Gomes

DNA Markers for Portuguese Olive Oil Fingerprinting

The certification of olive oil has led to the definition of Protected Denomination of Origin (PDO) producing regions in European countries. PDO products should be protected, and a solution could be by using DNA fingerprinting. In this work we evaluate the efficiency of RAPD, ISSR, and SSR molecular markers for olive oil varietal identification and their possible use in certification purposes. Twenty-three Portuguese olive oil samples (11 obtained monovarietal and 12 purchased commercial oils) were screened by means of two RAPD, four ISSR, and four SSR markers. The quality of amplified products was used to evaluate the reproducibility and the level of polymorphism. Principal component analysis was performed with DCENTER using unweighted pair group mathematical average (UPGMA) that allowed group formation according to olive oil varietal geographic origin.

RAPD and ISSR molecular markers in Olea europaea L.: Genetic variability and molecular cultivar identification

Thirty Portuguese and eight foreign olive (Olea europaea L.) cultivars were screened using Random Amplified Polymorphic DNA (RAPD) and Inter-Simple Sequence Repeat (ISSR) markers. Twenty RAPD primers amplified 301 reproducible bands of which 262 were polymorphic; and 17 ISSR primers amplified 204 bands of which 180 were polymorphic. The percentage of polymorphic bands detected by ISSR and RAPD was similar (88 and 87%, respectively). The genetic variability observed was similar in the Portuguese and foreign olive cultivars. Seven ISSR and 12 RAPD primers were able to distinguish individually all 38 olive cultivars. Twenty specific molecular markers are now available to be converted into Sequence Characterised Amplified Region (SCAR) markers. Relationships among Portuguese and foreign cultivars is discussed.

Assessing Genetic Diversity in Olea europaea L. Using ISSR and SSR Markers

Olea europaea L. is one of the most economically important crops in the Mediterranean area, and known for having large genetic variability. In order to assess the genetic diversity, DNA from 41 olive cultivars, present in the protected denomination of origin (PDO) region of Trás-os-Montes, was screened using inter simple sequence repeat (ISSR) and microsatellite (SSR) markers. Eleven ISSR primers amplified 135 reproducible bands of which 108 were polymorphic. The percentage of polymorphic bands detected by ISSR was 79%. The highest number of polymorphic bands was obtained by the use of primers UBC807 (15) and UBC809 (16). A total of 67 alleles were detected by six SSR primers, with an average of 11 alleles per primer. The number of alleles per locus ranged from five (ssrOeUaDCA05) to 15 (ssrOeUaDCA03). The observed heterozygosity ranged from 0.219 (ssrOeUaDCA05) to 0.900 (ssrOeUaDCA04), while the expected heterozygosity varied between 0.426 (ssrOeUaDCA05) and 0.887 (ssrOeUaDCA03). The polymorphism information content (PIC) values ranged from 0.392 (ssrOeUaDCA05) to 0.863 (ssrOeUaDCA03). The collection of primers selected gave a reasonable number of amplification products for the genetic diversity analysis. Based on the results, the genetic diversity among 41 olive cultivars is discussed. This study reveals the great importance of guaranteeing the differentiation of olive cultivars and their application for certification purposes.

Development of Colletotrichum acutatum on Tolerant and Susceptible Olea europaea L. cultivars: A Microscopic Analysis

Colletotrichum acutatum is a cosmopolitan and damaging plant pathogen of temperate, subtropical, and tropical fruits and causes anthracnose on olive (Olea europaea L.). Three olive cultivars showing a variable response to infection by C. acutatum were selected to a preliminary study of pathogen development. Fruit samples, from susceptible and tolerant cultivars, were taken at 0, 24, 48, 72, and 192xa0h after inoculation for a microscopic and histological study of the infection and colonization process. The aim of this study was to compare the infection process: conidial germination, germ tube and appressorium formation, hyphal growth, and mesocarp colonization in susceptible and tolerant olive cultivars as a condition for further exploration of disease development, which is required to develop cultivars with improved resistance to anthracnose. The rate of mesocarp colonization differed between the susceptible and tolerant cultivars, and both intracellular hemibiotrophy and subcuticular intramural necrotrophy were observed. Hemibiotrophic infection predominated in the moderately tolerant cultivar.

Evaluation of chemical and phenotypic changes in Blanqueta, Cobrançosa, and Galega during olive fruits ripening

Olea europaea L. presents a huge number of cultivars that differ in terms of end-use, and consequently in their chemical composition. The use of different phenotypic (fruit dimension, shape, and color) and chemical parameters (phenolic compounds, flavonoids, and antioxidant activity) during olive fruit maturation in three olive cultivars (Blanqueta, Cobrançosa, and Galega), grown in the Protected Denomination Region of Trás-os-Montes, revealed clear differences among the cultivars, indicating that they behave differently during the maturation process, Galega being the first to reach full maturity. All colorimetric parameters (L*, b*, C*, and H*) decreased with maturity. Galega cultivar presented the highest phenolic content in all the maturation stages analyzed (128.25–175.46 mg GAE g−1). Also, this cultivar presented the highest antioxidant activity in semi-green stage fruits (231.72 μmol Trolox g−1). The flavonoid content increased in all cultivars from the green drupe to black stage, with differential behavior between the cultivars within maturation stages. Moreover, a high correlation was found between the phenolic compounds content and the antioxidant activity levels.

High Resolution Melting (HRM) applied to wine authenticity

Wine authenticity methods are in increasing demand mainly in Denomination of Origin designations. The DNA-based methodologies are a reliable means of tracking food/wine varietal composition. The main aim of this work was the study of High Resolution Melting (HRM) application as a screening method for must and wine authenticity. Three sample types (leaf, must and wine) were used to validate the three developed HRM assays (Vv1-705bp; Vv2-375bp; and Vv3-119bp). The Vv1 HRM assay was only successful when applied to leaf and must samples. The Vv2 HRM assay successfully amplified all sample types, allowing genotype discrimination based on melting temperature values. The smallest amplicon, Vv3, produced a coincident melting curve shape in all sample types (leaf and wine) with corresponding genotypes. This study presents sensitive, rapid and efficient HRM assays applied for the first time to wine samples suitable for wine authenticity purposes.

Applying high-resolution melting (HRM) technology to olive oil and wine authenticity

Olive oil and wine production have a worldwide economic impact. Their market reliability is under great concern because of the increasing number of fraud and adulteration attempts. The need for a traceability system in all its extension is crucial particularly for the cases of olive oils and wines with certified labels, in which only a limited number of olives and grapevine varieties, respectively, are allowed in a restricted well-defined geographical area. Molecular markers have been vastly applied to the food sector, and in particular High-Resolution DNA Melting technology has been successfully applied for olive oil and wine authentication, as part of the traceability system. In this review, the applications of HRM and their usefulness for this sector considering, Safety, Security and Authenticity will be reviewed. A broad overview of the HRM technique will be presented, focusing on the aspects that are crucial for its success, in particular the new generation of fluorescent dsDNA dyes used for amplicon detection and quantification, and the data analysis. A brief outlook on the olive oil and wine authenticity procedures, based on new DNA technology advances, and in which way this may influence the future establishment of a traceability system will be discussed.

Critical Review on the Significance of Olive Phytochemicals in Plant Physiology and Human Health

Olive oil displays remarkable organoleptic and nutritional features, which turn it into a foodstuff appreciated by consumers, and a basic component of the Mediterranean diet. Indeed, the noticed benefits of including olive oil in the diet have been assigned to the presence of diverse bioactive compounds with different molecular structures. These compounds confer a wide range of biological properties to this food matrix, including the prevention of distinct human diseases as well as the modulation of their severity. The most relevant bioactive compounds present in olive oil correspond to benzoic and cinnamic acids, phenolic alcohols and secoiridoids, and also flavonoids. Over the last decades, several studies, devoted to gaining a further insight into the relative contribution of the separate groups and individual compounds for their biological activities, have been conducted, providing relevant information on structure–activity relationships. Therefore, this paper critically reviews the health benefits evidenced by distinct phenolic compounds found in olive oils, thus contributing to clarify the relationship between their chemical structures and biological functions, further supporting their interest as essential ingredients of wholesome foods.

Alternative SNP detection platforms, HRM and biosensors, for varietal identification in Vitis vinifera L. using F3H and LDOX genes

The wine sector requires quick and reliable methods for Vitis vinifera L. varietal identification. The number of V. vinifera varieties is estimated in about 5,000 worldwide. Single Nucleotide Polymorphisms (SNPs) represent the most basic and abundant form of genetic sequence variation, being adequate for varietal discrimination. The aim of this work was to develop DNA-based assays suitable to detect SNP variation in V. vinifera, allowing varietal discrimination. Genotyping by sequencing allowed the detection of eleven SNPs on two genes of the anthocyanin pathway, the flavanone 3-hydroxylase (F3H, EC: 1.14.11.9), and the leucoanthocyanidin dioxygenase (LDOX, EC 1.14.11.19; synonym anthocyanidin synthase, ANS) in twenty V. vinifera varieties. Three High Resolution Melting (HRM) assays were designed based on the sequencing information, discriminating five of the 20 varieties: Alicante Bouschet, Donzelinho Tinto, Merlot, Moscatel Galego and Tinta Roriz. Sanger sequencing of the HRM assay products confirmed the HRM profiles. Three probes, with different lengths and sequences, were used as bio-recognition elements in an optical biosensor platform based on a long period grating (LPG) fiber optic sensor. The label free platform detected a difference of a single SNP using genomic DNA samples. The two different platforms were successfully applied for grapevine varietal identification.

Sorting out the value of spectroscopic tools to assess the Colletotrichum acutatum impact in olive cultivars with different susceptibilities

The olive tree (Olea europaea L.) can be affected by Colletotrichum acutatum, causing a loss of yield and quality of the final products, whilst the incidence of this fungal infection depends on several factors, including cultivar susceptibility. Thus, the effect of C. acutatum infection in cultivars displaying different susceptibilities to this fungal disease (‘Galega Vulgar’ ‐ susceptible, ‘Cobrançosa’ ‐ moderately susceptible, ‘Picual’ ‐ tolerant) has been assessed through spectrophotometric methods and HPLC, while the FTIR spectra of the cuticles have been concomitantly registered, resorting to the ATR accessory.