Establishing and Optimizing AFLP Amplification Reaction System of Shiraia Bambusicola

摘要

DNA of Sharaia bambuiscola was extracted by the improved method of CTAB. The several key factors affecting the effect of DNA digestion and the PCR selective amplification such as the time of DNA digestion, the times of the per-amplified dilute products, the mount of the selective amplification primer, Taq concentration and dNTPs concentration were optimized and trialed with establishment of an optimized AFLP reaction system of S. bambusicola. The best time of digestion DNA with double endonucleases (EcoR I and MseI) was 3h. The optimized selection amplification system was 25μl reaction mix containing 2.5μl the 20 times of the per-amplified dilute products, 2.5μL 10× buffer(with Mg~(2+)), 10mmol/L primer each 1.5μL, 2.5U·μL~(-1) Taq polymerase 0.7μL, 10mmol/L dNTP 0.5μL, and 15.8μL ddH_2O. Stable and clean DNA finger print can be obtained and 3 pairs of AFLP primers with good genetic diversity were selected according to the optimized reaction system. The results will be an effective protocol for further studying the genetic stucture and differentiation, phylogenetic trees, host specificity and artificial cultivation of Shiraia bambusicola population.