The total genomic DNA of the two parents and the individual
progeny plants was isolated from 0.5 g of leaf tissue according to
the DNA trap method developed by DNA Technology Laboratory,
Kasetsart University, Kamphaeng Saen, Thailand. The PCR reaction
was performed in a 10 ml reaction mixture containing 2 ml of
template DNA (50 ng), 1 ml of 10 PCR buffer, 0.8 ml of 25mM
MgCl2 (final concentration 2 mM), 2 ml of 1 mM dNTPs (final
concentration 0.2 mM), 0.4 ml of 5mM forward and reverse
primers (final concentration 0.2 mM) and 0.5 ul of Taq DNA
polymerase (final concentration 0.5 units). The volume was raised
to 10 ml with distilled water. The sample was covered with one
drop of mineral oil. The PCR reaction was initiated by denaturation
at 94 8C for 3 min followed by 35 cycles of 94 8C for 30 s, 55 8C for
30 s and 72 8C for 1 min and 30 s. A final 5 min incubation at 72 8C
was allowed for the completion of primer extension.