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Establishment of Astragalus adsurgens' ISSR-PCR Optimal Conditions with Orthogonal Optimization Method

CHEN Zhi-hong et al (Center of Genetic Resources of Livestock,Foul and Herbage,China Animal Husbardry & Veterinary Institute,Beijing 100094)  
Orthogonal design is applied to the ISSR-PCR conditions optimization in this paper.Two orthogonal experiments were made to investigate the amplification efficiency of different amplification conditions.As a result,a satisfactory ISSR technique system for Astragalus adsurgens with desirable repeatability and polymorphic bands was established.In a total volume of 20?μl ISSR-PCR system,it contained 1×buffer,0.2?mmol/L dNTP,0.3?μmol/L Primer,2.5?mmol/L Mg~(2+),1U Taq DNA polymerase and 2.5?ng/μl template DNA.The optimal annealing temperature of this primer for ISSR-1PCR reaction was proposed by gradient PCR and it was 54.1?℃.The result provided standardizing ISSR-PCR program for the analysis of genetic diversity of Astragalus adsurgens.
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