澳洲坚果SCoT反应体系的建立及应用

Establishment and Application of SCoT Amplification System for Macadamia

摘要:

为建立澳洲坚果(Macadamia spp.)的SCoT最适反应体系,用单因素设计方法对影响SCoT-PCR反应体系的主要影响因素进行了优化筛选,并构建了澳洲坚果种质的指纹图谱。结果表明,澳洲坚果的SCoT最适反应体系为:反应体系总体积为20 μL,包含2.5 mmol L^-1 Mg²⁺, 0.3 mmol L^-1 dNTPs, 0.8 μmolL^-1引物, 40 mgL^-1模板DNA和1.0 U Taq DNA聚合酶。最适退火温度为50℃。该反应体系对澳洲坚果种质具有良好的稳定性、可靠性和较高的分辨率。选用多态性良好的单引物SC5 和引物组合SC34+SC39,可以将12份澳洲坚果种质完全区分开,每份种质都各有独特的指纹图谱,置信概率达到99.988%。构建的SCoT反应体系可为澳洲坚果种质鉴定和遗传多样性分析等提供技术支持。

Abstract:

SCoT-PCR amplification system of macadamia (Macadamia spp.) was optimized and established by using single factor design, such as Mg²⁺, dNTPs, primer, Taq DNA polymerase and DNA template, and DNA fingerprint maps of macadamia germplasms were constructed. The results showed that final volume of optimum reaction system was 20 μL, including 2.5 mmol L^-1 Mg²⁺, 0.3 mmol L^-1 dNTPs, 0.8 μmolL^-1 primer, 1.0 U Taq DNA polymerase, 40 mgL^-1 template DNA, and 2 μL 10 × buffer with ddH₂O water. The suitable annealing temperature of primers was 50℃. The optimum SCoT-PCR system was shown to be steady and reliable. The 12 germplasms of macadamia could be absolutely identified by primer SC5 and SC34 + SC39 with 99.988% probability of confidence, every germplasm of macadamia had a unique fingerprint map. It showed that the optimum SCoT-PCR system could be applied effectively in germplasm identification and genetic diversity analysis of macadamia.