【作者】 董军刚；

【导师】 何蓓如；

【作者基本信息】 西北农林科技大学 ， 作物遗传育种， 2010， 博士

【摘要】 细胞质雄性不育是油菜杂种优势利用的重要途径。生态型细胞质雄性不育系可以实现一系两用,且恢复源广泛,利于配制杂交种,在育种上具有很大的应用前景。然而,合适的育性转化条件是生态型雄性不育系利用的关键因素。本文以温敏细胞质雄性不育系533S为不育源,利用杂交选育的方法育成新的温敏细胞质雄性不育系417S,并对其育性转化的光温条件、育性的遗传、细胞质类型等进行了研究。取得的主要结果如下:1.417S与其不育源的异同417S与其不育源的不育性状类似,表现对高温敏感。杂交结果表明417S的细胞质与源不育系细胞质相同。与不育源相比较,417S在育性转换条件、品质性状和农艺学性状上取得了明显的改良。具体表现在:417S的育性转换温度高于源不育系,这使得417S的制种区更易寻找,不育系的硫苷含量由原来的高硫苷(125.6μmol/g饼)改良为低硫苷(24.2μmol/g饼),含油量提高近6个百分点,株高降低了14.6cm,抗倒伏性明显增强。2.417S的育性类型、育性转换敏感期及育性转换指标通过人工控温、控光试验,对417S的育性敏感类型做了分析。结果表明,417S属温敏型不育系。日自交结实率与日平均气温相关分析表明,417S的育性敏感期为单花开花前3-12天,敏感期长度为10d左右。当开花前3-12天日平均温度低于17.0℃时,417S所开的花为雄性不育花,单株自交结实率很低,而高于20.5℃时, 417S开的花表现雄性可育,自交结实正常。当温度处于17.0-20.5℃之间时,417S表现为半不育,有微量花粉产生,自交可部分结实,为育性转换温度。3.417S温敏不育性的遗传通过测交、杂交和回交结合人工控温试验和RAPD方法对417S的温敏不育性的遗传进行了分析。结果表明:417S属质核互作型不育系,其温敏不育性受不育细胞质和2对主效隐性重叠基因控制,同时还受数目不定的微效多基因修饰。在大量测交基础上,仍未能从现有的品种或资源中筛选出保持系。所有测交的品种对417S均表现不同程度的育性恢复。正反交结果表明,1102C可能有着与417S不同的细胞质。通过RAPD分析,找到了1个与恢复基因连锁的分子标记BA392-400bp,该标记与恢复基因的遗传图距为6.0cM,同时在分子水平上初步验证了417S的遗传规律。4.417S线粒体基因组的PCR分析用25对引物对7个甘蓝型油菜线粒体DNA进行PCR分析,在7个材料中共扩增出116条带,共24种带型。平均每对引物在1个材料中扩增出0.7条带。多数引物扩增产物为单一条带。其中,有7对引物在参试的材料中无差异;2对引物(P18、P19,pol cms特异引物)只在pol cms不育系中出现一条带,表明417S与pol CMS不同;有5对引物可将417S和nap CMS区别开,分别涉及线粒体的atp1,atp9,nad4L,orf263和orf222-nad5c-orf139五个基因区域。UPGMA聚类分析结果表明,参试的7个材料可分为3类,第一类包括材料417S和nap CMS,第二类包括1102C、Bronowski和ZS9,第三类包括pol CMS和Westar。417S与nap CMS的育性表现最为相似,可能有着近似的细胞质起源。nap CMS的保持系与1102C(pol CMS系统的恢复系)分为一类,这与遗传试验结果1102C可能为417S的保持系相一致。5.RFLP杂交分析用7种探针atp1、atp6、atp8、atp9、cob、cox1和cox3结合5种内切酶(EcoR I,EcoR V,HindⅢ, BamH I, Pst I)检测4种甘蓝型油菜417S、pol、nap和ogu cms mtDNA的多态性。在25种有效探针/酶组合中,3个组合在检测的4个不育系间无多态性;1个组合cox1/Hind III能将4种不育细胞质彼此区分开来,417S产生了1条4.5kb的杂交带,pol CMS产生了6.6和4.3kb的两条带,而nap CMS产生了1条6.6kb的条带,ogu CMS产生了1条4.3kb的条带;8个探针/酶组合能将417S细胞质同pol, ogu, nap不育细胞质区分开来;7个探针/酶组合可将pol, nap和ogu cms两两区分开来。在25种有效的线粒体探针/酶组合中,有19种探针/酶组合检测到417S同其它3种不育细胞质的差异,有17个探针/酶组合可将417S与nap CMS分开。结果表明生态雄性不育系417S与pol、nap、ogu CMS在线粒体DNA水平是不同的,为不同的细胞质类型。6.417S三个扩增片段序列分析将3个orf222/nad5/orf139位点特异引物对417S mtDNA的扩增产物测序,删除冗余序列后,P23、P24、P25扩增产物大小分别为669bp,537bp和806bp。NCBI网站BLAST比对结果表明:P23扩增产物与线粒体全基因组中nad5和orf222序列存在2个碱基差异,与陕2A和pol CMS不育相关基因orf224存在100个碱基差异。P24扩增产物与nad5序列存在5个碱基的差异,与甘蓝型油菜的atp6基因序列存在7个碱基差异,与品种Isuza-natane的atp6基因存在12个碱基的差异。P25扩增产物与线粒体全基因组中nad5序列完全一致,与orf222序列存在7个碱基的差异。将P23和P25扩增序列进行拼接后BLAST比对结果表明,对417S的orf222位点的扩增产物与nap CMS不育基因序列存在7个碱基的差异,主要表现为碱基的插入。其中,在orf222序列的4829、5033bp处分别插入了一个碱基C,在5082处插入了2个碱基CC,在4828和5351bp处分别插入了一个碱基G,而在5444bp处缺失了1个碱基A。这一结果表明,417S与nap CMS在nap CMS不育相关基因orf222/nad5/orf139序列上存在一定差异,属于不同的细胞质不育类型。更多还原

【Abstract】 Cytoplasmic male sterility(CMS)is a prominent approach to utilizing heterosis in rapeseed. The photo-thermo-sensitive cytoplasmic male sterility (PTMS) can be used to produce hybrid seeds according to the two-line hybrid system model. The PTMS system has been the focus of attention increasingly for its widely restoring ability, easy to screen combinations and to multiple male sterile line by self-pollination. However, it plays the key role that a PTMS line possess available index of fertility conversion for hybrid seed production. Attempts were made in this paper to develop a novel available PTMS line through hybridization breeding by using the PTMS line 533S as the resource of sterility gene, and to study genetics, ecology, molecular characterization of novel PTMS line developed. The main results obtained are as following:1. Comparison 417S with its original sterility417S was similar to its original sterility in fertility expression. Both were sensitive to higher temperature. The results of reciprocal cross between 417S and its original showed that 417S shared same cytoplasm with its original. By comparison with its original, 417S made a magnificent progress in quality and agronomic traits because glucosinolates content of 417S is 24.2μmol/g cake while its original is 125.6μmol/g cake, oil content increased by 6 percent approximately, plant height decreased by 14.6 centimeter, and possessing a good lodging resistance. It is easy to find a propagating area than its original sterility because the fertility altering temperature of 417S is higher than its original sterility.2. Type of fertility altering, critical stage for fertility, and index of fertility alteringThe results of experiments under artificial temperature, photoperiod regime and multi-location environment showed that 417S was a thermo-sensitive male sterile line. The critical stage for fertility of 417S was 3-12 days prior to blossoming when the buds was about 2-3 mm in length because the daily seed-setting rate was highly positive correlative (r=0.906) with the average temperature per day during this period. The hierarchical cluster analysis indicated that the critical air temperature of 417S was 17.0-20.5℃, namely, the 417S exhibits male sterility, when 417S could not produce seed by self-pollinating bagged at average air temperature below 17.0℃but became male fertile over 20.5℃when 417S produce seeds as normal. 3. Studies on inheritance of 417S and screening of molecular markerThe results of test cross, cross and backcross combined with artificial environments and RAPD analysis showed that the thermo-sensitive sterility of 417S is controlled by interaction between cytoplasmic genes and two pair major recessive overlapping genic genes with modification by uncertain minor genes. A maintainer has not been developed based on results of test cross with a great deal of varieties or breeding lines. All testers could restore the sterility of 417S at different restore ability. Of which, 1102C may be a different cytoplasm with 417S based on F1, F2 populations of reciprocal cross. The RAPD marker was screen through BSA (Bulked segregant analysis) method. A marker (BA392-400bp) was linked with the restoring genes for the thermo-sensitive male sterile gene (6.0cM), and could be used as marker linked to thermo-sensitive male sterile restoring gene originated from 417S.4. PCR analysis of 417S mtDNA genomePCR analysis of mtDNA genome from 7 varieties or breeding lines with 25 primer pairs specific for genes of mtDNA rapeseed was performed and yielded 116 bands, i.e. 24 type of band, in all samples. Majority of primer pair could amplify one band at average of 0.7 strip of band in each sample. Of them, seven primer pairs have no polymorphism among the seven samples. 417S is distinguished from pol CMS because primer pair 18 and 19, specific to pol CMS mtDNA gene orf224/atp6, yielded only one band in pol CMS lines, but no product in other samples. There are five primer pairs could distinguish 417S from nap CMS by mitochondrial atp1, atp9, nad4L, orf263, orf222-nad5-orf139 gene regions. The results of cluster analysis with unweighted pair group method with arithmatic mean method (UPGMA)based on PCR profiles showed that seven cytoplasm tested were grouped three clusters. Cluster I, consisting of 417S and nap CMS, Cluster II, consisting of 1102C, bronowski and ZS9, and Cluster III, consisting of pol CMS and Westar, which accorded with the results of genetics testing that cytoplasm of 417S is different from 1102C.5. RFLP analysisRFLP analysis of the mitochondrial DNA of the 4 CMS lines, i.e. 417S, pol, nap and ogu CMS, combining 7 probes originated from mitochondrial genes of rapeseed with 5 endonuclease, EcoR I, EcoR V, BamH I, Hind III and Pst I (Takara), was performed. Of 25 combinations, three combinations of probe/enzyme have no polymorphism among 4 samples tested. One combination of probe/enzyme, cox1/Hind III could distinguish the 4 CMS each other. One band (4.5kb) in 417S, two band(6.6kb and 4.3kb) in pol CMS , one band(6.6kb) in nap CMS, and one band(4.3kb) in ogu CMS were observed. 8 combinations of probe/enzyme could distinguished 417S from others, 7 combinations of probe/enzyme could distinguish pol, nap, ogu CMS each other. 17 combinations of probe/enzyme could distinguish 417S from nap CMS, 19 combinations of probe/enzyme could powerfully distinguish the 417S from others. These results definitely demonstrated the 417S hold a new type of sterile cytoplasm different from pol, nap and ogu CMS. This provides molecular evidence for the cytoplasmic heterogeneity.6. Sequences analysis of 417SThree fragments with size of 669bp, 537bp, 806bp respectively amplified from three primer pairs specific to orf222/nad5c/orf139 gene were cloned, sequenced and assembled. The results of alignment via BLAST procedure on NCBI showed that there are two base pairs(bp) differences between sequence of 417S amplified with primer pair P23 and nad5 gene from mitochondrial gene sequence(position 44279-44947) and orf222 gene(position 4151-4819) in nucleotide, 100 base pair differences between 417S and pol CMS in gene orf224. There are 5 bp differences between sequence of 417S from primer pair P24 and nad5 (position 37157-37694), 7 bp differences from atp6 gene of B.napus, 12 bp differences from atp6 gene of Var. Isuza-natane. Sequence of 417S from primer pair 25 was identical to nad5 (position 43514-44319) while 7 bp differences from orf222 (position 4779-5580) gene of B.napus. After assembled fragments of primer pair 23 and 25 via DNASTAR software, seven base pair differences was observed between 417S and orf222 gene, which mostly they are base insertion. The results indicated that 417S and nap CMS were distinct.更多还原