Optimization of SRAP-PCR system for Portunus trituberculatus based on orthogonal experimental design

SRAP is a novel molecular marker with high polymorphism. It is reliable in amplification, easy to operate and has a high universality in primer pairs. SRAP technique has been widely used for studies from plants to aquatic animals in the fields of genetic diversity, germplasm identification, genetic map construction and so on. Based on an orthogonal experimental design, we optimized the five factors of SRAP-PCR amplification system for Portunus trituberculatus (TaqDNA polymerase, Mg²⁺, template, dNTPs and primer) from four levels. The results show that the effects of these factors on PCR reaction from strong to weak is as follows: template (purity 1.75~1.90, concentration 10~70 ng · μL^-1), Mg²⁺ (1.60~2.00 mmol · L^-1), dNTPs (0.10~0.40 mmol · L^-1), TaqDNA polymerase (0.50~2.00 U) and primer (0.10~0.40 μmol · L^-1).The optimal SRAP-PCR reaction system (25 μL) can be summarized as follows: 0.50 U TaqDNA polymerase, 2.20 mmol · L^-1 Mg²⁺, 10 ng DNA template, 0.20 mmol · L^-1 dNTPs and 0.20 μmol · L^-1 primer. This optimized system may be helpful in the studies of genetic diversity and sex-linked markers for P.trituberculatus.