Introduction

Apple scab, caused by the fungal pathogen Venturia inaequalis (Cooke) G.Wint., is one of the most devastating diseases for apples (Malus x domestica Borkh) in temperate zones with humid growing seasons. Most of the commercial apple cultivars are susceptible to the disease, and growers have to spray 20–30 times with fungicides in a season. The use of resistant cultivars could reduce the cost to the growers and may also contribute to a cleaner environment and to a reduction of fungicide residuals on apples for consumers.

The most widely used apple scab resistance gene is Vf from Malus floribunda 821. However, in the meantime, V. inaequalis strains have been detected that are able to overcome the Vf resistance (Parisi et al. 1993). Especially in northwestern Europe, these strains are present and have spread around (Parisi et al. 2006). As a result, several orchards consisting of Vf-cultivars have to be sprayed like orchards with susceptible cultivars (Trapman 2006). For durable resistance, several resistance genes should be accumulated (pyramiding). Fortunately, new loci, which include both major genes and quantitative trait loci (QTLs) that confer resistance to a broad spectrum of V. inaequalis strains, have been discovered in Malus (Calenge et al. 2004; Schmidt and Van de Weg 2005; Gessler et al. 2006; Gardiner et al. 2006). Until now, 11 major apple scab resistance genes have been mapped (Table 1). Some of them are considered as ephemeral resistance genes, acting only against a very narrow spectrum of races of V. inaequalis. Molecular markers linked to these genes are available (Gessler et al. 2006), and in this context, marker-assisted selection could be a useful tool in order to accelerate the breeding programs, for example, selecting the parents for the future crosses.

Table 1 Major apple scab resistance genes that have been positioned on the genetic linkage map of Malus
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In this work, we report the identification and mapping of a new qualitative apple scab resistance gene named Vd3 from the selection “1980-015-25” of the apple breeding program of Plant Research International in Wageningen, the Netherlands. This gene provides resistance to the highly virulent EU-NL-24 strain of race 7 of V. inaequalis, which has overcome the resistance from Vf and Vg.

Material and methods

Plant material and DNA extraction

For the mapping of Vd3, we used the population 2000-012C (Table 2). This population is a part of the population 2000-012 comprising 894 individuals and derived from the cross between the scab-resistant selection “1980-015-025” and the susceptible selection “1973-001-041.” In the resistance tests, the cultivars “Elstar,” “Priscilla,” “Gala,” and “Golden Delicious”; the selections “D3,” “1980-015-025,” and “1973-001-041”; and some individuals of the population 2000-012 carrying only Vd3 were used. For pedigree analysis, the apple cultivars Elstar and Priscilla and the selections D3, 1972-010-33, and 1980-015-025 were tested with the simple sequence repeat (SSR) CH-Vf1. DNA extraction was performed from unfolded leaves of apple as described for the Diversity Arrays Technology (DArT) technique (Jaccoud et al. 2001) in http://www.diversityarrays.com.

Table 2 The segregating population 2000-012C used in this study
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Evaluation of scab resistance

Scab resistance was evaluated after tunnel tests with mist evaporation, where the four youngest leaves of six replicates of the progeny 2000-012 containing Vd3 only and reference cultivars (Elstar, D3, Priscilla, Gala, Golden Delicious, 1980-015-025, and 1973-001-041) (Table 3) were inoculated with a monoconidial suspension of the nine different races of V. inaequalis (105 conidia/ml) used in the disease tests (Table 3). These isolates belong to the European collection of V. inaequalis from the “Durable Apple Resistance in Europe” project (Lespinasse et al. 2000). Plants were incubated for 48 h at 20°C and 100% relative humidity, and then transferred to a greenhouse with a relative humidity of 85–90%. Disease symptoms were assessed macroscopically after 14–17 days, indicating the presence (susceptible plant) or absence (resistant plant) of sporulation.

Table 3 Strains of V. inaequalis used in the disease tests, and their sporulation on cultivars and selections containing Vf, V25 or Vd3
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The population 2000-012C was inoculated a few weeks after emergence of the young seedlings. For mapping purposes, the strain EU-NL-24 was used. V25 confers resistance to this strain. This hampered mapping of the other resistance gene, i.e., Vd3. Therefore, the 143 plants that contained V25 were discarded based on flanking markers of V25. The remaining 92 plants were used for mapping Vd3. EU-NL-24 is capable of overcoming the Vf gene (Parisi et al 2004) and combines the virulences of races 6 and 7 (Calenge et al. 2004). Disease symptoms were assessed macroscopically after 14–17 days and rated in eight classes indicating the amount of sporulation as follows: class 0, 0% of sporulation; class 1, 1–2% sporulation; class 2, 2–5% sporulation; class 3, 5–10% sporulation; class 4, 10–25% sporulation; class 5, 25–50% sporulation; class 6, 50–75% sporulation; class 7, 75–100% sporulation. This scale is similar to that reported by Durel et al. (2003), which was adapted from Croxall et al. (1952). The two youngest inoculated leaves were scored for sporulation.

DArT markers

DArT markers were produced by Diversity Arrays Technology (Yarralumla, Australia) as described in Wenzl et al. (2004) and Wittenberg et al. (2005).

SSR markers

All of the SSRs in linkage group (LG) 1 available at the High-quality Disease Resistant Apples for a Sustainable Agriculture (HiDRAS) database (http://www.hidras.unimi.it/) were selected. In total, nine SSRs were screened in the population 2000-012C (Table 4). SSR amplifications were performed in a GeneAmp® PCR System 9700 thermal cycler (Applied Biosystems, Foster City, CA, USA) in a final volume of 20 μl, containing 75 mM Tris–HCl, pH 8.8; 20 mM (NH4)2SO4; 1.5 mM MgCl2; 0.2 mM of each dNTP; 0.5 μM of fluorescent dye-labeled forward primer (Hex or 6-Fam, Biolegio, Nijmegen, the Netherlands); 0.5 μM of reverse primer; 20 ng of genomic DNA; and 1 U of SuperTaq DNA polymerase (HT Biotechnology, Cambridge, UK) using the following temperature profile: 94°C for 2 min 30 s, then 34 cycles of 94°C for 30 s, 50°C for 30 s, and 72°C for 1 min, finishing with 72°C for 5 min. Samples were analyzed on an ABI PRISM 3700 DNA Analyzer (Applied Biosystems) and scored with GENOTYPER version 3.6 (Applied Biosystems).

Table 4. SSRs tested in 1980-015-025 parent, their position in genetic maps of Discovery (Silfverberg-Dilworth et al. 2006) and 1980-015-025, and the χ 2 statistical analysis of the segregation for monogenic inheritance
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Testing of specific markers linked to other scab resistance genes in LG1

RAPD marker P-136

The Va-linked RAPD marker P-136 reported by Hemmat et al. (2003) was also analyzed in the population 2000-012C to test its association with Vd3. RAPD amplification was performed in a GeneAmp® PCR System 9700 thermal cycler (Applied Biosystems) as described in Hemmat et al. (2003). PCR products were separated on 2% agarose gels (Hispanagar, Burgos, Spain).

Vf2ARD marker

The Vf2ARD marker developed by Boudichevskaia et al. (2008) was tested in the population 2000-012C. The PCR was performed in a GeneAmp® PCR System 9700 thermal cycler (Applied Biosystems) as described in Boudichevskaia et al. (2008). PCR products were separated on 1% agarose gels (Hispanagar).

Linkage analysis

The linkage analysis was carried out using JoinMap 4.0 software (Van Ooijen 2006) with the Kosambi mapping function (Kosambi 1944) used to convert recombination units into genetic distances. LGs were established using as threshold a minimum logarithm of odds of 6.0 and a recombination frequency lower than 0.4. The Vd3 gene was mapped as a dominant gene based on the phenotype data (1 for resistance and 0 for susceptibility). The genetic linkage map was constructed for the resistant parent following the “two-way pseudo test-cross” model of analysis (Grattapaglia and Sederoff 1994) and setting a “cross-pollinator” data type.

Results

Evaluation of scab resistance

The population 2000-012C, segregating for Vf, V25, and Vd3, was used to evaluate the resistance against the monocodial strain of V. inaequalis EU-NL-24, capable of overcoming the resistance conferred by the Vf gene. The other strains used in the disease test overcame the resistance conferred by the Vd3 gene (Table 3). The resistance donor was the selection 1980-015-025, which contains the Vf, V25, and Vd3 genes. This selection was heterozygous for the Vd3 trait, and the susceptible parent was homozygous recessive. From 14 to 17 days after inoculation, plants were scored for scab resistance and classified into eight classes based on symptoms (see “Material and methods” section). As the monoconidial strain is virulent to the Vf resistance, two effective major resistance genes were left, i.e., V25 and Vd3. V25 and Vd3 inherited independently. For the purpose of mapping Vd3 precisely, 143 progeny plants carrying the V25 gene were discarded using markers that flanked this gene tightly (unpublished data). The sporulation of the remaining seedlings (N = 92) is depicted in the histogram in Fig. 1. The resistance reaction observed in the plants carrying Vd3 was a hypersensitive pit type and chlorotic reactions.

Fig. 1
figure 1

Histogram showing the number of young seedlings of population 2000-012C belonging to each of the sporulation classes. The sporulation refers to percentage sporulating area in the upper two inoculated leaves

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Based on this histogram, those nonsporulating plants were designated as resistant, while those sporulating plants were designated as susceptible. On the basis of this scoring, 41 out of the 92 F1 individuals were considered resistant and 51 susceptible (Table 2). Segregation of resistance and susceptibility in this cross fit the ratio of 1:1, based on the Chi-square test (χ 2 = 1.08) using a significance level of p value of 0.05, indicating a monogenic inheritance. The hypothesis of two independent major dominant genes involved in the resistance was also tested, but it was rejected due to the high values of the Chi-square test (18.71) (Table 2).

Construction of the LG1 of 1980-015-025

This new gene could be mapped on LG1, closely to Vf, but on the homologous chromosome, so in repulsion to the CH-Vf marker (Vinatzer et al. 2004). For further mapping of Vd3, SSRs in LG1 were tested. Seven out of nine SSRs screened were polymorphic in the mapping population, and five of them were incorporated to the 1980-015-025 map (Table 4). In the case of the DArT markers, out of 234 markers from the resistant parent and absent in the susceptible parent, five were mapped on LG1. Other markers linked to scab resistance genes in LG1 were tested in population 2000-012C. With the first one, the RAPD marker P-136 linked to the Va gene according to Hemmat et al. (2003) should give the band of 700 bp, but this band was not present in the population 2000-012C (data not shown). The second marker was the marker Vf2ARD developed by Boudichevskaia et al. (2008) based on the sequence of HcrVf2 (Vinatzer et al. 2001) in the apple accessions “Antonovka,” “Realka,” and “Discovery.” It was mapped in a similar location to the north of Vf, as reported by these authors (Fig. 2), but the marker was also present in the susceptible parent of our population (data not shown).

Fig. 2
figure 2

Genetic maps of Vd3. a LG1 of 1980-015-025 showing the estimated position of Vf and Va genes according to Gessler et al. (2006). b Map position of Vd3 in LG1. c Map position of Vd3 after removing the phenotype information of the three genotype–phenotype incongruent (GPI) plants

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At first, LG1 was created only with SSR and DArT markers (Fig. 2a). This map covers a distance of 58.9 cM comprising 11 loci. The SSRs mapped maintain the colinearity with the Discovery map of Silfverberg-Dilworth et al. (2006) and with the “Florina” map reported by Broggini et al. (2009). Finally, Vd3 was mapped on LG1 as a dominant heterozygous gene, using the resistance data of the segregating population (Fig. 1). In the first location, this gene mapped between the SSR CH-Vf1 and the DArT 67005F17 at a distance of 2.9 cM from the Vf locus (Fig. 2b). After careful observation of the resistant/susceptible phenotype and genotype of the markers around Vd3, it was observed that three progeny plants (one belonging to class 1 and two to class 2) showed genotype–phenotype incongruence (GPI) (Gygax et al. 2004). These plants, classified as susceptible, showed either marker alleles in coupling with the resistance allele. In this case, by removing the resistance data of these plants, Vd3 was mapped again between the markers CH-Vf1 and 67005F17, but the distance to the Vf locus was reduced to 1.0 cM (Fig. 2c). Exclusion of the GPI plants did not change the order of the markers, and the length of the LG1 was only reduced 0.2 cM.

Pedigree analysis of 1980-015-025 and resistance source of Vd3

Figure 3 shows the parents and grandparents of the selection 1980-015-025. The male parent Priscilla was used as donor of the Vf resistance. This could be confirmed with the 159-bp allele from CH-Vf1 linked to HcrVf1 (Vinatzer et al. 2004). This band was only present in the Vf cultivar Priscilla and in 1980-015-025.

Fig. 3
figure 3

SSR and resistance test of the pedigree of 1980-015-025. For the SSR CH-Vf1, the numbers indicate the different alleles of the marker in base pairs and for the strain EU-NL-24. S indicates the presence of sporulation and R indicates the absence of sporulation. na indicates nonassessed

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As D3 appeared to be completely resistant in the field, this founder was used by the breeders of Plant Research International as a donor of additional resistance. They crossed it with the susceptible cultivar Elstar. Elstar and Priscilla are susceptible to the strain EU-NL-24 (Table 3) and, thus, lack the Vd3 gene. However, D3 is resistant to EU-NL-24 (Table 3). This suggests that Vd3 comes from D3. This could be confirmed with the alleles of the CH-Vf1 SSR, as shown in Fig. 3: Vf cosegregates with the 159-bp band of the SSR, whereas Vd3 cosegretates with the 129-bp band. The latter band originates from the source of resistance D3.

Discussion

Resistance screening

The correct placement of the threshold between resistance and susceptibility is very important to determine the resistance inheritance model and the position of the resistance gene in the linkage map. The selection of the threshold used in this work was due to the degree of sporulation observed from class 1 to class 7 and the absence of V. inaequalis spores in class 0 (Fig. 1). Comparison with the genetic markers indicates that this threshold gives good results, apart from three seedlings with 1–5% of sporulation. This indicates that Vd3 usually provided complete resistance, but 3% of the seedlings showed a low level of sporulation in spite of the presence of Vd3. We did not observe any healthy plants lacking Vd3. This implies that we had no plants that escaped from inoculation.

Linkage map

An accurate phenotyping of the resistance/susceptible data in the progenies is essential for the correct placement of the gene of interest in the linkage map. Two positions are proposed for the Vd3 gene according to the inclusion or exclusion of GPI plants of the analysis (Fig. 2b, c). In both situations, the gene is mapped between the same markers, and the order of the markers in the map is not altered, but in the case of the exclusion of the GPI plants, the distance between Vd3 and the Vf locus was reduced from 2.9 to 1 cM. As was reported by other authors (Patocchi et al. 1999a; Gygax et al. 2004; Erdin et al. 2006), we found difficulties determining the correct position of Vd3, as the phenotypic data were not as precise as the molecular data. During the analysis of the population 2000-012C, we classified three plants (3%) as susceptible, but the flanking marker alleles were in coupling with the resistance. One susceptible plant without Vf showed from 2% to 5% sporulation. As we classified 2–5% sporulation as susceptible and absence of Vd3, this plant would lack both Vf and Vd3. This is the only indication of a recombination between the Vf locus and the Vd3 locus. The flanking markers confirm this recombination event around the Vf locus in this plant. This single plant is responsible for positioning Vd3 just south of Vf, and not at the Vf locus itself or north of Vf. If the sporulation of 2–5% was regarded as resistant and the presence of Vd3 or all of the plants in classes 1 and 2 were discarded, then Vd3 would have been positioned in the same bin as the Vf locus. We conclude that there is evidence that Vd3 is south of Vf, but for a firm proof, additional observations are needed.

Patocchi et al. (1999a), during the fine mapping of the Vf gene, classified about 9% of the plants as resistant, but they showed the alleles in repulsion to Vf in the flanking markers. The exclusion of the phenotype data of these plants was necessary in the correct mapping of the gene as they demonstrated later with the map-based cloning of the Vf gene (Patocchi et al. 1999b). In the mapping of the Vbj gene, Gygax et al. (2004) found 13 resistant plants, out of a population of 173 individuals, that did not have the marker alleles in coupling with the gene and 12 susceptible ones that had them. Finally, Erdin et al. (2006), during the mapping of the Vb gene in the population Golden Delicious × “Hansen’s baccata #2,” identified six GPI plants. After the exclusion of the resistance data of these plants, the tension that they observed in the map disappeared. Patocchi et al. (1999a) suggested two hypotheses to explain this situation; the first one is that these plants are double recombinants, and the second one is that they were wrongly classified. As reported by these authors (Patocchi et al. 1999a; Gygax et al. 2004; Erdin et al. 2006), the presence of double recombinants is quite unlikely because of the distance of the flanking markers in coupling with Vd3. In our case, we expect a frequency of double recombinants of 0.6% and not the 3% observed. The hypothesis of the wrong classification could be more probable supported by the fact that these plants were classified in class 1 (one plant) and class 2 (two plants), which means from 1% to 5% of sporulation. Erdin et al (2006) suggested that the presence of susceptible plants showing the marker alleles in coupling with the resistance could be because of the presence of modifiers that might reduce the effect of the resistance gene.

The presence in a small genomic region of Vd3, the Vf locus composed of 4R (resistance)-genes (HcrVf1, HcrVf2, HcrVf3, and HcrVf4) (Vinatzer et al. 2001), and the Vf2ARD (Boudichevskaia et al. 2008) could indicate the presence of a gene cluster of Vf-like sequences. This is supported by the fact that Broggini et al. (2009) found two SSRs developed from BAC clones containing Vf-like sequences in the vicinity of the Vf locus. Another cluster of R-genes against V. inaequalis was found by Bus et al. (2004) in LG2. This cluster comprises four major genes, as well as several race-specific QTLs (Bus et al. 2004). This grouping of R genes in clusters is found frequently in plants. The biological reason could be the generation of novel resistances through the unequal crossover between different genes (Hammond-Kosack and Jones 1997).

Vd3 is a new apple scab resistance gene

The genetic position of Vd3 in LG1 permitted us to show that Vd3 is novel compared to all of the previously identified resistance genes against V. inaequalis, including Vf, Va, and Vf2ARD genes (Patocchi et al. 1999a; Hemmat et al. 2003; Boudichevskaia et al. 2008). In the case of the Vf gene, the resistance spectra demonstrated that Vd3 is not Vf. While the Vf gene has a wide resistance spectrum conferring resistance to the V. inaequalis races 1 to 5 (Mc Hardy 1996), in our disease tests, Vd3 only confers resistance to the EU-NL-24 strain (race 7) capable of overcoming the resistance provided by Vf (Table 3).

Regarding the Va gene, although also mapped in LG1, the position is different to that reported for Vd3. Gessler et al. (2006) reported the positions of the Va gene mapped by Zini (2005), who used the cultivar “Freedom” as the resistance donor. This cultivar is carrying the Vf gene and another one coming from an unspecified Antonovka clone. In the two locations reported, the Va gene is outside the interval between the SSRs Hi12c02 and CH05g08 and, in both cases, at a distance of about 25 cM from Vf (Zini 2005). This distance is in agreement with that reported previously by Hemmat et al. (2003) using as resistance donor “Antonovka PI 172633.” On the contrary, Vd3 is close to Vf (Fig. 2) and is within the region flanked by those SSRs. Using the RAPD marker P-136 described by Hemmat et al. (2003), no results were obtained (data not shown). The band of 700 bp reported by these authors as linked to the Va gene was not present in the population 2000-012C. Therefore, it was not possible to test the association between P-136 and Vd3. Moreover, the reaction types of both genes indicate that they are not the same, as Va induces a hypersensitive pit-type reaction (Dayton and Williams 1968), whereas Vd3 induces a chlorosis reaction too. Finally, taking into account the pedigree analysis showed in Fig. 3, we can say that the source of Vd3 (D3) does not come from an Antonovka accession. The 129-bp allele coming from D3 does not occur in the Antonovka accessions tested by Vinatzer et al. (2004) or in Antonovka 34.16 (unpublished data from HiDRAS database).

Recently, Boudichevskaia et al. (2008) reported the mapping of sequences homologous to the Vf genes identified by Vinatzer et al. (2001) (HcrVf genes). The candidate gene Vf2ARD, developed on the basis of the divergences in the sequence of the C1 subdomain of the HcrVf2 gene present in the apple accessions Antonovka, Realka, and Discovery, was mapped to the north of the Vf locus at a distance of 1.9 cM from the marker CH-Vf1 (Boudichevskaia et al. 2008). Using the primers developed by these authors, we mapped the Vf2ARD gene in a similar location but at a distance between 2.5 and 3 cM, depending on the map (Fig. 2). So Vd3, which is south of the Vf locus, is located at a distance of about 5 cM from Vf2ARD. Moreover, this marker was also present and mapped in the susceptible parent of our population (data not shown). So, we can discard that Vd3 and Vf2ARD were the same gene.

Bénaouf and Parisi (2000) detected in the cross Golden Delicious × M. floribunda 821, in addition to the Vf resistance, another locus for resistance descending from M. floribunda 821. They named it Vfh, but they did not map it. According to Parisi et al. (2004), EU-NL-24 can sporulate on M. floribunda 821. This indicates that the Vfh gene is not the same as the Vd3 gene.

Calenge et al. (2004) mapped QTLs for scab resistance using different strains of V. inaequalis. They also used EU-NL-24. The QTLs for EU-NL-24 were not mapped in the vicinity of the Vf locus in the progeny of the cross derived from Discovery, an English cultivar partially resistant to V. inaequalis, and “TN10-8,” a partially resistant hybrid derived from “Schmidt’s Antonovka PI 172632.”

In conclusion, we present in this work a new apple scab resistance gene that is only 1 cM south of the Vf locus. The V. inaequalis strain EU-NL-24 is virulent to Vf but avirulent to Vd3 cultivars. However, Vd3 has not been effective against the majority of other V. inaequalis strains we used in our disease tests. The Vd3 gene is south of the Vf locus, in contrast to previously mapped HcrVf-like sequences.