Introduction

Tomato powdery mildew caused by Oidium neolycopersici has recently been recognized as a worldwide emerging pathogen on tomato (Jones et al. 2001; Kiss et al. 2001). Several dominant genes have been characterized, which confer resistance to O. neolycopersici and originate from wild tomato species (Bai et al. 2005; Kozik 1993; Lindhout et al. 1994). The exploitation of these genes in plant breeding is limited due to linkage drag phenomena, consisting in unfavourable wild alleles closely linked to the resistance loci (Zeven et al. 1983; Young and Tanksley 1989). In addition, resistance conferred by the dominant Ol-genes is based on race-specific hypersensitive response (HR), thus is likely not durable. In contrast, the recessive gene ol-2, located around the centromere of chromosome 4 (De Giovanni et al. 2004), derives from the germplasm of the cultivated tomato species, Solanum lycopersicum (Ciccarese et al. 1998), and confers race-non-specific resistance via papilla formation, a non-HR-based mechanism (Bai et al. 2005).

The introduction of ol-2 resistance into tomato cultivars via traditional breeding methods is complicated by its recessive nature, as the selection of the ol-2 allele needs to be tested in the selfing progeny of each backcross generation. Although marker assisted selection (MAS) with co-dominant molecular markers would allow the breeder to perform the selection of the ol-2 allele in backcross generations, the low genetic variation within S. lycopersicum (Miller and Tanksley 1990) hindered the identification of polymorphic co-dominant markers linked to the resistance locus. So far, mainly amplified fragment length polymorphism (AFLP®) markers have been identified for the ol-2 region (De Giovanni et al. 2004; Ricciardi et al. 2007). In practical breeding, AFLP® markers cannot offer efficient MAS as they are dominant and require advanced equipment. Moreover, AFLP® markers are patented and thus not utilizable by most breeding companies. Although several efforts were made, the conversion of ol-2 linked AFLP® markers into PCR markers failed (Ricciardi et al. 2007).

Similarly to ol-2, the barley gene mlo (Büschges et al. 1997) is recessively inherited and confers durable resistance to barley powdery mildew (Blumeria graminis f. sp. hordei) through the apposition of papillae at the cell wall (Piffanelli et al. 2002). The similarity of ol-2 and mlo resistances for both genetic (recessive) and phytopathological (papilla-based mechanism) characteristics may indicate that ol-2 confers mlo-based durable resistance.

In the present work, several attempts were carried out in order to obtain new simple PCR co-dominant markers linked to ol-2, allowing selection of the resistance via MAS. Aiming at ol-2 positional cloning, a segregating F2 population of 316 individuals was analyzed to generate an integrated map of the ol-2 region, including all the dominant AFLP® markers reported so far. Bacterial artificial chromosome (BAC) library screening and Fluorescence in situ Hybridization (FISH) were used to investigate the possibility that ol-2 resistance is mediated by a mlo-based mechanism.

Materials and methods

Plant material

A F2 mapping population of 316 individuals was used, which was derived from a cross between the resistant F3 line R28 and the susceptible S. lycopersicum cv. Moneymaker (MM). The F3 line R28 (homozygous for the ol-2 allele) was developed from a cross between the resistant line LC-95 of S. lycopersicum var. cerasiforme and the susceptible S. lycopersicum cv. Super Marmande (SM) (Ciccarese et al. 1998).

Fungal material and disease test

The O. neolycopersici used in this study was collected from infected tomato plants in The Netherlands. One-month old plants were inoculated with a suspension of conidia as described by Bai et al. (2003). The inoculated plants were grown in a greenhouse compartment at 20 ± 2°C with 70 ± 15% relative humidity (RH) and day-length of 16 h. Disease evaluation was carried out twice, 12 and 15 days post-inoculation (dpi), on the basis of presence or absence of fungal sporulation.

Marker analysis

Total DNA was extracted from plant leaves 2 days before inoculation by using a rapid cetyl trimethyl ammonium bromide (CTAB) isolation method (Brugmans et al. 2003).

Seven simple sequence repeat (SSR) markers on tomato chromosome 4 were chosen among the ones published by Suliman-Pollatschek et al. (2002) (Table 1). PCR reactions (10 μl) contained 50 ng of genomic DNA, 1× PCR buffer, 0.5 pmol of each forward and reverse primer (one of which was labelled with IRD700 or IRD800), 0.2 mM dNTPs and 0.5 units of Taq-polymerase in demi water. PCR conditions were: 1 cycle at 94°C for 3 min followed by 30 cycles of 30 s at 94°C, 45 s at the optimal annealing temperature, 45 s at 72°C, and a final extension of 7 min at 72°C. Amplified products were scored on 6% polyacrylamide gel with a LICOR 4200 DNA sequencer and on 2.5% agarose gel stained with ethidium bromide.

Table 1 Markers tested to reveal polymorphisms between the parental lines Solanum lycopersicum cv. MM and R28
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The cleaved amplified polymorphic sequence (CAPS) marker U3-2HhaI (Table 1) was obtained by designing primers on the SCAU31500 sequence characterized amplified region (SCAR) sequence reported by De Giovanni et al. (2004) with the software Primer3 (Rozen and Skaletsky 2000) and digesting the amplification products with different restriction enzymes. PCR and digestion were carried out according to the conditions reported by Bai et al. (2003).

Nine markers with known sequence mapped on tomato chromosome 4 and two tomato expressed sequence tag (EST) sequences (cLEC-80-N18 and cTOC-20-K10) homologous to the barley mlo gene were selected from the Sol Genomics Database (http://www.sgn.cornell.edu, Table 1). Locus specific primers were designed to amplify genomic DNA as described by Bai et al. (2003). All the amplification products were subsequently digested with several restriction enzymes as described by Bai et al. (2004). AFLP® fingerprints of the ol-2 linked markers identified by De Giovanni et al. (2004) and Ricciardi et al. (2007) were generated on a LICOR 4200 DNA sequencer as described by Bai et al. (2003).

Linkage analysis

The software JoinMap 3.0 (Van Ooijen and Voorrips 2001) was used to generate an integrated linkage map of the ol-2 chromosomal region. A logarithm of odds (LOD) score of 4.0 was used as linkage threshold and the Kosambi mapping function was used to convert recombination frequency into genetic map distance.

BAC screening and fluorescence in situ hybridization

The HindIII BAC library of S. lycopersicum cv. Heinz 1706 (Budiman et al. 2000) was screened for the presence of the tomato mlo-homologous EST sequences cLEC-80-N18 and cTOC-20-K10 and the ol-2 linked SCAR sequence U3-2. The screening was performed by PCR with corresponding primers and conditions presented in Table 1.

FISH of the BACs 184L18 and 205G16 on pachytene complements was performed as described by Zhong et al. (1999) and Chang (2004). In brief: flower buds of tomato cv. Tiny Tim containing anthers at meiotic prophase I were fixed in ethanol–acetic acid (1:3). After digesting the cell walls with a pectolytic enzyme mix, cells were spread in acetic acid 45% and post-fixed in ethanol–acetic acid (3:1). BAC clones 184L18 and 205G16 were directly labelled with FITC-dUTP and Cy3.5-dCTP, respectively. The probes were hybridized on the cell complements together with excess of Cot100 genomic DNA for blocking repetitive DNA sequences. The slides were counterstained with DAPI and studied under a fluorescence microscope with a high-sensitive CCD camera. Image capturing and analysis followed the procedures as described by Chang (2004).

Results

Disease test

A disease test was performed on the 316 individuals of the F2 population of MM × R28. Ten MM and ten R28 plants were used as susceptible and resistant controls, respectively. Ten days post-inoculation all the MM plants showed powdery mildew sporulation on the upper side of the leaves, whereas no symptoms could be detected on R28 individuals. The F2 population was rated for resistance at 12 and 15 dpi. In total, there were 240 susceptible and 76 resistant F2 individuals, which fit into the expected 3:1 ratio for a single recessive gene model (χ2 = 0.152).

An integrated map for the chromosomal region harbouring ol-2

Aiming to fine-map the ol-2 gene, we designed primers for nine chromosome 4 specific sequences located in the region bearing ol-2 (Table 1). All the primer pairs amplified monomorphic fragments between MM and R28, which were then digested with different restriction enzymes. Unfortunately, no CAPS polymorphism was detected.

SSR markers have been reported to be powerful in detecting polymorphisms within S. lycopersicum. Therefore, we selected seven SSRs mapped on chromosome 4 and two of them, Tom316 and Tom332, revealed length polymorphisms between MM and R28 on 6% polyacrylamide gel. Polymorphism of the SSR marker Tom316 was also detectable on 2.5% agarose gel (Fig. 1).

Fig. 1
figure 1

SSR marker Tom316 tested on the susceptible (S) parent Moneymaker (MM), the resistant (R) parent R28 and eight F2 individuals. Co-dominant length polymorphism is revealed on both 6% polyacrylamide gel (a) and 2.5% agarose gels (b). Lane L (ladder) points the molecular weight (bp)

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All the ol-2 linked markers previously identified were used to search for polymorphisms between MM and R28. We failed to amplify the SCAU31500 SCAR sequence with the primers published by De Giovanni et al. (2004), thus new primers were designed. The amplification product, named U3-2, was subsequently digested and converted into a co-dominant CAPS marker, U3-2HhaI. Three out of the eight ol-2 linked AFLP® markers so far reported, E34M61-270, E40M56-90 and E41M32-390, showed polymorphism between MM and R28.

By screening the F2 population of MM and R28 with the three polymorphic AFLP® markers, the CAPS marker U3-2HhaI and the two SSR Tom316 and Tom332, a map of the ol-2 chromosomal region was constructed and integrated with the ol-2 maps reported by De Giovanni et al. (2004) and Ricciardi et al. (2007) (Fig. 2). Remarkably, almost identical segregation patterns were observed for nearly all the markers and the resistance, indicating that the ol-2 gene is located in a chromosomal region characterized by recombination suppression.

Fig. 2
figure 2

Integrated linkage map of the Ol-2 locus region on tomato chromosome 4. Genetic distances were estimated by using Kosambi’s mapping function

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Physical co-localisation of the ol-2 gene and tomato mlo-homologue(s)

As both the genetic and phytopathological features of the ol-2-associated resistance mimic the well-characterized mlo-based resistance in barley, it is logical to argue that ol-2 is likely a mlo homologue. By inspecting the tomato EST collection, two tomato ESTs (cLEC-80-N18 and cTOC-20-K10) were identified that are homologous to barley mlo. Unfortunately, genetic mapping of these two ESTs in the F2 population was hampered by the lack of polymorphism between MM and R28. To visualize the relative chromosomal location of the ol-2 gene and tomato mlo-homologue(s), we first screened the Heinz BAC library with PCR primers amplifying the tomato mlo-homologous ESTs cLEC-80-N18 and cTOC-20-K10 (Table 1). Two BAC clones, 184L18 and 205G16, were identified, which were positive for both the ESTs. These two BAC clones were further inspected for the presence of the ol-2 linked SCAR sequence U3-2. Remarkably, both BACs were positive for the U3-2 sequence, suggesting the co-localization of the ol-2 gene with mlo homologue(s). In addition to the expected PCR fragment of 1200 bp for the U3-2 sequence, other amplification products were obtained, indicating that the two BACs contain repetitive sequences.

FISH of 184L18 and 205G16 BACs on pachytene chromosomes of tomato revealed in both cases major signals on chromosome 4, in the heterochromatin block of the short arm near the centromere (Fig. 3). In spite of blocking the BAC probes with Cot100 repetitive DNA, we also found small signals in heterochromatic regions of other chromosomes, which indicate that the BACs are rich in pericentromeric heterochromatic specific dispersed repeats. Repeat signals were more abundant for 184L18 (Fig. 3). As the ol-2 gene is located on chromosome 4 around the centromere (De Giovanni et al. 2004), the presence of tomato mlo-homologue(s) in the pericentromeric heterochromatin of the same chromosome further suggests that ol-2 is a mlo-homologue.

Fig. 3
figure 3

FISH signals of the BAC clones 184L18 (labelled with FITC-dUTP) and 205G16 (labelled with Cy3.5-dCTP). The BACs display small and variable signals, close together, in the heterochromatic area near the centromere of chromosome 4. 205G16 gives a major focus next to the centromere and few minor signals more distally, whereas 184L18 produces a variable number of smaller foci in the whole pericentromeric heterochromatic region. Some of the green and red signals overlap giving yellow fluorescence. Both BACs also give some background signals in heterochromatin areas of other chromosomes, due to cross hybridization of the repetitive sequences in the labelled BAC probe

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Discussion

Although several powdery mildew resistance genes have been reported, most of the current commercial tomato cultivars are susceptible to powdery mildew. The ol-2 source of resistance, which originates from the cultivated tomato species S. lycopersicum, might allow circumvention of linkage drag problems which are encountered during the transfer of wild tomato resistance genes into cultivated backgrounds. On the other hand, the genetic relatedness between the donor parent and elite genotypes complicates the identification of polymorphic markers to be used for MAS breeding programmes. This was demonstrated by the failure in trying to convert several sequences located on the ol-2 chromosomal region into PCR markers.

Suliman-Pollatschek et al. (2002) reported percentages of polymorphism within S. lycopersicum were 52% for SSR, 10.8% for AFLP® and 5% for RFLP markers. The higher variation of SSR loci was demonstrated by the present study, in which two out of seven SSR markers tested revealed length polymorphisms between MM and R28.

The three markers identified in this work can be easily obtained (the SSR marker Tom316 requires a simple PCR reaction and visualization on agarose gel) and are not patented, thus can be freely and conveniently applied for MAS in breeding programmes. Furthermore, their co-dominant nature will allow breeders to select directly for the presence of the recessive ol-2 allele.

In this study, BAC library screening and FISH analyses were used to demonstrate the co-localization of an ol-2 linked marker with mlo orthologue(s) in the pericentromeric heterochromatic region of the short arm of tomato chromosome 4. Amplification of the U3-2 sequence by using BAC DNA as a template was invariably accompanied by the presence of multiple PCR amplification. However, when genomic DNA was used as PCR template, only one PCR fragment was observed. It seems that primer mismatches occur during PCR amplification only with the BAC DNA as template. Multiple PCR amplifications obtained by using the U3-2 primer pair suggests that the BACs contain repetitive sequences, which are known to occur with very high frequency in heterochromatic regions. The abundance of repetitive sequences also explains the presence of multiple weak hybridization signals obtained with FISH analyses. On the other hand, the presence of discrete PCR fragments with the expected molecular weight for the two-tomato mlo ESTs suggests the presence of only one intact mlo-like gene sequence. Although heterochromatin has been classically depicted as a gene poor region of the genome, our findings are in accordance with a growing body of evidence which shows that genes resident in heterochromatin can indeed encode essential functions (Nagaki et al. 2004; Rossi et al. 2007).

In tomato, the actual ratio of genetic and physical distance varies considerably depending on the chromosomal region. In the Tm-2a region, close to the centromere of chromosome 9, 1 cM corresponds to more than five million base pairs (Pillen et al. 1996), whereas for the chloronerva gene region, located in the euchromatin of chromosome 1, the ratio of genetic to physical distance is only 160 kb per 1 cM (Ling et al. 1999). By determining frequency and distribution of recombination nodules on tomato synaptonemal complexes, Sherman and Stack (1995) observed a much lower frequency of recombination nodules in heterochromatic regions around the centromeres compared to euchromatin. Occurrence of recombination suppression near the centromere is reported also for other plant species (Tanksley et al. 1992; Drouaud et al. 2006). Therefore, the observed clustering of ol-2 linked markers can well be explained by their localization in the pericentromeric heterochromatin of chromosome 4. The low ratio between genetic and physical distance does not favour ol-2 positional cloning, as it could require the screening of very large segregating populations and extensive chromosome walking.

In addition to the similarity of ol-2 and mlo resistances for both genetic (recessive) and phytopathological (papilla-based mechanism) characteristics, BAC screening and FISH data in this study indicated the genetic and cytogenetic co-localization of the ol-2 gene with tomato mlo-homologue(s). All of this evidence strongly suggests that ol-2 is likely a mlo homologue. We are currently following a homology-based cloning approach for ol-2 isolation as an alternative to positional cloning.