Characterization of the Fusarium wilt resistance Fom-2 gene in melon
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- Oumouloud, A., Mokhtari, M., Chikh-Rouhou, H. et al. Mol Breeding (2012) 30: 325. doi:10.1007/s11032-011-9622-6
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The melon gene Fom-2, which confers resistance to Fusarium oxysporum f.sp. melonis (Fom) races 0 and 1, has been previously characterized by map-based cloning, and it encodes a protein with a nucleotide binding site (NBS) and leucine-rich repeats (LRRs). Here, we used the primer Fom2-LRR1639 to clone and sequence a partial LRR region of the Fom-2 gene in 11 melon accessions resistant to Fusarium wilt from various geographic regions. Our work revealed that the structure of the partial LRR domain is highly conserved between eight of these resistant accessions and is similar to the resistant allele in the previously characterized PI-161375 line. Conversely, PI-124111 is a unique line that presents the same resistant allele that was previously described in the MR-1 line. The accession Cum-355 presents a protein that differs from that encoded by both the resistant lines PI-161375 and MR-1. This result suggests that Cum-355 has a new resistant allele of Fom-2 that determines the same specificity. Importantly, based on the sequence of the Fom-2 LRR domain, two sequence characterized amplified region (SCAR) markers, Fom2-R408 and Fom2-S342, were developed for Fom-2 resistant and susceptible alleles, respectively. These allele-specific PCR markers could be used as co-dominant markers when their primer pairs were combined in a multiplex PCR reaction. The specificity of these functional markers (FM) was validated on a set of 27 genotypes representing several melon types. These FM markers are expected to enhance the reliability and cost-effectiveness of marker-assisted selection for the Fom-2 gene in melon.
KeywordsFusarium oxysporum f. sp. melonis Race Allele Leucine-rich repeats Functional markers
Fusarium wilt, which is caused by the soil-borne fungus Fusarium oxysporum f.sp. melonis Snyder and Hans (Leach and Currence 1938) (Fom), causes important yield losses in melon (Cucumis melo L.) crops worldwide (Mas et al. 1981; Sherf and MacNab 1986; Zitter 1999). Four physiological races of Fom designated 0, 1, 2 and 1.2 have been identified based on their ability to differentially infect melon hosts (Risser et al. 1976).
The single dominant genes Fom-2 and Fom-1 confer resistance to races 0, 1 and 0, 2, respectively (Risser et al. 1976). A third gene, Fom-3, controls resistance to races 0 and 2 in cultivar Perlita-FR (Zink and Gubler 1985), though some controversy exists as to its possible allelism with Fom-1 (Risser 1987). Recently, Oumouloud et al. (2010) reported a new recessive gene, fom-4, in the Tortuga melon line that confers resistance to races 0 and 2. Fom isolates classified as race 1.2 are able to induce disease in melon lines carrying the resistance genes described above and were further divided into pathotype 1.2Y, which induces yellowing symptoms before the death of the plants, and pathotype 1.2W, which produces wilting and death without yellowing symptoms. Herman and Perl-Treves (2007) reported a near complete resistance to Fom race 1.2 in the melon breeding line BIZ, which has two complementary recessive genes.
Recently, several disease-resistance genes (R genes) that confer resistance to diverse pathogens have been cloned and their structures deduced from a wide range of plant species (Ingvardsen et al. 2008) using a combination of molecular tools and genetic maps, mainly map-based cloning or transposon tagging strategies (Dong et al. 2001; Yi et al. 2002; Martin et al. 2003). These R-gene products can be grouped into five classes based on their structural features (Dangl and Jones 2001). The most abundant class of genes encodes intracellular proteins (R proteins) that contain leucine-rich-repeat (LRR) and nucleotide-binding-site (NBS) domains and are likely located in the cytoplasm. NBS-LRRs can be divided into two classes based on the presence of a TIR domain (Toll and interleukin receptor-like sequence) or a coiled-coil motif (non-TIR) in their N-terminus.
In melon, the resistance gene Fom-2 was isolated by a map-based cloning strategy using the (Vedrantais × PI 161375) population (Joobeur et al. 2004). Two additional populations have been derived from two F7 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 have 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 the non-TIR domain of NBS-LRR R proteins. In contrast to most members of this class, no evidence of CC structure was found in the N terminal of the Fom-2 protein. More recently, Wang et al. (2011) revealed detailed features characteristics of this NBS-LRR R protein through Pfam analysis. They proposed two significant Pfam-A match structures, one of which is a NB-ARC domain, and the other a LRR-1 type. Another seven possible LRR-1s were observed at the C terminal of the Fom-2 protein that conformed with the consensus motif LxxLxxLxxLxx (N/C/T)x(x)L that is observed in cytoplasmic R-gene products (Jones and Jones 1997). In addition, the Fom-2 protein has one Sfi1 C (spindle body associated protein C-terminus) domain and EAF (ELL-associated factor) family.
Furthermore, nucleotide sequence analysis of the partial LRR domain of the Fom-2 gene revealed that the amino acid sequences from the susceptible cultivars (Védrantais, Ananas Yokneum and Durango) were identical to each other; however, when compared with the amino acid sequences deduced from the resistant genotypes (MR-1 and PI 161375) 25 amino acids out of 541 appeared to be different. The sequences of the LRR fragment from the resistant lines MR-1 and PI 161375 were identical with the exception 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 (Joobeur et al. 2004).
Sequence diversity of R genes has been studied in several different crops (Mondragon-Palomino et al. 2002). In Arabidopsis, Bakker et al. (2006) found that, in general, R genes have higher nucleotide diversity, a greater number of non-synonymous mutations and more recombination than the background genome. In L. perenne, one single nucleotide polymorphism (SNP) was found every 10 bp in the LRR region when comparing two sequences in the most divergent haplotypes (Xing et al. 2007). Genetic mechanisms such as point mutation, recombination and unequal crossing-over have all been suggested as potential causes for genetic variation within R genes.
Several alleles have been identified for some of the cloned resistance genes in different crops (Ingvardsen et al. 2008). In cases where disease susceptibility is due to a single mutation, functional markers (FMs), such as a SNP marker, might be developed based on the differences found (Andersen and Lubberstedt 2003). These FMs reside within the target genes themselves and can therefore be used with great reliability and efficiency to identify disease-resistant alleles in a breeding program. However, the usefulness of such markers might be limited until more alleles are sequenced. The objectives of this study were (1) to reveal additional resistant alleles of the Fom-2R gene in melon germplasm, and (2) to develop fast PCR-based FMs for Fom-2-mediated resistance against Fom races 0 and 1 with the aim of facilitating melon breeding programs.
Materials and methods
Melon accessions (Cucumis melo L.) used for the molecular analysis: their origin, horticultural type, and reaction to Fom race 1
Reaction to Fom race 1a
Piel de Sapo
Bola de Oro
C-87 × Ch-T
Cum-334 × Ch-T
Inoculation and DNA extraction
All accessions were inoculated with the isolate Fom0123, belonging to race 1 of Fom, using the inoculation method described in Oumouloud et al. (2010). For each accession, 20 plants were inoculated and maintained in a greenhouse at 28/20°C (day/night) for 3 weeks. The lines Charentais-Fom2, resistant to races 0 and 1, and Charentais-Fom-1, resistant to races 0 and 2, were used as resistant and susceptible controls, respectively. Moreover, 12 seedlings per accession remained as uninoculated controls. At the end of this period the presence of any Fusarium wilt symptoms was noted to classify each plant as either susceptible or resistant. Plants that died, or those showing any Fusarium wilt symptoms, were recorded as susceptible. Plants that did not show any Fusarium wilt symptoms at all were longitudinally cut and their vascular tissue examined; if it was browning the plant was recorded as susceptible, and if not it was noted as resistant.
Primer design and DNA amplification
Sequences of primers used for the genomic DNA amplification, their annealing temperatures (Tm) and the original sequence code
The primers Fom2-LRR1639 were designed to amplify the sequence of the partial LRR domain of Fom-2. Because specific sequences for disease resistance and disease susceptibility exist in this domain, one specific pair of primers (Fom2-R408) was designed for the resistance allele and another (Fom2-S342) for the susceptibility allele.
Genomic DNA was amplified using the same conditions as described in Oumouloud et al. (2008) except that the number of amplification cycles was reduced to 30 and the annealing temperature was optimized for each specific pair of primers (Table 2). Multiplex PCR was conducted in a 20-μl reaction containing 40 ng of genomic DNA, 20 mM of Tris–HCl (pH 8.4), 50 mM of KCl, 2 mM of MgCl2, 100 μM each of dATP, dGTP, dCTP and dTTP (Invitrogen, Carlsbad, CA, USA), 0.1 μM of each of the four primers and 0.6 units of Taq DNA Polymerase (Invitrogen). PCR reactions were performed in a thermocycler (model 9700; Perkin-Elmer Corp., Norwalk, CT, USA) as follows: pre-denaturation for 2 min at 94°C followed by 30 cycles of polymerization reaction each consisting of a denaturation step for 45 s at 94°C, an annealing step for 1 min at 62°C and an extension step for 2 min at 72°C. Finally, the samples were incubated for 5 min at 72°C.
Amplification products were separated by gel-electrophoresis on 2% agarose gels in 1 × TAE (90 mM Tris–Acetate and 2 mM EDTA, pH 8.0), stained with ethidium bromide at 50 ng/μl and visualized under UV light with an image analysis system (GelDoc2000, Bio-Rad, Hercules, CA, USA).
Cloning and sequencing of LRR fragments
Fragments amplified from individual plants of the 10 resistant accessions and C-211 were precipitated with ammonium acetate following the protocol detailed in Oumouloud et al. (2008) and diluted in sterile water. Three purified LRR fragments per accession were cloned into the plasmid pGEM-T Easy (Promega Corporation, Madison, WI, USA). JM109 High Efficiency Competent Cells (Promega) were transformed with these plasmid vectors (Sambrook et al. 1989), and the plasmids were purified using QIAprep kits (Qiagen). Sequencing of the cloned fragments was conducted by the Secugen S.L. (Centro de Investigaciones Biológicas, CSIC, Madrid, Spain).
For each accession, the consensus nucleotide sequence was obtained by aligning partially overlapping sequences derived from the three amplified samples. Nucleotide sequence analysis and translation to the corresponding amino acid sequence were performed using the BioEdit program ver. 5.0.6 (Hall 1999). To detect specific resistance alleles, the LRR sequences of the 10 accessions were compared to the sequences of the resistant and susceptible melon lines (Joobeur et al. 2004). Similar searches were performed with the BLAST program on the National Centre for Biotechnology Information (NCBI) website (www.nbci.nlm.nih.gov/BLAST).
Identification of Fusarium wilt resistance in melon
Typical symptoms of Fusarium wilt were observed in all inoculated plants of the Charentais-Fom1 line as early as 7 days after inoculation; 21 days after inoculation all these plants had died. However, all inoculated plants of the Charentais-Fom2 line proved to be resistant to Fom race 1. These results indicate that no susceptible seedling escaped detection in our tests.
The primers Fom2-LRR1639 amplified a single band with the expected size (1,639 bp) in all resistant accessions except C-211. The fragment generated from this accession has an estimated size of 2,711 bp.
Searches of the GenBank database using the BLASTN algorithm revealed that the accession C-211 presented the same allele as the susceptible breeding line BL. When the nucleotide sequence of the LRR fragment from the accession C-211 was compared with the resistant lines PI-161375 and MR-1, the two sequences were essentially identical for the first 1,017 bp (94% similarity) and for the last 553 bp (97% similarity). However, the middle region clearly differed from that of the two previously characterized resistant lines (only 86% similarity). Moreover, the deduced amino acid sequence of the C-211 accession revealed a clear difference from that of MR-1 and PI-161375.
Development and application of allele-specific PCR markers
The resistant and susceptible alleles of Fom-2 were also reliably identified using a multiplex PCR amplification protocol in which the two primer pairs described above were used in a single reaction. This technique was shown to be able to detect the two different fragments, corresponding to disease resistance or susceptibility, in both heterozygote accessions and hybrid crosses. Validation using a collection of 27 genotypes showed that the SCARs are 100% predictive of the presence or absence of the Fom-2 gene. Among the 27 tested genotypes, 13 were identified as being Fusarium wilt resistant, including 10 homozygous and three heterozygous genotypes (Fig. 4). Our results from the screening of 27 melon accessions clearly demonstrate the usefulness of these allele-specific SCARs in marker-assisted selection of Fom-susceptible and -resistant Fom-2R gene alleles, with important implications for breeding strategies to introduce resistant Fom-2R gene alleles into melon cultivars susceptible to races 0 and 1 of Fom.
Fom, which causes Fusarium wilt in melons, is considered one of the least controllable pathogens because, once the soil is infested with it, this fungus can persist by colonizing non-susceptible hosts and by producing durable chlamydospores (Banihashemi and DeZeeuw 1975; Schippers and Van Eck 1981). An effective primary control measure for this pathogen involves the use of resistant melon cultivars (Martyn and Gordon 1996). Because these cultivars are generally not effective against all races of Fom, a common breeding strategy has been to pyramid several R genes into one cultivar.
Here, we reported the cloning and sequencing of a partial LRR region of the gene Fom-2 in 11 resistant melon accessions from various geographic regions. This was accomplished using the primers Fom2-LRR1639, which we developed from the previously sequenced LRR region of Fom-2 and designed to amplify the sequence of the partial LRR domain of that gene.
We have identified three alleles of the Fom-2R gene (Figs. 1, 2). Our results revealed that the structure of the partial LRR domain is highly conserved in eight of the tested genotypes (C-40, C-41, C-87, Cum-241, Cum-334, Cum-190, Charentais-Fom2, and Kirkagaç) and is similar to the resistant allele characterized in the PI-161375 line. The PI-124111 accession is the only genotype we tested that presents the same resistant allele as that described in the MR-1 melon line. This result could be explained by the ancestral ties existing between the PI-124111 and MR-1 lines (Monforte et al. 2003), as the MR-1 line is considered a muskmelon breeding line derived from an inbred line of PI-124111 (Thomas 1986). Finally, the accession Cum-355 expresses a protein that differs from that of both the resistant lines PI-161375 and MR-1 (Fig. 2). This result suggests that the Cum-355 accession would have a new resistant allele of Fom-2 that determines the same specificity. The resistant alleles described here provide the opportunity to improve the durability of resistance to Fom race 2 by combining different Fom-2 resistance alleles within a single melon genotype.
The accession C-211 was previously shown to exhibit a high level of resistance to Fom race 1.2 (Oumouloud et al. 2009). Resistance to race 1.2 seems to be quantitative and polygenically inherited, and this type of resistance is usually thought to be race non-specific. In fact, Risser and Rode (1973) have described, in the accessions Kogani Nashi Makuwa and Ogon 9, a race non-specific resistance to race 1.2 that was also effective against races 0, 1 and 2. Also, the F1 hybrid line Adir, derived from the race 1.2 resistant line Biz, was shown to be resistant to all four races of Fom (Herman and Perl-Treves 2007). Moreover, Perchepied et al. (2005) co-localized the quantitative trait locus (QTL) fomX1.1, which confers resistance to race 1.2, with the gene Fom-2. The presence of both quantitative and qualitative resistance genes in the genomic region of a single melon line suggests that the C-211 line could present the Fom-2 susceptible allele, like the breeding line BL, and the resistance to race 1.2 seen in this accession could be mediated by the QTL fomX1.1.
Taken together, these results indicate that Fom-2 may belong to an R-gene group that shows low levels of polymorphism. Similar results have been reported for other R genes including Pi-d2 (Chen et al. 2006), Cm-eIF4E (Nieto et al. 2006), RB (Song et al. 2003) and Xa26/Xa3 (Xiang et al. 2006), where only two alleles, a resistant and a susceptible allele, have been identified.
To date, marker-assisted selection for Fom-2 has involved markers that are only linked to this resistance gene within the genetic background they were developed in, and which risk being separated from the trait by recombination (Wang et al. 2000; Burger et al. 2003; Oumouloud et al. 2004). Importantly, here, we present two simple and efficient SCAR and AS-PCR markers, Fom2-R408 and Fom2-S342. The AS-PCR primer pairs can be combined in a multiplex PCR reaction because they have similar annealing temperatures but major differences in the size of the amplified fragments (Fig. 4); hence, these SCARs can be used as co-dominant markers. Both SCARs were developed based on the functional nucleotide polymorphisms detected between the susceptible and resistant Fom-2 alleles, and they are defined as functional markers (FMs). Such markers were first documented in plants by Andersen and Lubberstedt (2003) and have been recently developed for several cloned R genes. The FMs are highly predictive of phenotype as they target the functional polymorphism within a desired gene and overcome the problem of recombination/linkage.
Recently, Wang et al. (2011) reported two cleaved amplified polymorphic sequence (CAPS) markers and AS-PCR markers based on SNPs at the LRR region of the Fom-2 gene. To confirm the applicability of these markers, 34 melon cultivars were genotyped with CAPS markers (CAPS-3F and CAPS-3R) and Xba I. Their results showed a perfect correlation between the CAPS marker genotype and the observed phenotype. From a practical point of view, these CAPS marker assays require both PCR amplification and restriction enzyme digestion, and this extra step could be a disadvantage in a large-scale breeding program. The co-dominant SCAR markers reported here are simpler to analyze, and they resulted in good identification of the genotypes tested. From a sample of 27 melon genotypes, we identified three heterozygous and 24 homozygous genotypes, and resistant accessions were either homozygous or heterozygous genotypes (Fig. 4). These germplasms can be efficiently utilized in melon wilt resistance breeding programs where resistance can be determined in crosses between homozygous resistant and susceptible germplasms.
This study was funded by the grant AGL2008-05687-C02-02 from the Spanish Ministry of Education and Science (MEC). A. Oumouloud acknowledges a fellowship from the IAECI (Spanish Foreign Affairs Ministry).