石榴ISSR-PCR反应体系的优化研究*

 以酸绿籽石榴为试材,对ISSR反应体系中的各个主要影响因子进行优化筛选。结果表明：25 μL　ISSR反应体系各组分的最适浓度分别为：1×buffer（不含Mg²⁺),1.5 mmol/L Mg²⁺,0.2 mmol/L dNTPs,TaqDNA聚合酶1.5 U,引物0.3 μmol/L,模板40ng。反应程序为：94 ℃预变性4 min,94 ℃变性45 s,54 ℃退火1 min,72 ℃延伸2 min,40个循环,最后72 ℃延伸10 min。该体系的建立为从DNA分子水平进一步研究石榴种质资源遗传多样性、系统分类、品种鉴定奠定基础。

Optimizations of the Inter-simple Sequence Repeat Reaction System in Pomegranate

The optimization of the main influential factors of ISSR reaction system in pomegranate (Punica granatum L),Suanlvzi cultivar was studied. The results showed that the optimal concentrations of components in 25 μL reaction system were 1xBufer(without Mg²⁺),1.5 mmol/L Mg²⁺,0.2 mmol/L dNTPs,1.5 U Taq DNA polymerase,0.3 μmol/L primer and 30 ng template DNA,respectively. The PCR programme was designed as followingpre-denaturing at 94 ℃ for 5 min,denaturing at 94 ℃ for 35 s,annealing at 36 ℃ for 35 s,extending at 72 ℃ for l min,total 40 cycles,then extending at 72 ℃ for 5 min.This system can be used in the further studies of genetic diversity,systematic classification and identification of P.granatum L. at DNA Level.