萝卜基因组DNA的RAMP-PCR体系优化

以萝卜为材料，对基因组DNA的RAMP分析体系中的Mg²⁺、dNTPs和引物浓度进行优化。分别设计3个浓度梯度：Mg²⁺为 0.75、1.5、3.0 mmol·L^-1；dNTPs为 0.05、0.15、0.3 mmol·L^-1；引物为 0.065、0.2、0.4 μmol·L^-1，并对合适退火温度进行研究。筛选出的RAMP优化体系为(20 μL):dNTPs 0.15 mmol·L^-1，Mg²⁺ 1.5 mmol·L^-1，引物0.2～0.4 μmol·L^-1，DNA 10 ng，Taq E 0.8U；PCR扩增程序为94℃ 3 min，94℃ 1 min，45℃ 1 min，72℃ 1.5 min，42个循环, 72℃ 8 min。运用此体系，进行引物组合筛选，并对7个萝卜品种的遗传多样性与品种鉴定进行RAMP标记分析。

Optimization of RAMP-PCR reaction system in genomic DNA of radish (Raphanus sativus)

Genomic DNA of Radish was analyzed by optimizing the concentration of Mg²⁺, dNTPs and primer in Random Amplified Microsatellite Polymorphism (RAMP) system. Three different concentration gradients were set respectively, Mg²⁺ 0.75，1.5，3.0 mmol·L^-1；dNTPs 0.05，0.15，0.3 mmol·L^-1；primer 0.065，0.2，0.4 μmol·L^-1, and the suitable annealing temperature was also screened. An optimization of RAMP reaction system for radish genomic DNA is（20 μL）: dNTPs 0.15 mmol·L^-1，Mg²⁺ 1.5 mmol·L^-1，primer 0.2～0.4 μmol·L^-1，DNA 10 ng，Taq E 0.8U.The amplification protocol was: 94℃ 3 min，42 cycles for 1 min at 94℃，1 min at 45℃, 1.5 min at 72℃, followed by a final extension of 8 min at 72℃. The suitable system was applied in screening the primer combinations, and the genetic diversity of seven radish varieties was analyzed in RAMP marker system.