Integration of Innovative Technologies in the Agri-Food Sector: The Fundamentals and Practical Case of DNA-Based Traceability of Olives from Fruit to Oil.

Several socio-economic problems have been hidden by the COVID-19 pandemic crisis. Particularly, the agricultural and food industrial sectors have been harshly affected by this devastating disease. Moreover, with the worldwide population increase and the agricultural production technologies being inefficient or obsolete, there is a great need to find new and successful ways to fulfill the increasing food demand. A new era of agriculture and food industry is forthcoming, with revolutionary concepts, processes and technologies, referred to as Agri-food 4.0, which enables the next level of agri-food production and trade. In addition, consumers are becoming more and more aware about the origin, traceability, healthy and high-quality of agri-food products. The integration of new process of production and data management is a mandatory step to meet consumer and market requirements. DNA traceability may provide strong approach to certify and authenticate healthy food products, particularly for olive oil. With this approach, the origin and authenticity of products are confirmed by the means of unique nucleic acid sequences. Selected tools, methods and technologies involved in and contributing to the advance of the agri-food sector are presented and discussed in this paper. Moreover, the application of DNA traceability as an innovative approach to authenticate olive products is reported in this paper as an application and promising case of smart agriculture.

Comparative Analysis and Structural Modeling of Elaeis oleifera FAD2, a Fatty Acid Desaturase Involved in Unsaturated Fatty Acid Composition of American Oil Palm.

American oil palm (Elaeis oleifera) is an important source of dietary oil that could fulfill the increasing worldwide demand for cooking oil. Therefore, improving its production is crucial and could be realized through breeding and genetic engineering approaches aiming to obtain high-yielding varieties with improved oil content and quality. The fatty acid composition and particularly the oleic/linoleic acid ratio are major factors influencing oil quality. Our work focused on a fatty acid desaturase (FAD) enzyme involved in the desaturation and conversion of oleic acid to linoleic acid. Following the in silico identification and annotation of Elaeis oleifera FAD2, its molecular and structural features characterization was performed to better understand the mechanistic bases of its enzymatic activity. EoFAD2 is 1173 nucleotides long and encodes a protein of 390 amino acids that shares similarities with other FADs. Interestingly, the phylogenetic study showed three distinguished groups where EoFAD2 clustered among monocotyledonous taxa. EoFAD2 is a membrane-bound protein with five transmembrane domains presumably located in the endoplasmic reticulum. The homodimer organization model of EoFAD2 enzyme and substrates and respective substrate-binding residues were predicted and described. Moreover, the comparison between 24 FAD2 sequences from different species generated two interesting single-nucleotide polymorphisms (SNPs) associated with the oleic/linoleic acid contents.

SNP discovery and structural insights into OeFAD2 unravelling high oleic/linoleic ratio in olive oil.

Fatty Acid Desaturase 2 (FAD2), a key enzyme in the fatty acid biosynthesis pathway, is involved in the desaturation and conversion of oleic acid to linoleic acid. Therefore, it plays a crucial role in oleic/linoleic acid ratio and the quality of olive oil. DNA sequencing of 19 FAD2 genes from a set of olive oil varieties revealed several single-nucleotide polymorphisms (SNPs) and highlighted associations between some of the SNPs and saturated fatty acids contents. This was further confirmed by SNP-interaction and machine learning approach. Haplotype diversity analysis led to the discovery of three highly polymorphic SNPs and four haplotypes harboring differential oleic/linoleic acid ratios. Moreover, a combination of molecular modeling and docking experiments allowed a deeper and better understanding of the structure-function relationship of the FAD2 enzyme. Sequence patterns and variations involved in the regulation of the FAD2 activity were also identified. Furthermore, S82C and H213N substitutions in OeFAD2 make the Oueslati variety more interesting in terms of fatty acid profile and oleic acid level.

Tunisian Table Olive Oil Traceability and Quality Using SNP Genotyping and Bioinformatics Tools.

To enhance and highlight the authentication and traceability of table olive oil, we considered the analysis of 11 Tunisian table olive cultivars based on seven SNP molecular markers (SOD, CALC, FAD2.1, FAD2.3, PAL70, ANTHO3, and SAD.1) localized in six different genes. Accordingly, we assessed the potential genotype-phenotypes links between the seven SNPs, on the one hand, and the quantitative and qualitative parameters, on the other. The obtained genotypes were analyzed with computational biology tools based on bivariate analysis, multinomial logistic regression, and the Bayesian networks modeling. Obtained results showed that PAL70 SNP marker was negatively influenced by the phenol rate (r = -0.886; p <0.001), the oxidative stability (r = -0.884; p <0.001), traducing a direct effect of the PAL70 genotype deviations on the proportion of total phenol for each variety. Additionally, we revealed a significant association of SAD.1 marker with the content of the linolenic unsaturated fatty acids (C18:3; p=0.046). Moreover, SAD.1 was positively correlated with the saturated stearic acid C18:0 (r = 0.644; p = 0.032) based on multinomial logistic regression and Bayesian networks modeling, respectively. This research work provides better understanding and characterization of the quality of Tunisian table olive and supplies a significant knowledge and data information for table olive traceability and breeding.

Nutraceutical potentialities of Tunisian Argan oil based on its physicochemical properties and fatty acid content as assessed through Bayesian network analyses.

BACKGROUND: Argan oil is traditionally produced by cold pressing in South-western Morocco where rural population uses it as edible oil as well as for its therapeutic properties which give them in counterpart valuable income. Given the economical interest of this oil, several attempts of fraudulency have been registered in the world global market leading to loss of authenticity. Our purpose is to launch a program of Tunisian Argan oil valorization since trees from this species have been introduced sixty years ago in Tunisia. The first step was thus to characterize the physicochemical properties and determine the chemical composition of Tunisian Argan oil in order to assess its quality. METHODS: Physicochemical parameters of oil quality were determined according to the international standard protocols. Fatty acid content analysis of Argan oils was performed by gas chromatography coupled to mass spectrophotometry. A comparative study was realized among Tunisian, Moroccan and Algerian samples differing also by their extraction procedure. The impact of geographical localisation on the fatty acids composition was studied by statistical and modeling Bayesian analyses. RESULTS: Physicochemical parameters analysis showed interestingly that Tunisian Argan oil could be classified as extra virgin oil. Argan oil is mainly composed by unsaturated fatty acids (80%), mainly oleic and linoleic acid (linoleic acid was positively influenced by the geographical localization (r = 0.899, p = 0.038) and the P/S index (r = 0.987, p = 0.002)) followed by saturated fatty acids (20%) with other beneficial compounds from the unsaponifiable fraction like polyphenols and carotenoids. Together with fatty acid content, these minor components are likely to be responsible for its nutraceutical properties and beneficial effects. CONCLUSION: Tunisian Argan oil displayed valuable qualitative parameters proving its competitiveness in comparison with Moroccan and Algerian oils, and could be therefore considered as extra virgin edible oil for nutraceutical purposes as well as for cosmetic use.

The grapevine VvWRKY2 gene enhances salt and osmotic stress tolerance in transgenic Nicotiana tabacum.

Our study aims to assess the implication of WRKY transcription factor in the molecular mechanisms of grapevine adaptation to salt and water stresses. In this respect, a full-length VvWRKY2 cDNA, isolated from a Vitis vinifera grape berry cDNA library, was constitutively over-expressed in Nicotiana tabacum seedlings. Our results showed that transgenic tobacco plants exhibited higher seed germination rates and better growth, under both salt and osmotic stress treatments, when compared to wild type plants. Furthermore, our analyses demonstrated that, under stress conditions, transgenic plants accumulated more osmolytes, such as soluble sugars and free proline, while no changes were observed regarding electrolyte leakage, H2O2, and malondialdehyde contents. The improvement of osmotic adjustment may be an important mechanism underlying the role of VvWRKY2 in promoting tolerance and adaptation to abiotic stresses. Principal component analysis of our results highlighted a clear partition of plant response to stress. On the other hand, we observed a significant adaptation behaviour response for transgenic lines under stress. Taken together, all our findings suggest that over-expression of VvWRKY2 gene has a compelling role in abiotic stress tolerance and, therefore, would provide a useful strategy to promote abiotic stress tolerance in grape via molecular-assisted breeding and/or new biotechnology tools.

First study of correlation between oleic acid content and SAD gene polymorphism in olive oil samples through statistical and bayesian modeling analyses.

BACKGROUND: Virgin olive oil is appreciated for its particular aroma and taste and is recognized worldwide for its nutritional value and health benefits. The olive oil contains a vast range of healthy compounds such as monounsaturated free fatty acids, especially, oleic acid. The SAD.1 polymorphism localized in the Stearoyl-acyl carrier protein desaturase gene (SAD) was genotyped and showed that it is associated with the oleic acid composition of olive oil samples. However, the effect of polymorphisms in fatty acid-related genes on olive oil monounsaturated and saturated fatty acids distribution in the Tunisian olive oil varieties is not understood. METHODS: Seventeen Tunisian olive-tree varieties were selected for fatty acid content analysis by gas chromatography. The association of SAD.1 genotypes with the fatty acids composition was studied by statistical and Bayesian modeling analyses. RESULTS: Fatty acid content analysis showed interestingly that some Tunisian virgin olive oil varieties could be classified as a functional food and nutraceuticals due to their particular richness in oleic acid. In fact, the TT-SAD.1 genotype was found to be associated with a higher proportion of mono-unsaturated fatty acids (MUFA), mainly oleic acid (C18:1) (r = - 0.79, p < 0.000) as well as lower proportion of palmitic acid (C16:0) (r = 0.51, p = 0.037), making varieties with this genotype (i.e. Zarrazi and Tounsi) producing more monounsaturated oleic acid (C18: 1) than saturated acid. These varieties could be thus used as nutraceuticals and functional food. CONCLUSION: The SAD.1 association with the oleic acid composition of olive oil was identified among the studied varieties. This correlation fluctuated between studied varieties, which might elucidate variability in lipidic composition among them and therefore reflecting genetic diversity through differences in gene expression and biochemical pathways. SAD locus would represent an excellent marker for identifying interesting amongst virgin olive oil lipidic composition.

OGDD (Olive Genetic Diversity Database): a microsatellite markers' genotypes database of worldwide olive trees for cultivar identification and virgin olive oil traceability.

Olive (Olea europaea), whose importance is mainly due to nutritional and health features, is one of the most economically significant oil-producing trees in the Mediterranean region. Unfortunately, the increasing market demand towards virgin olive oil could often result in its adulteration with less expensive oils, which is a serious problem for the public and quality control evaluators of virgin olive oil. Therefore, to avoid frauds, olive cultivar identification and virgin olive oil authentication have become a major issue for the producers and consumers of quality control in the olive chain. Presently, genetic traceability using SSR is the cost effective and powerful marker technique that can be employed to resolve such problems. However, to identify an unknown monovarietal virgin olive oil cultivar, a reference system has become necessary. Thus, an Olive Genetic Diversity Database (OGDD) (http://www.bioinfo-cbs.org/ogdd/) is presented in this work. It is a genetic, morphologic and chemical database of worldwide olive tree and oil having a double function. In fact, besides being a reference system generated for the identification of unkown olive or virgin olive oil cultivars based on their microsatellite allele size(s), it provides users additional morphological and chemical information for each identified cultivar. Currently, OGDD is designed to enable users to easily retrieve and visualize biologically important information (SSR markers, and olive tree and oil characteristics of about 200 cultivars worldwide) using a set of efficient query interfaces and analysis tools. It can be accessed through a web service from any modern programming language using a simple hypertext transfer protocol call. The web site is implemented in java, JavaScript, PHP, HTML and Apache with all major browsers supported. Database URL: http://www.bioinfo-cbs.org/ogdd/.

Classical and Bayesian predictions applied to Bacillus toxin production.

Bacillus thuringiensis is a bacterium with unusual properties that make it useful for pest control in ecoagriculture. It can form a parasporal crystal containing polypeptides (also called delta-endotoxins). These entomopathogenic toxins are made during the stationary phase of the bacterial growth cycle and were initially characterized as an insect pathogen. Nowadays, the use of saturated two-level designs is very popular. This method is especially used in industrial applications where the cost of experiments is expensive. Standard classical approaches are not appropriate to analyze data from saturated designs. It is due to the fact that they only allow to estimate the main factor effects and cannot assure enough freedom degrees to estimate the error variance. In this paper, we propose the use of empirical Bayesian procedures to get inferences for data obtained from saturated designs, inspired from Hadamard matrices. The proposed methodology is illustrated by assuming a dataset to prove the model robustness. The comparison between the two studied mathematical techniques, based on mean square error values (MSE), revealed that Bayesian method (BM) was more accurate than least square method (LSM): for example, the results showed that 2002 and 2000.7 mg/l for experimental and predicted (BM) data were obtained against 2002 and 1991 mg/l for experimental and predicted (LSM) data. This suggested method could be generalized in several application fields in biological sciences.