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本研究以云南地方品种羊角洋芋为材料,采用单因素与L16(45)正交设计相结合的方法,对影响RSAPPCR反应体系的Mg2+浓度、Taq酶量、d NTPs浓度、模板DNA量和引物浓度进行优化,综合选取得到最佳反应体系。结果表明:马铃薯RSAP-PCR最佳反应体系为:20μL体系中,Mg2+浓度4.00 mmol/L,Taq酶2.00 U,d NTPs浓度0.45 mmol/L,模板DNA 60 ng,引物浓度0.50 mmol/L。应用该体系对退火温度进行优化,得到RSAP的两步最佳退火温度分别为35℃和51℃。利用10个马铃薯品种验证所得PCR体系,证明该体系稳定,可靠,重复性好,可为今后利用RSAP分子标记评价马铃薯的遗传多样性、开展品种选育、构建遗传图谱提供技术支持。
In this study, Yunnan local cultivars Cinnamomum angustifolia were used as materials. The effects of Mg2 + concentration, Taq enzyme concentration, dNTPs concentration, amount of template DNA and primer concentration on RSAPPCR reaction system were studied by the combination of single factor and L16 (45) orthogonal design. Optimize, comprehensive selection to get the best reaction system. The results showed that the optimal reaction system for potato RSAP-PCR was as follows: the concentration of Mg2 + was 4.00 mmol / L, the concentration of Taq enzyme was 2.00 U, the concentration of dNTPs was 0.45 mmol / L, the template DNA was 60 ng, and the primer concentration was 0.50 mmol / L in 20 μL system. The system was used to optimize the annealing temperature. The two-step RSAP annealing temperature was 35 ℃ and 51 ℃ respectively. The PCR system verified by 10 potato varieties showed that the system was stable, reliable and reproducible. It could provide technical support for the future application of RSAP molecular marker to evaluate the genetic diversity of potato, breed breeding and construct genetic map.