Toward a Sturdy Genetic Map of Melon (Cucumis melo L.)

Marc Oliver and Jordi García-Mas

Departament de Genètica Vegetal, IRTA, 08340-Cabrils, Spain

Ana Isabel López

Estación Experimental 'La Mayora', CSIC, 29750-Algarrobo Costa, Spain

Humberto Gómez-Paniagua

Semillas Fitó S. A. Selva de Mar, 111, 08019-Barcelona, Spain

M. Carmen de Vicente

Departament de Genètica Vegetal, IRTA, 08340-Cabrils, Spain

Additional index words. RFLP, RAPD, inter-SSR, AFLP, genetic mapping

Abstract. We report the process of constructing a molecular linkage map of melon in a segregating population between the Korean line PI 161375 and the Spanish variety 'Piel de Sapo'. Both parents were assayed for polymorphism with RFLP, RAPD, AFLP and Inter-SSR markers. At present, our map has twelve linkage groups, the same as the basic chromosome number of the species (Cucumis melo L.). The objective is to construct a sturdy map based mostly on codominant markers which allows the transfer of information to other segregating populations carrying genes of agronomic interest.

Melon (Cucumis melo L.) is a vegetable crop of great economic importance mainly in tropical and subtropical climates. For years, the improvement of this crop has pointed to the increase of fruit quality and the control of certain reproductive characters which helped obtaining hybrid seed (gynoecy, monoecy and male sterility). Despite the fact that fruit quality traits are of critical importance, the main problems related to melon production are based on the heavy losses due to the incidence of diseases. To fight against them, a number of chemical treatments are used with low success. As a consequence, it appears that the most important strategy to control those problems is one that undertakes the addition of genetic resistance, reducing at the same time the use of potentially toxic pesticides.

Germplasm evaluation for resistance to diseases has revealed the existence of genotypes with resistance to several diseases and also several types of resistance for the same disease (Gómez-Guillamón and Torés, 1992; Gómez-Guillamón et al., 1994). Commercially speaking, the goal is the introgression of different resistances in the same individual, a task which can be pursued by tradi

tional methods of selection though often difficult to apply. Information relating linkage between resistance genes and other characters of morphological nature has been scarce until recently (Pitrat, 1991), or at least insufficient to carry out this work with the desired success. Hence the use of molecular markers seems the best choice.

In melon, studies with molecular markers are quite recent. Polymorphism between melon lines has been tested to clarify genetic relationships and the organization of taxa (Baudracco and Pitrat, 1994; Katzir et al., 1995, 1996, 1997; Kovalski et al., 1995). Different types of molecular markers used in melon include RFLP, RAPD, SSR, inter-SSR, and AFLP.

More recently, two genetic maps of melon have been published. Baudracco-Arnas and Pitrat (1996) constructed a map with RFLP and morphological markers, disease resistance genes, and mostly RAPD markers. Wang et al. (1997) issued a genetic map built with a majority of AFLP markers. In both cases, the number of linkage groups obtained was higher than twelve, the basic chromosome number of the species, so that the level of completion of those maps might be considered still low. On the other hand, both maps

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have been made with a preponderance of dominant markers (RAPD, AFLP). Those markers, very useful to speed up the process of map construction, when applied to linkage analysis of an F2 population provide less information than codominant markers and often result in recombination distances larger than expected.

Because the availability of a sturdy map is the most important requisite to venture in the application of molecular markers to breeding and in different projects of molecular genetics, we considered our objective to be the construction of a genetic map of melon, taking advantage of different types of molecular markers, but enhancing the use of codominant markers.

Materials and methods

Plant material. Ninety-three individuals of an F2 population derived from intraspecific cross between the Korean line PI 161375, as the female parent, and a line of the Spanish melon 'Piel de Sapo' (PS), as the male parent, were used in this study. This population segregates for genes controlling resistance to virus (melon necrotic spot virus, MNSV; cucumber mosaic virus, CMV; zucchini yellow mosaic virus, ZYMV) and a gene controlling resistance to aphid-mediated virus transmission (Vat). In addition, the population segregates for desirable horticultural traits inherited from PS.

The F2 individuals were germinated and maintained in vitro as a permanent source of leaf tissue for DNA extraction. The assay of the F2 plants for all types of markers was made on an individual basis.

DNA extraction. Total genomic DNA was isolated from young leaves as previously described (Doyle and Doyle, 1990) with a few modifications intended to improve the quality of the isolated DNA: two consecutive extractions with chloroform and one additional washing step before the final precipitation of DNA with 5 m NaCl for 20 min at ­20 °C to remove polysaccharides.

RFLP markers. Probes used in this study came from different genomic and cDNA libraries: 1) 23 melon cDNA clones were provided by Michel Pitrat (INRA, Montfavet) and were used previously for the construction of a melon map

(Baudracco-Arnas and Pitrat, 1996); 2) a Hind III genomic library was constructed on pBluescript from total DNA of the melon PS line; 3) a lambda -Zap cDNA library was obtained from RNA extracted from young leaves of the melon line PI 161375.

Total DNA from the parents and the F2 individuals was digested with the following restriction enzymes: BstNI, DraI, EcoRI, EcoRV, and HindIII. Probes were radiolabeled with the random hexamer method (Sambrook et al. 1989) and hybridized with the DNA membranes.

RAPD markers. The protocol for RAPD reactions was adapted from that of Williams et al. (1990). The reaction mixture (25 mL) was composed of 1¥ buffer (50 mm KCl, 10 mm Tris-HCl pH 8.3, 1.5 mm MgCl2, 0.01% gelatin), 0. 44 mm MgCl2, 0.1 mm dNTPs, 30 ng of primer, 1.5 U Taq polymerase (Perkin Elmer-Applied Biosystems) and 10 ng template DNA. Amplifications were performed in a Perkin Elmer 9600 thermocycler with preheating 1 min at 94 °C, and 45 cycles of 10 s at 94 °C, 10 s at 35 °C and 1 min 10 s at 72 °C. A final extension step was added for 2 min at 72 °C.

Inter-ssr. The protocol used with the inter-SSR primers was adapted from Gupta et al. (1994). The reaction mixture (25 mL) was composed of 1¥ buffer (50 mm KCl, 10 mm Tris-HCl pH 8.3, 1.5 mm MgCl2, 0.01% gelatin), 0.125 mm dNTPs, 30 ng of primer, 2 U Taq polymerase (Perkin Elmer-Applied Biosystems) and 50 ng template DNA. Amplifications were performed in a Perkin Elmer 480 thermocycler with preheating 2 min at 94 °C, and 35 cycles of 1 min at 94 °C, 1 min at the annealing temperature specified for each primer as the melting temperature (Tm = 2(A+T) + 4(G+C) ­ 5), and 1 min at 72 °C. The final extension step was performed for 5 min at 72 °C.

AFLP. Total DNA was double digested with EcoRI and MseI following the protocol of Vos et al. (1993) with a few modifications. The resultant fragments were ligated with adaptors specific for either one of the EcoRI and MseI restriction sites. A preselective amplification was carried out using as primers the sequences of the adaptors with an additional nucleotide (at the 3' end downstream from the restriction site). A second amplification was performed using combinations of

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Table 1. Number of molecular markers in 12 linkage groups.

Linkage Recombination

group RFLP RAPD Inter-SSR AFLP Total distance (cM)

1 5 2 2 1 10 61

2 7 3 0 1 11 110

3 9 3 0 5 17 90

4 4 4 0 3 11 77

5 6 2 0 0 8 61

6 3 3 2 6 14 71

7 3 0 0 0 3 6

8 7 7 0 2 16 88

9 6 3 0 3 12 74

10 3 0 0 3 6 31

11 7 3 1 6 17 143

12 6 6 0 6 18 110

Total 66 36 5 36 143 922

Unlinked 4 14 9 27

fluorescently dyed yellow, green or blue labeled EcoRI primers and unlabeled MseI primers (Perkin Elmer-Applied Biosystems) with three selective nucleotides. The resultant labeled fragments were run by capillary electrophoresis on an Abi Prism 310 automated DNA sequencer (Perkin Elmer-Applied Biosystems). This system allows the simultaneous analysis of three-color labeled primer combinations plus a red-labeled size standard using the Genescan Analysis software (v. 2.0).

Data analysis. Segregation data of all markers used were scored independently by two people. Mapmaker v. 3.0 (Lander et al. 1987; Lincoln et al., 1992) was used for analyses of linkage between markers. The parameters established to detect linkage were a minimum LOD score of 3.0 and a maximum recombination distance <30 cM in Kosambi units.

Results and discussion

Polymorphism. One-hundred-seventy loci were analyzed in the progeny of PI 161375 x 'Piel de Sapo' (Table 1). Of these, 70 were RFLPs (from 3 different origins), 50 RAPDs, 5 inter-SSRs, and 45 AFLPs.

Probes for RFLPs were differently polymorphic depending on their origin: 68% of the probes already mapped in a cross between the PI 161375 and 'Charentais' (Baudracco-Arnas and Pitrat, 1996) were useful in our population with the specified restriction enzymes. From the genomic

library of melon, 20% of the probes were polymorphic while 37% were repetitive DNA. When probes came from the leaf cDNA library we obtained 58% polymorphic and 1% repetitive probes.

The selection of RAPDs was based on a previous study (data not shown) carried out to choose the parents for the mapping population. In that RAPD survey, the level of polymorphism detected between the PI 161375 and 'Piel de Sapo' was 60%. Out of 500 primers assayed, we selected the best 50 polymorphic primers by repeating these PCR reactions at least three times and observing consistent and reproducible results.

Data about polymorphism of AFLPs in the parents of our population is quite scarce at this moment. From experiments with eighth primer combinations we found that the average number of total bands per combination is 60 and that the average polymorphism is about six bands per combination.

To find five polymorphic inter-SSR bands we tested 16 primer sequences; hence for this type of marker the percent polymorphism found in our population was 25% because one of the primers rendered two useful bands.

Segregation types. Most of the loci studied segregated according to the ratio 1:2:1 (94% RFLPs) or 1:3 (89% AFLPs, 89% RAPDs, and 100% inter-SSRs). In total, only 8% of the loci (4 RFLPs, 4 RAPDs, and 3 AFLPs) showed distorted segregations in relation to the expected 3:1 or 1:2:1 ratios.

Linkage map. The melon map of the popula

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tion PI 161375 x 'Piel de Sapo' contains so far 143 loci which could be assigned to 12 linkage groups (GI to GXII) number which agrees with the basic chromosome number of the species (Table 1). The results presented here are, however, too preliminary to conclude that these linkage groups correspond exactly with the twelve chromosomes of the melon genome. The number of markers per linkage group ranges from 3 (GX) to 18 (GXII) with an average of 12 markers per group. Twenty-seven of the markers were unlinked (Table 1). Additional markers could lead to the merger of small linkage groups, and at the same time give rise to the appearance of new ones.

Those loci whose segregations present a departure from the expected are scattered in different linkage groups. All types of markers (RFLP, etc. ) are randomly distributed, without any particular pattern or clustering. Some linkage groups do not contain RAPD, Inter-SSR or AFLP markers. Interestingly, none of the linkage groups lacks RFLP markers, the only codominant marker included in the study.

The estimated length of the melon genome with the linkage groups shown here is 922 cM (Kosambi). This number is far from the estimated total length of the melon genome as calculated by Ramachandran and Seshadri (1986; 2364 cM) using cytological data and also short of the map distances by Baudracco-Arnas and Pitrat (1996; 1390 cM) and Wang et al. (1997; 1942 cM). This fact strengthens the possibility of many changes to our map, not only an increase in size but also rearrangements of linkage groups.

This work is being continued by adding more codominant markers (mostly RFLPs). When the map is completed we expect first to transfer the information to other populations which are segregating for different characters (disease resistances, fruit quality, etc.), and to use it in the short term for marker assisted selection and in the long term for map-based cloning of genes of interest.

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