甜叶菊随机扩增多态性DNA技术优化及亲缘关系研究

以甜叶菊为试材,对影响其随机扩增多态性DNA标记反应体系的7个因子进行优化,同时对来自国内外的12个品种进行亲缘关系分析。结果表明,20μL的优化体系包括:双蒸水13.6μL,10×Buffer(含15mmol/LMgCl2)溶液3μL,2.5mmol/L的dNTPS1.2μL,10μmol/L的引物1μL,20ng的模板DNA1μL,1UTaq聚合酶;热循环程序为:94℃预变性4min,94℃变性30s,35℃退火30s,72℃延伸1min,40个循环,最后72℃延伸7min。聚类图结果表明,品种间的遗传距离变异范围为0.12～0.88,在遗传距离为0.37时,8个引物可将12个品种分为四大类,来自日本的2个品种与国内品种关系较远。

Study on optimization of random amplified polymorphic DNA reaction system and genetic relationship in Stevia rebaudiana Bertoni

Optimization of 7 factors of random amplified polymorphic DNA(RAPD) reaction system and relationship analysis of 12 kinds of cultivars in Stevia rebaudiana Bertoni were studied.The results indicated that,optimization system of 20μL included:13.6μL of ddH2O,3μL of 10×Buffer with 15mmol/L MgCl2,1.2μL of 2.5mmol/L dNTPS,1μL of 10μmol/L Primer,1μL of 20ng DNA and 1unit of Taq polymerase;thermal cycle program was:1 cycle of denaturing for 4min at 94℃,40 cycles of denaturing for 30s at 94℃,annealing for 30s at 35℃,primer extension for 1 min at 72℃,and the last cycle of extension for 7min at 72℃.The result of clustering showed that the genetic distance among 12 cultivars ranged from 0.12 to 0.88,and could be classified into four groups when the genetic distance was 0.37,while two cultivars from Japan was far away from Chinese cultivars.