Study on Optimization of ISSR Reaction System for Carrot

Key words: 
• Daucus carota L. var. sativa DC. /
• ISSR /
• optimization

Abstract: The improved CTAB method was used to extract genomic DNA of Daucus carota L. var. sativa.,which were used as the template for ISSR-PCR. A optimal ISSR-PCR reaction system suitable to Daucus carota L. var. sativa DC. was established via single factor experiments. The optimal ISSR-PCR system was that 25 μL reaction solution contained 2.5 μL 10×PCR buffer,2.5—3.0 mmol·L^-1 Mg²⁺,200μmol·L^-1 dNTPs,1.5U Taq polymerase,0.5 μmol·L^-1 primers,20ng DNA template.The optimized amplification program was that pre-denaturing at 94 ℃ for 4min,then denaturing at 94 ℃ for 40 s,primer annealing at 48 ℃—58 ℃ for 45 s,extension at 72 ℃ for 2 min,for 35 cycles,extension at 72 ℃ for 8min. The products were stored at 16 ℃. 24 effective primers were screened out.