Establishment and optimization of an ISSR-PCR reaction system for Crassostrea hongkongensis
To establish an ISSR-PCR reaction system in Crassostrea hongkongensis, the factors influencing ISSR-PCR system were explored with the orthogonal design and main parameters comparison test, based on the genomic DNA of C. hongkongensis. The optimized ISSR-PCR system （25 μL） was determined as follows： 1 xBuffer, 2.5 mmol/L Mg^2＋, 0.2 mmol/L dNTPs, 0.2 gmol/L primer, 1.2 U Taq DNA polymerase, and 20 ng DNA template. The reaction program included an initial denaturation for 5 minutes at 94℃, 35 cycles of denaturation for 1 minutes at 94℃, annealing for 1 minutes at 48-54℃, extension for 1.5 minutes at 72℃, and a final extension of 10 minutes at 72℃. The clear, reproducible, polymorphic products were obtained by the established ISSR-PCR reaction system. The results lay a foundation for the analysis of genetic diversity of C. hongkongensis by ISSR marker.
Crassogtrea hongkongensisISSR-PCRreaction system optimization
国家科技期刊平台
NSTL
万方数据
维普
