Cucurbit Genetics Cooperative Report 23:32-36 (article 10) 2000
Takeo Saito
Research Coordination Section, National Research Institute of Vegetables, Ornamental Plants & Tea, Ano, Mie, 514-2392 Japan
Masami Morishita
Kurume Branch, National Research Institute of Vegetables, Ornamental Plants & Tea, Jurume, Fukuka, 839-8503 Japan
Masashi Hirai
Department of Vegetable Breeding, National Research Institute of Vegetables, Ornamental Plants & Tea, Ano, Mie, 514-2392 Japan
Introduction. Many sources of resistance to Sphaerotheca fuliginea in melon (Cucumis melo L.) are currently available and a number of resistant genes have been identified (3) and are listed by Pitrat (4). In this report, the inheritance of powdery mildew and aphid resistance in the breeding line ‘PMAR No.5’ (introduced to Japan from the University of California USA in 1981) (7) has been studied. furthermore we have screened random amplified polymorphic DNA (RAPD) markers linked to these resistance genes.
Materials and Methods.The F1, F2 and backcross generations (BC-S = F1x Susceptible parent and BC-R – F1 x Resistant parent) from the crosses between ‘PMR No.5’ and ‘Harukei No. 3’ were obtained. ‘PMR No.5’ is a cantaloupe type and resistant to powdery mildew and aphids. ‘Harukei No.3’ is an Earl;s Favorite type and susceptible to them.
A leaf-disk assay for powdery mildew r4esistance was carried out as described (2). Race 1 of S. fuliginea was isolated from field grown
Har8ukei No. 3′. After seven days sporulation was recorded on a scale of 0 (= no sporulation) to 7 (= entire disk covered with heavy sporulation). The plants were grouped in three categories for X2 analysis, based on disease ratings: resistant (score 0), intermediate (score 1) and susceptible (scores 3, 5 and 7).
At the 1-2 leaf stage, ten to fifteen aphids were placed on each plant for a mass infection test (6). After seven days we checked leaf-curling. The plants were grouped in two categories for X2 analysis: resistant (no leaf-curling) and susceptible (leaf-curling).
Genomic DNA was extracted from young true leaves by using the plant DNA extraction kits, Nucleon Phytopure (Scotlab, Scotland), according to the manufacture’s protocol. The PCR protocol was adapted from that of Yui et al. (8). Four DNA bulks from the F2 population were used for a bulked segregant analysis (PMR – 10 powdery mildew resistant plants, PMS – 6 susceptible plants, AR – 10 aphid resistant plants, and AS – 10 aphid susceptible plants). A total of 1412 primers were screened for detection of RAPD between the PMR and PMS, and between the AR and AS. Unique fragments successfully amplified in the resistant bulk were named after the primer name with their size in base parts.
Results and Discussion. Powdery mildew resistance. All the F1 plants and BC-R were resistant and the observed segregations fitted well with 13 resistant: 2 intermediate; 1 susceptible in the F2, and 2:1:1 in the BC-S (Table 1). Segregation ratios suggested a digenic control of a completely and an incompletely dominant genes, in which the former is epistatic over the later.
Melon resistance to powdery mildew has been studied for a long time. The genetics, however, remain confusing. One of the reasons for the confusion is the different categorization of resistance among authors.
By grouping in three categories (resistant, intermediate and susceptible), ‘PMR No. 5’ is found to have two resistance genes. To clarify whether these two genes are the same or different to those reported by other authors, allelism tests are needed.
A total of 60 primers successfully amplified unique fragments in the PMR bulk. For example, the marker, WE-43640 amplified with the WE-43 primer (Table 2), was frequently observed in the PMR individuals and was scarcely found in the PMS ones in the F2 population (Fig. 1). By further analysis, a total of 4 fragments were selected as candidates of linked markers. The segregation of these fragments in the BC-S population (120 plants) was further examined. The markers, WE-43640 , OPX-151100 and UBC411770 , were found to be linked to a completely dominant resistance gene at a distance of 43.4, 43.3 and 49.8 cM, respectively (Figure 2). The maker UBC475970 was found to be linked to an incompletely dominant resistance gene at a distance of 47.5 cM (Figure 3).
Aphid resistance.The data produced evidence for a dominant monogenic control in ‘PMR No.5’ as all the plants in the F1 and BC-R were resistant and the observed segregation fit a 3:1 resistant to susceptible ratio in the F2 and a 1:1 ratio in the BC-S (Table 3). Yoshida and Iwanaga (6) also reported that the flat and curled phenotypes were controlled by a single gene (Ag) with flat leaves being dominant.
A total of 16 primers successfully amplified unique fragments in the aphid resistant bulk. By further analysis, the marker, UBC401800 , was found to e linked to the aphid resistance gene at a distance of 30.2 cM (Figure 4).
In this report, four RAPD markers linked to the PMR genes and one marker linked to the AR gene were found by bulked segregant analysis. However, the distance between the resistance gene and the marker is 30 to 50 cM. There could be several reasons why more tightly linked RAPD markers were not found. One possibility is the presence of other resistance genes with minor effects that make it difficult to correctly identify plants for the appropriate bulks. Baudracco-Arnas & Pitrat (1) and Wang et al. (5) also concluded that RAPD analysis was not the best solution or melon map construction because of skewed segregation.
Presently we are trying to identify DNA markers more tightly linked to powdery mildew resistance genes and aphid resistance gene.
Table 1. Observed segregation for powdery mildew resistance and the goodness of fit test.
| Pedigree | Number of plants | Expected y ratio | x2 | P | ||
| Rz | I | S | ||||
| PMR No.5 | 37 | 0 | 0 | 37:0:0 | ||
| Harukei No. 3 | 0 | 0 | 53 | 0:0:53 | ||
| F1 | 50 | 0 | 0 | 50:0:0 | ||
| F2 | 164 | 24 | 12 | 13:2:1 | 0.074 | 0.964 |
| BC-S | 56 | 27 | 37 | 2:1:1 | 2.200 | 0.333 |
| BC-R | 116 | 0 | 0 | 116:0:0 | ||
z R = resistant, 1 + intermediate, S = susceptible.
y The genetic model tested is one completely dominant gene and another incompletely dominant gene, with the former being epistatic to the later.
Table 2. RAPD markers for powdery mildew resistance (PMR) and aphid resistance (AR) in Cucumis melo L.
| RAPD marker | Trait | Size (bp) | Primer | Sequence of primer | Origin of primer |
| WE-43640 | PMR (completely dominant) | 640 | WE-43 | 5’ACTCACAAATTG-3′ | Wako Pure Chemical Ind. Ltd. |
| OPX-151100 | PMR (completely dominant) | 1100 | OPX-15 | 5’CAGACAAGCC-3′ | Operon Technologies, Inc. |
| UBC411770 | PMR (completely dominant) | 770 | UBC-411 | 5′-GAGGCCCGTT-3′ | University of British Columbia |
| UBC475970 | PMR (incompletely dominant) | 970 | UBC-475 | 5′-CCAGCGTATT-3′ | University of British Columbia |
| UBC401800 | AR | 800 | UBC-401 | 5′-TAGGACAGTC-3′ | University of British Columbia |
Table 3. Segregation and goodness of fit test of aphid resistance in Cucumis melo L.
| Pedigree | Number of plantsz | Expectedy ratio | x2 | P | |
| R | S | ||||
| PMR No.5 | 10 | 0 | 10:1 | ||
| Harukei No. 3 | 0 | 20 | 0:20 | ||
| F1 | 40 | 0 | 40:0 | ||
| F2 | 145 | 51 | 3:1 | 0.1096 | 0.947 |
| BC-S | 35 | 25 | 1:1 | 1.667 | 0.435 |
| BC-R | 60 | 0 | 60:0 | ||
z R = resistant, S = susceptible.
y A single, dominant gene model.
Literature Cited
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