摘要
利用随机扩增多态性DNA (randomamplifiedpolymorphicDNA ,RAPD)技术获得与大麻性别连锁的分子标记。将 10株雄性大麻或 10株雌性大麻的单个DNA样品等量混合分别组成雄性或雌性DNA池(DNA pool) ,以提供具有相同遗传背景的雌、雄性DNA样品。每个随机引物分别用三个不同的循环程序进行PCR扩增。在 30个随机引物中 ,用引物S40 1扩增得到一条约 2 .5kb雄性多态性片段。对该片段进行了克隆和序列分析 ,并根据序列分析结果将上述RAPD分子标记转化为重复性和特异性更好的SCAR(sequencecharacterizedamplifiedregions)
In this study the random amplified polymorphic DNA (RAPD) technique was employed with the objective of finding markers linked to sex determination in hemp. Ten male individual DNA samples or ten female individual DNA samples were prepared and contributed to two DNA pools: one was male DNA pool and another female DNA pool, with the intention of providing a common genetic background for each pool and leaving the sex linked materials as the primary difference between the two pools. A total of 30 10 mer primers (Table 1) was tested with at least three different PCR cycling procedures. A male associated fragment with a length of about 2.5 kb (Figs.1 and 2) was generated with S401 primer. The fragment was cloned and sequenced (Fig.3). In order to convert the RAPD marker into SCAR (sequence characterized amplified regions) marker, one 20 mer and another 22 mer specific primers were constructed and used for PCR amplifying. The male linked dominant SCAR marker was obtained (Fig.4), which would favor the screening of all gynoecious hemp lines.
基金
国家重大基础研究项目 (G19990 1170 0 )资助
