油葵SRAP-PCR反应体系的建立与优化  

Establishment and Optimization of SRAP-PCR Reaction System in Sunflower

作　　者：于志贤[1] 侯建华[1] 

机构地区：[1]内蒙古农业大学农学院,呼和浩特010010

出　　处：《中国农学通报》2013年第33期296-301,共6页Chinese Agricultural Science Bulletin

基　　金：内蒙古自然科学基金重点项目"油用向日葵抗旱相关性状的QTL分析研究"(2010Zd06);国家自然科学基金项目"向日葵抗(耐)旱相关性状基因/QTL定位及分析"(31160288)

摘　　要：为建立油葵SRAP-PCR的反应体系,采用单因素试验法,对Mg2+、dNTPs、引物浓度、Taq DNA聚合酶、模板DNA分别设置5～7个水平梯度,筛选出适宜的用量范围,以此为基础,再通过L16(45)正交试验设计,对影响SRAP-PCR的5个因素进行优化,建立了油葵SRAP-PCR的最佳反应体系:20μL体系中含10×Buffer 2μL,Mg2+2.75 mmol/L,dNTPs 0.18 mmol/L,Taq DNA聚合酶1.25 U,正反引物各0.3μmol/L,模板DNA 60 ng,最佳退火温度为52.2℃。用22份油葵材料对该体系进行验证,结果显示扩增条带清晰、多态性高,说明该体系稳定可靠,可有效的用于油葵种质资源的鉴定、遗传图谱构建等研究。In this study,one-factor experimental design had been used to establish the SRAP-PCR reaction system of sunflower,which including several different gradients of Mg2 ＋,dNTPs,primer concentration,Taq DNA Polymerase,template DNA and filter out the suitable range,on this basis,then optimize the five factors which influence the SRAP-PCR through the orthogonal experimental design of L16（45）. Eventually brought about an optimized system for sunflower,which was as follows：20 μL capacity of reaction system contains 10×Buffer 2 μL,Mg2＋2.75 mmol/L,dNTPs 0.18 mmol/L,Taq DNA Polymerase 1.25 U,forward primer and reverse primer 0.3 μmol/L,template DNA 60 ng,the best annealing temperature 52.2℃. Finally,22 varieties of sunflower were used to test the optimized PCR reaction system stability. The results showed that,the bands of the amplification clear and high polymorphism,so the system was reliable which could be applied in the germplasm identification and the genetic map construction of sunflower effectively.

关 键 词：油葵 SPAP-PCR 反应体系 

分 类 号：S565.5[农业科学—作物学]