| To screen the optimum ISSR-PCR reaction system for potato, the experiment was conducted by using
orthogonal design with 4 levels of 5 factors (i.e. rTaq DNA polymerase, Mg2+, DNA, dNTPs and primers). Results showed
that the optimal reaction system of ISSR-PCR was as follows: 0.5 U rTaq DNA polymerase, 2.5 mmol/L Mg2+, 75 ng DNA,
200 mol/L dNTPs and 0.8 mol/L primer in 25 L reaction volume. And the PCR reaction effects was in the order of
Mg2+ > rTaq DNA polymerase> dNTPs = primer> DNA. Then, 39 ISSR primers were screened by using the optimal reaction
system, and 15 primers with clear band, more band number and good repeatability were selected. Finally, the optimized
annealing temperatures of the 15 primers were obtained by gradient PCR. |
