Citrullus lanatus (Thunb.) Matsum. & Nakai - Cucurbit Breeding ...

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number of amplification reactions needed and allowed an initial screen of polymorphic markers for linkage analysis. Once polymorphic markers were identified among the DNA bulks, they could be used for a mapping experiment on the entire segregating populations, thus increasing the chances of finding linked markers for the trait of interest with fewer reactions. Even though this technique was developed for RFLP markers, it can be easily adapted to any molecular marker. The objective of this study was to optimize methodologies and protocols for the identification of molecular markers linked to resistance to gummy stem blight in watermelon. In addition, we tested 355 primers (176 SSR, 15 ISSR, 68 EST-based, and 96 RAPD) for polymorphism among the resistant and susceptible parents of our F 2 populations, segregating for resistance to gummy stem blight. We used BSA to identify candidate molecular markers linked to resistance, and mapped them on our F 2 populations. Materials and Methods Germplasm and Crosses In the experiment, we used two families developed from the two crosses PI 189225 × 'NH Midget' and PI 482283 × 'NH Midget'. PI 189225 and PI 482283 (resistant parents) were C. lanatus var. citroides. 'NH Midget' (susceptible parent) was C. lanatus var. lanatus. 'NH Midget' was obtained from commercial seed stocks and the plant introduction (PI) accessions were obtained from the Southern Regional Plant Introduction Station at Griffin, Georgia. For each family, we developed four generations (P aS 1, P bS 1, F 1, F 2, F 3) in the greenhouses at North Carolina State University in Raleigh, North Carolina. Inoculum Preparation Originally, the isolate of D. bryoniae was obtained from diseased cucumber tissues harvested from naturally-infected plants in Charleston, South Carolina in 1998. In the fall of 2001, we reisolated the strains of D. bryoniae from watermelon plants that were artificially inoculated with the isolates in our greenhouses using the technique described here. Pycnidia were identified with a dissecting microscope (20×) and transferred to Petri plates containing potato dextrose agar (PDA) (25 ml/Petri plate). Isolates were selected from the first 133
subculture on artificial medium based on macroscopic observations: colonies dark in color and showing concentric circles of growth were kept and transferred to fresh PDA. Cultures that did not appear contaminated by other fungi or bacteria were transferred to a medium containing 25% PDA to stimulate abundant sporulation. Finally, we observed pycnidia, pseudothecia and spores to verify that their shape and size matched those of D. bryoniae as published (Zitter et al., 1996). For long-term storage (Dhingra and Sinclair, 1995), we transferred the fungus onto a disk of sterile filter paper (Whatman #2, 70 mm diameter) sitting over a layer of PDA in a Petri plate, subcultured the fungus for 2 to 4 weeks, dehydrated the filter paper disk and the mycelium for 12 to 16 hours at room temperatures (24±3 °C) under a sterile, laminar flow hood, cut the filter paper into squares (5×5 mm), and stored them in sterile test tubes in a refrigerator (3±1 °C) in the dark. D. bryoniae was grown in Petri plates containing 25 mL of 50% PDA. We incubated infected Petri plates for two to four weeks at 24±2 °C under alternating periods of 12 hours of fluorescent light (40 to 90 µmol•m -2 •sec -1 PPFD) and 12 hours of darkness until pycnidia formed. For the inoculations, we prepared a spore suspension by flooding the culture plates with 10 mL of sterile, distilled water, and gently scraping the surface of the agar with an L-shaped sterile glass rod to remove the spores from the mycelia. We filtered the liquid from each pan through four layers of sterile cheesecloth to remove dislodged agar and some mycelia. The final pH of the inoculum was not adjusted. We measured spore concentration with a hemacytometer and adjusted to a concentration of 5•10 5 spores•mL -1 by adding deionized water. Tween 20 (0.06 g•L -1 ) was added to the inoculum to keep the spores well dispersed in the inoculum solution (Song et al., 2004). Cultural Practices Seeds were planted in 72-square-cell plug flats for all generations and families. Seedlings from the F 2 seeds were transplanted into the greenhouse in black polyethylene potting bags (vol ≈ 11 L). We used a soilless mix (Canadian sphagnum peat moss, perlite, vermiculite, processed pine bark) and fertigated as needed using a 10:10:10 NPK soluble fertilizer. F 2 plants in the pollination greenhouse were sprayed with fungicide and insecticide as needed, starting only after the collection of leaves for DNA extraction. For each of the F 2 plants, we grew a single runner on trellises and, starting after the collection of leaves for DNA extraction, we removed laterals and self-pollinated open flowers daily by hand. Fruit were harvested 40 days after pollination. Seeds 134
Page 1 and 2:
Abstract GUSMINI, GABRIELE. Inherit
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Biography Gabriele Gusmini was born
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Currently, Gabriele is planning his
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Table of Contents LIST OF TABLES...
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Germplasm and Crosses..............
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GENERAL INTRODUCTION LIST OF TABLES
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GENERAL INTRODUCTION LIST OF FIGURE
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HERITABILITY AND GENETIC VARIANCE E
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Brief History of Watermelon Breedin
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absence of trichomes on the leaves
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The genetics of the flesh color in
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The primary nematode species attack
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1975), but no gene has been found f
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Henderson, W.R., G.H. Scott, and T.
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Shimotsuma, M. 1963. Cytogenetical
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Table 1. Watermelon cultivars with
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Figure 2. Cultivar Allsweet, releas
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Figure 4. Watermelon seeds size: 1
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Introduction The inheritance of rin
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stripes. In his study, Shimotsuma c
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Diamond'), GG mm = mottled dark gre
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Shimotsuma, M. 1963. Cytogenetical
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Figure 2. Cultivars Japan 6 and Chi
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Figure 4. Cultivars Crimson Sweet (
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Abstract The watermelon [Citrullus
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The rind (skin) colors and patterns
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two hybrids are the original canary
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Names and symbols for new genes pro
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Salmon Yellow and Red Flesh During
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During our experiments we did not f
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Poole, C.F. and P.C. Grimball. 1945
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Table 2. Single locus goodness-of-f
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Table 6. Continued. z Generation To
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Figure 1. Intermittent stripes in '
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Figure 3. Moons and stars induced o
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Figure 5. Examples of unexpected fl
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Abstract High yield is a major goal
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States appears to be narrow, with m
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y using only two replications from
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value=0.0001), indicating that cult
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Levi, A., C.E. Thomas, A.P. Keinath
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Table 1. Analysis of variance (degr
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Table 2. Continued. Yield z Fruit s
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Table 2. Continued. Yield z Fruit s
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w L/D = length/diameter ratio measu
Page 95 and 96:
Abstract The cultivated watermelon
Page 97 and 98: dominance, and epistatic effects we
Page 99 and 100: thumped (Maynard, 2001). Weights we
Page 101 and 102: The giant-fruited parents had a lar
Page 103 and 104: Literature Cited Brar, J.S. and K.S
Page 105 and 106: Table 1. Phenotypic variances by ge
Page 107 and 108: Table 2. Variance and heritability
Page 109 and 110: t h 2 n = narrow-sense heritability
Page 111 and 112: Table 3. Continued. Effective Facto
Page 113 and 114: Figure 1. Each of the three cultiva
Page 115 and 116: CHAPTER FIVE HERITABILITY AND GENET
Page 117 and 118: fungus that is seed-borne (Lee et a
Page 119 and 120: the technique described herein. Pyc
Page 121 and 122: 9 = plant dead. Plants with a disea
Page 123 and 124: The possible gain from selection pe
Page 125 and 126: Based on our data, Norton may not h
Page 127 and 128: Gusmini, G., T.C. Wehner, and G.J.
Page 129 and 130: Sherbakoff, C.C. 1917. Some importa
Page 131 and 132: Table 1. Single locus goodness-of-f
Page 133 and 134: Table 1. Continued. z Generation To
Page 135 and 136: w Expected was the hypothesized seg
Page 137 and 138: on leaves only; 5 = some leaves dea
Page 139 and 140: on leaves only; 5 = some leaves dea
Page 141 and 142: z Data are ratings from four famili
Page 143 and 144: CHAPTER SIX PRELIMINARY STUDY OF MO
Page 145 and 146: fungus that is seed-borne (Lee et a
Page 147: approach was successful in assembli
Page 151 and 152: We extracted DNA using an extractio
Page 153 and 154: products using the Genescan ® -500
Page 155 and 156: (DNA/Polysaccharides) and 260/280 (
Page 157 and 158: Future Research Based on our prelim
Page 159 and 160: Gusmini, G., T.L. Ellington, and T.
Page 161 and 162: Perin, C., L.S. Hagen, V. De Conto,
Page 163 and 164: Table 2. Analysis of variance for t
Page 165 and 166: 029 v 1 . 3 3 . . 3 4 3 3 . . . + B
Page 167 and 168: Table 3. Continued. 071 v 1 3 . 3 4
Page 169 and 170: Table 3. Continued. 113 2 4 5 5 4 3
Page 171 and 172: Table 3. Continued. 155 2 4 5 4 . 4
Page 177 and 178: Figure 1. Agarose gel electrophores
Page 179 and 180: 1 5 10 15 20 25 30 35 + + + + + + +
Page 181 and 182: The genetics of rind color (or patt
Page 183 and 184: were not successful, even though se
Page 185 and 186: Breeding Methods Traits of interest
Page 187 and 188: alternative to biotechnology and te
Page 189 and 190: The solution to these problems is n
Page 191 and 192: Cucurbit Gene List Committee. 1979.
Page 193 and 194: Henderson, W.R., G.H. Scott, and T.
Page 195 and 196: Matthews, P. (ed.). 1993. The guinn
Page 197 and 198: Provvidenti, R. 1991. Inheritance o
Page 199 and 200:
Souza, F.F.d., M.A.d. Queiroz, and
Page 201:
Wright, S. 1968. The genetics of qu
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