海菜花ISSR-PCR反应体系的优化与引物筛选

Optimization of ISSR-PCR Reaction System and Primer Selection of Ottelia acuminate

为建立海菜花ISSR-PCR最优反应体系并筛选出适宜的ISSR-PCR引物,本研究应用L_(16)(3^(4))正交设计试验和单因素实验,筛选ISSR-PCR反应体系中主要参数(引物,2×Taq Master Mix,模板DNA)的最适浓度。结果表明,海菜花最适ISSR-PCR反应体系为引物0.22 ng,2×Taq Master Mix 1.5 U/25μL、模板DNA24 ng。通过最优反应体系进行引物筛选,获得19条适宜海菜花扩增的ISSR引物,并利用10个地理居群的海菜花DNA对该体系进行验证。本研究对海菜花后续的遗传多样性、遗传图谱构建提供参考。

In order to establish the optimal ISSR-PCR reaction system and select ISSR-PCR primers of Ottelia acuminate,this study applied L_(16)(3^(4))orthogonal design experiment and single factor experiment to screen the optimal concentration of the main parameters(primers,2×Taq Master Mix,template DNA)which had influence on the ISSR-PCR reaction system.The results revealed that the best ISSR-PCR reaction system consisted of primers0.22 ng/25μL,2×Taq Master Mix 1.5 U/25μL,along with template DNA 24 ng/25μL.19 ISSR primers suitable for O.acuminata amplification were obtained through optimal reaction system primer screening.The system was verified by DNA for 10 geographical populations of O.acuminata.This study provides a reference for the subsequent genetic diversity and genetic map construction of O.acuminata.