摘要
[目的]优化玉叶金花SSR-PCR扩增反应体系。[方法]以红纸扇为材料,采用正交设计和单因素试验方法,从Mg^(2+)、dNTPs、TaqDNA聚合酶、引物、模板DNA浓度以及退火温度4个方面对玉叶金花SSR-PCR体系进行优化,并采用4对SSR引物和4种玉叶金花植物样品进行验证。[结果]玉叶金花20μL的SSR-PCR反应最优体系为Mg^(2+)浓度1.50 mmol/L、d NTPs浓度0.150 mmol/L、引物浓度0.45μmol/L、TaqDNA聚合酶2.00 U、模板DNA 15 ng、退火温度53.4℃。[结论]该反应体系的扩增条带清晰、稳定、重复性好,可用于玉叶金花的SSR分子标记开发、物种分子鉴定、遗传多样性分析和谱系关系构建等相关研究。
[Objective]To optimize SSR-PCR amplification reaction system for Mussaenda by orthogonal design.[Method]The orthogonal design and single factor test were adopted to optimize the SSR-PCR system using M. erythrophylla genomic DNA as template. Six factors including the concentration of Mg^（2＋）,d NTPs,Taq DNA polymerase,primer,DNA template and annealing temperature were tested separately in this system,which was further verified by four primers and samples of four Mussaenda species.[Result]The optimized SSR-PCR reaction system condition was obtained in a 20 μL system containing 1. 50 mmol/L Mg^（2＋）,0. 150 mmol/L d NTPs,0. 45 μmol/L SSR primer,2. 00 U Taq DNA polymerase and 15 ng DNA template,and the annealing temperature 53. 4 ℃.[Conclusion]With clear,stable and reproducible bands,the system can be used for SSR maker development,germplasm identification,genetic diversity and relationship analyses.
作者
郑艳
胡章立
陈涛
ZHENG Yan, HU Zhang-li , CHEN Tao(1. College of Lite Sciences and Oceanography, Shenzhen University, Shenzhen, Guangdong 518060 ;2. Shenzhen Fairy Lake Botanieal Garden, CAS, Shenzhen, Guangdong 51800)
出处
《安徽农业科学》
CAS
2018年第13期98-103,共6页
Journal of Anhui Agricultural Sciences
基金
深圳市技术创新计划技术开发项目(CXZZ20140903154251302)
深圳市城管局科研项目(201803)
