一种优化的胡杨高效多重(12重)SSR体系(英文)

High Throughput SSR Multiplex Kits (12-plex) for Euphrates' Poplar,Populus euphratica (Salicaceae)

微卫星多重PCR方法是一种非常经济并且高通量的基因分型技术。本研究在耐干旱、盐碱的胡杨(Populus euphratica)中开发出一套荧光标记的12重微卫星工作体系。该体系包含12条表达序列标签微卫星(EST-SSR)引物,其中3条设计于NCBI,另外9条设计于二代的转录组序列。利用该多重微卫星体系可在单一的PCR反应体系中成功扩增出12条表达序列标签的微卫星短序列片段,并在胡杨的3个自然居群96个个体中对该体系进行了验证,结果显示该体系具有很高的稳定性及多样性。同时,在杨属的5个派7个种中对其通用性进行了检验,显示这些引物具有很高的通用性,成功扩增率为79%。本研究中提供的12重多重PCR结合本实验已经公开发表的2个8重体系对揭示胡杨及其他杨树的进化历史具有重要的作用。最后,本研究认为引物的选择,扩增效率,哑等位基因的检测是多重体系开发过程中最为关键的步骤。

Multiplex PCR of microsatellite is a cost-effective and high-throughput technique of genotyping. We developed a new 12-plex PCR kit for Populus euphratica, the only tree species in desert area ranging from Western China to Mediterranean coast. Three primers were designed for the expressed sequence tags （ESTs） sequences from the NCBI database and the other nine primers were designed based on the EST sequences of P. euphratica obtained by Solexa. The multiplex kit was tested by 96 samples from three natural populations. The results showed sufficient amplification stability and high polymorphism. All the 12 loci used in this kit showed a high transferability （79%） in other seven species from five sections of the genus. The new 12-plex kit combined with the two eight multiplex kits we had developed in previous studies, should be useful to reveal the genetic mechanism and evolution history of the P. euphratica and related species. During the research, we found that primers selection, amplification efficiency, null allele detection are the essential parts of the multiplex kit development.