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以毛花猕猴桃种质资源为试验材料,通过正交实验设计对影响ISSR反应体系各个主要因素进行优化。研究结果表明,最佳ISSR-PCR反应体系为:总体积为20μL,2μL buffer(无Mg2+),1 mmol/L Mg2+,1 U Taq酶,0.25 mmol/L d NTPs,1μmol/L引物,80 ng模版DNA;扩增程序为94℃3 min;94℃30 s,50℃~60℃退火45 s,72℃1 min(40个循环);72℃7 min,12℃保存;引物UBC842的最佳退火温度为58℃。利用该体系,用引物UBC835对46份野生毛花猕猴种质资源进行扩增,扩增结果表明建立的ISSR-PCR反应体系重复性好,稳定性强。
Using the germplasm resources of kiwifruit germplasm as the experimental material, the main factors influencing the ISSR reaction system were optimized by orthogonal experiment design. The results showed that the optimal ISSR-PCR reaction system was as follows: the total volume was 20μL, 2μL buffer (without Mg2 +), 1mmol / L Mg2 +, 1U Taq enzyme, 0.25mmol / L dNTPs, 1μmol / The template DNA was amplified by reverse transcription polymerase chain reaction (PCR) at 94 ℃ for 3 min. After annealing at 94 ℃ for 30 s, annealing at 50 ℃ ~ 60 ℃ for 45 s and annealing at 72 ℃ for 1 min (40 cycles) The annealing temperature is 58 ° C. Using this system, the primers of UBC835 were used to amplify the germplasm of 46 wild rhesus macaque germplasms. The amplification results showed that the established ISSR-PCR reaction system had good reproducibility and stability.