摘要
以小麦抗条锈病基因Yr5的供体亲本Triticumspeltaalbum作为对照 ,对近等基因系Yr5 6×AvocetS和感病亲本AvocetS进行RAPD分析。扩增产物用 4%变性PAGE分离 ,银染显色。在变性PAGE上可以检测到 50～ 10 0条带 ,是琼脂糖凝胶电泳的 5倍以上。筛选了 2 40个随机引物 ,发现 2 3条稳定的多态性DNA片段 ,初步检测表明其中6条与Yr5基因具有连锁性。用 12 1株AvocetS和Yr5 6×AvocetS杂交制备的F2 代分离群体进一步进行的遗传连锁性检测表明 ,多态性DNA片段S13 2 0 2 0 7和S13 4 83 6 3 分别与Yr5基因完全连锁和紧密连锁。结果表明 ,用变性PAGE分离PCR产物并结合银染显色 ,提高了小麦RAPD分析的多态性水平 。
RAPD analysis was performed between a near-isogenic line (NIL) Yr5/6×Avocet S carrying the resistance gene Yr5 against wheat stripe rust and its susceptible parent Avocet S,using the Yr5 gene donor parent Triticum spelta album as control.Amplified DNA fragments were separated on 4% denaturing PAGE (polyacrylamide gel electrophoresis) and displayed by silver staining.Fifty to 100 bands were detected,5 folds more than those revealed on agarose gels.A total of 240 random primers were screened,and 23 reproducible polymorphic DNA fragments were found,out of which 6 polymorphic bands appeared to be linked to Yr5 gene.Genetic linkage was tested on 121 segregating F_2 plants derived from a cross between Avocet S and Yr5/6×Avocet S.It was showed that the polymorphic DNA fragment S1320_207 was completely linked to Yr5 gene,and S1348_363 closely linked to Yr5 gene.The results suggested that using denaturing PAGE-silver staining could increase the level of DNA polymorphisms detected in wheat and also improve the repeatability of RAPD analysis.
基金
国家"863"计划项目 (编号 :2001AA211051)
国家重点基础研究发展规划项目(编号:G2000016202)资助
关键词
PAGE
RAPD
小麦
条锈病
抗病基因
Yr5
polyacrylamide gel electrophoresis(PAGE)
random amplified polymorphic DNA(RAPD)
wheat
stripe rust
resistance gene
Yr5
