棉花细菌人工染色体的荧光原位杂交(BAC-FISH)技术

细菌人工染色体荧光原位杂交(BAC-FISH)技术是植物染色体识别、物理作图等分子细胞遗传学研究的重要工具,但对于某些物种尤其是多倍体植物,由于大量重复序列的存在等问题,使得该技术应用受到很大的限制.通过选择棉花分子遗传图中高重组区的微卫星位点(simple sequence repeats,SSR)标记的策略,筛选到不含或含有少量重复序列的细菌人工染色体(BAC)克隆,同时,在通用FISH技术程序基础上,通过改进发根、变性、洗脱条件等步骤,构建出适合于棉花的BAC-FISH技术,简化了操作流程的同时,获得稳定的杂交结果及较高的检出率;并通过将一随机获得的BAC进行染色体的物理定位,进一步引入双探针、双色及重复杂交技术,显示了该技术的成熟与良好的应用前景和价值.

Fluorescence In situ Hybridization of Bacterial Artificial Chromosome in Cotton

BAC-FISH (bacterial artificial chromosome and fluorescence in situ hybridization) is a powerful tool for molecular cytogenetics such as chromosome identification and physical mapping in plant. Due to the large proportion of repetitive sequence occurring in some plant species,especially the polyploidy species, the application of BAC-FISH was hindered severely. Molecular markers from a high-density genetic map of tetraploid cotton were used to screen BAC libraries. Protocols of BAC-FISH in cotton mitotic and meiotic cells were introduced by some modifications such as root tip preparation, slide denaturation and post-hybridization washing according to previous protocols. The dual-color, dual-probe and repeated FISH were also introduced by applying them to physical mapping a random BAC clone on the corresponding chromosome.