兰属SRAP-PCR反应体系的建立与优化

为获得兰属清晰的SRAP标记图谱，对兰属SRAP-PCR反应体系进行了初步探讨，建立了扩增多态性高、重复性好、带型清晰的SRAP-PCR反应体系。最佳反应体系：在30 μL反应总体系中，Mg2+ 2.2 mmol/L、dNTPs 0.8 mmol/L、DNA模板150 ng、DNA聚合酶2.0 U，上、下游引物各1.5 μmol/L；扩增程序：在94 ℃预变性4 min，反应前5个循环在94 ℃变性1 min、35 ℃复性1 min、72 ℃延伸1 min的条件下运行，随后的30个循环复性温度提高至55 ℃，最后72 ℃延伸5 min．

Establishment and Optimization of SRAP-PCR Reaction System in Cymbidium

To obtain clear Sequence-related amplified polymorphism(SRAP) fingerprints of Cymbidium,the factors influencing SRAP analysis were studied.A reliable,effective and reproductive PCR reaction system for detecting SRAP was developed.Each 30 μL PCR reaction mixture consisted of 2.2 mmol/μL of Mg2+,0.8 mmol/L of dNTPs,150 ng of genomic DNA,1.5 μmol/L of primer and 2.0 unit of Taq polymerase.Samples were subjected to thermal profile for amplification in an oven thermocycler: 4 min of denaturing at 94 ℃,five cycles of three steps,1 min of denaturing at 94 ℃,1 min of annealing at 35 ℃ and 1 min of elongation at 72 ℃,in the following 30 cycles the annealing temperature was increased to 55 ℃,with a final elongation step of 5 min at 72 ℃.