Introduction

Melon (Cucumis melo L.) is an important horticultural fruit crop in tropical and subtropical regions, but it is also grown extensively in temperate zone countries. World production of cantaloupes and other melons in 2009 was about 26 million tons (www.fao.org). As many other crops, melon is susceptible to numerous foliar and root fungal pathogens that induce disease and reduce yield and fruit quality. Among these, Fusarium wilt is caused by a soil-borne pathogen, Fusarium oxysporum Schlechtend: Fr. f. sp. melonis (H.N. Hansen) W.C. Snyder & H.N. Hans (Fom). This fungus survives in the soil as chlamydospores, and is capable of colonizing crop residues and roots of most crops grown in rotation with melon (Gordon et al. 1989). Therefore, crop rotation has only provided limited protection against melon Fusarium wilt (MFW) disease (Crino et al. 2007).

Soil disinfection using various chemical products mainly methyl bromide (Cebolla et al. 2000) was a traditional practice to control Fom in greenhouses. Because of environmental and human health concerns (Brimner and Boland 2003), this fumigant was banned in industrialized countries. Developing countries have a different methyl bromide control schedule mandating a 20 % reduction in 2005 and total phase-out by 2015 (Gullino et al. 2003).

Soil solarization is another effective strategy to reduce soil inoculum and control wilt disease (Tamietti and Valentino 2006), but is not readily applicable for intensive vegetable farming systems, where time required to solarize the soil is very limited. Furthermore, soil solarization is often limited by local climate constraints such as temperature and relative humidity (Shlevin et al. 2004). Grafting of melons onto resistant rootstocks is also considered a promising practice to control soil-borne diseases in vegetables, particularly for MFW (Cohen et al. 2002; King et al. 2008). However, the added cost still limits its feasibility only to melon varieties with great economic value.

The use of resistant cultivars, therefore, is probably the most effective and practical means of controlling MFW. The success of breeding programs for MFW resistance is influenced by many factors, including: the nature of the pathogen and diversity of virulence in the population; availability, diversity and type of genetic resistance; or the effectiveness of methods and tools, such as molecular markers, used for assessing plant resistance.

The objective of this paper is to review the present state of knowledge on the MFW disease, the existing sources of genetic resistance, and the availability and usefulness of molecular markers and quantitative trait loci (QTL) linked to resistant to Fom. Such knowledge of is needed to develop resistant cultivars to this disease.

Pathogen and pathogenesis of MFW

Species, races and vegetative compatibility groups

Fusarium oxysporum Schlechtendahl emend. Snyder and Hansen is a cosmopolitan species (Booth 1971) comprising both pathogenic and nonpathogenic isolates (Gordon and Martyn 1997). The pathogenic isolates of F. oxysporum cause Fusarium wilt on several agricultural crops, and are accordingly subdivided into formae speciales (Snyder and Hansen 1940; Baayen 2000).

One of the economically more important and destructive f. speciales is the causal agent of MFW, F. oxysporum f. sp. melonis Snyder and Hansen (Leach and Currence 1938). The first report of MFW in New York was in 1930 (Chupp 1930), and it later has been found in many melon-growing areas worldwide, including America (Leach and Currence 1938), Europe, Asia (Quiot et al. 1979; Sherf and Macnab 1986), and Africa (Schreuder et al. 2000). The nature of the diversity comprised within Fom has direct bearing on the prospects for MFW control through genetic resistance; therefore a range of approaches are typically employed for the characterization of Fom isolates.

In general, to define genetic relationships within f. speciales, F. oxysporum strains have been grouped into vegetative compatibility groups (VCGs) based on their ability to form heterokaryons (Puhalla 1985). Strains that belong to the same VCG normally have identical alleles at their compatibility loci, enabling the exchange of nuclear material (Glass et al. 2000). In Fom, Jacobson and Gordon (1990a) identified eight VCGs: 0130, 0131, 0132, 0133, 0134, 0135, 0136 and 0137, in a worldwide collection of Fom isolates. Also, various DNA-based tools have been used to separate Fom into a number of clonal lineages that more or less correspond to their VCG grouping (Jacobson and Gordon 1990b; Namiki et al. 1998).

Currently, variation in virulence within a f. speciales has been categorized by assigning pathotypes to pathogenic races. Races are defined by their differential interaction with host genotypes (Armstrong and Armstrong 1978), which, in some cases, are cultivars known to carry one or more major genes for resistance. Fom isolates have been divided into four common races designated as 0, 1, 2 and 1,2 (Table 1), based on pathogenicity on three melon differential cultivars, ‘Charentais-T’, ‘Doublon’ and ‘CM-17187’ (Risser et al. 1976). The resistance genes effective against the respective races have been characterized in these differential cultivars: ‘Doublon’ and ‘CM-17187’ possess single dominant resistance genes, Fom-l and Fom-2, respectively; ‘Charentais-T’ has no known resistance gene. The race nomenclature corresponds to the resistance genes that are overcome (Risser et al. 1976). Race 1,2 overcomes these two resistance genes and is further divided into pathotype 1,2y, which induces leaf yellowing symptoms before the death of the plants, and 1,2w, which causes wilting and death without prior yellowing symptoms (Bouhot 1981). More recently, race 1,2 has spread through the world (Veloso et al. 2000; Perchepied and Pitrat 2004; Herman and Perl-Treves 2007). The spread of this race has become a problem for melon cultivation, and the development of genotypes with resistance to multiple races of the pathogen represents a major objective in melon breeding programs. Until recently, only a few cultivars (hybrids) tolerant to race 1,2 have been available commercially, and they are mainly used as rootstocks.

Table 1 Classification of Fusarium oxysporum f. sp. melonis races based on Risser et al. (1976)
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Origin of pathogenic races

In general terms, the appearance of a new Fom race, as might be evidenced by a previously resistant cultivar succumbing to the disease, could be explained in one of two ways: either the new race was introduced from another geographic area, or it originated locally. The latter scenario may be explained either by derivation from a preexisting race, through selection pressure for virulence by extensive spread of resistant cultivars, or by selecting from the local population of non-pathogenic strains of F. oxysporum. A good example of introduction from distant areas is Fom race1/VCG 0134, that is known to occur in Europe (Jacobson and Gordon 1990a), Central Asia (Mohammadi et al. 2004), North America (Jacobson and Gordon 1990a), and South Africa (Schreuder et al. 2000). Molecular markers, including mitochondrial (Jacobson and Gordon 1990b) and nuclear DNA haplotypes (Appel and Gordon 1995), confirm that VCG 0134 corresponds to a clonal lineage. Thus, identified infestations of race 1/VCG 0134 in California (Gwynne et al. 1997), Maryland (Jacobson and Gordon 1990a), New York (Zuniga and Zitter 1993), and South Africa are very likely the result of introduction from areas where the race was already established.

Although movement of strains as a result of human activities is clearly dominant in the establishment of new infestations of MFW, de novo origin of pathogenic forms is also possible. Several lines of evidence support this view. First, in these f. speciales, there is a complex relationship between pathogenic races and VCGs (or clonal lineages). That is, a given race may be associated with more than one VCG and some VCGs are associated with multiple races. For example, VCG 0134 of Fom is associated with all four known races. Furthermore, races 0, 1, and 1,2, are all associated with identical mtDNA (Jacobson and Gordon 1990b) and nuclear DNA haplotypes (Schroeder and Gordon 1993). The close relationship between these three different races may indicate that relatively simple genetic changes can lead to a change in cultivar specificity, i.e. one pathogenic race can give rise to another.

Factors such as co-evolution with the plant host and the spread of virulence determinants via processes such as parasexuality, heterokaryosis, and sexual recombination have also been implicated in the evolution of this pathogen (Gordon and Okamoto 1992; Guadet et al. 1989). Although parasexuality and heterokaryosis are known to occur in F. oxysporum (Beckman and Roberts 1995), sexual fruiting structures have never been observed in the species and only indirect evidence for sexual recombination has been detected (Snyder and Hansen 1940).

Molecular methods for Fom detection and quantification

Current methods used for Fom detection include visual observation of disease symptoms, fungal mycelia and spores (Baayen 2000), as well as isolation of the fungus using a selective culture medium (Komada 1975). Because the selective medium is not species- or strain-specific, proper identification can be time-consuming and non-definitive, and results are not immediately available due to the time required for fungal growth. Molecular methods based on polymerase chain reaction (PCR), such as real-time PCR, offer advantages over classical detection methods because they are sensitive, reliable, and quick (Schena et al. 2004).

More recently, López-Mondéjar et al. (2012) developed and validated a real-time PCR method that allowed detection and quantification of Fom in asymptomatic melon seedlings and in their organic substrates. This technique allowed a sensitive and rapid detection of Fom in melon plant material and substrate as soon as 48 h after inoculation, compared with 5–6 days required by the culture-dependent techniques.

At present, however, no molecular tools are available to discriminate among Fom races and therefore further work will be necessary to develop new DNA-based technology for this purpose. In tomato, assays for xylem-secreted effector-transcripts of F. oxysporum f. sp. lycopersici were shown to correctly diagnose the fungal race (Lievens et al. 2009); such effectors, however, are yet to be discovered in f. sp. melonis. More recently, 75 cDNA fragments with differential expression between the races 1 and 1,2 were identified (Sestili et al. 2011). Such transcripts could provide markers for race identification, but this would require further studies.

Epidemiology and defense responses

Fom infects the root system, wherein it progresses through the epidermis, cortex and endodermal tissues and penetrates the xylem vessels through the pits. From this stage on, the fungus uses the xylem for upward movement and establishment throughout the plant (Bishop and Cooper 1983). Whilst in the xylem, the mycelium sporulates, and microconidia are carried upwards by the xylem stream. At vessel ends, conidia germinate and the secondary mycelium penetrates the next vessel.

Plant infection by F. oxysporum is therefore a complex process that comprises several stages of host–pathogen interaction: recognition of the host roots and adsorption; penetration of hyphae through the different root tissues; penetration and progression in the xylem; and adaptation to the internal plant environment. To be successful, the fungus must overcome different plant defense responses at each stage (Beckman and Roberts 1995; Di Pietro et al. 2003; Michielse and Rep 2009). During the final stage of infection, the fungus secretes lytic enzymes and toxins that lead to disease symptoms, including necrotic lesions, chlorosis and wilting. Melon resistance sources have been identified and genetically characterized, but the defense mechanisms that confer resistance remain elusive.

The interaction and dynamics of Fom race 1,2 colonization in a susceptible melon cultivar ‘Ein Dor’ versus resistant melon line BIZ has been documented using green fluorescent protein (GFP) as a marker (Zvirin et al. 2010). At 1–2 days post-inoculation, the fungus grew on the root epidermis and adhered to epidermal cell borders. By day 4, the mycelium crossed the cortex and endodermis through narrow pores in cell walls and reached xylem vessels, where it sporulated and produced secondary hyphae that grew upwards. Race 1,2 colonized the resistant plant’s vascular system, but the incidence of seedling infection was lower than in susceptible ‘Ein Dor’, suggesting stronger defense responses in BIZ expressed at the pre-xylem stage of infection. Infection of the vascular system of BIZ was slower; at 11 days post-inoculation, race 1,2 only colonized the lower hypocotyl of BIZ, whilst the upper hypocotyls of ‘Ein Dor’ were already infected. Thus, Zvirin et al. (2010) established that resistant plants were not immune to the pathogen, but were able to quantitatively inhibit its progression by expressing an efficient defense response. They also demonstrated that transcript levels of phenylalanine ammonia lyase, chitinase and hydroperoxide lyase were induced to a greater extent in the resistant line.

Pretreatment of melon plants with dinitroaniline herbicides markedly increased their resistance to Fusarium wilt (Grinstein et al. 1976; Lotan-Pompan et al. 2007). Cohen et al. (1986) reported that the dinitroaniline herbicides trifluralin and dinitramine were the most effective for inducing resistance to Fom and suggested that reduction of wilt symptoms is associated with a reduction in ethylene production. In addition, higher glutathione levels following dinitroaniline treatment have been suggested to confer protection against Fom (Bolter et al. 1993).

Lotan-Pompan et al. (2007) identified, using suppression subtractive hybridization and cDNA-AFLP, seven genes whose expression is associated with resistance to Fom race 2 following trifluralin treatment. Furthermore, the authors demonstrated that expression of four stress-related and up-regulated genes was enhanced when the plants were subjected to salinity stress, suggesting that trifluralin induces a general stress response which protects the plant against Fusarium wilt.

Resistance to Fom races 0, 1 and 2

Sources of resistance to Fom races 0, 1 and 2

The Fom-1 gene, originally identified in melon cultivar ‘Doublon’, confers high-level of resistance to races 0 and 2, whereas the Fom-2 gene, originally identified in melon cultivar ‘CM17187’, confers high resistance to races 0 and 1 (Risser et al. 1976). To date, these genes have been extensively used in melon breeding and were already introduced to the majority of modern melon cultivars. However, it is desirable to have additional resistance sources available, because future adaptation of the pathogen could render specific resistance genes ineffective. Therefore, many studies searched for novel melon resistance sources to Fom (Zink 1983; Champaco et al. 1992; Pitrat et al. 1996; Álvarez et al. 2005). These studies revealed that, in the melon germplasm, resistance to Fom races 0, 1 and 2 is more frequent than that to Fom race 1,2. Resistance traits were found in melon accessions belonging to different botanical varieties of C. melo L. from different geographical origins, mostly areas of greater melon genetic diversity: the Iberian Peninsula, Far East and Middle East. Pitrat et al. (1996) screened an extensive collection of C. melo for resistance to Fom and found that 14.7 % of the 353 accession tested were resistant to both races 0 and 2, and 13.8 % were resistant to races 0 and 1. In another study, Álvarez et al. (2005) found new resistance sources to Fom races 0, 1 and 2 in a collection of 139 accessions from different geographical origins. Resistance to race 1 was less common than resistance to the other two races, since only four accessions showed race 1 resistance, whereas twelve were resistant to races 0 and 2.

Genetics of resistance to Fom races 0, 1 and 2

The first genetic studies on Fom resistance were carried out by Messiaen et al. (1962). They described Fom resistance among French ‘Cantaloupe Charentais’ genotypes and selected open pollinated cultivars homogeneous for resistance, such as ‘Doublon’ and ‘Védrantais’. This resistance was conditioned by a dominant gene called Fom-1 (Messiaen et al. 1962). Screening of the genetic resources led to the discovery of an independent dominant gene (Fom-2) in some accessions from the Far-East (CM 17187). This protection, however, was short-term, since it was overcome by novel Fom strains of race 1,2 (Risser et al. 1969).

Later, Zink and Gubler (1985) also described a dominant gene, Fom-3, as responsible for resistance to races 0 and 2 in melon line Perlita-FR. Fom-3 is tightly linked to Fom-1 and the two are suspected to be allelic. Risser (1987) affirmed that resistance in Perlita-FR is controlled by Fom-1 and the susceptible plants detected in the F2 generation of the cross Perlita-FR × ‘Doublon’, used to test for allelism, could result from residual segregation occurring in the Perlita-FR parent used by Zink and Gubler (1985).

Recently, Oumouloud et al. (2010) reported a new recessive gene (fom-4) that confers, together with Fom-1, resistance to races 0 and 2 in the Tortuga melon line. Similar to the other f. oxysporum F. speciales, that have exclusively asexual reproduction and little potential for gene flow, Fom presents only a low risk to overcome major resistance genes (McDonald and Linde 2002). However, this situation may change because of the extensive commercial cultivation of melons carrying the resistance controlled by Fom-1 and Fom-2.

The use of different resistance genes such as fom-4 could provide higher levels of resistance to Fom races 0 and 2; Tortuga may therefore constitute a new alternative source of resistance. In addition, resistance controlled by more than one gene might increase its durability (Khetarpal et al. 1998). Thus, better protection against race 2 could be achieved by combining two resistant genes, Fom-1 and fom-4. The combined use of the identified molecular markers linked to Fom-1 gene (Oumouloud et al. 2009) and the ones linked to fom-4 we are developing at this moment (Oumouloud et al. 2012) would be very useful in marker assisted selection for introducing the two resistance genes into the melon varieties.

Molecular markers linked to Fom-1 and Fom-2 genes

The expression of MFW symptoms following artificial inoculation is affected by the virulence of the pathogen isolates (Namiki et al. 1998), the genetic background of the plant (Mas et al. 1981), and environmental factors such as temperature and light intensity (Cohen et al. 1996; Burger et al. 2003). Furthermore, some genetically susceptible plants may escape wilting following standard inoculation. Escapes as well as ambiguous symptom expression in controlled inoculation tests result in selection of false negatives that reduce selection progress for resistance to MFW. Molecular markers linked to genes conferring resistance to Fom have the potential to reduce or eliminate this problem, by ensuring that the chromosome segment that carries the resistance gene is selected and maintained even when the inoculation test is not very reliable, or strictly on the basis of the presence of the marker without disease testing.

Inoculation with non-pathogenic Fom races may confer cross-protection (Mas et al. 1981; Alabouvette and Couteaudier 1992; Freeman et al. 2001). Thus, inoculation with one pathogenic race could impede subsequent selection of the same plant for resistance to an additional race in a sequential testing series. It was found, in fact, that melon plants that survived inoculation with race 1 did not show any disease symptoms following subsequent inoculation with race 2 (Chikh-Rouhou et al. 2006). Hence, molecular markers enable simultaneous selection of genes for resistance to two or more Fom races.

Development of molecular markers to the Fom-2 gene

The bulk segregant analysis (BSA) strategy, described by Michelmore et al. (1991), was used to detect random amplified polymorphic DNA (RAPD) markers linked to Fom-2 (Wechter et al. 1995). These fingerprint markers have been transformed to more stable and convenient single-locus ones, such as cleaved amplified polymorphic sequences (CAPS; Zheng et al. 1999), or sequence characterized amplified region (SCAR; Zheng and Wolf 2000).

Combining the BSA strategy with amplified fragment length polymorphism (AFLP) methodology, Wang et al. (2000) found some AFLP markers linked to the Fom-2 locus, and converted them into co-dominant SCAR markers, designated ‘AM’ and ‘FM’ (Table 2). The usefulness of these SCAR markers for indirect selection of Fom-2 was confirmed across 24 melon accessions from diverse race 1 resistant and susceptible origins. These and other markers are regularly used by melon breeders and served as starting points for map-based cloning of the Fom-2 gene (see below).

Table 2 Single-locus markers linked to Fom-2 gene
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Cloning and characterization of Fom-2 gene

The Fom-2 resistance gene was isolated by a map-based cloning strategy using a population derived from the cross ‘Vedrantais’ × PI 161375 (Joobeur et al. 2004). Two additional populations have been derived from two F7 recombinant inbreed lines (RILs) segregating for Fom-2. A BAC contig was built from the MR-1 library (Luo et al. 2001), and the sequencing of two overlapping partial BAC clones identified three candidate genes. Research efforts concentrated on one of these genes because of its high similarity to resistance genes of the NBS–LRR class (Joobeur et al. 2004).

The putative Fom-2 gene is 3 kb long and contains an uninterrupted open reading frame predicted to encode a 1,073 amino acid polypeptide that includes the different features of non-TIR NBS–LRR proteins. In contrast to most members of this class, no evidence of CC structure was found in the N terminus of the Fom-2 protein.

Additionally, Wang et al. (2011) described, using the Pfam software (http://pfam.sanger.ac.uk/), the detailed characteristics of this NBS–LRR protein. The protein harbours two significant Pfam-A match structures, an NB-ARC domain, and an LRR-1 domain. Another seven possible LRR-1s were observed at the C terminus of the Fom-2 protein, that conformed with the consensus motif LxxLxxLxxLxx (N/C/T)x(x)L observed in cytoplasmic R-gene products (Jones and Jones 1997). The Fom-2 protein has also one Sfi1 C (spindle body associated protein C-terminus) domain and an EAF (ELL-associated factor) domain.

Joobeur et al. (2004) revealed that the amino acid sequences from three susceptible cultivars (‘Védrantais’, ‘Ananas Yokneum’ and ‘Durango’) were identical to each other; however, when compared with the amino acid sequences deduced from resistant genotypes (MR-1 and PI 161375), 25 amino acids out of 541 were different. The sequences of the LRR fragment from the two resistant lines were identical, except of three nucleotides. These differences resulted in the substitution of the amino acid residues V and K in MR-1 by M and E in PI 161375, respectively. So far, however, functional validation of the Fom-2 gene by transgenic complementation/silencing of resistance, or by TILLING, has not been reported. A preliminary study using transgenic roots of composite melon plants reported the expression of the Fom-2 promoter fragment along the vascular tissues (Normantovich et al. 2012). Using the same system to express the Fom-2 coding sequence in a Fom-susceptible background resulted in partial resistance in most, but not all experiments (Normantovich et al. 2012).

Recently, Oumouloud et al. (2012) reported the cloning and sequencing of a partial LRR fragment of Fom-2 from 11 melon resistant accessions from various geographic regions. They identified three alleles of Fom-2 and their results revealed that the structure of the Fom-2 LRR domain is highly conserved, since 8 of the 11 resistant genotypes showed similar alleles to the resistant one characterized in the PI 161375 line. Conversely, PI 124111 was the only line that presented the same resistant allele previously described in MR-1. This could be explained by the ancestral relations between the two lines (Monforte et al. 2003), as the MR-1 breeding line was derived from PI 124111 (Thomas 1986). Finally, accession Cum-355 carried a novel resistance allele that differs from both PI 161375 and MR-1.

The information generated from the Fom-2 LRR region sequences allowed systematic development of “Functional Markers” that were developed based on the nucleotide polymorphisms detected between the susceptible and resistant Fom-2 alleles (Wang et al. 2011; Oumouloud et al. 2012). Such markers were first documented in plants by Andersen and Lubberstedt (2003) and have been recently developed for several cloned R-genes. They are highly predictive of phenotype as they target the functional (“causal”) polymorphism within a desired gene and overcome the problem of recombination between marker and trait.

In this context, Wang et al. (2011) reported two CAPS markers, representing allele-specific markers based on SNP in the LRR region of Fom-2 (Table 2). In a parallel study, Oumouloud et al. (2012) developed two simple and efficient SCARs, Fom2-R408 and Fom2-S342, that represent a pair of allele-specific markers (Table 2). The Fom2-R408 primers amplify a single band of 408 bp only in the resistant genotypes, whereas the Fom2-S342 primers amplify a 342 bp band only in the susceptible ones. The two primer pairs can be combined in a multiplex PCR reaction, providing together a co-dominant marker. Such SCARs resulted in good identification of 27 resistant genotypes representing several melon horticultural types, enhancing the reliability and cost effectiveness of marker assisted selection for the Fom-2 gene.

Molecular markers linked to Fom-1 gene

The first molecular markers linked to this gene, were reported by Brotman et al. (2002, 2005) using two recombinant inbred line populations developed at “Institut National de la Recherche Agronomique-France” (INRA). Based on BSA, Brotman et al. (2005) identified a RAPD marker that generated a 1,235-bp fragment linked to the Fom-1 gene in repulsion phase. This RAPD was converted to a CAPS marker designated as 62-CAPS (Table 3). This marker mapped at a distance of 0.7 and 6.3 cM from Fom-1 in the two populations, respectively. An additional marker, based on cloned resistance gene homologues (RGH), was designated NBS-1 (Brotman et al. 2005), transformed into a CAPS marker (Table 3), and mapped at 2.8 cM from Fom-1.

Table 3 DNA markers linked to Fom-1 gene
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Testing 62-CAPS and NBS1–CAPS in a set of 14 genotypes (seven resistant to Fom race 2 and seven susceptible) revealed a complex situation. The markers did not separate all the genotypes according to their resistance phenotype; instead, they seemed to group the accessions according to their horticultural or botanical groups, with group-specific marker haplotypes.

In a study that combined BSA and RAPD markers, Oumouloud et al. (2008) identified three molecular markers (B17649, V01578 and V061092) linked to the Fom-1 locus, using the F2 generation from the cross Charentais-Fom1 (Fom race 2 resistant) × TRG-1551 (susceptible). The polymorphic fragments were cloned, sequenced and converted to SCAR markers SB17645, SV01574 and SV061092 respectively (Table 3). The markers SB17645 and SV01574 amplified DNA fragments of 645 and 574 bp, respectively, in the resistant parent, while SV061092 amplified a 1,092 bp fragment only in the susceptible parent. Markers SB17645 and SV01574 were linked in coupling phase to Fom1, at 3.5 and 4 cM, respectively, whereas SV061092 was more distant (15.1 cM). The application of these three markers across 24 melon Fom race 2 resistant and susceptible accessions from diverse origins revealed different marker haplotypes and supported multiple, independent origins of this resistance trait. Nevertheless, since the markers are not very tightly linked to the gene, recombination could also explain cases of incorrect genotyping.

In a different study, four AFLP markers linked to Fom-1 (GTC/ATG-260, TCG/GGT-400, TAG/GCC-470 and GTG/ACC) have been identified using the BSA approach (Tezuka et al. 2009). These markers were mapped in 125 F2 individuals derived from the cross between line MR-1 (Fom race 2 resistant), and the susceptible Japanese line P11. Markers GTC/ATG-260 and TCG/GGT-400 were linked in coupling phase to Fom-1 at 0.5 and, 4.9 cM respectively, whereas TAG/GCC-470 was linked in repulsion to the resistant allele at 0 cM. The fourth marker, GTG/ACC, was considered co-dominant and mapped at 10.3 cM from Fom-1. TAG/GCC-470 was converted to a SCAR designated S-TAG/GCC-470 that amplified a single fragment (347 bp) only from P11 and the susceptible bulks (Table 3). Sequencing of regions flanking TCG/GGT-400 allowed the development of two CAPS markers, C-TCG/GGT-400 and CAPS2 (Table 3).

The usefulness of these DNA markers in determining the Fom-1 genotype was tested with several fixed lines and commercial F1 melon hybrids. The results, like the ones discussed above, differed among horticultural types. S-TAG/GCC-470, GTC/ATG-260 and C-TCG/GGT-400 corresponded well to the Fom-1 genotype in var. reticulatus and cantalupensis but not in vars. chinensis, conomon and makuwa.

Tezuka et al. (2011) developed a SCAR marker (S-MRGH9) and three CAPS markers (CAPS3, C-MRGH12 and C-MRGH13) based on the published BAC 31O16 sequence (Van Leeuwen et al. 2005), which contains a cluster of melon RGH and maps around the Fom-1 locus (Table 3).

These markers were mapped using the same F2 population (Tezuka et al. 2009), as well as a collection of 104 recombinant inbred lines derived from the cross MR-1 × P11. A set of 70 melon genotypes, that included 43 fixed lines and 27 commercial F1 hybrids were used to test the usefulness of the above markers. According to the authors, C-TCG/GGT-400 seems suitable for Fom-1 genotyping in var. cantalupensis, while CAPS2, NBS1-CAPS, C-MRGH12 and 62-CAPS are suitable, with few exceptions, for genotyping accessions from vars. chinensis, conomon and makuwa.

The variety-dependent usefulness of DNA markers might indicate that there is no linkage disequilibrium between Fom-1 and the markers, and crossing over has occurred in some cultivars and lines. Another explanation is that the R gene cluster around the Fom-1 locus is very polymorphic; the four alleles detected by C-MRGH13 (Tezuka et al. 2011) could also reflect such polymorphisms.

Characterization of Fom-1 gene

The Fom-1 gene was isolated by a map-based cloning strategy (Brotman et al. 2012). A backcross (‘Védrantais’ × PI 414723) × PI 414723 population of 1190 individuals was screened for recombination events within the interval delimited by markers NBS1 and 62-CAPS flanking the Fom-1 locus. In parallel, all BC1S1 families were inoculated with Fom race 2. The region was sequenced using libraries, and progenies displaying recombination events were genotyped by additional BAC-based markers. The results indicated that Fom-1 was separated by a single recombinant from marker RG9-2 (Table 3), residing in exon 2 of the NB-LRR homologue, RGH9. On its other side, Fom-1 was flanked by marker RG-G (Table 3) that maps between RGH8 and RGH9. This identified RGH9 as the Fom-1 gene. The sequence analysis revealed that this gene belongs to the TIR–NB–LRR type. Further studies will be required to elucidate the molecular polymorphism between resistant and susceptible alleles and provide functional validation of the Fom-1 gene action.

Resistance to Fom race 1,2

Sources of resistance to Fom race 1,2

As previously stated, many resources of high resistance to race 0, 1, and 2 have been reported, and incorporated into commercial melon cultivars; nevertheless, no gene that confers complete resistance to either race 1,2w or 1,2y has been reported, although partial resistance has been found.

The first report of melon resistance to Fom race 1,2 was recorded by Risser and Rode (1973) from accessions Ogon-9 and Piboule. Later, Pitrat et al. (1996) established that sources of resistance to race 1,2 are restricted to a few Far-Eastern accessions belonging to C. melo subsp. agrestis. When screening melon accessions from different geographical origins for resistance to race 1,2, they found that only 3 % of the 271 accessions tested showed some resistance, all of them from the Far East. The resistance to race 1,2 described in accessions Ogon-9 and Piboule allowed breeding of partially resistant lines, such as Isabelle (Risser and Rode 1973), two doubled-haploid lines, Nad-1 and Nad-2 (Ficcadenti et al. 2002), and some commercial hybrids.

Additionally, Hirai et al. (2002) selected two melon rootstocks cultivars ‘Dodai No. 2’ and ‘Dodai No.1’ that showed partial resistant to the race 1,2y. Also, Herman and Perl-Treves (2007) reported a near complete resistance to race 1,2 of Fom in the breeding line BIZ, and showed that BIZ definitely had a higher level of resistance than Isabelle. An F1 hybrid, ‘Adir’, derived by crossing BIZ with a non-resistant counterpart, displayed good field resistance to race 1,2. ‘Adir’ also was used as a rootstock for the susceptible melon cultivar ‘Ophir’, and provided good protection against MFW (Horev 2002).

High resistance levels to race 1,2 have been described in three Japanese melon accessions (Kogane Nashi Makuwa, C-211, and C-40), and useful levels in one Russian (C-160) and two Spanish (C-300 and Mollerusa-7) accessions (Oumouloud et al. 2009). Chikh-Rouhou et al. (2010) screened 110 melon accessions from diverse origins for resistance to race 1,2 and reported partial resistance to this race within a Portuguese accession BG-5384 belonging to var. cantalupensis. Matsumoto et al. (2011) found high-level of resistance to race 1,2y in 34 of 76 accessions from 7 of 11 wild Cucumis spp. Nevertheless, this high-level resistance to race 1,2y could not be introduced into melon cultivars, because of strong reproductive barriers between melon and the wild Cucumis spp.

Genetics of resistance to Fom races 1,2

Resistance to Fom race 1,2 is complex and appears to be under polygenic control. Perchepied and Pitrat (2004) reported the mode of inheritance of resistance to Fom race 1,2 in the partially resistant line Isabelle using a RIL population derived from an F1 hybrid between Isabelle, and the susceptible cultivar, ‘Védrantais’. The authors confirmed that resistance to race 1,2 in Isabelle is under polygenic control, and established that the heritability of resistance was high (0.72–0.96), and the minimum number of effective factors controlling the resistance ranged between 4 and 14. They also showed that some RILs were significantly more susceptible than ‘Védrantais’; this result may indicate transgressive segregation, which implies that the susceptible parent ‘Védrantais’, may also have alleles for resistance.

Herman and Perl-Treves (2007) studied the nature of Fom race 1,2 resistance in the F2 and backcross generations from the cross between BIZ and PI 414723. Segregation of the resistance response in these populations supported a model in which two complementary, recessive genes, designated fom-1,2a and fom-1,2b, are required to obtain full resistance. Recently, Chikh-Rouhou et al. (2011) determined the mode of inheritance of resistance to Fom race 1,2 in lines BG-5384, Shiro Uri Okayama, Kogane Nashi Makuwa and C-211. The F1, F2, and reciprocal backcross generations from the crosses between the above four resistant accessions and ‘Piel de Sapo’, a Fom race 1,2 susceptible melon, were analyzed by inoculation with isolates belonging to the 1,2y and 1,2w pathotypes. The distribution of the area under the disease progress curve (AUDPC) values in the F2 and BC generations after inoculation with both pathotypes of race 1,2 of Fom was continuous, suggesting quantitative inheritance of resistance. Broad-sense heritabilities ranged from 0.48 to 0.59. Such relatively moderate values could be explained by the existence of several factors involved in the resistance and the epistatic effects detected in this study. Additive, dominance, and epistatic effects were significant in all crosses, which indicates that the resistance is under complex genetic control in the four accessions. Additive effects were always positive and significant, regardless of the pathotype, indicating that it should be possible to increase resistance levels by accumulating resistance genes.

QTL mapping

Following the above reported inheritance study, Perchepied et al. (2005) performed QTL-analysis of the resistance to Fom 1,2 in breeding line Isabelle using the same 120 RIL population described above. They identified a total of nine QTL located on five linkage groups, most of them at linkage group ends, together explaining between 41.9 and 66.4 % of the total variation. Most of these QTL appear to be recessive. The resistant alleles of seven QTL originated from the partially resistant parent Isabelle, whereas resistance alleles of two QTL came from the susceptible parent, ‘Védrantais’. This agreed with the observed significant transgression towards susceptibility (Perchepied and Pitrat 2004).

Among the QTL, fomIII.1 and fomVI.1 were specific for the Y pathotype, while fomV.2 and fomXII.1 were only identified following inoculation with pathotype W strains. On the other hand, five QTL, fomIII.2, fomIII.3, fomV.1, fomXI.1, and fomXII.1, were effective against both pathotypes Y and W. These results suggested that partial resistance to Fom race 1,2 is governed by pathotype-shared loci, as well as by pathotype-specific loci.

QTL fomV.2 co-localized with the resistance genes Vat, which confers resistance to aphid colonization and virus transmission, and Pm-w for powdery mildew resistance, within a cluster of resistance gene homologs (RGH; Brotman et al. 2002; Garcia-Mas et al. 2001). QTL fomXI.1 co-localized with the resistance gene Fom-2 (marker AM), which confers resistance to Fom races 0 and 1, and with the RGH sequence NBS3 (Brotman et al. 2002). The Fom-2 gene itself has not been reported to contribute to race 1,2 resistance, therefore the loci implicated in resistance to Fom race 1,2 and races 0 and 1 may be different, but tightly linked. The presence of both quantitative and qualitative resistance genes in the same genomic regions suggests that QTL may correspond to allelic variation of qualitative resistance genes with intermediate phenotypes (Robertson 1989).

More recently, analysis of the melon genome sequence revealed that the resistance genes were organized in clusters. In particular, 79 R-genes were located within 19 genomic clusters, 16 with genes belonging to the same family. In the genomic interval of less than 135 kb, 7 TIR–NBS–LRR genes were clustered and located at the region harbouring the Fom-1 resistance gene (Garcia-Mas et al. 2012).

Herman et al. (2008) reported a preliminary QTL analysis for resistance to Fom race 1,2 in the breeding line BIZ using a set of 154 F3 families derived from the cross between this line and PI 414723. A major recessive QTL for resistance to Fom race 1,2 originated from Biz was detected in linkage group 2 (Gonzalo et al. 2005) that doesn’t harbour the QTL described by Perchepied et al. (2005). The authors hypothesized that BIZ and Isabelle might carry different loci for resistance and not just different alleles in similar loci.

Conclusion

Two dominant resistance genes, Fom-1 and Fom-2, control resistance to races 0 and 2, and 0 and 1, respectively. Many sources of resistance to these races have been found in several accessions belonging to different melon botanical varieties and the resistance was introduced to many melon commercial cultivars. Many molecular markers, linked to Fom-1 and Fom-2, potentially useful for marker-assisted selection have been identified. The Fom-2 gene was cloned and characterized, and SNP-based functional markers associated with resistance to race 1 were developed. These markers are expected to enhance the reliability and cost effectiveness of marker-assisted selection for the Fom-2 gene in melon.

Regarding the Fom-1 gene, several molecular markers linked to this gene have been reported; nevertheless, their usefulness was variety-dependent. The recent cloning of this gene, which encodes a TIR–NB–LRR protein, now paves the way to its full molecular characterization.

The new recessive gene fom-4, reported in Tortuga, should be studied in depth since it could prevent future breaking of resistance. Development of markers associated with fom-4 is under way, and will be used for pyramiding genes to further increase the durability of resistance to race 2.

Sources of high resistance to Fom race 1,2 predominantly come from Far Eastern accessions belonging to C. melo subsp. agrestis such as Ogon-9 or Kogani Nashi Makuwa. These accessions are organoleptically different from the cultivated melons. Resistance to race 1,2 is complex, and appears to be controlled by multiple recessive genes, except in breeding line BIZ, where it was reported to be controlled by two recessive genes. To date, QTL associated with resistance to race 1,2 have been reported only in the lines Isabelle and BIZ. The availability of the melon genome sequence (Garcia-Mas et al. 2012) and the use of newer technologies such as DNA microarrays will accelerate genome mapping and tagging of new QTL associated with resistance to race 1,2. These QTLs could be used to transfer the resistance into high yielding melon genotypes and combine different QTL with major resistance genes to the other races.