菠菜性别相关的ISSR标记

菠菜为雌雄异株植物,用CTAB法提取其雌、雄株成株幼嫩叶片DNA,分别构建雌、雄株DNA池,以之为模板,用已优化的ISSR体系扩增,在74条ISSR引物中,I62扩增出一条约1 200bp雌性连锁标记,回收纯化该特异扩增片段,将其连接于pUCm-T载体,转化进大肠杆菌JM109菌株,并检测及测序。回收克隆和测序后发现该片段全长1 176bp,富含AT,AT占57.0%。根据测序结果设计1对25bp的特异引物将这个雌性连锁的ISSR标记转化为稳定性和特异性更好的SCAR标记。该特异引物对随机选取的雌雄菠菜单株进行PCR扩增,在雌株中均有1 176bp的特异条带,而雄株中均无。此特异条带的获得为菠菜性别相关基因的克隆奠定基础。

Development of a ISSR Marker Linked to Sex in Spinacia oleracea L.

The DNA was extracted from mature Spinacia oleracea L. separately by the method of CTAB, then used as template after mixed by gender to be amplified with the optimized ISSR reaction condition. A total of 74 primers were tested with the same PCR cycling procedures. Only a female-associated fragment with a length of about 1 200 bp was generated with I62 primer. The I62 fragment was extracted from agarose gel and cloned into pUCm-T vector, and then transformed into E.coli JM109. It was sequenced after test. Results showed that this sequence was 1 176 bp, and was abundant in AT (57.0%). In order to convert the ISSR marker into SCAR marker, 25 bp specific primers were constructed and used for PCR amplifying. The specific primers were tested in female and male Spinacia oleracea L. individuals. Result showed that the primer pair could amplify a specific fragments of 1 176 bp in female individuals and the male individuals were none. This means that the specific fragment could be used as a base to clone sex-linked gene of Spinacia oleracea L.