【作者】 肖勇；

【导师】 孟金陵；

【作者基本信息】 华中农业大学 ， 发育生物学， 2010， 博士

【摘要】 由于不同物种之间巨大的遗传差距,种间杂交往往能产生很强的杂种优势,但是由于种间杂交后代一般为非整倍体,因而不育或者育性极差。为了将种间杂种优势应用油菜种子产量的提高上,一些新型甘蓝型油菜被创建,如：利用白菜型油菜的Ar基因组部分替换甘蓝油菜的An基因组,或者分别用白菜型油菜的Ar亚基因组和埃塞俄比亚芥的Cc亚基因组部分替换甘蓝型油菜的An/Cn亚基因组。新型甘蓝型油菜与常规甘蓝型油菜杂交所产生的亚基因组杂种,在种子产量方面表现出很强的杂种优势。但是由于先前培育新型甘蓝型油菜外源含量偏低,变异偏窄,致使培育的亚基因组杂种在适应性或者某些农艺性状存在一定的缺陷,也阻碍了亚基因组杂种的推广应用。为了克服先前培育的亚基因组杂种存在的不足,本研究致力于构建一个新型甘蓝型油菜种质资源库,该资源库包含几百份埃塞俄比亚芥和白菜型油菜品种的遗传变异,并且其基因组基本上由白菜型油菜的Ar亚基因组和埃塞俄比亚芥的Cc亚基因组组成(不含或者少量含有甘蓝型油菜的An/Cn基因组成分)。我们通过两步来完成这个新型甘蓝型油菜ArArCcCc基因库的构建,首先是以少量的白菜型油菜和大量的埃塞俄比亚芥品种为亲本(29份白菜型油菜和110份埃塞俄比亚芥品种亲本)构建一个Cc亚基因组多样性群体(Cc Polymorphic population, CcP群体),在创建CcP群体的过程中,一共获得了562份5倍体中间型单株(这部分工作由江莹芬,田恩堂和陈伦林完成),组成CcP群体F1世代。对49个5倍体单株细胞学分析显示：84%的花粉母细胞在减数分裂过程中,都会发生染色体丢失现象。原位杂交结果表明,在减数分裂中期先行以及后期落后的染色体大部分为Bc基因组染色体。考察五倍体的分枝数,每角果粒数,育性以及一些品质性状。结果显示,CcP-F1群体育性差,分枝多,营养优势强；此外其品质性状类似于埃塞俄比亚芥亲本(硫甙和芥酸含量高而含油量低等特点)。五倍体后代染色体数目复杂,为了从F2群体中筛选染色体数目为38条的单株,调查大约6,000个F2单株的形态学,大约有1,137个F2单株形态学类似于常规甘蓝型油菜,对这些单株进行了染色体数目的检测,结果显示：染色体数目从27条到46条呈连续分布,其中有641个F2单株染色体数目为38条。我们也考察了F2群体的农艺和品质性状,结果显示：对比五倍体单株,F2群体的分枝数减少,但育性得到了很大的改善,硫甙和芥酸含量显著降低而含油量有所提高。从F2群体中,选择了981个单株,收获这些单株自交结实的种子,构成了大约包含10,000个单株的F。群体。从F。世代每个株系选择一个育性最好的单株套袋自交,组建F4群体。我们从细胞学、遗传变异、农艺性状以及杂种优势的表现对F4群体进行了评估：细胞学结果显示：F4单株花粉育性正常,大部分单株可育花粉粒所占的比例在90%以上；此外其花粉母细胞减数分裂正常,几乎没有染色体丢失现象发生；我们对100个F4单株进行染色体数目的检测,发现其染色体数目都为38条,对其中的10个单株进行GISH (Genome in situ hybridization)分析,并未发现B基因染色体的存在,但是在两个新型甘蓝型油菜单株中,其花粉母细胞的某个染色体上存在较小的杂交信号,推测可能由于B基因组染色体片段整合到A/C基因组上所致。应用48对SSR引物分析了F4群体、自然甘蓝型油菜,以及埃塞俄比亚芥和白菜型油菜亲本的遗传多样性。结果显示,创建的F4群体的遗传多样性丰富于自然甘蓝型油菜以及埃塞俄比亚芥和白菜型油菜亲本。主成分分析显示：F4单株的遗传基础与常规甘蓝型油菜有所不同,被聚为不同的两类。考察了F4群体的农艺性状,如：每角果粒数,千粒重,分枝数,硫甙,芥酸,含油量等。结果显示：农艺性状变异广泛,含油量最低为19.91%,而最高可以达到51.2%；千粒重最轻只有1.94 g,而最重可达到7.12 g；油酸含量最低为2.6%,最高可达到76.8%,等。利用100份F4单株配置亚基因组杂种。结果显示：无论在生物学产量和种子产量上都表现了很强的杂种优势。此外,我们尝试性地分析了遗传距离与杂种优势之间的关系。另外,以大量的白菜型油菜品种和少量的埃塞俄比亚芥品种为亲本(235份白菜型油菜和7份埃塞俄比亚芥品种亲本)构建一个Ar亚基因组多样性群体(ArPolymorphic population, ArP群体),其过程与Ccp群体的构建类似。2008年,我们将CcP-F5群体与ArP-F3群体混合种植在一块组建成新型甘蓝型油菜ArArCcCc种质资源库,并与带有显性核不育的新型甘蓝型油菜按照9：1的比例相隔种植,进行首轮的轮回选择；在2009年,我们从这个种质资源库中选择650个具有优异农艺性状的新型甘蓝型油菜单株,与带有显性核不育的新型甘蓝型油菜按照1：1的比率种植；经过两轮的轮回选择某些新型甘蓝型油菜的综合性状得到了一定的改良。更多还原

【Abstract】 Interspecific hybridization always exhibits good performance in heterosis because of enormous genetic difference between different species. But hybrids from the cross between species are generally aneuploids so that their fertility is very bad. In order to exploit the interspecies (intersubgenome) heterosis in rapeseed, new type B. napus were created by substituting A genome of B. rapa to A genome of B. napus in part, or A genome of B. napus and C genome of B. carinata to A/C genome of B. napus separately in part. Strong heterosis in seed yield was observed for intersubgenome hybrid between new type B. napus and traditional B. napus. But it was difficult to develop these intersubgenome hybrids into commercial varieties because of low introgressed rate from Ar/Cc and limited genetic variation for the new type B. napus.For solving the two problems, it is necessary to construct a new type B. napus gene pool, containing genetic variation of several hundreds of B. carinata and B. rapa cultivars. Moreover, the genome of individual from the gene pool was substantially constituted by ArArCcCc. We report the latest work progress here. At first,110 B. carinata cultivars as female parents were used to cross 29 B. rapa cultivars as male parents for constructing Cc Polymorphic population (CcP population).562 pentaploid plants were synthesized to form Cc polymorphic F1 population (CcP-F1 population).Cytological analysis demonstrated about 84% of the pollen mother cells from 49 observed pentaploid plants showed various abnormalities associated with the chromosomes lost at different stages of meiosis. GISH analysis showed that most of the lost chromosomes were likely from the mono B genome. We investigated CcP-F1 population for their agronomic traits, including fertility, branch number, seed number per pod and quality traits. The pentaploid plants characterized bad fertility, strong biomass.CcP-Fj population were open-pollinated in an isolated net to produce CcP-F2 population consisted of about 6000 plants. Based on cytological analysis for pentaploid plants, it is difficult to predict chromosome number of F2 plants. In order to screen out the F2 plants with 38 chromosomes,1,137 F2 plants were selected for counting chromosome number because of their morphology similar to tradition B. napus. The number of chromosomes in the selected F2 population still varied from 27 to 46; the most abundant class contained the plants with 38 chromosomes that were expected from meiosis observation of the pentaploid. Moreover, F2 population was also evaluated for their agronomic traits. Comparing with pentaploid plants, plants from F2 population showed improved fertility, higher oil content, lower glucosinolate and erucic acid content, etc.Nine hundred eighty one plants from F2 population were harvested to produce F3 population consisted of about 10,000 plants. The 958 F3 plants with the best fertility were selected (without chromosome checking) to produce an F4 population with the same size as the F3 generation.F4 population was evaluated for their cytogenetic, genetic variation, agronomic traits and heterosis.The majority of detected plants from F4 population show improved pollen fertility which was found to be more than 90%. It was observed that 99.5% of the pollen mother cells (2,741 in total) exhibited normal meiosis behaviour almost without chromosome losing at different stages of meiosis. All of the checked somatic cells from F4 plants exhibited expected 38 chromosomes. GISH analysis was employed to detect whether B genome chromosomes have been completely lost in pollen mother cells of 10 pentaploid plants:no any hybridization signal was detected in 8 of 10 pentaploid plants; intact B genome chromosome was not detected but a few B genome DNA fragment were integrated into A/C genome in 2.Genetic diversity and population structure for 100 new type B. napus individuals along with their original parental species,10 cultivars of B. rapa (AA),55 accessions of B. carinata (BBCC), and 26 accessions of traditional B. napus with worldwide origins, were evaluated by 253 polymorphic bands amplified with 48 SSR primers. From the results, wider genetic diversity was found in F4 individuals than in traditional B. napus, as well as their original parents. Moreover, principal component analysis showed F4 population had been genetically difference with tradition B. napus as a result of introgression from Ar/Cc.We investigated F4 population for their agronomic traits, i.e. seed number/pod, weight/1,000 seed, branch number, glucosinolate, erucic acid, oil content, and so on. The results demonstrated that extensive diversity for agronomic was observed in F4 population: oil content varied from 19.91% to 51.2%, weight/1,000 seed between 1.94 g and 7.12 g, oleic acid ranged from 2.6% to 76.8%.One hundred F4 individuals were used to cross traditional B. napus to yield intersubgenomic hybrids. Strong intersubgenomic heterosis was observed either in biomass or in seed yield. The analysis was done to illustrate the relationship between heterosis and genetic distance. Moreover, seven accessions of B. carinata as female parents were used to cross 235 cultivars of B. rapa as male parents to construct an Ar polymorphic population (ArP population). The synthesis process that constructed ArP population was the same as CcP population.In 2008, we joined CcP-F5 and ArP-F3 population into ArArCcCc gene pool. Recurrent selection was carried out by growing new type B. napus from the gene pool and improved dominant genic sterile line at a ratio of 9:1. In 2009,650 new type B. napus plants with excellent agronomic traits were selected to grow together with improved dominant genic sterile line at a ratio of 1:1. Some new type B. napus plants were improved in agronomic traits by the two rounds of recurrent selection.更多还原