杉木种子随体染色体SCAR标记的建立

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

摘要:

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RAPD标记OPB07-18₉₀₇转化为SCAR标记。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。

Abstract:

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 E. 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.