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.

Ethylene is a gaseous hormone that coordinates several important physiological processes resulting in the final fruit quality in apple. Due to its genetic heterozygousity, apple offers the possibility to exploit the natural allelic variation existing at the level of two genes involved in the ethylene biosynthetic pathway, Md-ACS1 and Md-ACO1. The allelism of these two genes can be exploited for the advanced selection of genotypes characterized by low ethylene production, with a consequent extended postharvest storability. The impact of these two elements in ethylene control was verified using two specific apple populations ('GDxBr' and 'FjxMG') presenting distinct allelotype configurations. In the first progeny, two QTLs were identified in genetic positions corresponding with these genes, and for the first time the association of a QTL for ethylene production with Md-ACO1 in apple was established. However, the analysis carried out on the second population, homozygous for the allele of Md-ACS1 associated with a low ethylene production, suggested the possible involvement of other genetic elements in the regulation of ethylene production. In the end we also present, in parallel to the GC assessment, a novel methodology based on proton transfer reaction-time of flight-mass spectrometry (PTR-ToF-MS) for a more reliable, fast and efficient monitoring of the ethylene production in apple. (C) 2013 Elsevier B.V. All rights reserved.

Among the fruit tree species, the European pear (Pyrus communis L.) has the most stable cultivar structure. Although the selection activity in the last several centuries has produced several hundred cultivars, only a few pear cultivars are currently grown. Within the Pomoideae (Pyrinae) there are 22 Pyrus species, along with another ten or so that have been variously described and assignable to synonyms of the more important species. Perhaps the most widely known species, if not the most widely cultivated, is P communis L. The European pear is essentially the only Pyrus species currently grown in Europe while in North America both the European and the Oriental pear are grown. The European pear and its ancestral species, P pyraster Burgsd., grow wild throughout Europe, and it was here where it was domesticated as early as 300 BC. The pear and apple appear to be amphidiploid or allotetraploid species, i.e., those formed by the gametic union of two Rosaceae species of 8 and 9 chromosomes. The high level of genetic recombination combined with selection for fruit size, appearance, flavor, postharvest storability, and resistance to pathogens and diseases has resulted in a diverse array of cultivars. There has been major advances in fruit appearance (shape, color, attractiveness), size, ripening season (summer and fall are predominant) and postharvest traits. Much effort is being invested by researchers to find resistance genes to the main biotic adversities of pear: the fire blight bacterium (Erwinia amylovora), the European pear psylla (Cacopsilla pyri), which is the vector of the phytoplasma causing pear decline, the scab causing fungi Venturia pyrina, and the black spot fungus Stemphylium vesicarium.

European pear, as well as its close relatives Japanese pear and apple, exhibits S-RNase-based gametophytic self-incompatibility. The male determinant of this self-incompatibility mechanism is a pollen-expressed protein containing an F-box domain; in the genera Petunia (Solanaceae), Antirrhinum (Plantaginaceae), and Prunus (Rosaceae), a single F-box gene determines the pollen S. In apple and Japanese pear, however, multiple S-locus F-box genes were recently identified as candidates for the pollen S, and they were named S-locus F-Box Brothers. These genes were considered good candidates for the pollen S determinant since they exhibit S-haplotype-specific polymorphisms, pollen-specific expression, and linkage to the S-RNase . In the present study, S-locus F-Box Brothers homologs have been cloned from two of the most agronomically important European pear varieties, “Abbé Fétel” (S 104-2 /S 105 ) and “Max Red Bartlett” (S 101 /S 102 ), and they have been mapped on a genetic linkage map developed on their progeny. Our results suggest that the number of F-box genes linked to the S-locus of the European pear is higher than expected according with previous reports for apple and Japanese pear, since up to five genes were found to be linked to a single S-haplotype. Moreover, two of these genes exhibited an incomplete linkage to the S-RNase , allowing the identification of low-frequency recombinant haplotypes, generated by a crossing-over event between the two genes. These F-box genes are most likely placed in close proximity of the S-locus but do not belong to it, and they can thus be excluded from being responsible for the determination of pollen S function.

The recent analysis of the S-locus region of apple and Japanese pear, two species of Pyrinae (Rosaceae), suggested multiple and different F-box genes (called SFBBs) as candidates for the male determinant (pollen S) of RNase-based gametophytic self-incompatibility in these two species. Here, we followed a phylogenetic approach to take advantage of the pattern of molecular evolution of the S-locus of Pyrinae in characterizing SFBB homologs belonging to S-haplotypes of apple and three species of Pyrus (European, Japanese, and Chinese pears). Our results suggested that the S-locus region of Pyrinae contains no less than six SFBB members and that its structure seems to be rather conserved between apple and pear species. In accordance with the prevailing theory on S-haplotype evolution, the pollen S is expected to have coevolved with the S-RNase and to show some common features derived from the long-term evolution under frequency-dependent balancing selection, i.e., high sequence diversity, evidence of positive selection, and shared ancestral polymorphisms. Using this conceptual framework, we present evidence that some SFBB genes may be better candidates for pollen S in Pyrinae than others. Overall, the SFBB genes analyzed exhibited much lower sequence diversity than their associated S-RNases; likewise, they showed little or no evidence of positive selection. However, evidence of coevolution with the S-RNase clearly emerged for two of them. Altogether our results suggested different evolutionary histories for different SFBBs putatively derived from their distinct involvement in self-incompatibility.

This work presents a simple in vitro system to study physiological, biochemical and molecular changes occurring in a pear callus (Pyrus communis L., cv. Beurré Bosc) grown in close proximity to spatially separated undifferentiated homologous (pear) or heterologous (quince; Cydonia oblonga Mill., East Malling clone C) cells in its neighboring environment. After a 7-day co-culture period, the presence of heterologous cells produced negative effects on the pear callus, whose relative weight increase and adenylate energy charge decreased by 30 and 24%, respectively. Such behavior was associated with a higher O(2) consumption rate (+125%) which did not seem to be coupled to adenosine triphosphate synthesis. Analyses of alternative oxidase and enzymatic activities involved in reactive oxygen species (ROS) detoxification strongly suggested that the higher O(2) consumption rate, measured in the pear callus grown in the heterologous combination, may probably be ascribed to extra-respiratory activities. These, in turn, might contribute to generate metabolic scenarios where ROS-induced oxidative stresses may have the upper hand. The increase in the levels of 2-thiobarbituric acid reactive metabolites, considered as diagnostic indicators of ROS-induced lipid peroxidation, seemed to confirm this hypothesis. Moreover, reverse transcription polymerase chain reaction analysis revealed that the expression levels of a few senescence-associated genes were higher in the pear callus grown in the heterologous combination than in the homologous one. Taken as a whole, physiological and molecular data strongly suggest that undifferentiated cells belonging to a pear graft-incompatible quince clone may induce an early senescence-like status in a closely co-cultured pear callus.

Apple fruit are well known for their storage life, although a wide range of flesh softening occurs among cultivars. Loss of firmness is genetically coordinated by the action of several cell wall enzymes, including polygalacturonase (PG) which depolymerizes cell wall pectin. By the analysis of ‘Fuji’ (Fj) and ‘Mondial Gala’ (MG), two apple cultivars characterized by a distinctive ripening behaviour, the involvement of Md-PG1 in the fruit softening process was confirmed to be ethylene dependent by its transcript being down-regulated by 1-methylcyclopropene treatment in MG and in the low ethylene-producing cultivar Fj. Comparing the PG sequence of MG and Fj, a single nucleotide polymorphism (SNP) was discovered. Segregation of the Md-PG1SNP marker within a full-sib population, obtained by crossing Fj and MG, positioned Md-PG1 in the linkage group 10 of MG, co-located with a quantitative trait locus (QTL) identified for fruit firmness in post-harvest ripening. Fruit firmness and softening analysed in different stages, from harvest to post-storage, determined a shift of the QTL from the top of this linkage group to the bottom, where Md-ACO1, a gene involved in ethylene biosynthesis in apple, is mapped. This PG–ethylene-related gene has beeen positioned in the apple genome on chromosome 10, which contains several QTLs controlling fruit firmness and softening, and the interplay among the allelotypes of the linked loci should be considered in the design of a marker-assisted selection breeding scheme for apple texture.

BACKGROUND:Fruit development, maturation and ripening consists of a complex series of biochemical and physiological changes that in climacteric fruits, including apple and tomato, are coordinated by the gaseous hormone ethylene. These changes lead to final fruit quality and understanding of the functional machinery underlying these processes is of both biological and practical importance. To date many reports have been made on the analysis of gene expression in apple. In this study we focused our investigation on the role of ethylene during apple maturation, specifically comparing transcriptomics of normal ripening with changes resulting from application of the hormone receptor competitor 1-methylcyclopropene.RESULTS:To gain insight into the molecular process regulating ripening in apple, and to compare to tomato (model species for ripening studies), we utilized both homologous and heterologous (tomato) microarray to profile transcriptome dynamics of genes involved in fruit development and ripening, emphasizing those which are ethylene regulated.The use of both types of microarrays facilitated transcriptome comparison between apple and tomato (for the later using data previously published and available at the TED: tomato expression database) and highlighted genes conserved during ripening of both species, which in turn represent a foundation for further comparative genomic studies. The cross-species analysis had the secondary aim of examining the efficiency of heterologous (specifically tomato) microarray hybridization for candidate gene identification as related to the ripening process. The resulting transcriptomics data revealed coordinated gene expression during fruit ripening of a subset of ripening-related and ethylene responsive genes, further facilitating the analysis of ethylene response during fruit maturation and ripening.CONCLUSION:Our combined strategy based on microarray hybridization enabled transcriptome characterization during normal climacteric apple ripening, as well as definition of ethylene-dependent transcriptome changes. Comparison with tomato fruit maturation and ethylene responsive transcriptome activity facilitated identification of putative conserved orthologous ripening-related genes, which serve as an initial set of candidates for assessing conservation of gene activity across genomes of fruit bearing plant species.

‘Max Red Bartlett’ is a red bud mutation of the yellow pear (Pyrus communis L.) cultivar ‘Williams’ (known as ‘Bartlett’ in North America). Anthocyanins are the most important pigments for red colour in fruits. Synthesis of anthocyanins is mediated by a number of well-characterized enzymes that include chalcone synthase (CHS), flavanone-3-hydroxylase (F3H), dihydroflavonol-4-reductase (DFR), anthocyanidin synthase (ANS), and UDP-glucose:flavonoid-3-O-glucosyltransferase (UFGT). Expression of the genes encoding these five enzymes was examined in pear fruit skin in order to elucidate the molecular mechanism for red coloration. In addition, the gene PcMYB10, encoding an R2R3 MYB transcription factor involved in anthocyanin biosynthetic pathway regulation, was isolated from both ‘Williams’ and ‘Max Red Bartlett’. Analysis of the deduced amino acid sequence suggests that this gene is an ortholog of anthocyanin regulators known in other plant species. Its expression level was significantly higher in ‘Max Red Bartlett’ (red pear) compared with the original yellow variety ‘Williams’. Although the map position of PcMYB10 corresponds to that of MdMYBa and MdMYB10, which control pigmentation of apple fruit skin, PcMYB10 is not directly responsible for red versus yellow colour in the two pear varieties, as the mutation underlying this difference maps to a different region of the pear genome.