Functional analysis and expression profiling of <Emphasis Type="Italic">HcrVf1</Emphasis> and <Emphasis Type="Italic">HcrVf2</Emphasis> for development of scab resistant cisgenic and intragenic apples

Apple scab resistance genes, HcrVf1 and HcrVf2, were isolated including their native promoter, coding and terminator sequences. Two fragment lengths (short and long) of the native gene promoters and the strong apple rubisco gene promoter (P_MdRbc) were used for both HcrVf genes to test their effect on expression and phenotype. The scab susceptible cultivar ‘Gala’ was used for plant transformations and after selection of transformants, they were micrografted onto apple seedling rootstocks for scab disease tests. Apple transformants were also tested for HcrVf expression by quantitative RT-PCR (qRT-PCR). For HcrVf1 the long native promoter gave significantly higher expression that the short one; in case of HcrVf2 the difference between the two was not significant. The apple rubisco gene promoter proved to give the highest expression of both HcrVf1 and HcrVf2. The top four expanding leaves were used initially for inoculation with monoconidial isolate EU-B05 which belongs to race 1 of V. inaequalis. Later six other V. inaequalis isolates were used to study the resistance spectra of the individual HcrVf genes. The scab disease assays showed that HcrVf1 did not give resistance against any of the isolates tested regardless of the expression level. The HcrVf2 gene appeared to be the only functional gene for resistance against Vf avirulent isolates of V. inaequalis. HcrVf2 did not provide any resistance to Vf virulent strains, even not in case of overexpression. In conclusion, transformants carrying the apple-derived HcrVf2 gene in a cisgenic as well as in an intragenic configuration were able to reach scab resistance levels comparable to the Vf resistant control cultivar obtained by classical breeding, cv. ‘Santana’.     

Identification of functional apple scab resistance gene promoters

Apple scab (Venturia inaequalis) is one of the most damaging diseases affecting commercial apple production. Some wild Malus species possess resistance against apple scab. One gene, HcrVf2, from a cluster of three genes derived from the wild apple Malus floribunda clone 821, has recently been shown to confer resistance to apple scab when transferred into a scab-susceptible apple variety. For this proof-of-function experiment, the use of the 35S promoter from Cauliflower mosaic virus was reliable and appropriate. However, in order to reduce the amount of non-plant DNA in genetically modified apple to a minimum, with the aim of increasing genetically modified organism acceptability, these genes would ideally be regulated by their own promoters. In this study, sequences from the promoter region of the three members of the HcrVf gene family were compared. Promoter constructs containing progressive 5 deletions were prepared and used for functional analyses. Qualitative assessment confirmed promoter activity in apple. Quantitative promoter comparison was carried out in tobacco (Nicotiana glutinosa) and led to the identification of several promoter regions with different strengths from a basal level to half the strength of the 35S promoter from Cauliflower mosaic virus.     

Identification of a leucine-rich repeat receptor-like serine/threonine-protein kinase as a candidate gene for <Emphasis Type="Italic">Rvi12 (Vb)</Emphasis>-based apple scab resistance

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.     

Genetics of resistance in apple against <Emphasis Type="Italic">Venturia inaequalis</Emphasis> (Wint.) Cke

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.     

Molecular characterization of cisgenic lines of apple ‘Gala’ carrying the Rvi6 scab resistance gene

Using resistance genes from a crossable donor to obtain cultivars resistant to diseases and the use of such cultivars in production appears an economically and environmentally advantageous approach. In apple, introgression of resistance genes by classical breeding results in new cultivars, while introducing cisgenes by biotechnological methods maintains the original cultivar characteristics. Recently, plants of the popular apple ‘Gala’ were genetically modified by inserting the apple scab resistance gene Rvi6 (formerly HcrVf2) under control of its own regulatory sequences. This gene is derived from the scab‐resistant apple ‘Florina’ (originally from the wild apple accession Malus floribunda 821). The vector used for genetic modification allowed a postselection marker gene elimination to achieve cisgenesis. In this work, three cisgenic lines were analysed to assess copy number, integration site, expression level and resistance to apple scab. For two of these lines, a single insertion was observed and, despite a very low expression of 0.07‐ and 0.002‐fold compared with the natural expression of ‘Florina’, this was sufficient to induce plant reaction and reduce fungal growth by 80% compared with the scab‐susceptible ‘Gala’. Similar results for resistance and expression analysis were obtained also for the third line, although it was impossible to determine the copy number and TDNA integration site–such molecular characterization is requested by the (EC) Regulation No. 1829/2003, but may become unnecessary if cisgenic crops become exempt from GMO regulation.     

The role of Schmidt ‘Antonovka’ in apple scab resistance breeding

??Antonovka?? has long been recognised as a major source of scab (Venturia inaequalis) resistance useful for apple breeding worldwide. Both major gene resistances in the form of the Rvi10 and Rvi17 and quantitative resistance, collectively identified as VA, have been identified in different accessions of ??Antonovka??. Most of the ??Antonovka?? scab resistance used in apple-breeding programmes around the world can be traced back to Schmidt ??Antonovka?? and predominantly its B VIII progenies 33,25 (PI 172623), 34,6 (PI 172633), 33,8 (PI 172612) and 34,5 (PI 172632). Using genetic profile reconstruction, we have identified ??common ??Antonovka?? ?? as the progenitor of the B VIII family, which is consistent with it having been a commercial cultivar in Poland and the single source of scab resistance used by Dr. Martin Schmidt. The major ??Antonovka?? scab resistance genes mapped to date are located either very close to Rvi6, or about 20?C25?cM above it, but their identities need further elucidation. The presence of the 139?bp allele of the CH-Vf1 microsatellite marker known to be associated with Rvi17 (Va1) in most of the ??Antonovka?? germplasm used in breeding suggests that it plays a central role in the resistance. The nature and the genetic relationships of the scab resistance in these accessions as well as a number of apple cultivars derived from ??Antonovka??, such as, ??Freedom??, ??Burgundy?? and ??Angold??, are discussed. The parentage of ??Reglindis?? is unclear, but the cultivar commercialised as ??Reglindis?? was confirmed to be an Rvi6 cultivar.     

Introgression of the Vf source of scab resistance and distribution of linked marker alleles within the Malus gene pool

A chromosomal region originating from Malus floribunda 821 confers Vf scab resistance to many isolates of Venturia inaequalis. Twelve DNA markers located in this region were used to scan the equivalent of 31 cM in 98 Malus accessions. This allowed a molecular diagnosis of a source of resistance in apple germplasm with the aid of pedigree information, and in the context of a limited marker survey representing other chromosomes. At least five marker alleles were present in all scab-resistant breeding selections or varieties arising from M. floribunda. The validity of findings based on RAPD markers was confirmed with SCAR assays and Southern-hybridisation experiments. The order of markers determined in previous mapping studies was confirmed and sets of recombinants identified that establish reliable fine-mapping orders within 0.7 cM of the resistance locus. None of the marker alleles were present in the accessions that are either susceptible or possess weak polygenic resistance to scab. The presence of some alleles corresponding to those present at least 5.3 cM from Vf in M. floribunda was detected in some accessions. Other major sources of scab resistance do not appear to possess alleles in common with the Vf region, which will simplify future allelism tests. The results are discussed in the context of the introgression of resistance loci together with marker-assisted selection. The use of breeding pedigrees to assist in fine-scale mapping and map-based cloning is discussed. Received: 16 February 1999 / Accepted: 11 March 1999     

Expression profiling in HcrVf2-transformed apple plants in response to Venturia inaequalis

Apple scab resistance is one of the most well-characterized plant–pathogen interactions in a woody plant species. While the HcrVf2 gene from the wild apple Malus floribunda 821 has proved capable of conferring scab resistance to the susceptible cv. Gala after genetic transformation, its identification represents only the first step in understanding the molecular mechanisms and, hence, the network of genes underlying the defence response. We used a PCR-based suppression subtractive hybridization to identify apple genes that are differentially expressed after Venturia inaequalis inoculation. Subtractive hybridization was performed between cDNA from challenged leaves of HcrVf2-resistant transgenic Gala and susceptible cv. Gala plants. A library of 523 unigenes was constructed and characterized by assigning a putative function via comparison with public databases. This set of pathogen-modulated apple genes includes many defence-related genes and is therefore an important source of information for understanding the molecular basis of the Malus–V. inaequalis interaction. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The development of a cisgenic apple plant

Cisgenesis represents a step toward a new generation of GM crops. The lack of selectable genes (e.g. antibiotic or herbicide resistance) in the final product and the fact that the inserted gene(s) derive from organisms sexually compatible with the target crop should rise less environmental concerns and increase consumer's acceptance. Here we report the generation of a cisgenic apple plant by inserting the endogenous apple scab resistance gene HcrVf2 under the control of its own regulatory sequences into the scab susceptible apple cultivar Gala. A previously developed method based on Agrobacterium-mediated transformation combined with a positive and negative selection system and a chemically inducible recombination machinery allowed the generation of apple cv. Gala carrying the scab resistance gene HcrVf2 under its native regulatory sequences and no foreign genes. Three cisgenic lines were chosen for detailed investigation and were shown to carry a single T-DNA insertion and express the target gene HcrVf2. This is the first report of the generation of a true cisgenic plant.     

A Phenotypic,Molecular and Biochemical Characterization of the First Cisgenic Scab-Resistant Apple Variety ‘Gala’

Scab resistance is one of the most important goals of apple breeding, typically achieved by time-consuming and expensive conventional breeding techniques. Cisgenesis, which is the genetic modification of a recipient organism with genes from a crossable—sexually compatible—organism, is a promising tool for plant breeding to develop disease resistance in a rapid way. A cisgenic, scab-resistant line of the apple variety ‘Gala’ expressing the native apple scab resistance gene Rvi6 (formerly HcrVf2) under control of its own regulatory sequences has been recently developed. In this paper, we present the results from a phenotypic, molecular and biochemical evaluation of clonal replicates of this line (C11.1.53). The phenotype (shoot length, shoot diameter, internode length, number of leaves, leaf length and leaf width) of C11.1.53 was compared to that of the Gala parental background over a period of 108 days. Only a few statistically significant differences were detected, which are probably due to small differences in the quality of the budwood used for grafting rather than effects related to the presence of the cisgene. As the expression of a resistance gene can affect the downstream cascade of plant defence responses, a selection of apple defence-related genes was analyzed by quantitative real-time PCR analysis. These genes are also known as major allergen genes in apple. Even if three out of ten apple allergen genes tested in the leaves differed in the cisgenic line compared to both Gala (background) and ‘Florina’ (the variety from which the Rvi6 gene was cloned), using 2D-PAGE, we were unable to find any significant difference in the expressed proteomes of the leaves of C11.1.53 compared to Gala. Results are discussed in the context of a possible use of cisgenic lines for fruit crop improvement.     

Cloning and functional characterization of the Rvi15 (Vr2) gene for apple scab resistance

Apple scab, caused by Venturia inaequalis, is a serious disease of apple. Previously, the scab resistance Rvi15 (Vr2) from the accession GMAL 2473 was genetically mapped, and three candidate resistance genes were identified. Here, we report the cloning and functional characterization of these three genes, named Vr2-A, Vr2-B, and Vr2-C. Each gene was cloned with its native promoter, terminator and introns, and inserted into the susceptible apple cultivar ‘Gala’. Inoculation of the plants containing Vr2-A and Vr2-B induced no resistance symptoms, but abundant sporulation. However, inoculation of the plants harboring Vr2-C showed a hypersensitive response with clear pinpoint pits, and no or very little sporulation. We conclude that Vr2-C is the Rvi15 (Vr2) gene. This gene belongs to the Toll and mammalian interleukin-1 receptor protein nucleotide-binding site leucine-rich repeat structure resistance gene family. The proteins of this gene family reside in the cytoplasm, whereas V. inaequalis develops in the apoplast, between the epidermis and cuticle, without making haustoria. The spatial separation of the recognizing resistance protein and the pathogen is discussed. This is the second cloned gene for apple scab resistance, and out of these two the only one leading to a symplastic protein.     

A major resistance gene from Russian apple ‘Antonovka’ conferring field immunity against apple scab is closely linked to the <Emphasis Type="Italic">Vf</Emphasis> locus

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.     

Screening apples for OPD20/600 using sequence-specific primers

Apple scab, caused by Venturia inaequalis (Cke.) Wint., is the most serious disease of apple trees in many areas of the world. Resistance to V. inaequalis, derived from the small-fruited species Malus floribunda 821, is determined by a major dominant gene, Vf. Using random decamer primers, we identified a RAPD marker, OPD20/600, which is linked to the Vf gene. OPD20/600 was then cloned and sequenced. Sequence-specific primers based on the marker were used to further screen M. floribunda 821, 7 scab-susceptible apple cultivars, 10 scab-resistant apple cultivars, and 28 scab-resistant Coop selections. The sequence-specific primers allowed identification of polymorphisms of OPD20/600 based on the presence or absence of a single band. The advantages of sequence-specific primers over decamer primers for developing genetic markers are discussed.     

Towards improvement of marker assisted selection of apple scab resistant cultivars: <Emphasis Type="Italic">Venturia inaequalis</Emphasis> virulence surveys and standardization of molecular marker alleles associated with resistance genes

Molecular breeding for pathogen resistance faces two major problems that delay its widespread adoption, resistance breakdown and difficulties in unambiguously identifying the alleles of the markers associated with specific resistance genes. Since the breakdown of the Rvi6 (Vf) gene in the Northern part of Europe breeders have intensified the search for new resistance sources to be introduced into their breeding programs. Alternative major genes to Rvi6 are available (e.g. Rvi2, Rvi4, Rvi5, Rvi10; Rvi11, Rvi12, Rvi13, and Rvi15, respectively Vh2, Vh4, Vm, Va, Vbj, Vb, Vd, Vr2 according to the old apple scab resistance gene nomenclature) but, with few exceptions (i.e., Rvi4, Rvi5 and, Rvi13), they have so far not been incorporated in commercial varieties. Pyramiding, i.e., combining several of these major resistance genes (R-genes) in individual plants, is one of the most promising strategies currently available to develop apple cultivars with durable apple scab resistance. But, which genes are the best suited to produce such new cultivars? Although the most interesting genes are surely those whose resistance so far has not been broken by the pathogen, genes with resistance that has been overcome coupled with only limited spread of the virulence may also be used in the pyramiding process. However, obtaining information on whether an R-gene is overcome and if so, the extent of the spread of the virulence is difficult and time consuming. Furthermore, often such reports are not up-to-date and the correctness of the data is difficult to verify. To solve these problems, the initiative “Monitoring of Venturia inaequalis virulences” has been proposed. The monitoring is based on a network of orchards of selected differential hosts. Incidence and severity of scab on these genotypes will be collected yearly; and after validation, the data will be published through the homepage of the project (www.vinquest.ch). Here, we present an outline of this initiative. A second major obstacle for broad adoption of marker assisted selection is the lack of tools to align marker analyzes performed in different laboratories to unambiguously identify the alleles linked to specific resistances. The identification of the alleles of the markers in coupling with the resistance genes is often very difficult, if the same genotype used to develop the markers is not simultaneously analyzed. In this paper we present an approach to standardize the size of the alleles in coupling with the resistance genes, using easily accessible cultivars. The proposed procedure has been applied to selected markers for the apple scab resistance genes Rvi2, Rvi4, Rvi5, Rvi6, Rvi11, Rvi12, Rvi13, Rvi14 and Rvi15 (respectively Vh2, Vh4, Vm, Vf, Vbj, Vb, Vd, Rvi14 and Vr2 according to the old nomenclature).     

Mapping and functional analysis of four apple receptor-like protein kinases related to <Emphasis Type="Italic">LRPKm1</Emphasis> in <Emphasis Type="Italic">HcrVf2</Emphasis>-transgenic and wild-type apple plants

The Malus–Venturia inaequalis interaction is the most studied plant–pathogen interaction involving a woody species. Besides the cloning of an apple scab resistance gene HcrVf2, several sequences have been recently identified that are modulated after pathogen recognition in Vf-resistant genotypes. Among these, there is a putative leucine-rich repeat receptor-like protein kinase from the apple scab-resistant cv. Florina, named LRPKm1 that is induced after V. inaequalis inoculation and salicylic acid treatment. In this work, the isolation, characterization, and mapping of four new genes belonging to the LRPKm multigene family are reported. According to their cumulative expression profiles in HcrVf2-transgenic and wild-type apple plants treated with V. inaequalis, LRPKm genes have been divided in two groups. LRPKm1 and LRPKm3, giving a response related to the presence of HcrVf2, are probably involved in the recognition of pathogen-derived signals. LRPKm2 and LRPKm4, with an expression profile unrelated to the HcrVf2 gene, are putatively involved in the plant basal defense. Furthermore, we have localized LRPKm proteins at the cytological level in the plasma membrane of epidermal cells in resistant genotypes following pathogen challenge, thus confirming software predictions and molecular results. The possible involvement of LRPKm proteins in apple scab resistance and in the plant basal defense makes them attractive for a better comprehension of the molecular mechanisms of the signal transduction pathways after pathogen recognition.     

Vf scab resistance of Malus

The apple production in temperate regions with spring rains, the Scab caused by the fungus Venturia inaequalis is the most important constraint. To produce spotless apples and avoid damage that develops during storage, growers apply fungicide on a regular or weather-determined basis. All major apple cultivars are highly susceptible to this disease. To limit the need for fungicide applications, apple breeders are currently introgressing disease resistance from wild Malus accessions into commercial lines. The first attempts to do this were made 100 years ago. As apples are self-incompatible, pseudo-backcrossing is used to eliminate unwanted traits from wild Malus and select new cultivars that are attractive to both producers and consumers. This process, from the first cross of a commercial cultivar with a wild, disease-resistant Malus, is extremely long due to apple’s long juvenile phase, the need for more than seven backcross steps and the high heterozygosity of this genus. Therefore, most of today’s scab-resistant cultivars rely on a single introduction of scab resistance from Malus floribunda 821, referred to as Vf. In this paper, we trace the history of Vf from its initial identification through its use in breeding and commercial production. We sum up the literature describing how and where Vf resistance has been overcome by new pathotypes of V. inaequalis. Finally, we describe the current knowledge of the genes behind Vf resistance, its mode of action and the use of Vf genes in gene technology.     

Phenotypic Reaction and Genetic Analysis Using AFLP-derived SCARs for Resistance to Apple Scab

Six sequence‐characterized amplified region (SCAR) markers linked to the apple scab resistance gene Vf were evaluated for their utility in marker‐assisted selection (MAS) in apple breeding. Of the six SCARs used in this study, ACS‐6 was located left of the Vf gene, ACS‐7 and ACS‐9 co‐segregated with Vf, and ACS‐8, ACS‐4, ACS‐5 were located right of the Vf gene. Three families derived from crosses between scab‐resistant and scab‐susceptible cultivars, including ‘Liberty’ × ‘Deljub’, ‘Liberty’ × ‘Delcorf’, and ‘Florina’ בDelcorf’, previously screened for scab resistance following greenhouse inoculation with the fungal pathogen Venturia inaequalis, were genotyped and compared with phenotypic reactions to scab infection in the field. For each family, a subset progeny of 30 seedlings (propagated onto Malling 9 rootstock and of 7 years old) was selected based on fungal sporulation according to the following scheme. Ten seedlings with no visible scab sporulation on leaves were given phenotypic scores of 0 (deemed resistant); 10 seedlings with moderate scab sporulation were given phenotypic scores of 1.0 (deemed moderately resistant); and 10 seedlings with heavy sporulation were given phenotypic scores of 2.0 (deemed susceptible). DNA was isolated from leaf tissue collected from all 90 seedlings, parents and Malus floribunda 821, the original source of the Vf gene, and screened with all six SCARs. All six SCARs were present in the two scab‐resistant parents, ‘Liberty’ and ‘Florina’, and M. floribunda 821; while, the two scab‐susceptible parents, ‘Deljub’ and ‘Delcorf’, lacked all SCARs. All SCARs were either present or absent in varying numbers of seedlings in each progeny with phenotypic ratings of either 0 (resistant) or 1.0 (moderately resistant); while all seedlings with phenotypic ratings of 2.0 (susceptible) lacked all SCARs. The inconsistencies between phenotypic scab ratings and SCAR marker data are discussed.     

High-resolution genetic map of the <Emphasis Type="Italic">Rvi15</Emphasis> (<Emphasis Type="Italic">Vr2</Emphasis>) apple scab resistance locus

The Rvi15 (Vr2) apple scab resistance locus found in the GMAL 2473 accession has been previously mapped to the top of the Linkage Group 2 (LG2) by analyzing 89 progeny plants of a cross between ‘Idared’ and GMAL 2473. A new population of 989 progeny plants, derived from a cross between ‘Golden Delicious’ and GMAL 2473, has been analyzed with the two SSR markers CH02c02a and CH02f06, previously found to be associated with Rvi15 (Vr2), and with two published markers derived from NBS sequences (ARGH17 and ARGH37) estimated to map close to the Rvi15 (Vr2) locus. ARGH17 and ARGH37, were found to be the closest markers to the resistance locus, bracketing it within an interval of 1.5 cM. The SSRs mapped one on each side of Rvi15 (Vr2). CH02f06 mapped at 2.9 cM from ARGH37 while CH02a02a mapped at 1.7 from ARGH17. The position of Rvi15 (Vr2) respect to CH02a02a indicates that Rvi15 (Vr2) and Rvi4 (Vh4), a second apple scab gene mapped on the top of LG2, are two different resistance genes. In order to develop even more tightly linked markers to Rvi15 (Vr2), ARGH17 was used as the starting point for chromosome walking through the Rvi15 (Vr2) homolog region of the cv. ‘Florina’. A single ‘Florina’ BAC clone, 36I17, was sufficient to span the homologous locus in the new population’s recombinant progeny. Sequencing of the 36I17 BAC clone allowed identifying seven putative ORFs, including two showing a TIR-NBS-LRR structure. Ten additional markers could be developed mapping within a 1.8 cM interval around the Rvi15 (Vr2) resistance gene. ARGH17 and GmTNL1 markers, the latter also derived from NBS-LRR resistance gene homolog sequence, are the closest markers to Rvi15 (Vr2) bracketing it within a 0.5 cM interval. The availability of 12 markers within the Rvi15 (Vr2) region, all within a small physical distance (kbp) in ‘Florina’, suggests that cloning of the Rvi15 (Vr2) apple scab resistance gene from GMAL 2473 will be possible.