摘要
【目的】寻找黑麦基因组中新的重复序列作为特异PCR标记。【方法】以普通小麦中国春、川农18、育成品系R111、绵阳11为对照,以荆州黑麦、秦岭黑麦、非洲黑麦、森林黑麦为材料,用RAPD法筛选到黑麦基因组中的一个高拷贝DNA片段OPD15940,将OPD15940输入到NCBI的BLAST框中进行比对。根据OPD15940设计特异PCR引物D15F和D15R,利用这对引物对小麦族物种进行扩增,验证OPD15940的特异性。进而利用原位杂交技术定位pScD15940在染色体上的位置。【结果】序列比对后发现OPD15940与重复序列Sukkula中近60个53bp的小片段有较高的同源性,但又不同于Sukkula,是一类新的重复序列。通过特异PCR确定仅含黑麦染色质的物种能扩增出OPD15940,因而OPD15940为黑麦所特有。原位杂交结果显示除端部区域外,pScD15940弥散状分布在黑麦整套染色体上。【结论】OPD15940可以作为分子标记检测导入到小麦背景中的黑麦染色体。
[Objective] New repetitive sequence as specific molecular marker for rye genome was developed. [Method]Using common wheat Chinese Spring, R25, Rill and MYll as control, we screened genome-specific repetitive DNA sequence from Secale cereale cv. Jingzhou rye, S. cereale. L. Qingling rye, S. africanum and S. silvestre. A high copy specific DNA segment OPD15940 of rye genome was obtained by using random amplified polymorphic DNA （RAPD） analysis. Based on the sequence of OPD15940, a pair of specific primer D15F and D15R were designed to validate the particularity of OPD15940with the related species in Triticeae. Furthermore, the fluorescence in situ hybridization （FISH） was used to localize the OPD15940 on rye chromosomes. [Results] The Blast search for NCBI database revealed that OPD15940 had high homology with 60 small DNA segments of 53 bp-long Sukkula-like sequence. However, OPD15940 was not characteristic of Sukkula retrotransposon and it was therefore a kind of new repetitive DNA sequence. PCR result indicated that OPD15940could be merely detected in materials containing rye chromosome （s） but not in any other tested Triticeae species. The FISH result suggested that OPD 15940 was specifically hybridized throughout all rye chromosomes arms except for the terminal regions. [Conclusion] OPD15940 was a new repetitive sequence and can be used as a genome-specific DNA marker for detection Secale chromosome in wheat background.
出处
《中国农业科学》
CAS
CSCD
北大核心
2007年第8期1587-1593,共7页
Scientia Agricultura Sinica
基金
国家自然科学基金(30170502和30170579)
国家高科技发展研究计划("863"计划
2001AA241034)
教育部新世纪优秀人才资助项目(NCET-06-0810)
四川省教育厅重点项目(2004A020)
关键词
黑麦
重复序列
原位杂交
特异PCR
Rye
Repetitive DNA sequence
in situ hybridization
Specific PCR
