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RAPD Markers Linked to Morphological and Disease Resistance Traits in Squash Rebecca N. Brown and James R. Myers Department of Horticulture, Oregon State University, Corvallis, OR 97331 USA Molecular markers linked to phenotypic traits can be useful to breeders. The most useful types of markers are those which allow for selection in seedlings instead of mature plants, or which permit selection for disease resistance without inoculating the entire segregating population. Sometimes markers are developed for a specific trait of interest using bulked segregant analysis or near-isogenic lines. However, markers linked to morphological and disease resistance traits are often detected while constructing genetic maps. Saturated or nearly saturated genetic maps exist for a number of vegetable species, including cucumber and melon. Cucurbita has not been extensively mapped; the map published by Lee et al. (2) contained only five linkage groups and no morphological traits. The Random Amplified Polymorphic DNA (RAPD) marker – trait linkages reported here are part of a larger RAPD map of an interspecific Cucurbita pepo x C. moschata cross. The entire map will be published elsewhere. RAPD markers are useful for making genetic maps in minor crops, as they are much less costly than Restriction Fragment Length Polymorphisms (RFLPs) or Simple Sequence Repeats (SSRs). However, they do have some recognized drawbacks. They are dominant markers. Reproducibility can be poor, particularly of complex banding patterns. Perhaps most significantly, RAPD markers cannot be readily transferred between populations, as they are identified by size rather than sequence. Thus RAPDs are most useful for marker assisted selection when they are tightly linked to a disease resistance gene or other trait which has been transferred from a single donor line into many different populations. Many of the difficulties with the reproducibility of markers can be solved by sequencing the RAPD band and creating a longer, more specific primer that identifies a Sequence Characterized Amplified Region (SCAR). Material and Methods: Plant material. The mapping population consisted of 162 BC1 individuals from a cross between a commercial yellow straightneck (C. pepo) inbred and C. moschata ‘Nigerian Local’. The yellow squash was the Cucurbit Genetics Cooperative Report 24:91-93 (2001) recurrent parent. ‘Nigerian Local’ is resistant to zucchini yellow mosaic virus (ZYMV) and has been much used as a source of resistance in summer squash. The yellow squash inbred is resistant to powdery mildew. Morphological traits included precocious yellow fruit (the B gene), fruit flesh color, the intensity of fruit color, warts, mottled leaves, fruit shape, and spiny stems. The population segregated for resistance to the two diseases and for the morphological traits described above. Assessment of Phenotype. Plants in the mapping population and five individuals from each parental line were grown to maturity in the greenhouse in Corvallis, Oregon from September through January, 1999-2000. All morphological data were collected using simple observation. Powdery mildew resistance was determined based on the severity of symptoms after extensive natural infection. Attempts were made to control the mildew only on the ‘Nigerian Local’ plants, which were known to be extremely susceptible. Virus inoculation with ZYMV was done on mature plants using a high-pressure paint sprayer to apply the inoculum directly to the growing points. We inoculated mature plants so that the virus symptoms would not interfere with data collection on the other morphological traits. One month after inoculation plants were visually rated for virus symptoms and tissue was collected for Enzyme Linked Immunosorbant Assay (ELISA) testing. DNA Extraction and RAPD Analysis. DNA was extracted from newly expanded true leaves using a CTAB –chloform:isoamyl method (1). Following extraction the DNA was further purified by running each sample through a spin column re-precipitating the DNA (Genemate PCR Pure kit). This was done to remove salts and other compounds that were interfering with the Taq polymerase; it permitted us to reduce the amount of Taq in the reaction by 50%. DNA was quantified with a DyNAQuant fluorometer (Hoechst) and working solutions were standardized at 5 ng/ul DNA. Parents were screened with 378 10-mer primers from Operon and the University of British Columbia. 91
Those primers that had strong bands present in Nigerian Local but not in the yellow squash were used to amplify the mapping population. Because the mapping population was the BC1 to the yellow squash parent, bands present in that parent did not segregate in the progeny. Reaction mixtures contained 30 ng of plant genomic DNA, 1.5 mM MgCl2, 0.1 mM dNTPs, 3 pmole of primer (Operon), 0.5 units of Taq DNA polymerase (Promega), and 1X Taq buffer in a total volume of 15 ul. The amplification program was 2 min. at 94°C followed by 5 cycles of 5 seconds at 94°C, 1 min. at 37°C, 30 sec. at 54°C, a 7 min. ramp to 72°C, and 2 min. at 72°C and 30 cycles of 5 seconds at 94°C, 1 min. at 37°C, 30 sec. at 54°C, 2 min. at 72°C ending with 15 min. at 72°C. Because of the size of the population (162 BC1 individuals, 5 samples from each parental line, and parental and H20 controls, for a total of 178 samples) it was necessary to amplify the population in two batches for each primer. Parental and H2O controls were included in each batch, to enable us to detect any lack of repeatability between batches. In the interest of speed, four thermocyclers (an MJR-100, a Perkin-Elmer 9600 and two Perkin-Elmer 9700s) were used, but both batches for each primer were run on the same machine. In addition, the amplification programs were adjusted to be as identical as possible on the four machines. The PCR products were separated on 1.5% agarose (Seakem LE from FMC or Ultrapure from Gibco) in 0.5% TBE buffer. Size was determined with a 100-bp ladder (Promega). The gels were stained with ethidium bromide and recorded on Polaroid 667 film. Only strong, repeatable bands were scored. Results: The yellow squash parent had smooth, intense yellow preanthesis fruit, which matured golden yellow. Peduncles were yellow and the fruit flesh was white. Leaves were uniform green. It was resistant to powdery mildew, but susceptible to ZYMV. The Nigerian Local parent had warty, intense black-green fruit and white flesh. Other plants in the population had white fruit or mottled dark- and lightgreen fruit. The leaves were mottled with silver. It was resistant to ZYMV but highly susceptible to powdery mildew. The F1 plant had warty fruit, which was bicolor for intense yellow/intense green, and a green peduncle. The BC1 progeny segregated for bicolor, peduncle color, color intensity, orange or Cucurbit Genetics Cooperative Report 24:91-93 (2001) white flesh, wartiness, mottled leaves, powdery mildew susceptibility and ZYMV susceptibility. The morphological traits associated with markers were two affecting fruit color (including the economically important precocious yellow B gene), mottled leaf, and a virus resistance gene complementary to ZYMV resistance (Zym). RAPD markers are identified by the primer and the size of the band in base pairs. Map units are Kosambi centimorgans. The precocious yellow gene (B) from the yellow squash parent was linked in repulsion to Operon I101700 at 27.1 cM. This linkage is somewhat questionable both because it is loose, and because I101700 and the RAPD markers most closely linked to it show distorted segregation, while B segregated 1:1 as expected. Operon H14600 and UBC 4891200 flanked the dominant mottled leaf allele (M) from Nigerian Local. H14600 is 13 cM from M, and 4891200 is 16.3 cM away. The actual linkages may be tighter, as the mottle-leaf trait was under-expressed in our low-light greenhouse. A gene controlling the intensity of mature fruit color is flanked by Operon B14750 17.8 cM away on one side and Operon G17700 9.7 cM away on the other. Both markers are linked to the fruit color gene in repulsion. A complementary gene for ZYMV resistance is 26.7 cM from Operon L141050. They are linked in coupling. The primary resistance gene Zym is epistatic to this gene, whose phenotype is a positive ELISA score for a symptomless plant one month after inoculation with ZYMV. We are currently conducting progeny tests to further understand this new gene. Allelism tests have not been done to determine its relationship to Zym-2 and Zym-3 (3). Other genes which have been mapped but remain unlinked are powdery mildew resistance (pm), orange fruit flesh, Ep, Zym, and warts (Wt). Discussion: These five marked loci are only a beginning. They are not likely to be of great use in marker assisted selection because of the poor transferability of RAPDs, linkages in repulsion to the traits in C. pepo, and loose linkages. However, we hope to add additional markers to the squash map, including SSR markers, which are highly transferable. Progeny testing currently under way will tell us more about the new ZYMV resistance locus, and bulked segregant analysis may let us find markers which are more tightly linked. These could 92
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Cucurbit Genetics Cooperative Coope
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The Cucurbit Genetics Cooperative (
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Georgia, Statesboro, GA, “Evaluat
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Cucurbit Genetics Cooperative Finan
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Table 1. The composition of culture
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Prezygotic barriers have previously
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Cucumber Inbred Line USDA 6632E Jac
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Multiple Shoot Induction from the S
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Table 3. Effects of combinations of
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concentration of NAA (0.1 mg/l) wit
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eplicate. Shoot differentiation and
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Table 2. Shoot induction efficiency
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Figure 1. The viability of Cucumis
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Gynogenesis in a Dihaploid Line of
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Resistant Blister Reaction to Powde
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Table 1. Analysis of variance for p
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Fusarium Wilt Resistance in Eight I
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aised PM seedlings and plants, alth
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Table 1. F2 single factor segregati
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of the loci (e.g., M7675, U13831, A
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Table 1. Percentage of RAPD band pr
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Inodorus Primer Casaba Honeydew Cha
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Frequency of RAPD Polymorphisms in
Page 51 and 52: BC551 76.6 92.9 38.5 20.5 38.9 42.2
Page 53 and 54: Powdery Mildew: An Emerging Disease
Page 55 and 56: Table 1 (continued).z Cultivar Coun
Page 57 and 58: Disease Severeity Rating Figure 1.
Page 59 and 60: In the present study C. lanatus ent
Page 61 and 62: Lakes, NJ) was a 0.5 cc (50 unit) s
Page 63 and 64: Characterization of a New Male Ster
Page 65 and 66: Table 1. Progeny test of male steri
Page 67 and 68: Progeny Diploid Year Season generat
Page 69 and 70: elatively small during its developm
Page 71 and 72: content had no significant effect o
Page 73 and 74: Stars-N-Stripes 26.3 12.0 35 8.5 5.
Page 75 and 76: 11. Mozumder, B.K.G, N.E. Caroselli
Page 77 and 78: Table 1. Vine length of watermelon
Page 79 and 80: Survey of Watermelon Trialing Metho
Page 81 and 82: assigned to adjacent plots (Table 2
Page 83 and 84: Variation among tropical pumpkin cu
Page 85 and 86: Table 1. Aggressiveness of powdery
Page 87 and 88: A large variability of aggressivene
Page 89 and 90: Table 1: Observed reaction of melon
Page 91 and 92: Same Gene for Bush Growth Habit in
Page 93 and 94: Relationship between Fruit Shape an
Page 95 and 96: Table 3. Seed yield per fruit and s
Page 97 and 98: The four cultivar-groups differed m
Page 99 and 100: Lee et al. (17) developed a RAPD ma
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Page 105 and 106: Genetic Variability in Pumpkin (Cuc
Page 107 and 108: those obtained by the ANOVA and the
Page 109 and 110: Agarose discs were prepared as desc
Page 111 and 112: Table 4. Effect of different densit
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Page 115 and 116: Table 1. Components used for cultur
Page 117 and 118: 6. Jia, Shi-rong, You-ying Fu and Y
Page 119 and 120: the preceding one or two days (depe
Page 121 and 122: Gene List 2001 for Cucumber Jiahua
Page 123 and 124: Ar - Anthracnose resistance. One of
Page 125 and 126: de I determinate habit. Short vine
Page 127 and 128: gi - ginkgo. Leaves reduced and dis
Page 129 and 130: Mdh-4 Mdh- 3 Malate dehydrogenase-4
Page 131 and 132: h from 'Gy 2 cp cp', 'Spartan Salad
Page 133 and 134: wf - White flesh. Intense white fle
Page 135 and 136: M16056 Cotyledon cDNA Encoding ribu
Page 137 and 138: (for CsPK2.2) AB001590 Hypocotyl RN
Page 139 and 140: Caruth, T. F. 1975. A genetic study
Page 141 and 142: John, C. A. and J. D. Wilson. 1952.
Page 143 and 144: Ohkawa, J., A. Shinmyo, M. Kanchana
Page 145 and 146: Shimizu, S., K. Kanazawa, and A. Ka
Page 147 and 148: Youngner, V. B. 1952. A study of th
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Page 151 and 152: Denlinger, Phil Mt. Olive Pickle Co
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Keita, Sugiyama Kurume Branch, Natl
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germplasm evaluation and conservati
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Sarfatti, Matti Hazera Ltd., Resear
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Alabama Fenny Dane Arkansas Ted Mor
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Covenant and By-Laws of the Cucurbi
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ARTICLE IX. General Prohibitions No
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