大豆SCoT分子标记技术体系的优化、验证及检测

Optimization,verification and detection of SCoT molecular marker system in soybean

采用L25(56)正交试验和单因素试验2种方法对影响大豆SCoT-PCR反应的Mg2+浓度、dNTPs浓度、Taq DNA聚合酶用量、引物浓度和模板DNA用量等因素进行优化,优化后的20μL大豆SCoT-PCR体系为:Mg2+浓度2.0mmol·L-1、Taq聚合酶用量1.5U、引物浓度0.250μmol·L-1、dNTPs浓度0.2mmol·L-1、DNA模板用量30ng;退火温度最适为50.4℃。运用18个大豆品种验证,该反应体系稳定、可靠,并从82条引物中筛选出条带清晰、多态性较好的32条引物。利用大豆品种吉育75、本地黑豆及其10株F2对该反应体系进行了初步的遗传验证,结果显示,杂交后代植株中出现了双亲的位点和亲本位点的缺失。该反应体系的建立为大豆种质遗传多样性分析、遗传连锁图谱构建及分子标记辅助育种提供了新的技术手段。

Start codon targeted polymorphism （SCoT） is a novel gene targeted marker technique.L25 （56） orthogonal experiment and single factor experiment design were applied to optimize SCoT-PCR system of soybean in five factors as Mg2＋,dNTPs,primer,Taq DNA polymerase and template DNA.The results showed that the optimized system was as follows：a total volume of 20μL including 2.0 mmol · L-1 Mg2＋,0.2 mmol · L-1 dNTPs,0.250μmol · L-1 primer,1.5U Taq polymerase,30ng DNA template.The most suitable annealing temperature of primers was 50.4℃.The optimized SCoT-PCR system was tested on eighteen soybeans,and the result was stable and reliable.From the 82 primer combinations tested,32 were selected with clear band patterns and abundant polymorphism.Amplifications and genetic verification were carried out on Jiyu 75,local black soybean and progenies from Jiyu 75 × local black soybean.The results showed polymorphism bands and common bands from parents and progeny.The system provided a new technology for evaluation of genetic diversity,construction of genetic linkage map and molecular marker assisted breeding for the soybean.