燕麦SCoT分子标记体系的优化及引物筛选

Optimization of System of SCoT-PCR and Selection of Primers in Oat

【目的】本研究对影响燕麦目标起始密码子多态性聚合酶链式反应(SCoT-PCR)的4个因素(模板DNA、引物、Mix混合液用量及退火温度)进行了优化,并通过最优体系进行了多态性引物的筛选。【方法】以4份燕麦品种DNA为模板,采用L_(9)(3^(3))正交试验确定模板DNA、引物、Mix混合液用量的最优反应体系,在48~54℃温度范围设置8个梯度进行退火温度筛选,并通过最优体系对80条引物进行了多态性筛选和验证。【结果】优化后的反应体系总体积为20μl,包含2μl模板DNA(10 ng·μl^(-1))、2μl引物(10μmol·L^(-1))、9μl Mix和7μl ddH_(2)O。筛选出了扩增效果和多态性较好的36条引物及各自的最佳退火温度。【结论】成功优化建立燕麦SCoT-PCR扩增体系,该体系的建立将为燕麦品种遗传多样性分析、品种指纹图谱构建以及分子标记辅助育种等提供重要的参考依据。

【Objective】The study aimed to optimize the four factors(template DNA,primer,mix and annealing temperature)that affected the SCoT-PCR(start codon target polymorphism)system of oat,and screen the polymorphic primers through the optimal system.【Method】Four oat varieties DNA were used as the template,and the orthogonal experiment of L_(9)(3^(3))was designed to determine the optimal reaction system of template DNA,primer and mix.Then,eight gradients in the temperature range of 48-54℃ was set for annealing temperature screening,and a total of 80 primers were screened and verified by the optimal system.【Result】The total volume of the optimized reaction system was 20μl,including 2μl template DNA(10 ng·μl^(-1)),2μl primer(10μmol·L^(-1)),9μl mix and 7μl ddH_(2)O.A total of 36 primers with good amplification and polymorphism and their optimal annealing temperature were screened out.【Conclusion】Finally,the SCoT-PCR system in oat was established successfully,which would provide an important reference for genetic diversity analysis,genetic map construction,molecular assisted breeding and other aspects of oat varieties.