【作者】 王小艳；

【导师】 张恩慧；

【作者基本信息】 西北农林科技大学 ， 园艺植物种质资源学， 2010， 硕士

【摘要】 本研究以甘蓝Ogura胞质雄性不育系CMS451及保持系B451与甘蓝隐性核雄性不育两用系RGms03-632为材料,用RAPD和SRAP标记分析了两种不同类型雄性不育基因,获得了适宜于甘蓝的RAPD和SRAP反应体系和三个与不育基因连锁的差异条带,其主要结果如下:1.以甘蓝品种幼苗为试材,分别采用CTAB法,SDS法,Urea法提取其基因组DNA,结果表明,SDS法是提取甘蓝基因组DNA的最佳方法。2.优化了甘蓝适宜的RAPD反应体系,总反应体积为25μL,含有2.0 mmol/L MgCl2,0.2 mmol/L dNTPs,0.56μmol/L primer,0.5U Taq DNA聚合酶,20ng DNA,2.5μl,10×buffer,剩余的用ddH2O补充。3.优化了甘蓝适宜的SRAP反应体系,总反应体积为25μL,含有2.4 mmol/L MgCl2,0.3 mmol/L dNTPs,0.1μmol/L primer,1.5 U Taq DNA聚合酶,30ng DNA,2.5μL10×buffer,剩余的用ddH2O补充。4.经过RAPD引物筛选标记甘蓝Ogura胞质不育基因,发现S20-495在不育池中扩增出特异条带。在NCBI上比对此特异条带序列,发现它与芜菁自交不亲和系SRK-46,SP11-46,SLG-46的基因全序列同源性达78%。经过RAPD引物筛选甘蓝隐性核雄性不育两用系RGms03-632,在不育池中扩增出S1301-639稳定的差异条带,经Joinmap ver3.0分析标记结果与田间调查的差异,得其与不育基因遗传距离为8.854cM。在NCBI上比对测序结果,发现它与甘蓝自交不亲和系S-15的SP11A基因全序列的同源性达78%。5.SRAP标记甘蓝隐性核雄性不育两用系RGms03-632,发现Me6Em8组合引物在两混合池中扩增出稳定的特异条带,其条带大小在250-500bp之间,对其进行单株验证,用Joinmap ver3.0分析标记结果与田间调查的差异,计算出此条带与不育基因的遗传距离为22.548cM。更多还原

【Abstract】 In this research, analysing sterile gene cytoplasmic male sterile of Oguar cabbage CMS451 and maintainer line B451, recessive male sterile line RGms03-632 by RAPD and SRAP ,obtained the reaction systems of RAPD and SRAP which were suitable to cabbage and three specific bands which linked to sterile gene in cabbage.The main results are as follow:1. The genomic DNA from the variety of cabbage was extracted with CTAB,SDS Urea,respectively.Results showed SDS was optimal genomic DNA extraction method.2. RAPD reaction system of cabbage was optimized,total volume was 25μL, including 2.0mmol/LMgCl2,0.2mmol/LdNTPs,0.56μmol/Lprimer,0.5uTaq,DNA20ng,2.5μL10×buffer the rest was supplied with ddH2O.3 SRAP reaction system of cabbage was optimized,total volume was25μL, including 2.4mmol/LMgCl2,0.3 mmol/LdNTPs, 0.1μmol/L primer, 1.5 uTaq, DNA30ng, 2.5μL 10×buffer,2.5μL10×buffer,the rest was supplied with ddH2O.4. Ogura CMS cabbage was studied By RAPD. Results showed that S20-495 was believed to be the specific band in sterile pool.Though NCBI ,S20-495 sequence has higher alignment score with the SRK-46,SP11-46,SLG-46 gene sequence up to78%.Recessive male sterile line RGms03-632 was studied By RAPD. Results showed that S1301-639 was believed to be the specific band in sterile pool. The Genetic distance was 8.854cM identified by Joinmap ver3.0 software.Though NCBI ,S1301-639 sequence has higher alignment score with with Brassica self-incompatibility line S-15, SP11A gene sequence up to 78%.5. Recessive male sterile line RGms03-632 was studied By SRAP. Results showed that a pair of primer Me6Em8 was believed to be the specific band in sterile pool,the band was between 250-500bp. The Genetic distance was 22.548cM identified by Joinmap ver3.0 software.更多还原