Optimization and establishment of SRAP-PCR reaction system for Nelumbo nucifera Gaertn

Using Nelumbo nucifera Gaertn ‘Xinhong’ leaves as the experimental material,by the orthogonal experiment design L16(45),five factors including concentration for Mg2+ and dNTPs,dosage for TaqDNA polymerase,primer and template DNA in SRAP-PCR reaction system were optimized,and the optimization SRAP-PCR reaction system suitable for lotus were also established.The result showed that the optimal SRAP-PCR reaction system was as follows:total volume 10 μL,including 2.0 mmol·L-1 Mg2+,300 μmol·L-1 dNTPs,0.5 U TaqDNA polymerase,4 μmol·L-1 primer,50 ng DNA and 10×PCR Buffer.The order of each factor in different levels affecting the result of PCR was:TaqDNA polymerase,Mg2+,primer,dNTPs,DNA.The optimal SRAP-PCR reaction system was identified by means of genomic DNA from 48 varieties of N.nucifera Gaertn and the amplification pattern with clear band and rich polymorphism was obtained.It is concluded that the SRAP-PCR reaction system is steady and reliable.