Establishment and optimization of the amplified fragment length polymorphism reaction system of Carthamus tinctorins L. genome / 第二军医大学学报

ABSTRACT

Objective: To study the factors affecting amplified fragment length polymorphism (AFLP) of Carthamus tinctorins L. genome, and to develop and optimize the AFLP reaction system, laying a foundation for Marker-assisted breeding of Carthamus tinctorins L.. Methods: Cetyl-trimethyl-ammonium-bromide (CTAB) method was used to extract the genomic DNA of Carthamus tinctorins L., whose concentration and purity were determined by UV spectrophotometry. The genomic DNA was digested by restricted enzyme and ligated with one-step method or two-step method so as to find out which one is more suitable. Then the products were prepared with different dilution multiples (5, 10, 15, 20, 25 and 30) for pre-amplification and with different concentrations of gradients (10, 25, 50, 75, 100, 150 and 200) for selected amplification. Then they were subjected to PAGE electrophoresis and silver-staining after denaturing at 95°C for 8 min. Results: The AFLP reaction system for Carthamus tinctorins L. genome included the following steps: the genomic DNA was precipitated with alcohol by CTAB method to avoid contamination by any inhibitors; the DNA was completely digested by Mse I and EcoR I at 37°C for 3 hours. Two-step method was superior to one-step method in this AFLP system. The digestion time was 3 hours at 37°C and ligated at 16°C over night, with Buffer 2 (NEB Company) as the specific buffer. Furthermore, the optimal dilution was 25 folds for pre-amplification and 75 folds for selected amplification. In this reaction system, the strips in PAGE electrophoresis were clear and less decomposed. Conclusion: The AFLP reaction system in this experiment is suitable for silver-stained AFLP of Carthamus tinctorins L. analysis.