茶花品种SSR指纹图谱分型技术反应体系优化

建立茶花品种分子指纹图谱对茶花品种鉴别、品种选育和深入实施品种产权保护等具有重要意义。为筛选适用于茶花品种SSR指纹图谱构建的引物及其最优反应体系，采用梯度退火温度和正交试验设计的方法，对4对SSR引物反应条件进行了研究。结果表明，2对引物扩增出目的条带，其中引物MSCjaF25最适退火温度52.9℃，10 mL体系中各组分含量为Mg²⁺ 1.2 mmol/L、dNTPs 0.2 mmol/L、Taq DNA聚合酶0.5 U、引物0.2 mmol/L以及模板DNA 40 ng时图谱质量最优；引物MSCjaF37最适退火温度58.2℃，10 mL体系中各组分含量为Mg²⁺ 0.8 mmol/L、dNTPs 0.1 mmol/L、Taq DNA聚合酶1.0 U、引物0.3 mmol/L以及模板DNA 40 ng时，图谱质量最优。以12个茶花品种进行扩大试验证明，优化的体系重复性和稳定性良好。研究为茶花品种分子指纹图谱的构建奠定重要基础。进一步分析显示，全面开展茶花品种分子指纹图谱研究还面临着筛选更多通用SSR引物的挑战。

The Study on SSR-PCR Fingerprints Map Classification for Camellia japonica Cultivars

Molecular fingerprints map is very important to the identification, breeding and intellectual property right protection of Camellia japonica cultivars. To screen applicable SSR primers and the corresponding optimizing reaction system for the construction of SSR-PCR fingerprints map, graded annealed temperature and orthogonal diagram experimental design were employed to evaluate the reaction conditions for 4 pairs of SSR primer. The results showed that: clear and unambiguous electrophoresis bands with designed size of amplified production presented only with primers MSCjaF25 and MSCjaF37 under the amplifying condition of annealing at 52.9℃, 1.2 mmol/L Mg²⁺, 0.2 mmol/L dNTPs, 0.5 U Taq DNA polymerase, 0.2 mmol/L primers and 40 ng DNA template in 10 mL reaction system and annealing at 58.2℃, 0.8 mmol/L Mg²⁺, 0.1 mmol/L dNTPs, 1.0 U Taq DNA polymerase, 0.3 mmol/L primers and 40 ng DNA template in 10 mL reaction system, respectively. Amplifying of 12 Camellia japonica cultivars using primers MSCjaF25 and MSCjaF37 made pure confirmation to the repeatability and stability of these optimized reaction conditions. Further analysis revealed that screening more SSR primers had been the facing challenge for molecular fingerprints map study on Camellia japonica cultivars.