Response Surface Optimization of ISSR-PCR Reaction for Genetic Study on Phyllanthus Emblica

  Objective  ISSR-PCR reaction system for genetic study on Phyllanthus emblica germplasms was optimized.

  Methods  A mixed DNA template composed of genomic DNA of P. emblica germplasms came from Myanmar, India, Guangdong, Yunnan, and Fujian was obtained. The single factor test and response surface analysis were used to optimize the ISSR-PCR reaction conditions including primer concentration, amount of 2×Taq Master Mix, DNA template amount, and annealing temperature.

  Result  The primer concentration and addition amount of 2×Taq Master Mix had a greater impact on the amplification than did the DNA template amount. A significant interaction between the primer concentration and DNA template amount was found. The optimized system with a primer concentration of 0.4 μmol·L⁻¹, a 2×Taq Master Mix of 13 μL, and a DNA template concentration of 30 ng was established that achieved a low relative error of 9.39% in predicting the theoretical response. Within the annealing temperature between 50.5 ℃ and 52.7 ℃, increasing temperature improved the number and quality of bands. When the annealing temperature is 50.5-52.7 ℃, the number of strips increases, and the quality of the strips becomes better with the increase of temperature. The annealing temperature is 52.7 ℃ to obtain good diversity and clear bands.

  Conclusion  The optimized ISSR-PCR reaction system was established applying a primer concentration of 0.4 μmol·L⁻¹, a 2×Taq Master Mix of 13 L, a DNA template concentration of 30 ng at the annealing temperature of 52.7 ℃ for 35 amplification cycles. The methodology could be used to study the genetic diversity and relationship of P. emblica germplasms.