Fabrizio Costa

We report a high-quality draft genome sequence of the domesticated apple (Malus × domestica). We show that a relatively recent (>50 million years ago) genome-wide duplication (GWD) has resulted in the transition from nine ancestral chromosomes to 17 chromosomes in the Pyreae. Traces of older GWDs partly support the monophyly of the ancestral paleohexaploidy of eudicots. Phylogenetic reconstruction of Pyreae and the genus Malus, relative to major Rosaceae taxa, identified the progenitor of the cultivated apple as M. sieversii. Expansion of gene families reported to be involved in fruit development may explain formation of the pome, a Pyreae-specific false fruit that develops by proliferation of the basal part of the sepals, the receptacle. In apple, a subclade of MADS-box genes, normally involved in flower and fruit development, is expanded to include 15 members, as are other gene families involved in Rosaceae-specific metabolism, such as transport and assimilation of sorbitol.

Worldwide, grapes and their derived products have a large market. The cultivated grape species Vitis vinifera has potential to become a model for fruit trees genetics. Like many plant species, it is highly heterozygous, which is an additional challenge to modern whole genome shotgun sequencing. In this paper a high quality draft genome sequence of a cultivated clone of V. vinifera Pinot Noir is presented.We estimate the genome size of V. vinifera to be 504.6 Mb. Genomic sequences corresponding to 477.1 Mb were assembled in 2,093 metacontigs and 435.1 Mb were anchored to the 19 linkage groups (LGs). The number of predicted genes is 29,585, of which 96.1% were assigned to LGs. This assembly of the grape genome provides candidate genes implicated in traits relevant to grapevine cultivation, such as those influencing wine quality, via secondary metabolites, and those connected with the extreme susceptibility of grape to pathogens. Single nucleotide polymorphism (SNP) distribution was consistent with a diffuse haplotype structure across the genome. Of around 2,000,000 SNPs, 1,751,176 were mapped to chromosomes and one or more of them were identified in 86.7% of anchored genes. The relative age of grape duplicated genes was estimated and this made possible to reveal a relatively recent Vitis-specific large scale duplication event concerning at least 10 chromosomes (duplication not reported before).Sanger shotgun sequencing and highly efficient sequencing by synthesis (SBS), together with dedicated assembly programs, resolved a complex heterozygous genome. A consensus sequence of the genome and a set of mapped marker loci were generated. Homologous chromosomes of Pinot Noir differ by 11.2% of their DNA (hemizygous DNA plus chromosomal gaps). SNP markers are offered as a tool with the potential of introducing a new era in the molecular breeding of grape.

A new set of 148 apple microsatellite markers has been developed and mapped on the apple reference linkage map Fiesta x Discovery. One-hundred and seventeen markers were developed from genomic libraries enriched with the repeats GA, GT, AAG, AAC and ATC; 31 were developed from EST sequences. Markers derived from sequences containing dinucleotide repeats were generally more polymorphic than sequences containing trinucleotide repeats. Additional eight SSRs from published apple, pear, and Sorbus torminalis SSRs, whose position on the apple genome was unknown, have also been mapped. The transferability of SSRs across Maloideae species resulted in being efficient with 41% of the markers successfully transferred. For all 156 SSRs, the primer sequences, repeat type, map position, and quality of the amplification products are reported. Also presented are allele sizes, ranges, and number of SSRs found in a set of nine cultivars. All this information and those of the previous CH-SSR series can be searched at the apple SSR database ( http://www.hidras.unimi.it ) to which updates and comments can be added. A large number of apple ESTs containing SSR repeats are available and should be used for the development of new apple SSRs. The apple SSR database is also meant to become an international platform for coordinating this effort. The increased coverage of the apple genome with SSRs allowed the selection of a set of 86 reliable, highly polymorphic, and overall the apple genome well-scattered SSRs. These SSRs cover about 85% of the genome with an average distance of one marker per 15 cM.

Suberin, a polymer composed of both aliphatic and aromatic domains, is deposited as a rough matrix upon plant surface damage and during normal growth in the root endodermis, bark, specialized organs (e.g., potato [Solanum tuberosum] tubers), and seed coats. To identify genes associated with the developmental control of suberin deposition, we investigated the chemical composition and transcriptomes of suberized tomato (Solanum lycopersicum) and russet apple (Malus x domestica) fruit surfaces. Consequently, a gene expression signature for suberin polymer assembly was revealed that is highly conserved in angiosperms. Seed permeability assays of knockout mutants corresponding to signature genes revealed regulatory proteins (i.e., AtMYB9 and AtMYB107) required for suberin assembly in the Arabidopsis thaliana seed coat. Seeds of myb107 and myb9 Arabidopsis mutants displayed a significant reduction in suberin monomers and altered levels of other seed coat-associated metabolites. They also exhibited increased permeability, and lower germination capacities under osmotic and salt stress. AtMYB9 and AtMYB107 appear to synchronize the transcriptional induction of aliphatic and aromatic monomer biosynthesis and transport and suberin polymerization in the seed outer integument layer. Collectively, our findings establish a regulatory system controlling developmentally deposited suberin, which likely differs from the one of stress-induced polymer assembly recognized to date.

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.

Recently the coupling of proton transfer reaction ionization with a time-of-flight mass analyser (PTR-TOF-MS) has been proposed to realise a volatile organic compound (VOC) detector that overcomes the limitations in terms of time and mass resolution of the previous instrument based on a quadrupole mass analysers (PTR-Quad-MS). This opens new horizons for research and allows for new applications in fields where the rapid and sensitive monitoring and quantification of volatile organic compounds (VOCs) is crucial as, for instance, environmental sciences, food sciences and medicine. In particular, if coupled with appropriate data mining methods, it can provide a fast MS-nose system with rich analytical information. The main, perhaps even the only, drawback of this new technique in comparison to its precursor is related to the increased size and complexity of the data sets obtained. It appears that this is the main limitation to its full use and widespread application. Here we present and discuss a complete computer-based strategy for the data analysis of PTR-TOF-MS data from basic mass spectra handling, to the application of up-to date data mining methods. As a case study we apply the whole procedure to the classification of apple cultivars and clones, which was based on the distinctive profiles of volatile organic compound emissions.

Texture of apple fruit originates from anatomic traits related to cell wall architecture and is one of its most important quality characteristics, thus there is the desire to better understand the different factors which contribute to apple texture. Here we present a novel approach based on the simultaneous profiling of the mechanical and acoustic response of the flesh tissue to compression, using a texture analyzer coupled with an acoustic device. The methodology was applied to a 86 different apple cultivars, measured after two months postharvest cold storage and characterised by 16 acoustic and mechanical parameters. Statistical treatment of the data with principal component analysis (PCA) allowed for the identification of three groups of variables, the mechanical ones being clearly distinguished from the acoustic ones. Moreover, the distribution of the apple cultivars in the multivariate PCA plot allowed characterisation of the cultivars according to their textural performance. Each cultivar was analyzed also with non-destructive vis/NIR spectroscopy in order to determine impartially the ripening stage. Sensory evaluation by panellists was performed on a selected group of cultivars and sensory data correlated with the acoustic-mechanical data. The results demonstrate the good performance of our combined acoustic-mechanical strategy in measuring apple crispness as it is perceived by human senses.

Next-generation DNA sequencing (NGS) produces vast amounts of DNA sequence data, but it is not specifically designed to generate data suitable for genetic mapping. Recently developed DNA library preparation methods for NGS have helped solve this problem, however, by combining the use of reduced representation libraries with DNA sample barcoding to generate genome-wide genotype data from a common set of genetic markers across a large number of samples. Here we use such a method, called genotyping-by-sequencing (GBS), to produce a data set for genetic mapping in an F1 population of apples (Malus × domestica) segregating for skin color. We show that GBS produces a relatively large, but extremely sparse, genotype matrix: over 270,000 SNPs were discovered but most SNPs have too much missing data across samples to be useful for genetic mapping. After filtering for genotype quality and missing data, only 6% of the 85 million DNA sequence reads contributed to useful genotype calls. Despite this limitation, using existing software and a set of simple heuristics, we generated a final genotype matrix containing 3967 SNPs from 89 DNA samples from a single lane of Illumina HiSeq and used it to create a saturated genetic linkage map and to identify a known QTL underlying apple skin color. We therefore demonstrate that GBS is a cost-effective method for generating genome-wide SNP data suitable for genetic mapping in a highly diverse and heterozygous agricultural species. We anticipate future improvements to the GBS analysis pipeline presented here that will enhance the utility of next-generation DNA sequence data for the purposes of genetic mapping across diverse species.

Blueberries are 'soft fruit', a name which underlies the impact that fruit texture plays in their marketability. Worldwide blueberry production has been increasing in the last years, and they are now the second most economically important soft fruit after strawberry. During pre- and postharvest ripening, fruit softening represents the most dramatic change, leading to important losses in quality during storage. To support the FEM breeding program toward the creation of new blueberry accessions with superior texture, we used a novel texture analyzer TAXTplus to improve the phenotyping resolution, a fundamental step for a more reliable and precise selection of the most suitable ideotypes. In this work change in the mechanical profile of the blueberry cultivar 'Brigitta Blue' was initially assessed during berry development, maturation and postharvest ripening, revealing important textural variations. In addition, this novel equipment was exploited to compare the fruit texture behavior at two specific stages, maturity and after postharvest cold storage, on a set of 49 different blueberry and hybrids cultivars and selections. A multivariate statistical approach, including principal component analysis, was employed to analyze the dataset related to the textural parameters, according to their performance. Finally a storage index, originally defined for apple fruit texture, was adopted to depict the different storage potential of a set of blueberry cultivars.

In 2010, a major scientific milestone was achieved for tree fruit crops: publication of the first draft whole genome sequence (WGS) for apple (Malus domestica). This WGS, v1.0, was valuable as the initial reference for sequence information, fine mapping, gene discovery, variant discovery, and tool development. A new, high quality apple WGS, GDDH13 v1.1, was released in 2017 and now serves as the reference genome for apple. Over the past decade, these apple WGSs have had an enormous impact on our understanding of apple biological functioning, trait physiology and inheritance, leading to practical applications for improving this highly valued crop. Causal gene identities for phenotypes of fundamental and practical interest can today be discovered much more rapidly. Genome-wide polymorphisms at high genetic resolution are screened efficiently over hundreds to thousands of individuals with new insights into genetic relationships and pedigrees. High-density genetic maps are constructed efficiently and quantitative trait loci for valuable traits are readily associated with positional candidate genes and/or converted into diagnostic tests for breeders. We understand the species, geographical, and genomic origins of domesticated apple more precisely, as well as its relationship to wild relatives. The WGS has turbo-charged application of these classical research steps to crop improvement and drives innovative methods to achieve more durable, environmentally sound, productive, and consumer-desirable apple production. This review includes examples of basic and practical breakthroughs and challenges in using the apple WGSs. Recommendations for "what's next" focus on necessary upgrades to the genome sequence data pool, as well as for use of the data, to reach new frontiers in genomics-based scientific understanding of apple.

Fruit ripening is a complex physiological process in plants whereby cell wall programmed changes occur mainly to promote seed dispersal. Cell wall modification also directly regulates the textural properties, a fundamental aspect of fruit quality. In this study, two full-sib populations of apple, with ‘Fuji’ as the common maternal parent, crossed with ‘Delearly’ and ‘Pink Lady’, were used to understand the control of fruit texture by QTL mapping and in silico gene mining. Texture was dissected with a novel high resolution phenomics strategy, simultaneously profiling both mechanical and acoustic fruit texture components. In ‘Fuji×Delearly’ nine linkage groups were associated with QTLs accounting from 15.6% to 49% of the total variance, and a highly significant QTL cluster for both textural components was mapped on chromosome 10 and co-located with Md-PG1, a polygalacturonase gene that, in apple, is known to be involved in cell wall metabolism processes. In addition, other candidate genes related to Md-NOR and Md-RIN transcription factors, Md-Pel (pectate lyase), and Md-ACS1 were mapped within statistical intervals. In ‘Fuji×Pink Lady’, a smaller set of linkage groups associated with the QTLs identified for fruit texture (15.9–34.6% variance) was observed. The analysis of the phenotypic variance over a two-dimensional PCA plot highlighted a transgressive segregation for this progeny, revealing two QTL sets distinctively related to both mechanical and acoustic texture components. The mining of the apple genome allowed the discovery of the gene inventory underlying each QTL, and functional profile assessment unravelled specific gene expression patterns of these candidate genes.

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.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.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.

Texture is a principal quality factor and represents one of the main priorities in apple postharvest management and breeding programs designed for the creation of new ideotypes defined by a better fruit quality and extended storability. The apple panorama is characterized by a great variability of texture performance due to specific functional regulation and genetic control of the physiological machinery devoted to the degradation of the polysaccharide architecture of the middle lamella/cell wall structure. In this work we present an investigation of texture dynamics in apple, in terms of variation of several texture components dissected over two months of postharvest storage. Apple texture was assessed at harvest and after storage, by acquiring both mechanical and acoustic profiles in a collection of 83 apple cultivars. The general texture variability, illustrated over the reduced hyperspace defined by principal components, revealed a different variety distribution between the two stages. Time evolution plots and the novel storage index presented here highlighted that each single texture component behaves differently, as seen by some cultivars (i.e. 'Fuji') having a more favourable acoustic response after postharvest. The dissected fruit texture dynamics assessed in a set of reference apple cultivars are discussed.

Abstract Background Apple is a widely cultivated fruit crop for its quality properties and extended storability. Among the several quality factors, texture is the most important and appreciated, and within the apple variety panorama the cortex texture shows a broad range of variability. Anatomically these variations depend on degradation events occurring in both fruit primary cell wall and middle lamella. This physiological process is regulated by an enzymatic network generally encoded by large gene families, among which polygalacturonase is devoted to the depolymerization of pectin. In apple, Md-PG1 , a key gene belonging to the polygalacturonase gene family, was mapped on chromosome 10 and co-localized within the statistical interval of a major hot spot QTL associated to several fruit texture sub-phenotypes. Results In this work, a QTL corresponding to the position of Md-PG1 was validated and new functional alleles associated to the fruit texture properties in 77 apple cultivars were discovered. 38 SNPs genotyped by gene full length resequencing and 2 SSR markers ad hoc targeted in the gene metacontig were employed. Out of this SNP set, eleven were used to define three significant haplotypes statistically associated to several texture components. The impact of Md-PG1 in the fruit cell wall disassembly was further confirmed by the cortex structure electron microscope scanning in two apple varieties characterized by opposite texture performance, such as ‘Golden Delicious’ and ‘Granny Smith’. Conclusions The results here presented step forward into the genetic dissection of fruit texture in apple. This new set of haplotypes, and microsatellite alleles, can represent a valuable toolbox for a more efficient parental selection as well as the identification of new apple accessions distinguished by superior fruit quality features.

The hydrophobic cuticular membrane of land plants performs a number of important roles during fruit development, including protection from a range of abiotic and biotic stresses. The components of the fleshy fruit cuticle are synthesized and secreted from the epidermal cells. While the biosynthetic and transport pathways of the cuticle have been thoroughly investigated for a number of decades, the regulatory mechanisms allowing fine tuning of cuticle deposition are only now beginning to be elucidated. Transcription factors belonging to the APETALA2, homeodomain-leucine zipper IV, and MYB families have been shown to be important regulators of both cuticle biosynthesis and epidermal cell differentiation, highlighting the connection between these processes. The involvement of MADS-box transcription factors demonstrates the link between fruit ripening and cuticle deposition. Epigenetic and post-transcriptional regulatory mechanisms also play a role in the control of cuticle biosynthesis, in addition to phytohormones, such as abscisic acid, that have been shown to stimulate cuticle deposition. These various levels of genetic regulation allow the plant constantly to maintain and adjust the cuticle in response to environmental and developmental cues.

The outer epidermal layer of apple fruit is covered by a protective cuticle. Composed of a polymerized cutin matrix embedded with waxes, the cuticle is a natural waterproof barrier and protects against several abiotic and biotic stresses. In terms of apple production, the cuticle is essential to maintain long post-harvest storage, while severe failure of the cuticle can result in the formation of a disorder known as russet. Apple russet results from micro-cracking of the cuticle and the formation of a corky suberized layer. This is typically an undesirable consumer trait, and negatively impacts the post-harvest storage of apples. In order to identify genetic factors controlling cuticle biosynthesis (and thus preventing russet) in apple, a quantitative trait locus (QTL) mapping survey was performed on a full-sib population. Two genomic regions located on chromosomes 2 and 15 that could be associated with russeting were identified. Apples with compromised cuticles were identified through a novel and high-throughput tensile analysis of the skin, while histological analysis confirmed cuticle failure in a subset of the progeny. Additional genomic investigation of the determined QTL regions identified a set of underlying genes involved in cuticle biosynthesis. Candidate gene expression profiling by quantitative real-time PCR on a subset of the progeny highlighted the specific expression pattern of a SHN1/WIN1 transcription factor gene (termed MdSHN3) on chromosome 15. Orthologues of SHN1/WIN1 have been previously shown to regulate cuticle formation in Arabidopsis, tomato, and barley. The MdSHN3 transcription factor gene displayed extremely low expression in lines with improper cuticle formation, suggesting it to be a fundamental regulator of cuticle biosynthesis in apple fruit.

The epidermis of aerial plant organs is the primary source of building blocks forming the outer surface cuticular layer. To examine the relationship between epidermal cell development and cuticle assembly in the context of fruit surface, we investigated the tomato (Solanum lycopersicum) MIXTA-like gene. MIXTA/MIXTA-like proteins, initially described in snapdragon (Antirrhinum majus) petals, are known regulators of epidermal cell differentiation. Fruit of transgenically silenced SlMIXTA-like tomato plants displayed defects in patterning of conical epidermal cells. They also showed altered postharvest water loss and resistance to pathogens. Transcriptome and cuticular lipids profiling coupled with comprehensive microscopy revealed significant modifications to cuticle assembly and suggested SlMIXTA-like to regulate cutin biosynthesis. Candidate genes likely acting downstream of SlMIXTA-like included cytochrome P450s (CYPs) of the CYP77A and CYP86A subfamilies, LONG-CHAIN ACYL-COA SYNTHETASE2, GLYCEROL-3-PHOSPHATE SN-2-ACYLTRANSFERASE4, and the ATP-BINDING CASSETTE11 cuticular lipids transporter. As part of a larger regulatory network of epidermal cell patterning and L1-layer identity, we found that SlMIXTA-like acts downstream of SlSHINE3 and possibly cooperates with homeodomain Leu zipper IV transcription factors. Hence, SlMIXTA-like is a positive regulator of both cuticle and conical epidermal cell formation in tomato fruit, acting as a mediator of the tight association between fruit cutin polymer formation, cuticle assembly, and epidermal cell patterning.

In this study, we assessed the main bioactive compounds of a broad apple germplasm collection, composed by 247 accessions of wild (97) and domesticated (150) species. Among the stilbenes, trans- and cis-piceid were found to be ubiquitary components of both wild and cultivated apples. Apple was suggested to be the second dietary source of resveratrols. Results confirmed that the selection pressure of breeding and domestication did not uniformly affect all the phytochemicals contained in apples. For instance, organic acids (malic and ascorbic acid) and some phenolics (stilbenes, hydroxycinnamic acids, and dihydrochalcones) were significantly influenced by selection, while some relevant flavonoids (flavonols and flavan-3-ols) and triterpenoids (ursolic, oleanolic, and betulinic acids) were not. This comprehensive screening will assist in the selection of Malus accessions with specific nutraceutical traits suitable to establish innovative breeding strategies or to patent new functional foods and beverages.

In terms of the quality of minimally processed fruit, flesh browning is fundamentally important in the development of an aesthetically unpleasant appearance, with consequent off-flavours. The development of browning depends on the enzymatic action of the polyphenol oxidase (PPO). In the 'Golden Delicious' apple genome ten PPO genes were initially identified and located on three main chromosomes (2, 5 and 10). Of these genes, one element in particular, here called Md-PPO, located on chromosome 10, was further investigated and genetically mapped in two apple progenies ('Fuji x Pink Lady' and 'Golden Delicious x Braeburn'). Both linkage maps, made up of 481 and 608 markers respectively, were then employed to find QTL regions associated with fruit flesh browning, allowing the detection of 25 QTLs related to several browning parameters. These were distributed over six linkage groups with LOD values spanning from 3.08 to 4.99 and showed a rate of phenotypic variance from 26.1 to 38.6%. Anchoring of these intervals to the apple genome led to the identification of several genes involved in polyphenol synthesis and cell wall metabolism. Finally, the expression profile of two specific candidate genes, up and downstream of the polyphenolic pathway, namely phenylalanine ammonia lyase (PAL) and polyphenol oxidase (PPO), provided insight into flesh browning physiology. Md-PPO was further analyzed and two haplotypes were characterised and associated with fruit flesh browning in apple.

Fruits stored at low temperature can exhibit different types of chilling injury. In apple, one of the most serious physiological disorders is superficial scald, which is characterized by discoloration and brown necrotic patches on the fruit exocarp. Although this phenomenon is widely ascribed to the oxidation of α-farnesene, its physiology is not yet fully understood. To elucidate the mechanism of superficial scald development and possible means of prevention, we performed an integrated metabolite screen, including an analysis of volatiles, phenols and lipids, together with a large-scale transcriptome study. We also determined that prevention of superficial scald, through the use of an ethylene action inhibitor, is associated with the triggering of cold acclimation-related processes. Specifically, the inhibition of ethylene perception stimulated the production of antioxidant compounds to scavenge reactive oxygen species, the synthesis of fatty acids to stabilize plastid and vacuole membranes against cold temperature, and the accumulation of the sorbitol, which can act as a cryoprotectant. The pattern of sorbitol accumulation was consistent with the expression profile of a sorbitol 6-phosphate dehydrogenase, MdS6PDH, the overexpression of which in transgenic Arabidopsis thaliana plants confirmed its involvement in the cold acclimation and freezing tolerance.

Fruit quality features resulting from ripening processes need to be preserved throughout storage for economical reasons. However, during this period several physiological disorders can occur, of which superficial scald is one of the most important, due to the development of large brown areas on the fruit skin surface. This study examined the variation in polyphenolic content with the progress of superficial scald in apple, also with respect to 1-MCP, an ethylene competitor interacting with the hormone receptors and known to interfere with this etiology. The change in the accumulation of these metabolites was further correlated with the gene set involved in this pathway, together with two specific VOCs (Volatile Organic Compounds), α-farnesene and its oxidative form, 6-methyl-5-hepten-2-one. Metabolite profiling and qRT-PCR assay showed these volatiles are more heavily involved in the signalling system, while the browning coloration would seem to be due more to a specific accumulation of chlorogenic acid (as a consequence of the activation of MdPAL and MdC3H), and its further oxidation carried out by a polyphenol oxidase gene (MdPPO). In this physiological scenario, new evidence regarding the involvement of an anti-apoptotic regulatory mechanism for the compartmentation of this phenomenon in the skin alone was also hypothesized, as suggested by the expression profile of the MdDAD1, MdDND1 and MdLSD1 genes. The results presented in this work represent a step forward in understanding the physiological mechanisms of superficial scald in apple, shedding light on the regulation of the specific physiological cascade.

Fruit quality represents a fundamental factor guiding consumers’ preferences. Among apple quality traits, volatile organic compounds and texture features play a major role. Proton Transfer Reaction-Time of Flight-Mass Spectrometry (PTR-ToF-MS), coupled with an artificial chewing device, was used to profile the entire apple volatilome of 162 apple accessions, while the fruit texture was dissected with a TAXT-AED texture analyzer. The array of volatile compounds was classed into seven major groups and used in a genome-wide association analysis carried out with 9142 single nucleotide polymorphisms (SNPs). Marker–trait associations were identified on seven chromosomes co-locating with important candidate genes for aroma, such as MdAAT1 and MdIGS. The integration of volatilome and fruit texture data conducted with a multiple factor analysis unraveled contrasting behavior, underlying opposite regulation of the two fruit quality aspects. The association analysis using the first two principal components identified two QTLs located on chromosomes 10 and 2, respectively. The distinction of the apple accessions on the basis of the allelic configuration of two functional markers, MdPG1 and MdACO1, shed light on the type of interplay existing between fruit texture and the production of volatile organic compounds.

Fruit texture is a complex feature composed of mechanical and acoustic properties relying on the modifications occurring in the cell wall throughout fruit development and ripening. Apple is characterized by a large variation in fruit texture behavior that directly impacts both the consumer's appreciation and post-harvest performance. To decipher the genetic control of fruit texture comprehensively, two complementing quantitative trait locus (QTL) mapping approaches were employed. The first was represented by a pedigree-based analysis (PBA) carried out on six full-sib pedigreed families, while the second was a genome-wide association study (GWAS) performed on a collection of 233 apple accessions. Both plant materials were genotyped with a 20K single nucleotide polymorphism (SNP) array and phenotyped with a sophisticated high-resolution texture analyzer. The overall QTL results indicated the fundamental role of chromosome 10 in controlling the mechanical properties, while chromosomes 2 and 14 were more associated with the acoustic response. The latter QTL, moreover, showed a consistent relationship between the QTL-estimated genotypes and the acoustic performance assessed among seedlings. The in silico annotation of these intervals revealed interesting candidate genes potentially involved in fruit texture regulation, as suggested by the gene expression profile. The joint integration of these approaches sheds light on the specific control of fruit texture, enabling important genetic information to assist in the selection of valuable fruit quality apple varieties.

Proton Transfer Reaction-Mass Spectrometry (PTR-MS) in its recently developed implementation based on a time-of-flight mass spectrometer (PTR-ToF-MS) has been evaluated as a possible tool for rapid non-destructive investigation of the volatile compounds present in the metabolome of apple cultivars and clones. Clone characterization is a cutting-edge problem in technical management and royalty application, not only for apple, aiming at unveiling real properties which differentiate the mutated individuals. We show that PTR-ToF-MS coupled with multivariate and data mining methods may successfully be employed to obtain accurate varietal and clonal apple fingerprint. In particular, we studied the VOC emission profile of five different clones belonging to three well known apple cultivars, such as 'Fuji', 'Golden Delicious' and 'Gala'. In all three cases it was possible to set classification models which can distinguish all cultivars and some of the clones considered in this study. Furthermore, in the case of 'Gala' we also identified estragole and hexyl 2-methyl butanoate contributing to such clone characterization. Beside its applied relevance, no data on the volatile profiling of apple clones are available so far, our study indicates the general viability of a metabolomic approach for volatile compounds in fruit based on rapid PTR-ToF-MS fingerprinting.

More than other characteristics, the sensory properties of fruit and vegetables, mainly related to texture aspects, are the most important drivers of consumer preferences. In this work, the development, evaluation and application of a sensory characterisation method is presented for 21 apple (Malus × domestica Borkh.) varieties, which were stored for 2 months in cold storage. Furthermore, the change of the sensory profiles was evaluated after 1, 2 and 4 months postharvest storage. Sensory analysis was based on a quantitative descriptive method: a trained panel of 13 judges evaluated the apple samples according to 15 sensory attributes relating to texture, odour, flavour and appearance. The method was validated through the analysis of panel consonance and consistency. Univariate and multivariate analyses showed that sensory attributes can be used to discriminate among different cultivars and highlight the perceivable changes in fruit quality induced by postharvest practises.

A combined approach for perceptible quality profiling of apples based on sensory and instrumental techniques was developed. This work studied the correlation between sensory and instrumental data, and defined proper models for predicting sensory properties through instrumental measurements. Descriptive sensory analysis performed by a trained panel was carried out during two consecutive years, on a total of 27 apple cultivars assessed after two months postharvest storage. The 11 attributes included in the sensory vocabulary discriminated among the different apple cultivars by describing their sensory properties. Simultaneous instrumental profiling including colorimeter, texture analyser (measuring mechanical and acoustic parameters) and basic chemical measurements, provided a description of the cultivars consistent with the sensory profiles. Regression analyses showed effective predictive models for all sensory attributes (Q2 ≥ 0.8), except for green flesh colour and astringency, that were less effective (Q2 = 0.5 for both). Interesting relationships were found between taste perception and flesh appearance, and the combination of chemical and colorimeter data led to the development of an effective prediction model for sweet taste. Thus, the innovative sensory-instrumental tool described here can be proposed for the reliable prediction of apple sensory properties.

The aroma trait in apple is a key factor for fruit quality strongly affecting the consumer appreciation, and its detection and analysis is often an extremely laborious and time consuming procedure. Molecular markers associated to this trait can to date represent a valuable selection tool to overcome these limitations. QTL mapping is the first step in the process of targeting valuable molecular markers to be employed in marker-assisted breeding programmes (MAB). However, a validation step is usually required before a newly identified molecular marker can be implemented in marker-assisted selection. In this work the position of a set of QTLs associated to volatile organic compounds (VOCs) was confirmed and validated in three different environments in Switzerland, namely Wädenswil, Conthey and Cadenazzo, where the progeny 'Fiesta×Discovery' was replicated. For both QTL identification and validation, the phenotypic data were represented by VOCs produced by mature apple fruit and assessed with a Proton Transfer Reaction-Mass Spectrometer (PTR-MS) instrument. The QTL-VOC combined analysis performed among these three locations validated the presence of important QTLs in three specific genomic regions, two located in the linkage group 2 and one in linkage group 15, respectively, for compounds related to esters (m/z 43, 61 and 131) and to the hormone ethylene (m/z 28). The QTL set presented here confirmed that in apple some compounds are highly genetically regulated and stable across environments.

Abstract ASSIsT (Automatic SNP ScorIng Tool) is a user-friendly customized pipeline for efficient calling and filtering of SNPs from Illumina Infinium arrays, specifically devised for custom genotyping arrays. Illumina has developed an integrated software for SNP data visualization and inspection called GenomeStudio® (GS). ASSIsT builds on GS-derived data and identifies those markers that follow a bi-allelic genetic model and show reliable genotype calls. Moreover, ASSIsT re-edits SNP calls with null alleles or additional SNPs in the probe annealing site. ASSIsT can be employed in the analysis of different population types such as full-sib families and mating schemes used in the plant kingdom (backcross, F1, F2), and unrelated individuals. The final result can be directly exported in the format required by the most common software for genetic mapping and marker–trait association analysis. ASSIsT is developed in Python and runs in Windows and Linux. Availability and implementation: The software, example data sets and tutorials are freely available at http://compbiotoolbox.fmach.it/assist/. Contact: eric.vandeweg@wur.nl

Apple (Malus x domestica Borkh.) is a model species for studying the metabolic changes that occur at the onset of ripening in fruit crops, and the physiological mechanisms that are governed by the hormone ethylene. In this study, to dissect the climacteric interplay in apple, a multidisciplinary approach was employed. To this end, a comprehensive analysis of gene expression together with the investigation of several physiological entities (texture, volatilome and content of polyphenolic compounds) was performed throughout fruit development and ripening. The transcriptomic profiling was conducted with two microarray platforms: a dedicated custom array (iRIPE) and a whole genome array specifically enriched with ripening-related genes for apple (WGAA). The transcriptomic and phenotypic changes following the application of 1-methylcyclopropene (1-MCP), an ethylene inhibitor leading to important modifications in overall fruit physiology, were also highlighted. The integrative comparative network analysis showed both negative and positive correlations between ripening-related transcripts and the accumulation of specific metabolites or texture components. The ripening distortion caused by the inhibition of ethylene perception, in addition to affecting the ethylene pathway, stimulated the de-repression of auxin-related genes, transcription factors and photosynthetic genes. Overall, the comprehensive repertoire of results obtained here advances the elucidation of the multi-layered climacteric mechanism of fruit ripening, thus suggesting a possible transcriptional circuit governed by hormones and transcription factors.

Texture is a complex trait and a major component of fruit quality in apple. While the major effect of MdPG1, a gene controlling firmness, has already been exploited in elite cultivars, the genetic basis of crispness remains poorly understood. To further improve fruit texture, harnessing loci with minor effects via genomic selection is therefore necessary. In this study, we measured acoustic and mechanical features in 537 genotypes to dissect the firmness and crispness components of fruit texture. Predictions of across-year phenotypic values for these components were calculated using a model calibrated with 8,294 SNP markers. The best prediction accuracies following cross-validations within the training set of 259 genotypes were obtained for the acoustic linear distance (0.64). Predictions for biparental families using the entire training set varied from low to high accuracy, depending on the family considered. While adding siblings or half-siblings into the training set did not clearly improve predictions, we performed an optimization of the training set size and composition for each validation set. This allowed us to increase prediction accuracies by 0.17 on average, with a maximal accuracy of 0.81 when predicting firmness in the 'Gala' × 'Pink Lady' family. Our results therefore identified key genetic parameters to consider when deploying genomic selection for texture in apple. In particular, we advise to rely on a large training population, with high phenotypic variability from which a 'tailored training population' can be extracted using a priori information on genetic relatedness, in order to predict a specific target population.

The low temperature normally applied to prevent fruit decay during the storage of apples, can also triggers the onset of a chilling injury disorder known as superficial scald. In this work, the etiology of this disorder and the mechanism of action of two preventing strategies, such as the application of 1-MCP (1-methylcyclopropene) and storage at low oxygen concentration in 'Granny Smith' and 'Ladina' apple cultivars were investigated. The metabolite assessment highlighted a reorganization of specific metabolites, in particular flavan-3-ols and unsaturated fatty acids, while the genome-wide transcriptomic analysis grouped the DEGs into four functional clusters. The KEGG pathway and GO enrichment analysis, together with the gene-metabolite interactome, showed that the treatment with 1-MCP prevented the development of superficial scald by actively promoting the production of unsaturated fatty acids, especially in 'Granny Smith'. 'Ladina', more susceptible to superficial scald and less responsive to the preventing strategies, was instead characterized by a higher accumulation of very long chain fatty acids. Storage at low oxygen concentration stimulated a higher accumulation of ethanol and acetaldehyde together with the expression of genes involved in anaerobic respiration, such as malate, alcohol dehydrogenase and pyruvate decarboxylase in both cultivars. Low oxygen concentration, likewise 1-MCP, through a direct control on ethylene prevented the onset of superficial scald repressing the expression of PPO, a gene encoding for the polyphenol oxidase enzyme responsible of the oxidation of chlorogenic acid. Moreover, in 'Granny Smith' apple, the expression of three members of the VII subgroups of ERF genes, encoding for elements coordinating the acclimation process to hypoxia in plants was observed. The global RNA-Seq pattern also elucidated a specific transcriptomic signature between the two cultivars, disclosing the effect of the different genetic background in the control of this disorder.

Abstract Fleshy fruits of angiosperms are organs specialized for promoting seed dispersal by attracting herbivores and enticing them to consume the organ and the seeds it contains. Ripening can be broadly defined as the processes serving as a plant strategy to make the fleshy fruit appealing to animals, consisting of a coordinated series of changes in color, texture, aroma, and flavor that result from an intricate interplay of genetically and epigenetically programmed events. The ripening of fruits can be categorized into two types: climacteric, which is characterized by a rapid increase in respiration rate typically accompanied by a burst of ethylene production, and non-climacteric, in which this pronounced peak in respiration is absent. Here we review current knowledge of transcriptomic changes taking place in apple (Malus × domestica, climacteric) and grapevine (Vitis vinifera, non-climacteric) fruit during ripening, with the aim of highlighting specific and common hormonal and molecular events governing the process in the two species. With this perspective, we found that specific NAC transcription factor members participate in ripening initiation in grape and are involved in restoring normal physiological ripening progression in impaired fruit ripening in apple. These elements suggest the existence of a common regulatory mechanism operated by NAC transcription factors and auxin in the two species.

A series of 21 multiplex (MP) polymerase chain reactions containing simple sequence repeat (SSR) markers spanning most of the apple genome has been developed. Eighty-eight SSR markers, well distributed over all 17 linkage groups (LGs), have been selected. Eighty-four of them were included in 21 different MPs while four could not be included in any MPs. The 21 MPs were then used to genotype approximately 2,000 DNA samples from the European High-quality Disease-Resistant Apples for a Sustainable agriculture project. Two SSRs (CH01d03 and NZAL08) were discarded at an early stage as they did not produce stable amplifications in the MPs, while the scoring of the multilocus (ML) SSR Hi07d11 and CN44794 was too complex for large-scale genotyping. The testing of the remaining 80 SSRs over a large number of different genotypes allowed: (1) a better estimation of their level of polymorphism; as well as of (2) the size range of the alleles amplified; (3) the identification of additional unmapped loci of some ML SSRs; (4) the development of methods to assign alleles to the different loci of ML SSRs and (5) conditions at which an SSR previously described as ML would amplify alleles of a single locus to be determined. These data resulted in the selection of 75 SSRs out of the 80 that are well suited and recommended for large genotyping projects.

Apple is a fleshy fruit distinguished by a climacteric type of ripening, since most of the relevant physiological changes are triggered and governed by the action of ethylene. After its production, this hormone is perceived by a series of receptors to regulate, through a signaling cascade, downstream ethylene related genes. The possibility to control the effect of ethylene opened new horizons to the improvement of the postharvest fruit quality. To this end, 1-methylcyclopropene (1-MCP), an ethylene antagonist, is routinely used to modulate the ripening progression increasing storage life. In a recent work published in The Plant Journal, the whole transcriptome variation throughout fruit development and ripening, with the adjunct comparison between normal and impaired postharvest ripening, has been illustrated. In particular, besides the expected downregulation of ethylene-regulated genes, we shed light on a regulatory circuit leading to de-repressing the expression of a specific set of genes following 1-MCP treatment, such as AUX/IAA, NAC and MADS. These findings suggested the existence of a possible ethylene/auxin cross-talk in apple, regulated by a transcriptional circuit stimulated by the interference at the ethylene receptor level.

Mango is heavily affected by rapid off-tree ripening progression with an excessive fruit softening that eventually limits its marketability. The comprehension of the physiological events occurring in the post-climacteric/senescent phase can contribute to the identification of possible solutions for the improvement of shelf life. In this study, the ripening process of mangoes cv Keitt was monitored for two weeks in a simulated commercial shelf life, after shipping from Brazil to Italy, measuring both ripening-associated parameters (firmness, soluble solid content, ethylene production) and non-destructive method based on Vis/NIR spectroscopy (the ripening index IAD). Moreover, the expression pattern of thirteen genes related to different ripening aspects such as ethylene biosynthesis (ACS, ACO), perception (ETR, ERS) and signaling (EIN2, ERF), in combination with genes responsible for cell wall metabolism (PG14, PG21, EXP, PEL, CEL) and carotenoids accumulation (PSY, NCED) were assessed. The results highlighted a specific gene signature characterizing the post-climacteric softening and the senescence onset. Moreover, the non-destructive IAD has proven to be an effective non-destructive monitoring system of the fruit ripening in mango.

To elucidate the physiology underlying the development of superficial scald in pears, susceptible "Blanquilla" fruit was treated with different compounds that either promoted (ethylene) or repressed (1-methylcyclopropene and lovastatin) the incidence of this disorder after 4 months of cold storage. Our data show that scald was negligible for the fruit treated with 1-methylcyclopropene or lovastatin, but highly manifested in untreated (78% incidence) or ethylene-treated fruit (97% incidence). The comparison between the fruit metabolomic profile and transcriptome evidenced a distinct reprogramming associated with each treatment. In all treated samples, cold storage led to an activation of a cold-acclimation-resistance mechanism, including the biosynthesis of very-long-chain fatty acids, which was especially evident in 1-methylcyclopropane-treated fruit. Among the treatments applied, only 1-methylcyclopropene inhibited ethylene production, hence supporting the involvement of this hormone in the development of scald. However, a common repression effect on the PPO gene combined with higher sorbitol content was found for both lovastatin and 1-methylcyclopropene-treated samples, suggesting also a non-ethylene-mediated process preventing the development of this disorder. The results presented in this work represent a step forward to better understand the physiological mechanisms governing the etiology of superficial scald in pears.

The knowledge of the molecular mechanisms underlying fruit quality traits is fundamental to devise efficient marker-assisted selection strategies and to improve apple breeding. In this study, cDNA microarray technology was used to identify genes whose expression changes during fruit development and maturation thus potentially involved in fruit quality traits. The expression profile of 1,536 transcripts was analysed by microarray hybridisation. A total of 177 genes resulted to be differentially expressed in at least one of the developmental stages considered. Gene ontology annotation was employed to univocally describe gene function, while cluster analysis allowed grouping genes according to their expression profile. An overview of the transcriptional changes and of the metabolic pathways involved in fruit development was obtained. As expected, August and September are the two months where the largest number of differentially expressed genes was observed. In particular, 85 genes resulted to be up-regulated in September. Even though most of the differentially expressed genes are involved in primary metabolism, several other interesting functions were detected and will be presented.

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.

Ethylene is a gaseous hormone playing a fundamental role in many systems and its detection and quantification is often crucial for several research fields. In the present work we investigated the possibility to employ Proton Transfer Reaction-Mass Spectrometry (PTR-MS) instruments modified by a Selective Reagent Ionization (SRI) upgrade to detect ethylene in real time with high sensitivity. Compared to commercial laser-based instruments the performance of SRI-MS is similar in terms of detection limits and superior as far as response time and dynamic range are concerned. We showed that, with SRI coupled to a time-of-flight (ToF) mass analyzer, absolute ethylene determination is possible. We experimentally determined the reaction rate coefficients for ethylene reactions with H3O+, O2+ and NO+ as primary ions in the SRI-MS drift tube and proved that in the case of O2+ such reaction proceeds at collision rate, while in the case of H3O+ and NO+ the reaction is less efficient. Reaction product yields and their eventual dependence on the buffer gas were investigated. New product ions that were not previously reported were found. We concluded that among the explored instrumental set-ups, the preferable one for ethylene detection with SRI-ToF-MS is O2+ mode, considering the C2H4+ ion signal at m/z 28.0307. We finally showed that SRI-MS is a powerful tool for ethylene investigations in biological samples.

Abstract The aroma profile in apple was investigated during artificial processing operated with a device imitating the human consumption. The system, composed by a “chewing device” coupled with a Proton Transfer Reaction – Time of Flight-Mass Spectrometry (PTR-ToF-MS), allowed an accurate dynamic volatile organic compound (VOC) fingerprinting suitable to study the volatile kinetics of three apple cultivars (‘Golden Delicious’, ‘Fuji’, and ‘Granny Smith’) during shelf-life ripening. The obtained results demonstrate the complementarity between the dynamic VOC assessment during “mastication” and the usual static headspace analysis. The great advantage of such analytical approach was the possibility to study the kinetics of the volatiles released during eating and the possibility to consider their concentration similar to in vivo condition resulting to an improved characterization of the aroma profile. Moreover, differences in textural properties of apple flesh revealed a possible direct role of the cell wall architectural structure in the regulation of VOC release during consumption. This strategy may be ideal for VOC assessment addressed to investigate fruit quality aspects impacting the consumer appreciation.

Fruits are nowadays considered important suppliers of anti-oxidant molecules. Apples are particularly rich in phenolic compounds, non-nutritional phytochemicals that play active roles in controlling severe chronic diseases. In this work, 19 phenolic compounds were investigated in both skin and pulp tissues of seven apple accessions across the Malus genus collected at two stages: during fruit development and at harvest. The primary difference in phenolic concentration between wild and domesticated accessions, especially in the pulp, could be explained by the larger growth rate of the domesticated varieties. The proposed dilution effect was also confirmed through the observation of the increased content of procyanidin B2+B4 and phloridzin in russet-skinned apples, known to have higher concentrations of these compounds. The metabolite screening was also accompanied by the expression analysis of 16 polyphenolic genes showing, for nine elements, a higher expression at harvest than during fruit development. Finally, a polyphenolic comparison with red-fleshed apples was also carried out, underlying a larger amount of procyanidins and quercetin-3rhamnoside in the white-fleshed accessions. The results presented and discussed in this work suggest that specific white-fleshed apples, especially with russeted-skin, may play an important role in ameliorating the nutraceutical potential of apple fruit.

Apple fruit quality is strongly influenced by the interplay between juiciness and texture. To better decipher the complexity underneath the control of such quality traits, a multidisciplinary approach combining the mechanic and acoustic profiling of texture, juice analysis, cell morphology, sensory and genetic analysis was carried out. The analyses were conducted after 1.5 months of cold storage on fourteen accessions employed in novel breeding schemes for texture and juiciness. The food matrix structure was exploited focusing on both the cell morphology (employing an optical microscope) and the intercellular space (using an X-ray computed micro-tomography scanner). The mechanical and acoustic properties of texture were profiled with a texture analyser, while the juice was extracted using a mechanical press. In parallel to the analytical assessments, fruit texture, juiciness and flavour were also evaluated by sensory analysis. The results highlighted a positive correlation between cell shape and the intercellular volume. Apple accessions distinguished by round cells were characterized by a reduced intercellular space, while cell with an angular cell shape had a higher intercellular space. While the cell shape was associated with juiciness, the firmness response was more influenced by cell size. The interplay between cellular morphology and juiciness was also investigated together with the allelotype variability of a genetic marker designed for MdPG1, a polygalacturonase gene known to control the regulation of fruit texture in apple. The highest juiciness was found in apples with both a high fraction of round cells and the presence of the MdPG1 allele associated with low softening rates. The elucidation of the role of cellular morphology in the control of fruit texture and juiciness, and their association with the MdPG1 alleles, provided valuable information for a more detailed and informative analysis of fruit quality, enabling a more precise characterization and selection of superior apple accessions.

The ripening of climacteric fruits, such as apple, is represented by a series of genetically programmed events orchestrated by the action of several hormones. In this study, we investigated the existence of a hormonal crosstalk between ethylene and auxin during the post-harvest ripening of three internationally known apple cultivars: 'Golden Delicious', 'Granny Smith' and 'Fuji'. The normal climacteric ripening was impaired by the exogenous application of 1-methylcyclopropene (1-MCP) that affected the production of ethylene and the physiological behaviour of specific ethylene-related quality traits, such as fruit texture and the production of volatile organic compounds. The application of 1-MCP induced, moreover, a <i>de-novo</i> accumulation of auxin. The RNA-Seq wide-transcriptome analysis evidenced as the competition at the level of the ethylene receptors induced a cultivar-dependent transcriptional re-programming. The DEGs annotation carried out through the KEGG database identified as most genes were assigned to the plant hormone signaling transduction category, and specifically related to auxin and ethylene. The interplay between these two hormones was further assessed through a candidate gene analysis that highlighted a specific activation of GH3 and ILL genes, encoding key steps in the process of the auxin homeostasis mechanism. Our results showed that a compromised ethylene metabolism at the onset of the climacteric ripening in apple can stimulate, in a cultivar-dependent fashion, an initial <i>de-novo</i> synthesis and de-conjugation of auxin as a tentative to restore a normal ripening progression.

In the present study the relationships between apple texture and rheological parameters were evaluated by means of multivariate analysis. In particular, reference apple cultivars were used to assess fruit crispness of two new apple breeding populations. Principal Component Analysis and Linear Discriminant Analysis were initially applied to the rheological data collected on reference cultivars for which experimental crispness scores were determined. Principal Component Analysis enhanced the interpretation of the relationships between crispness and rheological variables, while Linear Discriminant Analysis showed a clear trend of separation among the different apple cultivars according to their crispness score. Finally, the new apple populations were projected in the multivariate model in order to predict their crispness. These predicted scores of apple crispness are expected to have a relevant impact to the further investigation of apple quality, since they can be exploited to better describe the general fruit quality as well as the detection of genomic regions and causal genes involved in the control of fruit texture in apple.

To decipher the transcriptomic regulation of the on-tree fruit maturation in pear cv. 'Abate Fetel', a RNA-seq transcription analysis identified 8939 genes differentially expressed across four harvesting stages. These genes were grouped into 11 SOTA clusters based on their transcriptional pattern, of which three included genes upregulated while the other four were represented by downregulated genes. Fruit ripening was furthermore investigated after 1 month of postharvest cold storage. The most important variation in fruit firmness, production of ethylene and volatile organic compounds were observed after 5 days of shelf-life at room temperature following cold storage. The role of ethylene in controlling the ripening of 'Abate Fetel' pears was furthermore investigated through the application of 1-methylcyclopropene, which efficiently delayed the progression of ripening by reducing fruit softening and repressing both ethylene and volatile production. The physiological response of the interference at the ethylene receptor level was moreover unraveled investigating the expression pattern of 12 candidate genes, initially selected to validate the RNA-seq profile. This analysis confirmed the effective role of the ethylene competitor in downregulating the expression of cell wall (PG) and ethylene-related genes (ACS, ACO, ERS1, and ERS2), as well as inducing one element involved in the auxin signaling pathway (Aux/IAA), highlighting a possible cross-talk between these two hormones. The expression patterns of these six elements suggest their use as molecular toolkit to monitor at molecular level the progression of the fruit on-tree maturation and postharvest ripening.

The aim of this research was to investigate the antioxidant kinetics of different apple varieties, a red-flesh variety (‘R201’), a non-browning (‘Majda’), and a ‘Golden Delicious’. Kinetic approaches for antioxidant activity provide more detailed information than conventional assays by examining both the quantity and velocity of active molecules in their reaction with radicals. In this study, DPPH• stopped flow method was applied to study the antioxidant activity and capacity of the three apples varieties, allowing the determination of the reaction rates. The results show that the antioxidant activity of ‘R201’ was not significantly different from ‘Golden Delicious’. Instead, the activity of the non-browning variety was 20 times higher than the others according to the DPPH• kinetic approach, despite having the lowest phenolic content. To further understand their reactivity, the antioxidant molecules were identified using HPLC-HRMS/MS coupled with a coulometric array detector, which validated the DPPH• kinetics. This analysis also found differences in the phenolic profile of the three varieties, attributed to the enhanced antioxidant activity of 'Majda' to its high content of ascorbic acid. Overall, the research highlights that the antioxidant behavior of apples primarily depends on the velocity of the antioxidants rather than the amount of phenolic compounds.

Abstract After harvest fruit are stored to preserve the quality features established during the on-tree development and maturation, ensuring thus a continuous availability of fresh fruit on the market. For certain fruit species like apple, storage can last for almost a year, especially when coupled with several strategies, such as the reduction of the oxygen concentration or the application of ethylene competitor molecules, like 1-methylcyclopropene (1-MCP). To guarantee the maintenance of the highest quality, the monitoring of the physiological processes ongoing during the postharvest ripening is compelling. For this purpose, 16 genes belonging to key fruit ripening pathways, such as the ethylene and the sugar/fermentation metabolism, have been chosen as potential markers for the molecular characterization of the major changes occurring in the fruit during storage. Among these genes, ACS, PPO, PG1, RAP2-like , and ADH exhibited the most significant differential expression across the various samples. Based on the transcriptional pattern, this set of genes constitutes a valuable molecular tool for a precise and reliable RNA-based monitoring of the postharvest ripening progression and fermentation process in apples. PPO , together with S6PDH , were furthermore employed to inspect the onset of the superficial scald in apple and resulted to correlate with the evaluation of the incidence of this disorder and the accumulation of the sugar alcohol sorbitol, known to play important protecting roles to chilling injuries. The assessment of the transcriptional signature of these elements can facilitate the development of gene expression markers suitable for a more informed investigation of the physiological progression of the postharvest ripening in apples, ultimately leading to the promotion of high-quality stored apples, extending storage time while minimizing postharvest disorders and fruit loss.

Citation: Romero I, Fortes AM and Costa F (2024) Editorial: Omics technologies and fruit postharvest quality. Front. Plant Sci. 15:1404708. doi: 10.3389/fpls.2024.1404708

High throughput arrays for the simultaneous genotyping of thousands of single-nucleotide polymorphisms (SNPs) have made the rapid genetic characterisation of plant genomes and the development of saturated linkage maps a realistic prospect for many plant species of agronomic importance. However, the correct calling of SNP genotypes in divergent polyploid genomes using array technology can be problematic due to paralogy, and to divergence in probe sequences causing changes in probe binding efficiencies. An Illumina Infinium II whole-genome genotyping array was recently developed for the cultivated apple and used to develop a molecular linkage map for an apple rootstock progeny (M432), but a large proportion of segregating SNPs were not mapped in the progeny, due to unexpected genotype clustering patterns. To investigate the causes of this unexpected clustering we performed BLAST analysis of all probe sequences against the 'Golden Delicious' genome sequence and discovered evidence for paralogous annealing sites and probe sequence divergence for a high proportion of probes contained on the array. Following visual re-evaluation of the genotyping data generated for 8,788 SNPs for the M432 progeny using the array, we manually re-scored genotypes at 818 loci and mapped a further 797 markers to the M432 linkage map. The newly mapped markers included the majority of those that could not be mapped previously, as well as loci that were previously scored as monomorphic, but which segregated due to divergence leading to heterozygosity in probe annealing sites. An evaluation of the 8,788 probes in a diverse collection of Malus germplasm showed that more than half the probes returned genotype clustering patterns that were difficult or impossible to interpret reliably, highlighting implications for the use of the array in genome-wide association studies.

Fleshy fruits are generally stored before consumption, especially apple, during which several diseases and disorders can occur. Mechanical handling and transportation may cause general wounding, leading to dramatic quality decay and fruit loss, thus to a negative economic impact. Although the majority of the research addressing fruit quality has mainly focused on the flesh, the multi-layered peel tissue complex represents the first natural barrier of a fruit. In this study, a texture analyzer was employed to investigate the peel contribution on the general fruit firmness of apple, assessing a germplasm collection composed by 65 accessions after two months of cold storage. This device generated a mechanical force–displacement profile from which a set of analytical parameters was derived. A comparative analysis between intact and peeled portions performed on each single fruit, enabled the characterization of the contribution of the peel on fruit firmness. By analysing the phenotypic variance of these analytical parameters within the apple collection by a multivariate statistical approach, two clusters were identified, amongst which a specific set of apple cultivars was distinguished by a superior peel contribution. The parameters here isolated and employed are finally proposed as novel traits with a potential impact for breeding, and suitable for the characterization and selection of novel accessions with an enhanced postharvest potential due to an improved mechanical performance of the peel.

Loquat (Eriobotrya japonica) fruit marketability is affected by the incidence and severity of purple spot (PS), a pre-harvest physiological disorder showing an evident skin discoloration with depressed surface. Despite its impact in limiting the cultivation and economic potential of loquat, the etiology of this disorder is still poorly understood. To this end, our study aimed to investigate and disclose possible mechanisms underlying PS development. The intensity and severity of PS in three loquat cultivars ('Morphitiki', 'Karantoki' and 'Obusa') was phenotypically monitored during successive on-tree fruit developmental stages. 'Obusa' fruits harvested at commercial maturity stage showed the highest incidence of purple spot (58.6%), while 'Morphitiki' fruits did not show any symptoms. 'Karantoki' fruits demonstrated an intermediate response, with 31.3% of the fruit being affected. Thereafter, fruits with 30–50% PS severity were selected and used for further analysis; peel tissue was removed from both symptomatic and asymptomatic tissue of the same fruit for all examined cultivars. 'Karantoki' fruit with PS were characterized by the highest accumulation of total soluble sugars, sucrose, glucose and fructose contents, while the concentration of these primary metabolites was the lowest in asymptomatic fruit of 'Obusa', exception made for the sucrose. The incidence of PS was also transcriptionally investigated by assessing the mRNA profile of important genes involved in polyphenolic (PAL1, PAL2 and PPO1) and carbohydrate (CWI2, CWI3, SPS1, SPS2, NI2, NI3, SuSy, HXK, FRK and VI) pathway. The enhanced expression levels of CWI3 and VI genes in symptomatic fruit of the highly susceptible cultivar 'Obusa' highlight a cultivar-specific type of response. Notably, SuSy registered significantly suppressed levels in symptomatic tissue of both 'Obusa' and 'Karantoki'. To what extent PPO is associated with PS incidence and whether the etiology of the disorder can be assigned to an oxidative process triggered and coordinated by its action need to be further elucidated. The aforementioned genes are suggested to be further examined as potential markers towards a more sophisticated and informed characterization of purple spot detection in loquat fruit.

The aim of this paper is to apply a methodology towards the sharing of responsibility between the utility and the consumer with reference to the occurrence of harmonic voltage distortions at the point of common coupling (PCC). The approach is based on the measured values of harmonic voltage and current as well as the supply and load harmonic impedance information. In addition to the general method principles, the paper focuses a specific application involving a real industrial installation, fed by 230 kV and having a large amount of rectifiers. The results are then used to verify the proposal consistency regarding the sharing of the responsibilities between the utility and the industry as far as the harmonic voltage distortion is concerned. The proposed process finds sustenance during the implementation of mitigation procedures with sights to the attendance of the standards of quality established by the regulating agencies.

Volatile secondary metabolites represent major and complex components of fruit flavor and odor. However, limited information about their genetic control is available. Quantitative trait loci (QTLs) affecting fruit volatile emission are usually identified by integrating genetic and metabolic data on germplasm collections or breeding materials. Proton Transfer Reaction—Time of Fligh—Mass Spectrometry (PTR-ToF-MS) is a novel fast and high sensitivity analytical method for VOC (Volatile Organic Compound) detection. Recently we effectively employed PTR-ToF-MS for studying the genetic control of ester emission in apple, although a complete methodology allowing full potential QTL investigations is still missing. In this work, for the first time, a new double clustering data mining strategy to fruit volatilome data produced by PTR-ToF-MS was applied. We used this new methodology in order to perform a QTL mapping investigation related to apple VOCs on a full-sib apple population (‘Golden Delicious’ × ‘Scarlet’) assessed during shelf-life ripening, resulting in the identification of several QTLs located on seven chromosomes and associated mainly with ethylene, estragole, carbonyl, alcohol, ester and sesquiterpene emission. Although the proposed methodology is applied to apple VOCs, potential for analogous investigations in other fruit species is discussed.

The molecular and biochemical events underlying the onset of superficial scald in two pear cultivars with different susceptibility (‘Blanquilla’ and ‘Conference’) was investigated in fruit untreated and treated with lovastatin, 1-MCP or ethylene. ‘Conference’ pears were characterized by higher content of flavonols and linolenic acid (18:3), two metabolites related to chilling injury resistance. In this cultivar, the expression level of three genes belonging to the ascorbate glutathione pathway (APX, DHAR and MDHAR) were constitutively over-expressed, highlighting the role that endogenous antioxidant potential played in scald control. In the scald-susceptible cultivar (‘Blanquilla’) the lovastatin treatment, in contrast to 1-MCP, effectively prevented superficial scald development and α-farnesene production without affecting fruit ripening. Moreover, lovastatin stimulated an increased production of ethanol and oleic + cis vaccenic acid (18:1), both compounds being also involved in cold stress tolerance. In both cultivars, and in contrast to 1-MCP, lovastatin did not impair the expression level of the genes devoted to ethylene production (ACO, ACS) and perception (ERS1, ERS2). As a consequence, the expression levels of the genes involved in texture modifications (PG1) and volatile emission (LOX, HPL, ADH and AAT) were maintained in lovastatin-treated samples allowing the fruit to reach an adequate final quality. The results from this study are discussed to highlight the complex regulatory network underlying superficial scald development in different pear cultivars.

Fruit flesh browning is a natural oxidative phenomenon occurring after cutting or processing. This event, manifesting dark-brown coloration, can seriously limit the production and marketability of fresh cut fruit products. Antioxidant compounds can partially prevent this phenomenon, but in turn they can affect the quality and the organoleptic properties of fresh products. In this work we have investigated, through a multidisciplinary approach, the regulation of a natural non-browning phenotype of a particular apple cultivar ‘Majda’ in comparison with the browning reference apple cultivar ‘Golden Delicious’. The results highlight that the non-browning phenotype of ‘Majda’ was controlled by multiple mechanisms, particularly distinguished by a different concentration of chlorogenic acid and the expression of the polyphenol oxidase (MdPPO) gene. The metabolite assessment and gene expression profiling also disclosed the involvement of organic acids and glutathione in the prevention of oxidative browning phenomenon. The peculiar non-browning behavior of ‘Majda’ was also verified by a DPPH• kinetic approach that demonstrated the higher antioxidant activity of this apple cultivar. The results presented in this work proposed ‘Majda’ as a potential parental candidate to be considered for breeding activity oriented to select novel non-browning accessions addressed to ameliorate the sustainability of pre-cut fresh products.

Modern agriculture depends on a coordinated system to evaluate, introduce, distribute and maintain germplasm, since plant germplasm is the base for a productive agriculture. It is estimated that 90% of the germplasm collections are stored as seeds, of which 40% are cereals. Medicinal plants are particularly difficult to store as seeds, due to the lack of knowledge of their reproduction biology and seed behavior. Besides, there are a large number of important tropical and subtropical medicinal plants species which produce recalcitrant seeds that quickly lose viability and do not survive desiccation, hence conventional seed storage strategies are not possible. There is also a number of other import species that are sterile or do not easily produce seeds, or seeds are highly heterozygous and clonal propagation is preferred to conserve elite genotypes. Although field genebanks provide easy access to conserved material for use, they have a risk of destruction by natural calamities, pest and diseases. For this reason, safety duplicates of the living collections are established using alternate strategies of conservation and it is in this area that biotechnology contributed significantly by providing complementary in vitro conservation options through tissue culture techniques. In vitro conservation also offers other distinct advantages. For example, the material can be maintained in a pathogen-tested state, thereby facilitating safer distribution and germplasm exchange. Further, the cultures are not subject to environmental disturbance. The National Genetic Resources and Biotechnology Research Center (CENARGEN), was created by the Brazilian Organization for Agricultural Research (EMBRAPA), an institution linked to the Ministry of Agriculture, in order to coordinate and organize all activities related to genetic resources in Brazil, including genetic resources of medicinal plants. The in vitro collection of medicinal plants of CENARGEN is constituted by at least 395 accessions of five genus: Mentha (74), Lippia (47), Pfaffia (15), Stevia (16) and Cochlospermum (10). Experiments have shown that in vitro shoot cultures stored at temperature in the range of 18-20°C on half strength Murashige and Skoog medium nutrient with 2% sucrose reduce the plant growth and significantly extend the subculture intervals of accessions to fresh medium. We conclude that this in vitro conservation system can be greatly useful for conservation and exchange of genetic resources of these medicinal plants.

In vascular plants the cell-to-cell interactions coordinating morphogenetic and physiological processes are mediated, among others, by the action of hormones, among which also short mobile peptides were recognized to have roles as signals. Such peptide hormones (PHs) are involved in defense responses, shoot and root growth, meristem homeostasis, organ abscission, nutrient signaling, hormone crosstalk and other developmental processes and act as both short and long distant ligands. In this work, the function of CTG134, a peach gene encoding a ROOT GROWTH FACTOR/GOLVEN-like PH expressed in mesocarp at the onset of ripening, was investigated for its role in mediating an auxin-ethylene crosstalk. In peach fruit, where an auxin-ethylene crosstalk mechanism is necessary to support climacteric ethylene synthesis, CTG134 expression peaked before that of ACS1 and was induced by auxin and 1-methylcyclopropene (1-MCP) treatments, whereas it was minimally affected by ethylene. In addition, the promoter of CTG134 fused with the GUS reporter highlighted activity in plant parts in which the auxin-ethylene interplay is known to occur. Arabidopsis and tobacco plants overexpressing CTG134 showed abnormal root hair growth, similar to wild-type plants treated with a synthetic form of the sulfated peptide. Moreover, in tobacco, lateral root emergence and capsule size were also affected. In Arabidopsis overexpressing lines, molecular surveys demonstrated an impaired hormonal crosstalk, resulting in a re-modulated expression of a set of genes involved in both ethylene and auxin synthesis, transport and perception. These data support the role of pCTG134 as a mediator in an auxin-ethylene regulatory circuit and open the possibility to exploit this class of ligands for the rational design of new and environmental friendly agrochemicals able to cope with a rapidly changing environment.

SummaryPlant disease-resistance genes show highly conserved regions corresponding to characteristic amino acid domains. These conservative structures make it possible to isolate resistance gene analogs (RGAs) by PCR using degenerate or heterologous primers. Putative apricot (‘Stark Early Orange’) RGAs were amplified, cloned and characterized using endonucleases; several divergent cloned-PCR products have been sequenced. The gene bank database screening showed a certain degree of homology with known resistance genes or RGA sequences. Specific primers designed on the basis of the hyper-variable regions of the sequences identified have been used to identify molecular markers for Sharka resistance. After screening of a panel of apricot genotypes (six resistant, ten susceptible and one tolerant), one primer pair (SEOBT101) appears to be associated with Sharka resistance.

The correlation of light availability and fruit quality traits for apple fruits (Malus × domestica Borkh.) within the canopy in two training systems, Slender Spindle and Bi-axis, were tested on nine-year-old trees of 'Gala' grafted on M9 rootstock. Three trees were chosen for each training system and twelve fruits from each tree were selected in different canopy positions and labeled after fruit set. The light availability for each fruit was measured using a digital camera equipped with a fisheye lens. At harvest, fruit size, over color and quality traits were assessed for each monitored fruit. The results indicated a large variability of fruit light availability within the canopy depending on fruit position. The upper and outer fruit positions for both training systems resulted in significantly higher light compared to the lower and inner positions. The percentage of fruit skin color and the soluble solids content (SSC) were influenced by the canopy layers. Fruits in the upper tree positions had a higher percentage of over color and higher SSC, while no differences were found in the inner and outer positions. Fruit position didn't affect fruit size, ripening stage, and flesh firmness. The two training systems resulted in no differences for light availability or fruit quality traits.

Functional markers, a new class of molecular marker, are based on the polymorphism present within the gene. We report the allelic variability and allele mining of markers obtained on four genes controlling specific traits. In order to investigate particular aspects of fruit quality, four functional markers for the ACO and ACS genes, which are involved in the ethylene pathway, and for the expansin and polygalacturonase genes, which play a key role in fruit softening, were positioned on the linkage maps of 'Fuji' x 'Mondial Gala' and 'Prima' x 'Fiesta' and evaluated for their involvement in phenotype from the 'Fuji' x 'Braeburn' cross. The four markers are: 1) Md-ACO1 mapped on linkage group 10 on the border of the 5% confidence region of a known QTL for firmness (King et al., 2000); 2) Md-ACS1 mapped on LG15, but it did not appear associated with any known QTL for firmness. Offspring homozygous for allele Md-ACS1-2 always showed low levels of ethylene synthesis and extremely good retention of fruit firmness and shelf - life. 3) Md-EXPDCA1 mapped on L01 at approximately 9 cM of the Vf gene was strongly correlated to a known QTL for crispiness and juiciness; and 4) Md-PG1 also mapped on LG10 and SNP polymorphism were associated with fruit softening.

In terms of the quality of minimally processed fruit, flesh browning is fundamentally important in the development of an aesthetically unpleasant appearance, with consequent off-flavours.The development of browning depends on the enzymatic action of the polyphenol oxidase (PPO).In the 'Golden Delicious' apple genome ten PPO genes were initially identified and located on three main chromosomes (2, 5 and 10).Of these genes, one element in particular, here called Md-PPO, located on chromosome 10, was further investigated and genetically mapped in two apple progenies ('Fuji x Pink Lady' and 'Golden Delicious x Braeburn').Both linkage maps, made up of 481 and 608 markers respectively, were then employed to find QTL regions associated with fruit flesh browning, allowing the detection of 25 QTLs related to several browning parameters.These were distributed over six linkage groups with LOD values spanning from 3.08 to 4.99 and showed a rate of phenotypic variance from 26.1 to 38.6%.Anchoring of these intervals to the apple genome led to the identification of several genes involved in polyphenol synthesis and cell wall metabolism.Finally, the expression profile of two specific candidate genes, up and downstream of the polyphenolic pathway, namely phenylalanine ammonia lyase (PAL) and polyphenol oxidase (PPO), provided insight into flesh browning physiology.Md-PPO was further analyzed and two haplotypes were characterised and associated with fruit flesh browning in apple.

To preserve quality features and ensuring availability of fresh fruit on the market, apples need to be stored after harvest. The low temperature applied during storage, beside avoiding important fruit loss, can also promote the development of serious chilling injury disorders, such as superficial scald. One of the strategies largely employed to prevent the development of this phenomenon is the control of the storage atmosphere by lowering down the oxygen concentration. In this work, a multifaceted survey was carried out to investigate transcriptome variation together with three categories of metabolites (phenolics, lipids and volatile organic compounds ) in fruit of 'Granny Smith' apple cultivar stored in both static and dynamic controlled hypoxia atmosphere for five and seven months, respectively. The global transcriptome survey identified a core set of differentially expressed genes in three main functional groups, revealing as the duration of storage had an important effect in the coordination of gene expression. The effect of the length of storage was furthermore highlighted by the DEG-network analysis that identified a distinct number and type of transcriptomic hubs. Samples characterized by the development of superficial scald were distinguished by a higher concentration of chlorogenic acid and a higher expression of phenylalanine ammonia lyase (PAL) and polyphenol oxidase (PPO) in a time-dependent fashion. The prevention of this disorder was instead related to distinctive re-programming events, involving the accumulation of specific antioxidant types of metabolites, very long chain fatty acids (VLCFAs) and the expression of genes coordinating a hypoxia acclimation process, such as RAP2-like and plant cysteine oxidase (PCO).

Abstract This study aimed to evaluate, through infrared thermography, the water status of melon crop during the fruiting phase and the productivity relationships resulting from fixed deficit irrigation (FDI) and regulated deficit irrigation (RDI) in two cultivation cycles. Nine irrigation treatments based on ETc (%) were applied, comprising four FDI treatments (T1- 125%; T2-100%; T3-75%; T4-50% throughout the cycle) and five RDI treatments (T5, T6, T7, T8, and T9) with replacements varying by 25% of ETc at stages I, II, III, and IV of the melon crop. Data on plant temperature (Tc) and air temperature (Ta) at 8:00, 12:00, and 16:00 h, leaf water potential (ΨF), gas exchange ( gs and E ), and soil moisture (SMO) were obtained at 45 days after transplanting in cycle I and at 42 days after transplanting in cycle II. Using the Tc and Ta data, thermographic indices ∆T canopy−air , CWSI, and IG were calculated. After harvesting, productivity and water use efficiency (WUE) were determined. Gas exchange did not strongly correlate with water replacement for FDI treatments in both conducted cycles. The ∆T canopy−air , CWSI, and IG indices significantly correlated with ΨF and SMO at the monitored times; however, 12:00 h was found to be the most suitable time for thermographic monitoring. Productivity and WUE were higher in the RDI treatments, with the T6 treatment (50% in phase I, 100% in phase II, and 75% in phases III and IV) standing out. Correlations between WUE and thermographic indices showed better results for RDI in cycle I, with both indices at 8 h and 12 h. In cycle II, better correlations were presented by FDI at both monitored.

Single-nucleotide polymorphisms (SNPs) have been recognized as the marker of choice in genetic studies on mapping populations due to their suitability for high throughput genome-wide genotyping. Several SNP genotyping platforms and related software for genotype calling are commercially available, including Illumina Infinium platform and the Genome Studio software from Illumina. Although the SNP genotyping can benefit of a high automation rate, SNP-allele calling is far from being fully automatized and, equally important, a common approach for filtering of well performing markers has not been published yet. Moreover, the application of a 20K array on diploid apple learned that around 7% of the markers were incorrectly called due to limitations in the genotyping software. All this made the genotyping a timeconsuming procedure with an unnecessary high error rate. In this poster we present a semi-automatized, pipeline for SNP filtering and calling of progenies of mapping populations based on Excel using Genome Studio derived data as input. This pipeline efficiently distinguishes between robust informative markers and markers that are problematic or truly monomorphic. It also resolves miss-callings that are due to the presence of paralogous loci, null alleles, or of additional SNP at the primer site. The final set of SNP markers are grouped based on their segregation pattern and genotype calls are adjusted to JoinMap format. Completion of the pipeline from start to finish takes just 2-3 hours. The pipeline has been calibrated by the use of three mapping populations and has been applied on another ten populations. It showed to be highly efficient, as 99.7% of the filtered polymorphic markers mapped easily. This work was performed in the framework of the EU-FruitBreedomics project, a central goal of which is to accelerate and increase the efficiency of Rosaceous cultivar release through the delivery of molecular markers that are of use for marker assisted breeding

Fruit texture and long storage life are key features defining apple eating-quality. Changes in flesh firmness, as a combination of such traits as crispiness, hardness and rate of softening, are part of the physiological process of ripening. The relationship between some cell-wall disassembly enzymes and ethylene production in climacteric fruit (mainly tomato) has been widely demonstrated. In this work we studied this physiological correlation in apple using a semi-quantitative RT-PCR technique on cv. 'Mondial Gala' fruits differing in firmness and ethylene production according to the considered ripening stage. Allele-specific primers were designed on specific apple homologue sequences for ACO and ACS ripening genes involved in ethylene biosynthesis and for isoforms of expansin and polygalacturonase that play a key role in fruit softening. A different expression of ACO and ACS genes was found depending on ethylene production; expansin and polygalacturonase expression patterns agree with the general model of softening (an early stage, expansin-dependent, and a later one, poligalacturonase-dependent), proposed for climacteric fruits.

At FEM-IASMA we initiated an innovative integrated breeding program, combining the information derived by the sequencing of the heterozygous Golden Delicious apple genome with traditional breeding activities. Our main goal is the creation of novel apple varieties characterised by a high quality and resistant to major pathogen diseases. Marker technology can improve the long and laborious selection process, allowing for marker assisted selection. The anchoring of the assembled genome to our genetic maps will enable the identification of the most favourable markers closely related to the traits of interest, aiding in efficient selection of the next generation seedlings.

The Slender Spindle, one of the most used training systems for apple, was compared with the Bi-axis, an innovative training system, to determine their effect on fruit quality and fruit distribution within the canopy on 'Gala' cultivar. Two levels of crop loads were also introduced to observe their effects through chemical and manual thinning. Fruit position in the canopy was monitored during summer using different software to draw in 3D the tree architecture. Also, fruits were monitored during the growing season for their growth and maturation. At the end of the season, fruits were harvested, graded and analyzed for their quality traits. Fruit position had a strong influence on fruit quality. The fruits in the layers were significantly different. In the upper part of the tree, fruits had a higher percentage of over color and bigger fruit size compared to the middle and the lower part of the canopy. No significant differences were found between the two training systems. Crop load influenced fruit quality in both training systems in the same way on fruit size and over color. The lowest crop load treatment had a higher percentage of over color and bigger fruit size compared to the higher crop load.

Fruit quality is represented by a series of genetically controlled features that change throughout the entire ontogenic life. Among the several quality traits, texture plays a crucial role, impacting both consumers’ appreciation and postharvest performance. In order to decipher its regulation a multidisciplinary approach was employed. Initially, the texture performance was measured with a high resolution phenotyping device, represented by a texture analyzer equipped with an acoustic device. In the first attempt to dissect the fruit texture genetic control, two QTL mapping strategies were used. The first approach employed six bi-parental families linked by a common pedigree scheme, known as pedigree based analysis. The joint analysis of the phenotypic and genotypic data set through a Bayesian statistics identified a series of genomic regions related to both mechanical and acoustic signatures. These regions were further validated with a genome wide association study approach, which considered a much larger phenotypic and genotypic variation. To complement the genetic information, a whole transcriptome analysis was also carried out. To this end, two microarray platforms were designed and used to unravel the functional machinery ongoing during the fruit development and ripening phases, especially with regards to the plant hormone ethylene. In this study, the role of this hormone was dissected applying 1-MCP, a molecule competing with ethylene at receptor level. The combination of these resources provides a valuable source of information, essential to step forward in the comprehension of the genetic and physiological regulation of the fruit texture in apple. This knowledge would enable, in a close future, a more accurate and precise selection of the most favourable and valuable new apple accessions distinguished by a superior fruit quality.

Abstract Texture plays a major role in the determination of fruit quality in apple. Due to its physiological and economic relevance, this trait has been largely investigated, leading to the fixation of the major gene PG1 controlling firmness in elite cultivars. To further improve fruit texture, the targeting of an undisclosed reservoir of loci with minor effects is compelling. In this work, we aimed to unlock this potential with a genomic selection approach by predicting fruit acoustic and mechanical features as obtained with a TA.XT plus texture analyzer in 537 individuals genotyped with 8,294 SNP markers. The best prediction accuracies following cross-validations within the training set (TRS) of 259 individuals were obtained for the acoustic linear distance (0.64). Prediction accuracy was further improved through the optimization of TRS size and composition according to the test set. With this strategy, a maximal accuracy of 0.81 was obtained when predicting the synthetic trait PC1 in the family ‘Gala × Pink Lady’. We discuss the impact of genetic relatedness and clustering on trait variability and predictability. Moreover, we demonstrated the need for a comprehensive dissection of the complex texture phenotype and the potentiality of using genomic selection to improve fruit quality in apple. Highlight A genomic selection study, together with the optimization of the training set, demonstrated the possibility to accurately predict texture sub-traits valuable for the amelioration of fruit quality in apple.