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棉花学报  2012 

亚洲棉7号染色体分离及文库构建

DOI: 1002-7807(2012)05-0379-07, PP. 379-385

Keywords: 亚洲棉,显微切割,微克隆

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Abstract:

采用前低渗、酶解、后低渗和盖片轻压相结合的方法,建立了激光法分离二倍体亚洲棉石系亚1号单条染色体技术。单染色体经去蛋白、酶切和PCR扩增后,产物以Southern杂交、简单重复序列(Simplesequencerepeats,SSR)引物扩增和荧光原位杂交技术(Fluorescenceinsituhybridization,FISH)进行验证,构建了棉花单条染色体文库。克隆片段长度在150~1000bp,平均为550bp;文库包含1.38×105个克隆,覆盖染色体长度1倍左右,空载率为1%,滴度为1.3×106pfu·mL-1,单一拷贝和低拷贝比例达到59%以上,为该条染色体分子标记的筛选、重要基因克隆和定位、遗传图谱的饱和奠定基础。

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