作者：吴鑫,雷天刚,何永睿,刘小丰,许兰珍,彭爱红,陈善春*
作者单位：西南大学园艺园林学院,西南大学柑桔研究所,中国农业科学院柑桔研究所,国家柑桔品种改良中心
摘要：通过对PCR反应程序、反应体系(DNA模板量、PCR反应体积、Mg2+浓度、dNTP浓度、Taq酶用量、引物量)、电泳检测方法的系统优化,建立了柑桔SRAP-PCR和ISSR-PCR体系;以此进行大规模引物筛选,从而建立了柑桔SRAP和ISSR分子标记技术体系。SRAP-PCR:25μL体系,模板DNA25ng,Tris-HCl10mmol/L,KCl50mmol/L,Mg2+1.2mmol/L,dNTP120μmol/L,Taq酶1.5U,引物0.4μmol/L,反应程序为94℃预变性5min,35个循环(94℃30s,47℃1min,72℃1min),72℃延伸10min;ISSR-PCR:25μL体系,模板DNA25ng,Tris-HCl10mmol/L,KCl50mmol/L,Mg2+1.6mmol/L,dNTP200μmol/L,Taq酶1U,引物0.8μmol/L。筛选出稳定性好、多态性高的24对SRAP引物和13条ISSR引物。
关键词：柑桔,SRAP,ISSR,建立,优化
Title: Establishment and Optimization of SRAP and ISSR Marker System in Citrus
Authors: Wu Xin,Lei Tian-gang,He Yong-rui,Liu Xiao-feng,Xu Lan-zhen,Peng Ai-hong
Abstract: Here we have established SRAP-PCR and ISSR-PCR system in Citrus by optimization of PCR reaction program, reaction system (template DNA, reaction volume, Mg2+, dNTP, Taq DNA polymerase and primer) and electrophoresis detection experiments. Based on these reaction systems, a mass of primers have been screened. In the end, we got optimum SRAP and ISSR marker systems in Citrus. SRAP-PCR system: template DNA 25 ng, Tris-HCl 10 mmol/L, KCl 50 mmol/L, Mg2+ 1.2 mmol/L, dNTP 120 μmol/L, Taq DNA polymerase 1.5 U, primer 0.4 μmol/L in 25 μL system, reaction program: initial denaturation step for 5 min at 94℃, followed by 35 cycles at 94℃ for 30 s, 47℃ for 1 min and 72℃ for 1 min, followed by a final extension step at 72℃ for 10 min; ISSR-PCR system: template DNA 25 ng, Tris-HCl 10 mmol/L, KCl 50 mmol/L, Mg2+ 2.0 mmol/L, dNTP 200 μmol/L, Taq DNA polymerase 1 U, primer 0.8 μmol/L in 25 μL system. 24 pairs of SRAP primers and 13 ISSR primers, with high stability and polymorphism, has been obtained.
Key words: Citrus； SRAP； ISSR； Establishment； Optimization；
文献注录：吴鑫,雷天刚,何永睿,刘小丰,许兰珍,彭爱红,陈善春*. 柑桔SRAP和ISSR分子标记技术体系的建立与优化 [J]. 分子植物育种. 2008, 01: 170-176.
基金: 科技部科研院所社会公益研究专项(2004DIB45147);市科技攻关重点项目(CSTC,2006AB1009);国家863项目(2006AA100108-4-12-4)资助
报/刊名：《分子植物育种》，发表于2008 / 第 01 期。
文献类型： [J] （文献级别：核心刊物）
页码：第 170-176 页 / 共7 页