Valentina Di Rienzo

Traceability of PDO Olive Oil “Terra di Bari” Using High Resolution Melting

The aim of the research was to verify the applicability of microsatellite (SSR) markers in High Resolution Melting (HRM) analysis for the identification of the olive cultivars used in the “Terra di Bari” PDO extra virgin olive oil. A panel of nine cultivars, widespread in Apulia region, was tested with seventeen SSR primer pairs and the PCR products were at first analysed with a Genetic Analyzer automatic sequencer. An identification key was obtained for the nine cultivars, which showed an unambiguous discrimination among the varieties constituting the “Terra di Bari” PDO extra virgin olive oil: Cima di Bitonto, Coratina, and Ogliarola. Subsequently, an SSR based method was set up with the DCA18 marker, coupled with HRM analysis for the distinction of the Terra di Bari olive oil from non-Terra di Bari olive oil using different mixtures. Thus, this analysis enabled the distinction and identification of the PDO mixtures. Hence, this assay provided a flexible, cost-effective, and closed-tube microsatellite genotyping method, well suited to varietal identification and authentication analysis in olive oil.

Traceability of Italian Protected Designation of Origin (PDO) table olives by means of microsatellite molecular markers.

The aim of this work was to develop a DNA microsatellite-based method of analysis to allow traceability of the three Italian Protected Designation of Origin (PDO) table olives in comparison with fruits of another seven highly diffused table olive cultivars. The analyses were carried out by using 16 primer pairs, with a mean of five different alleles detected per primer set, and power of discrimination from 0.56 to 0.90. Allelic error rates in the range of 0-3.8% were observed. By combining data from the most reliable and highly informative microsatellites (DCA3, DCA16, DCA17, DCA18, UDO-043, and GAPU101), it was possible to identify the PDO fruits over the panel of 10 cultivars, with the probability of a chance match between different cultivars as low as 10(-9) and with 0.5% error rate. The amplification profile is independent of environmental and processing conditions and is helpful to verify the authenticity of PDO samples.

A Rapid Assay to Detect Toxigenic Penicillium spp. Contamination in Wine and Musts

Wine and fermenting musts are grape products widely consumed worldwide. Since the presence of mycotoxin-producing fungi may greatly compromise their quality characteristics and safety, there is an increasing need for relatively rapid “user friendly” quantitative assays to detect fungal contamination both in grapes delivered to wineries and in final products. Although other fungi are most frequently involved in grape deterioration, secondary infections by Penicillium spp. are quite common, especially in cool areas with high humidity and in wines obtained by partially dried grapes. In this work, a single-tube nested real-time PCR approach—successfully applied to hazelnut and peanut allergen detection—was tested for the first time to trace Penicillium spp. in musts and wines. The method consisted of two sets of primers specifically designed to target the β-tubulin gene, to be simultaneously applied with the aim of lowering the detection limit of conventional real-time PCR. The assay was able to detect up to 1 fg of Penicillium DNA. As confirmation, patulin content of representative samples was determined. Most of analyzed wines/musts returned contaminated results at >50 ppb and a 76% accordance with molecular assay was observed. Although further large-scale trials are needed, these results encourage the use of the newly developed method in the pre-screening of fresh and processed grapes for the presence of Penicillium DNA before the evaluation of related toxins.

Chemical and Molecular Characterization of Crude Oil Obtained by Olive-Pomace Recentrifugation

In oil-mills, olive-pomace recentrifugation is a common way to reduce pomace moisture and, at the same time, to recover the oil therein. According to current rules, the obtained oil is defined as “crude olive-pomace oil.” The aim of this work is to verify the effect of recentrifugation on specific chemical and molecular parameters of the crude olive-pomace oil, by comparing it with the corresponding virgin olive oil obtained from the same olive lots. In particular, the following were considered: (i) the polar compounds of the oils that include compounds originated from oxidative and hydrolytic degradation, analyzed by high-performance size exclusion chromatography (HPSEC), and (ii) the profile of DNA microsatellite molecular markers that was analyzed by using the High Resolution Melting (HRM) technique. The obtained results evidenced the significantly higher hydrolytic degradation of crude olive-pomace oil, compared with the corresponding virgin olive oil, but at an extent unlikely able to allow the detection of fraudulent admixtures with virgin olive oils. In addition, the findings demonstrated the feasibility of the application of the HRM analysis of DNA microsatellites to crude olive-pomace oil, able to reveal the alteration of the declared varietal profile of a virgin olive oil sample by simply checking the HRM curve profiles.

GBS-derived SNP catalogue unveiled wide genetic variability and geographical relationships of Italian olive cultivars

Information on the distribution of genetic variation is essential to preserve olive germplasm from erosion and to recover alleles lost through selective breeding. In addition, knowledge on population structure and genotype–phenotype associations is crucial to support modern olive breeding programs that must respond to new environmental conditions imposed by climate change and novel biotic/abiotic stressors. To further our understanding of genetic variation in the olive, we performed genotype-by-sequencing on a panel of 94 Italian olive cultivars. A reference-based and a reference-independent SNP calling pipeline generated 22,088 and 8,088 high-quality SNPs, respectively. Both datasets were used to model population structure via parametric and non parametric clustering. Although the two pipelines yielded a 3-fold difference in the number of SNPs, both described wide genetic variability among our study panel and allowed individuals to be grouped based on fruit weight and the geographical area of cultivation. Multidimensional scaling analysis on identity-by-state allele-sharing values as well as inference of population mixtures from genome-wide allele frequency data corroborated the clustering pattern we observed. These findings allowed us to formulate hypotheses about geographical relationships of Italian olive cultivars and to confirm known and uncover novel cases of synonymy.

Genetic flow among olive populations within the Mediterranean basin

Background The olive tree is a typical crop of the Mediterranean basin where it shows a wide diversity, accounting for more than 2,600 cultivars. The ability to discriminate olive cultivars and determine their genetic variability is pivotal for an optimal exploitation of olive genetic resources. Methods We investigated the genetic diversity within 128 olive accessions belonging to four countries in the Mediterranean Basin (Italy, Algeria, Syria, and Malta), with the purpose of better understanding the origin and spread of the olive genotypes across Mediterranean Basin countries. Eleven highly polymorphic simple sequence repeat (SSR) markers were used and proved to be very informative, producing a total of 179 alleles. Results Cluster analysis distinguished three main groups according to their geographical origin, with the current sample of Maltese accessions included in the Italian group. Phylogenetic analysis further differentiated Italian and Maltese olive accessions, clarifying the intermediate position of Maltese accessions along the x/y-axes of principal coordinate analysis (PCoA). Model-based and neighbor clustering, PCoA, and migration analysis suggested the existence of two different gene pools (Algerian and Syrian) and that the genetic exchange occurred between the Syrian, Italian and Maltese populations. Discussion The close relationship between Syrian and Italian and Maltese olives was consistent with the historical domestication and migration of olive tree from the North Levant to eastern Mediterranean basin. This study lays the foundations for a better understanding of olive genetic diversity in the Mediterranean basin and represents a step toward an optimal conservation and exploitation of olive genetic resources.

Rapid identification of tomato Sw-5 resistance-breaking isolates of Tomato spotted wilt virus using high resolution melting and TaqMan SNP Genotyping assays as allelic discrimination techniques

In tomato, resistance to Tomato spotted wilt virus (TSWV) is conferred by the dominant gene, designated Sw-5. Virulent Sw-5 resistance breaking (SRB) mutants of TSWV have been reported on Sw-5 tomato cultivars. Two different PCR-based allelic discrimination techniques, namely Custom TaqMan™ SNP Genotyping and high-resolution melting (HRM) assays, were developed and compared for their ability to distinguish between avirulent (Sw-5 non-infecting, SNI) and SRB biotypes. TaqMan assays proved to be more sensitive (threshold of detection in a range of 50–70 TSWV RNA copies) and more reliable than HRM, assigning 25 TSWV isolates to their correct genotype with an accuracy of 100%. Moreover, the TaqMan SNP assays were further improved developing a rapid and simple protocol that included crude leaf extraction for RNA template preparations. On the other hand, HRM assays showed higher levels of sensitivity than TaqMan when used to co-detect both biotypes in different artificial mixtures. These diagnostic assays contributed to gain preliminary information on the epidemiology of TSWV isolates in open field conditions. In fact, the presented data suggest that SRB isolates are present as stable populations established year round, persisting on both winter (globe artichoke) and summer (tomato) crops, in the same cultivated areas of Southern Italy.