基于毛细管电泳技术的牡丹SRAP-PCR反应体系优化及引物筛选

以牡丹叶片DNA为模板,对SRAP-PCR反应程序进行研究,确定适合的反应程序,即94℃预变性5 min;94℃变性1 min,33℃退火1 min,72℃延伸1 min,共5个循环;随后94℃变性1 min,52℃退火1 min,72℃延伸1 min,共35个循环;最后72℃延伸10 min。基于毛细管电泳技术,采用正交设计L18(37)对牡丹SRAP-PCR反应体系的5因素(Taq酶,Mg2+,模板DNA,dNTP,引物)3个水平进行了优化,构建了牡丹SRAP最佳反应体系:模板DNA 50 ng,dNTP 0.25 mmol/L,Mg2+浓度2.5 mmol/L,引物浓度0.4μmol/L,TaqDNA聚合酶0.5 U,总体积为25μL。各因素对扩增结果影响程度均不同:dNTPs>引物>DNA模板>Mg2+>Taq酶。运用该体系从756个SRAP引物组合中筛选出多态性好、条带清晰的26个引物组合,并证明了该体系稳定可靠。该体系的建立以及引物组合的确定为今后利用SRAP分子技术进行牡丹的相关研究奠定了科学基础。

Optimization of SRAP-PCR System and Selection of Primers for Paeonia suffruticosa Andr. Based on Capillary Electrophoresis

In this study,the procedure of SRAP-PCR was determined : an initial denaturing step was performed at 94 ℃ for 5 min;followed by 5 cycles at 94 ℃ for 1 min,33 ℃ for 1 min and 72 ℃ for 1 min;followed by 35 cycles at 94 ℃ for 1 min,50 ℃ for 1 min,72 ℃ for 1 min,and a final extension at 72 ℃ for 10 min.The orthogonal design was used to optimize SRAP-PCR system with five factors at three levels respectively based on capillary electrophoresis.The results showed that the optimized SRAP reaction mixtures for Paeonia suffruticosa Andr.:(total volume 25 μL) 50 ng DNA template,0.25 mmolL dNTP,2.5 mmol/L Mg2+,0.4 μmol/Lprimer,0.5 U Taq DNA polymerase.The concentration of dNTP had the greatest effect and DNA template had the least effect on the result.And twenty-six pairs of primers were selected with abundant polymorphism above 70% from 756 pairs of primers.The optimized SRAP-PCR system and pairs of primers could be applied to related research of Paeonia suffruticosa Andr.