【作者】 李季；

【导师】 杨光圣；

【作者基本信息】 华中农业大学 ， 作物遗传育种， 2012， 博士

【摘要】 9012AB是我国上世纪90年代选育的一类甘蓝型油菜新型细胞核雄性不育两型系,它利用临保系成功克服了其它核不育系统在制种过程中需要拔除50%可育株的难题。早期遗传分析表明,9012AB雄性不育性状受三对独立的基因(BnMs3, BnMs4和BnRf)互作控制(陈凤祥等,1998)。而最近的研究发现,BnMs4与BnRf等位,从而将三基因互作修正为双基因控制的隐性核不育(董发明等,2010)。为揭示该不育系雄性不育发生的分子机理,在前期标记定位的基础上,本研究重新构建了与9012AB遗传背景不同的三个定位群体(PopB, PopC和PopD),并通过图谱整合实现了核不育基因Bnms3的精细遗传和物理定位,采用图位克隆和比较基因组学相结合的方法,最终成功分离BnMs3基因。主要结果如下：1.通过对与9012AB遗传背景不同的三个新定位群体8,000余单株的分析,将原来与目标基因BnMs3共分离的标记SCR2和9W21定位于基因的同一侧,分别包含有2个和1个重组单株；而SCR1仍然与BnMs3共分离。MT7则位于BnMs3的另一侧,包含有1个重组单株。根据JBnB004A17的序列在9W21和SCRl之间设计23对SSR引物,其中重组标记BSR73的鉴定进一步将BnMs3的候选区间缩小至39kb。2.利用能有效区分甘蓝和白菜亚基因组的标记DM1检测前期构建的BAC克隆重叠群显示,候选克隆JBnB004A17来源于甘蓝型油菜A10染色体,对应BnMs3的同源区。因而利用该标记扩增BnMs3连锁的片段为探针重新筛选BAC文库并获得12个阳性BAC克隆。指纹图谱分析和标记分型表明,BnMs3候选区间可由6个来源于C9染色体的BAC克隆构建成一个完整的重叠群。测序其中可能包含目标位点的克隆JBnB043L23,获得其约118kb插入的序列。3.基于JBnB043L23和JBnB004A17在标记BSR73和MT7定位区段的差异序列设计新的SCAR和SSR引物,检测BnMs3所位于的甘蓝亚基因组片段的多态性。利用各定位群体的重组单株分析,标记RCP137和RCP145最终将BnMs3限定在JBnB043L23上约9.3kb的物理区段内。4.基因预测显示,9.3kb的候选区间存在一个完整的开放读码框(ORF2)和另外两个读码框(ORF1和ORF3)的部分序列。候选基因遗传转化实验证实ORF1为BnMs3的目标基因,其DNA序列及预测的编码蛋白均与拟南芥基因At5g16620(Tic40)高度同源,因此将BnMs3命名为BnaC9.TlC40.a,突变体Bnms3命名为bnaC9.tic40.a-2,位于A10染色体的高度同源基因则命名为BnaA10.TIC40.a。序列分析表明BnaC9.TIC40.a含有14个外显子,编码一个包含455个氨基酸残基的蛋白,具有丝氨酸／脯氨酸富集结构域、跨膜结构域、TPR结构域和Sti1p/Hop/Hip结构域等4个保守结构域。RT-PCR的分析表明,BnaC9.TIC40.a在9012B的1-5级雄蕊、根、茎、叶和花瓣等各个组织中均存在表达,bnaC9.tic40.a-2和BnaA10.TIC40.a在9012B和9012A的各个组织中亦存在表达。5.从BnMs3野生型材料(9012F),Bnms3突变型材料(9012A和临保系T45),以及两个同源BAC克隆中分别分离两侧最近标记锚定的序列,比较发现9012A中候选区间序列与来源于C9染色体的目标克隆JBnB043L23的相似性仅为79.1%,但与来源于A10染色体的同源克隆JBnB004A17之间相似性却为85.5%。相反,9012F中候选区间序列与JBnB043L23具有93.3%的相似性,与JBnB004A17之间相似性仅为80.8%。该数据暗示着9012A中Bnms3等位基因型很可能是由甘蓝型油菜中A10与C9染色体间的同源重组(homologous recombination)而致。而来源于临保系T45的候选区间序列与JBnB043L23之间的相似度高达99.9%,表明其内的Bnms3等位基因型是由BnMs3等位基因型的少量碱基自然变异所致。因此,不育基因型Bnms3存在两种不同的单倍型。6.临保系T45与野生型材料Tapidor之间在BnaC9.TIC40.a内部只存在2bp的缺失变异,根据该处碱基变异设计的InDel标记RCP170L/R能够很好的区分临保系来源的Bnms3与其它所有材料,表明标记RCP170L/R为临保系T45中Bnma3单倍型的功能标记。而基于9012A和野生型材料Tapidor及9012F中BnaC9.TIC40.a序列差异处开发的共显性标记RCP170L2-3/R2能够很好的区分9012A与其他所有材料。因此,原则上,上述两个标记可准确的鉴别任何分离群体中BnMs3位点的基因型。更多还原

【Abstract】 A novel recessive genic male sterility line,9012AB, has been characterized by generating a complete male-sterile population through pollinating9012A male-sterile plants with the temporary maintainers. Preliminary genetic analyses suggested that three loci (BnMs3, BnMs4and BnRf) are responsible for the male sterility in9012AB (Chen et al.,1998). Recent study, however, suggests that the BnMs4locus should be allelic to BnRf, turning the trigenic inheritance model to a digenic one (Dong et al.,2010). In this research, we constructed three new populations with different genetic compositions (Pop B, Pop C and Pop D, respectively) to isolate the BnMs3locus, on basis of the fine genetic mapping of BnMs3(He et al.,2008). With the integration of classic map-based cloning strategy and comparative genomics, we successfully cloned the gene, BnMs3. Main results are as follows:1. Genetyping of more than8,000individuals among the new populations showed that BnMs3, still cosegregated with SCR1, can be restricted by two previously BnMs-cosegregated markers9W21and MT7. each with one recombination event. Hereby, we designed new SSR primers according to the sequence between9W21and SCR1on BAC clone JBnB004A17. The development of a new marker (BSR73) on the same side as9W21further reduced the candidate region of BnMs3to a39-kb fragment on JBnB004A17.2. We used DM1, a BnMs3-linked marker which can effectively differentiate the A and C subgenome in B. napus, to identify the BAC clone JBnB004A17. It was revealed that the resulting fragment corresponds to the band in B.rapa, and is different from the polymorphic band associated with BnMs3in the mapping polulations, suggesting that JBnB004A17is actually originated from a BnMs3paralogous region in the A subgenome. The BnMs3-linked fragment amplified by DM1from Tapidor was therefore employed to screen the BAC library again. PCR and Southern blotting analysis found that6BAC clones from C subgenome possibly cover the BnMs3candidate region. One BAC clone from them, JBnB043L23, was then shotgun sequenced, harvesting an insertion fragment of118kb.3. High sequence similarity was observed on a large scale between JBnB043L23and JBnB004A17, while extensive variations including fragment insertion/deletion and massive SNPs were also revealed. According to the sequence differences between the two homologous BAC clones, SCAR primers specifically binding to JBnB043L23as well as SSR primers were designed from the61-kb region delimited by marker BSR73and MT7. Population analysis showed that massive primers located in this region could generate good polymorphism. Assay of recombinantion individuals with these markers led to our final delimitation of BnMs3to a9.3-kb physical region, restricted by closest flanking markers RCP145and RCP137.4. Gene prediction analysis of the9.3-kb fragment identified only one complete ORF (ORF2) and partial genomic sequences of its two flanking genes (ORF1and ORF3). Complementation test indicated that the ORF1, restoring the fertility of the male-sterile9012A plants, is the target gene of BnMs3. We designed BnMs3as BnaC9.TIC40.a, since ORF1was highly homologous with Arabidopsis gene At5g16620(Tic40), and the mutant of9012A was braC9.tic40.a-2. Consistently, the A10paralogue of BnMs3was referred to BnaA10.TIC40.a. The allele of BnaC9.TIC40.a from Tapidor includes14exons, and is predicted to encode a protein with455amino acids, which contains four conserved domains, i.e.. a Ser/Pro-rich domain, transmembrane domain. TPR domain and Stilp/H op/Hip domain. RT-PCR analysis showed that BnaC9. TIC40.a can be expressed in all the tissues tested in9012B, including stamens with diffenerent sizes, roots, stems, petals and leafs. Additionly, whether in9012A and9012B, bnaC9.1ic40.a-2and BnaA10. TIC40.a were found to be expressed in all the tissues tested.5. The genomic sequences corresponding to the9.3-kb BAC fragment were individually isolated from9012A,9012F and T45. Comparison of these fragments among different lines showed that9012A has an85.5%similarity with JBnB004A17, much higher than that with JBnB043L23(79.1%). In contrast,9012F has a93.3%similarity with JBnB043L23, while its similarity with JBnB004A17is only80.8%. In most cases,9012A was found to have an identical sequence with JBnB004A17in the sites polymorphic between JBnB004A17and JBnB043L23, whereas9012B is closely identical to JBnB043L23. However, the sequence similarity in the candidate region between T45and JBnB043L23was99.9%. Thus, we proposed that the Bnms3allele in9012A was originally caused by an occasional homeologous reciprocal recombination between chromosome A10and C9, whereas the Bnms3allele from T45was most likely to be derived from a spontaneous mutation. So, there are two Bnms3alleles with distinctive origins. 6. We designed an InDel marker according to the2-bp nucleotide variation between T45and Tapidor in BnaC9.TIC40.a. RCP170L/R can accurately differentiate the BnMs3allele from all the inbred lines (OP cultivars) from the Bnms3allele in T45, indicating it is a functional marker for the Bnms3locus in T45. We also developed another codominant marker RCP170L2-3/R2according to the differences between9012A and9012B of BnaC9.TIC40.a, which can successfully differentiate the Bnms3allele in9012A from all the other materials with a wild-type BnMs3allele as well as T45. In principle, utilization of these two markers can accurately trarget the genotype of the BnMs3locus in any segregation generation involved in RGMS breeding.更多还原