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本文以苹果为试材,研究了影响苹果ISSR-PCR反应的相关因素,并筛选了苹果ISSR-PCR引物。结果表明,在25μL ISSR-PCR反应体系中,适宜的引物和模板浓度分别为0.8μmol/L和20 ng。适宜的退火温度可减少非特异扩增,提高PCR产物的多态性和差异性,退火温度大部分比Tm值低2~4℃。从112个引物中筛选出17个多态性较好的引物,共扩增出113个位点,其中多态性位点74个,所占比例为65.5%。
In this paper, the apple as a test material, the impact of apple ISSR-PCR reaction related factors, and selected apple ISSR-PCR primers. The results showed that the suitable primer and template concentrations were 25 μmol / L and 20 ng in 25 μL ISSR-PCR reaction system respectively. Appropriate annealing temperature can reduce the nonspecific amplification and improve the polymorphism and difference of PCR products. The annealing temperature is mostly 2 ~ 4 ℃ lower than the Tm value. Thirteen primers with good polymorphism were screened out from 112 primers, and a total of 113 loci were amplified, of which 74 were polymorphic, accounting for 65.5%.