Establishment and Application of SCoT Amplification System for Macadamia

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.

Key words:Macadamia;SCoT;Single factor experiment;System optimization;DNA fingerprint map