Development of a hybrid apple population: MAS-based testing for Vf gene detection and preliminary field evaluation of its scab resistance

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 genes involved in the response to Venturia inaequalis and salicylic acid treatment

Apple scab caused by Venturia inaequalis is the most important fungal disease of apples (Malus × domestica). Currently, the disease is controlled by up to 15 fungicide applications to the crop per year. Resistant apple cultivars will help promote the sustainable control of scab in commercial orchards. The breakdown of the Rvi6 (Vf) major-gene based resistance, the most used resistance gene in apple breeding, prompted the identification and characterization of new scab resistance genes. By using a large segregating population, the Rvi12 scab resistance gene was previously mapped to a genetic location flanked by molecular markers SNP_23.599 and SNP_24.482. Starting from these markers, utilizing chromosome walking of a Hansen’s baccata #2 (HB2) BAC-library; a single BAC clone spanning the Rvi12 interval was identified. Following Pacific Biosciences (PacBio) RS II sequencing and the use of the hierarchical genome assembly process (HGAP) assembly of the BAC clone sequence, the Rvi12 resistance locus was localized to a 62.3-kb genomic region. Gene prediction and in silico characterization identified a single candidate resistance gene. The gene, named here as Rvi12_Cd5, belongs to the LRR receptor-like serine/threonine-protein kinase family. In silico comparison of the resistance allele from HB2 and the susceptible allele from Golden Delicious (GD) identified the presence of an additional intron in the HB2 allele. Conserved domain analysis identified the presence of four additional LRR motifs in the susceptible allele compared to the resistance allele. The constitutive expression of Rvi12_Cd5 in HB2, together with its structural similarity to known resistance genes, makes it the most likely candidate for Rvi12 scab resistance in apple.

BackgroundScab, caused by the fungus Venturia inaequalis, is one of the most important diseases of cultivated apple. While a few scab resistance genes (R genes) governing qualitative resistance have been isolated and characterized, the biological roles of genes governing quantitative resistance, supposed to be more durable, are still unknown. This study aims to investigate the molecular mechanisms involved in the partial resistance of the old Belgian apple cultivar ‘Président Roulin’ against V. inaequalis.ResultsA global gene expression analysis was conducted in ‘Président Roulin’ (partially resistant) and in ‘Gala’ (susceptible) challenged by V. inaequalis by using the cDNA-AFLP method (cDNA-Amplified Fragment Length Polymorphism). Transcriptome analysis revealed significant modulation (up- or down-regulation) of 281 out of approximately 20,500 transcript derived fragments (TDFs) in ‘Président Roulin’ 48 hours after inoculation. Sequence annotation revealed similarities to several genes encoding for proteins belonging to the NBS-LRR and LRR-RLK classes of plant R genes and to other defense-related proteins. Differentially expressed genes were sorted into functional categories according to their gene ontology annotation and this expression signature was compared to published apple cDNA libraries by Gene Enrichment Analysis. The first comparison was made with two cDNA libraries from Malus x domestica uninfected leaves, and revealed in both libraries a signature of enhanced expression in ‘Président Roulin’ of genes involved in response to stress and photosynthesis. In the second comparison, the pathogen-responsive TDFs from the partially resistant cultivar were compared to the cDNA library from inoculated leaves of Rvi6 (HcrVf2)-transformed ‘Gala’ lines (complete disease resistance) and revealed both common physiological events, and notably differences in the regulation of defense response, the regulation of hydrolase activity, and response to DNA damage. TDFs were in silico mapped on the ‘Golden Delicious’ apple reference genome and significant co-localizations with major scab R genes, but not with quantitative trait loci (QTLs) for scab resistance nor resistance gene analogues (RGAs) were found.ConclusionsThis study highlights possible candidate genes that may play a role in the partial scab resistance mechanisms of ‘Président Roulin’ and increase our understanding of the molecular mechanisms involved in the partial resistance against apple scab.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1043) contains supplementary material, which is available to authorized users.

Swiss and more generally European apple (Malus × domestica) production is hampered by several diseases, the most destructive being fire blight, caused by Erwinia amylovora. On the other hand, there are apple scab, caused by Venturia inaequalis and powdery mildew, caused by Podosphaera leucotricha, which represent the major phytosanitary problems. Classical breeding has produced many scab and mildew resistant cultivars and efforts to breed also fire blight resistant cultivars are currently undertaken. Marker assisted selection (MAS) increases efficiency by allowing early non-destructive screening of seedlings and identifying genotypes showing pyramids of resistance genes. If the development of markers for MAS was the primary goal of genetic analysis in the 1990s, identification and cloning of resistance genes is now the goal. The first and until now the sole resistance gene which has been isolated and transformed into a susceptible apple cultivar is the gene HcrVf2 (Rvi6), responsible for the Vf scab resistance present in most classically bred scab resistant cultivars. Much effort is currently spent in the identification and positional cloning of other apple genes conferring resistance to apple scab and fire blight. In our labs, we identified the putative scab resistance gene Rvi15 and two fire blight resistance genes namely from ‘Evereste’ and Malus × robusta 5. The functionality of these candidate genes is currently under scrutiny by complementation experiments. However, the final goal is the creation of a product, e.g., an improved apple cultivar that is resistant to scab and fire blight. The ideal product would have advantages to the environment and producer, and should raise as little concern as possible with consumers. To accomplish this ‘ideal product’, we opted for the cisgenic approach by introducing the scab resistance gene HcrVf2 with its own regulatory sequences into the highly susceptible apple cultivar, ‘Gala’, through Agrobacterium transformation. All marker genes were eliminated after transformation. Similarly, we are currently introducing into both the readily developed cisgenic ‘Gala’ and in the untransformed ‘Gala’ the putative Malus own fire blight resistance gene candidates, aiming at both proof of functionality of the identified candidates and possibly at rapid development of a fire blight and scab resistant cisgenic apple.

This article presents the results of DNA marker-assisted selection of apple tree varieties and breeding material obtained in the North Caucasian Region Research Institute of Horticulture and Viticulture for several genes – Rvi2, Rvi3, Rvi5, and Rvi15 – determining scab resistance (Venturia inaequalis (Cooke) G. Winter). The aim was to estimate the polymorphism of Rvi2, Rvi3, Rvi5, and Rvi15 scab resistance genes in representatives of Malus Mill. of domestic selection with the purpose of identifying the most prospective genotypes for further breeding. The research objects included apple-tree genotypes of different genetic origin. The following genotypes were used as controls for identification of DNA-marker alleles of target genes: Malus pumila R12740-7A (gene Rvi2); Q71 (gene Rvi3); Malus atrosanguinea 840 (gene Rvi5); and GMAL2473 (gene Rvi15). The research was conducted using the facilities of the Collective Use Center “Research and Breeding Collection of Genetic Resources of Horticultural Crops”, Krasnodar, using conventional research methods. DNA extraction was conducted using a modified STAB method previously developed by researchers of the North Caucasian Region Research Institute of Horticulture and Viticulture. Various degrees of the prevalence of resistance genes in the sample were revealed. The most frequent genes were found to be Rvi3 (45.95 % of carriers identified) and Rvi15 (43.24 %). The prevalence of Rvi2 was 27.02 %. The least frequent gene was found to be Rvi5 (2.70 %). Among the studied apple varieties, carriers of 2-3 scab resistance genes were identified, including Vesta (RVI2, RVI3, RVI15) and late winter Margo (RVI2, RVI15). The 12/1-21-63 (Golden Delicious (4×)×2034 (F2 M. floribunda×Golden Delicious))×Modi) hybrid family demonstrated the highest number of carriers of several scab resistance genes. Application of DNA marker-assisted technology made it possible to identify 4-hybrid forms containing three target resistance genes in the genome: Rvi3, Rvi2, Rvi15 – 17/1-6-1 (Karmen×Gemeni), 17/2-6-7(12/1- 21-63×Modi), 17/1-7-17 (12/1-20-56×Fujion); Rvi3, Rvi5, Rvi15 – 17/1-6-73 (12/1-21-63×Modi). The identified carriers of several target genes can be used in further breeding for long-term resistance against Venturia inaequalis (Cooke) G. Winter.

Apple scab, caused by Venturia inaequalis, is a major fungal disease worldwide. Cultivation of scab-resistant cultivars would reduce the chemical footprint of apple production. However, new apple cultivars carrying durable resistances should be developed to prevent or at least slow the breakdown of resistance against races of V. inaequalis. One way to achieve durable resistance is to pyramid multiple scab resistance genes in a cultivar. The choice of the resistance genes to be combined in the pyramids should take into account the frequency of resistance breakdown and the geographical distribution of apple scab isolates able to cause such breakdowns. In order to acquire this information and to make it available to apple breeders, the VINQUEST project (www.vinquest.ch) was initiated in 2009. Ten years after launching this project, 24 partners from 14 countries regularly contribute data. From 2009 to 2018, nearly 9,000 data points have been collected. This information has been used to identify the most promising apple scab resistance genes for developing cultivars with durable resistance, which to date are: Rvi5, Rvi11, Rvi12, Rvi14, and Rvi15. As expected, Rvi1, together with Rvi3 and Rvi8, were often overcome, and have little value for scab resistance breeding. Rvi10 may also belong to this group. On the other hand, Rvi2, Rvi4, Rvi6, Rvi7, Rvi9, and Rvi13 are still useful for breeding, but their use is recommended only in extended pyramids of ≥3 resistance genes.

Apple scab, caused by Venturia inaequalis, is a destructive fungal disease of major apple cultivars worldwide, most of which are moderately to highly susceptible. Thus, development of scab resistant cultivars is one of the highest priorities of apple breeding programs. The principal source of resistance for breeding programs has been the scab resistance gene Rvi6 that originated from the Japanese crabapple Malus floribunda (Sieb.) sel. 821. Isolates of V. inaequalis able to overcome Rvi6 have been identified in Europe, but have not yet been reported on the American continents. We recently discovered scab infection on M. floribunda 821 trees in a research orchard at Geneva, NY, U.S.A., where approximately 10% of the leaves bore profusely sporulating apple scab lesions, many of which had coalesced to cover entire leaves. We observed both chlorosis, typical to Rvi6, and pinpoint pitting symptoms typical to failed infections by V. inaequalis on hosts bearing the Rvi7 gene. We assessed genetic diversity and population genetic structure of 11 V. inaequalis isolates in total, of North American and European origin, isolated from M. floribunda 821, 'Nova Easygro', 'Golden Delicious', TSR33T239, 'Schone van Boskoop', and 'Prima', using 16,321 genome-wide SNPs. Population genetic structure and PCA separated the isolates into distinct European and U.S. groups. The forgoing suggests that the new Rvi6 virulent isolates emerged within U.S. populations, rather than being transported from Europe. The complete resistance breakdown in M. floribunda 821 but not in descendant cultivars, which kept their field resistance, suggests that durable resistance to apple scab will require a more comprehensive understanding of Rvi6 mediated resistance in diverse genetic backgrounds.

The effect of long‐term N‐supply on growth, scab resistance and phenolic compounds in the leaves of two apple cultivars was studied. The different pools of phenylpropanoids (hydroxycinnamic acids, dihydrochalcones) and flavonoids (flavonols, catechins, procyanidins) were quanitfied by HPLC from non‐infected and inoculated leaves representing different ontogenetic stages. Scab incidence was also evaluated. Strictly following the carbon‐nutrient‐balance hypothesis, apple trees responded to high N‐supply with increased shoot growth and with a reduced accumulation of total phenolic compounds in their leaves. This was shown for the cultivar ‘Golden Delicious’, which is susceptible to the scab disease caused by Venturia inaequalis, and for the resistant cultivar ‘Rewena’. Whereas high N‐fertilization increased the susceptibility of ‘Golden Delicious’, it did not decrease the resistance of ‘Rewena’ despite of the pronounced reduction of phenolic concentrations. Thus, a simple C trade off between growth‐related metabolism and secondary metabolism cannot solely explain changes in defensive potential.

Apple scab, caused by the fungal pathogen Venturia inaequalis, is one of the most severe diseases of apple worldwide. It is the most studied plant-pathogen interaction involving a woody species using modern genetic, genomic, proteomic and bioinformatic approaches in both species. Although 'Geneva' apple was recognized long ago as a potential source of resistance to scab, this resistance has not been characterized previously. Differential interactions between various monoconidial isolates of V. inaequalis and six segregating F1 and F2 populations indicate the presence of at least five loci governing the resistance in 'Geneva'. The 17 chromosomes of apple were screened using genotyping-by-sequencing, as well as single marker mapping, to position loci controlling the V. inaequalis resistance on linkage group 4. Next, we fine mapped a 5-cM region containing five loci conferring both dominant and recessive scab resistance to the distal end of the linkage group. This region corresponds to 2.2 Mbp (from 20.3 to 22.5 Mbp) on the physical map of 'Golden Delicious' containing nine candidate nucleotide-binding site leucine-rich repeat (NBS-LRR) resistance genes. This study increases our understanding of the complex genetic basis of apple scab resistance conferred by 'Geneva', as well as the gene-for-gene (GfG) relationships between the effector genes in the pathogen and resistance genes in the host.

BackgroundBreeding for resistance to apple scab (caused by Venturia inaequalis), the most devastating fungal disease of apples, relies on genetic resources maintained in germplasm collections.MethodsTo identify new sources of scab resistance, we evaluated 177 Malus accessions, including 27 primary and 13 hybrid Malus species from diverse geographical origins, in an orchard at Geneva, New York. We also screened a differential host set for 2 years to monitor for changes in the effectiveness of ten known scab resistance genes, which allowed us to confirm the presence of virulent pathogen races in the orchard.ResultsWe found that ~ 37% of the wild Malus accessions and domesticated cultivars were resistant to apple scab in the field. Several of these accessions were unrelated to sources of previously known resistance genes and are promising for apple scab genetic research and resistance breeding. Cultivars carrying the Rvi6 (Vf) gene from Malus floribunda clone 821, e.g. ‘Liberty’ or ‘Florina’, remained resistant despite the breakdown of Rvi6. ‘Demir’, a Malus hybrid from Turkey, and ‘Chisel Jersey’, a traditional English hard cider cultivar, showed fewer symptoms than the Rvi6 resistant cultivar ‘Prima’. Races 1 to 7 and 9 of V. inaequalis were present in the orchard, but no scab was observed on the indicator host accessions for races 11 and 12.ConclusionsDetailed and systematic screening of Malus germplasm identified resistant and moderately resistant donor accessions based on resistance reaction types. These accessions are promising for use in future genetic studies to identify novel sources of scab resistance alleles for apple breeding to develop cultivars with durable apple scab resistance.

A major scab resistance gene called Va1 was identified in the Russian apple cultivar ‘Antonovka’ (accession APF22) conferring scab resistance under conditions of natural scab infection in the field. After scab scorings over a period of 3 years, a 1:1 segregation was observed in the mapping population 04/214 (‘Golden Delicious’ × ‘Antonovka’). The Va1 resistance gene provides sufficient broad spectrum resistance that is of use in apple resistance breeding and has been assigned Rvi17 according the proposal for a new scab nomenclature (Bus et al., Acta Horticulturae 814:739–746, 2009). Analysis of simple sequence repeats (SSRs) located on the apple linkage group (LG) 1 showed that the Va1 locus is closely linked (1 cM) to SSR CH-Vf1 known to cosegregate with the Vf locus. A tight genetic association was also observed between a specific cleaved amplified polymorphic sequence marker (ARD-CAPS) developed from the HcrVf paralog Vf2ARD present in ‘Antonovka’, but there is no indication yet for a causal relationship with Vf2ARD. Although the whole race spectrum of Va1 is still unknown, it was obvious that it acts against the scab races 6 and 7 which are able to overcome the resistance of Malus floribunda 821. A second resistance factor (named Va2) was studied by race 1-specific scab tests based on grafted 04/214 clones. A 1:1-segregation ratio was observed, too, but 18 “phenotypic recombinants” were found after comparisons with the field scab data of the same genotypes. Va2 was mapped on LG 1 with a genetic distance of about 15 cM above CH-Vf1. The positions of the newly identified ‘Antonovka’ scab resistance factors are compared with previously reported Va mapping approaches and published results from quantitative trait loci analyses performed with different ‘Antonovka’ genotypes.

Apple (Malus × domestica) is the third important fruit in terms of production and consumption worldwide. Apple scab caused by Venturia inaequalis is the most devastating disease of apple. In the apple-growing regions, many fungicides are sprayed to control the disease leading to increase in the production cost. Development of scab-resistant cultivars is the long-lasting solution to control the disease. In apples, more than 20 major scab resistance genes have been identified in various cultivars and few wild relatives. Of all these genes, Rvi6 derived from Malus floribunda has been most extensively used in different breeding programs. Gene for gene interactions of these resistance genes with the avirulence genes from V. inaequalis have been understood in many cases. QTL-based polygenic resistance has also been characterized in apple. Nucleotide Binding Site Leucine-Rich Repeats (NBS-LRR) have been identified from the apple genome and many of them have been characterized from the scab resistance region. Molecular markers associated with most of the major scab resistance genes have been identified and their position has been mapped on different linkage groups. Marker-assisted selection (MAS) can be helpful in speeding up and accurately identifying the scab-resistant parents and progeny. Pyramiding of several major resistance genes can be undertaken for more durable resistance against apple scab. The present paper reviews the Malus-Venturia pathosystem, current status of knowledge about scab resistance genes, and their application in breeding against apple scab.

Apple scab is a disease caused by the fungus Venturia inaequalis, which leads to significant economic losses in apple production especially in temperate regions. Breeding programmes are attempting to introgress scab resistance genes from wild apple into commercial cultivars to control the disease. Most of the commercially available scab-resistant varieties to date rely on the Rvi6 (Vf) resistance gene from Malus floribunda 821. The evolution of new pathotypes of V. inaequalis, which have caused the breakdown of Rvi6-based resistance, at least in northern Europe, highlights the need for the characterisation and pyramiding of scab resistance genes from different sources for durable disease resistance. In this study, the scab resistance gene Rvi12 from Malus baccata ‘Hansen’s baccata #2’ was confirmed as mapping to apple linkage group 12 in the cross ‘Gala’ × ‘Hansen’s baccata #2’ in an interval between SSR markers Hi02d05 and CH02h11b. Using the ‘Golden Delicious’ genome sequence, novel SSR markers and SNPs were identified in the Rvi12 mapping interval and mapped in an extended mapping population of 1,285 plants. Rvi12 was fine-mapped to an interval spanning 958 kb of the ‘Golden Delicious’ genome sequence. The 18 SNPs fine-mapped to the Rvi12 interval were screened in eight apple breeding founders, and for 16 of the 18 SNPs, the alleles linked in coupling with the Rvi12 resistance locus were found only in ‘Hansen’s baccata #2’. The SNPs identified will thus be useful for the efficient identification of apple genotypes carrying the Rvi12 resistance locus.

The presence of genes for resistance to scab (Venturia inaequalis) and powdery mildew (Podosphaera leucotricha) was studied using molecular markers in a sample of 279 apple cultivars from the Czech collection of apple genetic resources. The sample comprised 37 cultivars supposed to have the Vf gene for scab resistance, 97 reference world cultivars and 145 old and local cultivars. Six PCR molecular markers for the scab resistance genes Vf, Vm, Vbj, Vr and Vh and three PCR molecular markers for the powdery mildew resistance genes Pl-w, Pl-1 and Pl-d were used. The marker for the major scab resistance gene Vf was detected in all cultivars supposed to have Vf, except in Romus 1, and in the three small-fruited cultivars Malus Evereste, Golden Gem and Hilleri. The markers of the Vr and Vh scab resistance genes were detected in 22 cultivars in combination with the marker for Vf, in 56 reference world cultivars and in 82 old and local apple cultivars. PCR molecular markers for one or two of the powdery mildew resistance genes were detected in the small-fruited cultivars Malus Evereste, Golden Gem, prof. Sprengeri and Hilleri; and in the larger fruited cultivars Hagloe Crab, Borovinka and Tita Zetei. We did not find markers for the scab resistance genes Vm and Vbj in any of the studied cultivars. They are absent also in the remaining part of the Czech collection of apple genetic resources. PCR molecular markers are useful tools for the identification of resistance genes within apple germplasm collections and can be used to increase the number of sources for disease resistance in breeding programmes.