Establishment and optimization of sequence-related amplified polymorphism amplification system for Carthamus tinctorius L / 第二军医大学学报

ABSTRACT

Objective: To study the factors influencing the amplification of sequence-related amplified polymorphism (SRAP) system in Carthamus tinctorius L. and to establish a stable SRAP reaction system, laying a foundation for molecular marker assistant breeding of Carthamus tinctorius L. Methods: Cetyltrimethylammonium bromide method was used to extract the genomic DNA of Carthamus tinctorius L. Twenty-seven tests with 3 factors at 3 levels were designed, the conditions including Taq polymerase concentrations (0.02, 0.04, 0.06 U/μl). dNTP concentrations (0.15, 0.25, 0.30 mmol/L) and Primer concentrations (0.15, 0.30, 0.45 μmol/L); another 8 tests were designed based on the single factor of Mg2+ concentrations (0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 mmol/ L). Twenty ng DNA template was added into 25 μl SRAP reaction system; the system was optimized and agarose electrophoresis was used for determination. Results: A SRAP reaction system for Carthamus tinctorius L. was established. In a 25 μl reaction system, Taq polymerase was 0.02 U /μl, dNTP was 0.25 mmol/L, Primer was 0.30 μmol/L, and Mg2+ was 3.0 mmol/ L. Target bands increased after optimization, with good reproducibility, and the amplification results were satisfactory. Conclusion: The SRAP reaction system in this experiment is suitable for analysis of Carthamus tinctorius L.