刺五加AFLP分子标记技术体系的建立

Establishment of AFLP Analysis System for Acanthopanax senticosus

用改良的的试剂盒法提取刺五加叶片的基因组DNA,可以获得高质量的刺五加基因组DNA,DNA降解较少、污染低、电泳条带清晰,可以用于PCR扩增反应,可以被内切酶EcoRⅠ和MseI彻底消化。通过优化酶切反应、引物连接、预扩增、选择扩增,建立了优化的刺五加AFLP分析技术体系,从64对引物中筛选到5对高效扩增引物,并利用这5对引物在6个刺五加种源中获得了89个多态性AFLP分子标记。建立了利用红外荧光检测技术和Li-Cor4300DNA分析系统,进行刺五加AFLP分析的分子标记技术。

The improved method of DNA isolation was used to extract genome DNA from leaves of Acanthopanacis senticosl,and higher quality genome DNA was obtained,that of strips in agarose electrophoresis were clear and less decomposed,could be used for PCR.These DNA samples with high purification were intact without containing inhibitor of enzyme reaction and could be completely digested by restriction enzymes such as MseI and PstI.The system of amplified fragment length polymorphisms（AFLP） technique was established successfully by processes like restriction enzymes digesting reaction,preamplification,and selective amplification.The fingerprintings of Acanthopanacis senticosl were obtained by these processes,and the reproducibility was high,five pairs of primers with high polymorphism and powerful distinctiveness were selected from sixty-four pairs of primers,and got 89 polymorphism markers for AFLP analysis of Acanthopanacis senticosl.The molecular technique for AFLP analysis of Acanthopanacis senticosl were established through detection of IRDye labels by Li-Cor4300 DNA analyzer.