To provide technical support for construction of genetic linkage map and molecular markers assisted breeding of sunflower, an optimized AFLP reaction system using double-digestion of Pst Ⅰ and Mse Ⅰ endonuclease has been established. The extraction method of genomic DNA, the DNA digestion and ligation system and several key factors affecting the PCR selective amplification are optimized. In addition, the primer combination which suits for analysis of AFLP is also screened in sunflower. The results show that the high quality of genomic DNA as PCR template can be extracted with the improved CTAB method, and that the DNA digestion and ligation can be used as a one-step process with the reaction time of 14 hours. In the optimized selection amplification system of total volume of 20 μL, the Mg²⁺ concentration is 2 mmol/L, dNTPs concentration is 0.3 mmol/L, the primers concentration is 0.6 mmol/L and the products of pre-amplification for selective amplification are diluted to 30 times. Moreover, 48 pairs stable and polymorphisms primer have been selected for AFLP analysis in sunflower.

sunflower  /  AFLP  /  establishment and optimization of the system  /  screening of primer pairs