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MareyMap plot of TxE linkage map. Vertical bars indicate the putative position of the centromere. The solid line represents the recombination rate plotted along the 8 pseudomolecules calculated using the cubic spline method. (PDF 236Â kb)

Anchoring of the peach scaffolds to the three genetic maps. Colored bars represent the 8 linkage groups: pink for PxF, purple for TxE and blue for CxA. WGS scaffolds were positioned in each pseudomolecule (Pp01 to Pp08) with the corresponding genetic markers and are depicted in three different colors (dark blue, pink and pale blue); genetic markers are in the same colors of the corresponding WGS scaffolds. The zero (0) denotes the six scaffolds placed with random orientation along the pseudomolecules. The asterisk (*) indicates the two scaffolds with random order. The crosshatch (#) indicates the two scaffolds with the wrong order in Peach v2.0 that need to be inverted in a future release. (PDF 495Â kb)

MareyMap plot of CxA linkage map. Vertical bars indicate the putative position of the centromere. The solid line represents the recombination rate plotted along the 8 pseudomolecules calculated using the cubic spline method. (PDF 233Â kb)

MareyMap plot of PxF linkage maps (including the F1 and recurrent parent maps). Vertical bars indicate the putative position of the centromere. The solid line represents the recombination rate plotted along the 8 pseudomolecules calculated using the cubic spline method. (PDF 405Â kb)

The power to identify, map and quantify QTL underlying complex traits may substantially increase when considering multiple segregating progenies simultaneously. The relatedness among these progenies can be fully exploited when the pedigree is known and the ancestors are available for genotyping. Previously, we successfully used the lexQTL software to map QTL in such a Pedigree Based Analysis (PBA) approach to a large pedigreed population in apple with only 87 SSR markers (Bink et al. 2014). The current availability of high-throughput SNP genotyping infrastructures allows marker genotyping at much higher densities, which will contribute to higher mapping resolution and more accurate QTL characterization but also poses challenges to the QTL mapping software. Within the framework of the projects SCRI-RosBREED (#2009-51181-05808) and EU-FP7 FruitBreedomics (#FP7- 65582), we have improved and extended functionality of the FlexQTL software to better handle large numbers of SNP markers with regard to computation time, the phasing of the lowly-informative SNPs in complex pedigrees (e.g., inbreeding loops). Furthermore visualization tools were added to inspect recombination events. Furthermore, highly informative multi-allelic haplotypes can now be built comprising consecutive low informative SNPs, which will reduce computational effort without losing information from the high density marker data. The progress and challenges on utilizing high density SNP data in Rosaceae crops will be highlighted via a case study, i.e., applying the new version of FlexQTL software to fruit firmness trait on a large 20K genotyped apple population as available from the FruitBreedomics project.

Apple consumption is highly recomended for a healthy diet and is the most important fruit produced in temperate climate regions. Unfortunately, it is also one of the fruit that most ofthen provoks allergy in atopic patients and the only treatment available up to date for these apple allergic patients is the avoidance. Apple allergy is due to the presence of four major classes of allergens: Mal d 1 (PR-10/Bet v 1-like proteins), Mal d 2 (Thaumatine-like proteins), Mal d 3 (Lipid transfer protein) and Mal d 4 (profilin). In this work new advances in the characterization of apple allergen gene families have been reached using a multidisciplinary approach. First of all, a genomic approach was used for the characterization of the allergen gene families of Mal d 1 (task of Chapter 1), Mal d 2 and Mal d 4 (task of Chapter 5). In particular, in Chapter 1 the study of two large contiguos blocks of DNA sequences containing the Mal d 1 gene cluster on LG16 allowed to acquire many new findings on number and orientation of genes in the cluster, their physical distances, their regulatory sequences and the presence of other genes or pseudogenes in this genomic region. Three new members were discovered co-localizing with the other Mal d 1 genes of LG16 suggesting that the complexity of the genetic base of allergenicity will increase with new advances. Many retrotranspon elements were also retrieved in this cluster. Due to the developement of molecular markers on the two sequences, the anchoring of the physical and the genetic map of the region has been successfully achieved. Moreover, in Chapter 5 the existence of other loci for the Thaumatine-like protein family in apple (Mal d 2.03 on LG4 and Mal d 2.02 on LG17) respect the one reported up to now was demonstred for the first time. Also one new locus for profilins (Mal d 4.04) was mapped on LG2, close to the Mal d 4.02 locus, suggesting a cluster organization for this gene family, as is well reported for Mal d 1 family. Secondly, a methodological approach was used to set up an highly specific tool to discriminate and quantify the expression of each Mal d 1 allergen gene (task of Chapter 2). In aprticular, a set of 20 Mal d 1 gene specific primer pairs for the quantitative Real time PCR technique was validated and optimized. As a first application, this tool was used on leaves and fruit tissues of the cultivar Florina in order to identify the Mal d 1 allergen genes that are expressed in different tissues. The differential expression retrieved in this study revealed a tissue-specificity for some Mal d 1 genes: 10/20 Mal d 1 genes were expressed in fruits and, indeed, probably more involved in the allergic reactions; while 17/20 Mal d 1 genes were expressed in leaves challenged with the fungus Venturia inaequalis and therefore probably interesting in the study of the plant defense mechanism. In Chapter 3 the specific expression levels of the 10 Mal d 1 isoallergen genes, found to be expressed in fruits, were studied for the first time in skin and flesh of apples of different genotypes. A complex gene expression profile was obtained due to the high gene-, tissue- and genotype-variability. Despite this, Mal d 1.06A and Mal d 1.07 expression patterns resulted particularly associated with the degree of allergenicity of the different cultivars. They were not the most expressed Mal d 1 genes in apple but here it was hypotized a relevant importance in the determination of allergenicity for both qualitative and quantitative aspects of the Mal d 1 gene expression levels. In Chapter 4 a clear modulation for all the 17 PR-10 genes tested in young leaves of Florina after challenging with the fungus V. inaequalis have been reported but with a peculiar expression profile for each gene. Interestingly, all the Mal d 1 genes resulted up-regulated except Mal d 1.10 that was down-regulated after the challenging with the fungus. The differences in direction, timing and magnitude of induction seem to confirm the hypothesis of a subfunctionalization inside the gene family despite an high sequencce and structure similarity. Moreover, a modulation of PR-10 genes was showed both in compatible (Gala-V. inaequalis) and incompatible (Florina-V. inaequalis) interactions contribute to validate the hypothesis of an indirect role for at least some of these proteins in the induced defense responses. Finally, a certain modulation of PR-10 transcripts retrieved also in leaves treated with water confirm their abilty to respond also to abiotic stress. To conclude, the genomic approach used here allowed to create a comprehensive inventory of all the genes of allergen families, especially in the case of extended gene families like Mal d 1. This knowledge can be considered a basal prerequisite for many further studies. On the other hand, the specific transcriptional approach make it possible to evaluate the Mal d 1 genes behavior on different samples and conditions and therefore, to speculate on their involvement on apple allergenicity process. Considering the double nature of Mal d 1 proteins, as apple allergens and as PR-10 proteins, the gene expression analysis upon the attack of the fungus created the base for unravel the Mal d 1 biological functions. In particular, the knowledge acquired in this work about the PR-10 genes putatively more involved in the specific Malus-V. inaequalis interaction will be helpful, in the future, to drive the apple breeding for hypo-allergenicity genotype without compromise the mechanism of response of the plants to stress conditions. For the future, the survey of the differences in allergenicity among cultivars has to be be thorough including other genotypes and allergic patients in the tests. After this, the allelic diversity analysis with the high and low allergenic cultivars on all the allergen genes, in particular on the ones with transcription levels correlated to allergencity, will provide the genetic background of the low ones. This step from genes to alleles will allow the develop of molecular markers for them that might be used to effectively addressed the apple breeding for hypo-allergenicity. Another important step forward for the study of apple allergens will be the use of a specific proteomic approach since apple allergy is a multifactor-determined disease and only an interdisciplinary and integrated approach can be effective for its prevention and treatment.

Climate changes resulting from increases in temperature and air pollution influence pollen allergenicity, responsible for the dramatic raise in respiratory allergies. Allergies are a complex of symptoms derived from altered IgE-mediated reactions of the immune system towards substances known as allergens. Allergic sensibilization can be of food or respiratory origin; many allergens have been identified in pollens or fruits of different plant species and allergic cross-reactivity can occur in a patient reacting to similar allergens from different origins. Although the link between climate change and pollen allergenicity is proven, the underlying mechanism is little understood. Transglutaminases (TGases), enzymes able to post-translationally modify proteins, are activated under stress and involved in some inflammatory responses, enhancing the activity of pro-inflammatory phospholipase A2, (PLA2). A calcium-dependent TGase activity has been identified in the apple pollen cell wall, raising the possibility that TGase may have a role in the modification of allergens and, when pollen arrives in contact with the human mucosa, the enzyme could be responsible in the transamidation of proteins as PLA2. In Europe a great number of people suffers from apple fruit allergy, but little is known on fruit/pollen cross-allergenicity. Apple fruit allergies are mainly due to four different classes of allergens: Mal d 1, 2, 3, 4. Here we have investigated the presence of Mal d allergens also in pollen to clarify its allergenic potential using a RT-PCR approach both on ungerminated and germinated pollen and we show our initial effort on clarification of climate changes effects on the expression of Mal d allergens. This multidisciplinary approach, aimed to establish the scientific basis for future studies on pollen-food cross allergenicity is supported by the grant “Crossallergenicity” Progetto Strategico d’Ateneo E.F. 2006, University of Bologna.

Le proteine allergeniche della mela presentano delle caratteristiche comuni tra loro: - Tutte le proteine allergeniche sono codificate da famiglie geniche composte da un numero variabile di geni, gli isoallergeni, di cui per ognuna esitono numerose varianti alleliche. Questa presenza ridondante di sequenze Mal d nel genoma del melo, ed in particolare su cromosomi omologhi, sembra essere dovuta alla natura allopoliploide del melo e questo fa anche supporre che il numero di isoallergeni noti sia destinato a crescere ulteriormente. - Tre classi di proteine su quattro (Mal d 1, Mal d 2 e Mal d 3) appartengono alle proteine PR e quindi sono in qualche modo correlate alla difesa della pianta da vari tipi di stress, sia biotici che abiotici. - Alcuni fattori comuni a molti allergeni come la stabilità a bassi pH, alle alte temperature e a proteolisi o la capacità di trasportare lipidi o altri ligandi potrebbero influenzarne l’allergenicità. Nonostante l’aumento delle conoscenze in questi ultimi anni, non è stato ancora chiarito quali isoallergeni siano effettivamente espressi nel frutto e quali siano i veri responsabili della reazione allergica. Questo è un punto chiave nel problema delle allergie alla mela ma anche alla frutta e agli ortaggi in generale. Nella impostazione della ricerca su cui abbiamo qui riferito, si è scelto un approccio multidisciplinare che prevede l’integrazione, a fianco delle biotecnologie, di discipline come l’allergologia e la pomologia (Figura 8). Attraverso questi studi si tenterà di associare ad ogni locus genico un determinato potenziale allergenico e quindi, individuando le sequenze di isoallergeni associate ad un basso grado di allergenicità, si potrà anche orientare il miglioramento genetico del melo verso l’ottenimento di varietà a bassa allergenicità. Una volta individuati dei marker per la bassa allergenicità, si potranno cercare genotipi interessanti per questo carattere anche esplorando le collezioni dell’antico germoplasma del melo. Un aspetto però da non sottovalutare è la doppia faccia della medaglia ovvero il ruolo delle proteine responsabili dell’allergenicità; molte di queste, infatti, sono coinvolte nella risposta della pianta ad attacchi di patogeni e altri stress. Questa considerazione rende inevitabile la preoccupazione che l’auspicata riduzione dell’allergenicità, possa comportare una maggiore suscettibilità agli stress indesiderabile per ovvi motivi. Per ora è solamente un interrogativo. Considerando i notevoli effetti benefici delle mele sulla salute degli individui e considerando il notevole interesse che, negli ultimi anni, il mercato riserva alla nutraceutica e agli alimenti correlati al benessere, non è da escludere che lo studio del carattere riguardante l’ipoallergenicità dei frutti possa essere interessante non solo per quanto riguarda la salute umana ma anche per un possibile riscontro economico

Apple, the most common fruit in European and North American diets, can cause allergic reactions in susceptible individuals, and genes for four families of apple allergens have been identified to date: Mal d 1, Mal d 2, Mal d 3, and Mal d 4. Our remit was to evaluate the effects of genotype, tissue, and stage of fruit development on the expression of these allergen genes and, hence, on the potential allergenicity of apple fruit.Transcript levels were investigated in fruit of the apple cultivars, ‘Gala’ and ‘Florina’, using quantitative Real-Time PCR.The resulting patterns of allergen gene expression differed, with Mal d 1 and Mal d 2 being the most highly expressed in the skin and flesh of ripe fruit, respectively. Overall, ‘Florina’ fruit showed higher levels of expression than ‘Gala’ fruit for all allergens tested.

Allergies are a complex of symptoms derived from altered IgE-mediated reactions of the immune system towards substances known as allergens. Apple and hazelnut allergens have been identified in pollens or fruits. Apple allergies are due to four different classes of allergens (Mal d 1, 2, 3, 4), whose allergenicity is related both to genotype and tissue specificity. Building by Homology technique was applied to predict the 3D structures of Mal d 1-4. Starting from the comparison of these results leading to understand the different ways of reactions of the Malds, we have studied the answer of sensitised individuals to 11 apple genotypes, separating fruits skin positive answers from those to the pulp through Skin Prick Test and prick-to-prick test, evidencing different allergic responses to the different cultivar. Climate changes resulting from increases in temperature and air pollution influence pollen allergenicity, responsible for the dramatic raise in respiratory allergies (hay fever, bronchial asthma, conjunctivitis). Although the link between climate change and pollen allergenicity is proven, the underlying mechanism is little understood. Transglutaminases (TGases), a class of enzymes able to post-translationally modify proteins, are activated under stress and involved in some inflammatory responses, enhancing the activity of pro-inflammatory phospholipase A2, suggesting a role in allergies. Aero allergenic (hazelnut. Corylus avellana) and enthomophylus (apple. Malus domestica) pollen exposed to climate changes (different temperatures, relative humidity (rH), acid rain at pH 5.6 and copper pollution (3.10 µg/l)) showed an increase in pollen surface TGase activity in Corylus, while Malus pollen TGase was not significantly affected. Several pollen proteins were post-translationally modified, including the activation of mammalian sPLA2, thus potentially altering pollen allergenicity and inflammation. Western blotting of pollen TGase revealed its induced expression after exposure to stressors, while detection of in situ TGase activity and morphological examination indicated pollen damage and release of TGase. Grants:Crossallergenicity Progetto Strategico d’Ateneo E.F. 2006,University of Bologna

Premessa/obiettivi: Sono state descritte 4 famiglie di allergeni in melo (Mal d 1, 2, 3, 4 [1]) e sintomi clinici associati [2-3]. Un diverso grado di allergenicità è riportato in letteratura sia in relazione al genotipo [4] che al tessuto analizzato: alcuni allergeni sembrano localizzarsi soprattutto a livello della buccia [5]. Scopo dello studio è determinare la risposta di pazienti allergici alla mela attraverso SPT. Materiali e Metodi: 19 soggetti, giunti consecutivamente presso l’Ambulatorio di Allergologia Pediatrica con allergia alimentare alla mela (prevalentemente come sindrome orale allergica), sono stati sottoposti a SPT per estratto di mela (Lofarma) e per 11 genotipi di melo (Florina, Durello, Ohrin, Red Chief, Jonagold, Jonathan, Topaz, Fiesta, Fuji, Gala, Golden), condotti separatamente per buccia e polpa (prick-to-prick). Risultati: Sono stati considerati positivi pomfi di diametro maggiore-uguale a quello dell’istamina (hys) dello stesso paziente. Le polpe di Durello e Ohrin sono risultate meno sensibilizzanti, causando percentualmente la minore positività (rispettivamente 54.5% e 57.9%) e una reazione mediamente inferiore a hys (SPT/hys < 1). Invece, la maggior parte dei pazienti ha mostrato una forte reazione alla polpa di Jonathan e Jonagold (positività > 73.7%, SPT/hys > 1,36). Le bucce di Durello e Jonagold mostrano una minore reazione, rispettivamente con percentuale di positività di 54.5% e 57.9% e SPT/hys di 0.71 e 1.0. Una forte reazione è invece presente per i genotipi Gala (positività = 94.7%, SPT/hys = 1.43) e Fiesta (positività = 78.9%, SPT/hys = 1.46). La somma del numero di positività dei pomfi risultati dalla buccia (144) è lievemente maggiore rispetto al numero di positività della polpa (136). Tuttavia, esiste una variabilità di risposta fra polpa e buccia in diversi genotipi, specie per Ohrin: dei 18 soggetti che hanno presentato almeno 1 positività tra polpa e buccia, solo in 8 casi entrambi i tessuti erano positivi. Discussione: I risultati suggeriscono che vi siano differenze allergeniche nelle diversi cultivar, tuttavia ciò è complicato dalla variabilità legata alla risposta dei pazienti. Bibliografia: 1. Fernandez-Rivas e coll. JACI 2006; 118: 481-8. 2. Marzban e coll. Plant Science 2005; 169: 387-94. 3. Sancho e coll. Allergy 2005;60: 1262-8. 4. Bolhaar e coll. JACI 2005;116: 1080-6. 5. Borges e coll. Plant Physiology and Biochemistry 2006;44: 535-42.

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.

Freshly consumed apples (Malus domestica L. Borkh) can cause allergic reactions because of the presence of four classes of allergens. Knowledge of the genetic factors affecting the allergenic potential of apples would provide important information for the selection of hypoallergenic genotypes, which can be combined with the adoption of new agronomical practices to produce fruits with a reduced amount of allergens. In the present research, a multiple analytical approach was adopted to characterize the allergenic potential of 24 apple varieties released at different ages (pre- and post-green revolution). A specific workflow was set up including protein quantification by means of polyclonal antibodies, immunological analyses with sera of allergic subjects, enzymatic assays, clinical assessments on allergic patients, and gene expression assays on fruit samples. Taken as a whole, the results indicate that most of the less allergenic genotypes were found among those deriving from selection processes carried out prior to the so-called "green revolution".

International audience; The development of molecular markers linked to specific traits is now routine practice, but the gap between genomics and breeding often delays their application. In the frame of the FP7 European project FruitBreedomics, apple pilot studies were designed to exploit the project's outcomes towards the practical application of marker-assisted breeding (MAB) programs. The aim of this pilot study was to develop an outsourcing genotyping pipeline, which will provide access to the single nucleotide polymorphism (SNP) markers analysis for breeding companies without an internal DNA lab. The process from seed sowing to genotypic and phenotypic seedling selection was optimized. KASP (TM) (competitive allele-specific PCR) genotyping assays were developed for a number of major resistance genes for apple scab (Rvi2, Rvi4, Rvi6, and Rvi15); powdery mildew (Pl2); and rosy apple aphid (Dp-fl). In addition, KASP (TM) assays for the genes Md-ACS1, Md-ACO1, and Md-PG1 involved in fruit quality (firmness, texture, and storability) were also developed. The pilot study demonstrated the efficacy of the SNP-based selection strategy, especially for those programs dealing with traits not easily assessable in vivo, such as pyramided resistances and fruit quality traits.

International audience; Quantitative trait loci (QTL) mapping approaches rely on the correct ordering of molecular markers along the chromosomes, which can be obtained from genetic linkage maps or a reference genome sequence. For apple (Malus domestica Borkh), the genome sequence v1 and v2 could not meet this need; therefore, a novel approach was devised to develop a dense genetic linkage map, providing the most reliable marker-loci order for the highest possible number of markers. The approach was based on four strategies: (i) the use of multiple full-sib families, (ii) the reduction of missing information through the use of HaploBlocks and alternative calling procedures for single-nucleotide polymorphism (SNP) markers, (iii) the construction of a single backcross-type data set including all families, and (iv) a two-step map generation procedure based on the sequential inclusion of markers. The map comprises 15 417 SNP markers, clustered in 3 K HaploBlock markers spanning 1 267 cM, with an average distance between adjacent markers of 0.37 cM and a maximum distance of 3.29 cM. Moreover, chromosome 5 was oriented according to its homoeologous chromosome 10. This map was useful to improve the apple genome sequence, design the Axiom Apple 480 K SNP array and perform multifamily-based QTL studies. Its collinearity with the genome sequences v1 and v3 are reported. To our knowledge, this is the shortest published SNP map in apple, while including the largest number of markers, families and individuals. This result validates our methodology, proving its value for the construction of integrated linkage maps for any outbreeding species.