杉木种子随体染色体SCAR标记的建立

回收、纯化由引物OPB07(5’-GGTGACGCAG-3’) OPB18(5’-CCACAGCAGT-3’)扩增而得的杉木(Cunninghamia lanceolata(Lamb.)Hook)种子随体染色体特异性RAPD(随机扩增的DNA多态性分析)片段OPB07-18907,将该片段克隆至pUCm-T载体,转化受体菌后筛选出阳性克隆,并对插入片段进行测序,根据序列特点设计两对SCAR(序列特异性扩增区)引物,PCR结果显示,这两对引物的4种组合都可以扩增出属于随体染色体的特征带,适宜退火温度为57℃。成功将特异RAPD标记OPB07-18907转化为稳定的SCAR标记。开发随体染色体SCAR标记目的是:一方面能在分子水平上鉴定微分离的杉木随体染色体,另一方面,也可以将杉木已构建的遗传图谱中连锁群与染色体进行对应。探讨了该SCAR标记对杉木核型分析的作用。

Generation of SCAR Markers for Identification of Satellite Chromosome from Cunninghamia lanceolata Seeds

To generate markers of sequence characterized amplified regions (SCAR), we recovered and purified a fragment OPB07-18907 from satellite chromosome of Cunninghamia lanceolata (Lamb.) Hook, using random amplified polymorphic DNA (RAPD) pairwise primers OPB07 (5'-GGTGACGCAG-3') and OPB18 (5'-CCACAGCAGT-3' ). This fragment was then cloned into pUCm-T vector, and transferred into Escherichia coli.DH5α. Positive colonies were identified for sequencing. Two SCAR primer pairs were designed according to the sequences of OPB07-18907. Based on information of the fragment, the SCAR marker was produced for satellite chromosome. The products of SCAR-PCR indicated that all primer combinations could be used to amplify specific bands of satellite chromosome. In addition, we found that 57℃ was the optimal annealing temperature for the use of these primers. We developed the SCAR marker of satellite chromosome for identifying the microdissected satellite chromosome at molecular level, and correlating a genetic linkage group of C. lanceolata with a specific chromosome. We also discussed the implementation of SCAR marker in karyotype polymorphism in C. lanceolata.