大葱雄性不育分子标记辅助选择的研究

Studies on Molecular Marker-assisted Selection for Sterile Line and Maintainer Line in Welsh Onion (Allium fistulosum L.)

大葱雄性不育在杂种优势利用中具有重要价值 ,传统方法选育不育系效率低 ,分子标记辅助育种可提高育种效率。本研究试图建立大葱雄性不育辅助育种的技术体系 ,加快大葱不育系、保持系的育种进程。利用RAPD技术分析了多态性片段S132 80 0 、S3896 0 、S72 30 0 、S73110 0 、S2 0 0 2 4 0 0 与大葱育性的连锁关系 ,重组率分别为 0、 7 5 %、 0、 4 2 %、 0。其中S132 80 0 、S2 0 0 2 4 0 0 能在多数品种中区分N、S胞质 ,且重组率接近 0 ,用于胞质鉴定具有很高的准确率 ,因而具有较高的利用价值。以S132 80 0 、S2 0 0 2 4 0 0 为探针对不育系和保持系mtDNA的酶切片段进行了Southern杂交分析 ,结果表明它们在N、S胞质中存在多态性 ,预示着它们可能是与大葱CMS相关的片段。鉴于RAPD标记的应用有一定局限性 ,进一步对特异片段S132 80 0 、S2 0 0 2 4 0 0 进行了克隆、测序和SCAR标记转化 ,其中S132 80 0 成功转化为SCAR标记 ,而S2 0 0 2 4 0 0 转化后多态性消失。为进一步降低成本 ,简化了SCAR扩增产物的检测技术 ,初步建立了大葱不育系、保持系分子标记辅助选择的技术体系。研究表明 ,SCAR产物加入EB直接检测存在一定误差 ,而通过快速电泳可以快速、准确地鉴定单株的胞质类型。RAPD标记和SCAR标记在育种?

Cytoplasmic male sterility(CMS) plays an important role in welsh onion heterosis utilization. Selection based on the markers would be more efficient provided there was tightly linkage between the marker and the trait of interest. In this study, we tried to develop technique system for markers assisted selection to facilitate breeding for male-sterile and maintainer line, finally shorten breeding years and accelerate step to heterosis utilization in welsh onion. Linkage analysis was made between S13 2800 , S38 960 , S72 300 , S73 1100 , S200 2400 and cytoplasmic types, whose recombination ranged from 0 to 7.5%. S13 2800 and S200 2400 , markers with no recombination found, could distinguish N and S cytoplasm in several cultivars. And great veracity and value would be achieved if the markers were utilized to cytoplasm identification in CMS breeding. Southern blotting analysis testified polymorphism between N and S cytoplasm using S13 2800 and S200 2400 as probe and restricted fragments of mtDNA from both sterile line and maintainer line, which indicated the two fragments be correlated with CMS in welsh onion. Furthermore, they were cloned to pGEM-T-easy vector, sequenced and converted to SCAR markers considering limiting factors of RAPD markers. Fortunately S13 2800 was successfully converted to SCAR marker and the new marker could differentiate cytoplasmic types correctly. But it failed to display polymorphism when S200 2400 was converted to SCAR marker. Simplified detecting technique of SCAR amplified products was then made to decrease the cost. The applications of RAPD and SCAR markers were expected to accelerate breeding progress and enhance breeding efficiency of sterile and maintainer line in welsh onion.