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Cannabidiolic acid (CBDA) and trans-Δ

Cannabis sativa L. has been long cultivated for its narcotic potential due to the accumulation of tetrahydrocannabinolic acid (THCA) in female inflorescences, but nowadays its production for fiber, seeds, edible oil and bioactive compounds has spread throughout the world. However, some hemp varieties still accumulate traces of residual THCA close to the 0.20% limit set by European Union, despite the functional gene encoding for THCA synthase (THCAS) is lacking. Even if some hypotheses have been produced, studies are often in disagreement especially on the role of the cannabichromenic acid synthase (CBCAS). In this work a set of European Cannabis genotypes, representative of all chemotypes, were investigated from a chemical and molecular point of view. Highly specific primer pairs were developed to allow an accurate distinction of different cannabinoid synthases genes. In addition to their use as markers to detect the presence of CBCAS at genomic level, they allowed the analysis of transcriptional profiles in hemp or marijuana plants. While the high level of transcription of THCAS and cannabidiolic acid synthase (CBDAS) clearly reflects the chemical phenotype of the plants, the low but stable transcriptional level of CBCAS in all genotypes suggests that these genes are active and might contribute to the final amount of cannabinoids.

Cannabis sativa L. is an annual species cultivated since antiquity for different purposes. While, in the past, hemp inflorescences were considered crop residues, at present, they are regarded as valuable raw materials with different applications, among which extraction of the essential oil (EO) has gained increasing interest in many fields. The aim of the present study is the evaluation of the yield and the chemical composition of the EO obtained by hydrodistillation from eleven hemp genotypes, cultivated in the same location for two consecutive growing seasons. The composition of the EOs was analyzed by GC–MS, and then subjected to multivariate statistical analysis. Sesquiterpenes represented the main class of compounds in all the EOs, both in their hydrocarbon and oxygenated forms, with relative abundances ranging from 47.1 to 78.5%, the only exception was the Felina 32 sample collected in 2019, in which cannabinoids predominated. Cannabinoids were the second most abundant class of compounds, of which cannabidiol was the main one, with relative abundances between 11.8 and 51.5%. The statistical distribution of the samples, performed on the complete chemical composition of the EOs, evidenced a partition based on the year of cultivation, rather than on the genotype, with the exception of Uso-31. Regarding the extraction yield, a significant variation was evidenced among both the genotypes and the years of cultivation.

The recent discovery of the novel heptyl phytocannabinoids cannabidiphorol (CBDP) and Δ9-tetrahydrocannabiphorol (Δ9-THCP) raised a series of questions relating to the presence and abundance of these new unorthodox compounds in cannabis inflorescence or derived products. As fresh inflorescence contains mainly their acid precursors, which are not commercially available, an ad hoc stereoselective synthesis was performed in order to obtain cannabidiphorolic acid (CBDPA) and Δ9-tetrahydrocannabiphorolic acid (THCPA) to be used as analytical standards for quantitative purposes. The present work reports an unprecedented targeted analysis of both pentyl (C5) and heptyl (C7) CBD- and THC-type compounds in forty-nine cannabis samples representing four different chemotypes. Moreover, the ultrahigh performance liquid chromatography coupled to high-resolution mass spectrometry-based method was applied for the putative identification of other heptyl homologs of the most common phytocannabinoid acids, including cannabigerophorolic acid (CBGPA), cannabichromephorolic acid (CBCPA), cannabinophorolic acid (CBNPA), cannabielsophorolic acid (CBEPA), cannabicyclophorolic acid (CBLPA), cannabitriophorolic acid (CBTPA), and cannabiripsophorolic acid (CBRPA).

Cannabis sativa is traditionally classified according to five chemotypes based on the concentration of the main phytocannabinoids tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabigerol (CBG). However, cannabis chemovars and varieties very often present similar concentrations of such phytocannabinoids but different chemical profiles, which is unavoidably translated into different pharmacological effects when used for therapeutic purposes. For this reason, a more refined approach is needed for chemovar distinction, which is described in this study and named phytocannabinomics. The classification was achieved by a comprehensive characterization of the phytocannabinoid composition, by liquid chromatography coupled to high-resolution mass spectrometry untargeted metabolomics for the detection of over a hundred phytocannabinoids, and data analysis by chemometrics for chemovars differentiation. The method was developed on fifty cannabis varieties, grown under the same conditions, and was validated to discriminate between the standard chemotypes by partial least squares discriminant analysis. Then, the method was extended to consider the entire chemical variety of the cannabis accessions, by an unsupervised approach based on the principal component analysis. The latter approach clearly indicated several new subgroups within the traditional classifications, which arise from a unique composition of the minor phytocannabinoids. The existence of these subgroups, which were never described before, is of critical importance for evaluating the pharmacological effects of cannabis chemovars.

Silybum marianum (L.) Gaertn. is an important medicinal plant belonging to Mediterranean flora. The medicinal properties of the species are mainly due to silymarin, a combination of different flavonolignans contained in the fruit. As for silymarin, so far a wide variability of possible S. marianum chemotypes has been described. In the present study the flavonolignan profile of 40 different S. marianum wild accessions was analysed at both population and single plant level, further extending the analysis to progenies derived from crosses between parental lines with different chemotypes. The results of this work indicate that S. marianum wild populations can be composed either of individuals with the same chemotype, or heterogeneous mixtures of individuals characterized by different chemotypes. Only three chemotypes (A, B and C) have been identified among Italian wild populations. Based on data collected we furthermore propose that chemotype C is the result of the hybridization between A and B chemotypes. If assessed at single plant level, chemotypes are extremely stable therefore evidencing a strong genetic control of silymarin biosynthetic pathway. Chemotypes A and B are present in all the analysed regions and no clear correlation between chemotypes and geographic features has been found. In conclusion, this work provides a general procedure for the characterization of different and stable chemotypes, for a deeper understanding of silymarin biosynthetic pathway, and in order to implement S. marianum breeding programmes aiming to improve silymarin quality.

L-Kynurenine (KYN) and kynurenic acid (KYNA) are products of the metabolism of L-tryptophan (TRP) in the central nervous system of animals, but they are not commonly found in plants. In particular, KYNA is known for its interesting pharmacological properties (anti-oxidative, anti-inflammatory, hypolipidemic, and neuroprotective), which suggest a potential functional food ingredient role. The three compounds were identified in samples of Cannabis sativa L. by means of high-performance liquid chromatography coupled to high-resolution mass spectrometry using an untargeted metabolomics approach. Their concentrations were evaluated using a targeted metabolomics method in three organs of the plant (roots, stem, and leaves) in soil at two different growth stages and in hydroponics conditions. The distribution of TRP, KYN and KYNA was found tendentially higher in leaves compared to stem and roots and changed over time. Moreover, the levels of KYNA found in this study are unprecedentedly high compared to those found so far in other plant species, suggesting that Cannabis sativa L. could be a promising alternative source of this metabolite.

Very recently, the genome of the modern durum wheat cv. Svevo was fully sequenced, and its assembly is publicly available. So, we exploited the opportunity to carry out an in-depth study for the systematic characterization of the γ-gliadin gene family in the cv. Svevo by combining a bioinformatic approach with transcript and protein analysis. We found that the γ-gliadin family consists of nine genes that include seven functional genes and two pseudogenes. Three genes, Gli-γ1a, Gli-γ3a and Gli-γ4a, and the pseudogene Gli-γ2a* mapped on the A genome, whereas the remaining four genes, Gli-γ1b, Gli-γ2b, Gli-γ3b and Gli-γ5b, and the pseudogene Gli-γ4b* mapped on the B genome. The functional γ-gliadins presented all six domains and eight-cysteine residues typical of γ-gliadins. The Gli-γ1b also presented an additional cysteine that could possibly have a role in the formation of the gluten network through binding to HMW glutenins. The γ-gliadins from the A and B genome differed in their celiac disease (CD) epitope content and composition, with the γ-gliadins from the B genome showing the highest frequency of CD epitopes. In all the cases, almost all the CD epitopes clustered in the central region of the γ-gliadin proteins. Transcript analysis during seed development revealed that all the functional γ-gliadin genes were expressed with a similar pattern, although significant differences in the transcript levels were observed among individual genes that were sometimes more than 60-fold. A progressive accumulation of the γ-gliadin fraction was observed in the ripening seeds that reached 34% of the total gliadin fraction at harvest maturity. We believe that the insights generated in the present study could aid further studies on gliadin protein functions and future breeding programs aimed at the selection of new healthier durum wheat genotypes.

Apple scab, caused by the apoplastic fungal pathogen Venturia inaequalis , is one of the most damaging apple diseases worldwide and scab resistance is still one of the most important goals in apple breeding programmes. Improving knowledge of defence systems against pathogens through the identification of resistance and defence-related genes enables the development of molecular tools that can be used for assisted-selection of elite varieties. The apple scab Rvi6 (formerly HcrVf2 ) resistance gene has been extensively used to breed resistant varieties, and several defence-related genes activated downstream have recently been identified. In this work, subtractive hybridisation was performed in order to collect cDNA differentially expressed in the resistant Rvi6 cultivar ‘Florina’ after challenge with the fungus and treatment with salicylic acid. A total of 97 unique sequences were identified, and most of them were successfully annotated and located on the apple genome in silico . The expression profiles of twelve candidate genes were analysed using quantitative real-time PCR in both incompatible and compatible Malus - V.inaequalis interactions. The responses induced by the two treatments differed significantly, suggesting that salicylic acid might be not involved in apple scab defence mechanisms. Some candidate genes are involved in well-known defence strategies against apoplastic fungal pathogens, such as the reinforcement and maintenance of cell wall integrity and fungal growth hindrance. Others are presumed to play a role in the signal transduction pathway, in a similar way to that known for other pathosystems. Finally, potential mechanisms involved in the defence response against apple scab are proposed.

The use of natural compounds to preserve fruit quality and develop high value functional products deserves attention especially in the growing industry of processing and packaging ready-to-eat fresh-cut fruit. In this work, potential mechanisms underlying the effects of postharvest biofumigation with brassica meal-derived allyl-isothiocyanate on the physiological responses and quality of ‘Hayward’ kiwifruits were studied. Fruits were treated with 0.15 mg L−1 of allyl-isothiocyanate vapours for 5 h and then stored in controlled atmosphere (2% O2, 4.5% CO2) at 0 °C and 95% relative humidity, maintaining an ethylene concentration

The pink ear rot is one of the most damaging maize diseases, caused by the mycotoxigenic fungal pathogen, Fusarium verticillioides. The application of biological control agents, like antagonistic and/or resistance inducer microorganisms, is an option to reduce fungal infection and kernel contamination in a sustainable and environmentally friendly way. It is well known that Trichoderma species are non-pathogenic fungi able to antagonize plant pathogens and to induce systemic resistance in plants. The present work aimed to verify if Trichoderma spp., applied to maize kernels, affect the plant growth and induce systemic responses to F. verticillioides. Besides, the capability to reduce fumonisin concentration in liquid cultures was investigated. Two T. gamsii (IMO5 and B21), and one T. afroharzianum (B75) isolates, selected both for antagonism and for the ability to reduce root infections, significantly reduced the endophytic development of the stem-inoculated pathogen, compared to the control. The mechanisms of action appeared to be strain-specific, with IMO5 enhancing transcript levels of marker genes of Induced Systemic Resistance (ZmLOX10, ZmAOS, and ZmHPL) while B21 enhancing marker genes of Systemic Acquired Resistance (ZmPR1 and ZmPR5), as evinced by measuring their expression profiles in the leaves. Moreover, IMO5 promoted plant growth, while B21 was able to significantly reduce the fumonisin content in a liquid medium. The results of this work give new evidence that the seed application of T. gamsii is a promising tool for controlling F. verticillioides to be integrated with breeding and the adoption of good agricultural practices.

Quantitative reverse transcription PCR is a sensitive technique for evaluating transcriptional profiles in different experimental datasets. To obtain a reliable quantification of the transcripts level, data normalization with stable reference genes is required. Stable reference genes are identified after analysis of their transcripts profile in every new experiment and species of interest. In Silybum marianum, a widely cultivated officinal plant, only few gene expression studies exist, and reference genes for RT-qPCR studies in the diverse plant tissues have never been investigated before. In this work, the expression stability of 10 candidate reference genes was evaluated in leaves, roots, stems and fruits of S. marianum grown under physiological environmental condition. The stability values for each candidate reference gene were calculated by four canonical statistical algorithms GeNorm, NormFinder, Bestkeeper and ΔCt method in different subsets of samples, then they were ranked with RefFinder from the most to the least suitable for normalization. Best combinations of reference genes are finally proposed for different experimental data sets, including all tissues, vegetative, and reproductive tissues separately. Three target genes putatively involved in important biosynthetic pathway leading to key metabolites in the fruits of milk thistle, such as silymarin and fatty acids, were analyzed with the chosen panels of reference genes, in comparison to the ones used in previous papers. To the best of our knowledge, this is the first report on a reliable and systematic identification and validation of the reference genes for RT-qPCR normalization to study gene expression in S. marianum.

Plant secondary metabolic pathways are finely regulated by the activity of transcription factors, among which members of the bHLH and MYB subfamilies play a main role. Cannabis sativa L. is a unique officinal plant species with over 600 synthesized phytochemicals having diverse scale-up industrial and pharmaceutical usage. Despite comprehensive knowledge of cannabinoids&rsquo, metabolic pathways, very little is known about their regulation, while the literature on flavonoids&rsquo, metabolic pathways is still scarce. In this study, we provide the first genome-wide analysis of bHLH and MYB families in C. sativa reference cultivar CBDRx and identification of candidate coding sequences for these transcription factors. Cannabis sativa bHLHs and MYBs were then classified into functional subfamilies through comparative phylogenetic analysis with A. thaliana transcription factors. Analyses of gene structure and motif distribution confirmed that CsbHLHs and CsMYBs belonging to the same evolutionary clade share common features at both gene and amino acidic level. Candidate regulatory genes for key metabolic pathways leading to flavonoid and cannabinoid synthesis in Cannabis were also retrieved. Furthermore, a candidate gene approach was used to identify structural enzyme-coding genes for flavonoid and cannabinoid synthesis. Taken as a whole, this work represents a valuable resource of candidate genes for further investigation of the C. sativa cannabinoid and flavonoid metabolic pathways for genomic studies and breeding programs.

Tree Genetics & Genomes, 5 (2), ISSN:1614-2950, ISSN:1614-2942

Interveinal chlorosis and leaf margin wrinkling are widespread symptoms of Cannabis sativa. They are traditionally attributed to the so-called hemp streak virus (HSV), but its existence has not been demonstrated yet. To our knowledge, no molecular investigation has so far been performed in order to identify the causal agent of this symptomatology, we therefore decided to use traditional and molecular virology techniques to better characterize symptoms and pursue the etiological agent. No pathogenic virus was found by using targeted PCR reactions and by RNA sequencing, whereas we were able to detect the Cannabis cryptic virus (CanCV) with both techniques. We, therefore, developed an RT-qPCR assay based on a CanCV-specific TaqMan probe and applied it to a wide range of symptomatic and symptomless plants, using a two-step (for quantification), or a one-step (for fast detection) protocol. Both symptoms and the virus were only shown to be transmitted vertically and did not pass via mechanical inoculation or grafting, though we could not find any cause-effect correlation between them. In fact, the virus was found in all the tested hemp samples, and its abundance varied greatly between different accessions and individuals, independently from the presence and severity of symptoms. The suggestion that hemp streak is caused by a virus is therefore questioned. Some abiotic stresses seem to play a role in triggering the symptoms but this aspect needs further investigation. For breeding purposes, a selection of parental plants based on the absence of symptoms proved to be efficient in containment of the disease.

Allergy to freshly consumed apple fruits is often associated to pollinosis and manifested as oral allergy syndrome (OAS). The allergenic properties of apple varieties differ greatly, spanning from low allergenic to high allergenic varieties. The knowledge of the genetic determinants for allergenicity has been of great interest in scientific community for several years, but the molecular mechanisms involved are still little understood. Here, factors putatively involved in allergenicity were investigated at biochemical and molecular level in pollen and in fruits of apple varieties differing in their allergenic potential. Among putative sensitizing factors, transglutaminase (TGase) and phospholipase A2(PLA2) were considered together with reactive oxygen species (ROS) and known apple allergen genes, with particular attention devoted to the Mal d 1 gene family, the most important one in sensitization. We found that the expression of some allergen genes and the activities of TGase, PLA2and ROS producing enzyme are lower in the hypo-allergenic variety ‘Durello di Forlì’ in comparison with the high-allergenic genotypes ‘Gala’ and ‘Florina’. These results highlight correlations among allergen expressions, enzymatic activities and apple cultivars; these data underline the possibility that some of them could be used in the future as markers for allergenicity.

Background The availability of the peach genome sequence has fostered relevant research in peach and related Prunus species enabling the identification of genes underlying important horticultural traits as well as the development of advanced tools for genetic and genomic analyses. The first release of the peach genome (Peach v1.0) represented a high-quality WGS (Whole Genome Shotgun) chromosome-scale assembly with high contiguity (contig L50 214.2 kb), large portions of mapped sequences (96%) and high base accuracy (99.96%). The aim of this work was to improve the quality of the first assembly by increasing the portion of mapped and oriented sequences, correcting misassemblies and improving the contiguity and base accuracy using high-throughput linkage mapping and deep resequencing approaches. Results Four linkage maps with 3,576 molecular markers were used to improve the portion of mapped and oriented sequences (from 96.0% and 85.6% of Peach v1.0 to 99.2% and 98.2% of v2.0, respectively) and enabled a more detailed identification of discernible misassemblies (10.4 Mb in total). The deep resequencing approach fixed 859 homozygous SNPs (Single Nucleotide Polymorphisms) and 1347 homozygous indels. Moreover, the assembled NGS contigs enabled the closing of 212 gaps with an improvement in the contig L50 of 19.2%. Conclusions The improved high quality peach genome assembly (Peach v2.0) represents a valuable tool for the analysis of the genetic diversity, domestication, and as a vehicle for genetic improvement of peach and related Prunus species. Moreover, the important phylogenetic position of peach and the absence of recent whole genome duplication (WGD) events make peach a pivotal species for comparative genomics studies aiming at elucidating plant speciation and diversification processes. Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-3606-9) contains supplementary material, which is available to authorized users.

Apple germplasm collections are increasingly appreciated as a repository for the genetic improvement of species, and their evaluation is an essential prerequisite for their utilization in apple breeding. A set of 418 apple genotypes, including 383 accessions from the Italian germplasm and 35 International cultivars as reference, was analyzed using 15 SSRs with the aim of assessing the genetic diversity within this panel of varieties, evaluating relationships among them and determining their genetic structure. Genetic analyses performed by Bayesian model-based clustering revealed a clear differentiation of two major groups (G1 and G2). Local Italian accessions were grouped mainly in G2 while all except one of the reference cultivars were found in G1. Each of these two clusters has been further divided into two subgroups by a nested approach. These results were confirmed by factorial correspondence (FCA) and molecular variance (AMOVA) analyses. A core collection of 55 accessions, representative of the Italian apple germplasm and capable of retaining all the 238 SSR alleles detected on 192 unique genotypes, was established by the M-strategy method. The Italian apple germplasm represents an important source of genetic diversity which can be used, in addition to other characterized European germplasm collections, to optimize the efficiency of genome-wide association studies aimed at identifying the genomic regions controlling major horticultural traits.

Author details Wageningen UR Plant Breeding, Plant Research International, Droevendaalsesteeg 1, Wageningen PB 6708, The Netherlands. Department of Fruit Tree and Woody Plant Sciences, University of Bologna, Viale Fanin 46, Bologna 40127, Italy. Department of Paediatrics, University of Bologna, Via Massarenti 11, Bologna 40138, Italy. Present address: Consiglio per la Ricerca e la sperimentazione in Agricoltura-Centro di Ricerca per le Colture Industriali, via di Corticella 133, Bologna 40128, Italy.

cDNA-AFLP analysis for transcript profiling has been successfully applied to study many plant biological systems, particularly plant-microbe interactions. However, the separation of cDNA-AFLP fragments by gel electrophoresis is reported to be labor-intensive with only limited potential for automation, and the collection of differential bands for gene identification is even more cumbersome. In this work, we present the use of dHPLC (denaturing high performance liquid chromatography) and automated DNA fragment collection using the WAVE(®) System to analyze and recover cDNA-AFLP fragments. The method is successfully applied to the Malus-Venturia inaequalis interaction, making it possible to collect 66 different transcript-derived fragments for apple genes putatively involved in the defense response activated by the HcrVf2 resistance gene. The results, validated by real time quantitative RT-PCR, were consistent with the plant-pathogen interaction under investigation and this further supports the suitability of dHPLC for cDNA-AFLP transcript profiling. Features and advantages of this new approach are discussed, evincing that it is an almost fully automatable and cost-effective solution for processing large numbers of samples and collecting differential genes involved in other biological processes and non-model plants.

European populations exhibit progressive sensitisation to food allergens, and apples are one of the foods for which sensitisation is observed most frequently. Apple cultivars vary greatly in their allergenic characteristics, and a better understanding of the genetic basis of low allergenicity may therefore allow allergic individuals to increase their fruit intake. Mal d 1 is considered to be a major apple allergen, and this protein is encoded by the most complex allergen gene family. Not all Mal d 1 members are likely to be involved in allergenicity. Therefore, additional knowledge about the existence and characteristics of the different Mal d 1 genes is required. In the present study, we investigated the genomic organisation of the Mal d 1 gene cluster in linkage group 16 of apple through the sequencing of two bacterial artificial chromosome clones. The results provided new information on the composition of this family with respect to the number and orientation of functional and pseudogenes and their physical distances. The results were compared with the apple and peach genome sequences that have recently been made available. A broad analysis of the whole apple genome revealed the presence of new genes in this family, and a complete list of the observed Mal d 1 genes is supplied. Thus, this study provides an important contribution towards a better understanding of the genetics of the Mal d 1 family and establishes the basis for further research on allelic diversity among cultivars in relation to variation in allergenicity. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11032-011-9588-4) contains supplementary material, which is available to authorized users.

Summary To evaluate differences in skin prick test response to apple cultivars in patients with apple allergy, 19 patients (10 adults and 9 children) underwent prick-to-prick skin prick test with eleven commercial and noncommercial apple cultivars, and evaluation of specific IgE to apple and recombinant apple allergens Mal d 1 and Mal d 3. The results show that different reactions might be evoked in a single patient by different apple cultivars and also separately for the peel and the pulp of a single cultivar. The cultivars were ranked according to their allergenicity level, being Jonathan, Jonagold, Golden Delicious and Fiesta the most allergenic when considering the pulp and Gala, Fiesta and Golden Delicious for the peel. Further investigations are needed to clarify if a single patient can be allergic only to well-defined apple cultivars and which allergometric tests are necessary to ascertain this.

Apple scab resistance is one of the most well-characterized plant-pathogen interactions in woody plant species. After the cloning of the apple scab resistance gene HcrVf2, we aimed at identifying the network of genes that are differentially expressed after pathogen challenge by cDNA-AFLP. As electrophoretic analyses are labour-intensive with only limited potential for automation and the recovery of DNA fragments from gels is cumbersome, we investigated the possibility of performing the cDNA-AFLP analysis by d-HPLC (denaturing high performance liquid chromatography) and automating the DNA fragments collection using the Transgenomic WAVE® System. Direct sequencing of the collected DNA fragments was also carried out and our preliminary results are reported.

Main conclusion Major metabolic pathways and genes affected by low-temperature treatment were identified and a thorough picture of the early transcriptional changes in sugar beet plantlets upon cold stress was given. Sugar beet (Beta vulgaris L.) is an important source of sugar and bioethanol production in temperate areas worldwide. In these areas, plantlet survival and sucrose yield of mature plants can be seriously limited by low temperatures, especially when plantlets are exposed to freezing temperatures (below 0 °C) at the early developmental stages. This frequently occurs when the crop is sown in early spring or even in autumn (autumn sowing) to escape drought at maturity and pathogen outbreaks. The knowledge of molecular responses induced in plantlets early upon exposure to low temperature is necessary to understand mechanisms that allow the plant to survive and to identify reactions that can influence other late-appearing traits. In this work, a wide study of sugar beet transcriptome modulation after a short exposure to a cold stress, mimicking what is experienced in vivo by young plantlets when temperature drops in the early spring nights, was carried out by high-throughput sequencing of leaves and root RNAs (RNA-Seq). A significant picture of the earliest events of temperature sensing was achieved for the first time for sugar beet: the retrieval of a great amount of transcription factors and the intensity of modulation of a large number of genes involved in several metabolic pathways suggest a fast and deep rearrangement of sugar beet plantlets metabolism as early response to cold stress, with both similarities and specificities between the two organs.

Scab resistance is one of the most important goals of apple breeding, typically achieved by time-consuming and expensive conventional breeding techniques. Cisgenesis, which is the genetic modification of a recipient organism with genes from a crossable—sexually compatible—organism, is a promising tool for plant breeding to develop disease resistance in a rapid way. A cisgenic, scab-resistant line of the apple variety ‘Gala’ expressing the native apple scab resistance gene Rvi6 (formerly HcrVf2) under control of its own regulatory sequences has been recently developed. In this paper, we present the results from a phenotypic, molecular and biochemical evaluation of clonal replicates of this line (C11.1.53). The phenotype (shoot length, shoot diameter, internode length, number of leaves, leaf length and leaf width) of C11.1.53 was compared to that of the Gala parental background over a period of 108 days. Only a few statistically significant differences were detected, which are probably due to small differences in the quality of the budwood used for grafting rather than effects related to the presence of the cisgene. As the expression of a resistance gene can affect the downstream cascade of plant defence responses, a selection of apple defence-related genes was analyzed by quantitative real-time PCR analysis. These genes are also known as major allergen genes in apple. Even if three out of ten apple allergen genes tested in the leaves differed in the cisgenic line compared to both Gala (background) and ‘Florina’ (the variety from which the Rvi6 gene was cloned), using 2D-PAGE, we were unable to find any significant difference in the expressed proteomes of the leaves of C11.1.53 compared to Gala. Results are discussed in the context of a possible use of cisgenic lines for fruit crop improvement.

WiFi Direct introduces new opportunities to deploy real opportunistic networks through users' mobile devices. However, its original specification does not take into account all the parameters that can emerge from an opportunistic network scenario, not only in terms of technical requirements (e.g., available resources and connectivities) but also of users characteristics and profiles, which can heavily influence the system's performances and devices' interactions. In this work we investigate the feasibility of creating opportunistic networks on top of WiFi Direct framework by analyzing the protocol's performances in real scenarios with a variable number of mobile devices. Experimental results show the times required to form a group of variable size and the best configurations to support opportunistic networking operations and upper layer applications.

Analisi comparativa degli allergeni e degli enzimi coinvolti in reazioni allergiche in polline di diverse cultivars di Malus domestica Rosa Anna Iorioa, Roberta Parisb, Giulia Pagliaranib, Stefano Tartarinib, Giampaolo Riccic, Donatella Serafini-Fracassinia and Stefano Del Ducaa* aDepartment of BiGeA, University of Bologna, via Irnerio 42, 40126, Bologna, Italy. stefano.delduca@unibo.it b Department of Fruit Tree and Woody Plant Sciences, University of Bologna, Viale Fanin 46, Bologna 40127, Italy cDepartment of Medical Science of the Gozzadini Children's Hospital Sant'Orsola-Malpighi, University of Bologna, via Massarenti 11, 40138, Bologna, Italy. L'allergia alla mela è un problema frequente per un numero crescente di cittadini europei: è uno dei frutti che più comunemente causa problemi di sensibilizzazione allergica, esistono diverse cultivars con livelli variabili di allergenicità (la cui sintomatologia clinica è nota) e la sua coltivazione copre un ruolo importante nell’agricoltura europea, specialmente in quella italiana. Per quanto riguarda l’allergia al polline di melo esistono pochi riferimenti in letteratura probabilmente perché, non essendo un polline anemofilo ma entomofilo, è presente in minore quantità in atmosfera e quindi anche se allergenico è difficilmente responsabile di allergie respiratorie; si sono comunque osservati casi frequenti tra gli agricoltori che stanno a contatto con colture arboree. Un fenomeno diffuso è la cosiddetta sindrome betulla-frutta-verdura, dove i pazienti sensibilizzati all'allergene maggiore di polline di betulla Bet v 1, cross-reagiscono con proteine omologhe presenti in frutta e verdura, come la mela. Generalmente, i sintomi sono limitati alla cavità orale, la cosiddetta sindrome orale allergica (SOA). L'allergenicità alla mela è un fenomeno complesso a causa del numero elevato di determinanti genetici e fattori ambientali, ma sono stati pubblicati alcuni studi sulla caratterizzazione genetica e genomica degli allergeni. Nel genoma del melo sono state identificate quattro classi principali di allergeni, chiamate Mal d 1, 2, 3 e 4, organizzate in famiglie multigeniche contenenti diverse isoforme differenzialmente espresse nella frutta. Ad oggi, solo poche varietà di mele sono riconosciute per caratteristiche di ipoallergenicità. Lo scopo del nostro lavoro è stato quello di caratterizzare e confrontare l’allergenicità del polline di diverse cultivars di Malus domestica, molto diffuse nella regione del mediterraneo - Gala, Florina, Golden Delicious, Durello - in modo da poterle classificare in funzione del grado di allergenicità (iperallergeniche, mediamente allergeniche o ipoallergeniche). Lo studio di espressione genica è stato condotto sulle seguenti proteine: PR10-like protein (Mal d 1), thaumatin-like protein (TLP, Mal d 2), non specific lipid transfer proteins (nsLTP, Mal d 3) e profilina (Mal d 4); inoltre è stato valutato anche il profilo di espressione di enzimi quali fosfolipasi A2 (PLA2) e transglutaminasi (TGase), coinvolti nella reazione allergica. Per ogni famiglia genica sono state analizzate diverse isoforme grazie all’impiego di coppie di primer specifici disegnati per ogni sottofamiglia e scelti in accordo con il loro profilo di espressione genetica nel frutto della mela. Gli studi biochimici tramite Western blots con anticorpi diretti contro gli allergeni e gli enzimi coinvolti nella reazione infiammatoria ed i saggi ELISA-modificati con l’uso di siero di pazienti allergici al frutto hanno fornito elementi interessanti sulla ricerca di agenti sensibilizzanti in polline. Questi dati sono in linea con quelli pubblicati sulla classificazione dei frutti, dove Gol¬den e Gala sono riportati come iperallergenici; un dato innovativo è invece la ca¬ratterizzazione del potenziale allergenico di Florina, iperallergenica, e di Durel¬lo, ipoallergenico, su cui i dati bibliografici sono scarsi anche per il frutto. Proseguendo questa strada sperimentale e con approfondimenti ulteriori si potrebbe essere in grado di produrre alimenti a bassa allergenicità le cui caratteristiche dichiarate darebbero un valore aggiunto all’alimento.

Organic farming practices are commonly thought to reduce the environmental impact of agriculture and to preserve the naturalness of the products. Herein, we report the effect of crop management practices on nutritional and toxicological value of potato tubers. Comparative studies are often controversial and the results are dependent on genotype and methodological approach. Targeted analysis and “omics” strategies are discussed, pointing at the nutritional aspects and the corresponding biological and molecular processes involved. Organic farming supporters still do not accept the use of genetic modification to produce new varieties suited for organic agriculture and crop improvement by genetic engineering still sparks hot debate among various scientific and social factions whose major concern is the possible existence of unintended effects both on human and world health. In this context, the advent of “new plant breeding techniques” has reignited the discussion on genetic engineering and on the compatibility of the new technologies with an eco-friendly agriculture. Could cisgenic and genome-edited potatoes be new good options for organic agriculture? We discuss how these approaches can be used to address food security challenges and to overcome specific problems based on the biological characteristics of potato tubers, producing new varieties that can improve farmers’ profit with a lower impact on public opinion. However, political, ethical, and social fears will probably persist much longer, mainly in Italy, historically a fiercely anti-GM country with a European leadership in organic food production and export.

Genetic transformation of fruit tree species is a promising tool to improve one specific trait of a cultivar without changing its overall characteristics. However, Genetically Modified (GM) foods are not accepted worldwide; in Europe there is persistent suspicion of GM crops, and supermarkets often display the label “GM-free”. One of the main health safety concerns about GM foods relates to allergenicity. Fruits of the Rosaceae family, mainly apples, are reported as the plant food most frequently involved in allergic reactions. To date four major families of apple allergens are reported, i.e., Mal d 1, Mal d 2, Mal d 3 and Mal d 4, each including several genes called also isoforms. Mal d 1, -2 and -3 are also known as pathogenesis-related (PR) proteins, while Mal d 4 is classified as profilin with a putative actin-binding role. Within the European ISAFRUIT project the potential risk of increased allergenicity in GM plants was investigated through the study of allergen genes expression in HcrVf2 transgenic scab resistant lines, in comparison with the isogenic cultivar and a classically bred cultivar with the same resistance gene.