Hort. Environ. Biotechnol. 54(2):134-140. 2013. DOI 10.1007/s13580-013-0156-1
Research Report
Inheritance of Resistance to Powdery Mildew in the Watermelon and Development of a Molecular Marker for Selecting Resistant Plants 1
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Kwang-Hwan Kim , Sung-Gyu Ahn , Ji-Hyun Hwang , Young-Mi Choi , Hyun-Shik Moon , and Young-Hoon Park1* 1
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Department of Horticultural Bioscience, Pusan National University, Miryang 627-706, Korea Deptartment of Forest Environmental Resources, Gyeongsang National University, Jinju 660-701, Korea *Corresponding author:
[email protected]
Received October 28, 2012 / Revised December 13, 2012 / Accepted February 8, 2013 GKorean Society for Horticultural Science and Springer 2013
Abstract. The race of watermelon powdery mildew (PM) present in South Korea was confirmed, and genetic inheritance of resistance to this race was determined from a cultivars ‘Arka Manik’. Bioassay tested on a set of melon differential genotypes indicated that the race of P. xathii predominating in South Korea was either belonged to race 1W or another race different from race 2W. Inheritance of PM resistance was studied from F2, F2:3, and reciprocal backcross generations derived from ‘Arka Manik’ (resistant cultivar) × HS3355 (susceptible line). The segregation ratios indicated that resistance to PM in ‘Arka Manik’ is conditioned by a single incompletely dominant gene (Pm1.1). Bulked segregant analysis (BSA) of F2 plants using random amplification of polymorphic DNA (RAPD) identified one polymorphic polymerase chain reaction (PCR) band (OP-R483) and a close linkage (3.6 cM) was observed between resistance and OP-483. The marker OP-483 was converted to cleaved amplified polymorphic sequence (CAPS-G/C) and a single nucleotide polymorphism (SNP) marker (MCA-A/G) using melting curve analysis. MCA-A/G marker genotyping of a wide range of susceptible breeding lines revealed that the marker allele of ‘Arka Manik’ was unique in breeding populations. Additional key words: fungal pathogen, high-throughput genotyping, PCR-based marker, Podosphaera xanthii
Introduction Powdery Mildew (PM) of cucurbit crops is a fungal disease caused by Podosphaera xanthii (castagne) U. Braun & N. Shishkoff as a predominant agent. The optimum condition for sporulation of the pathogen is a relatively high temperature of 25-30Gand > 98% humidity. The infected plants show symptoms of mycelial and condial development on the entire plants including petioles stems, cotyledons, and leaves. Severely affected plants result in chlorotic leaves, decreased canopy, and reduced fruit quality and yield (Keinath and Dubose, 2004; McGrath and Tomas, 1996). Two pathogenically distinct races, 1W and 2W, were reported from watermelon in the United States, where watermelon PM has emerged as an important disease problem (Davis et al., 2001, 2007; Tetteh et al., 2010). Race confirmation in the watermelon has been carried out using a set of differential
melon cultivars (Davis et al., 2007). Several cultivars and wild-type species highly resistant to these races have been identified in the U.S. (Davis et al., 2006b, 2007; Tetteh et al., 2010). However, the genetic inheritance of the resistances has not been well-studied (Davis et al., 2002). In South Korea, watermelon is a major vegetable crop that accounts for a farm production value of $9.4 million and a cultivation area of 20,756 ha in 2009. Until recently, PM was not a serious disease for watermelon production in Korea. However, outbreaks of PM are continuously increasing, as greenhouse cultivation increases and climate change creates more subtropical environment. Generally in South Korea, the occurrence of PM is observed, regardless of cultivation practice or production location (Lee et al., 2010). The disease is controlled mainly by repeated applications (6-7 times) of fungicides throughout the growing season, due to a lack of resistant cultivars.
Electronic supplementary material: The online version of this article (doi:10.1007/s13580-013-0156-1) contains supplementary material, which is available to authorized users.
Hort. Environ. Biotechnol. 54(2):134-140. 2013.
In an effort to develop resistant cultivars, 120 watermelon accessions were screened and 23 cultivars, including IT207182, were identified to be moderately resistant to PM (Lee et al., 2010). ‘Arka Manik’, an open-pollinated cultivar introduced from India (Rai et al., 2008) and PI254744 were also found to be highly resistant to PM in South Korea. To date, however, the race confirmation of P. xanthii present in South Korea and investigation of inheritance of the resistance have not been undertaken. Molecular markers tightly linked to disease resistance genes can be useful for marker-assisted selection (MAS) that facilitates the breeding process by selecting resistant plants based on their marker genotype (Collard and Mackill et al., 2008; Moose and Mumm et al., 2008; Tester et al., 2010). Bulked segregant analysis (BSA) is an efficient approach to screen the markers linked to a target trait, especially under circumstances where genetic mapping is not easily applicable (Chantret et al., 2000; Giovanni et al., 2004; Huang and Roder, 2004; Michelmore et al., 1991). To our knowledge, no publicly available molecular marker for MAS of PM resistance in watermelon has been reported. The present study was carried out with the objectives of confirming the pathotype of P. xanthii present in watermelon production fields in South Korea, determining the genetic inheritance of the resistance to PM in ‘Arka Manik’, and developing molecular markers tightly linked to the resistance for MAS. The results provide useful information that is practically applicable to breeding programs aimed at the development of new watermelon cultivars with high resistance to PM.
Materials and Methods 3ODQW 0DWHULDO Dfferential set of melon (Cucumis melo L.) cultivars including PMR 1, PMR 6, PMR 45, and ‘Topmark’ were used to ascertain the race of P. xanthii infecting watermelons in South Korea. For inheritance analysis of resistance to PM, the resistant cultivar ‘Arka Manik’ (P1) and the susceptible inbred line HS3355 A (P2) were used. These parental lines were crossed to produce the F1 generation. F1 plants were self-pollinated to produce F2 generations and F3 families (F2:3), or were crossed to both parents to produce the reciprocal backcross (BC) families BC1-P1 (F1 × ‘Arka Manik’) and BC1-P2 (F1 × HS3355). All crosses and production of the generations were conducted in the greenhouse at Pusan National University. ,QRFXODWLRQ DQG 'LVHDVH $VVHVVPHQW For race identification, eight plants for each differential melon cultivars were evaluated. Seeds were sown in a 50-cell plastic tray containing commercial soil mixture and placed
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on benches in the aforementioned greenhouse operating at temperatures of 25-30Gduring the day and 15-20Gduring the night, and a humidity higher than 50%. Each inoculum was prepared by rinsing with distilled water heavily infected watermelon leaves collected from watermelon production fields in Gyeonggi province, South Korea. Inoculation was performed twice at 14 and 21 days after seeding, with the first inoculation timed to the first-to-second true-leaf stage. 3 A suspension of 5 × 10 sporangia of P. xanthii per mL was sprayed on each seedling to runoff using a plastic hand sprayer. To study the inheritance of resistance in watermelon, disease assessment was performed at a seedling stage (2-leaf). For disease evaluation, 112 F2:3 families derived by self-pollinating F2 plants, 31 BC1-P1, and 31 BC1-P2 plants were used. In the F2:3 progeny test, a completely randomized block design was used. Two blocks were tested, and eight plants for each F2:3 family were replicated for each block. Plant management and inoculation in the greenhouse were performed following the same methods for the experiment of race identification with melons. Disease severity was assessed using three rating classes depending on the level of sporulation as follows: 3 (resistance, R) = the resistance level of ‘Arka Manik’, no disease symptom at seedling stage, very slight colony development at juvenile stage; 5 (intermediate resistance, IR) = the resistance level of F1, few colonies present, moderate level of sporulation; 7 (susceptibility, S) = the susceptibility level of HS3355, highly profuse sporulation over the entire plants. For the genetic model of inheritance, Chi-square tests on phenotypic data were conducted to determine goodness of fit of observed to expected Mendelian segregation ratio in the F2:3 and reciprocal BC generations. 0ROHFXODU 0DUNHU 'HYHORSPHQW To develop a molecular marker tightly linked to PM resistance, the BSA approach was implemented. Based on the results of progeny test for PM resistance, DNA samples from 13 highly resistant F2 plants were isolated and mixed together in an equal amount. The same was done for 16 highly susceptible F2 plants. Genomic DNA extraction from plants was conducted as described previously (Kim et al., 2010). Random amplified polymorphic DNAs (RAPDs) were evaluated to identify polymorphisms between the bulked resistant and susceptible DNA samples. For the RAPD procedure, 1,000 10-mer Operon primers were tested by following methods described previously (Je et al., 2009). For cloning and sequencing of PCR amplicons, DNA fragments were eluted from an agarose gel, cloned into a T&A cloning vector, and sequenced by the dye termination method using an ABI3730 capillary DNA sequencer (Applied
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Kwang-Hwan Kim, Sung-Gyu Ahn, Ji-Hyun Hwang, Young-Mi Choi, Hyun-Shik Moon, and Young-Hoon Park
Biosystems, Foster City, CA, USA). More detailed description for methods can be found in Kim et al. (2010). PCR primer design and sequence alignment were performed using Primer3 v. 2.0 and ClustalW software, respectively. PCR amplifications for cleaved amplified polymorphic sequence (CAPS) marker, CAPS-G/C, were conducted in a total volume of 20 ȝL containing 20 ng of genomic DNA, each forward and reverse primer at 0.3 ȝM, 1 × PCR buffer, 0.2 mM dNTPs, and 0.6 U of Taq polymerase (Solgent, Daejeon, Korea) with the following touchdown cycling profile: 1 cycle of 5 min at 95, 10 cycles of 15 s at 95, 30 s at 60G(decreasing in steps of 0.5/cycle for cycles 2-10) and 30 s at 72, and 1 min at 72G, followed by 35 cycles of 15 s at 95, 30 s at 55, and 1 min. at 72. PCR products were digested with a restriction enzyme CviKI-1 (New England BioLabs, Ipswich, MA, USA) following the manufacturer’s instructions. Specific primers and hybridization probes for melting curve analysis (MCA) of the MCA-A/G marker were obtained ® from TIB MOLBIOL (Berlin, Germany). Anchor probe of a pair of hybridization probes was labeled with fluorescein, which served as the donor fluorophore. The other sensor ® probe was labeled with LightCycler Red 640, serving as the acceptor dye. PCR reactions were performed in a ® LightCycler 1.5 apparatus (Roche Diagnostics, Mannheim, ® Germany) using LightCycler FastStart DNA Master HybProbes (Roche Diagnostics, Mannheim, Germany). The reactions were carried out in a final volume of 10 ȝL containing 2 ȝL ® of 10× LightCycler FastStart DNA Master HybProbes , 3 mM MgCl2, 0.5 ȝM of each of the primers, 0.225 ȝM of the corresponding hybridization probes, and 2 ȝL of the sample DNA. The initial cycling conditions were 10 min at 95, followed by 40 cycles of 5 s at 95, 10 s at 62, and 15 s
at 72. After a denaturation step of 30 s, the melting curves were obtained from 40G to 85G with a temperature -1 transition of 0.2·s . Finally, all samples were cooled to 40Gfor 30 s. During the melting curve analysis, temperaturedependent hybridization of the sequence-specific hybridization probes to single-stranded DNA was monitored. Fluorescence resonance energy transfer (FRET) occurred from the excited fluorescein dye to the detection dye LC-Red640 if both probes were bound.
Results 5DFH ,GHQWLILFDWLRQ DQG ,QKHULWDQFH RI WKH 5HVLVWDQFH To confirm the watermelon race of PM present in South Korea, inocula collected from Gyeonggi province were tested on a set of melon differential cultivars. Among PMR 1, PMR 6, PMR 45, and ‘Top mark’, ‘Top mark’ was susceptible, while other cultivars did not show any disease symptoms after infection. The result of the bioassay showed that the inocula had the same pathogenic reaction known for race 1W (McGrath and Tomas, 1996). Inheritance of PM-resistance was studied with different generations derived from a cross between a resistant cultivar ‘Arka Manik’ and a susceptible inbred HS3355. Bioassay at the seedling stage showed that ‘Arka Manik’ was highly resistant (R, DSR = 3), ‘HS3355’ was highly susceptible (S, DSR = 7), and their F1 was intermediately resistant (IR, DSI = 5). In segregating generations, each individual plant could be categorized into the phenotype of two parents or F1, and was scored as DSR 3, 5, or 7. The segregation ratios observed in F2:3 and reciprocal backcross generations indicated that a simple genetic control for the resistance to PM was involved in ‘Arka Manik’ (Table 1). Chi-square
Table 1. Segregation for resistance to powdery mildew in F2, reciprocal BC1, and F2:3 generations for crosses of resistant cultivar ‘Arka Manik’ (P1) and susceptible line HS3355 (P2). *HQHUDWLRQ] )
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Hort. Environ. Biotechnol. 54(2):134-140. 2013. 2
(X ) statistic was tested against the Mendelian inheritance model for single incompletely dominant gene for the resistance. For reciprocal backcross generations BC1-P1 and BC1-P2, the segregation ratio of 1 (R):1 (IR) and 1 (S):1 (IR) was observed, respectively. In the F2:3 generation, mean DIS scores of each of 112 families segregated in a 1 (R):2 (IR):1 (S) ratio (see supplementary material). In addition, segregation for resistance was observed in the progeny test of 52 F2:3 families, which scored a mean DSR 4.1-5.9 (IR). These results indicated that PM resistance in ‘Arka Manik’ is conditioned by a single incompletely dominant gene, namely Pm1.1. 0ROHFXODU 7DJJLQJ RI 30 5HVLVWDQFH BSA of F2 plants using RAPD, amplified fragment length polymorphism, and sequence-related amplified polymorphism was conducted to develop a DNA marker for selection of PM resistance. For BSA, genomic DNAs from 13 resistant and 16 susceptible F2 plants based on progeny testing were extracted and pooled. In total, 5,493 RAPDs were scored and one unambiguously polymorphic PCR band (OP-R483) was identified from an Operon RAPD primer. This polymorphic band was absent from bulked DNA samples of susceptible plants, but was present in bulked DNA samples of resistant plants. This RAPD marker was tested again on each individual plant used in BSA (Fig. 1A). The polymorphic band was present for all 13 resistant plants. For the 16 susceptible plants, the band was absent from 14 plants but was present in two plants (617-080 and 094) (Table S1) indicating that recombination occurred between the resistance gene and marker locus in these plants. All F2 plants were tested to confirm the frequency of
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recombination between resistance and the RAPD marker (OP-R483), and approximately 3.6% recombination frequency (3.6 cM) was observed; except for the F2 plants 617-080 and 094, there were two additional plants that appeared to mismatch with the phenotyping results in their F2:3 progeny test. 617-078 was intermediately resistant to PM, but showed the marker for susceptibility. 617-088 was susceptible, but showed the marker for resistance (OP-R483). All others that were resistant or intermediately resistant harbored the OP-R483 marker. Due to dominant characteristic of this RAPD marker, heterozygosity could not be defined for F2 parents that corresponded to 52 F2:3 families showing segregation for resistance and intermediate resistance (Table S1). 0DUNHU &RQYHUVLRQ IRU &$36 DQG +7613 To convert into a simple codominant marker, the PCR band of OP-R483 was cloned and the DNA sequence was characterized. Based on this DNA sequence, a set of primers (SCAR1-F and SCAR1-R) closely located next to the RAPD primer sequence was designed and the corresponding locus (OP-S483) from the susceptible parent HS3355 was amplified and sequenced. Sequence alignment between OP-R483 and OP-S483 revealed five single nucleotide mismatches or polymorphisms (SNPs) (Fig. 2), which were useful for designing CAPS markers (Fig. 1B) or MCA marker using fluorescently labeled DNA probe (Fig. 3). Using these SNPs, we evaluated the CviKI-1 enzyme restriction pattern for CAPS and allele discrimination method by MCA with ® the two probe chemistries (HybProbe ) that is based on FRET (Fig. 3). These CAPS (CAPS-G/C) and MCA (MCA-A/G) markers
Fig. 1. Agarose gel image showing genotyping of F2 plants segregating for powdery mildew resistance by using an Operon RAPD primer (A) and a CAPS marker CAPS-G/C (B). CAPS-G/C marker was developed based on the polymorphic RAPD band (OP-483) indicated by arrow, which cosegregated at a high frequency with the PM resistance. M, 100-bp DNA size marker; F2, F2 plants used for genotyping.
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Kwang-Hwan Kim, Sung-Gyu Ahn, Ji-Hyun Hwang, Young-Mi Choi, Hyun-Shik Moon, and Young-Hoon Park
Fig. 2. DNA sequence alignment of PCR amplicons produced by primer set SCAR1 from PM-resistant ‘Arka Manik’ (AM) and PM-susceptible HS3355 (HS). The forward and reverse primer regions are indicated in open gray boxes. Single nucleotide polymorphisms (SNPs) are shown in alphabets. The SNP in the restriction enzyme site of CviKI-1 used for designing CAPS-G/C was indicated by black box. The SNP and sites for anchor and sensor probes used for developing MCA-A/G are indicated by red color and arrows, respectively, All nucleotide sequences that were identical between HS and AM are shown as dashes(-).
were used to genotype the two parents and 112 F2 plants. The genotype of all samples were consistent between CAPS and MCA, and agreed with the data previously determined by the RAPD marker OP-R483; all F2 plants that showed the RAPD marker for susceptibility showed the marker genotype for susceptibility in this SNP evaluation, whereas F2 plants that were resistant by RAPD evaluation were either resistant or heterozygous. Cosegregation between phenotype based on F2:3 progeny test and codominant marker genotype are summarized in Table 2. In comparison to the RAPD marker, these codominant markers revealed more F2 plants that showed mismatch in phenotype and marker genotype. Among 22 F2 plants that were scored as resistant, 17 showed homozygous marker genotype for resistance, whereas five (617-011, 035, 038, 065, and 110) were heterozygous. For 52 intermediately resistant F2 plants, 49 displayed the heterozygous marker genotype, except for two (617-026 and 036) plants that showed homozygosity for the resistance and one plant (617-078) that showed homozygosity for the susceptibility. For 38 susceptible F2 plants, 35 plants were homozygous for the susceptibility, while three plants (617-080, 088, and 094) were heterozygous; 617-080 and 094 were the plants that were susceptible, but showed RAPD marker for resistance
Fig. 3. Melting curve analysis (MCA) of the A/G mismatch (Fig. 2) that is tightly linked to powdery mildew (PM) resistance by using MCA-A/G marker. A) Genotyping of PM-resistant ‘Arka Manik’, PM-susceptible HS3355, and their F1 progeny. B) Genotyping of F2 progenies C) Genotyping of ‘Arka Manik’ and diverse breeding lines susceptible to PM. Polymorphic genotypes and their melting curves are indicated by different colors and arrows. Table 2. Frequencies of cosegregation between powdery mildew (PM)-resistance and markers CAPS-G/C and MCA-A/G. 3KHQRW\SH] 5
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Hort. Environ. Biotechnol. 54(2):134-140. 2013.
in the BSA. These plants turned out to be heterozygous at the marker locus. The marker usability in other crosses was evaluated by assessing the marker polymorphism between ‘Arka Manik’ and various watermelon germplasms susceptible to PM. These germplasms included 18 inbred lines or open pollination varieties from different countries of origin and 40 inbred lines (data not shown) that were developed by private companies in South Korea and used as important sources for breeding. For CAPS-G/C, two domestic and six foreign inbred lines showed the marker genotype of ‘Arka Manik’, and all others showed marker for susceptibility. However, for the MCA-A/G marker, all watermelon germplasms tested showed a melting peak for susceptibility (Fig. 3C), indicating that this SNP in ‘Arka Manik’ is very unique and most of the susceptible watermelon germplasms do not possess this allele from ‘Arka Manik’. Our results indicate that our markers can be widely used for various parental cross combinations in MAS, because the marker genotype for the susceptible allele is highly conserved among various susceptible breeding sources. Especially, MCA-A/G can be a good choice for MAS in terms of its discrimination power for the resistance allele from ‘Arka Manik’ and applicability to high-throughput ® genotyping: this marker can be run on the LightCycler that provides rapid PCR on a 384-well plate and allows simultaneous amplification and analysis in approximately 2 h without any additional steps after amplification. This reduces the likelihood of contamination of PCR products, makes the process simpler, and streamlines the workflow, which are all highly desirable for MAS in watermelons.
Discussion According to previous studies (Davis et al., 2001, 2006a) with melon differentials, only two watermelon PM races, 1W and 2W, have been revealed based on pathogenically distinct reactions. In our study, the melon differential ‘Topmark’ was susceptible, while PMR 1, 6, and 45 did not show any disease symptoms after infection. This result was in accordance with the reaction pattern of race 1W to melon differentials (McGrath and Thomas, 1996). Thus, the disease response test proved that the pathotype of P. xathii predominant in South Korea is either race 1W or a new race different from race 2W. Recently, watermelon germplasm with higher levels of resistance have been reported (Davis et al., 2007) for race 1W. In this screening, eight of 1,573 accessions demonstrated high plant resistance. These accessions included PI lines of two Citrullus species (C. lanatus var. lanatus, and C. colocynthis) and one related species of Praecitrullus fistulosus collected from Zimbabwe, India, and several other
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countries. Using a breeding process like backcross, resistance genes from these accessions can be introduced into breeding lines and cultivars with commercially useful genetic background can be realized. However, the breeding process can be complicated and time-consuming when linkage drags exist between the resistance and unadapted horticultural traits in the exotic breeding resources (Brown et al., 1989; Zamir, 2001). ‘Arka Manik’, a resistance source used in this study, is an open pollinated variety with multiple disease resistance and commercial values, which is genetically homogeneous (Rai et al., 2008). Our recent genetic diversity study (Hwang et al., 2011) showed that this variety was cross-related at the molecular level to commercial watermelon cultivars and advanced breeding lines collected from Asia including South Korea. Since ‘Arka Manik’ demonstrated adapted traits and simple genetic control of PM, it can be desirably used as a donor parent of the resistance gene in a breeding program. Intensive screening of watermelon germplasm for resistance to PM race 1 (Davis et al., 2006b, 2007) and 2 (Tetteh et al., 2010) was reported in the United States. High levels of resistance have been found from PI525088 and a resistant pure breeding line PI525088-PMR was derived by successive self-pollination and selection of PI525088. The inheritance of race W1 PM resistance in PI525088 was found to be multigenic (Davis et al., 2002). Although the phenotype of a relatively high number (11) of F2 plant did not match to their marker genotype, it should be noted that most of these plants (8 of 11) harbored heterozygous marker genotype. This implies that the mismatches was possibly due to the mean DSI values for these heterozygous plants that were scored weighted toward resistance or susceptibility by disease escape or plant sampling error. If this is the case, the linkage association of the marker to PM-resistance locus can be tighter then the value that is calculated by the data above. In conclusion, the watermelon PM pathotype prevalent in South Korea shows a similar pathogenic reaction known for race 1W in different melon hosts. The Indian open pollination cultivar ‘Arka Mank’ was resistance to this PM race and displayed a single incompletely dominant gene effect. BSA identified a RAPD marker that is tightly linked to PM resistance in ‘Arka Manik’. This RAPD locus was successfully converted to a codominant high-throughput marker for MAS by using MCA on a real-time PCR platform. To our knowledge, this is the first DNA marker reported for MAS of PM resistance in watermelon. Our information will significantly improve the breeding process and expedite the development of new watermelon cultivars with PM resistance. Currently, we are developing an intraspecies genetic map to locate the locus Pm1.1. This work includes the sequencing of full transcripts from ‘Arka Manik’ and a susceptible inbred
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line and identification of extended sequence tag SNPs. Acknowledgments: This research was supported by a grant (code: 0636-20110007) from the Vegetable Breeding Research Center through R&D Convergence Center Support Program, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.
Literature Cited Brown, A.H.D., G.J. Lawrence, M. Jenkin, J. Douglass, and E. Gregory. 1989. Linkage drag in backcross breeding in barley. J. Hered. 80:234-239. Chantret, N., P. Sourdille, M. Roder, M. Tavaud, M. Bernard, and D. Doussinault. 2000. Location and mapping of the powdery mildew resistance gene MIRE and detection of a resistance QTL by bulked segregant analysis (BSA) with microsatellite in wheat. Theo. Appl. Genet. 100:1217-1224. Collard, B.C.Y. and D.J. Mackill. 2008. Marker-assisted selection: An approach for precision plant breeding in the twenty-first century. Phil. Trans. R. Soc. B. 363:557-572. Davis, A.R., C.E. Thomas, A. Levi, B.D. Bruton, S.D. Pair, and C.E. Thomas. 2001. Powdery mildew: An emerging disease of watermelon in the United States. Cucurbit Genet. Coop. Rpt. 24:42-48. Davis, A.R., C.E. Thomas, A. Levi, B.D. Bruton, and S.D. Pair. 2002. Watermelon resistance to powdery mildew race 1, p. 192-198. In: D.N. Maynard (ed.). Cucurbitaceae ’02. ASHS Press, Alexandria, VA. Davis, A.R., A. Levi, T. Wehner, and M. Pitrat. 2006a. PI 525088-PMR, a melon race 1 powdery mildew resistant watermelon line. HortScience 41:1527-1528. Davis, A.R., A. Tetteh, T. Wehner, A. Levi, and M. Pitrat. 2006b. Watermelon resistance to powdery mildew race 1 and race 2. p. 412-420. In: G.J. Holmes (ed.). Proc. Cucurbtaceae 2006. Universal Press, Raleigh, NC. Davis, A.R., A. Levi, A. Tetteh, T. Wehner, V. Russo, and M. Pitrat. 2007. Evaluation of watermelon and related species for resistance to race 1W powdery mildew. J. Amer. Soc. Hort. Sci. 132:790-795. Giovanni, C.D., P. Dell’Orco, A. Bruno, F. Ciccarese, C. Lotti, and L. Ricciardi. 2004. Identification of PCR-based marker (RAPD, AFLP) linked to a novel powdery mildew resistance gene (ol-2)
in tomato. Plant Sci. 166:41-48. Hwang, J., S. Ahn, J. Oh, Y. Choi, J. Kang, and Y. Park. 2011. Functional characterization of watermelon (Citrullus lanatus L.) EST-SSR by Gel electrophoresis and high resolution melting analysis. Scientia Hort. 130:715-724. Huang, X. and M. Roder. 2004. Molecular mapping of powdery mildew resistance genes in wheat: A review. Euphytica 137: 203-223. Je, H.J., Y.O. Park, S.C. Kim, J.H. Hwang, Y.J. Lee, B.G. Son, and Y.H. Park. 2009. Evaluation of genetic relationships among persimmon cultivars introduced and indigenous in Korea using RAPD. Kor. J. Hort. Sci. Technol. 27:448-455. Keinath, A.P. and V.B. Dubose. 2004. Evaluation of fungicides for prevention and management of powdery mildew on watermelon. Crop Protection 23:35-42. Kim, J.K., D.C Ahn, H.J. Oh, K.H. Kim, Y.M. Choi, S.Y. Oh, N.J. Kamg, B.R. Jeong, Z.H. Kim, and Y.H. Park. 2010. Skewed inheritance of EST-SSR alleles in reciprocal crosses of cut roses. Kor. J. Hort. Sci. Technol. 28:618-626. Lee, W.M., S.H. Son, D.K. Park, H.J. Lee, Y.C. Huh, and I.H. Cho. 2010. Selection of watermelon germplasm with disease resistance to powdery mildew (Sphaerotheca fusca). Kor. J. Hort. Sci. Technol. 28(Suppl. II):73. (Abstr.) McGrath, M.T. and C.E. Thomas. 1996. Powdery mildew, p. 28-30. In: T.A. Zitter, D.L. Hopkins, and C.E. Thomas (eds.). Compendium of cucurbit disease. The Amer. Phytopathol. Soc. St. Paul, MN. Michelmore, R.W., I. Paran, and R.V. Kesseli. 1991. Identification of markers linked to disease resistance genes by bulked segregant analysis: A rapid method to detect markers in specific genomic regions by using segregating populations. Proc. Natl. Acad. Sci. USA 88:9828-9832. Moose, S.P. and R.H. Mumm. 2008. Molecular plant breeding as the foundation for 21st century crop improvement. Plant Physiol. 147:969-977. Rai, M., S. Pandey, and S. Kumar. 2008. Cucurbit research in India: A retrospect. Cucurbitaceae proceedings of the IXth EUCARPIA meeting on genetics and breeding of Cucurbitaceae (Pitrat M, ed), INRA, Avignon(France), May 21-24, 2008. Tester, M. and P. Langridge. 2010. Breeding technologies to increase crop production in a changing world. Science 327:818-822. Tetteh, A.Y., T.C. Wehner, and A.R. Davis. 2010. Identifying resistance to powdery mildew race 2W in the USDA-ARS watermelon germplasm collection. Crop Sci. 50:933-939. Zamir, D. 2001. Improving plant breeding with exotic genetic libraries. Nature 2:983-989.
