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

in a 10 µL reaction mixture containing 20.0 mM Tris Hydroxymethyl Aminomethane (Tris-HCl, pH 8.8), 0.1%
Triton-X-100, 2.0 mM MgSO 4, 10.0 mM KCl, 10.0 mM (NH 4) 2SO 4, 250µM dATP, dCTP, dGTP, and dTTP
(Promega U.S., Madison, Wisconsin), 1.25 µM primer, one unit of Taq DNA Polymerase (New England
Biolabs®, Beverly, Massachusetts), and ~15 ng of template DNA. PCR amplification reactions were repeated
for 40 cycles for SSR and EST-based markers and 35 cycles for ISSR markers (SSR/EST-based: 15 s
denaturation at 92.0 °C, 15 s annealing at 52.0 °C, 120 s elongation at 72.0 °C; ISSR: 25 s denaturation at
94.0 °C, 60 s annealing at 50.0 °C, 120 s elongation at 72.0 °C).
The 25 µL reaction mixture used for PCR amplification reactions of RAPD markers contained
20.0 µM NaCl, 50.0 mM Tris Hydroxymethyl Aminomethane (Tris-HCl, pH 9.0), 1% Triton-X-100, 0.01%
gelatin, 1.6 mM MgCl 2, 200 µM dATP, dCTP, dGTP, and dTTP (Promega U.S., Madison, Wisconsin), 0.2 µM
primer, seven units of Taq DNA Polymerase (New England Biolabs®, Beverly, Massachusetts), and ~25 ng of
template DNA. PCR amplification reactions were repeated for 50 cycles (40 s denaturation at 93.5 °C, 70 s
annealing at 48.0 °C, 120 s elongation at 72.0 °C).
Gel Electrophoresis
We separated amplification products by electrophoresis on agarose gel (1.5% agarose in 1× Tris-
Acetate-EDTA (TAE) for SSR and EST-based markers, 2.0% agarose in 1× TAE for ISSR and RAPD
markers), using 1× TAE as running buffer. The gel contained ethidium bromide (0.67 µg/mL) to stain the DNA
fragments. We visualized DNA fragments on a UV trans-illuminator. We calculated the molecular weights of
the amplification products using the 100 bp DNA ladder (Promega U.S., Madison, Wisconsin).
Amplification products of SSR and EST-based markers that did not show polymorphism on agarose
gel were also analyzed by electrophoresis in acrylamide gel. An ABI PRISM ® 377 DNA Sequencer (Applied
Biosystems, Foster City, California) was used. The acrylamide gel used was the Long Ranger ® Singel ® Pack
(Cambrex Bio Science, Rockland, Maine). We fluorescently labeled DNA fragments with the 6-FAM
(fluorescein) fluorophore. 6-FAM had the adaptor AAC AGC TAT GAC CAT GA at the 3' end. 6-FAM was
incorporated in the PCR reaction buffer (0.25 µM). We calculated the molecular weights of the amplification
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