辣椒SRAP-PCR反应体系主要影响因素的单因素和正交设计优化

Single Major Influencing Factor and It's Orthogonal Optimized Design of SRAP-PCR in Hot Pepper (Capsicum annum L.)

SRAP标记是一种新的分子标记技术,建立和优化所研究物种的SRAP反应体系是应用SRAP标记的技术关键。本研究以3个辣椒品种为试验材料,采用单因素试验设计对辣椒SRAP-PCR反应体系的5个主要影响因子(Taq酶、Mg2+、模板DNA、dNTP和引物浓度)设置8个不同的水平梯度,筛选出比较适宜的Mg2+、dNTP、Primer、Taq酶、模板DNA浓度的范围。在此基础上,进一步采用L16(45)正交设计对辣椒SRAP-PCR反应体系的五因素在四个水平上进行优化实验,得出了如下的优化体系:25μl体系中包含1.5mmol/L的Mg2+、0.2mmol/L的dNTP、0.1μmol/L的单条引物、1U的Taq酶和60ng的模板DNA。经用24个辣椒材料进行扩增验证,该体系检测的结果重复性好,稳定性强。因此,本试验所获得的优化体系适用于辣椒的SRAP分子标记研究。

SRAP （Sequence-related amplified polymorphism） is a new molecular marker technology. It is critical to establish and optimize its PCR reaction system for application to target species. In this experiment, using three hybrids of hot pepper as materials, an optimization experiment with single factor design, which was involved in five factors of Taq DNA polymerase, Mg^2＋, primer, dNTP, and DNA template with eight concentration levels each, was conducted to screen their feasible concentration range. On this basis, another one with orthogonal design of E16 （45） was carried out,and eventually brought about an optimized system as follows： 25μl of reaction system should contain 1.5 mmol/L of MgCl2, 1 U of Taq DNA polymerase ,0.2 mmol/L of dNTP mixture,0.1μmol/L of each primer, and 60 ng of template DNA. This system was tested by using 24 variaties of hot peper to be relatively stable and reproducible. Accordingly, it was suitable for SRAP analysis of hot pepper.