Genetic Mapping of the Scab Resistance Gene in Cucumber

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Shengping Zhang Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 10081 China

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Han Miao Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 10081 China

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Xing-fang Gu Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 10081 China

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Yuhong Yang Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 10081 China

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Bingyan Xie Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 10081 China

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Xiaowu Wang Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 10081 China

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Sanwen Huang Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 10081 China

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Yongchen Du Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 10081 China

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Rifei Sun Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 10081 China

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Todd C. Wehner Department of Horticultural Science, North Carolina State University, Raleigh, NC 27695-7609

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Abstract

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 (Abul-Hayja 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 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−2.

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 × 106 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:
DEU1

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 μL of the PCR reaction mix contained 8.02 μL of double-distilled water (ddH2O), 1.5 μL of 10× buffer, 0.2 μL of dNTPs (10 mm), 0.08 μL of Taq DNA polymerase (10 U/μL), 0.6 μL of primer F (50 ng·μL−1), 0.6 μL of primer R (50 ng·μL−1), and 4.0 μL of DNA (10 ng·μL−1). 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 μL 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).

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).

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).

Table 1.

Results

Cucumber scab resistance of the RILs.

In the results for 2007, the DI for parental lines 9110Gt and 9930 were 17.5 and 83.6, respectively. The resistance of 9110Gt and the susceptibility of 9930 to cucumber scab were confirmed. The F1 generation had a DI of 20.0, and was judged to be resistant. There were 72 plants in the RIL population resistant to cucumber scab with a DI < 45.0, and 76 plants susceptible to cucumber with a DI > 45.0 (Fig. 1). The segregation of resistance and susceptibility fit a ratio of 1:1 (Table 2).

Table 2.

Segregation ratios of resistance to cucumber scab in the 9110Gt × 9930 cucumber recombinant inbred line (RIL) population during 2007 and 2008. The segregation of resistance and susceptibility fit a ratio of 1:1.

Table 2.
Fig. 1.
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.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 135, 1; 10.21273/JASHS.135.1.53

In 2008, line 9110Gt had a DI = 12.8 in the artificial inoculation, and was confirmed to be highly resistant to scab. The DIs for line 9930 and the F1 generation were 76.9 and 20.0, respectively. There were 71 plants in the RIL population resistant to cucumber scab with a DI < 45.0, and 77 plants susceptible to cucumber with a DI > 45.0 (Fig. 1). The segregation of resistance and susceptibility fit a ratio of 1:1 (Table 2).

The results from 2007 and 2008 were consistent except for one line of RILs. This line was identified as moderately resistant with a DI of 38.9 in 2007. However, it was rated susceptible with a DI of 45.7 in 2008. The reason may be due to environmental conditions. Analysis of the data for parental lines, the F1 generation, and the RIL population confirmed that a single dominant gene controlled resistance to cucumber scab.

SSR marker analysis and genetic mapping.

Molecular analysis performed on 9110Gt and 9930 using the SSR method resulted in identification of 320 primers generating polymorphic amplicons from the total of 2531 pairs of SSR primers. The polymorphic rate was 12.6%; 251 SSR markers showed polymorphism in the RIL population. SSR markers linked with the Ccu gene based on JoinMap, version 3.0, were identified, and were found to be on cucumber chromosome 2. There were 33 SSR loci and Ccu in the linkage group spanning 87.9 cM of distance, with an average genetic interval of 2.59 cM. The shortest and the longest genetic intervals were 0.3 and 11.4 cM, respectively. The location of Ccu was at 77.4 cM, with flanking markers SSR03084 (R: CAG ACC CTG AAG CGG ATA AA; L: GAC AAG GGA TTC ATC CGA GA) and SSR17631 (R: TTC CCT AAG TAG TGA CGG ATT TTT; L: TTG ATT CCA ATT TCA TTA CTT TTC A) linked to the Ccu gene with genetic distances of 0.7 and 1.6 cM, respectively (Fig. 2).

Fig. 2.
Fig. 2.

Linkage of SSR markers to Ccu gene in cucumber mapped on chromosome 2. There were 33 SSR loci and Ccu in the linkage group spanning 87.9 cM of distance, with an average genetic interval of 2.59 cM. The location of Ccu was at 77.4 cM, with flanking markers SSR03084 and SSR17631 were linked to the Ccu gene with genetic distances of 0.7 and 1.6 cM.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 135, 1; 10.21273/JASHS.135.1.53

Flanking markers linked to Ccu gene.

The veracity of the two SSR markers SSR03084 and SSR17631 tightly linked to the Ccu gene was tested using 59 diverse inbred lines and hybrids. For SSR03084, there were 14 inbred lines and hybrids (D7, D13, D16, D28, D31, D32, D33, D34, D37, D38, D39, D56, D57, and D58) with the same band (about 195 bp) as resistant (maternal) parent 9110Gt, and the rest of 45 inbred lines and hybrids with the same band (about 205 bp) as the susceptible (paternal) parent 9930 (Fig. 3). The result was identical to the artificial inoculation identification, except for D56. D56 was identified as susceptible to cucumber scab, but had the same special band (about 195 bp) as 9110Gt. Thus, the accuracy rate for SSR03084 was 98.3%. As for SSR17631, the result of SSR analysis for the 59 diverse inbred lines and hybrids was identical to the artificial inoculation identification, except for D4. D4 should have the same band (about 205 bp) as 9930, but it had the same band (about 195 bp) as 9110Gt. Thus, the accuracy rate for SSR17631 was also 98.3%. Therefore, SSR03084 and SSR17631 were reliable SSR markers to be used to screen cucumber scab resistance resources in molecular breeding.

Fig. 3.
Fig. 3.

SSR analysis for 59 diverse cucumber inbred lines and hybrids using SSR03084. The polymorphic amplicon of 195 bp was present in resistant lines and 9110Gt, whereas the polymorphic amplicon of 205 bp was in susceptible lines and 9930. Lane M: DNA molecular weight marker (fragment lengths are given in bps); Lane P1: resistant line 9110Gt, 195-bp amplicon present; Lane P2: susceptible line 9930, 205-bp amplicon present; Lane 1 to Lane 42: D1 to D42; Lane 43: blank; Lane 44 to Lane 59: D43 to D58; Lane 60: blank; Lane 61: D59; 195-bp amplicon was present in D7, D13, D16, D28, D31, D32, D33, D34, D37, D38, D39, D56, D57, D58; 205-bp amplicon was present in the rest of the 45 materials. The two arrows indicate the 195-bp amplicon.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 135, 1; 10.21273/JASHS.135.1.53

Discussion

In cucumber, many important disease resistance traits are inherited as single genes (Pierce and Wehner, 1990). Cucumber scab resistance is controlled by a single dominant gene (Abul-Hayja et al., 1975; Andeweg, 1956; Bailey and Burgess, 1934; Vakalounakis, 1993; Wehner, 2005). This study confirmed the previously published conclusion with the expected 1:1 segregation ratio in a RIL population.

There was one line of the RILs that was identified as moderately resistant, with a DI of 38.9 in 2007 and was identified as susceptible with DI of 45.7 in 2008. This line had a band (about 190 bp) consistent with maternal line 9110Gt (resistant to cucumber scab) by the SSR analysis. Thus, this line should be resistant to cucumber scab. Environmental conditions may have changed its reaction in 2008.

The use of molecular markers for selection in cucumber is difficult because of its narrow genetic base. The level of polymorphism in commercial cucumber is low (Kennard et al., 1994), similar to self-pollinated (Apuya et al., 1988; Helentjaris et al., 1986) rather than cross-pollinated species (Figdore et al., 1988; Helentjaris et al., 1986). In this study, the rate of polymorphism was 12.6% in 9110Gt and 9930. Among the cucumber disease resistance traits already mapped are resistances to downy mildew [Pseudoperonospora cubensis (Horejsi et al., 2000)] and to zucchini yellow mosaic virus (Park et al., 2000). We screened SSR markers SSR03084 (0.7 cM) and SSR17631 (1.6 cM) that were tightly linked to the cucumber scab resistance gene Ccu, and Ccu was mapped onto chromosome 2 of cucumber. SSR03084 has the smallest genetic distance from Ccu of any SSR marker identified thus far.

MAS efficiency depends on the recombination fraction and linkage phase of the marker to the target gene (Staub et al., 1996b). MAS is more efficient when using flanking rather than single markers (Horejsi et al., 2000). The flanking markers SSR03084 and SSR17631 reported here have the potential for use in MAS. The veracity of the two SSR markers was tested using 59 inbred lines and hybrids. The accuracy rates for SSR03084 and SSR17631 were the same, at 98.3%. If the two markers were used together in MAS, the accuracy rate would be increased to 98.9%. SSR analysis was also conducted for the 148 RIL population using SSR03084 and SSR17631. As for SSR03084, the result was identical to the artificial inoculation identification, except for one line, and the accuracy rate was 99.3%. However, there were six lines with a result not identical to the DI when SSR analysis was conducted using SSR17631. Thus, the accuracy rate decreased to 95.9% for SSR17631 in the RIL population. The reason was related to the genetic distance between the SSR marker and the Ccu gene. If both SSRs were used to analyze the 148 RIL population, the accuracy rate would increase to 100%. Thus, better correlation to disease resistance can be got when both SSRs are used. These markers will be used in marker-assisted breeding projects and for map-based cloning of the resistance gene.

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  • 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.

  • Linkage of SSR markers to Ccu gene in cucumber mapped on chromosome 2. There were 33 SSR loci and Ccu in the linkage group spanning 87.9 cM of distance, with an average genetic interval of 2.59 cM. The location of Ccu was at 77.4 cM, with flanking markers SSR03084 and SSR17631 were linked to the Ccu gene with genetic distances of 0.7 and 1.6 cM.

  • SSR analysis for 59 diverse cucumber inbred lines and hybrids using SSR03084. The polymorphic amplicon of 195 bp was present in resistant lines and 9110Gt, whereas the polymorphic amplicon of 205 bp was in susceptible lines and 9930. Lane M: DNA molecular weight marker (fragment lengths are given in bps); Lane P1: resistant line 9110Gt, 195-bp amplicon present; Lane P2: susceptible line 9930, 205-bp amplicon present; Lane 1 to Lane 42: D1 to D42; Lane 43: blank; Lane 44 to Lane 59: D43 to D58; Lane 60: blank; Lane 61: D59; 195-bp amplicon was present in D7, D13, D16, D28, D31, D32, D33, D34, D37, D38, D39, D56, D57, D58; 205-bp amplicon was present in the rest of the 45 materials. The two arrows indicate the 195-bp amplicon.

  • Abul-Hayja, Z. , Williams, P.H. & Whelan, E.D.P. 1975 Independence of scab and bacterial wilt resistance and ten seedling markers in cucumber HortScience 10 423 424

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Andeweg, J.M. 1956 The breeding of scab-resistant frame cucumbers in the Netherlands Euphytica 5 185 195

  • Apuya, N. , Frazier, B.L. , Keim, P. , Roth, E.J. & Lark, K.G. 1988 Restriction fragment length polymorphisms as genetic markers in soybean, Glycine max (L.) Merr. Theor. Appl. Genet. 75 889 901

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Bailey, R.M. & Burgess, I.M. 1934 Breeding cucumbers resistant to scab Proc. Amer. Soc. Hort. Sci. 32 474 476

  • Bradeen, J.M. , Staub, J.E. & Wye, C. 2001 Towards an expanded and integrated linkage map of cucumber (Cucumis sativus L.) Genome 44 111 119

  • Cavatorta, J. , Moriarty, G. & Henning, M. 2007 ‘Marketmore 97’: A monoecious slicing cucumber inbred with multiple disease and insect resistances HortScience 42 707 709

    • Crossref
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Shengping Zhang Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 10081 China

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Han Miao Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 10081 China

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Xing-fang Gu Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 10081 China

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Yuhong Yang Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 10081 China

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Bingyan Xie Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 10081 China

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Xiaowu Wang Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 10081 China

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Sanwen Huang Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 10081 China

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Yongchen Du Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 10081 China

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Rifei Sun Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 10081 China

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Todd C. Wehner Department of Horticultural Science, North Carolina State University, Raleigh, NC 27695-7609

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Contributor Notes

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: guxf@mail.caas.net.cn.

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  • 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.

  • Linkage of SSR markers to Ccu gene in cucumber mapped on chromosome 2. There were 33 SSR loci and Ccu in the linkage group spanning 87.9 cM of distance, with an average genetic interval of 2.59 cM. The location of Ccu was at 77.4 cM, with flanking markers SSR03084 and SSR17631 were linked to the Ccu gene with genetic distances of 0.7 and 1.6 cM.

  • SSR analysis for 59 diverse cucumber inbred lines and hybrids using SSR03084. The polymorphic amplicon of 195 bp was present in resistant lines and 9110Gt, whereas the polymorphic amplicon of 205 bp was in susceptible lines and 9930. Lane M: DNA molecular weight marker (fragment lengths are given in bps); Lane P1: resistant line 9110Gt, 195-bp amplicon present; Lane P2: susceptible line 9930, 205-bp amplicon present; Lane 1 to Lane 42: D1 to D42; Lane 43: blank; Lane 44 to Lane 59: D43 to D58; Lane 60: blank; Lane 61: D59; 195-bp amplicon was present in D7, D13, D16, D28, D31, D32, D33, D34, D37, D38, D39, D56, D57, D58; 205-bp amplicon was present in the rest of the 45 materials. The two arrows indicate the 195-bp amplicon.

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