Optimization of RGA-PCR System of Sea-island Cotton by Uniform Design and Orthogonal Design

Uniform design and orthogonal design were applied to optimize RGA-PCR system of seaisland cotton.Four influencing factors on RGA-PCR,including Mg~(2+),d NTP,primer and Taq DNA polymerase concentration were tested using uniform design and orthogonal design.A suitable RGA-RCR system for sea-island cotton was established as follows:each 10ul RGA-PCR amplification reaction solution was consisted of 1×Buffer 2.5 mmol/L Mg~(2+),0.25 mmol/L dNTP,1.0μmmol/L primer,0.375U Taq DNA polymerase and 20ng template DNA.Each factor had a different effect on the results of PCR.Mg~(2+) concent ration had the greatest effect and Taq DNA polymerase had the least effect.The optimized RGA-PCR system was tested on sea-island cotton andshown to be steady and reliable.10 primer combinations were selected with abundant polymorphism from 22 primer combinations.The optimized RGA-PCR system and polymorphism primer combinations could be applied to research of sea-island cotton.