Brazil produced 330,000 metric tons of melons in 2005, principally in the Northeast region where one of the most important melon pathogens is the powdery mildew fungus Podosphaera xanthii. The disease is controlled mainly by incorporating single dominant resistance genes into commercial hybrids. We report on linkage analysis of the Pm-1 resistance gene, introgressed from the AF125Pm-1 Cantalupensis Charentais-type breeding line into the yellow-fleshed melon (Group Inodorus) breeding line AF426-S by backcrossing to produce the resistant line AF426-R, and the amplified fragment length polymorphism (AFLP) marker M75/H35_155 reported to be polymorphic between AF426-S and AF426-R. Segregation analysis of M75/H35_155 using a backcross population of 143 plants derived from [AF426-R x AF426-S] x AF426-S and screened for resistance to P. xanthii race 1 produced a recombination frequency of 4.9%, indicating close linkage between M75/H35_155 and Pm-1. Using the same segregating population, the M75/H35_155 marker had previously been reported to be distantly linked to Prv¹, a gene conferring resistance to papaya ringspot virus-type W. Since M75/H35_155 is linked to Prv¹ at a distance of 40.9 cM it is possible that Pm-1 and Prv¹ are also linked.
AFLP marker; Cucumis melo; disease resistance; linkage analysis; powdery mildew
An AFLP marker linked to the Pm-1 gene that confers resistance to Podosphaera xanthii race 1 in Cucumis melo
Ana Paula Matoso Teixeira; Fatima Aparecida da Silva Barreto; Luis Eduardo Aranha Camargo
Departamento de Entomologia, Fitopatologia e Zoologia Agrícola, Escola Superior de Agricultura Luiz de Queiroz, Universidade de São Paulo, Piracicaba, São Paulo, Brazil
Send correspondence to Send correspondence to: Luis Eduardo Aranha Camargo Departamento de Entomologia , Fitopatologia e Zoologia Agrícola, Escola Superior de Agricultura Luiz de Queiroz, Universidade de São Paulo Av. Pádua Dias 11 13418-900 Piracicaba , SP, Brazil E-mail: email@example.com
Brazil produced 330,000 metric tons of melons in 2005, principally in the Northeast region where one of the most important melon pathogens is the powdery mildew fungus Podosphaera xanthii. The disease is controlled mainly by incorporating single dominant resistance genes into commercial hybrids. We report on linkage analysis of the Pm-1 resistance gene, introgressed from the AF125Pm-1 Cantalupensis Charentais-type breeding line into the yellow-fleshed melon (Group Inodorus) breeding line AF426-S by backcrossing to produce the resistant line AF426-R, and the amplified fragment length polymorphism (AFLP) marker M75/H35_155 reported to be polymorphic between AF426-S and AF426-R. Segregation analysis of M75/H35_155 using a backcross population of 143 plants derived from [AF426-R x AF426-S] x AF426-S and screened for resistance to P. xanthii race 1 produced a recombination frequency of 4.9%, indicating close linkage between M75/H35_155 and Pm-1. Using the same segregating population, the M75/H35_155 marker had previously been reported to be distantly linked to Prv1, a gene conferring resistance to papaya ringspot virus-type W. Since M75/H35_155 is linked to Prv1 at a distance of 40.9 cM it is possible that Pm-1 and Prv1 are also linked.
Key words: AFLP marker, Cucumis melo, disease resistance, linkage analysis, powdery mildew.
Melon (Cucumis melo L, Cucurbitales, Cucurbitaceae) is of great economic importance in Brazil where about 330 thousand metric tons are produced per year (FNP, 2005), principally in the Northeast region due to the adoption of advanced technology and the favorable climate of this region which allows melons to be grown practically throughout the year. This region benefits from the fact that the harvest occurs during September through March during the off-season in Spain, the most important European producer. More than half the melon yield from the Brazilian Northeast is destined for export, with about U$ 108 million US dollars worth of melons, equivalent to over 204 thousand tons of fruit, having been exported in 2007 (Portal do Agronegócio, 2007).
The fungus Podosphaera xanthii (Castagne) U. Braun & Shishkoff (2000) (Erysiphales, Erysiphaceae) is the causal agent of powdery mildew in Brazil. The first report of the destructive effects of this pathogen in melon crops was in 1925 in California (Jahn et al., 2002). This pathogen can infect plants in protected crops as well as in the field, with high temperature and low humidity increasing the intensity of infection. In Northeast Brazil this disease is more frequent at the end of the vegetative cycle, causing additional losses by shortening the harvesting time and affecting the quality of late fruits due to intensive exposure to sunlight as a consequence of defoliation (Rego, 1995, Viana et al., 2001).
All resistance genes to P. xanthii present in American, European and Japanese melon varieties derive from germplasm collected in India, which is one of the centers of origin of melons (Pitrat et al., 1998, cited by Jahn et al., 2002). According to Pitrat (1998), there are 18 sources of resistance genes against P. xanthii, and 10 resistance genes described in the literature that differ from each other not only according to their origin but also in their specificity relative to the pathogen race and mode of action (Pitrat, 2002). The Pm-1 gene from PI78374 confers resistance to P. xanthii race 1 and was introduced into the Cinco cultivar (Thomas and Webb, 1982). It is a dominant-resistance gene commonly used in melon breeding programs aimed at obtaining varieties adapted to the Brazilian Northeast, in view of the occurrence of P. xanthii race 1 in that region (Reis and Buso, 2004).
Molecular markers linked to resistance genes are commonly used in marker-assisted breeding, especially when the trait under consideration is controlled by a single gene. In melon, for example, there have been reports of linkage between molecular markers and resistance genes to Fusarium oxysporum f. sp. melonis (Wechter and Dean, 1998; Karsies et al., 2000; Brotman et al., 2005), zucchini yellow mosaic virus (ZYMV: Danin-Poleg et al., 2002), melon necrotic spot virus (MNSV: Baudracco-Arnas and Pitrat, 1996), cucumber mosaic virus (CMV; Dogimont et al., 2000), and papaya ringspot virus-W (PRSV-W: Brotman et al., 2005; Teixeira and Camargo, 2006). In addition, two candidate resistance genes have been identified by positional cloning, one conferring resistance to race 2 of F. oxysporum f. sp. melonis (Joobeur et al., 2004) and another conferring resistance to MNSV (Nieto et al., 2006).
Although several studies exist on resistance genes against melon powdery mildew, there are few published studies dealing with linkage analyses. Working with the melon line WMR29, Pitrat (1991) reported linkages among a resistance gene to races 1 and 2 of P. xanthii, the Fn (flaccida necrosis) gene, that confers resistance to ZYMV strain F, and the Vat gene, that confers resistance to virus transmission by the aphid Aphis gossypii. Pitrat (1991) also described linkage between a resistance gene to race 2 of P. xanthii in the C. melo line PI 414723 and the morphological marker "halo cotyledons". Finally, Perchepied et al. (2005) mapped one major quantitative trait loci (QTL) conferring resistance to P. xanthii race 3 and another to race 5. Since the allelic relationships of powdery mildew resistance genes are not clear, it is not known if one of these QTL corresponds to the Pm-1 gene.
We conducted a linkage analysis between the C. melo amplified fragment length polymorphism (AFLP) marker M75/H35_155 and the Pm-1 resistance gene using a segregating population from a cross between two near-isogenic C. melo lines differing from each other not only with regard to the presence of Pm-1 but also with respect to the presence of Prv1, a resistance gene to PRSV-W (Teixeira and Camargo, 2006). The M75/H35_155 marker was previously identified in this C. melo population by Teixeira and Camargo (2006) as being distantly linked (40.9 cM) to the Prv1 gene. The present work tested the linkage hypothesis between M75/H35_155 and Pm-1 due to the highly-isogenic nature of the parental lines.
The resistant C. melo line AF125Pm-1 was used as the donor of Pm-1. This line is a Cantalupensis Charentais type, derived from the self-pollination of C. melo line Cinco which has resistance genes to several pathogens, including the P. xanthii resistance gene Pm-1 and the PRSV-W resistance gene Prv1. We introduced the Pm-1 gene into the susceptible (S) recurrent C. melo line AF426-S during six backcrosses to produce the resistant (R) line AF426-R, thus lines AF426-S and AF426-R are near-isogenic lines (NILs). A backcross population of 143 plants (BC1F1) resulting from the [AF426-S x AF426-R] x AF426-S cross was used for segregation analysis between the M75/H35_155 AFLP marker and the Pm-1 gene.
To analyze the reaction of BC1F1 plants to P. xanthii race 1, plants were grown in a growth chamber at 25 ºC to 30 ºC with a relative humidity varying from 80 to 90% and a 12 h photoperiod provided by 20W daylight type fluorescent lamps (Sylvania, Brazil) providing 18.000 to 20.000 lux. To assess susceptibility to P. xanthii race 1 we collected leaf samples from each plant 26 days after emergence. The samples were disinfested with 70% (v/v) alcohol for a few seconds, rinsed with distilled water and then four 1.5 cm diameter leaf-disks were cut from each leaf and placed in petri dishes. With the aid of an eyelash glued to a pasteur pipette, the leaf disks were inoculated by transferring a small number of conidia and conidiophores of isolate R1 of P. xanthii (provided by Sakata Seeds Sudamerica Ltda., Brazil) maintained on cotyledons of the cucumber (Cucumis sativus L) Safira. After inoculation the plates were maintained in a growth chamber at 20 ± 2 ºC, 70% relative humidity under a 12-hour photoperiod provided by 4 20W daylight type fluorescent lamps (Sylvania, Brazil). Leaf-disks from the parent lines and from the AF125Pm-1 donor line were inoculated as controls in addition to leaf-disks from C. melo plants from the differential varieties Hales Best Jumbo, PMR 45, PMR 5, PMR 6 and Edisto, which were used to confirm the race identity of isolate R1 according to a standard classification system (Thomas, 1978). Symptoms were evaluated 12 days after inoculation, using a stereoscopic microscope. Plants whose leaf-disks showed signs of the pathogen (mycelium, conidiophores, and conidia) on their surface were considered susceptible.
We extracted DNA from leaves of the plants when they were about 20 days old according to the method of Hoisington et al. (1994). Amplification of the M75/H35_155 marker (155 bp) was made as described by Teixeira and Camargo (2006) after DNA digestion with the MseI and HindIII (Invitrogen, USA) enzymes followed by amplification with the selective primers HindIII-ACA and MseI-GTA (Invitrogen,USA).
Proportions of genotype classes for the marker and for the Pm-1 gene were tested using the chi-squared test. The recombination frequency between the marker and the resistance gene was used to estimate the genetic distance between both.
As expected, parental line AF426-S showed symptoms of susceptibility while no development of the pathogen was observed on lines AF426-R and AF125Pm-1. The frequency of resistant and susceptible plants in the backcross population did not differ from the expected frequency for segregation of a dominant resistance gene in a backcross population (Table 1), confirming the type of inheritance expected in this cross.
Segregation of marker M75/H35_155 was also dominant and monogenic, as expected (Table 1). Linkage analysis between M75/H35_155 and Pm-1 revealed linkage between both at a distance of 4.9 cM. Only 7 recombinants plants were found and all were susceptible with the presence of the M75/H35_155 fragment, which excludes the possibility that these plants were not inoculated with P. xanthii (i.e. disease escapes). Since M75/H35_155 is distantly linked to the Prv1 gene as previously described by Teixeira and Camargo (2006) which, in turn, is located in linkage group IX of the melon reference map (Périn et al., 2002; Brotman et al., 2002), it is possible that Pm-1 is also located in this group. This could be tested by using more markers from group IX, but the probability of finding a polymorphic marker in the presumed interval between Prv1 and Pm-1 would be low due to the highly isogenic nature of the parental lines. An alternative would be to map marker M75/H35_155 in the reference map, if possible.
Because P. xanthii is a pathogen present in various producing regions worldwide, use of resistant plant cultivars is the most suitable control strategy (Perchepied et al., 2005). The importance of the Pm-1 gene stems from the fact that P. xanthii race 1 frequently occurs in production fields in the Brazilian Northeast (Reis and Buso, 2004). Therefore, molecular markers linked to the Pm-1 gene are important tools for the development of resistant varieties. Further efforts should be made to convert the AFLP marker M75/H35_155 into a simpler and specific type of marker such as a sequence characterized amplified region (SCAR) based marker or a cleaved amplified polymorphic sequences (CAPS) marker. This is the first report of a marker linked to the Pm-1 gene.
The authors thank Sakata Seeds Sudamerica Ltda. for providing material and technical support. This research was also supported by the State of São Paulo Science Foundation (FAPESP) through grants 02/09915-7 and 04/12422-8.
Received: June 6, 2007; Accepted: October 1, 2007.
Associate Editor: Márcio de Castro Silva Filho
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