杂交油菜“蜀杂6号”特异序列扩增标记(SCAR)建立及其在杂种鉴定中的应用

Development of SCAR Markers to Authenticate the Hybrid Seed Lots of Shuza No.6( Brassica napus L.)

用 10 0个随机引物对“蜀杂 6号”父、母本进行 RAPD扩增 ,选出 5个能在父、母本中产生多态性扩增的引物。在杂种 F1代中验证它们的特异性和稳定性 ,从中筛选出能从杂种 F1代中稳定扩增“蜀杂 6号”父、母本特异标记的随机引物 GE2 0 4和 GE2 2 2 ,并对它们扩增的父、母本特异标记片段进行克隆和测序。根据测序结果设计的特异序列扩增引物 ,将“蜀杂 6号”的父、母本 RAPD标记转化为序列特异的 SCAR标记。通过对“蜀杂 6号”父、母本和 30 0个杂种 F1代单株的父、母本特异序列扩增标记 (SCAR)的检测 ,建立了用 SCAR- PCR检测杂交油菜“蜀杂 6号”杂种纯度的方法 ,并逐一对照大田杂种一代生产种的表现型 。

Five primers, with which polymorphism bands could be amplified between tw o DNA pools of the parents of hybrid rapeseed, Shuza No.6 , were screened out fro m 100 RAPD primers. Then these five primers were used to amplify the total DNAs of F 1 to determine their specificity and stability. Several maternal specific an d paternal specific DNA bands were amplified with GE204 and GE222, in the DNA pools of Shuza No.6 and its parents. respectively In order to convert the RA PD markers into more stable SCAR (Sequence characterized amplified region) markers, the maternal specific marker GE204 500 and paternal specific marker GE22 2 300 w ere cloned and sequenced respectively. When SCAR primers designed according to t he sequences of GE204 500 and GE222 300 were mixed and used to amplify DNAs of Shu za No.6 and its parents, two distinctive bands, one of which was maternal spec ific and the other was paternal specific, appeared in all hybrid plants, but on ly one parent specific band in maternal or paternal plants. The reliability of this result was confirmed by the phenotypes of hybrid seed lots in the f ield.