籽用西瓜SCoT-PCR反应体系优化及引物筛选
Optimization for SCoT-PCR System and Primer Selection of Seed-used Watermelon

采用L25(56)正交试验设计，对影响目标起始密码子多态性-聚合酶链式反应(SCoT-PCR)的5个因素(Taq酶用量、Mg2+浓度、模板 DNA 用量、dNTPs 浓度和引物浓度) 进行优化试验，建立了籽用西瓜SCoT-PCR反应体系：Mg2+ 2.5 mmol·L-1、dNTPs 0.15 mmol·L-1、Taq酶 1.5 U、引物 0.5 μmol·L-1、模板DNA 20 ng，总体积20 μL。比较各因素对扩增反应的结果，其中以Mg2+浓度的影响最大，Taq酶用量的影响最小。应用22个籽用西瓜品种验证该体系稳定可靠，并从 41个 SCoT 引物中筛选出扩增条带清晰、多态性丰富的22个引物，并逐一筛选出最适退火温度。该反应体系的建立为今后利用SCoT标记技术对籽用西瓜种质遗传多样性评价、指纹图谱构建、种质鉴定、分子标记辅助选择育种等研究提供了新的技术手段。
An orthogonal design L25(56) was used to optimize SCoT-PCR (start codon targeted polymorphism) amplification system of seed-used watermelon in five factors such as Taq DNA polymerase, Mg2+, DNA template, dNTPs and primer concentrations).The results showed that the optimized system was 2.5 mmol·L-1 Mg2+ , 0.15 mmol·L-1 dNTPs, 1.5 U Taq polymerase, 0.5 μmol·L-1 primer, 20 ng DNA template in 20 μL mixture.Each factor had different effect on the results.The concentration of Mg2+ was the key factor affecting the SCoT-PCR system.The optimized SCoT-PCR system was tested on 22 seed-used watermelons，and the result was stable and reliable.From the 41 primer combinations tested, 22 were selected with clear band patterns and abundant polymorphism.The most suitable annealing temperature of primers was selected.The optional reaction system provide a new technology for evaluation of genetic diversity, construction of fingerprinting, germplasm identification and molecular marker assisted breeding on seed-used watermelon.