Breeding elite apple cultivars with scab resistance is a key global goal, as reliance on fungicides is unsustainable. The causal fungus, Venturia inaequalis, evolves rapidly, threatening cultivars with single-gene resistance. Since the 1980s, breeding programmes have introduced novel resistance sources via backcrossing. Here, we generated a haplotype-phased genome assembly of Russian apple R12740-7A and an Oxford Nanopore assembly of the Rvi2-resistance accession TSR34T15, enabling detailed dissection of the Rvi2 resistance locus. Fine-mapping using a ‘Royal Gala’ × TSR34T15 segregating family delimited Rvi2 to a narrow genomic interval, within which we identified a 10 041 bp long terminal repeat retrotransposon (LTR-RT) insertion—an insert-based structural variant (SV) strongly linked with Rvi2. Notably, this LTR-RT harbours an FPPS gene, a member of the farnesyl pyrophosphate/geranylgeranyl pyrophosphate (FPP/GGPP) synthase family, located 2 kb from a key candidate defence gene. Although the FPPS gene exhibits stable expression, its integration within the retrotransposon suggests a cis-regulatory role, potentially priming adjacent defence genes for robust up-regulation upon pathogen attack. We validated the marker derived from this SV in diverse germplasms and successfully implemented it in marker-assisted selection across extensive seedling cohorts. This marker will streamline the development of scab-resistant apple varieties.

Apple scab, caused by Venturia inaequalis, is the most economically important fungal disease impacting apple production globally. Most commercial apple cultivars are susceptible to scab, although several sources of genetic resistance have been identified. The availability of genetic markers tightly linked with the functional resistance alleles is one of the factors limiting the breeding of scab-resistant cultivars. 'Honeycrisp', one of the popular North American apple cultivars, is a source of Vhc1 scab resistance on linkage group 1, but the associated locus spans 12.6 cM and harbours over 200 genes. Here, we present the fine-mapping of the Vhc1 locus, the development of haplotype-specific simple sequence repeat (SSR) markers and the identification of candidate resistance alleles. Chromosome-level phased genome assemblies of the scab-resistant parent 'Honeycrisp' and scab-susceptible 'Gala' were used to design nine novel polymorphic markers, along with two previously reported markers, spanning the Vhc1 QTL region. An F1 population derived from the two cultivars was developed and was evaluated for scab resistance across two consecutive years using the V. inaequalis isolate Vi-19-004 collected from Malus floribunda 821. Broad-sense heritability was estimated at 0.66 across experiments. Phenotype-genotype association analysis fine-mapped a moderate-effect apple scab resistance QTL, explaining 5.2%-11.8% of phenotypic variance, to a 4.2-cM genetic interval and a corresponding 0.85-Mb physical region. SSR markers mdCu_2500, mdCu_3000 and CH-Vf1 collocated with the Vhc1 QTL and can be used in future marker-assisted selection studies for Vhc1. Six LRR-encoding genes underlying the QTL region with enhanced expression upon inoculation with V. inaequalis were identified as scab resistance candidate alleles. These newly developed markers and candidate genes will accelerate the development of 'Honeycrisp' based scab-resistant cultivars.

Malus genomic resources with improved quality are becoming an integral part of candidate gene identification and functional validation. Recently, we released a reference-based phased chromosome-level genome assembly of ‘Antonovka’ 172670B Malus domestica cultivar from the ‘Antonovka’ group known for its disease resistance and abiotic stress tolerance. However, reference-based assemblies have limited accuracy in candidate alleles identification along extended genomic loci, as these may contain contigs from different haplotypes. Here, we provide an improved high-quality version of the ‘Antonovka’ 172670B genome. This assembly is fully phased as it integrates chromosome conformation capture sequencing and parental SNP data binning for de novo scaffolding and post-assembly phasing of pseudochromosomes into haplotypes, respectively. It shows improved quality scores and completeness of 59.8 and 96.7

Apple (Malus × domestica Borkh.) production is threatened by scab, caused by the fungus Venturia inaequalis. One defense mechanism of apple trees against fungal pathogens such as V. inaequalis is the biosynthesis of antifungal compounds. Amongst these, phenolic compounds are particularly hypothesized to correlate with scab resistance, thereby offering a putative route to breed new apple cultivars with enhanced resistance. To characterize the involvement of phenolics in scab resistance, as conferred by either the Rvi6 resistance gene or through increased somatic ploidy, we monitored the phenolics profile in the leaves of apple genotypes harboring Rvi6 or increased ploidy that show enhanced levels of scab resistance compared to susceptible genotypes. Our study revealed differences in total and specific phenolic contents across the tested genotypes with significant correlation to Rvi6-based resistance and a minor effect of polyploidy herein. In particular, procyanidin dimer levels appeared positively correlated with the level of resistance, indicating a putative functional role in scab resistance. In contrast, the majority of other phenolics were negatively correlated with the resistance. Finally, our study did not identify a significant correlation between reduced phloridzin:flavanol ratio and Rvi6 resistance. These findings are discussed in the context of the role of phenolic metabolism in apple scab resistance.

Wild Malus species harbor untapped genetic diversity to advance apple breeding, particularly for disease resistance and stress tolerance. However, existing marker panels, developed mainly using Malus domestica accessions, introduce ascertainment bias and limit detecting rare variants in wild species. We developed and validated a medium-density and cost-effective pan-generic 3 K apple DArTag panel optimized to capture genome-wide variation across the Malus genus. The panel was constructed using conserved, syntenic, and collinear genomic blocks identified within the core genome of 13 Malus accessions for cross-species transferability. The panel was validated across three bi-parental mapping populations totaling 593 progeny. Across these populations, 2461–3234 SNP markers were polymorphic and 1482–2620 were informative. Each population contained over 900 multiallelic micro-haplotype loci, with several hundred loci exhibiting three or four distinct haplotypes. Markers were uniformly distributed across all 17 chromosomes, each containing between 60 and 230 informative SNPs. The panel was further evaluated on 174 diverse germplasm accessions from 20 Malus species. It exhibited strong cross-species transferability, exceptionally low rates of missing data (< 0.5

Diffuse apple scab symptoms on the abaxial leaf surface are caused by the fungal pathogen Venturia inaequalis and occur even in well-managed orchards. The disease is challenging to manage, as the symptoms emerge on aging leaves later in the season and only develop on some leaves, suggesting the involvement of intrinsic defense mechanisms. We studied the development of diffuse abaxial scab symptoms on 'Jonagold' leaves in an orchard at three different shoot positions and at four different time points during the growing season to identify leaf-specific defense mechanisms. We identified infection moments by tracking the ascospore load and formation of new shoot leaves and determined the period required for proliferation of diffuse abaxial scab symptoms. Then, we correlated gene expression and phenolics profiles with the V. inaequalis DNA content to identify those inhibiting scab proliferation in asymptomatic leaves. Leaves in the middle of shoots showed higher symptom expression compared with shoot base and tip leaves. Disease inhibition in asymptomatic leaves was negatively correlated with several defense-related genes and phenolics. Altogether, our observations highlight that diffuse abaxial scab is one of the key challenges in apple growing and that the leaf ontogenic status during the infection process affects the development of these symptoms, with putative regulation by phenolic metabolism and ontogeny-related defense genes.

Crop wild relatives of perennial fruit crops have a wealth of untapped genetic diversity that can be utilized for cultivar development. However, barriers such as linkage drag, long juvenility, and high heterozygosity have hindered their utilization. Advancements in genome sequencing technologies and assembly methods, combined with the integration of chromosome conformation capture have made it possible to construct high-quality reference genomes. These genome assemblies can be combined into pan-genomes, capturing inter- and intraspecific variations across coding and non-coding regions. Pan-genomes of perennial fruit crops are being developed to identify the genetic basis of traits. This will help overcome breeding challenges, enabling faster and more targeted development of new cultivars with novel traits through breeding and biotechnology.

Apple and citrus are perennial tree fruit crops that are vital for nutritional security and agricultural economy and to achieve the Sustainable Development Goals of the United Nations. Apple scab and fire blight, along with Huanglongbing, canker, and tristeza virus, stand out as their most notorious diseases and annually destabilize fruit supply. An environmentally sound approach to managing these diseases is improving tree resistance through breeding and biotechnology. Perennial fruit tree germplasm collections are distributed globally and offer untapped potential as sources of resistance. However, long juvenility, specific pollination and flowering habits, and extensive outcrossing hinder apple and citrus breeding. Advances in breeding approaches including trans- and cis-genesis, genome editing, and rapid-cycle breeding, which, in addition to conventional crossbreeding, can all facilitate accelerated integration of resistance into elite germplasm. In addition, the global pool of available sources of resistance can be characterized by the existing genetic mapping and gene expression studies for accurate discovery of associated loci, genes, and markers to efficiently include these sources in breeding efforts. We discuss and propose a multitude of approaches to overcome the challenges of breeding for resistance in woody perennials and outline a technical path to reduce the time required for the ultimate deployment of disease-resistant cultivars.

Wild Malus species flourished in North America long before Europeans introduced domesticated apples. Malus coronaria and M. ioensis are native to the mid-western and eastern United States, while M. angustifolia and M. fusca grow in the southeast and west, respectively. They offer disease resistance, climate and soil adaptability, and horticultural traits for apple breeding. However, their utilization remains limited due to insufficient genomic resources and specific genetics. We report high-quality phased chromosome-scale assemblies of M. coronaria and M. ioensis, generated using long-read and conformation capture sequencing. Phylogenetic and synteny analysis indicated high relatedness between these 2 genomes and previously published genome of M. angustifolia, and lower relatedness with M. fusca. Gene family-based pangenome of North American Malus identified 60,211 orthogroups containing 340,087 genes. Genes involved in basic cellular and metabolic processes, growth, and development were core to the existence of these species, whereas genes involved in secondary metabolism, stress response, and interactions with other organisms were accessory and are likely associated with adaptation to specific environments. Structural variation hotspots were mostly overlapping with high gene density. This study offers novel native North American Malus genome resources that can be used to identify genes for apple breeding and understand their evolution and adaptation.

Apples are one of the most valued tree fruit crops around the world. Currently, a few highly popular and economically successful apple cultivars dominate the commercial production and serve as main genetic contributors to the development of new apple cultivars. This limited level of genetic diversity, grown as a clonally propagated monoculture renders the apple industry vulnerable to the wide range of weather events, pests, and pathogens. Wild apple species are an excellent source of beneficial alleles for the wide range of biotic and abiotic stressors challenging apple production. However, the biological barriers of breeding with small-fruited wild apples greatly limit their use. Using a closely related wild species of apple such as Malus sieversii can improve the efficiency of breeding efforts and broaden the base of available genetics. M. sieversii is the main progenitor of the domesticated apple, native to Central Asia. The similarity of fruit morphology to domesticated apples and resistances to abiotic and biotic stresses makes it appealing for apple breeding programs. However, this important species is under threat of extinction in its native range. Preserving the wild apple forests in Central Asia is vital for ensuring the sustainable protection of this important genetic resource. The insufficient awareness about the complete range of challenges and opportunities associated with M. sieversii hinders the maximization of its potential benefits. This review aims to provide comprehensive information on the cultural and historical context of M. sieversii, current genetic knowledge for breeding, and the conservation challenges of wild apple forests.

Apple scab disease, caused by the fungus Venturia inaequalis, endangers commercial apple production globally. It is predominantly managed by frequent fungicide sprays that can harm the environment and promote the development of fungicide-resistant strains. Cultivation of scab-resistant cultivars harboring diverse qualitative Rvi resistance loci and quantitative trait loci associated with scab resistance could reduce the chemical footprint. A comprehensive understanding of the host-pathogen interaction is, however, needed to efficiently breed cultivars with enhanced resistance against a variety of pathogenic strains. Breeding efforts should not only encompass pyramiding of Rvi loci and their corresponding resistance alleles that directly or indirectly recognize pathogen effectors, but should also integrate genes that contribute to effective downstream defense mechanisms. This review provides an overview of the phenotypic and genetic aspects of apple scab resistance, and currently known corresponding defense mechanisms. Implementation of recent "-omics" approaches has provided insights into the complex network of physiological, molecular, and signaling processes that occur before and upon scab infection, thereby revealing the importance of both constitutive and induced defense mechanisms. Based on the current knowledge, we outline advances toward more efficient introgression of enhanced scab resistance into novel apple cultivars by conventional breeding or genetic modification techniques. However, additional studies integrating different "-omics" approaches combined with functional studies will be necessary to unravel effective defense mechanisms as well as key regulatory genes underpinning scab resistance in apple. This crucial information will set the stage for successful knowledge-based breeding for enhanced scab resistance.

Apple scab, a fungal disease caused by Venturia inaequalis, leads to losses in both yield and fruit quality of apples (Malus domestica Borkh.). Most commercial apple cultivars, including those containing the well-characterized Rvi6-scab-resistance locus on linkage group (LG) 1, are susceptible to scab. HcrVf2 and HcrVf1 are considered the main paralogs of the Rvi6 locus. The major apple scab-resistance loci Vhc1 in "Honeycrisp" and Rvi17 in "Antonovka," were identified in close proximity to HcrVf2. In this study, we used long-read sequencing and in silico gene sequence characterization to identify candidate resistance genes homologous to HcrVf2 and HcrVf1 in Honeycrisp and Antonovka. Previously published chromosome-scale phased assembly of Honeycrisp and a newly assembled phased genome of Antonovka 172670-B were used to identify HcrVf2 and HcrVf1 homologs spanning Vhc1 and Rvi17 loci. In combination with 8 available Malus assemblies, 43 and 46 DNA sequences highly homologous to HcrVf2 and HcrVf1, respectively, were identified on LG 1 and 6, with identity and coverage ranging between 87-95 and 81-95%, respectively. Among these homologs, 2 candidate genes in Antonovka and Honeycrisp haplome A are located in close physical proximity to the scab-resistance marker Ch-Vf1 on LG 1. They showed the highest identity and coverage (95%) of HcrVf2 and only minor changes in the protein motifs. They were identical by state between each other, but not with HcrVf2. This study offers novel genomic resources and insights into the Vhc1 and Rvi17 loci on LG 1 and identifies candidate genes for further resistance characterization.

Apple is an important fruit crop of temperate regions. The narrow genetic base of commercially cultivated apples has resulted in its vulnerability to a large number of fungal, bacterial, and viral pathogens. Apple breeders are always seeking new sources of resistance within the cross-compatible Malus species that can be deployed into elite genetic backgrounds. We have evaluated resistance to two major fungal diseases of apples: powdery mildew and frogeye leaf spot, using a germplasm collection of 174 Malus accessions to identify novel sources of genetic resistance. In 2020 and 2021, we evaluated these accessions for the incidence and severity of powdery mildew and frogeye leaf spot diseases at Cornell AgriTech, Geneva, New York, in a partially managed orchard. The severity and incidence of powdery mildew and frogeye leaf spot, as well as weather parameters were recorded in June, July, and August. Total incidence of powdery mildew and frogeye leaf spot infections increased from 33 to 38%, and 56 to 97% in 2020 and 2021, respectively. Our analysis showed that relative humidity and precipitation correlate with powdery mildew and frogeye leaf spot susceptibility. The predictor variables with highest impact to the variability of powdery mildew were accessions and relative humidity in May. A total of 65 Malus accessions were found to be resistant to powdery mildew, and only one accession showed moderate resistance to frogeye leaf spot. Several of these accessions belong to Malus hybrid species and domesticated apples and can therefore be potential sources of novel resistance alleles for apple breeding.

Apple (Malus x domestica Borkh.) production is significantly affected by apple scab disease caused by the fungus Venturia inaequalis. Currently, management of the disease is largely based on fungicide applications and less on the inherent resistance in apple cultivars carrying resistance (Rvi) genes. Moreover, the durability of both approaches is questionable, since new virulent strains with resistance to fungicides and/or Rvi genes can develop. Hence, a combination of complementary management strategies is needed to tackle apple scab. To broaden the management options, a viable approach might be the breeding for polyploid cultivars and their implementation in commercial orchards. Apple germplasm is substantially characterized by polyploidy, which throughout the evolution of angiosperms often led to improved adaptation to adverse climatic conditions and abiotic stresses. However, the role of polyploidy in conferring resistance to biotic challenges is less clear. Here, we show that autopolyploidy in two apple cultivars, i.e. the susceptible 'Gala', and the resistant 'Makali' which carries the Rvi6 scab resistance gene, can reduce severity of apple scab symptoms upon inoculation with two different V. inaequalis isolates. Leaves of tetraploid plants show reduced sporulation symptoms in comparison with their diploid counterparts upon infection with both pathogenic strains, attributed to a reduced presence of V. inaequalis. These results suggest that polyploidy enhances resistance of apple cultivars against different V. inaequalis isolates. However, the degree of the enhanced resistance depends on a plant-pathogen-specific interaction. The knowledge provided here offers a relevant framework for improving our understanding of polyploidy-enhanced resistance to biotic stress in plants and may set the stage for implementing polyploidy breeding for further genetic improvement of apple.

There is an urgent need for novel, efficient and environmentally friendly strategies to control apple scab (Venturia inaequalis), for the purpose of reducing overall pesticide use. Fructans are recently emerging as promising "priming" compounds, standing out for their safety and low production costs. The objective of this work was to test a fructan-triggered defense in the leaves of apple seedlings. It was demonstrated that exogenous leaf spraying can reduce the development of apple scab disease symptoms. When evaluated macroscopically and by V. inaequalis-specific qPCR, levan-treated leaves showed a significant reduction of sporulation and V. inaequalis DNA in comparison to mock- and inulin-treated leaves, comparable to the levels in fosetyl-aluminum-treated leaves. Furthermore, we observed a significant reduction of in vitro mycelial growth of V. inaequalis on plates supplemented with levans when compared to controls, indicating a direct inhibition of fungal growth. Variations in endogenous sugar contents in the leaves were followed during priming and subsequent infection, revealing complex dynamics as a function of time and leaf ontogeny. Our data are discussed in view of the present theories on sugar signaling and fructan-based immunity, identifying areas for future research and highlighting the potential use of fructans in apple scab management in orchards.

Hop (Humulus lupulus L.) is an important industrial plant providing ingredients for brewing and pharmaceutical industry worldwide. Its intensive production is challenged by numerous diseases. One of the most lethal and difficult to control is verticillium wilt, a vascular disease caused by the fungal pathogen Verticillium nonalfalfae. The disease can be successfully controlled by the host resistance. Despite various studies that already researched resistance mechanisms of hops, only limited number of resistance genes and markers that could be utilized for efficient resistance breeding has been identified. In this study we aimed to follow fungus colonization pattern and the differential expression of selected genes during pre-symptomatic period of susceptible (Celeia) and resistant (Wye Target) hop cultivars. Results of gene expressions and fungal colonisation of compatible and incompatible interactions with V. nonalfalfae suggest that the hop plant is challenged already at the very early fungal colonisation stages. In total, nine out of 17 gene targets investigated in our study resulted in differential expression between inoculated and control plants of susceptible and resistant cultivars. The difference was the most evident in stems at an early stage of colonisation (6 dpi), showing relatively stronger changes in targeted gene expression to infection in the resistant cultivar than in the susceptible one. Analysed gene targets are involved in the overall defence response processes of nucleic acid binding, signalling, protein ubiquitination, cell oxidative burst, hydroxylation, peroxidation, alternative splicing, and metabolite biosynthesis. The up-regulation of some genes (e.g. glycine-rich RNA-binding family protein, protein phosphatase, cysteine-rich receptor-like protein kinase, zinc finger CCCH domain-containing protein 40, cinnamic acid 4-hydroxylase, class III peroxidase, putative MAPK2, peroxiredoxin-2F) upon infection in incompatible interactions might reflect defence activation, restriction of disease spreading throughout the plant and successful response of resistant genotype.

Hop (Humulus lupulus L.) is grown mostly as flavouring and bittering ingredient for beer and is also appreciated in the herbal and cosmetic industry, as well as in pharmacology. Among several diseases that damage hop growing, the most devastating in European hop production is verticillium wilt, caused by the soil-borne fungal pathogen Verticillium nonalfalfae. Colonization pattern and differential expression of selected genes after artificial infection of susceptible and resistant hop cultivars with V. nonalfalafae in stems and roots have been analysed recently Švara et al., 2019. Here, we present the dataset related to verification of plant samples infections after artificial inoculation (fungi- and mock-inoculated). After inoculation plant samples were tested for the positive infection by PCR amplification of the V. nonalfalfae ITS DNA region with species specific primers developed and optimised for this purpose. For more insight please see the article “Temporal and spatial assessment of defence responses in resistant and susceptible hop cultivars during infection with Verticillium nonalfalfae”.

Development of new and durable strategies to improve the apple plant’s resistance against apple scab (Venturia inaequalis) remains a major challenge. Polyploids or organisms with three or more complete chromosome sets often possess properties superior to their diploid counterparts. Studies reported that polyploidy confers an increased resistance to biotic and abiotic stress factors. However, the potential use of polyploidy to increase disease resistance is still insufficiently investigated. We determined the influence of artificial genome doubling on the response of three Malus × domestica genotypes, with a variable level of susceptibility to apple scab. Based on visual symptom evaluation and real-time PCR quantification of the V. inaequalis DNA in apple leaves, an increased resistance was observed in the neotetraploid form of the monogenic resistant genotype compared to its diploid progenitor. No pronounced effects were observed comparing ploidy levels of the susceptible genotypes. These results suggest a potential role for polyploidisation in apple scab resistance, but it seems to depend on the degree of disease susceptibility of the genotype. Still, further research on the effects of polyploidy might offer potential in the development of new and durable resistance strategies.