摘要
以陕西省杂交油菜研究中心选育的单显性核不育油菜分离群体为材料,利用集群分离法(BSA)对该油菜单显性核不育基因进行了RAPD分析。在随机选取的300个10碱基随机引物中,引物S243(5'CTATGCCGAC3')在可育集团与不育集团间扩增出特异而可重复的1.5kb的多态性片段OPU-031500,而在细胞质雄性不育和其它核不育类型油菜中均未扩增出上述特异性片段,从而确证此RAPD标记OPU-031500片段是与甘蓝型油菜单显性核不育基因连锁的。将该多态性片段克隆并测序,发现其序列与拟南芥的一段DNA序列高度同源。根据同源序列及测序结果设计两对特异引物(P1/P2和P3/P4),引物P3/P4在可育系中可扩增到约1.5kb的单一特异片断,而在不育系中无带,从而将RAPD标记转化为稳定可靠的SCAR标记。
Bulked segregant analysis （BSA） was used to identify randomly amplified polymorphic DNA （RAPD） markers linked to the MS gene in mono-dominant GMS of rapeseed （Brassica napus L.）, which was bred by Hybrid Rapeseed Research Center of Shaanxi Province. A total of 300 ran- dom 10-mer oligonucleotide primers were screened on the DNA from fertile and sterile bulks. Primer S243 （5＇CTATGCCGAC3＇） gave identical 1.5 kb DNA polymorphic segment OPU-031500 in the bulk S, but not in the bulk F （Fig.2）. The DNAs from individual plants of each bulk and from their sister lines, which were generated from the same original crossing, were then screened with the primer S243, and the same results were obtained （Figs.3, 4）. Other types of GMS and CMS were analyzed using primer S243, and the specific 1.5 kb DNA segment was not found （Fig.5）. Therefore, the RAPD marker OPU-031500 is linked to the mono-dominant GMS trait in rapeseed. This RAPD marker OPU-031500 was cloned into a T-easy vector and sequenced. The sequence here obtained was highly homologous to one of the Arabidopsis DNA sequences. According to this DNA conserved region in different species, we designed a pair of specific primers P 1 （5＇ATGTCGCTGAGGCCG- AGCAC3＇） and P2 （5＇GGCACACTGTCACG- ATCCTTGG3＇） and amplified only one specific 2.3 kb DNA fragment in each bulk. There are two mutant loci between the two DNA fragments after sequencing. We designed another pair of specific primers P3 （5＇CTCCAGCAGCAGCAGC-AGCCT3＇） and P4 （5＇GCAGGAATGAGAA-CCGTAGG3＇） according to the DNA sequence at the mutant loci. A specific DNA segment was amplified only in the fertile line but not in the sterile line using the primers P3 and P4 （Fig.6）. Therefore the RAPD marker were converted into SCAR marker. Moreover, the SCAR marker detection method was improved （Fig.7）.
出处
《植物生理与分子生物学学报》
CAS
CSCD
北大核心
2006年第5期513-518,共6页
Journal Of Plant Physiology and Molecular Biology
基金
国家863计划项目(No.2001AA241111)
陕西省自然科学研究计划项目(No.2004C108)资助。~~
