【作者】 万正杰；

【导师】 傅廷栋；

【作者基本信息】 华中农业大学 ， 作物遗传育种， 2008， 博士

【摘要】 油菜是世界上重要的油料作物之一,油菜杂种优势已得到广泛应用,而细胞质雄性不育是油菜杂种优势最重要的途径。芥菜型油菜hau细胞质雄性不育是傅廷栋教授于1999年在华中农业大学发现的,通过回交转育已获得了稳定的油菜细胞质雄性不育系。本研究从育性调查、细胞学观察、恢保关系分析以及分子水平鉴定等方面对芥菜型油菜细胞质雄性不育hau CMS（Cytoplasmic male sterility）进行综合遗传分类;在遗传分类的基础上,通过线粒体DNA位点和RNA表达水平的差异分析,初步进行了hau CMS不育分子机理研究;利用hau CMS不育的稳定性,将不育细胞质转育到甘蓝型油菜和芸薹属十字花科蔬菜中;同时进行了恢复基因的筛选。主要研究结果如下:1.对芥菜型油菜细胞质hau不育系00-6-102A在武汉和兰州进行了6年12个回交世代的育性观察,hau CMS的不育性稳定彻底,雄蕊通常败育为小花瓣。不育株率和不育度均为100%,且不受环境条件的影响;将芥菜型细胞质转移到甘蓝型油菜6-W301和6-W307中,获得了同样稳定的甘蓝型油菜细胞质雄性不育系。2.在所有320份测交材料中,用14个来源不同的甘蓝型油菜已知恢复系、保持系材料分别与hau、pol、nap、ogu、tour等5种CMS进行恢保关系测定,鉴定测交F₁的育性,结果表明:hau CMS与nap、pol、ogu、tour胞质不育型具有明显不同的恢保关系。3.制作石蜡切片对hau CMS不育系00-6-102A和保持系00-6-102B进行了细胞学显微结构观察,hau CMS的花药败育发生于雄蕊原基分化时期。其特点是,败育时期很早,没有孢原细胞的分化,6枚雄蕊都败育为小花瓣,雄蕊偏离正常的分化轨道。Hau CMS的花药败育特点明显不同于以前研究过的油菜胞质不育类型（如ogu、pol、nap、tour CMS等）。4.用10个线粒体基因的40个探针/酶组合进行RFLP分析,总共有16个探针/酶组合（atp6/HindⅢ、atp6/EcoRI、atp6/EcoRV、atp9/BamHI、atp9/EcoRI和atpl/BamHI等）可以将hau CMS与pol、nap、ogu、tour CMS区分开。其中,5个探针/酶组合（atp1/BamHI、atp6/BamHI、orf222/EcoRI、Orf263-atp6/EcoRI、Drj222/HinaⅢ）能将5个CMS彼此区分开,可以作为鉴定油菜细胞质类型的标记使用。RFLP分析结果进一步证明,hau CMS在线粒体DNA水平上与pol、nap、ogu、tour不育胞质不同,是一种新的油菜不育胞质类型。5.在hau CMS育性分子机理研究方面,通过已经公布的甘蓝型油菜品种westar线粒体基因组序列和拟南芥线粒体基因组序列,PCR扩增获得34个油菜线粒体探针,对hau CMS不育系00-6-102A和保持系进行Realtime-PCR和Northern blot分析,结合线粒体DNA水平上的位点差异,获得6个可能与hauCMS不育相关的线粒体基因,经过PCR-walking和序列分析,其中位于atp6基因下游的一个新的开放阅读框orf288可能是与hau CMS密切相关的线粒体基因,其与已经公布的主要油菜细胞质雄性不育的基因区域不同。6.通过杂交和回交已将hau不育细胞质转移到芸薹属十字花科蔬菜（白菜、榨菜、雪里蕻、包心芥和红菜薹）中,获得稳定的蔬菜雄性不育系,且不育株率和不育度均为100%。7.用不同来源的320份芥菜型、甘蓝型油菜、埃芥及种间杂种与不育系测交,目前还未找到完全恢复的材料,只有2份部分恢复的材料,但还需要进一步测定。更多还原

【Abstract】 Rapeseed is one of the major oilseed crops.Utilization of rapeseed heterosis is widely applied.Cytoplasmic male sterility(CMS) is an important approach to exploit heterosis in this crop.The hau CMS(00-6-102A) was originally found as a spontaneous male sterile mutant in B.juncea in the experimental field of HAU in 1999.Stable CMS line was obtained by backcrossing.We adopted an integrated approach to classify the five types of CMS systems at morphological,cellular,genetic and molecular levels and confirmed that the hau CMS is different from others.Fertility mechanism of hau CMS was primarily confirmed by analysis of the Realtime-PCR,Northern blot and mitochondrial DNA Polymorphism As the sterility of hau CMS lines was stable and complete,this CMS was introgressed to B.napus and Cruciferous vegetables by hybridization.At the same time, the restorer lines were being screened.1.After twelve generations of backerossings with B.juncea 00-6-102B,a stable sterile B.juncea line(hau CMS) was established.The anthers in CMS plants were replaced by thickened petal-like structures and pollen grains were not detected. The"sterility degree" of 3,000 flowers was 100%and the "sterility rate" of 300 plants was also 100%in Lanzhou and Wuhan,indicating the stability of hau CMS in two different environments.This CMS was transferred to B.napus through interspecific hybridization. The resultant B.napus CMS was also absolute and stable when 6w-301 and 6w-307 were used as maintainers.2.Out of 320 materials of different origin,14 known resteror and maintainer lines in B.napus were selected for test-crossing with hau,pol,ogu,nap and tour CMS.The hau CMS line 6W-301A has the same nucleus genetic background as its B.napus maintainer line 6W-301 after eight backcross generations(BC8).The results of test-crossing with restorer lines indicated that fertility in the hau CMS was not restored by restorer lines from pol,ogu,nap and tour CMS systems.Therefore we concluded that hau CMS was a distinct type of cytoplasmic male sterility.3.Cytological observation of hau CMS anther development indicated the transverse section were thicker with increased cellular layers than normal petals and had no pollen sacs.The stamen primordium deviated from the normal polarization and formed petal primordium,which developed into petal-type structures at the stamen location.These characteristics differed from other CMS systems reported so far.The pollen abortion stage was earlier than other CMS types.The anther development of maintainer 00-6-102B was normal,and each anther had four normal pollen sacs in papilionaceous shape.The A-lines of pol,nap,tour,and ogu CMS systems formed anthers,but were devoid of functional pollen.4.The polymorphism of mitochondrial DNA was detected with mitochondrial probes atpl,orf222,atp6,atp9,cob,coxⅡ,Orf222-nad5-orf139,coxⅠ,atpa and Orf263-atp6.Out of 40 mitochondrial probe/enzyme combinations,16 combinations exhibited polymorphism such as atp6/HindⅢ,atp6/EcoRI,atp6/EcoRV,atp9/BamHI,atp9/EcoRI and atp1/BamHI.Hau CMS 00-6-102A showed different band patterns from the other four CMS lines and five probe/enzyme combinations alone distinguished the five CMS systems from each other.These results indicated that the mitoehondrial DNA of hau CMS 00-6-102A was different from those of pol,nap,ogu and tour CMS.5.By analysis of the Realtime PCR,Northern blot and mitochondrial DNA Polymorphism,six genes were possibly associated with fertility mechanism of hau CMS. Furthermore,PCR-walking and sequence analysis were conducted.The results showed that a new open reading frame ’ORF288’ probably associated with hau CMS was located at the downstream of atp6 gene.6.This CMS was introgressed to Cruciferous vegetables(such as B.juncea var.capitata Hort.ex Li,B.juncea var.multiceps Tsen et Lee,B.juncea Czern.et Coss.var.tsatsaiMao,B.rapa Linn.var.purpurea Bailey and B.rapa var.pekinensis ) by hybridization and backcross.The sterility of obtained CMS lines was stable and complete,in contrast to other four CMS types.7.Test crossing with 320 materials of different origins including B.juncea,B.napus, B.carinata and interspecific hybrids indicated material completely restoring the fertility of hau CMS was not found so far.The individuals obtained from test-crossing with two materials were partially fertile.更多还原