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The agronomic and economic success of a fruit-tree orchard is mainly determined by the choices made by the grower during the planting stage. A distinctive feature of fruit tree orchards is the very high investment cost for their establishment and the fluctuations in the revenue stream over their lifetime. Growers do not immediately make a profit after planting, and their success depends on the proper choice of fruit tree species, on orchard design and on the specific goals they choose to pursue. Moreover, profitability will also depend on a careful analysis of the economic, environmental, technological, social and infrastructural (transportation system, energetic resources, supply chain logistic) resources available. Today, most fruit tree growers opt to specialize and intensify their production. However, tree density (number of plants per hectare), length of the economically unproductive period, farming methods, production stability over time and many other factors, including ecological sustainability, should be taken into account by the grower when establishing an orchard.

This chapter describes the cherry’s origins, botanical classification (taxonomy) and domestication of the only two species cultivated for food and industrial processing: sweet and sour cherry. Cherry breeding programs worldwide have focused on trees, fruit quality traits and resistance to biotic and abiotic factors as well as on specific local characteristics. A detailed discussion of breeding techniques (crossbreeding, early selection, seedling screening and field assessment, embryo culture and mutagenesis) follows. A section is dedicated to sour cherry breeding, whose problems differ from those of the sweet cherry. We underline the importance of the knowledge and conservation of genetic resources for their use in genomic approaches. Then, discuss breeding strategies and the new traits that have been introduced in new genotypes through genomics. Germplasm biodiversity is analyzed in its phylogenetic context. Then, the molecular breeding approaches are extensively described with particular attention for gene mapping and the development of marker linked to monogenic and polygenic traits. A section is dedicated to the gametophytic incompatibility of the sweet cherry, with an updated summary of the research conducted to identify the 57 incompatibility groups (the cultivars for each of these are reported). Finally, we take into account other aspects related to breeding in respect to how functional genes affect some fruit characteristics, the strategies used after the cherry genome was sequenced and the potential of genetic engineering. The Appendix provides pomological profiles of 44 of the most important innovative cultivars, according to the descriptive standard of the Brooks and Olmo official lists with each accompanied by original photos to aid in their identification.

Gametophytic self-incompatibility (GSI) is the main mechanism that controls fertilization in many rosaceous species, including those belonging to the subtribe Pyrinae (formerly the Maloideae). In natural conditions, S-specificities are subject to frequency-dependent balancing selection; the genetic imprint left by this kind of selection is visible on the S-RNase gene in terms of high sequence diversity, evidence of positive selection, and shared ancestral polymorphisms: thus, some alleles have been maintained almost unaltered during evolution in different but related species, as is the case of Malus and Pyrus species. We have questioned whether the same expected features can be extended to whole S haplotypes, thus including not only the female S determinant (the S-RNase) but also its male counterpart, most likely provided by S-locus F-box Brothers (SFBB) genes - even though direct functional evidence is still needed. On the one side, coevolution between female and male S genes has been postulated several times, given that the generation of any new Sspecificity requires a coordinated change on both sides in order to maintain the full S-haplotypes functionality. But on the other side, recent models for S-RNase-based GSI suggest a key difference between the control of female and male S functions: while the first entirely depends on the single S-RNase gene, the second might likely be provided by multiple SFBB genes acting in a collaborative mode. Even though this hypothesis makes it necessary to re-discuss the mode of coevolution between the female and male S genes, it might provide a suitable explanation for the complex phylogenetic profiles of SFBB genes, which are only in part in agreement with that of the S-RNase. Phylogenetic and segregation analyses of S-locus genes, together with the increasing amount of genomic information available for apple and pear, provide a valuable tool for understanding both the molecular mechanism of SRNase-based GSI, and the complex evolutionary pattern of S-haplotypes.

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.

Fruit ripening can be considered as a complex set of biochemical and physiological changes occurring at the end of the developmental stage. Ripe fruit texture notably affects overall quality and consumer appreciation. Excessive softening limits shelf-life and storability, thereby increasing disease susceptibility and economic loss. Fruit softening is a process due to the depolymerisation of different polysaccharide classes, an event controlled by a synergic and coordinated action of several enzymes among which expansins play a fundamental role. To date, six expansin genes are known to be expressed during apple fruit ontogeny, from full bloom up to fruit ripening. We identified a novel expansin apple homolog (Md-Exp7) sharing high sequence similarity with specific-ripening expansin genes of other crops. A functional marker (Md-Exp7SSR) based on an SSR motif located within the untranslated region of the gene was developed and mapped on Linkage Group 1 of the apple and pear genomes in a region where one major apple QTL for fruit firmness had been previously identified. The allelic composition of 31 apple varieties for the SSR marker was associated with differences in fruit softening.

A heated debate is currently taking place in the scientific community about the use of two distinct breeding approaches: the conventional breeding (based on crossing and selection assisted by molecular-selective technologies such as MAS and MAB) and the breeding that uses the new techniques made available by the modern biotechnologies (an alternative or complementary way to reach goals that are not achievable by conventional breeding). This second approach allows the transfer of resistance genes to pathogens or to abiotic stresses (such as drought, salinity and weather anomalies) as well as the improvement of the fruit nutritional value without affecting the quality standards of cultivars. Today, in addition to the classic transgenic approach (GMOs), the new plant breeding technologies (NPBT), such as “genome editing” and cisgenesis, are available. The genome sequence of the main fruit tree species and the support of specific molecular markers for the identification of gene responsible for target traits made the breeding goals achievable in a much shorter time. Protocols for the analysis of molecular markers (SSR, SCAR, SNP) linked to monogenic and polygenic traits (eg. QTLs), such as fruit quality traits, their sensory features and the tree adaptability to environmental conditions are available. The new genotyping technologies allow the early selection at genome level in each single plant as well as the screening germplasm (ancient genetic heritage) looking for genes (or gene allelic variants) with the aim to recover traits that have been lost during crop evolution or because of an environmental selective pressure. Furthermore, the markers are useful to "build" customized fruits to prevent diseases or to improve fruit quality and storage. The potential offered by new plant breeding technologies to the current breeding of fruit tree species are certainly achievable by cisgenesis (but with the risk that the obtained plants will neither be admitted to field trials nor authorized for commercial release). As for genome editing (not to be confused with GMOs), the development of new technical CRISPR variants, adapted to fruit trees, is particularly crucial. In addition to the insertion of modification in the target regions and the guarantee of the absence of heterologous DNA, this will provide assurance on the variety genome preservation and the maintenance of the related fruit high quality standards.

Fruit tree species in the Rosaceae family have a gametophytic self-incompatibility system mediated by S-RNase; this mechanism provides specific recognition between pollen and pistil, mediated by female proteins (S-RNase) and male proteins (F-box proteins) codified by the single multigenic S locus. In recent years, above all due to developments in molecular biology, research in this sector has clarified many aspects relating to the structure and functioning of the S locus. The additional understanding has important applications, including molecular assays to determine the S genotype and assisted selection to obtain self-fertile varieties, already possible for many fruit tree species.

The Malus (apple) and Pyrus (pear) genera belong to the subtribe Pyrinae, formerly known as the subfamily Maloideae. Like many other species of the Rosaceae, they exhibit a gametophytic self-incompatibility (GSI) mechanism which is genetically determined by a single multigenic and highly polymorphic locus (S-locus). The S-locus controls pollen-pistil recognition through different genes; a stylar ribonuclease (S-RNase) is the female (pistil) determinant whereas its male (pollen) counterpart(s) might consist of one or more F-box proteins. The present study reports the characterization of S-locus F-box genes from a group of apple and pear (European, Japanese and Chinese pear) cultivars, selected on the basis of their S-allele composition. The S-locus structure and diversity in the Pyrinae is analyzed considering the number of F-box genes characterized for each S-haplotype, their genetic linkage to the S-RNase and their phylogenetic relationships within and among species. Our results provide new insight into the evolution of the S-locus within this taxonomic group, and the role that the different S-locus F-box genes might have in self-incompatibility. Peer Reviewed pear apple self-incompatibility S-RNase SFBB Published

Angiosperms are the most prevalent and evolutionarily advanced group of plants. A critical step for the success of flowering plants was the wide promotion of cross-fertilization as reproductive strategy. The Gametophytic Self-Incompatibility (GSI) system represents one of these strategies and it occurs in Rosaceous species (i.e. pome and stone fruits). Self-incompatibility is determined by haplotypes of the S-locus; the pistil specificity is controlled by an S locus-encoded ribonuclease (SRNase) and the pollen determinant is a protein containing an F-box domain (SFbox). S-RNases have been very well characterised thanks to the “genotyping of Salleles” (S-genotyping): this approach supported the definition of the inter-fertility groups among varieties of species in which self-incompatibility is diffused and allowed the identification of mutated alleles that occasionally result in self-fertile genotypes. In this paper all these aspects will be taken into account as well as the evolutionary differentiation of GSI in the Prunoideae (i.e. Prunus) with respect to the Pyrinae (i.e. Malus and Pyrus), that produced different S-locus structures and features in the two taxa; the roles of the pistil S and pollen S mutants in GSI breakdown and the possibility to overcome self-incompatibility by genetic engineering approach will be discussed.

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.

S-genotyping in self-incompatible fruit tree species is essential to plan crossing programs and design orchards. Molecular approaches are the best methods to determine the exact S-allele combination of cultivars either by identifying SRNase (the female determinant) or SFB (the male determinant). In apricot, PCR amplification of SFB often produces DNA fragments similar in length; therefore it is very difficult to identify polymorphisms on agarose gels. To overcome this bottleneck the SSCP (Single Strand Conformational Polymorphism) technique was applied to discriminate the allelic variants of this gene and to select the different related fragments which led to clone and sequence them. Combining the use of PCR and single-strand conformation polymorphisms (SSCP), seven sequences from the apricot genome were successfully identified and three new ones were proposed.

Self-incompatibility has been investigated in the cultivar ‘Abbé Fétel’ (Pyrus communis) in order to assign a role to transglutaminase (TGase), an enzyme able to post-translationally forming cross-links among proteins. This enzyme is localised in the pollen cytoplasm where it regulates the polymerisation of cytoskeleton proteins. An extracellular form is also necessary for pollen tube growth. In the self-pollinated style of Abbè Fétel (A x A, incompatible system), the activity of TGase increased when the pollen tube stopped its growth inside the style and the enzyme was immuno-localised around the tube tip. On the contrary in Abbé Fétel styles pollinated with Williams pollen (A x W, compatible system), TGase activity decreased during pollen germination. The TGase gene has been cloned and sequenced from Abbé Fétel pollen and style showing that it shares a high homology with a sequence of apple, whose genome has been recently published, which presents the typical TGase catalytic sequence, and with the TGase of Arabidopsis. This enzyme was expressed during pollen germination inside the style to a similar extent in both systems (A x A and A x W) showing that it is only the activity to have been stimulated by some factor dependent on SI. A molecular approach to clone one of the S-RNase alleles of the cv Abbé Fétel has been performed. This protein, supplied to pollen in the germination medium, was able to inhibit the growth of 50% of the tubes of the homologous pollen. This approach will allow us to verify if an interaction between the two enzymes will occur during SI, as TGase, at least in animal cells, is also able to act as disulphide isomerase modifying the RNase structure.