Optimization of Experiment Conditions and Primer Screening with ISSR Markers for Chimonanthus grammatus

The methods of CTAB,modified CTAB,SDS,High salt low pH,SDS-CTAB and SDS-Proteinase K were used to be compared in order to isolate high-quality genomic DNA from leaves of Chimonanthus grammatus.The results showed that the modified CTAB was the best one.By applying the single factor experiment with primer U811,a suitable ISSR reaction system for the analysis of genetic diversity of C.grammatus was established,namely 20 μL reaction system containing 1×PCR buffer,50 ng template DNA,0.6 μmol/L primer,1.5 mmol/L Mg²⁺,0.15 mmol/L dNTPs and 2.0 U Taq DNA polymerase.The reaction program was devised for 5 minutes of predenaturalization at 94℃,1 min of denaturalization at 94℃,45 seconds of anneal at 57.2℃,2 minutes of extension at 72℃,40 cycles,and 5 minutes of extension at 72℃ in the final cycle.One hundred ISSR primers were used to screen the suitable primers with 7 samples from different populations for assessing the genetic diversity of C.grammatus,of which 10 ISSR primers with high resolution and multiple polymorphic bands were screened.The total 74 ISSR bands were amplified with 10 primers,and produced 39 polymorphic bands.The percentage of polym-orphic bands was 52.7%.