狗牙根SRAP-PCR反应体系的优化

The Optimization of SRAP-PCR System of Bermuda Grass

本研究以狗牙根幼叶为为试材,采用单因子试验和正交设计试验2种方法,对SRAP-PCR反应体系进行优化试验,结果表明狗牙根25μLSRAP-PCR最佳反应体系为:1.5mmol/LMg2+、0.25mmol/LdNTP、0.4μmol/L引物、1.5UTaq酶和80ng模板DNA,最佳退火温度为50.6℃。运用该体系对30份狗牙根种质进行验证,电泳结果显示扩增条带清晰、多态性高,证明该体系稳定可靠,这一优化体系的建立为今后利用SRAP标记技术对狗牙根进行分子生物学研究提供了帮助。

In this paper, we attempted to establish the optimal SRAP-PCR amplification system of bermuda grass by employing single factor tests and orthogonal design approaches and using young leaves as tested material. According to our experiment, we established a SRAP-PCR system most suitable for bermuda grass, which came out total 25 μL reaction system containing 1.5 mmol/L Mg^2＋, 0.25 mmol/L dNTP, 0.4 μmol/L primer, 1.5 U Taq DNA polymerase, and 80 ng template DNA. The optimum annealing temperature was 50.6℃. We validated 30 bermuda grass germplasms by using this SRAP-PCR system, and the electrophoresis results showed that the amplification bands were clear and highly polymorphic, which proved that this system was stable and reliable. This optimized SRAP-PCR system might facilitate to be as one of the marker based PCR protocols applying for further molecular genetics research on bermuda grass.