川山茶ISSR-PCR及SSR-PCR优化体系建立及比较  

Optimization of Sichuan Camellia ISSR-PCR and SSR-PCR reaction systems

作　　者：刘家艳 权俊萍 沈薛洁 卢娟芳 田波 陈启航 

机构地区：[1]重庆市南山植物园管理处,重庆400065 [2]西南大学园艺园林学院,重庆400716

出　　处：《江苏林业科技》2016年第5期1-7,共7页Journal of Jiangsu Forestry Science & Technology

基　　金：重庆市科学技术委员会基础前沿研究项目(cstc2014jcyj A1669);重庆市园林局科技示范项目(园科字2013(9))

摘　　要：以川山茶品种"茶睡莲"为试验材料,以改良CTAB法提取高质量DNA,采用正交设计L16(45),探讨了Mg^(2+)、dNTPs、引物、TaqDNA聚合酶和模板DNA浓度对川山茶ISSR-PCR和SSR-PCR反应体系的影响。建立了相关优化体系:20μL的ISSR-PCR扩增体系中含20 ng模板DNA,2μL10×Buffer,2 mmol/L Mg^(2+),0.15 mmol/L dNTPs,0.6μmol/L引物和1 U TaqDNA聚合酶,扩增退火温度为55℃,35个循环;20μL的SSR-PCR扩增体系中含50 ng模板DNA,2.5 mmol/L Mg^(2+),0.05 mmol/L dNTPs,0.2μmol/L引物和0.5 U TaqDNA聚合酶,最佳退火温度为52℃,最佳循环数为38。应用该优化体系,分别用27个川山茶品种DNA进行了ISSR-PCR扩增和SSR-PCR扩增,结果显示,建立的优化体系具有较高稳定性。To obtain stable ISSR-PCR and SSR-PCR amplification systems of Camellia japonica,the optima were explored by the orthogonal design in 5 factors（ DNA template,Mg^（2＋）,dNTPs,primers and TaqDNA polymerase） at 4 levels with variety ‘Chashuilian ＇ as tested material. The experimental results showed that the optimized ISSR- PCR system for Camellia japonica ‘Chashuilian＇was established as follows,20 ng DNA template,2 μL 10×Buffer,2 mmol / L Mg^（2＋）,0. 15 mmol / L dNTPs,0. 6 μmol / L primer and 1 U TaqDNA polymerase and the optimal annealing temperature was 55 ℃ for UBC853 primer. And the optimized SSR- PCR system was established as follows,50 ng DNA template,2 μL 10 × Buffer,2. 5 mmol / L Mg^（2＋）,0. 05 mmol/L dNTPs,0. 2 μmol/L primer and 0. 5 U TaqDNA polymerase and the optimal annealing temperature was 52 ℃ for A55 primer. After 27 cultivars of Sichuan Camellia were used to test the stabilization of the optimized reaction system,respectively,the results indicated that the optimized reaction system was very stable.

关 键 词：川山茶 ISSR-PCR SSR-PCR 引物 退火温度 TAQDNA聚合酶 正交设计 优化体系 

分 类 号：S685.14[农业科学—观赏园艺]