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A new and optimized protocol, here called 6hDNA (i.e. a genomic DNA obtained by a six-hour extraction method), has been developed based on the traditional Cetyl-TrimethylAmmonium Bromide (CTAB) method. It allows a fast and easy isolation of genomic DNA from plant species, especially from those with high polyphenol and polysaccharide contents. Co-precipitation of polysaccharides was avoided by adding higher concentrations of selective precipitants of nucleic acid, CTAB 3% (w/v) and sodium chloride (NaCl) (1.42M). PolyVinylPyrrolidone (PVP) 1% (w/v) was applied to remove polyphenols as PCR inhibitors. Proteins were degraded by treatments of chloroform:isoamyl alchol (24:1) and phenol:chloroform:isoamyl alchol (25:24:1) and removed by centrifugation from plant extracts. The yield of total DNA from leaves of Vitis vinifera, Citrus sinensis and Olea europaea ranged from 42 to 980 ng μL-1 with A260/A280 ratio values between 1.6 and 2.06. The purity and integrity of the obtained DNA guarantees successful downstream applications including PCR and microsatellite markers. The use of lyophilized plant material and the reduced time of the total procedure make this new 6hDNA protocol more convenient when compared to the most common DNA isolation protocols, such as: "Doyle and Doyle", "Lodhi", "Li", or those using the DNAzol reagent and the Nucleospin Plant Minikit. [ABSTRACT FROM AUTHOR]

Citrus tristeza virus (CTV) is the pathogen causing tristeza diseases in several Rutaceae species and leading to significant economic damage to citrus worldwide. The Ctv locus provides broad spectrum resistance to CTV in Poncirus trifoliata L. Raf. This locus is present also in tolerant and susceptible species, so an epigenetic mechanism of Ctv expression regulation was proposed. Indeed, a difference in plant 24-nt sRNAs distribution corresponding to CTV.20 gene was previously observed in susceptible species following CTV infection. This gene, encoding for a plant virus movement-like protein, was investigated as a candidate gene for CTV susceptibility. Here, we show the presence of differences in methylation status of a specific region of CTV.20 in two susceptible species, sour and sweet orange, following CTV infection. On the contrary, no significant differences were observed in the tolerant Citrange carrizo following the infection. Moreover, a hypermethylation of the whole CTV.20 gene was observed in Citrange carrizo both healthy and infected, and in healthy sour and sweet orange. This preliminary study allows hypothesizing a possible role of methylation in regulation of CTV.20 expression involved in the CTV susceptibility. [ABSTRACT FROM AUTHOR]

The olive is a fruit tree species with a century-old history of cultivation in the Mediterranean basin. In Apulia (Southern Italy), the olive is of main social, cultural and economic importance, and represents a hallmark of the rural landscape. However, olive cultivation in this region is threatened by the recent spread of the olive quick decline syndrome (OQDS) disease, thus there is an urgent need to explore biodiversity and search for genetic sources of resistance. Herein, a genetic variation in Apulian olive germplasm was explored, as a first step to identify genotypes with enhanced bio-agronomic traits, including resistance to OQDS. A preselected set of nuclear microsatellite markers allowed the acquisition of genotypic profiles, and to define genetic relationships between Apulian germplasm and widespread cultivars. The analysis highlighted the broad genetic variation in Apulian accessions and the presence of different unique genetic profiles. The results of this study lay a foundation for the organization of new breeding programs for olive genetic improvement. [ABSTRACT FROM AUTHOR]

This study aimed to identify and evaluate the genetic diversity of olive trees in Jordan, a country located in the eastern Mediterranean, where olive domestication originated. For this purpose, a total of 386 olive trees were analyzed, including 338 collected from two surveys (JOCC-1 and JOCC-2) across seven regions, and 48 selected accessions from the Olive Germplasm Bank of Jordan (JGBOC). These trees underwent comprehensive phenotypic and molecular characterization using different tools. Significant differences in morphological traits were detected among tested regions using the Chi -square test. Principal components analysis revealed that fruit color change and growth habit as the most discriminating traits, segregating the trees into two groups, with the first group including the Kanabisi cultivar and the second group including the Kfari Baladi cultivar. Utilizing Kompetitive Allele Specific PCR assay, two sets of informative SNPs were used for the genetic diversity analysis. Cladograms were constructed using the maximum likelihood method, revealing a consistent pattern where two clades containing identical genotypes were observed to cluster with the Kfari Baladi or Kanabisi. In addition, the SNP data was used to perform a comparative analysis with the Worldwide Olive Germplasm Bank of Córdoba, which revealed 73 unreported olive genotypes from Jordan. Genetic structure analyses using Discriminant Analysis of Principal Components (DAPC) identified four clusters with distinctive patterns of relatedness among 149 unique accessions, including 52 olive accessions from various Mediterranean countries (IOCC-3). ADMIXTURE analysis revealed four genetic clusters, consistent with the clustering observed in DAPC and cladogram analysis, indicating a high level of genetic admixture among Jordanian olive germplasm. In conclusion, the results show that olive trees in Jordan are highly diverse, providing valuable information for future conservation and management plans., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Al-Kilani, Taranto, D’Agostino, Montemurro, Belaj, Ayoub, Albdaiwi, Hasan and Al-Abdallat.)

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., Competing Interests: CM is a founder member of the Spin Off of the University of Bari SINAGRI S.r.l.; the role of the company was to make available the instruments used in some analysis. The other authors declare no conflicts of interest.

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., Competing Interests: Authors LP, CP were employed by the Spin Off of the University of Bari SINAGRI S.r.l.; authors LP, CP, VR were employed by company Rete Italian Variety Club (IVC). CM is a founder member of the Spin Off of the University of Bari SINAGRI S.r.l.; CM and MMM were scientific consultants in kind for the company Rete Italian Variety Club (IVC). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declare that this study received funding from Rete Italian Variety Club (IVC). The funder had the following involvement in the study: study design, data collection and analysis., (Copyright © 2024 Piarulli, Pirolo, Roseti, Bellin, Mascio, La Notte, Montemurro and Miazzi.)

To accurately determine the spread of any pathogen, including plant viruses, a quick, sensitive, cost-effective, point-of-care diagnostic assay is necessary. Wheat spindle streak mosaic virus (WSSMV) is a Bymovirus, transmitted by the plasmodiophorid Polymyxa graminis Led, which causes yellow mosaic and reduces the grain yield in wheat. Currently, detection protocols for WSSMV use ELISA or more sensitive PCR-based approaches requiring specialized laboratory and personnel. A protocol for reverse transcription loop mediated isothermal amplification (RT-LAMP) has been developed and optimized for the rapid detection of viruses using crude extracts from wheat leaves. The protocol was specific for WSSMV detection, while no reaction was observed with SBCMV or SBWMV, the non-target viruses transmitted by the same vector. The RT-LAMP assay was shown to be as sensitive as the one-step WSSMV specific RT-PCR. The RT-LAMP assay can be performed under field conditions using a portable instrument, and can help the actual spread of WSSMV, an aspect of this virus not yet well understood, to be explored., Competing Interests: The authors declare no competing interests., (Copyright: © 2024 Marra et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Recent studies have demonstrated that fascial fibroblasts are susceptible to mechanical stimuli, leading to the remodeling of the extracellular matrix (ECM). Moreover, the extensive literature on Yes-associated protein (YAP) has shown its role in cell mechanics, linking cell properties, such as shape, adhesion, and size, to the expression of specific genes. The aim of this study was to investigate the presence of YAP in deep fascia and its activation after a mechanical stimulus was induced via a focal extracorporeal shockwave (fESW) treatment. Thoracolumbar fascia (TLF) samples were collected from eight patients (age: 30-70 years; four males and four females) who had undergone spine elective surgical procedures at the Orthopedic Clinic of University of Padova. YAP was measured in both tissue and TLF-derived fibroblasts through immunoblotting. COL1A1 and HABP2 gene expression were also evaluated in fibroblasts 2, 24, and 48 h after the fESW treatment. YAP was expressed in all the examined tissues. The ratio between the active/inactive forms (YAP/p-YAP) of the protein significantly increased in fascial fibroblasts after mechanical stimulation compared to untreated cells ( p = 0.0022). Furthermore, COL1A1 and HABP2 gene expression levels were increased upon treatment. These findings demonstrate that YAP is expressed in the deep fascia of the thoracolumbar region, suggesting its involvement in fascial mechanotransduction processes, remodeling, regeneration, and fibrogenesis. This study indicates, for the first time, that YAP is a "new player" in the mechanobiology of deep fascia.

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., Competing Interests: Author CM was employed by the company SINAGRI S.r.l. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Villano, Procino, Blaiotta, Carputo, D’Agostino, Di Serio, Fanelli, La Notte, Miazzi, Montemurro, Taranto and Aversano.)

Background: Pasta is a worldwide popular Italian food made exclusively of durum wheat. The choice of variety to be used to produce pasta is at the discretion of the producer based on the peculiar characteristics of each cultivar. The availability of analytical approaches for the tracking of specific varieties along the productive chain is becoming increasingly important to authenticate the pasta products and distinguish between fraudulent activities and cross-contaminations during the production process. Among the different methods, molecular approaches based on DNA markers are the most used for these purposes because of their ease of use and high reproducibility., Results: In the present study, we used an easy simple sequence repeats-based method to identify the durum wheat varieties used to produce 25 samples of semolina and commercial pasta comparing their molecular profile with those of the four varieties declared by the producer and other 10 durum wheat cultivars commonly used in pasta production. All of the samples showed the expected molecular profile; however, most of them present also a foreign allele indicating a possible cross-contamination. Moreover, we evaluated the accuracy of the proposed approach through the analysis of 27 hand-made mixtures with increasing amounts of a specific contaminant variety, allowing the estimation of the limit of detection of 5% (w/w)., Conclusion: We demonstrated the feasibility of the proposed method and its effectiveness in the detection of not declared varieties when these are present in a percentage equal to or higher than 5%. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry., (© 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.)

The Prunus genus encompasses a group of economically important and closely related crops, sharing an essentially common genome and, thereby, a high level of conserved and transferable microsatellite (SSR) loci. In Southern Italy, many of the local and/or neglected varieties are abandoned and at risk of extinction due to the high degree of urbanization and agricultural intensification, despite their value as genetic resources for crop improvement. This research aimed to genetically and morphologically characterize the traditional apricot ( P. armenica ) and peach ( P. persica ) germplasms collected in old family orchards. Most of the official descriptor categories were scored, thus revealing a rather high level of phenotypic variation in both collections. Genetic data allowed the discovery of diversity masked by morphological traits. Genotyping in 15 and 18 SSRs, eight of which were transferable across both species, showed an average polymorphic informativeness (PIC) of 0.44 and 0.59 for apricot and peach, respectively, and a total of 70 and 144 alleles. A reliable identification of each genotype was achieved, and the presence of possible mislabeling and/or erroneous denominations was solved. These results are encouraging for the valorization of the still poorly explored Italian Prunus germplasm, with significant economic consequences for bioresource conservation and management.

Soil-borne cereal mosaic virus (SBCMV) is a furovirus with rigid rod-shaped particles containing an ssRNA genome, transmitted by Polymyxa graminis Led., a plasmodiophorid that can persist in soil for up to 20 years. SBCMV was reported on common and durum wheat and it can cause yield losses of up to 70%. Detection protocols currently available are costly and time-consuming (real-time PCR) or have limited sensitivity (ELISA). To facilitate an efficient investigation of the real dispersal of SBCMV, it is necessary to develop a new detection tool with the following characteristics: no extraction steps, very fast results, and high sensitivity to allow pooling of a large number of samples. In the present work, we have developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) protocol with such characteristics, and we have compared it with real-time PCR. Our results show that the sensitivity of LAMP and real-time PCR on cDNA and RT-LAMP on crude extracts are comparable, with the obvious advantage that RT-LAMP produces results in minutes rather than hours. This paves the way for extensive field surveys, leading to a better knowledge of the impact of this virus on wheat health and yield.

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., Competing Interests: CM was employed by Spin Off Sinagri s.r.l. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Miazzi, Babay, De Vita, Montemurro, Chaabane, Taranto and Mangini.)

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 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.

Wine production represents an ancient human activity and one of the most economically important markets in Europe. Moreover, the health effects of grapes and related products have been largely demonstrated, and mostly depend on their richness in bioactive molecules such as flavonoid and non-flavonoid phenolic compounds. Italy has the highest global wine production and provides one of the richest grapevine germplasm in the Mediterranean area. In this paper, our attention was focused on the evaluation of the total phenol and anthocyanin content in five autochthonous Apulian grapevine cultivars, in both wines and their non-alcoholic extracts. Moreover, the potential antioxidant effects of the non-alcoholic wine extracts on the cell viability of Caco-2 and HeLa carcinoma cell lines were tested. Finally, for the most promising autochthonous selected cultivars (Negramaro, Nero di Troia and Susumaniello), comparative transcriptomic analysis in berries was performed using high-throughput sequencing technology., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

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.

Almond cultivation has great traditional and economic relevance in Southern Italy, especially in the Apulia region, where almond trees feature an ample and ancient varietal richness. To contrast the loss of plant genetic erosion and to safeguard the available bioresources, as well as to reinforce the local production, the regional Re.Ge.Fru.P. project aimed to re-evaluate, identify, and characterize the Apulian almond germplasm that is still uncharacterized and not jet studied using a dual (genetic and morphological) approach. Collection was conducted in the regional territory of 187 among the most widespread and minor or marginalized genotypes that were molecularly fingerprinted by means of 18 nuclear microsatellites (simple sequence repeats, SSRs). The high number of scored alleles reflected the great level of diversification within the Apulian germplasm, as also confirmed by neighbor joining and structure analysis, that clearly distinguished different genotype clusters. The phenotypic characterization using 17 morphological and phenological descriptors mirrored the genetic results, revealing a high degree of variability. The morphological traits with the best discriminatory ability were nut ventral suture, shell softness and shape and petal color. This work emphasizes the importance of recovering the genetic variability of Apulian almond germplasm, and the need to promote added value and enhance the local agri-food economy.

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.

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.