Metabolomics on Apple (Malus domestica) Cuticle-Search for Authenticity Markers.

The profile of secondary metabolites present in the apple cuticular layer is not only characteristic of a particular apple cultivar; it also dynamically reflects various external factors in the growing environment. In this study, the possibility of authenticating apple samples by analyzing their cuticular layer extracts was investigated. Ultra-high-performance liquid chromatography coupled with high-resolution tandem mass spectrometry (UHPLC-HRMS/MS) was employed for obtaining metabolomic fingerprints. A total of 274 authentic apple samples from four cultivars harvested in the Czech Republic and Poland between 2020 and 2022 were analyzed. The complex data generated, processed using univariate and multivariate statistical methods, enabled the building of classification models to distinguish apple cultivars as well as their geographical origin. The models showed very good performance in discriminating Czech and Polish samples for three out of four cultivars: "Gala", "Golden Delicious" and "Idared". Moreover, the validity of the models was tested over several harvest seasons. In addition to metabolites of the triterpene biosynthetic pathway, the diagnostic markers were mainly wax esters. "Jonagold", which is known to be susceptible to mutations, was the only cultivar for which an unambiguous classification of geographical origin was not possible.

Molecular Characterization of a Novel Enamovirus Infecting Raspberry.

Raspberry plants, valued for their fruits, are vulnerable to a range of viruses that adversely affect their yield and quality. Utilizing high-throughput sequencing (HTS), we identified a novel virus, tentatively named raspberry enamovirus 1 (RaEV1), in three distinct raspberry plants. This study provides a comprehensive characterization of RaEV1, focusing on its genomic structure, phylogeny, and possible transmission routes. Analysis of nearly complete genomes from 14 RaEV1 isolates highlighted regions of variance, particularly marked by indel events. The evidence from phylogenetic and sequence analyses supports the classification of RaEV1 as a distinct species within the Enamovirus genus. Among the 289 plant and 168 invertebrate samples analyzed, RaEV1 was detected in 10.4% and 0.4%, respectively. Most detections occurred in plants that were also infected with other common raspberry viruses. The virus was present in both commercial and wild raspberries, indicating the potential of wild plants to act as viral reservoirs. Experiments involving aphids as potential vectors demonstrated their ability to acquire RaEV1 but not to successfully transmit it to plants.

Clarithromycin Suppresses Apple Proliferation Phytoplasma in Explant Cultures.

Apple proliferation, caused by 'Candidatus Phytoplasma mali', is one of the most important economic threats in the field of apple production. Especially at a young age, infected trees can be affected by excessive bud proliferation and general decline. The fruit quality is also significantly reduced by this disease. To investigate treatment options, we applied a clarithromycin chemotherapy to infected in vitro cultures of 'Golden Delicious'. With increasing concentrations of clarithromycin in the media, the phytoplasma load decreased rapidly after one month of treatment, but phytotoxicity led to a pronounced mortality at 40 mg/L, which was the highest dose used in our experiment. Out of 45 initial explants, we obtained one negative mericlone and two mericlones with a concentration of phytoplasma DNA at the detection limit of PCR. The culture propagated from the mericlone that tested negative remained phytoplasma-free after 18 months of subculturing. Our results suggest the applicability of macrolide antibiotics against phytoplasma infections in vitro; however, it might be challenging to find the threshold zone where the concentration is sufficient for pathogen elimination, but not lethal for the plant material of different cultivars.

Elimination of Solanum nigrum ilarvirus 1 and Apple Hammerhead Viroid from Apple Cultivars Using Antivirals Ribavirin, Rimantadine, and Zidovudine.

Apple hammerhead viroid (AHVd) was detected in the apple cultivar 'Sampion' and in mixed infection with Solanum nigrum ilarvirus 1 (SnIV-1) in the cultivars 'Selena' and 'Jonagored Supra', using a high-throughput sequencing method. Experiments were conducted to eliminate both pathogens in apples using meristem tip cultures in combination with the antivirotics ribavirin, rimantadine, and zidovudine. Elimination of both pathogens was verified by repeated RT-PCR and qRT-PCR assays after 7-11 months. Elimination of SnIV-1 from all cultivars was successful with each of the three antivirotics at concentrations of 20, 40, and 80 mg L-1. Elimination of AHVd was also achieved, although less effectively and only with ribavirin in the concentration range of 20-160 mg L-1.

High-resolution genome-wide association study of a large Czech collection of sweet cherry (Prunus avium L.) on fruit maturity and quality traits.

In sweet cherry (Prunus avium L.), quantitative trait loci have been identified for fruit maturity, colour, firmness, and size to develop markers for marker-assisted selection. However, resolution is usually too low in those analyses to directly target candidate genes, and some associations are missed. In contrast, genome-wide association studies are performed on broad collections of accessions, and assemblies of reference sequences from Tieton and Satonishiki cultivars enable identification of single nucleotide polymorphisms after whole-genome sequencing, providing high marker density. Two hundred and thirty-five sweet cherry accessions were sequenced and phenotyped for harvest time and fruit colour, firmness, and size. Genome-wide association studies were used to identify single nucleotide polymorphisms associated with each trait, which were verified in breeding material consisting of 64 additional accessions. A total of 1 767 106 single nucleotide polymorphisms were identified. At that density, significant single nucleotide polymorphisms could be linked to co-inherited haplotype blocks (median size ~10 kb). Thus, markers were tightly associated with respective phenotypes, and individual allelic combinations of particular single nucleotide polymorphisms provided links to distinct phenotypes. In addition, yellow-fruit accessions were sequenced, and a ~ 90-kb-deletion on chromosome 3 that included five MYB10 transcription factors was associated with the phenotype. Overall, the study confirmed numerous quantitative trait loci from bi-parental populations using high-diversity accession populations, identified novel associations, and genome-wide association studies reduced the size of trait-associated loci from megabases to kilobases and to a few candidate genes per locus. Thus, a framework is provided to develop molecular markers and evaluate and characterize genes underlying important agronomic traits.

The Expanding Menagerie of Prunus-Infecting Luteoviruses.

Members of the genus Luteovirus are responsible for economically destructive plant diseases worldwide. Over the past few years, three luteoviruses infecting Prunus trees have been characterized. However, the biological properties, prevalence, and genetic diversity of those viruses have not yet been studied. High-throughput sequencing of samples of various wild, cultivated, and ornamental Prunus species enabled the identification of four novel species in the genus Luteovirus for which we obtained complete or nearly complete genomes. Additionally, we identified another new putative species recovered from Sequence Read Archive data. Furthermore, we conducted a survey on peach-infecting luteoviruses in eight European countries. Analyses of 350 leaf samples collected from germplasm, production orchards, and private gardens showed that peach-associated luteovirus (PaLV), nectarine stem pitting-associated virus (NSPaV), and a novel luteovirus, peach-associated luteovirus 2 (PaLV2), are present in all countries; the most prevalent virus was NSPaV, followed by PaLV. The genetic diversity of these viruses was also analyzed. Moreover, the biological indexing on GF305 peach indicator plants demonstrated that PaLV and PaLV2, like NSPaV, are transmitted by graft at relatively low rates. No clear viral symptoms have been observed in either graft-inoculated GF305 indicators or different peach tree varieties observed in an orchard. The data generated during this study provide a broader overview of the genetic diversity, geographical distribution, and prevalence of peach-infecting luteoviruses and suggest that these viruses are likely asymptomatic in peach under most circumstances.

Detection of Apple Hammerhead Viroid, Apple Luteovirus 1 and Citrus Concave Gum-Associated Virus in Apple Propagation Materials and Orchards in the Czech Republic and Hungary.

Grafting cultivars onto rootstocks is a widely used practice by the apple industry predominantly aimed at faster fruit bearing. Using high-throughput sequencing, we revealed the presence of recently described viral agents, namely apple hammerhead viroid (AHVd), apple luteovirus 1 (ALV-1), and citrus concave gum-associated virus (CCGaV), in germplasm collections and production orchards in the Czech Republic and Hungary. The HTS results were validated with RT-(q)PCR, and Northern blotting techniques. To obtain further insight about the presence of these agents, RT-PCR based surveys were carried out and showed their widespread presence alone or in mixed infections. The pathogens were present both in production areas and in feral samples. In addition, rootstock-to-scion transmission of ALV-1 and CCGaV was confirmed using commercial rootstock materials. Phylogenetic relationships based on partial sequences of distinct variants were also investigated. Furthermore, the rosy apple aphid was found to be ALV-1-positive, suggesting that it might be a potential vector of the virus.

Combining genetic resources and elite material populations to improve the accuracy of genomic prediction in apple.

Genomic selection is an attractive strategy for apple breeding that could reduce the length of breeding cycles. A possible limitation to the practical implementation of this approach lies in the creation of a training set large and diverse enough to ensure accurate predictions. In this study, we investigated the potential of combining two available populations, i.e., genetic resources and elite material, in order to obtain a large training set with a high genetic diversity. We compared the predictive ability of genomic predictions within-population, across-population or when combining both populations, and tested a model accounting for population-specific marker effects in this last case. The obtained predictive abilities were moderate to high according to the studied trait and small increases in predictive ability could be obtained for some traits when the two populations were combined into a unique training set. We also investigated the potential of such a training set to predict hybrids resulting from crosses between the two populations, with a focus on the method to design the training set and the best proportion of each population to optimize predictions. The measured predictive abilities were very similar for all the proportions, except for the extreme cases where only one of the two populations was used in the training set, in which case predictive abilities could be lower than when using both populations. Using an optimization algorithm to choose the genotypes in the training set also led to higher predictive abilities than when the genotypes were chosen at random. Our results provide guidelines to initiate breeding programs that use genomic selection when the implementation of the training set is a limitation.

SSR-Based Analysis of Genetic Diversity and Structure of Sweet Cherry (Prunus avium L.) from 19 Countries in Europe.

Sweet cherry (Prunus avium L.) is a temperate fruit species whose production might be highly impacted by climate change in the near future. Diversity of plant material could be an option to mitigate these climate risks by enabling producers to have new cultivars well adapted to new environmental conditions. In this study, subsets of sweet cherry collections of 19 European countries were genotyped using 14 SSR. The objectives of this study were (i) to assess genetic diversity parameters, (ii) to estimate the levels of population structure, and (iii) to identify germplasm redundancies. A total of 314 accessions, including landraces, early selections, and modern cultivars, were monitored, and 220 unique SSR genotypes were identified. All 14 loci were confirmed to be polymorphic, and a total of 137 alleles were detected with a mean of 9.8 alleles per locus. The average number of alleles (N = 9.8), PIC value (0.658), observed heterozygosity (Ho = 0.71), and expected heterozygosity (He = 0.70) were higher in this study compared to values reported so far. Four ancestral populations were detected using STRUCTURE software and confirmed by Principal Coordinate Analysis (PCoA), and two of them (K1 and K4) could be attributed to the geographical origin of the accessions. A N-J tree grouped the 220 sweet cherry accessions within three main clusters and six subgroups. Accessions belonging to the four STRUCTURE populations roughly clustered together. Clustering confirmed known genealogical data for several accessions. The large genetic diversity of the collection was demonstrated, in particular within the landrace pool, justifying the efforts made over decades for their conservation. New sources of diversity will allow producers to face challenges, such as climate change and the need to develop more sustainable production systems.

Micropropagation of Rosaceous Species SAM Grown in Temperate Climate.

The benefits of in vitro plant cultivation are mainly due to very high multiplication rate. Cultivation of plant material in vitro can be carried out during the whole year regardless of the time of the year or weather conditions. We create artificial conditions in the lab (heat, light, humidity), and we can regulate these conditions at any time. For the preservation of cultivar identity, we recommend establishing in vitro cultures from shoot tips usually larger than 0.2 mm. In practice, in vitro cultivation of plants uses these growth regulators to achieve organogenesis, for example, root formation, prolonged growth, or multiplication. During each subculture, these cultures are then transferred on a solid agar medium in the form of actively growing multiple shoots with a well-differentiated shoot tip containing meristematic area. Cytokinins are important for cell division and causes branching of plants. Auxins, both endogenous and exogenous, act at as a trigger for the differentiation and formation of root primordia. Morphological characteristics (formation of leaves or callus) and shoot development should be observed during in vitro multiplication and after transfer to ex vitro conditions.

Genome-Wide Association Mapping of Flowering and Ripening Periods in Apple.

Deciphering the genetic control of flowering and ripening periods in apple is essential for breeding cultivars adapted to their growing environments. We implemented a large Genome-Wide Association Study (GWAS) at the European level using an association panel of 1,168 different apple genotypes distributed over six locations and phenotyped for these phenological traits. The panel was genotyped at a high-density of SNPs using the Axiom®Apple 480 K SNP array. We ran GWAS with a multi-locus mixed model (MLMM), which handles the putatively confounding effect of significant SNPs elsewhere on the genome. Genomic regions were further investigated to reveal candidate genes responsible for the phenotypic variation. At the whole population level, GWAS retained two SNPs as cofactors on chromosome 9 for flowering period, and six for ripening period (four on chromosome 3, one on chromosome 10 and one on chromosome 16) which, together accounted for 8.9 and 17.2% of the phenotypic variance, respectively. For both traits, SNPs in weak linkage disequilibrium were detected nearby, thus suggesting the existence of allelic heterogeneity. The geographic origins and relationships of apple cultivars accounted for large parts of the phenotypic variation. Variation in genotypic frequency of the SNPs associated with the two traits was connected to the geographic origin of the genotypes (grouped as North+East, West and South Europe), and indicated differential selection in different growing environments. Genes encoding transcription factors containing either NAC or MADS domains were identified as major candidates within the small confidence intervals computed for the associated genomic regions. A strong microsynteny between apple and peach was revealed in all the four confidence interval regions. This study shows how association genetics can unravel the genetic control of important horticultural traits in apple, as well as reduce the confidence intervals of the associated regions identified by linkage mapping approaches. Our findings can be used for the improvement of apple through marker-assisted breeding strategies that take advantage of the accumulating additive effects of the identified SNPs.

Analysis of the genetic diversity and structure across a wide range of germplasm reveals prominent gene flow in apple at the European level.

BACKGROUND: The amount and structure of genetic diversity in dessert apple germplasm conserved at a European level is mostly unknown, since all diversity studies conducted in Europe until now have been performed on regional or national collections. Here, we applied a common set of 16 SSR markers to genotype more than 2,400 accessions across 14 collections representing three broad European geographic regions (North + East, West and South) with the aim to analyze the extent, distribution and structure of variation in the apple genetic resources in Europe. RESULTS: A Bayesian model-based clustering approach showed that diversity was organized in three groups, although these were only moderately differentiated (FST = 0.031). A nested Bayesian clustering approach allowed identification of subgroups which revealed internal patterns of substructure within the groups, allowing a finer delineation of the variation into eight subgroups (FST = 0.044). The first level of stratification revealed an asymmetric division of the germplasm among the three groups, and a clear association was found with the geographical regions of origin of the cultivars. The substructure revealed clear partitioning of genetic groups among countries, but also interesting associations between subgroups and breeding purposes of recent cultivars or particular usage such as cider production. Additional parentage analyses allowed us to identify both putative parents of more than 40 old and/or local cultivars giving interesting insights in the pedigree of some emblematic cultivars. CONCLUSIONS: The variation found at group and subgroup levels may reflect a combination of historical processes of migration/selection and adaptive factors to diverse agricultural environments that, together with genetic drift, have resulted in extensive genetic variation but limited population structure. The European dessert apple germplasm represents an important source of genetic diversity with a strong historical and patrimonial value. The present work thus constitutes a decisive step in the field of conservation genetics. Moreover, the obtained data can be used for defining a European apple core collection useful for further identification of genomic regions associated with commercially important horticultural traits in apple through genome-wide association studies.

Comparison of genetic diversity structure analyses of SSR molecular marker data within apple (Malus×domestica) genetic resources.

The aim of this study was to compare traditional hierarchical clustering techniques and principal coordinate analysis (PCoA) with the model-based Bayesian cluster analyses in relation to subpopulation differentiation based on breeding history and geographical origin of apple (Malus×domestica Borkh.) cultivars and landraces. We presented the use of a set of 10 microsatellite (SSR) loci for genetic diversity structure analyses of 273 apple accessions from national genetic resources. These SSR loci yielded a total of 113 polymorphic SSR alleles, with 5-18 alleles per locus. SSR molecular data were successfully used in binary and allelic input format for all genetic diversity analyses, but allelic molecular data did not reveal reliable results with the NTSYS-pc and BAPS softwares. A traditional cluster analysis still provided an easy and effective way for determining genetic diversity structure in the apple germplasm collection. A model-based Bayesian analysis also provided the clustering results in accordance to traditional cluster analysis, but the analyses were distorted by the presence of a dominant group of apple genetic resources owing to the narrow origin of the apple genome. PCoA confirmed that there were no noticeable differences in genetic diversity structure of apple genetic resources during the breeding history. The results of our analyses are useful in the context of enhancing apple collection management, sampling of core collections, and improving breeding processes.