【作者】 朱斌；

【导师】 李再云；

【作者基本信息】 华中农业大学 ， 作物遗传育种， 2016， 博士

【摘要】 由种间杂交进化产生异源多倍体是自然界物种形成的一条重要途径,其适应性要显著优于其二倍体祖先种。长期以来,揭示异源多倍体中祖先种基因组的起源及结构变化一直是众多研究者所关注的问题。从一个拥有较长进化史的天然异源多倍体中抽离出其亚基因组并使祖先种得以出现,为研究异源多倍体的基因组进化及互作提供了独特的机会。异源四倍体甘蓝型油菜(Brassica napus L.,AACC)是异源多倍体多倍化进程研究的模式植物。本研究中,通过远缘杂交诱导甘蓝型油菜中C亚基因组染色体的优先消除,抽离出了整个A亚基因组,进而重建了祖先种白菜型油菜(restituted B.rapa L.,RBR)。同时,以本实验室先前获得的一个RBR Oro做父本,与亲本甘蓝型油菜“Oro”进行杂交及连续回交,将C亚基因组的单条染色体附加到剥离的A亚基因组上,在同一背景下对C亚基因组进行解析。最终,通过C基因组染色体特异基因引物的PCR扩增及FISH方法鉴定出了全套的单体异附加系(MAALs)。RBR及MAALs的获得为甘蓝型油菜的基因组进化及基因组互作研究提供了新的思路及材料。本研究的主要结果如下:1.远缘杂交诱导RBR ZS11的建立以国内进行测序的甘蓝型油菜品种“中双11”与海甘蓝(Crambe abyssinica)进行大量的属间杂交,获得了4株染色体数为2n=29的F1杂种。C基因组的特异BAC为探针的FISH及染色体特异基因引物的PCR扩增证实了这些F1杂种(2n=29)中消除了一个完整的C亚基因组,而保留了全部的A亚基因组染色体,同时经海甘蓝基因组为探针的GISH分析,未检测到海甘蓝的染色体片段。随后在F1杂种与海甘蓝的回交后代中筛选到染色体数目为2n=22的单株,该单株经人工辅助自交后获得了染色体为2n=20的植株。这些植株的染色体配对方式与天然白菜的10个二价体及减数分裂后期I染色体以10:10分离一致,且经FISH检测并未发现C基因组染色体及片段,证实了这些材料为“中双11”的剥离白菜(RBR ZS11)。与天然白菜相比,RBR ZS11表现出一些特有的形态特征,例如生活力弱、易感病、叶片有毛,花型较小且花瓣皱缩,自交结实不正常,特别是冬天叶片贴地生长等。随机选取57对AFLP引物对4个F1植株、RBR ZS11及双亲进行遗传分析,各世代植株中均检测到一定数目的相对于甘蓝型油菜的缺失带、新增带及少量的海甘蓝特异带。以随机选取的32对AFLP引物对RBR ZS11及其它17份材料进行聚类分析,结果显示RBR ZS11与白菜型油菜“白油一号”聚为一类。2.甘蓝型油菜C亚基因组的解析为解析天然甘蓝型油菜的C亚基因组,以实验室先前获得的剥离白菜RBR Oro为父本,与供体甘蓝型油菜“Oro”进行杂交及连续回交。通过细胞学方法,共确定了646株BC1和BC2群体植株的染色体数目,其中有129(20.0%)株(2n=21)为可能的MAALs。利用已测序甘蓝型油菜“Darmor”的基因组信息设计、筛选了49对C基因组染色体特异的基因引物。经特异基因引物的PCR扩增,鉴定到全套的RBR Oro附加单条C亚基因组染色体的9个MAALs。随后以多个重复序列探针的BAC-FISH对MAALs植株进行分析,证实了染色体特异基因引物的可靠性。相比于亲本RBR Oro,MAALs表现了一些可能由附加C染色体决定的特异性状,例如AAC2的叶片蜡质。MAALs终变期的染色体配对以及雌、雄配子的传递率均有较大的差异,可能与附加的C基因组染色体与A基因组的同源性及甘蓝型油菜中调节染色体配对的机制有关。其中,AAC1、AAC2表现较高的异源配对三价体,且二者的雌、雄配子传递率显著高于其它MAALs,而AAC9的异源配对三价体频率及雌、雄配子传递率均最低,这可能与C9染色体携带有调控甘蓝型油菜染色体配对的一个主效位点(Pr Bn)相关。最后,借助于全套MAALs,通过设计特异的基因引物进行PCR扩增,将C亚基因组的22个较大的未定的Scaffolds锚定到了对应的C基因组染色体上,证实该套附加系在优化基因组信息上的特有作用。更多还原

【Abstract】 Allopolyploid derived from the interspecific hybridizations between related diploid ancestors is a vital way for speciation in nature,and they outcompete their progenitors to a wide range of climatic conditions.To unravel the origin and structure of the component genomes in these allopolyploids has been the goal of the extensive genetic researches over the long period.The extraction of the constituent subgenome(s)from a natural allopolyploid species of long history and then restitution of the progenitor(s)provides the unique opportunity to study the genome evolution and interplay.The allotetraploid oilseed rape(Brassica napus L.,AACC)has been taken as a model to decipher the allopolyploidization.In the present study,A-subgenome from the allotetraploid oilseed rape was extracted through inducing the preferential elimination of C-subgenome chromosomes in intertribal crosses and the progenitor B.rapa was restituted(RBR).Meanwhile,to dissect the C sub-genome in B.napus,one RBR(RBR Oro)obtained previously in our lab as pollen donor was crossed and backcrossed with parental B.napus(Oro)to generate a battery of aneuploids of A sub-genome added single C sub-genome chromosome.Finally,one complete set of monosomic alien additional lines(MAALs)of A sub-genome added one of nine C sub-genome chromosome was determined by PCR amplification of C chromosome specific gene makers and FISH analysis.Establishment of RBR and MAALs provide a unique avenue to explore the component sub-genome evolution and interplay in B.napus.The main results of this study are as follows:1.Restitution of RBR ZS11 induced by intertribal crossB.napus cultivar “Zhongshuang 11”,the first genotype which was selected for genome sequencing in China,was extensively pollinated by the crucifer Crambe abyssinica(2n=90)and four F1 hybrids were identified to have 2n=29.FISH with C genome specific probe and PCR amplification of C chromosome specific gene makers manifested that one copy of C sub-genome was eliminated and the complete A sub-genome was inherited in F1(2n=29)hybrid,moreover,no chromosome chromosomal fragments from of C.abyssinica was detected by GISH in these plants.Then,a plant with 2n=22 was obtained from the backcross between hybrid and C.abyssinica.Among the assisted self-pollination progenies from one BC1 plant with 2n=22,some plants with 2n=20 were harvested.These plants showed same chromosomes pairing with nature B.rapa as 10 bivalents and 10:10 segregation in meiotic cells,and FISH analysis furtherly confirmed no introgression of C chromosomal fragments,indicating extraction of A sub-genome(RBR ZS11)in B.napus “Zhuangshuang 11”.Comparing with those natural B.rapa,RBR ZS11 showed some specific features,such as decreased vigor,leaves with dense hairs,more susceptible to diseases,barrier of selfing seed-sets,small flowers with ridgy petals,and in particular,procumbent leaves in cold winter etc.By AFLP analysis with randomly selected 57 pairs of primers,the four hybrids and RBR ZS11 showed some deleted and novel bands for B.napus-specific fragments,as well as a bit of specific fragments for C.abyssinica.Phylogenetic analysis of RBR ZS11 and other 17 accessions by AFLP with randomly selected 32 pairs of primers demonstrated RBR ZS11 clustered with B.rapa “Baiyou 1 hao”.2.Dissection of C sub-genome in B.napusTo dissect the C-subgenome of natural allotetraploid B.napus,the parental B.napus cv.Oro was crossed and backcrossed with RBR Oro as pollen donor.Out of 646 BC1 and BC2 plants(2n=20-29)with chromosome numbers determined in the young ovary cells by cytological method,129(20.0%)had 2n=21 for putative monosomic alien addition lines(MAALs).Based on the genome sequences of B.napus genotype “Darmor”,49 pairs of chromosome-specific genes makers of C-subgenome were designed and screened.A complete set of MAALs of RBR Oro added one of nine C sub-genome chromosome was determined by PCR amplification of these specific gene makers.And then,BAC-FISH with several repeated sequences as probes were performed to confirm the reliablity of the gene markers in MAALs.MAALs expressed some peculiar features likely associated with each additional C-subgenome chromosome in comparison with parental RBR Oro,such as leaves and stems covered with much waxen powder in AAC2.These MAALs exhibited obviously different frequencies in homoeologous pairing and transmission of additional C-subgenome chromosomes,which were associated with the distinct degrees of their relatedness,and even with the possible genetic regulation for meiotic pairing evolved in B.napus.AAC1 and AAC2 showed the two highest percentages of PMCs with the allosyndetic trivalent and transmission rate of additional chromosome through male and female gametes,but AAC9 showed lowest allosyndetic trivalent and transmission rate,likely owing to the regulation of Pr Bn located on C9.Finally,22 large scaffolds undetermined for sequence assembly of B.napus were anchored to specific C-subgenome chromosomes using MAALs by PCR amplification of specific gene markers,indicating a large role of MAALs for improvement of B.napus genome information.更多还原