【作者】 刘俊；

【导师】 杨光圣；

【作者基本信息】 华中农业大学 ， 作物遗传育种， 2008， 博士

【摘要】 Rs1046AB是派生于宜3A的纯合型甘蓝型油菜显性细胞核雄性不育系,具有败育彻底、不育性稳定和恢复源广泛等特点。1985年,李树林等经过大量的遗传分析后,提出甘蓝型油菜显性核不育材料宜3A的育性受两对显性基因互作控制的遗传假说,其中一对为显性不育基因,另一对为显性上位抑制基因,当显性不育基因单独表达时可以导致雄性不育,而当显性上位抑制基因存在时,育性又可以恢复正常。最近宋来强等（2006）通过遗传分析表明利用一对复等位基因控制的模式来解释Rs1046AB的遗传更加合理,即Ms为显性不育基因,Mf为等位显性恢复基因,ms为正常可育位点,并且具有Mf＞Ms＞ms的显性遗传效应。以Rs1046AB为主要材料,洪登峰（2006）已利用一个192株的F₂群体对Rs1046AB的不育基因和恢复基因进行了初步定位。以此为基础,本研究中通过不育系Rs1046A和恢复系195-14A（温敏型细胞质雄性不育两用系）杂交构建F_2:3家系和F₂全不育株群体,利用分子标记技术筛选与不育基因Ms和恢复基因Mf连锁的分子标记,进一步开展显性核不育基因与恢复基因（Ms/Mf）的精细定位研究,取得的主要结果和结论如下:1.将3个AFLP标记E3M10、E1M13和S5T5（洪登峰,2006）成功转化为SCAR标记（依次命名为:SCD2、SCD7和SCD8）。,并结合SCAR标记SCE3（陆光远,2003）和SCHDF（洪登峰,2006）,进一步分析708个单株构成的F_2:3家系和987个单株的F₂全不育株群体,结果表明:SCD2、SCE3、SCD7和SCD8 4个SCAR标记全部位于目标基因一侧,距目标基因（Ms/Mf）的遗传距离依次为2.0cM、1.7cM、1.5cM、0.1cM,另外一个SCAR标记SCHDF位于目标基因的另一侧,且距目标基因的遗传距离为2.3cM,实现了对目标基因的准确定位。2.应用BSA法,结合AFLP技术,继续筛选了512对AFLP的引物组合,获得了4个与目标基因紧密连锁的AFLP标记。其中2个标记（P1M1、P1M4）为共显性标记,被定位于SCAR标记SCD7与SCD8之间。另外2个标记（P3M2、P12M6）为显性标记,与恢复基因连锁,分别位于目标基因两侧,前者被定位于SCAR标记SCD7和SCD8之间,后者被定位于标记SCHDF与目标基因之间,使目标基因的遗传距离进一步缩小。3.通过比较测序,将Song et al.,（2006）在甘蓝型油菜显性核不育系609AB中获得的不育基因两侧最近的SCAR标记SC6和SC9进行整合,并成功转化为既与不育基因连锁又与恢复基因连锁的SCAR标记,重新被命名为SC6D和SC9H。前者被定位于AFLP标记P12M6与目标基因之间,后者被定位于AFLP标记P3M2和P1M1/P1M4之间,且分别距目标基因的遗传距离均为1.0cM。结合上述AFLP标记和SCAR标记,在本研究中实现了对目标基因（Ms/Mf）的精细遗传定位。4.利用DH群体TN（Tapidor×Ningyou7）的遗传图谱将目标基因Ms/Mf定位于甘蓝型油菜的N8连锁群,并在N8连锁群上成功开发出一个与Ms/Mf基因连锁的共显性SSR标记HUA348,实现了目标基因精细定位图谱与已公布的甘蓝型油菜图谱的整合。5.根据目标基因区段与对应拟南芥同源区的信息（洪登峰,2006）,在目标基因两侧标记之间,设计18对特异引物（DFN1-DFN18）对目标区段进行扩增,结果除了DFN03、DFN12、DNA14和DFN18等4对引物外,其余14对引物都同时扩增出不育基因和恢复基因,对这14对引物进行比较测序分析之后再次设计引物,结果仍然没有开发出新的分子标记。6.以与目标基因紧密连锁的SCAR标记SCD8为探针进行Southern杂交,筛选甘蓝型油菜Tapidor BAC文库,获得了29个具有强杂交信号的BAC克隆。7.利用DFN1、DFN5、DFN8、DFN16和SCD8共5对引物通过PCR分析的方法对所获得的29个BAC克隆进行阳性验证,结果依据其中的18个克隆形成了可能覆盖Ms/Mf基因的BAC克隆重叠群。利用限制性内切核酸酶HindⅢ完全酶切29个BAC克隆,构建其指纹图谱,进一步验证了利用PCR分析法构建的BAC克隆重叠群。更多还原

【Abstract】 Rs1046AB is a dominant genic male sterility （DGMS） line derived from a spontaneous mutant of Yi-3A in Brassica napus. It has some advantages: such as complete and stable male sterility, widely spread of restorers and no negative cytoplasmic. The sterility of this mutant was previously regarded as to be conditioned by the interaction of a dominant male sterility gene Ms and its non-allelic dominant restorer gene Mf （or Rf in previous reports） （Li et al., 1985）. Recent genetic analyses, however, indicated that Ms and Mf may be allelic （Song et al., 2006）. Using an F₂ population （Rs1046A×195-14A） of 192 plants, Hong （2006） have constructed a primary linkage map of Ms/Mf genes. Based on these result, the present study emphasized on identifying DNA markers linked to Ms/Mf genes in the F_2:3 and sterile plants in F₂ populations constructed by crossing Rs1046A with a double haploid （DH） restorer line （19514A）. Main results of the present study are as follows:1. Three AFLP markers E3M10, E1M13 and S5T5 （Hong, 2006） were converted into SCAR markers successfully （designated as SCD2, SCD7 and SCD8）. Five SCAR markers, including SCE3 （Lu, 2003） and SCHDF （Hong, 2006） were used to analysis in F_2:3 population and all sterile plants in F₂ population, which including 708 individuals in former population and 987 individuals in latter population, respectively. As a result, four SCAR markers SCD2 , SCE3 , SCD7 and SCD8 all have been mapped in one side of the target genes （Ms/Mf）, with the genetic distances of 2.0cM, 1.7cM, 1.5cM and 0.1 cM, respectively, another SCAR marker SCHDF has been mapped in the other side of the target genes, with the genetic distance of 2.3cM.2. AFLP technology combined with bulked segregant analysis （BSA） was used to identify the genetic markers more tightly linked to Ms/Mf genes. Additional 512 primers combinations were screened and four AFLP markers linked with the Ms/Mf genes were obtained. Among them, two markers （P1M1 and P1M4） were co-dominant markers, they have been mapped between the SCAR markers SCD7 and SCD8. Another two markers （P3M2 and P12M6） were dominant markers, have been mapped in two sides of the target gene （Ms/Mf）, the former was between SCD7 and SCD8, and the latter was between SCHDF and target genes.3. Using comparative sequencing technology, two SCAR markers （SC6 and SC9） obtained by Song et al., 2006, which linked with the sterile gene Ms of 609AB in Brassica napus have been integrated in the linkage map of present study and converted into co-dominant markers, designated as SC6D and SC9H. SC6D was mapped between AFLP marker P12M6 and target genes, and SC9H was mapped between AFLP markers P3M2 and P1M1/P1M4, with the genetic distance of 1.0cM. Combining the above AFLP markers and SCAR markers, the target genes （Ms/Mf） have been finely mapped in a little genetic regions.4. Through comparing with the linkage map of DH （Tapidor×Ningyou7） population, the target genes {Ms/Mf) have been mapped on linkage group N8. One co-dominant SSR marker located on linkage group N8, HUA348, was confirm to linkage to the Ms/Mf genes.5. Based on the homologous region sequences of target genes （Ms/Mf） region and Arabidopsis homologous, 18 specific primers （DFN1-DFN18） have been designed to amplify Ms gene and Mf gene. As a result, 14 primers could have amplified Ms gene and Mf gene together, except for primers DFN3, DFN12, DFN14 and DFN18.6. Using the SCAR marker SCD8, linked with the restorer gene Mf as a probe screened the Tapidor BAC library by Southern hybridization, and 29 positive BAC clones were identified.7. Twenty-nine positive BAC clones were identified by four specific primers DFN1, DFN5, DFN8, DFN16 and SCAR marker SCD8, then, an overlap contigs maybe covered Ms/Mf genomic region were constructed by the 18 positive BAC clones in 29 positive BAC clones. 29 positive BAC clones were digested with HindIII, and a fingerprint of 29 positive BAC clones has been constructed. As a result, it is a further verification of the possible overlap contigs covered Ms/Mf genomic region were constructed by PCR amplification.更多还原