青天葵ISSR-PCR体系优化及引物筛选

Optimization of ISSR-PCR Reaction System and Primers Screening in Nerviliae fordii

目的:建立适合青天葵遗传差异分析的简单重复序列区间(ISSR)-聚合酶链式(PCR)反应体系。方法:采用正交试验设计对影响青天葵ISSR-PCR反应体系的5种因素(dNTP、模板DNA、引物、Mg2+和Taq DNA聚合酶)4个水平进行优化,并结合新复极差法,对试验中各单因素进行分析。结果:确立了青天葵最佳反应体系,即在20μL的总反应体积中含有10×PCR buffer 2μL,dNTP 225μmol·L-1,Mg2+2.5 mmol·L-1,引物0.4μmol·L-1,Taq DNA聚合酶1.0 U,模板DNA60 ng;从100条引物中筛选出16条扩增稳定、多态性丰富的ISSR引物。结论:建立的青天葵ISSR-PCR反应体系,经过24份青天葵样品检验,得出该体系稳定可靠,可用于青天葵遗传差异分析。

Objective：To establish a suitable inter-simple sequence repeat （ISSR）-polymerase chain reaction （PCR） system for analysis of genetic differences in Nerviliae fordii.Method：The ISSP-PCR was optimized using orthogonal design of five factors （dNTP,template DNA,primer,Mg2 ＋,Taq DNA polymerase） at four levels.Single factor analysis was conducted using Duncan＇s new multiple range method.Result：An optimum ISSR-PCR reaction system established for N.fordii was as follows：20 μL ISSR-PCR system contained 60 ng template DNA,2.0 μL of 10 × PCR buffer,1.0 U Taq polymerase,225 μmol ·L-1 dNTP mix,2.5 mmol ·L-1 of Mg2＋,0.4 μmol ·L-1 of primers.Sixteen ISSR primers with stable amplification and abundant polymorphism were selected from 100 ISSR primers.Conclusion：The optimized and established ISSR reaction system is stable and credible according to the testing results of 24 samples of N.fordii,which provides methodology basis for the genetic analysis of N.fordii.