摘要
荧光标记通用引物基因分型技术以其经济、实用的特点在微卫星标记研究中得到了较多应用。但传统以M13为通用引物的方法存在非特异性扩增条带多、基因分型质量差等问题。为加快微卫星基因分型新技术的推广和应用,提高林木分子遗传学研究水平,本研究参照Blacket等的方法,并结合微卫星多重PCR(multiplex PCR)技术,成功建立了杉木(Cunninghamia lanceolata)荧光标记通用引物多重PCR微卫星基因分型技术体系。新方法的关键在于应用了4个具有较高退火温度的通用引物和QIAGEN誖Multiplex PCR试剂盒,它们能有效减少非特异性扩增条带的产生,多重PCR体系的优化简单、快捷。通常只需调整通用引物的浓度和PCR产物稀释倍数,就能获得清晰、稳定的基因分型结果。研究表明,新方法不仅大幅度降低了实验成本,而且显著提高了微卫星基因分型效率。利用该技术实现了杉木EST-SSR标记位点的规模化开发,并开展了杉木种子园育种材料遗传多样性研究。本研究新技术的建立和应用可为杉木分子遗传学研究提供技术保障,也对其他物种的相关研究具有重要的参考价值。
Fluorescently labelled universal primers o f PCR fragments is an economical and effective technique,which has been widely used for microsatellite marker genotyping. But the traditional approach using the universal primer of M13 has been limited largely due to many non-specific amplification fragments and poor quality of genotyping. In order to accelerate the application of new methods on microsatellite genotyping and develop the research level of forest tree genetics, our studies established the genotyping method combined with fluorescently labelled universal primers and multiplex PCR. The new method has been used in the genotyping of Chinese fir(Cunninghamia lanceolata) successfully. The key factors of this method aimed at the application of a suit of four high-performance universal primers(having high annealing temperature) and the QIAGEN誖 Multiplex PCR kit,which could effectively reduce the non-specific amplification fragments, and made the optimization of multiplexP CR more simple and quickly. In the experiment, we only need to adjust the concentration of fluorescently labelled universal primers and the PCR products that the result of genotyping will clear and stable.The results showed that the new method would not only dramatically reduce project costs, but also significantly improve PCR efficiency of microsatellite marker genotyping. It has already been used successfully in large scale developmen t of Chinese fir EST-SSR markers and launched genetic diversity research on breeding materials in Chinese fir seed orchard. In this study, the establishment and application of new techniques will provide technical support for the molecular genetics research with Chinese fir, and also serve as reference for the studying in other species.
出处
《分子植物育种》
CAS
CSCD
北大核心
2016年第7期1795-1803,共9页
Molecular Plant Breeding
基金
国家林业局948国际引进项目(2015-4-18)
湖南省自然科学基金(10JJ2018)共同资助
关键词
杉木
微卫星
基因分型
荧光标记
多重PCR
Cunninghamia lanceolata
Microsatellite markers
Genotyping
Fluorescent labeling
Multiplex PCR
