Abstract:

The author took three kinds of fruit flies for materials, discussed the extraction of genomic DNA with high quality, studied on the effect of template DNA concentration, primer concentration, the amount of Taq DNA polymerase, dNTP concentration, annealing temperature and time on ISSR-PCR amplified results, established fruit flies’ general and stable ISSR-PCR reaction system. The result showed that the author obtained the fruit flies’ genomic DNA with high quality, established fruit flies’ general and stable ISSR-PCR reaction system that adapt to fruit flies: 2.5 μL 10×PCR Buffer, 50ng template DNA, 0.25 μmol/L primer, 0.5 U Taq DNA polymerase, 200 μmol/L dNTP and add ddH2O to 25 μL, made clear the ISSR-PCR amplification program: pre-denaturalized at 94℃ for 5 min, denaturalized at 94℃ for 30 s, annealed at 52.4℃ for 45 s, extended at 72℃ for 90 s, 36 cycles, at last extended at 72℃ for 7 min, then conserved at 4℃. The establishment of optimized system made up for the shortage of fruit flies’ traditional morphology observation, and laid a foundation for fast and accurate identification, population heterogeneity and analysis of genetic diversity.