地黄SCoT分子标记体系的建立和指纹图谱的构建

SCoT molecular marker system and fingerprint in Rehmannia glutinosa

该研究采用L_(25)(5~6)正交设计和单因素两种方法,对影响地黄SCoT-PCR反应的5个因素(模板DNA浓度,引物浓度,ddH_2O和Mix的用量以及退火温度)进行了优化。结果表明:优化后的反应体系总体积为25μL,含有8μL ddH_2O,1μL模板DNA(80 ng·μL^(-1)),1μL引物(8μmol·L^(-1))和15μL Mix,退火温度为45℃。运用30份地黄种质材料,对优化的SCoT-PCR正交体系进行多次重复验证,获得了多态性丰富、条带清晰的扩增图谱,证明该反应体系稳定可靠。利用该体系对32条SCoT引物进行两次筛选,得到14个扩增产物清晰、重复性好且多态性条带相对较高的引物。利用SCoT_4等5条引物构建了上述地黄2个种共30份种质的SCoT指纹图谱。利用这5个SCoT引物指纹图谱可将7个地黄常用栽培品种区分开。这表明SCoT分子标记体系适用于地黄主要品种亲缘关系及遗传多样性的研究,所构建的指纹图谱也为地黄常见的7个栽培品种的区分提供参考依据。

We used L 25 (5 6 ) orthogonal design and single factor method to optimize the five factors (template DNA concentration, primer concentration, ddH 2 O, Mix amount and annealing temperature) that affect the SCoT-PCR reaction of Rehmannia glutinosa . The results were as follows: The reaction system was a total volume of 25 μL containing 8 μL of ddH 2 O, 1 μL of template DNA (80 ng·μL^-1 ), 1 μL of primer (8 μmol·L ^-1 ) and 15 μL of Mix, and an annealing temperature of 45 ℃. The optimized SCoT-PCR orthogonal system was repeatedly verified by using 30 parts of Rehmannia germplasm materials, and the amplified spectrum with rich polymorphism and clear bands was obtained, which proves that the reaction system is stable and reliable. Using this system, 32 SCoT primers were screened twice, and 14 primers with clear, reproducible and relatively high polymorphic bands were obtained. Finally, SCoT fingerprints of 30 species of the above two species of Rehmannia were constructed by using five primers such as SCoT4. Using these five SCoT primer fingerprints, seven common cultivars of Rehmannia glutinosa can be distinguished. The study indicates that the SCoT molecular marker system is suitable for the study of genetic relationship and genetic diversity of the main varieties of Rehmannia glutinosa . The fingerprints constructed also provide reference for the differentiation of seven cultivars commonly found in Rehmannia glutinosa .