金樱子SRAP-PCR反应体系的建立与优化

以广西南宁产的金樱子叶片为材料,采用L16(45)正交试验设计,对SRAP-PCR反应体系中的Mg2+、dNTPs、TaqDNA聚合酶、引物和模板DNA用量5因素进行了优化。结果表明:适用于金樱子SRAP-PCR的最佳反应体系为:反应总体积10μL,包含2.0 mmol/L Mg2+、0.4 mmol/L dNTPs、0.5 UTaqDNA聚合酶、3μmol/L引物、25ng DNA及10×PCR Buffer;各因素对PCR反应影响由大到小依次为:Mg2+、DNA、dNTPs、引物、TaqDNA聚合酶。用7份不同来源的金樱子材料对优化的SRAP-PCR反应体系进行验证,均获得了条带清晰、多态性丰富的扩增图谱,证实了该体系的稳定可靠。

Establishment and optimization of SRAP-PCR reaction system for Rosa laevigata

The orthogonal design L16(45) was designed to optimize SRAP-PCR reaction system for leaf DNA of Rosa laevigata from Nanning of Guangxi province,with five factors(Mg2+,dNTP,primer,template DNA and Taq polymerase)at four levels respectively.The results showed that the suitable SRAP-PCR reaction system for R.laevigata was that total 10 μL reaction system including 2.0 mmol/L Mg2+,0.4 mmol/L dNTPs,0.5 U Taq polymerase,3 μmol/L primers,25 ng DNA and 10×PCR Buffer.And the order of factors which affect on the result of PCR were Mg2+,DNA,dNTPs,primers,and Taq polymerase.The optimal SRAP-PCR reaction system was identified by means of genomic DNA of R.laevigata from different places.And the amplification pattern with clear bandand and rich polymorphism was obtained.It is concluded that the SRAP-PCR reaction system is steady and reliable.