Genetic Mapping of the Scab Resistance Gene in Cucumber

Scab, caused by Cladosporium cucumerinum Ell. et Arthur, is a prevalent disease of cucumber (Cucumis sativus L.) worldwide. Scab can cause serious losses for cucumber production, especially in protected culture such as high tunnel production. Resistance to cucumber scab is dominant and is controlled by a single gene, Ccu. Breeding for resistant cultivars is the most efficient way to control the disease. Selection for resistance might be made easier if the gene were mapped to linked markers. Thus far, there are no tightly linked (genetic distance less than 1 cM) simple sequence repeat (SSR) markers for the Ccu gene, and no studies on mapping of the Ccu gene in cucumber using SSR markers. The objective of this study was to identify SSR markers for use in molecular breeding of scab resistance. In this study, we used a population of recombinant inbred lines (RILs). The population included 148 individuals derived from the cucumber inbred line 9110 Gt (Ccu Ccu) crossed with line 9930 (ccu ccu). The Ccu gene was mapped to linkage group 2, corresponding to chromosome 2 of cucumber. The flanking markers SSR03084 and SSR17631 were linked to the Ccu gene with distances of 0.7 and 1.6 cM, respectively. The veracity of SSR03084 and SSR17631 was tested using 59 diverse inbred lines and hybrids, and the accuracy rate for the two markers was 98.3%. In conclusion, two SSRs closely linked to scab resistance gene Ccu have been identified and can be used in a cucumber breeding program. Scab is a prevalent disease of cucumber worldwide. Scab can cause serious losses for cucumber production, especially in protected culture such as high tunnel production (Lee et al., 1997; Mao et al., 2008; Rego, 1994). Resistance to cucumber scab is dominant and is controlled by a single gene, Ccu (AbulHayja et al., 1975; Andeweg, 1956; Bailey and Burgess, 1934; Vakalounakis, 1993; Wehner, 2005). Researchers have reported on methods of identification (Xu and Zhu, 1994), mechanisms of pathogenicity (Li et al., 2001, 2008), and techniques for control (Li et al., 1998; Yuan, 1989) of cucumber scab. Genetic linkage of Ccu with other traits has also been studied (Mao et al., 2008; Vakalounakis, 1993; Vakalounakis and Klironomou, 1994). However, there are only a few reports on the molecular biology of scab on cucumber. Zhang et al. (2006) screened the amplified fragment length polymorphism (AFLP) marker, E20M64, linked to the Ccu gene, and reported a genetic distance of 4.83 cM. Sun et al. (2006) reported a simple sequence repeat (SSR) primer, CSWCT02B, that was linked to the Ccu gene with genetic distance of 28.7 cM. Bradeen et al. (2001) identified one restricted fragment length polymorphism (RFLP) marker, CMTC51 (0.5 cM), and one AFLP marker, E14M49-F-158-P2 (1.9 cM), linked to the Ccu gene using wide-based, merged maps. Thus far, no linkage of SSR markers to the Ccu gene has been reported, and no SSR marker with genetic distance less than 1 cM to the Ccu gene in cucumber has been identified. Scab can be efficiently controlled using resistant cultivars. Breeding for resistance was reported as early as 1934, but breeding work has continued throughout the world (Andeweg, 1956; Bailey and Burgess, 1934; Cavatorta et al., 2007; Li and Feng, 1994). However, phenotypic selection for scab resistance is difficult due to the large influence of environmental factors on development of the disease, and may require tests with multiple replications and years. Given these difficulties, cucumber breeders would benefit from a more efficient and more reliable scab resistance breeding procedure such as marker-assisted selection (MAS). With MAS, selection is on the marker rather than the disease rating. An efficient MAS system requires markers having tight linkage to Ccu, but combined with an assay that is easy to run. In previous studies, the markers linked to Ccu were RFLP and AFLP types, which are expensive, time consuming to measure, and involve the use of radioactive materials. Those marker types are not cost-efficient to use in molecular breeding. However, SSR markers are polymerase chain reaction (PCR)-based markers with codominant inheritance that are effective and easy to use in a breeding program. Development of SSR markers requires an investment before they can be used because genomic sequences have to be determined before primers can be designed and then used for mapping. Fortunately, the Cucumber Genome Initiative (CUGI), aimed at sequencing the genome of cucumber, was sponsored in China in 2007. Over 2000 pairs of highly polymorphic SSR markers from whole genome shotgun sequences were developed (Ren et al., 2009). In this article, a study of genetic mapping of the Ccu gene was Received for publication 14 Oct. 2009. Accepted for publication 24 Nov. 2009. The research was supported by the earmarked fund for Modern Agroindustry Technology Research System, by the Chinese Ministry of Agriculture (948 Program: 2008-Z42), by the Ministry of Science and Technology (2008BADB1B05, 973 program: 2009CB119004, 2006AA100108, 2006AA10Z1A8), and by the Key Laboratory of Horticultural Crops Genetic Improvement, Ministry of Agriculture. These authors contributed equally to this work. Corresponding author. E-mail: [email protected]. J. AMER. SOC. HORT. SCI. 135(1):53–58. 2010. 53 Table 1. Disease reaction of 59 diverse cucumber inbred lines and hybrids used to test the flanking markers linked to Ccu gene. Thirteen inbred lines and hybrids (D7, D13, D16, D28, D31, D32, D33, D34, D37, D38, D39, D57, and D58) were identified to be resistant (R), and the rest of 46 inbred lines and hybrids to be susceptible (S). Code Accession Source Ecotype S or R D1 Zhongnong No. 8 China Northern China type S D2 Zhongnong No. 9 China Japanese type S D3 Zhongnong No. 10 China Northern China type S D4 Zhongnong No. 12 China Northern China type S D5 Zhongnong No. 15 China Japanese type S D6 Zhongnong No. 16 China Northern China type S D7 Zhongnong No. 19 China European greenhouse type R D8 06W5 China Northern China type S D9 Zhongnong No. 118 China Northern China type S D10 Zhongnong No. 106 China Northern China type S D11 06W8 China Northern China type S D12 06W2 China Northern China type S D13 06W12 China Hybrid of northern China type and European type R D14 06P32 China Southern China type S D15 06P33 China Southern China type S D16 06W54 China European greenhouse type R D17 99246 China Northern China type S D18 016 China Northern China type S D19 228 China Northern China type S D20 05590 China Northern China type S D21 01316 China Northern China type S D22 05603 China Uncertain S D23 04939 China Northern China type S D24 05939 China Northern China type S D25 04348 China Northern China type S D26 0227 China Northern China type S D27 05905 China Northern China type S D28 06469 China European greenhouse type R D29 183 Japan Japanese type S D30 185 Japan Japanese type S D31 65G Europe European greenhouse type R D32 151G Europe European greenhouse type R D33 0.06524G Europe European greenhouse type R D34 0531G Europe European greenhouse type R D35 05925 China Uncertain S D36 05956 China Southern China type S D37 05958 China Southern China type R D38 05950 China Southern China type R D39 05909 China Hybrid of northern China type and European type R D40 05914 China Uncertain S D41 03486G China Northern China type S D42 05952 China Southern China type S D43 04870 China Southern China type S D44 05403 China Northern China type S D45 05372 China Northern China type S D46 05641 China Northern China type S D47 05634 China Northern China type S D48 06123 China Northern China type S D49 18930 China Northern China type S D50 06958 China Northern China type S D51 06959 China Northern China type S D52 Xintai mici China Northern China type S D53 Jinyan No.2 China Northern China type S D54 Zhongnong No. 21 China Northern China type S D55 Zhongnong No. 27 China Northern China type S D56 0313 China Northern China type S D57 Marketmore 76 U.S. American slicing type R D58 Manteo U.S. American processing type R D59 Xishuangbanna cucumber China Southern Asian type S 54 J. AMER. SOC. HORT. SCI. 135(1):53–58. 2010. conducted and SSR markers for the Ccu gene were identified that had tight linkage. Those SSR markers have been used in the selection of resistant germplasm. Materials and Methods MAPPING POPULATIONS. We used 148 F9 recombinant inbred lines (RILs) to map the Ccu gene. RILs were derived from the cross of two elite inbred lines, 9110Gt · 9930. Inbred 9110Gt is resistant to cucumber scab, and was derived from a cross of a European greenhouse line with a northern Chinese line. Inbred 9930 is susceptible to scab, and is a northern Chinese type for which the genome had been sequenced. DISEASE RESISTANCE SCREEN. The degree of resistance to cucumber scab for the RILs was tested in May 2007 and Dec. 2008 using an artificial inoculation method. The method involved soaking seeds in 5% sodium hypochlorite for 10 min, washing with tap water, and placing in a petri dish having two layers of filter paper pad for germination in incubator under the constant temperature of 28 C. When the radicle was 5 mm long, the seeds were sown individually into plastic pots filled with steam-sterilized propagation substrates. Propagation substrate was composed of peat, vermiculite, and soil from vegetable fields (2:1:1, volume basis) and was steam sterilized at 134 C for 30 min. Seedlings were placed in a growth chamber held at 25/20 C day/night with a 16-h photoperiod. Illumination was provided by high-pressure sodium lamps at 50 W m. Identification materials were arranged randomly with three replications of 10 plants each. Resistant cultivar Zhongnong No. 13 and susceptible cultivar Jinza No. 4 were used as controls. All seedings were inoculated, at the stage of fully open first true leaf, with a single strain of Cladosporium cucumerinum. Both sides of the leaves of the seedlings were sprayed uniformly using a hand-pumped sprayer to the point of leaf runoff. Inoculation concentration was 2 · 10 spores/mL. After inoculation, plants were kept in the dark at 100% relative humidity (RH) for 24 h, followed by 7 to 10 d at 60%/85% RH (day/night) at temperatures of 20 to 23 C. SYMPTOM ASSESSMENT. Disease resistance was rated 7 to 10 d after inoculation, and a disease index (DI), a weighted mean, was calculated according to the formula: DI = X ðThe number of plants with each disease rating3 Disease rating scaleÞ The whole number of investigated plants3 The highest disease rating scale 3100 The disease rating scale was as follow: 0 = absence of disease symptoms; 1 = few disease spots on leaf and no spots on stem; 2 = petiole shriveled, few disease spots on leaf, and small spots on stem; 3 = petiole shriveled, center of leaf wilted, sunken disease spots on stem; 4 = petiole shriveled, center of leaf dry and wilted, and sunken disease spots on stem; 5 = petiole and leaf dry and wilted and shrunken disease spots on stem. Scab resistance for each plant was determined using the DI. When the disease susceptible control (Jinza No. 4) reached DI > 45, the evaluation of identification efficiency was made. Seedlings rated 0 < DI # 15 were considered to be highly resistant, 15 < DI # 30 were considered to be resistant, 30 < DI # 45 were considered to be moderately resistant, 45 < DI # 65 were considered to be susceptible, and 65 < DI were considered to be highly susceptible. SSR MARKER ANALYSIS. DNA was extracted from young leaf tissue of the parental lines, F1, and each plant in the population of RILs using a CTAB extraction procedure (Maniatis et al., 1982; Staub et al., 1996a). DNA concentration was estimated on a 1% agarose gel with 1· TEA buffer stained with ethidium bromide. Each 15 mL of the PCR reaction mix contained 8.02 mL of double-distilled water (ddH2O), 1.5 mL of 10· buffer, 0.2 mL of dNTPs (10 mM), 0.08 mL of Taq DNA polymerase (10 U/mL), 0.6 mL of primer F (50 ng mL), 0.6 mL of primer R (50 ng mL), and 4.0 mL of DNA (10 ng mL). The PCR amplifications were performed using a GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA) as follows: 94 C/4 min, 35 cycles of 94 C/15 s, 55 C/15 s, 72 C/30 s, and 72 C/5 min, 16 C. Subsequently, 3 mL of the PCR product was employed for electrophoresis in a 6% polyacrylamide gel according to the method used by Sambrook and Russell (2001). A total of 2531 pairs of SSR primers were screened to identify polymorphisms between the parental lines (9110Gt and 9930) of the RIL population. The development of the SSR primers used in this study was described by Ren et al. (2009). PCRs using identified polymorphic SSR primers were conducted on DNA from individual plants of the RILs to collect data for genetic mapping analysis. GENETIC MAPPING. JoinMap program (version 3.0; Van Ooijen and Voorrips, 2001) was used to develop linkage maps. Marker segregation was analyzed for conformation to Mendelian ratios expected in the RILs using a chi-square test. A minimum logarithm of odds (LOD) of 4.0 was set as a threshold to relegate marker loci into linkage groups, to order markers, and to estimate interval distances (Kosambi function). Fig. 1. Frequency distributions of disease index to cucumber scab in the 9110Gt · 9930 cucumber recombinant inbred line population during 2007 and 2008. The parental line [9110Gt (susceptible) and 9930 (resistant)] values of disease index were indicated by arrows. J. AMER. SOC. HORT. SCI. 135(1):53–58. 2010. 55 TEST OF FLANKING SSR MARKERS LINKED TO CCU GENE. We used 59 diverse inbred lines and hybrids to test the veracity of flanking SSR markers linked to the Ccu gene. They included the northern Chinese type, southern Chinese type, European greenhouse type, American processing type, American slicing type, southern Asian type, Japanese type, and hybrids of northern Chinese type with European greenhouse type. Scab reaction had been measured previously, with 13 resistant and 46 susceptible (Table 1).