Isolation of genomic DNA and establishment of ISSR reaction system for Camellia crepnelliana Tutch

ObjectiveThe present study was undertaken to establish an ISSR-PCR amplification reaction system for Camellia crepnelliana Tutch for providing basis to further research.MethodThe young leaves of Camellia crepnelliana Tutch were used to isolate high-quality genomic DNA using the modified SDS method.The different conditions for ISSR reaction system were optimized.ResultThe extracted genomic DNA was of high quality as revealed by 1.8 to 2.0 absorbance ratio of wavelengths(260/280),did not show any degradation,and was found suitable for ISSR-PCR reaction.The components of the best ISSR-PCR amplification system in a total 25.0 μL reaction volume included 1.0 μL of template DNA (40 ng/μL),2.0 μL of dNTPs (2.5 mmol/L),0.2 μL of Taq DNA polymerase (5 U/μL),1.0 μμL primer (10 μmol/L),2.5 μL 10×PCR buffer(plus Mg2+).The PCR program was set to 5 min at 94℃ for pre-denaturing,followed by 40 cycles of 40 s at 94℃ (denaturation),40 s at 52℃ and 53℃ (annealing)and 90 s at 72℃ (extension),the final extension was set at 72℃ for 7 min.The optimized reaction system gave distinct DNA fingerprinting results.ConclusionThe established ISSRPCR amplification reaction system may be useful to assess the genetic variability and diversity in Camellia crepnelliana Tutch.