菌草SRAP-PCR体系优化及引物筛选OA

[Objective]SRAP-PCR system optimization and polymorphic SRAP primers identification for studying Juncao were conducted.[Method]Genomic DNA extracted from Juncao was used as a template in a combined single-factor and orthogonal experimentation to optimize the primer,Mg2+,template DNA,dNTP,and Taq DNA polymerase for SRAP-PCR analysis.A set of polymorphic SRAP primers was selected using 4 varieties of Juncao as targets.[Result]The optimized SRAP-PCR analysis required the concentrations of primer at 1.0 μmol·L-1,Mg2+at 2.50 mmol·L-1,template DNA at 2.5 ng·μL-1,dNTPs at 325 μmol·L-1,and Taq DNA polymerase at 0.35 U.These parameters affected the SRAP amplification efficiency in the order of primer＞template DNA＞Taq DNA polymerase＞dNTPs＞Mg2+.From 35 pairs of SRAP primers,18 with rich polymorphism and clear bands were selected.They had 3 to 10 polymorphic loci with an average polymorphism ratio of 77.36%.[Conclusion]The optimized SRAP-PCR system was highly stable and reliable.It could be applied satisfactorily for analyzing genetic diversity,studying germplasm conservation,and broadening utilization of Juncao.