Optimization of SRAP-PCR system on Vicia amoena using orthogonal design and selection of primers

Abstract

Abstract: An orthogonal design was used to optimize a SRAP-PCR system with 4 factors (Mg²⁺, dNTP, primer and Taq polymerase) at 3 levels plus the concentration of template DNA. The optimized SRAP-PCR system for Vicia amoena was: 2 μL 10×PCR buffer (Mg²⁺ free), 30 ng template DNA, Mg²⁺ 2.0 mmol/L, dNTP 0.2 mmol/L, primer 2.0 μmol/L, Taq DNA polymerase 1.5 U in a total of 25 μL reaction solution. Each factor had a different effect on the results of PCR. Primer concentration had the greatest effect and dNTP concentration had the least effect. The optimized SRAP-PCR system was tested on three V. amoena germplasm and was shown to be consistent and reliable. Twenty pairs of primer combinations with abundant polymorphisms were selected from 98 pairs of primer combinations. The optimized SRAP-PCR system and polymorphism primer combinations provide a basis for molecular genetic research on V. amoena.

CLC Number:

Q943

LIU Ying, WANG Xian-gou, ZHANG Ju-ming, LIU Fang. Optimization of SRAP-PCR system on Vicia amoena using orthogonal design and selection of primers[J]. Acta Prataculturae Sinica, 2012, 21(5): 325-330.

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