Molecular Traceability Approach to Assess the Geographical Origin of Commercial Extra Virgin Olive Oil.

Extra virgin olive oil (EVOO) is a precious and healthy ingredient of Mediterranean cuisine. Due to its high nutritional value, the interest of consumers in the composition of EVOO is constantly increasing, making it a product particularly exposed to fraud. Therefore, there is a need to properly valorize high-quality EVOO and protect it from fraudulent manipulations to safeguard consumer choices. In our study, we used a straightforward and easy method to assess the molecular traceability of 28 commercial EVOO samples based on the use of SSR molecular markers. A lack of correspondence between the declared origin of the samples and the actual origin of the detected varieties was observed, suggesting possible adulteration. This result was supported by the identification of private alleles based on a large collection of national and international olive varieties and the search for them in the molecular profile of the analyzed samples. We demonstrated that the proposed method is a rapid and straightforward approach for identifying the composition of an oil sample and verifying the correspondence between the origin of olives declared on the label and that of the actual detected varieties, allowing the detection of possible adulterations.

Breeding new seedless table grapevines for a more sustainable viticulture in Mediterranean climate.

The growing demand for sustainable and environmentally friendly viticulture is leading to a multiplication of breeding programs aimed at obtaining vines that are resistant to powdery mildew (PM) and downy mildew (DM), the two most damaging vine diseases. In Puglia, the most important Italian region for the production of table grapes, an extensive crossing program was launched in 2015 with 113 crosses, including elite table varieties, seedless varieties, and resistant varieties. The main seedling production parameters were measured for each cross. In particular, berries harvested as well as the number of seeds and seedlings obtained were considered. Approximately 103,119 seedlings were obtained and subjected to marker-assisted selection for seedlessness using the marker VvAGL11 and for resistance to PM and DM with appropriate markers. Approximately one third (32,638) of the progenies were selected as putative seedless and seventeen thousand five hundred-nine (17,509) were transferred to the field for phenotypic evaluation, including 527 seedless individuals putatively resistant, of which 208 confirmed to be resistant to DM, 22 resistant to PM, and 20 individuals that combined resistance and seedlessness traits. The work discusses the effects of parental combinations and other variables in obtaining surviving progeny and pyramiding genes in table grapes and provides useful information for selecting genotypes and increasing the efficiency of breeding programs for seedless disease-resistant grapes.

Genetic diversity and signature of divergence in the genome of grapevine clones of Southern Italy varieties.

Sexual reproduction has contributed to a significant degree of variability in cultivated grapevine populations. However, the additional influence of spontaneous somatic mutations has played a pivotal role in shaping the diverse landscape of grapevine agrobiodiversity. These naturally occurring selections, termed 'clones,' represent a vast reservoir of potentially valuable traits and alleles that hold promise for enhancing grape quality and bolstering plant resilience against environmental and biotic challenges. Despite their potential, many of these clones remain largely untapped.In light of this context, this study aims to delve into the population structure, genetic diversity, and distinctive genetic loci within a collection of 138 clones derived from six Campanian and Apulian grapevine varieties, known for their desirable attributes in viticulture and winemaking. Employing two reduced representation sequencing methods, we extracted Single-Nucleotide Polymorphism (SNP) markers. Population structure analysis and fixation index (FST) calculations were conducted both between populations and at individual loci. Notably, varieties originating from the same geographical region exhibited pronounced genetic similarity.The resulting SNP dataset facilitated the identification of approximately two hundred loci featuring divergent markers (FST ≥ 0.80) within annotated exons. Several of these loci exhibited associations with essential traits like phenotypic adaptability and environmental responsiveness, offering compelling opportunities for grapevine breeding initiatives. By shedding light on the genetic variability inherent in these treasured traditional grapevines, our study contributes to the broader understanding of their potential. Importantly, it underscores the urgency of preserving and characterizing these valuable genetic resources to safeguard their intra-varietal diversity and foster future advancements in grapevine cultivation.

Genotyping-by-Sequencing Defines Genetic Structure within the "Acquaviva" Red Onion Landrace.

Genetic structure and distinctive features of landraces, such as adaptability to local agro-ecosystems and specific qualitative profiles, can be substantially altered by the massive introduction of allochthonous germplasm. The landrace known as "Cipolla rossa di Acquaviva" (Acquaviva red onion, further referred to as ARO) is traditionally cultivated and propagated in a small area of the Apulia region (southern Italy). However, the recent rise of its market value and cultivation area is possibly causing genetic contamination with foreign propagating material. In this work, genotyping-by-sequencing (GBS) was used to characterize genetic variation of seven onion populations commercialized as ARO, as well as one population of the landrace "Montoro" (M), which is phenotypically similar, but originates from another cultivation area and displays different qualitative features. A panel of 5011 SNP markers was used to perform parametric and non-parametric genetic structure analyses, which supported the hypothesis of genetic contamination of germplasm commercialized as ARO with a gene pool including the M landrace. Four ARO populations formed a core genetic group, homogeneous and clearly distinct from the other ARO and M populations. Conversely, the remaining three ARO populations did not display significant differences with the M population. A set of private alleles for the ARO core genetic group was identified, indicating the possibility to trace the ARO landrace by means of a SNP-based molecular barcode. Overall, the results of this study provide a framework for further breeding activities and the traceability of the ARO landrace.

Comparative Genetic Analysis of Durum Wheat Landraces and Cultivars Widespread in Tunisia.

The durum wheat (Triticum turgidum L. ssp. durum Desf.) landraces constitute a useful natural germplasm to increase the genetic diversity in the modern durum cultivars. The Tunisian durum germplasm constitutes 28 accessions conserved in Genebank of Tunisia, which are still unexplored. In this study, a comparative genetic analysis was performed to investigate the relationships between the Tunisian durum lines and the modern cultivars and detect divergent loci involved in breeding history. The genetic diversity analyses carried out using nine morphological descriptors and the 25K single-nucleotide polymorphism (SNP) array allowed us to distinguish two groups of Tunisian landraces and one of durum cultivars. The analysis of molecular variance and diversity indices confirmed the genetic variability among the groups. A total of 529 SNP loci were divergent between Tunisian durum landraces and modern cultivars. Candidate genes related to plant and spike architecture, including FLOWERING LOCUS T (FT-B1), zinc finger CONSTANS, and AP2/EREBPs transcription factors, were identified. In addition, divergent genes involved in grain composition and biotic stress nucleotide-binding site and leucine-reach repeats proteins and disease resistance proteins (NBS-LRR and RPM) were found, suggesting that the Tunisian durum germplasm may represent an important source of favorable alleles to be used in future durum breeding programs for developing well-adapted and resilient cultivars.

Morphological and Eco-Geographic Variation in Algerian Wild Olives.

Algerian wild olives can represent an important resource for cultivated olive breeding, since they are characterized by great morphological variability. Moreover, they grow in different bioclimatic environments, including dry and hot climates, making the collections of wild olives a good source of abiotic stress resistance traits. Our study aimed to investigate the morphological diversity of 175 wild olive trees collected in North Algeria along with a wide range of different bioclimatic habitats for studying traits of olive accessions in relation to their different ecogeographical parameters. Wild olive trees were found in five different bioclimates areas spanning from humid to Saharan areas. They showed high variation in all traits, in particular fruit and stone weight, which expressed the highest coefficient of variation, and a high positive correlation between fruit weight/width. Cluster analysis separated the samples into two groups mostly based on fruit and stone size, while no relationship was observed with the area of sampling. Only the Saharan samples showed significantly different foliar and fruit characteristics compared to samples from other bioclimatic areas.

The Status of Genetic Resources and Olive Breeding in Tunisia.

The olive tree, an iconic symbol of the Mediterranean basin, is the object of growing international interest in the production of olive oil for the world food market. In Tunisia, which is the fourth-largest producer of olive oil in the world, the production of olives and olive oil is of great socio-economic importance. Cultivation is widespread from north to south, but it is carried out using traditional techniques that results in extremely irregular production levels. To maintain their competitiveness on the international market, Tunisian producers must improve the quality of the oil through breeding plans that enhance the rich genetic heritage that is still not adequately exploited. The objective of this review is to present the state of olive breeding in Tunisia, illustrating the opportunities available for a better use of the rich Tunisian genetic heritage, the challenges it must face, and the need to multiply the efforts for sustainability, even in the light of the challenges posed by climate changes.

Quantitatively Unraveling Hierarchy of Factors Impacting Virgin Olive Oil Phenolic Profile and Oxidative Stability.

A single phenolic group and even a compound play different roles in the sensory properties and stability of virgin olive oil (VOO), which in turn are strongly influenced by several factors. Understanding the causes of differences in phenolic compound composition and oxidative stability (OS) in VOOs is essential for targeted and timely harvest and processing while maintaining desired oil quality. The phenolic profile and OS of two monocultivar VOOs (Oblica and Leccino) grown in two geographical sites of different altitudes (coastal plain and hilly hinterland) were analyzed throughout the ripening period over two years. Concentration of secoiridoids was 30% higher in the Oblica than in the Leccino VOOs, which in turn had significantly higher values of OS. Both cultivars had more than twice as high concentrations of the two most abundant phenolic compounds, the dialdehyde form of decarboxymethyl oleuropein aglycone and the dialdehyde form of decarboxymethyl ligstroside aglycone, and OS values in a colder growing site of higher altitude. Among the studied monocultivar VOOs, the secoiridoid group did not behave equally during ripening. The hierarchy of different influencing factors was investigated using multivariate statistics and revealed: cultivar > geographical site > harvest period > growing season. In addition, the possibility of traceability of VOO using molecular markers was investigated by establishing SSR profiles of oils of the studied cultivars and comparing them with SSR profiles of leaves.

Current Status of Biodiversity Assessment and Conservation of Wild Olive (Olea europaea L. subsp. europaea var. sylvestris).

Oleaster (Olea europaea L. subsp. europaea var. sylvestris) is the ancestor of cultivated olive (Olea europaea L. subsp. europaea var. europaea) and it is spread through the whole Mediterranean Basin, showing an overlapping distribution with cultivated olive trees. Climate change and new emerging diseases are expected to severely affect the cultivations of olive in the future. Oleaster presents a higher genetic variability compared to the cultivated olive and some wild trees were found adapted to particularly harsh conditions; therefore, the role of oleaster in the future of olive cultivation may be crucial. Despite the great potential, only recently the need to deeply characterize and adequately preserve the wild olive resources drew the attention of researchers. In this review, we summarized the most important morphological and genetic studies performed on oleaster trees collected in different countries of the Mediterranean Basin. Moreover, we reviewed the strategies introduced so far to preserve and manage the oleaster germplasm collections, giving a future perspective on their role in facing the future agricultural challenges posed by climatic changes and new emerging diseases.

How to Choose a Good Marker to Analyze the Olive Germplasm (Olea europaea L.) and Derived Products.

The olive tree (Olea europaea L.) is one of the most cultivated crops in the Mediterranean basin. Its economic importance is mainly due to the intense production of table olives and oil. Cultivated varieties are characterized by high morphological and genetic variability and present a large number of synonyms and homonyms. This necessitates the introduction of a rapid and accurate system for varietal identification. In the past, the recognition of olive cultivars was based solely on analysis of the morphological traits, however, these are highly influenced by environmental conditions. Therefore, over the years, several methods based on DNA analysis were developed, allowing a more accurate and reliable varietal identification. This review aims to investigate the evolving history of olive tree characterization approaches, starting from the earlier morphological methods to the latest technologies based on molecular markers, focusing on the main applications of each approach. Furthermore, we discuss the impact of the advent of next generation sequencing and the recent sequencing of the olive genome on the strategies used for the development of new molecular markers.

Molecular Approaches to Agri-Food Traceability and Authentication: An Updated Review.

In the last decades, the demand for molecular tools for authenticating and tracing agri-food products has significantly increased. Food safety and quality have gained an increased interest for consumers, producers, and retailers, therefore, the availability of analytical methods for the determination of food authenticity and the detection of major adulterations takes on a fundamental role. Among the different molecular approaches, some techniques such as the molecular markers-based methods are well established, while some innovative approaches such as isothermal amplification-based methods and DNA metabarcoding have only recently found application in the agri-food sector. In this review, we provide an overview of the most widely used molecular techniques for fresh and processed agri-food authentication and traceability, showing their recent advances and applications and discussing their main advantages and limitations. The application of these techniques to agri-food traceability and authentication can contribute a great deal to the reassurance of consumers in terms of transparency and food safety and may allow producers and retailers to adequately promote their products.

Polyphenol oxidase genes as integral part of the evolutionary history of domesticated tetraploid wheat.

Studying and understanding the genetic basis of polyphenol oxidases (PPO)-related traits plays a crucial role in genetic improvement of crops. A tetraploid wheat collection (T. turgidum ssp., TWC) was analyzed using the 90K wheat SNP iSelect assay and phenotyped for PPO activity. A total of 21,347 polymorphic SNPs were used to perform genome-wide association analysis (GWA) in TWC and durum wheat sub-groups, detecting 23 and 85 marker-trait associations (MTA). In addition, candidate genes responsible for PPO activity were predicted. Based on the 23 MTAs detected in TWC, two haplotypes associated with low and high PPO activity were identified. Four SNPs were developed and validated providing one reliable marker (IWB75732) for marker assisted selection. The 23 MTAs were used to evaluate the genetic divergence (FST > 0.25) between the T. turgidum subspecies, providing new information important for understanding the domestication process of Triticum turgidum ssp. and in particular of ssp. carthlicum.

Applications of Microsatellite Markers for the Characterization of Olive Genetic Resources of Tunisia.

Among the countries of the Mediterranean Basin, Tunisia is located at the crossroad for the immigration of several civilizations over the last two millennia, becoming a strategic place for gene flow, and a secondary center of diversity for olive species. Olive is one of the principal crop species in Tunisia and now it strongly characterizes the rural landscape of the country. In recent years, collecting missions on farm and in situ were carried out by various institutes, with special emphasis given to ex situ collections serving as a reference for the identification of olive germplasm. Simple Sequence Repeats (SSRs) represent the easiest and cheapest markers for olive genetic fingerprinting and have been the tool of choice for studying the genetic diversity of this crop in Tunisia, to resolve cases of homonymy and synonymy among the commercialized varieties, to identify rare cultivars, to improve knowledge about the genetic variability of this crop, to identify a hot spot of olive biodiversity in the Tunisian oasis of Degache, and to enrich the national reference collection of olive varieties. The present review describes the state of the art of the genetic characterization of the Tunisian olive germplasm and illustrate the progress obtained through the SSR markers, in individuating interesting genotypes that could be used for facing incoming problems determined by climate changes.

Recovery, Assessment, and Molecular Characterization of Minor Olive Genotypes in Tunisia.

Olive is one of the oldest cultivated species in the Mediterranean Basin, including Tunisia, where it has a wide diversity, with more than 200 cultivars, of both wild and feral forms. Many minor cultivars are still present in marginal areas of Tunisia, where they are maintained by farmers in small local groves, but they are poorly characterized and evaluated. In order to recover this neglected germplasm, surveys were conducted in different areas, and 31 genotypes were collected, molecularly characterized with 12 nuclear microsatellite (simple sequence repeat (SSR)) markers, and compared with 26 reference cultivars present in the Tunisian National Olive collection. The analysis revealed an overall high genetic diversity of this olive's germplasm, but also discovered the presence of synonymies and homonymies among the commercialized varieties. The structure analysis showed the presence of different gene pools in the analyzed germplasm. In particular, the marginal germplasm from Ras Jbal and Azmour is characterized by gene pools not present in commercial (Nurseries) varieties, pointing out the very narrow genetic base of the commercialized olive material in Tunisia, and the need to broaden it to avoid the risk of genetic erosion of this species in this country.

Re.Ger.O.P.: An Integrated Project for the Recovery of Ancient and Rare Olive Germplasm.

The olive tree is one of the most important economic, cultural, and environmental resources for Italy, in particular for the Apulian region, where it shows a wide diversity. The increasing attention to the continuous loss of plant genetic diversity due to social, economic and climatic changes, has favored a renewed interest in strategies aimed at the recovery and conservation of these genetic resources. In the frame of a project for the valorization of the olive Apulian biodiversity (Re.Ger.O.P. project), 177 minor genotypes were recovered in different territories of the region. They were submitted to morphological, molecular, technological and phytosanitary status analysis in comparison with reference cultivars, then they were propagated and transferred in an ex situ field. All the available information was stored in an internal regional database including photographic documentation and geographic position. The work allowed obtaining information about the genetic diversity of Apulian germplasm, to clarify cases of homonymy and synonymy, to check the sanitary status, and to identify candidate genotypes useful both to set up breeding programs and to enrich the panel of olive cultivars available to farmers for commercial exploitation.

Serendipitous In Situ Conservation of Faba Bean Landraces in Tunisia: A Case Study.

Cultivation of faba bean (Vicia faba L.) in Tunisia is largely based on improved varieties of the crop. However, a few farmers continue to produce local cultivars or landraces. The National Gene Bank of Tunisia (NGBT) recently launched a collection project for faba bean landraces, with special focus on the regions of the North West, traditionally devoted to cultivating grain legumes, and where around 80% of the total national faba bean cultivation area is located. The seed phenotypic features of the collected samples were studied, and the genetic diversity and population structure analyzed using simple sequence repeat markers. The genetic constitution of the present samples was compared to that of faba bean samples collected by teams of the International Center for Agricultural Research in the Dry Areas (ICARDA) in the 1970s in the same region, and stored at the ICARDA gene bank. The results of the diversity analysis demonstrate that the recently collected samples and those stored at ICARDA largely overlap, thus demonstrating that over the past 50 years, little genetic change has occurred to the local faba bean populations examined. These findings suggest that farmers serendipitously applied international best practices for in situ conservation of agricultural crops.

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.

Polyphenol Oxidases in Crops: Biochemical, Physiological and Genetic Aspects.

Enzymatic browning is a colour reaction occurring in plants, including cereals, fruit and horticultural crops, due to oxidation during postharvest processing and storage. This has a negative impact on the colour, flavour, nutritional properties and shelf life of food products. Browning is usually caused by polyphenol oxidases (PPOs), following cell damage caused by senescence, wounding and the attack of pests and pathogens. Several studies indicated that PPOs play a role in plant immunity, and emerging evidence suggested that PPOs might also be involved in other physiological processes. Genomic investigations ultimately led to the isolation of PPO homologs in several crops, which will be possibly characterized at the functional level in the near future. Here, focusing on the botanic families of Poaceae and Solanaceae, we provide an overview on available scientific literature on PPOs, resulting in useful information on biochemical, physiological and genetic aspects.