Grapevine is one of the most relevant fruit crops worldwide, owing to its extensive distribution and considerable socio-economic significance. While the cultivated Eurasian species Vitis vinifera dominates global grape production, wild Vitis species from Asia and North America constitute essential genetic reservoirs, offering allelic diversity associated with tolerance or resistance to several biotic and abiotic stresses. Genotyping is a key tool in grapevine genetics, as it enables the assessment of genetic diversity, the elucidation of the molecular basis of agronomic and adaptive traits, and the implementation of marker-assisted selection. SNP (single nucleotide polymorphism) arrays provide an efficient genotyping tool, combining high-throughput capability, cost-effectiveness, and dense genome-wide marker coverage. Here, we report the development and validation of the Axiom®Vitis22K SNP array, implemented within a broader multi-species 70K SNP platform. The array includes informative SNPs from the GrapeReSeq 18K Vitis genotyping chip, SNPs and small InDels (insertions and deletions) putatively associated with phenotypic traits from literature, and novel SNPs obtained from the resequencing of 12 samples representing seven different grapevine species. Validation was performed by genotyping 144 genotypes from two diversity panels. Genotyping data were processed with the Axiom Analysis Suite and the newly developed AxioSAFE pipeline revealing a total of 10,314 robust polymorphic markers, together with 13 manually curated variants. These markers were successfully employed to study the genetic diversity and genetic relationships within the sample panels. Integration of genotypic and phenotypic data also enabled the validation of candidate SNPs associated with target traits such as flower sex type, seed content, berry color and taste. Overall, the results demonstrate the potential of the Vitis22K array to support large-scale genetic studies and breeding programs in grapevine.

Apple is one of the major cultivated fruit crops in temperate regions. To better support breeding programs and facilitate the development of improved cultivars, we generated a new haplotype-resolved version of the Golden Delicious genome, one of the major founders of many modern apple lineages. The assembly features the separation of the two haplotypes, with a total size of 647.3 Mb and 649.2 Mb, respectively. The phasing was accurately validated with 10,321 curated SNPs. Telomere-to-telomere continuity was verified by the analysis of telomeric sequence composition at the end of each chromosome. Gene prediction identified a total of 45,116 genes in haplotype 1 and 45,063 genes in haplotype 2. A pangenome analysis employing 6 haplotype resolved genomes identified both common and unique gene families. The availability of a phased genome enabled the assessment of genome-wide allelic specific expression. Our case study, focusing on Md-PG1 (a key regulator of fruit softening), revealed that the allelic form on haplotype 2 (GDH2-10g24673) was the dominant contributor to total gene expression. In addition, the phased genome also showed specific miRNA chromosomal distribution patterns, as well as a distinct methylation profile. Altogether, these genomic resources provide new insights into the allelic regulation of key agronomic traits and represent a valuable tool to accelerate apple breeding. ### Competing Interest Statement The authors have declared no competing interest.

Motivation:Genotyping datasets generated via the Thermo Fisher Axiom® array are generally big, as they comprise tens of thousands of markers and hundreds of individuals, and currently, no automatic data curation pipelines are available for this kind of data. This leaves researchers with only time-consuming manual analysis as the current standard for processing these complex genotyping datasets. There is a clear need for a more efficient, streamlined approach to handle the specific quality control challenges inherent in this platform. Results:AxioSAFE (Axiom SNP Assessment and Filtering Engine) is a semi-automatic computer tool for the curation of single nucleotide polymorphism (SNP) genotyping datasets generated via Thermo Fisher Axiom® array experiments. AxioSAFE provides an alternative methodology to cover a set of data curation operations, including steps such as a ploidy check, SNP filtering, Mendelian error analysis, and phasing. AxioSAFE identifies major occurrences of problematic SNPs and samples, including those not caught by the Axiom array default QC filters. Further functionality is included to let the user review identified problematic SNP classes. Availability and implementation:AxioSAFE is a Python program that can be either used via the command line interface or through a graphical user interface (GUI) and is provided as a Docker container available on DockerHub at https://hub.docker.com/r/lzspin/axiosafe, which includes all required libraries, software, and a tutorial dataset. The source code and documentation are available at https://bitbucket.org/lzspin/axiosafe/. The apple dataset used for the development of AxioSAFE is available at DOI: https://doi.org/10.5281/zenodo.18034024.

Primary metabolites are essential molecules playing a central role in plant biology and in the determination of fundamental fruit quality characteristics, such as sweetness and acidity. In this work, carbohydrates, organic and amino acids have been assessed with NMR for a large-scale screening in the apple flesh of an interspecific collection comprising 43 Malus species. Among the analyzed compounds, seven metabolites, with a Variable Importance in Projection score greater than 1, largely contributed to the distinction between wild Malus spp. and M. domestica accessions. The three most relevant compounds were sucrose, fructose and malic acid, each showing a distinct pattern. In general, sugars were more concentrated in the M. domestica group, while organic acids predominated in wild Malus species. Four amino acids were also detected, and although their overall contribution was smaller, they were more concentrated in M. domestica accessions. This metabolic survey elucidates the role of these specific primary metabolites in apple as domestication associated traits and provides a valuable resource for identifying promising accessions for modern breeding programs aimed at improving fruit quality.

Secondary metabolites play fundamental roles in apple, influencing the interaction with pollinators and frugivores for seed dispersal, contributing to fruit quality and promoting human health through their antioxidant property. Domestication and breeding have significantly re-shaped the apple metabolism, altering both aromatic profiles and nutritional properties. This study assessed the secondary metabolite variation in a comprehensive Malus spp. collection comprising 163 accessions belonging to 44 species. The profiling of phenolic and volatile organic compounds (VOCs), performed with Ultra-Performance Liquid Chromatography (UPLC) and Proton-Transfer-Reaction Time-of-Flight Mass Spectrometry (PTR-ToF-MS) instruments respectively, in both skin and pulp tissues, uncovered distinct metabolic patterns between wild and domesticated apples. This investigation underlined the higher concentration of these metabolites in the skin tissue and revealed a clear metabolic divergence between the two groups of Malus accessions. Wild Malus spp. accessions resulted particularly rich in specific polyphenols characterized by antioxidant activity, including catechin and procyanidins. Conversely, apples of Malus domestica accessions exhibited a more abundant VOC profile, particularly represented by esters associated with fruity aroma, enhancing sensory appeal. These findings provide a foundation for leveraging wild germplasm in breeding programs, and the identification of accessions with high polyphenolic concentration and desirable aromatic profiles offers valuable opportunities to improve the aromatic and nutraceutical properties of apple.

Fruit firmness and softening rate are two key quality parameters defined by the enzymatic disassembly of the polysaccharide architecture of the primary cell wall and middle lamella. Technological control of fruit ripening in pear, while extending shelf-life, can negatively affect general fruit quality. Therefore, genetic improvement of these properties can represent a valuable alternative. Two bi-parental populations were employed to dissect the genetic control of static and dynamic firmness traits, considering fruit firmness assessed at both harvest and after storage, by defining softening and storage index-derived parameters. Integrated QTL analysis was performed through a multi-parental cross design based on a Pedigree Based Analysis approach. This allowed the identification of specific QTL signatures distinguished by an increasing cumulative percentage of variability expressed from the harvest to the post-harvest stage and highlighted the presence of a major QTL on linkage group 3. The QTL intervals were distinguished by the presence of several classes of genes involved in the degradation of the cell wall, such as expansins, polygalacturonases, and pectate lyase. Haploblocks (HBs) derived by single SNPs also elucidated the role of HB-alleles as potential marker tools to assist in breeding programmes aimed at improving fruit firmness and softening, especially during post-harvest.

BACKGROUND:Among the Citrus species, lemon (Citrus limon Burm f.) is one of the most affected by the two-spotted spider mite (Tetranychus urticae Koch). Moreover, chemical control is hampered by the mite's ability to develop genetic resistance against acaricides. In this context, the identification of the genetic basis of the host resistance could represent a sustainable strategy for spider mite control. In the present study, a marker-trait association analysis was performed on a lemon population employing an association mapping approach. An inter-specific full-sib population composed of 109 accessions was phenotyped through a detached-leaf assays performed in modified Huffaker cells. Those individuals, complemented with two inter-specific segregating populations, were genotyped using a target-sequencing approach called SPET (Single Primer Enrichment Technology), the resulting SNPs were employed for the generation of an integrated genetic map.RESULTS:The percentage of damaged area in the full-sib population showed a quantitative distribution with values ranging from 0.36 to 9.67%. A total of 47,298 SNPs were selected for an association mapping study and a significant marker linked with resistance to spider mite was detected on linkage group 5. In silico gene annotation of the QTL interval enabled the detection of 13 genes involved in immune response to biotic and abiotic stress. Gene expression analysis showed an over expression of the gene encoding for the ethylene-responsive transcription factor ERF098-like, already characterized in Arabidopsis and in rice for its involvement in defense response.CONCLUSION:The identification of a molecular marker linked to the resistance to spider mite attack can pave the way for the development of marker-assisted breeding plan for the development of novel selection coupling favorable agronomical traits (e.g. fruit quality, yield) with a higher resistance toward the mite.

Heirloom Danish apple cultivars are historically and pomologically important, part of the cultural heritage, and have valuable adaptation to regional climate conditions. However, lack of information about their genetic identity and pedigree relatedness with other cultivars hampers proper cultivar identification, germplasm curation, genebank management, and future regional breeding efforts. Many Danish apple cultivars are maintained in the national collection “The Pometum”, maintaining around 850 apple accessions. Additional material is maintained in public or private Danish collections. However, no information exists regarding genotypic duplicates between these collections and germplasm collections in other countries, pedigree inferences across collections, and genotypically unique accessions at the genebank level. To provide such information, 976 accessions from Denmark were genotyped with simple sequence repeat (SSR) markers and the Illumina Infinium 20K single nucleotide polymorphism (SNP) array. The resulting genotypic data were compared to large databases of genotypic data from germplasm collections in multiple countries to identify genotypic duplicates and conduct pedigree reconstruction. The germplasm maintains 305 unique genotypic profiles which were not found in other germplasm collections. The study exposed previously unknown synonyms, accessions not true-to-type, and novel pedigree relationships involving accessions from multiple collection sites. The most frequent parents of Danish germplasm were ‘Hvid Vinter Pigeon’ and ‘Cox’s Orange Pippin’ whereas ‘Reinette Franche’ was the most common grandparent. The accession-level information will benefit germplasm curation, cultivar identification, genebank management, and future breeding efforts, and shed new light on cultivar history and origin.

Juglans regia (L.) is cultivated worldwide for its nutrient-rich nuts. In Italy, despite the growing demand, walnut cultivation has gone through a strong decline in recent decades, which led to Italy being among the top five net importing countries. To promote the development of local high-quality Italian walnut production, we devised a multidisciplinary project to highlight the distinctive traits of three varieties grown in the mountainous region Trentino (northeast of Italy): the heirloom 'Bleggiana', a second local accession called local Franquette and the French cultivar 'Lara', recently introduced in the local production to increase yield. The genetic characterization confirmed the uniqueness of 'Bleggiana' and revealed local Franquette as a newly described autochthonous variety, thus named 'Blegette'. The metabolic profiles highlighted a valuable nutritional composition of the local varieties, richer in polyphenols and with a lower ω-6/ω-3 ratio than the commercial 'Lara'. 'Blegette' obtained the highest preference scores from consumers for both the visual aspect and tasting; however, the volatile organic compound profiles did not discriminate among the characterized cultivars. The described local varieties represent an interesting reservoir of walnut genetic diversity and quality properties, which deserve future investigation on agronomically useful traits (e.g., local adaptation and water usage) for a high-quality and sustainable production.

Background Single nucleotide polymorphism (SNP) array technology has been increasingly used to generate large quantities of SNP data for use in genetic studies. As new arrays are developed to take advantage of new technology and of improved probe design using new genome sequence and panel data, a need to integrate data from different arrays and array platforms has arisen. This study was undertaken in view of our need for an integrated high-quality dataset of Illumina Infinium® 20 K and Affymetrix Axiom® 480 K SNP array data in apple ( Malus × domestica ). In this study, we qualify and quantify the compatibility of SNP calling, defined as SNP calls that are both accurate and concordant, across both arrays by two approaches. First, the concordance of SNP calls was evaluated using a set of 417 duplicate individuals genotyped on both arrays starting from a set of 10,295 robust SNPs on the Infinium array. Next, the accuracy of the SNP calls was evaluated on additional germplasm ( n = 3141) from both arrays using Mendelian inconsistent and consistent errors across thousands of pedigree links. While performing this work, we took the opportunity to evaluate reasons for probe failure and observed discordant SNP calls. Results Concordance among the duplicate individuals was on average of 97.1% across 10,295 SNPs. Of these SNPs, 35% had discordant call(s) that were further curated, leading to a final set of 8412 (81.7%) SNPs that were deemed compatible. Compatibility was highly influenced by the presence of alternate probe binding locations and secondary polymorphisms. The impact of the latter was highly influenced by their number and proximity to the 3′ end of the probe. Conclusions The Infinium and Axiom SNP array data were mostly compatible. However, data integration required intense data filtering and curation. This work resulted in a workflow and information that may be of use in other data integration efforts. Such an in-depth analysis of array concordance and accuracy as ours has not been previously described in the literature and will be useful in future work on SNP array data integration and interpretation, and in probe/platform development.

Within a span of the last two decades, sequencing technologies have rapidly evolved, allowing for the production of genome sequences from multiple angiosperm clades, including major crops and botanical models. Apple (Malus x domestica) is among the first crop genomes to be fully sequenced, thus contributing to expanded knowledge of genome structure, biological functions, trait physiology and inheritance, and leading to practical applications for crop improvement. Access to full genome sequences has also allowed for the development of new fields of investigation, such as epigenetics. With the advent of accurate and cheaper new sequencing technologies, together with high-throughput phenotyping methods, the next decade will probably see the development of projects based on whole epi/genome sequencing or resequencing, providing researchers with a firm foundation to investigate the dynamics involved in the development of traits of interest.

A wealth of previously unknown pedigree information has been generated for apple (Malus x domestica) through recent studies and an ongoing pedigree identification project using SNP array data. This information has been postulated to be useful in a number of ways for germplasm collections and breeding programs. For example, pedigree information is an important part of genetic characterization of material in germplasm collections. Germplasm collections often seek to preserve cultivars that currently lack commercial relevance but have regional or historical significance or are phenotypically interesting. Pedigree information is often limited on such material. With pedigree information, an accurate genetic structure of these collections can be conveyed to breeders who wish to incorporate novel germplasm into new cultivars. To illustrate ways in which this newly generated pedigree information can be used, we provide examples involving accessions from the germplasm collection Okowerk and the breeding association "apfel:gut e.V.", which focus in the development of apples for regional organic production. Both organizations are located in northern Germany. Despite focusing on regional apple cultivars and genetic diversity, many of their accessions and breeding lines are common, cosmopolitan, and foundational cultivars. However, the SNP-based analyses also reveal regionally specific founders, not true-to-type accessions, and genetically unique historical cultivars. This new information has benefitted these organizations by providing genetic characterization that was previously lacking, enabling them to better utilize these genetic resources. Additionally, this information will also be useful in the context of an ongoing larger scale apple pedigree reconstruction project.

Almond is appreciated for its nutraceutical value and for the aromatic profile of the kernels. In this work, an almond collection composed of 96 Sicilian accessions complemented with 10 widely cultivated cultivars was phenotyped for the production of volatile organic compounds using a proton-transfer time-of-flight mass spectrometer and genotyped using the Illumina Infinium ® 18 K Peach SNP array. The profiling of the aroma was carried out on fresh and roasted kernels enabling the detection of 150 mass peaks. Sixty eight, for the most related with sulfur compounds, furan containing compounds, and aldehydes formed by Strecker degradation, significantly increased during roasting, while the concentration of fifty-four mass peaks, for the most belonging to alcohols and terpenes, significantly decreased. Four hundred and seventy-one robust SNPs were selected and employed for population genetic studies. Structure analysis detected three subpopulations with the Sicilian accessions characterized by a different genetic stratification compared to those collected in Apulia (South Italy) and the International cultivars. The linkage-disequilibrium (LD) decay across the genome was equal to r 2 = 0.083. Furthermore, a high level of collinearity ( r 2 = 0.96) between almond and peach was registered confirming the high synteny between the two genomes. A preliminary application of a genome-wide association analysis allowed the detection of significant marker-trait associations for 31 fresh and 33 roasted almond mass peaks respectively. An accurate genetic and phenotypic characterization of novel germplasm can represent a valuable tool for the set-up of marker-assisted selection of novel cultivars with an enhanced aromatic profile.

Lemon (Citrus limon (L.) Burm. f.) is an evergreen tree belonging to the genus Citrus. The fruits are particularly prized for the organoleptic and nutraceutical properties of the juice and for the quality of the essential oils in the peel. Herein, we report, for the first time, the release of a high-quality reference genome of the two haplotypes of lemon. The sequencing has been carried out coupling Illumina short reads and Oxford Nanopore data leading to the definition of a primary and an alternative assembly characterized by a genome size of 312.8 Mb and 324.74 Mb respectively, which agree well with an estimated genome size of 312 Mb. The analysis of the transposable element (TE) allowed the identification of 2878 regions on the primary and 2897 on the alternative assembly distributed across the nine chromosomes. Furthermore, an in silico analysis of the microRNA genes was carried out using 246 mature miRNA and the respective pre-miRNA hairpin sequences of Citrus sinensis. Such analysis highlighted a high conservation between the two species with 233 mature miRNAs and 51 pre-miRNA stem-loops aligning with perfect match on the lemon genome. In parallel, total RNA was extracted from fruit, flower, leaf, and root enabling the detection of 35,020 and 34,577 predicted transcripts on primary and alternative assemblies respectively. To further characterize the annotated transcripts based on their function, a gene ontology and a gene orthology analysis with other Citrus and Citrus-related species were carried out. The availability of a reference genome is an important prerequisite both for the setup of high-throughput genotyping analysis and for functional genomic approaches toward the characterization of the genetic determinism of traits of agronomic interest.

Peach (Prunus persica (L.) Batsch) is one of the most produced and studied stone fruits. Many genetic and genomic resources are available for this species, including a high-quality genome. More recently, a new high-density Illumina peach Single Nucleotide Polymorphism (SNP) chip (9+9K) has been developed by an international consortium as an add-on to the previous 9K array. In the current study, this new array was used to study the genetic diversity and population structure of the National Peach Germplasm Collection of the Agrifood Research and Technology Centre of Aragon (CITA), located in Zaragoza (northern Spain). To accomplish this, 90 peach accessions were genotyped using the new peach SNP chip (9+9K). A total of 9796 SNPs were finally selected for genetic analyses. Through Identity-By-Descent (IBD) estimate analysis, 15 different groups with genetically identical individuals were identified. The genetic diversity and population structure elucidated a possible exchange of germplasm material among regions, mainly in the northern regions of Spain. This study will allow for more efficient management of the National Peach Germplasm Collection by classifying valuable individuals for genetic diversity preservation and will benefit forthcoming Genome-Wide Association Studies (GWAS) of commercially important fruit traits in peach.

Background: The release of the first reference genome of walnut (Juglans regia L.) enabled many achievements in the characterization of walnut genetic and functional variation. However, it is highly fragmented, preventing the integration of genetic, transcriptomic, and proteomic information to fully elucidate walnut biological processes. Findings: Here, we report the new chromosome-scale assembly of the walnut reference genome (Chandler v2.0) obtained by combining Oxford Nanopore long-read sequencing with chromosome conformation capture (Hi-C) technology. Relative to the previous reference genome, the new assembly features an 84.4-fold increase in N50 size, with the 16 chromosomal pseudomolecules assembled and representing 95% of its total length. Using full-length transcripts from single-molecule real-time sequencing, we predicted 37,554 gene models, with a mean gene length higher than the previous gene annotations. Most of the new protein-coding genes (90%) present both start and stop codons, which represents a significant improvement compared with Chandler v1.0 (only 48%). We then tested the potential impact of the new chromosome-level genome on different areas of walnut research. By studying the proteome changes occurring during male flower development, we observed that the virtual proteome obtained from Chandler v2.0 presents fewer artifacts than the previous reference genome, enabling the identification of a new potential pollen allergen in walnut. Also, the new chromosome-scale genome facilitates in-depth studies of intraspecies genetic diversity by revealing previously undetected autozygous regions in Chandler, likely resulting from inbreeding, and 195 genomic regions highly differentiated between Western and Eastern walnut cultivars. Conclusion: Overall, Chandler v2.0 will serve as a valuable resource to better understand and explore walnut biology.