作 者：
刘家艳;权俊萍;沈薛洁;卢娟芳;田波;陈启航
单 位：
西南大学园艺园林学院;重庆市南山植物园管理处
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
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。
译 名：
Optimization of Sichuan Camellia ISSR-PCR and SSR-PCR reaction systems
作 者：
LIU Jia-yan;QUAN Jun-ping;SHEN Xue-jie;LU Juan-fang;TIAN Bo;CHEN Qi-hang;Nanshan Botanical Garden Management Office;School of Horticulture and Landscape Architecture,Southwest University;
关键词：
Sichuan Camallia;;ISSR-PCR;;SSR-PCR;;Primer;;Annealing temperature;;TaqDNA polymerase;;Orthogonal design;;Optimized system
摘 要：
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.
