油葵SRAP-PCR反应体系的建立与优化

为建立油葵SRAP-PCR的反应体系，采用单因素试验法，对Mg2+、dNTPs、引物浓度、Taq DNA聚合酶、模板DNA分别设置5~7个水平梯度，筛选出适宜的用量范围，以此为基础，再通过L16(45)正交试验设计，对影响SRAP-PCR的5个因素进行优化，建立了油葵SRAP-PCR的最佳反应体系：20 μL体系中含10×Buffer 2 μL，Mg2+ 2.75 mmol/L，dNTPs 0.18 mmol/L，Taq DNA聚合酶1.25 U，正反引物各0.3 μmol/L，模板DNA 60 ng，最佳退火温度为52.2℃。用22份油葵材料对该体系进行验证，结果显示扩增条带清晰、多态性高，说明该体系稳定可靠，可有效的用于油葵种质资源的鉴定、遗传图谱构建等研究。

Establishment and Optimization of SRAP-PCR Reaction System in Sunflower

In this study,one-factor experimental design had been used to establish the SRAP-PCR reaction system of sunflower,which including several different gradients of Mg2 ＋,dNTPs,primer concentration,Taq DNA Polymerase,template DNA and filter out the suitable range,on this basis,then optimize the five factors which influence the SRAP-PCR through the orthogonal experimental design of L16（45）. Eventually brought about an optimized system for sunflower,which was as follows：20 μL capacity of reaction system contains 10×Buffer 2 μL,Mg2＋2.75 mmol/L,dNTPs 0.18 mmol/L,Taq DNA Polymerase 1.25 U,forward primer and reverse primer 0.3 μmol/L,template DNA 60 ng,the best annealing temperature 52.2℃. Finally,22 varieties of sunflower were used to test the optimized PCR reaction system stability. The results showed that,the bands of the amplification clear and high polymorphism,so the system was reliable which could be applied in the germplasm identification and the genetic map construction of sunflower effectively.