关键词: 澳洲坚果, ISSR, 体系优化

Abstract:

An one factor test was used to optimize ISSR-PCR amplification system on macadamia in four levels of five factors (Taq DNA polymerase，template DNA，dNTPs，primer and Mg²⁺，respectively) in this study. The results showed that the 25 μL reaction system consisted of 1×PCR buffer，1 U Taq DNA polymerase，20 ng template DNA，0.15 mmol·L^-1 dNTPs，0.25 μmol·L^-1primer and 2.5 mmol·L^-1 Mg²⁺. In addition，adding 0.4% formamide was able to reduce the background noise. The optimal PCR amplification process was as the following: 1 cycle initial denaturalization at 94 ℃ for 5 min，followed by 35 cycles，which included denaturalization at 94 ℃ for 30 s，annealing for 1 min，and extension at 72 ℃ for 2 min, and then extension at 72 ℃ for 7 min，and finally holding the samples at 4 ℃.

Key words: macadamia, ISSR, system optimization