Giulia Pagliarani

Expression profiling in HcrVf2-transformed apple plants in response to Venturia inaequalis

Apple scab resistance is one of the most well-characterized 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.

Genomic rearrangements and signatures of breeding in the allo-octoploid strawberry as revealed through an allele dose based SSR linkage map.

BackgroundBreeders in the allo-octoploid strawberry currently make little use of molecular marker tools. As a first step of a QTL discovery project on fruit quality traits and resistance to soil-borne pathogens such as Phytophthora cactorum and Verticillium we built a genome-wide SSR linkage map for the cross Holiday x Korona. We used the previously published MADCE method to obtain full haplotype information for both of the parental cultivars, facilitating in-depth studies on their genomic organisation.ResultsThe linkage map incorporates 508 segregating loci and represents each of the 28 chromosome pairs of octoploid strawberry, spanning an estimated length of 2050 cM. The sub-genomes are denoted according to their sequence divergence from F. vesca as revealed by marker performance. The map revealed high overall synteny between the sub-genomes, but also revealed two large inversions on LG2C and LG2D, of which the latter was confirmed using a separate mapping population. We discovered interesting breeding features within the parental cultivars by in-depth analysis of our haplotype data. The linkage map-derived homozygosity level of Holiday was similar to the pedigree-derived inbreeding level (33% and 29%, respectively). For Korona we found that the observed homozygosity level was over three times higher than expected from the pedigree (13% versus 3.6%). This could indicate selection pressure on genes that have favourable effects in homozygous states. The level of kinship between Holiday and Korona derived from our linkage map was 2.5 times higher than the pedigree-derived value. This large difference could be evidence of selection pressure enacted by strawberry breeders towards specific haplotypes.ConclusionThe obtained SSR linkage map provides a good base for QTL discovery. It also provides the first biologically relevant basis for the discernment and notation of sub-genomes. For the first time, we revealed genomic rearrangements that were verified in a separate mapping population. We believe that haplotype information will become increasingly important in identifying marker-trait relationships and regions that are under selection pressure within breeding material. Our attempt at providing a biological basis for the discernment of sub-genomes warrants follow-up studies to streamline the naming of the sub-genomes among different octoploid strawberry maps.

Simulated environmental criticalities affect transglutaminase of Malus and Corylus pollens having different allergenic potential

Increases in temperature and air pollution influence pollen allergenicity, which is responsible for the dramatic raise in respiratory allergies. To clarify possible underlying mechanisms, an anemophilous pollen (hazel, Corylus avellana), known to be allergenic, and an entomophilous one (apple, Malus domestica), the allergenicity of which was not known, were analysed. The presence also in apple pollen of known fruit allergens and their immunorecognition by serum of an allergic patient were preliminary ascertained, resulting also apple pollen potentially allergenic. Pollens were subjected to simulated stressful conditions, provided by changes in temperature, humidity, and copper and acid rain pollution. In the two pollens exposed to environmental criticalities, viability and germination were negatively affected and different transglutaminase (TGase) gel bands were differently immunodetected with the polyclonal antibody AtPng1p. The enzyme activity increased under stressful treatments and, along with its products, was found to be released outside the pollen with externalisation of TGase being predominant in C. avellana, whose grain presents a different cell wall composition with respect to that of M. domestica. A recombinant plant TGase (AtPng1p) stimulated the secreted phospholipase A2 (sPLA2) activity, that in vivo is present in human mucosa and is involved in inflammation. Similarly, stressed pollen, hazel pollen being the most efficient, stimulated to very different extent sPLA2 activity and putrescine conjugation to sPLA2. We propose that externalised pollen TGase could be one of the mediators of pollen allergenicity, especially under environmental stress induced by climate changes.

Genomic organisation of the Mal d 1 gene cluster on linkage group 16 in apple

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.

Cloning and expression of the major allergen genes in apple fruit

Summary 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.

A high-density, multi-parental SNP genetic map on apple validates a new mapping approach for outcrossing species

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.

Development of SNP-based assays for disease resistance and fruit quality traits in apple (Malus × domestica Borkh.) and validation in breeding pilot studies

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™ (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™ 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.

Erratum to: A qRT-PCR assay for the expression of all Mal d 1 isoallergen genes

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.