毛花猕猴桃种质资源ISSR反应体系的建立和优化

Establishment and Optimization of ISSR Reaction System of Kiwi Fruit(Actinidia eriantha) Germplasm

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。

Germplasm resources of Actinidia er iantha were estimated based on the Orthogonal experimental design to optimize main factors which had an influence on ISSR reaction system. The results showed that the optimum ISSR-PCR reaction system was 20 μL total volume including 2 μL buffer without Mg2+, 1 mmol/L Mg2+,1 U Taq enzyme, 0.25 mmol/L d NTPs,1 μmol/L primer, 80 ng DNA as template. Amplification program was94℃ for 3 min, then followed by 40 cycles each with denaturation at 94℃ for 30 s, anneal at 50℃~60℃ for 45 s,72℃ for 1 min, later at 72℃ for 7 min, in the end kept at 12℃. The best anneal temperature of primer UBC842 was 58℃. Primer UBC835 was used to amplify the germplasm resources of 46 wild ctinidia eriantha germplasm resources with this system. Amplification results indicated that the ISSR-PCR reaction system had good reproducibility and strong stability.