Optimization of PCR system in EST-SSR analysis of Phytophthora infestans.
Journal of Northeast Agricultural University (English Edition) ; 2010 [Vol.1] Pages:-
Abstract
Late blight caused by the oomycete Phytophthora infestans Montagne de Bary is a devastating disease on potato production. A total of six parameters affecting the PCR system in P. infestans were investigated based on the template DNA of the isolate HHO6-23 and EST-SSR primer pair PiO8N. The results showed that the optimal annealing temperature was 63°C, and the optimum PCR system of EST-SSR was 25 ng template DNA, 0.5 mmol.L-1 dNTPs, 2 µL 10 × Buffer (Mg2+ free), 1.75 mmol.L-1 MgCl2, 15 pmol primer, and 1.2 U Taq DNA polymerase in total 20µL reaction system. The PCR program was initial denaturation at 94°C for 2 mm, followed by 35 cycles of 94°C for 30 s, 63°C for 30 s, and 72°C for 30 s, then a final extension step was 72°C for 7 min, and held at 4°C. In addition, using the optimal PCR system, a total of 20 isolates of P. infestans were used for testing the stability and polymorphism of the PCR amplification. The clarity and abundant polymorphism indicated that this system was stable and suitable for researching the genetic diversity of P. infestans population.
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