| 摘要: |
| 该研究采用L25(56)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,ddH2O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25 μL,含有8 μL ddH2O,1 μL模板DNA(80 ng·μL-1),1 μL引物(8 μmol·L-1)和15 μL 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SCoT4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。 |
| 关键词: 目标起始密码子多态性(SCoT), 单因素试验, 正交设计, 引物筛选, 品种鉴定 |
| DOI：10.11931/guihaia.gxzw201808008 |
| 分类号:Q949.9, O413 |
| 文章编号:1000-3142(2019)05-0608-07 |
| Fund project:河南省自然科学基金(182300410018); 国家NSFC-河南人才培养联合基金(U1304304); 河南省教育厅科学技术研究重点项目(14B180028); 河南师范大学2016年度国家级项目培育基金(2016PL18); 大学生创新创业训练计划项目(0424, 0438)[Supported by the Henan Natural Science Foundation(182300410018); the National NSFC-Henan Talent Training Joint Fund(U1304304); Henan Provincial Department of Education Science and Technology Research Key Program(14B180028); Henan Normal University 2016 National Program Cultivation Fund(2016PL18); Undergraduate Innovation and Entrepreneurship Training Program(0424, 0438)]。 |
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| SCoT molecular marker system and fingerprint in Rehmannia glutinosa |
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YANG Ke1, ZHOU Yanqing1,2,3*, DUAN Hongying1, GUO Mengmeng1
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1. College of Life Sciences, Henan Normal University, Xinxiang 453007, Henan, China;2. Key Laboratory for Microorganisms and Functional
Molecules, Henan Normal University, Xinxiang 453007, Henan, China;3. Engineering Technology Research Center of Nursing and
Utilization of Genuine Chinese Crude Drugs, Henan Normal University, Xinxiang 453007, Henan, China
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| Abstract: |
| We used L25(56)orthogonal design and single factor method to optimize the five factors(template DNA concentration, primer concentration, ddH2O, 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 ddH2O, 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. |
| Key words: target initiation codon polymorphism(SCoT), single factor test, orthogonal design, primer screening, variety identification |
