【作者】 董发明；

【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】 利用杂种优势是提高油菜产量、增强抗耐性、缓解产量与品质矛盾的重要途径。隐性细胞核雄性不育具有不育性稳定彻底,恢复源广,无负胞质效应等优点,是一种具有较大应用潜力的授粉控制系统。然而,大部分隐性核不育材料无法完全保持,制种时必须拔除母本行中约50%的可育株。因此,应用隐性核不育系在大面积制种时成本高且风险较大。上世纪90年代,9012AB类型的隐性核不育通过引入临保系产生100%不育群体,为隐性核不育系统的利用开辟了一条新的途径。目前,该系统(或相似系统)已成为我国乃至世界油菜杂种优势利用的重要途径之一。本研究在系统的遗传分析和图谱定位的基础上,修正了9012AB的育性遗传模型。在此基础上,通过比较作图实现了BnRf位点的精细定位,并最终将其定位到一个BAC克隆13.8kb的物理区间内,主要结果如下：1.9012AB类型核不育育性遗传模式的修正。通过对9012AB及其临保系T45与来源不同的几个自交系和常规品种的遗传分析,发现之前被广泛接受的三对基因(Bnms3,Bnms4,Bnrf(?)或Bnesp)控制育性的遗传模式不能合理解释9012A与DH206A杂交后代的育性分离比率。基于这一事实,对9012AB的育性遗传模式进行了修正,即雄性不育只受Bnms3和BnRf两个位点控制,显性BnMs3为野生型可育基因,隐性Bnms3为突变不育基因；而原来假定的BnMs4位点实质上是BnRfa位点的一种复等位基因型BnRfa(恢复型),该位点的另外两种基因型分别为BnRfb(不育型)和BnRfc(可育型),三者的显隐性关系为BnRfa>BbRfb>BnRfc.按照这一遗传模式,9012AB可育株基因型为BnMs3ms3RfbRfb,不育株基因型为Bnms3ms3RfbRfb；临保系基因型为Bnms3ms3RfcRfc,恢复系基因型为BnMs3Ms3或Bn__RraRfa2.BnRf复等位遗传模式的分子标记验证。在来源于(9012A×DH206A)F2中可育株与9012A回交得到的育性分离比为1：1的F2BC1株系中,鉴定出基因型为Bnms3ms3RfaRfb的可育株。在该类型可育株继续与9012A回交得到育性分离比为1：1的群体RG206AB(RG206A,Bnms3ms3RfbRgbRG206B,Bnms3ms3RfaRfb)中,筛选获得了与BnRga等位基因型连锁的13个AFLP标记和5个SSR标记。标记整合发现,5个SSR标记都被定位于A7连锁群且同时与BnRfb基因型连锁,而3个由AFLP转化而成的SCAR标记亦与BnRfb存在连锁关系。在此基础上,利用BnRfb精细定位的标记实现了BnRfa的精细定位,并将BnRfa和BnRfb共定位到标记AT3G24240与AT3G23900之间,对应拟南芥共线性区域约50kb。该结果从分子标记水平上初步证实了Rf位点复等位遗传模式的可靠性,并为该位点物理图谱的整合奠定基础。3.含BnRf位点区域的物理图谱构建。利用BnRfb共分离的分子标记AT3G23870从9012A中扩增的特异序列为探针筛选甘蓝型油菜BAC克隆文库,获得16个阳性克隆。位点两侧最近的共显性标记AT3G23900及AT3G24240对候选克隆的PCR分析表明,BAC克隆JBnB089D05和JBnB134D11最可能包含目标位点。而源于JBnB089D05两侧末端序列开发的标记BES18和BES19进一步被定位到BnRf两侧,证实该克隆包含目标基因。从该BAC克隆上获得了BnRf两侧最近标记AT3G23900与AT3G23910-1之间13.8kb的序列,预测表明其包含3个完整的ORF及2个ORF的部分片段。更多还原

【Abstract】 The utilization of heterosis is an important approach to improve yield, resistance for biotic stress and abiotic stress, and alleviate yield and quality contradictions in rapeseed. As an effective pollination-control system, recessive genic male sterile (RGMS) lines are always regarded to possess several advantages, such as stable and thorough male sterility, almost no restriction of restorers and diverse sources of cytoplasplasm. However, a major drawback of most RGMS lines lies in the fact that the male-sterility can not be fully maintained. This shortcoming would inevitably cause a high labour cost for removal of the50%fertile from the mother line and a great risk of seed purity in commercial production of hybrids. This trouble has been basically solved with the introduction of a novel RGMS line9012AB, which has the capability of generating a complete male-sterile population by crossing9012A plants with the so-called temporary maintainers. In this study, we modified the hereditary mode on the inheritance of male sterility in9012AB, as a result of classical genetic analyses and molecular marker assay. Based on this result, we finely mapped the BnRf locus through map integration and comparative genomics, and finally delimited it into a13.8-kb DNA fragment of a BAC clone. The main results were as follows:1. Modification on the hereditary mode of male sterility in9012AB. By genetic analyses of the crosses between9012AB and its temporary maintainer line T45with some inbred lines and open-pollination (OP) cultivars, we found that the fertility segregation data cannot be united under the previous widely-accepted tri-genic hereditary mode on sterility inheritance. Instead, we proposed a new mode to interpret the fertility control in9012AB that it is conditioned by two individual loci, i.e. BnMs3and BnRf. At the BnMs3locus, the dominant allele can restore the male sterility caused by the recessive allele. The putative BnMs4locus is actually one allele of BnRf (BnRfa, the restoration allele), and the other two alleles are respectively BnRfb (the sterility allele from9012A) and BnRf (the wild type allele from TAM), with the dominance relationship of BnRfa>BnRfb>BnRf. According to this new mode, the genotypes of9012A,9012B and its temporary maintainer line T45are individually Bnms3ms3RfbRfb, BnMs3ms3Rfb Rfband Bnms3ms3RfcRfc. Similarly, the genotype of restoration lines is BnMs3Ms3__or__RfaRf 2. Molecular marker evidence on the multiple-allele BnRf locus. From the F2BC1families9012A×(9012A×DH206A)F2in which a fertility segregation ratio of1:1is observed, the fertile plants with the genotype of Bnms3ms3RfaRfb were identified. By successively backcrossing these plants with9012A, we generated a ferlity segregation population RG206AB (RG206A, genotyped as Bnms3ms3RfhbRfb;RG206B, genotyped as Bnms3ms3RfaRfb). Molecular marker screen among this population enabled us to yield13AFLP markers and5SSR markers associated with BnRf. Marker integration showed that all these5SSR markers are located on the A7linkage group of B. napus and linked to the allele of BnRfb. Similarly, three of the7AFLP-converted SCAR markers are also genetically close to BnRf. These results together drove us to finely map the BnRfa allele by using the previously identified markers from the high-resolution genetic map of BnRfb. As expected, both of the BnRf and BnRfb alleles were finally delimited to a region flanked by marker AT3G24240and AT3G23900, corresponding to a physical region of50kb in Arabidopsis chromosome3. This investigation on molecular markers around BnRfa and BnRf greatly supports the allelism of them at the BnRflocus.3. Physical mapping of the BnRf locus. We amplified the specific fragment from the genomic DNA of9012A by using the cosegregated marker AT3G23870. This PCR product as a hybridization probe fished16positive BAC clones from a B. napus BAC clone library, and. PCR analysis with the nearest flanking codominant markers (AT3G23900and AT3G24240) showed that BAC clones JBnB089D05and JBnB134D11are most likely to contain the candidate locus of BnRf. Subsequent mapping of the BAC end derived markers BES18and BES19on respective side of BnRf, further indicated that JBnB089D05is the target BAC clone which spans the BnRf locus. We successfully recovered the13.8-kb fragment between the closest flanking markers AT3G23870and AT3G23910-1from JBnB089D05. Gene prediction of the13.8-kb sequence showed that there are3complete ORFs and partial genomic sequences of2ORFs.更多还原