Construction of a contig of BAC clones spanning the region of the apple scab avirulence gene AvrVg

Fungal Genetics and Biology 44 (2007) 44–51 www.elsevier.com/locate/yfgbi

Construction of a contig of BAC clones spanning the region of the apple scab avirulence gene AvrVg G.A.L. Broggini a,¤, B. Le Cam b, L. Parisi b, C. Wu c, H.-B. Zhang c, C. Gessler a, A. Patocchi a a

Department of Plant Pathology, Institute of Integrative Biology (IBZ), ETH Zürich, 8092 Zürich, Switzerland b UMR PaVé, INRA, Centre d’Angers, BP 57, 49071 Beaucouzé, France c Department of Soil and Crop Sciences, Texas A&M University, College Station, TX 77843-2474, USA Received 20 April 2006; accepted 5 July 2006 Available online 10 August 2006

Abstract The ascomycete Venturia inaequalis, causal pathogen of apple scab, underlies a gene-for-gene relationship with its host plant apple (Malus spp.). ‘Golden Delicious’, one of the most common cultivated apples in the world, carries the ephemeral resistance gene Vg. Avirulence gene AvrVg, matching resistance gene Vg has recently been mapped on the V. inaequalis genome. In this paper, we present the construction of a BAC library from a V. inaequalis AvrVg isolate. The library is composed of 7680 clones, with an average insert size of 80 kb. By hybridization, it has been estimated that the library contains six haploid genome equivalents. Thus the V. inaequalis genome can be predicted to be approximately 100 Mb in size. A chromosome walk, starting from the marker VirQ5 co-segregating with AvrVg, has been performed using the BAC library. Twelve BAC clones were identiWed during four steps of the chromosome walking. The size of the resulting contig is t330 kb. © 2006 Elsevier Inc. All rights reserved. Keywords: Fungal-plant relationships; Pathogenicity factors; Ascomycetes; Phytopathogenic fungi; Gene sequencing; Genome analysis; Resistance; Virulence

1. Introduction The hemibiotrophic ascomycete Venturia inaequalis, the causal pathogen of apple scab on apple (Malus spp.), is a disease of great economical importance in most temperate climates. This pathogen underlies a gene-for-gene interaction with its host (Bénaouf and Parisi, 2000). According to the gene-for-gene concept, the recognition by the product of a plant gene (resistance gene) of a corresponding pathogen gene product (Avr gene) leads to resistance. Plant resistance genes (R genes) and their organization in the genome have been studied thoroughly, allowing them to be grouped into structural homologies among diVerent host species Abbreviations: SCAR, sequence characterized ampliWed regions; SSR, simple sequence repeats; PFGE, pulsed Weld gel electrophoresis; CAPS, cleaved ampliWed polymorphic sequences. * Corresponding author. Fax: +41 44 632 15 72. E-mail address: [email protected] (G.A.L. Broggini). 1087-1845/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.fgb.2006.07.001

(Hammond-Kosack and Jones, 1997; Kruijt et al., 2005; Martin et al., 2003). Several major apple scab resistance genes have been identiWed in apple (Dayton and Williams, 1968) and most of them mapped (Bus et al., 2005; Gygax et al., 2004; Hemmat et al., 2003; Maliepaard et al., 1998; Patocchi et al., 2004; Patocchi et al., 2005). One of them, HcrVf2 from the Vf region, has been cloned and has proven in complementation experiments to induce apple scab resistance (Belfanti et al., 2004). On the pathogen side, more and more fungal avirulence genes have been cloned in recent years. Avirulence genes from extracellular fungal pathogens often code for small cysteine rich proteins possessing a signal peptide for secretion (Rep, 2005). Despite these structural homologies among the proteins, the avirulence genes display only lowDNA sequence similarity. Up to now, no V. inaequalis avirulence gene has been cloned. A protein isolated from V. inaequalis, causes a hypersensitive response (HR), following inWltration, on the

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Accession No. 9AR2T196 (carrying the Vm resistance gene) but not on ‘Royal Gala’ susceptible to all known races (Win et al., 2003). However the gene coding for this protein has yet to be identiWed. The way V. inaequalis overcomes plant resistance is currently unknown. In other host– pathogen systems the resistance breakdown is caused by deletions, mutations or frame shifts in the avirulence genes or their promoters. In Cladosporium fulvum it was demonstrated that a single base mutation in the avirulence gene Avr4 led to virulence (Joosten et al., 1997), In Rynchosporium secalis, the virulence toward the barley lines carrying the Rrs1 resistance gene can be due to the loss of the complete avirulence gene Nip1 (Rohe et al., 1995). In Magnaportha grisea avirulence gene Avr-Pita, point mutations, insertions and deletions permit the fungus to overcome Pita resistance in rice (Orbach et al., 2000). The loss of avirulence in the pathogen is often associated with a penalty for the pathogen (Leach et al., 2001) and is explained by the assumption that besides the secondary function (avirulence) the gene may have a primary pathogenic function (Lauge and De Wit, 1998). The C. fulvum avirulence genes Ecp1 and Ecp2 are necessary for full virulence. However for other fungal Avr genes this was not observed (Rep, 2005). Knowledge about the primary function of Avr genes is important for development of strategies for durable resistances. In fact durability of resistance can be increased, by choosing and combining R genes that can only be overcome by a pathogen, whose loss of avirulence is linked to severe pathogen Wtness costs (Leach et al., 2001). Avirulence genes in several host–fungal pathogen systems have been cloned, principally by two methods: reverse genetics and map-based cloning. Five avirulence genes have been cloned by reverse genetics: Avr9, Avr4, Avr4E and Ecp2 counterpart of resistance genes Cf-9, Cf-4, Hcr-4E and Cf-Ecp2 in the tomato–C. fulvum system, and Nip1 gene of R. secalis causing avirulence on Rrs1 resistant barley (reviewed in Lauge and De Wit, 1998). Map-based cloning has been used to clone the seven fungal avirulence genes: M. grisea gene PWL2 (Sweigard et al., 1995), AVRPita (Orbach et al., 2000) and ACE1 (Bohnert et al., 2004); Ustilago hordei UhAvr1 (Linning et al., 2004); Melampsora lini AvrL567A and AvrL567B (Dodds et al., 2004) and Leptosphaeria maculans AvrLm1 (Gout et al., 2006). This method requires the construction of a genetic map with a high-number of markers mapping the region of interest. It also requires the construction of a genomic library allowing a chromosome landing or a chromosome walking in the region of interest to span the region of interest. ‘Golden Delicious’, an apple cultivar generally considered susceptible to apple scab, carries the ephemeral resistance gene Vg (Bénaouf and Parisi, 2000; Sierotzki and Gessler, 1998). Vg is a major resistance gene that has been overcome by some populations of V. inaequalis. Not surprisingly a monitoring study revealed that up to 87% of a pathogen collection was virulent on ‘Golden Delicious’, (Parisi et al., 2004). The Vg-gene was mapped by Calenge et al. (2004) on the distal end of linkage group 12. The

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counterpart of the resistance gene Vg in apple, is a single dominant avirulence gene, AvrVg, in V. inaequalis (Bénaouf and Parisi, 2000). The position of AvrVg has been identiWed on the V. inaequalis genome and several markers have been mapped in this region (Le Cam et al., 1999). SCAR markers VIRQ5 and SSR Vica 10/154 co-segregate with the AvrVg locus. whereas SSR Vitg 9/99 and SCAR P333 segregated on either side of the AvrVg locus (Le Cam unpublished data). The sequences of these markers are known and they are therefore a suitable starting point for chromosome walking. In this paper, we present the construction of a bacterial artiWcial chromosome (BAC) library of V. inaequalis, allowing an estimation of the genome size of V. inaequalis and the isolation of the region of AvrVg by chromosome walking. 2. Methods 2.1. Fungal strains and DNA extraction Venturia inaequalis isolates 301 (AvrVg¡) and 1066 (AvrVg+) were crossed at INRA Angers (Le Cam et al., 1999). The progenies of the cross were isolated (99 monoascosporic isolates) and grown on PDA (potato dextrose agar) plates at 20 °C. DNA was extracted following the protocol of Sierotzki et al. (1994) and diluted to a Wnal concentration of 1 ng/ul for PCR reactions. 2.2. BAC library construction The BAC library was constructed according to a procedure developed at the Texas A&M BAC center (Ren et al., 2005; Wu et al., 2004a; Wu et al., 2004b; Zhang, 2000). The mycelium of the V. inaequalis isolate 2.26 (AvrVg+) was grown as liquid cultures in PDB (potato dextrose broth) at 20 °C for four weeks. Nuclei were extracted according to Zhang (2000). Harvested nuclei were washed, re-suspended in the phosphate-buVered saline buVer (PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 2 mM KH2PO4), and brought the concentration of nuclei to 5– 10 £ 107 nuclei/ml. The nuclei suspensions was pre-warmed at 45 °C for 5 min in a water bath, mixed with an equal volume of 1% low-melting-point (LMP) agarose gel in PBS kept at 45 °C, and aliquoted in 100 l plug molds. The nuclei embedded in the LMP agarose plugs were lysed (Zhang et al., 1995), DNA puriWed in the agarose plugs and stored at 4 °C before use. To determine the desirable condition for partial digestion, each plug was cut into 12 slices, approximately equal in size, and incubated in 1 ml 1 £ reaction buVer (Gibco BRL, USA) containing 2 mM spermidine, 1 mM DTT (Sigma, USA), and 0.2 mg/ml BSA (New England Biolabs, USA) on ice for 2 h, with one buVer change after 1 h. Four slices of the plug were transferred into a 1.5 ml tube containing 100 l fresh reaction buVer plus 0.2–10 U BamHI. The reaction mixture was incubated on ice for another hour, then transferred into a 37 °C water bath and incubated for 8 min. The reaction was stopped

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immediately by adding 1/10 (v/v) of 0.5 M EDTA (pH 8.0) and analyzed by PFGE on a CHEF DRIII (Bio-Rad, USA) on a 1.0% (w/v) agarose gel at 6 V/cm, 11 °C, and 50 s pulse time for 18 h in 0.5 £ TBE (1 £ TBE: 89 mM Tris, 89 mM boric acid and 2 mM EDTA). The enzyme concentration that generated fragments with a majority ranging from 70 to 250 kb was selected for large-scale partial digestion. Large-scale partial digestion of HMW DNA for BAC library construction was carried out using Wve plugs and the BamHI concentration determined above (0.6 U/reaction). The plug slices containing partially digested DNA were subjected to a two-step size selection on a 1.0% (w/v) agarose gel at 6 V/cm, 11 °C, and 90 s pulse time for 18 h, followed by 4 V/cm, 11 °C, and 5 s pulse time for 6 h in 0.5 £ TBE. The gel fraction containing DNA fragments from 70 to 250 kb was excised. The DNA fragments were recovered from the gel by electroelution in dialysis tubing (12 000–14 000 Daltons molecular-weight exclusion, Gibco BRL, USA), using the CHEF DRIII at 6 V/cm, 11 °C, and 30 s pulse time for 4 h, followed by reversing the polarity for 90 s in 0.5 £ TBE. The eluted DNA fragments were dialyzed in the same tubing against 0.5 £ TE (5 mM Tris, 0.5 mM EDTA) for 3 h at 4 °C, with one 0.5 £ TE change per hour. The eluted DNA was collected carefully and measured in concentration by electrophoresis on a 0.8% (w/v) agarose gel and adjusted to 1–2 ng/l. One hundred microliters of the DNA fragments from the fraction were ligated into the cloning vector pECBAC1 at a molar ratio of 4 vector: 1 insert DNA using 5 U T4 DNA ligase (Gibco BRL, USA) at 16 °C for 12 h. Ligated DNA was transformed into Escherichia coli strain ElectroMAX DH10B competent cells (Gibco BRL, USA) by electroporation, using a Cell Porator System (Gibco BRL, USA). The setting conditions were 350 V, 330 F, low-ohms, and 4 k with fast charge. Transformed cells were transferred into 1 ml SOC medium and recovered at 37 °C for 1 h with gentle shaking. Recombinant transformants were selected on an LB agar (Gibco BRL, USA) plate containing 12.5 g/l chloramphenicol, 0.5 mM IPTG, and 50 g/ml X-gal. After a 32 h incubation at 37 °C, white colonies were randomly selected, and BAC DNA was isolated, digested with NotI, and subjected to size analysis by PFGE (Zhang, 2000; Zhang et al., 1996). The ligation that had a transformation eYciency of 200 or more white colonies/l ligation and that generated clones with the largest inserts was selected for library construction. White colonies were manually arrayed as individual clones in 384-well microtiter plates containing 50 l LB plus freezing broth with 12.5 mg/l chloramphenicol (Zhang, 2000; Zhang et al., 1996). After incubation at 37 °C for 14 h, the microtiter plates were stored at ¡80 °C. A GeneTAC G3 robotic workstation (Genomic Solutions Inc., USA) was used to spot the BAC library in double onto 8 £ 12 cm Hybond N+ Wlters (Amersham–Pharmacia, USA) in 3 £ 3 format, so that the high-density clone Wlters contained two spots of each clone from four 384-well microtiter plates (1536 £ 2 spots). The Wlters were processed according to Zhang et al. (1996) and Zhang (2000).

2.3. BAC DNA extraction BAC clones were grown in 100 ml Luria Broth cultures amended with 12.5 g chloramphenicol/ml and extracted using Qiagen Midi Plasmid extraction kit (QIAGEN GmbH, Germany) following the manufacturer’s protocol with the following modiWcations: for each solution P1, P2 and P3, 10 ml were used to produce the clear lysate. The column was equilibrated with 4 ml QBT solution and the plasmid eluted with 5 ml QF solution. After precipitation the plasmid was re-suspended in 300 l of ddH2O. No quantiWcation of the extracted plasmid was performed. 2.4. BamHI Wngerprints of BAC clones and estimation of the insert size For each sample 5 l DNA were digested with 1 U BamHI (New England Biolabs, USA), loaded on 0.8% agarose gels and run in 1 £ TBE (89 mM Tris, 89 mM boric acid, 2 mM EDTA). Gels were stained with ethidium bromide, photographed and blotted on Hybond N+ membranes (Amersham Biosciences, UK). To determine the insert size, 5 l of BAC-plasmid were digested with NotI (New England Biolabs, USA) digested and run on a PFGE using following conditions: 1 £ TBE, 1% agarose, 12.5 °C, 5–15 s switch time, 13 V/cm on a CHEF-DR II apparatus (Bio-Rad, USA). The insert sizes were determined using Lambda–DNA concatemers ladder (MidRange PFG Marker I, New England Biolabs, USA). 2.5. BAC end sequencing and mapping Sequencing reactions consisted of 4.5 l of extracted plasmid DNA, 1.5 l of 10 M primer T7 or Sp6long (Sp6 primer elongated at 3⬘ end by 4 bases 5⬘-ATTTAGGTGACACTATAGAATAC-3⬘) and 4 l BigDye Terminator Kit 3.1 (Applied Biosystems, USA). Ninety-nine cycles (30 s at 94 °C, 20 s at 50 °C and 4 min at 60 °C) were performed on a Gene Amp PCR System 9600 (Perkin Elmer Cetus, USA). Sequencing products were puriWed following the manufacturer’s protocol and loaded on an ABI PRISM®3100 Genetic Analyzer (Applied Biosystems, USA). PCR-primer pairs, with a Tm of 60 ° C, were then designed on the BAC-end sequences. PCR reactions were performed on a Gene Amp PCR System 9600 (Perkin Elmer Cetus, USA) in a volume of 15 l, containing 10 mM Tris–HCl pH 9.0, 1.5 mM MgCl2, 50 mM KCl, 0.1 mM dNTPs, 0.2 mM primers and 0.07 U/l Taq polymerase (New England Biolabs, USA) and 5 ng template DNA. PCR was carried out for 35 cycles of 94 °C for 30 s, 60 °C for 30 s and 72 °C for 1 min, with a Wnal extension of 10 min at 72 °C. If no length or presence/absence polymorphism was found between the two parents of the mapping cross, PCR products of both parents were sequenced, compared and searched for SNPs (single nucleotide polymorphisms) that were subsequently used to develop CAPS. Polymorphic markers were tested on the oVspring population to verify the progress of the chromosome walk.

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2.6. Chromosome walking strategy Chromosome walking was started by screening, by hybridization, the BAC library with two probes (Vitg9/99p and VIRQ5p) linked to the AvrVg gene. Clones hybridizing to one of these probes were tested by speciWc colony-PCR to verify the ampliWcation of the expected amplicon. BamHI Wngerprints of the clones conWrmed by PCR were hybridized with Vitg9/99p and VIRQ5p to conWrm their belonging to the contig under construction. The relative overlap of the BAC clones was assessed by comparing their BamHI Wngerprints. Insert-ends of all BAC clones were sequenced based on these sequences new PCR markers developed. Orientation of the clone extremities was assessed by testing the insert-end derived marker on all contig clones. The most protruding ends ampliWed only on the clones of origin. The markers developed on the most protruding ends of the contig were mapped; to conWrm the map position, to determine the direction of the chromosome walk and to assess the progress of the chromosome walk. Probes derived from markers developed on the protruding ends of the contig co-segregating with the AvrVg locus were used for the next chromosome walk-step. The walk continued until the markers derived from the two protruding ends no longer co-segregated with the AvrVg locus. 2.7. Hybridizations The BAC library Wlters were screened by radioactive hybridization as follows: nylon membranes were prehybridized overnight at 65 °C in hybridization buVer (0.5 M sodium phosphate pH 7.2, 7% SDS, 1% BSA, 1 mM EDTA and 0.2 mg/ml denatured salmon sperm DNA). Hybridization was performed overnight by adding 100 ng of radioactively labelled-32P (Nick Translation kit Invitrogen, UK) puriWed PCR product (Wizard SV Gel and PCR Clean-Up System, Promega, USA). Filters were then rinsed with 2 £ SSC (1 £ SSC: 0.15 M sodium chloride, 0.015 trisodium citrate) at room temperature and washed twice in 0.5 £ SSC, 0.1% SDS, once in 0.2 £ SSC, 0.1% SDS and once in 0.1 £ SSC, 0.1% SDS. All washes were conducted at 65 ° C for 20 min. The Wlters were exposed for 2–3 days to X-ray Wlm (Biomax MS, Kodak, USA) at ¡80 °C before development. The same procedure was used to perform the hybridization of the BamHI Wngerprints of the BAC clones. 3. Results 3.1. BAC library A BAC library composed of 7680 clones was constructed. The insert sizes, calculated on the basis of 40 BAC clones, ranged from 30 kb to 120 kb with an average size of 80 kb. Three probes VIRQ5p, Vitg9/99p and Vica10/154p (multi-locus), derived from the homonymous markers of the AvrVg region (Fig. 1(a)), were used to assess the number of haploid genome equivalents (identical to the average

47

number of positive clones per probe). The three probes hybridized to six, seven and eleven clones, respectively. The Wngerprints of the eleven clones that hybridized to Vica10/ 154p, allowed us to separate the clones into two groups of Wve and six clones. All Wve clones of the Wrst group additionally hybridized to the probe VIRQ5p suggesting that these clones are derived from the AvrVg region. Therefore six is the estimated number of haploid genome equivalents in the library. Multiplying the number of clones of BAC library (7680) by the average insert size (80 kb) and dividing by the number of haploid genome equivalents (6) the genome of V. inaequalis can be estimated to be about 102 Mb. 3.2. Chromosome walking The probes VIRQ5p and Vitg9/99p, derived from the homonymous markers, co-segregating and segregating from the locus AvrVg respectively, served as the starting point of the chromosome walking. Six clones hybridized to probe VIRQ5p (VIRQ-A1, VIRQ-A2, VIRQ-A3, VIRQA4, VIRQ-A5 and VIRQ-A6). VIRQ-A1 and VIRQ-A6 were found to be the only two clones required to cover the entire region isolated with VIRQ5 (clones VIRQ-A2, VIRQ-A3, VIRQ-A4 and VIRQ-A5 are sub-clones of the region spanned by VIRQ-A1 and VIRQ-A6). The ends of the BAC inserts, VIRQ-A1-Sp6 and VIRQ-A6-T7, were found to be the extremities of the contig. CAPS markers were developed on these ends and were mapped. Marker VIRQ-A1 Sp6 (digested by MaeIII) segregated from the avirulence locus, while marker VIRQ-A6-T7 (digested by BsaI) co-segregated with the AvrVg locus (Fig. 1(b)). Clones hybridizing with the probe Vitg9/99p did not overlap with the clone VIRQ-A1. Since the chromosome walk was oriented according to marker VIRQ-A1-Sp6, the clones hybridizing with the probe Vitg 9/99p are outside the region of interest and were not considered further. Two new BAC clones (VIRQ-B1 and VIRQ-B2) were isolated using VIRQ-A6-T7 as a probe. The new end of the contig was VIRQ-B2-Sp6. The mapping of the CAPS marker VIRQ-B2-Sp6 (digested by HaeIII) indicated that an additional step of chromosome walk was needed using the VIRQ-B2-Sp6 marker as probe (the CAPS marker co-segregates with AvrVg; Fig. 1(b)). In this step three BAC clones (VIRQ-C1, VIRQ-C2 and VIRQ-C3) hybridized to the probe VIRQ-B2-Sp6. The insert-ends of all three clones were sequenced. Comparison of these sequences with sequences from the BAC clones found in the previous steps (VIRQ-A6-T7 and VIRQ-B1-T7) allowed us to orient the extremities of these clones without developing markers. Clone extremity VIRQ-C1-Sp6 overlaps with VIRQ-B1-T7 by 400 bp. VIRQ-C3-T7 and VIRQ-C2-T7 are contiguous to VIRQ-B1-T7 as both lack the 400 bp fragment (Fig. 1(c)). Digestion by NotI showed that BAC clone VIRQ-C3 contained the largest insert (Fig. 2), suggesting that VIRQ-C3-Sp6 is the new protruding end of the contig. No polymorphisms were found among the parents of the

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G.A.L. Broggini et al. / Fungal Genetics and Biology 44 (2007) 44–51

Fig. 1. (a) Genetic map of the AvrVg region. Distances are expressed in number of recombinant individuals between two markers. Markers developed during the chromosome walking are shown in bold, all others were previously found to be linked to AvrVg by Le Cam et al. (1999). (b) BAC clone contig of the region of AvrVg. Arrows indicate the position of the markers developed from BAC end sequences. Pin heads indicate the four probes, used to screen the BAC library and to conWrm the correct overlap of clones. Dashed boxes identify the region of the contig where the corresponding probe hybridizes. The gray box shows the region of the contig displayed in Fig. 1(c). T D T7-end; S D Sp6-end of the BAC clone insert. (c) Alignment of the sequences of the extremities of the BAC clones VIRQ-A6-T7, VIRQ-B1-T7, VIRQ-C1-T7, VIRQ-C2-T7 and VIRQ-C3-T7. The comparison of the sequences obtained from BAC clone insert-ends allowed a fast orientation of the clone VIRQ-C1, -C2, and -C3 (see text).

mapping cross for the marker developed on BAC clone end VIRQ-C3-Sp6. We therefore used VIRQ-C3-Sp6 for an additional screening of the BAC library. The clone VIRQD1 was the only positive clone found. BAC end markers were developed and SCAR marker VIRQ-D1-T7 was mapped. The marker VIRQ-D1-T7 did segregate from the avirulence locus (Fig. 1(b)).

4. Discussion

3.3. Contig description

4.1. Genome size estimation

The contig spanning the region harboring AvrVg is covered by 12 clones (Fig. 3). The protruding end-derived markers VIRQ-A1-Sp6 and VIRQ-D1-T7 segregate from the AvrVg locus. The additional four markers developed on BAC insert-ends, all co-segregate with the avirulence locus AvrVg. The length of the AvrVg region spanned by the contig is about 330 kb. Five overlapping clones (VIRQ-A1, VIRQA6, VIRQ-B2, VIRQ-C3 and VIRQ-D1) comprise the minimal tiling path, spanning the complete region (Fig. 1(b)).

The estimated genome size of 102 Mb is about 2.5 times larger than the genome size reported for other ascomycetes. The genome size of Aspergillus nidulans (Galagan et al., 2005) was estimated to be 30 Mb, and that of M. grisea to be 42 Mb (Dean et al., 2005). Three probes (one of them hybridizing to two loci) all derived from the AvrVg region, were used for the estimation of the genome coverage. It is possible that the AvrVg region may be under represented in the BAC library. This may have led to an underestimation

A pioneering work has been performed for the genetics of Venturia inaequalis. For the Wrst time a BAC library of this fungus has been constructed, allowing an approximate estimation of its genome size and identiWcation of a chromosomal region spanning the avirulence locus AvrVg.

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Fig. 2. BAC-insert size estimation, by PFGE of Not I digestions of the BAC clones composing the contig spanning the region of AvrVg. The BAC clones are in same order as Fig. 1, which reXects the order of the clones in the contig. M: MidRange PFG Marker I, New England Biolabs. The sizes of the marker bands (in kb) are shown beside the corresponding band. The numbers above the bands indicate the estimated insert sizes in kb for the respective BAC clone.

of the whole chromosomes (Sun et al., 2001), re-association kinetics (Britten and Kohne, 1968; Couch et al., 1993), Xow cytometry (Vinogradov, 1994), real-time PCR based methods (Wilhelm et al., 2003) and the whole genome shotgun sequencing (Galagan et al., 2003, 2005). 4.2. Chromosome walking

Fig. 3. Comparison of BamH I Wngerprints of the 12 BAC clones composing the contig spanning the region of AvrVg. The BAC clones are in the same order as Figs. 1 and 2. M: Lambda-Hind III digest marker. The sizes of the marker-bands are indicated above the corresponding band.

of the genome equivalents and therefore an overestimation of the genome size. Under representation of BAC clones derived from the region of the avirulence gene AvrLm1 of Leptosphaeria maculans has been reported (Gout et al., 2006). As an alternative to the proposed method, the estimation of the V. inaequalis genome size could have been achieved by other methods like: pulse Weld electrophoresis

A four-step chromosome walk across the AvrVg region has been carried out resulting in a contig of BAC clones spanning 330 kb. Whole genome sequencing of several ascomycetes showed that the average gene density (expressed as average number of nucleotides per gene found) varies from 3600 nt/gene for Neurospora crassa (Galagan et al., 2003) to 2613 nt/gene for Aspergillus oryzae (Galagan et al., 2005). Considering these densities, the AvrVg region can be expected to contain up to 125 genes. However, indication of the presence of repetitive junk DNA within the AvrVg region is available. In fact some molecular markers, developed on sequences of BAC insertends containing non-coding sequences, ampliWed on other non-overlapping clones of the contig (data not shown). Therefore the predicted presence of 125 genes within the AvrVg region is probably an overestimation. In the region of the avirulence gene AvrLm1 of the ascomycetes Leptosphaeria maculans only 16 genes, clustered in two small regions and one standing alone, were found in a region of similar size (335 kb). Only by sequencing the whole region and analyzing the obtained sequences, can the eVective number of genes present in this region be determined. Regardless of the number of gene that could be identiWed in the AvrVg region, the potential number of candidates can be reduced by narrowing down the AvrVg region. This region is delimited by the markers, VIRQ-A1-Sp6 and VIRQ-D1-T7, segregating from the AvrVg gene. The reduction of the region can be achieved by producing polymorphic markers within the region delimited by the BAC clone

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extremities VIRQ-D1-T7 and VIRQ-B2-Sp6 (a region of about 100 kb) on one site of the contig and between the BAC clone extremities VIRQ-A1-Sp6 and VIRQ-A6-Sp6 (region of about 40 kb) on the other side. These are two pairs of markers segregating from and co-segregating with AvrVg on either side of the gene. First attempts in this direction have already been made, however with no success. No polymorphism among the parents of the cross was found on the extremities VIRQ-C1-T7, VIRQ-C2-Sp6, VIRQ-C3-Sp6 and VIRQ-D1-Sp6 on one side and VIRQA2-Sp6 and VIRQ-A3-Sp6 on the other side of the contig. To overcome this problem, we will sequence the whole 330 kb region and develop polymorphic markers within the intervals VIRQ-D1-T7/VIRQ-B2-Sp6 and VIRQ-A1-Sp6/ VIRQ-A6-Sp6. Additionally we will enlarge the mapping population, to increase the number of individuals showing recombination events in the AvrVg region. All open reading frames (ORFs) identiWed in the reduced AvrVg region, will be used in transformation experiments using one of the three methods available to transform V. inaequalis, such as, biolistic transformation (Parker et al., 1995), and the PEGor Agrobacterium-mediated methods (Fitzgerald et al., 2003). These techniques, which can be used for gene silencing (Fitzgerald et al., 2004) and complementation studies, will allow identiWcation of the AvrVg gene. Transformants could potentially be used to quantify Wtness penalties for silenced isolates. Acknowledgments We are grateful to Dr. Eve Silfverberg-Dilworth and to Rubik Sommerhalder for the critical reading of this manuscript. This work was supported by the SNF Grant No. 3100A0-100064/1. References Belfanti, E., Silfverberg-Dilworth, E., Tartarini, S., Patocchi, A., Barbieri, M., Zhu, J., Vinatzer, B.A., Gianfranceschi, L., Gessler, C., Sansavini, S., 2004. The HcrVf2 gene from a wild apple confers scab resistance to a transgenic cultivated variety. Proc. Natl. Acad. Sci. USA 101, 886–890. Bénaouf, G., Parisi, L., 2000. Genetics of host-pathogen relationships between Venturia inaequalis races 6 and 7 and Malus species. Phytopathology 90, 236–242. Bohnert, H.U., Fudal, I., Dioh, W., Tharreau, D., Notteghem, J.-L., Lebrun, M.-H., 2004. A putative polyketide synthase/peptide synthetase from Magnaporthe grisea signals pathogen attack to resistant rice. Plant Cell 16, 2499–2513. Britten, R.J., Kohne, D.E., 1968. Repeated Sequences in DNA. Science 161, 529. Bus, V.G.M., Rikkerink, E.H.A., van de Weg, W.E., Rusholme, R.L., Gardiner, S.E., Bassett, H.C.M., Kodde, L.P., Parisi, L., Laurens, F.N.D., Meulenbroek, E.J., Plummer, K.M., 2005. The Vh2 and Vh4 scab resistance genes in two diVerential hosts derived from Russian apple R12740-7A map to the same linkage group of apple. Mol. Breed. 15, 103–116. Calenge, F., Faure, A., Goerre, M., Gebhardt, C., Van de Weg, W.E., Parisi, L., Durel, C.E., 2004. Quantitative trait loci (QTL) analysis reveals both broad-spectrum and isolate-speciWc QTL for scab resistance in an apple progeny challenged with eight isolates of Venturia inaequalis. Phytopathology 94, 370–379.

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