Abstract:

L₉(3⁴) orthogonal experiment design including four factors of Mg²⁺, dNTP, Primer, Taq DNA polymerase was applied to optimize SRAP-PCR amplification system of Paspalum vaginatum, and the influence of different concentration of template DNA on the amplification were compared. The optimization of SRAP-PCR reaction system in Paspalum vaginatum was established. The results showed that the optimal SRAP-PCR reaction system was 10×PCR buffer 1μL, template DNA 50 ng, Mg²⁺ 2.50 mmol·L^-1, dNTP 150 μmol·L^-1, primer 0.4 μmol·L^-1 and TaqDNA polymerase 1.5 U in 10 μL total reaction system, respectively. Eighty-three pairs of primers with stable amplification and legible bands were screened form 100 SRAP primers through this system. Forty-four pairs of primers with rich polymorphism among 4 tested materials were screened from 83 pairs of primers. Optimization of SRAP-PCR system and primers screening in Paspalum vaginatum will provide technical assistance for the evaluation of genetic diversity, molecular marker assisted breeding and linkage map construction of Paspalum vaginatum.

Key words: Paspalum vaginatum, SRAP molecular marker, Orthogonal experiment, Optimization of system, Primers Screening

摘要：

以4个代表性海雀稗（Paspalum vaginatum）种质的叶片DNA为模板，采用L₉（3⁴）正交试验设计，对影响海雀稗SRAP-PCR反应的Mg²⁺、dNTP、引物和TaqDNA聚合酶的用量进行了优化，并比较了不同浓度模板DNA对扩增的影响，以确定适合海雀稗的SRAP-PCR最佳反应体系。结果表明：海雀稗SRAP-PCR最佳反应体系为10×PCR buffer 1 μL、模板DNA50 ng、Mg²⁺2.5 mmol·L^-1、dNTP150 μmol·L^-1、引物0.4 μmol·L^-1、TaqDNA聚合酶1.0 U，总体积10 μL。利用该反应体系从100对引物中筛选出可扩增清晰条带的引物83对，在83对引物中选出多态性丰富的引物44对。SRAP-PCR反应体系的优化及引物筛选，为今后利用SRAP标记技术进行海雀稗遗传多样性研究、基因定位、遗传图谱的构建以及分子标记辅助育种提供技术支持。

关键词: 海雀稗, SRAP标记, 正交实验, 体系优化, 引物筛选