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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.

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.

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.

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.

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.

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.

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.

The Malus–Venturia inaequalis interaction is the most studied plant–pathogen interaction involving a woody species. Besides the cloning of an apple scab resistance gene HcrVf2, several sequences have been recently identified that are modulated after pathogen recognition in Vf-resistant genotypes. Among these, there is a putative leucine-rich repeat receptor-like protein kinase from the apple scab-resistant cv. Florina, named LRPKm1 that is induced after V. inaequalis inoculation and salicylic acid treatment. In this work, the isolation, characterization, and mapping of four new genes belonging to the LRPKm multigene family are reported. According to their cumulative expression profiles in HcrVf2-transgenic and wild-type apple plants treated with V. inaequalis, LRPKm genes have been divided in two groups. LRPKm1 and LRPKm3, giving a response related to the presence of HcrVf2, are probably involved in the recognition of pathogen-derived signals. LRPKm2 and LRPKm4, with an expression profile unrelated to the HcrVf2 gene, are putatively involved in the plant basal defense. Furthermore, we have localized LRPKm proteins at the cytological level in the plasma membrane of epidermal cells in resistant genotypes following pathogen challenge, thus confirming software predictions and molecular results. The possible involvement of LRPKm proteins in apple scab resistance and in the plant basal defense makes them attractive for a better comprehension of the molecular mechanisms of the signal transduction pathways after pathogen recognition.

The interaction of apple genotypes and Venturia inaequalis, the causal agent of apple scab, is nowadays the most studied plant-pathogen interaction in a non-model woody plant. After the cloning of the apple scab resistance gene HcrVf2, the cascade of reactions induced after pathogen recognition is under investigation. To understand the gene networks that underlie plant defense responses, it is necessary to identify and characterize the genes responding to pathogen infection. Young leaf samples were collected from genetically modified ‘Gala’ plants carrying the HcrVf2 gene, at different times after inoculation with V. inaequalis. A cDNA-AFLP procedure, successfully applied to study plant-pathogen interactions, has been chosen in order to identify sequences (TDFs, transcript derived fragments) that are differentially expressed after pathogen inoculation. An optimized and highly reproducible cDNA-AFLP protocol was set up on PAGE, starting with an RNA extraction from apple leaves until gel band elution from polyacrylamide gels. The feasibility of this cDNA-AFLP protocol by the dHPLC for fragment separation in order to automatize all band elution steps will be discussed.

Apple scab resistance is one of the most wellcharacterized plant–pathogen interactions in a woody plant species. While the HcrVf2 gene from the wild apple Malus floribunda 821 has proved capable of conferring scab resistance to the susceptible cv. Gala after genetic transformation, its identification represents only the first step in understanding the molecular mechanisms and, hence, the network of genes underlying the defence response. We used a PCR-based suppression subtractive hybridization to identify apple genes that are differentially expressed after Venturia inaequalis inoculation. Subtractive hybridization was performed between cDNA from challenged leaves of HcrVf2-resistant transgenic Gala and susceptible cv. Gala plants. A library of 523 unigenes was constructed and characterized by assigning a putative function via comparison with public databases. This set of pathogen-modulated apple genes includes many defence-related genes and is therefore an important source of information for understanding the molecular basis of the Malus–V. inaequalis interaction.

Background - A considerable number of individuals suffer from oral allergy syndrome (OAS) to apple, resulting in the avoidance of apple consumption. Apple cultivars differ greatly in their allergenic properties, but knowledge of the causes for such differences is incomplete. Mal d 1 is considered the major apple allergen. For Mal d 1, a wide range of isoallergens and variants exist, and they are encoded by a large gene family. To identify the specific proteins/genes that are potentially involved in the allergy, we developed a PCR assay to monitor the expression of each individual Mal d 1 gene. Gene-specific primer pairs were designed for the exploitation of sequence differences among Mal d 1 genes. The specificity of these primers was validated using both in silico and in vitro techniques. Subsequently, this assay was applied to the peel and flesh of fruits from the two cultivars 'Florina' and 'Gala'. Results - We successfully developed gene-specific primer pairs for each of the 31 Mal d 1 genes and incorporated them into a qRT-PCR assay. The results from the application of the assay showed that 11 genes were not expressed in fruit. In addition, differential expression was observed among the Mal d 1 genes that were expressed in the fruit. Moreover, the expression levels were tissue and cultivar dependent. Conclusion - The assay developed in this study facilitated the first characterisation of the expression levels of all known Mal d 1 genes in a gene-specific manner. Using this assay on different fruit tissues and cultivars, we obtained knowledge concerning gene relevance in allergenicity. This study provides new perspectives for research on both plant breeding and immunotherapy.

Background Scab, caused by the fungus Venturia inaequalis, is one of the most important diseases of cultivated apple. While a few scab resistance genes (R genes) governing qualitative resistance have been isolated and characterized, the biological roles of genes governing quantitative resistance, supposed to be more durable, are still unknown. This study aims to investigate the molecular mechanisms involved in the partial resistance of the old Belgian apple cultivar ‘Président Roulin’ against V. inaequalis. Results A global gene expression analysis was conducted in ‘Président Roulin’ (partially resistant) and in ‘Gala’ (susceptible) challenged by V. inaequalis by using the cDNA-AFLP method (cDNA-Amplified Fragment Length Polymorphism). Transcriptome analysis revealed significant modulation (up- or down-regulation) of 281 out of approximately 20,500 transcript derived fragments (TDFs) in ‘Président Roulin’ 48 hours after inoculation. Sequence annotation revealed similarities to several genes encoding for proteins belonging to the NBS-LRR and LRR-RLK classes of plant R genes and to other defense-related proteins. Differentially expressed genes were sorted into functional categories according to their gene ontology annotation and this expression signature was compared to published apple cDNA libraries by Gene Enrichment Analysis. The first comparison was made with two cDNA libraries from Malus x domestica uninfected leaves, and revealed in both libraries a signature of enhanced expression in ‘Président Roulin’ of genes involved in response to stress and photosynthesis. In the second comparison, the pathogen-responsive TDFs from the partially resistant cultivar were compared to the cDNA library from inoculated leaves of Rvi6 (HcrVf2)-transformed ‘Gala’ lines (complete disease resistance) and revealed both common physiological events, and notably differences in the regulation of defense response, the regulation of hydrolase activity, and response to DNA damage. TDFs were in silico mapped on the ‘Golden Delicious’ apple reference genome and significant co-localizations with major scab R genes, but not with quantitative trait loci (QTLs) for scab resistance nor resistance gene analogues (RGAs) were found. Conclusions This study highlights possible candidate genes that may play a role in the partial scab resistance mechanisms of ‘Président Roulin’ and increase our understanding of the molecular mechanisms involved in the partial resistance against apple scab. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-1043) contains supplementary material, which is available to authorized users.