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/?l),dNTP concentrations(0.15,0.25,0.30 mmol/L)and Primer concentrations(0.15,0.30,0.45 ?mol/L);another 8 tests were designed based on the single factor of Mg~(2+) 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 ?l 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 ?l reaction system,Taq polymerase was 0.02 U/?l,dNTP was 0.25 mmol/L,Primer was 0.30 ?mol/L,and Mg~(2+) 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.