摘要
利用单因素梯度试验与正交试验相结合、方差分析与多重比较分析相结合的方法,对水仙简单重复序列—聚合酶链式反应(SSR-PCR)扩增反应体系中的4个因素(DNA、dNTPs、引物及Taq酶)进行优化分析,方差分析表明4个因素对SSR-PCR的影响均达到极显著水平,通过多重比较分析发现SSR引物和Taq酶对总体系的扩增结果影响最大。根据单因素梯度试验与正交试验结果,建立了适合水仙SSR标记分析的最优PCR反应体系:DNA模板2.5 ng·μL-1,10×PCR Buffer(含1.5 mmol·L-1Mg2+),dNTPs 0.25 mmol·L-1,上下游引物各0.50μmol·L-1,Taq酶1.00 U,总体积20μL。该优化体系的建立可为今后水仙SSR分析提供标准化的试验体系,为水仙种质鉴定、品种指纹图谱绘制、遗传多样性分析、群居遗传结构研究和遗传连锁图谱分析等领域提供技术支持。
Four factors including DNA , dNTPs, primers and Taq-polymerase in simple sequence repeat-polymerase chain reaction （ SSR-PCR） of Narcissus were optimized .The comparison of single factor gradient experiments and orthogonal experimental design as well as analysis of variance and multiple comparison analysis were used in optimization .Analysis of variances results showed that the factors influenced SSR-PCR significantly .It was found that SSR primers and Taq-polymerase were the major factors by multiple com-parison analysis .According to the results of single factor gradient experiments and orthogonal experimental design , the optimization PCR system was established.It contained 2.5 ng·μL^-1 DNA, 10 ×PCR Buffer （including 1.5 mmol· L^-1 Mg2＋）, 0.25 mmol· L^-1 dNTPs, 0.50 μmol· L^-1 forward and reverse primers, and 1.00 U Taq-polymerase.This PCR system may offer a standardized experiment to analysis of SSR , and technical support to identification of variety , varieties of fingerprint mapping , analysis of genetic diversity, and genetic structure of social and genetic linkage maps study of Narcissus.
出处
《福建林学院学报》
CSCD
北大核心
2014年第2期158-164,共7页
Journal of Fujian College of Forestry
基金
国家科技支撑计划项目(2007BAD07B00)
福建农林大学创新团队基金项目(cxtd12013)
福建省林业厅2013年花卉苗木品种引进与研发创新基金项目(闽林种站[2013]42号)
关键词
水仙
简单重复序列
体系优化
正交设计
多重比较
Narcissus
simple sequence repeat
system optimization
orthogonal design
multiple comparison
