【作者】 邹珺；

【导师】 孟金陵；

【作者基本信息】 华中农业大学 ， 发育生物学， 2009， 博士

【摘要】 由于长期的生殖隔离和人工选择,芸薹属植物中三个基本染色体组(A,B,C)在不同种内发生了深刻的变化。为了区分这种变化,本实验室提出用“亚基因组”来定义不同物种中的相同基因组,并用不同种拉丁文名的首写字母作为上标来区分不同的亚基因组。甘蓝型油菜(Brassica napus, AnAnCnCn)是世界上重要的油料作物之一,然而有限的栽培驯化历史和近期频繁的品种间杂交使得其遗传基础日益狭窄,杂种优势利用徘徊不前。为充分利用亚基因组间的分化差异进行油菜的遗传改良,本实验室通过种间杂交将白菜型油菜(B. rapa, ArAr)的Ar基因组和埃塞俄比亚芥(B.carinata, BcBcCcCc)的Cc基因组部分地替换了甘蓝型油菜的An和Cn基因组,创建出第一代“新型甘蓝型油菜”。将新型甘蓝型油菜与常规甘蓝型油菜配制的杂种称为“亚基因组间杂种”。已有的研究表明,用第一代新型甘蓝型油菜所配置的亚基因组间杂种具有较强的杂种优势,杂种优势的强弱与新型甘蓝型油菜中外源基因组成分的多少成正相关。在前期研究的基础上,本课题经分子标记辅助选择和多次的遗传重组,进一步提高了第一代新型甘蓝型油菜中Ar/Cc基因组成分,并在多种环境条件下选育优良单株,培育出Ar/Cc成分得到提高、农艺性状得到改良的第二代新型甘蓝型油菜。用第二代新型甘蓝型油菜与常规甘蓝型油菜测验品系配制的杂种,其生长势和种子产量在两年田间试验中均优于第一代新型甘蓝型油菜所配制的杂种,表现出更强的杂种优势。第二代新型甘蓝型油菜株系已经被国内育种家用于培育新型油菜不育系,被国际育种公司用于培育新的育种材料,还作为轮回亲本用于创建第三代新型甘蓝型油菜基因资源库,以进一步开拓油菜亚基因组间杂种优势。通过广泛分布在甘蓝型油菜遗传图谱上的117对SSR标记,对24份第一代新型新型甘蓝型油菜、79份第二代新型甘蓝型油菜、9份合成新型甘蓝型油菜的外源亲本、以及69份常规甘蓝型油菜品种进行遗传多样性分析。结果表明所有新型甘蓝型油菜与常规甘蓝型油菜品种差异明显,并且第二代新型甘蓝型油菜不同株系间遗传差异较第一代新型甘蓝型油菜株系间遗传差异更大。随着新型甘蓝型油菜中Ar/Cc成分的增加,它们与甘蓝型油菜亲本以及常规品种之间的遗传差异增大。用白菜型油菜和埃塞俄比亚芥同时改良甘蓝型油菜比单一用白菜型油菜引入的遗传变异更大,表明引入外源亚基因组成分同时改良甘蓝型油菜A、C基因组,是拓宽甘蓝型油菜遗传基础的有效途径。与常规品种相比,新型甘蓝型油菜(尤其是第二代新型甘蓝型油菜)每个SSR位点上含有更多的等位基因以及更为丰富的特异等位基因；新型甘蓝型油菜中原有连锁不平衡位点被打破,建立了更多新的连锁不平衡位点且与新型甘蓝型油菜的表型变异密切相关。这暗示着为创建新型甘蓝型油菜而开展的种间杂交使油菜基因组产生了深刻的变化。为了进一步剖析新型甘蓝型油菜基因组的结构变化及其对表型的影响,本研究以甘/白种间杂交(甘蓝型油菜品种华双3号X白菜型油菜品种天门油菜白)衍生的重组自交系群体(简称TH RIL)为材料,采用基于SSR、内含子以及反转录转座子序列设计的分子标记对该群体进行遗传作图并评价新型甘蓝型油菜内发生的序列变异。试验结果表明,TH RIL群体中不仅可以直接导入所有来自亚基因组间存在的序列变异,而且还产生了大量新的序列变异。这些新的序列变异绝大多数都是在创建新型甘蓝型油菜的种间杂交过程中遗传下来的,其中一半以上新变异的分离比例正常,但有相当一部分新变异可能因为竞争劣势或者优势而呈现偏分离,且在反转录转座子区域要比在SSR和内含子区域更易发生具有竞争优势的新变异。测序结果表明,SSR新序列变异主要表现在重复单位模体(motif)数目的变化,但这些模体的变化很有可能与基因的功能有关；新出现的反转录转座子标记代表的是新产生的反转录转座子位点,暗示着基因组内反转录转座子的激活而导致反转录转座子新拷贝的插入。在TH RIL遗传连锁图谱上定位了672个分子标记,检测到发生了各类序列变异的位点628个。将3个白菜型油菜遗传连锁图和2个常规甘蓝型油菜遗传连锁图分别进行了图谱整合。将整合遗传图谱与TH RIL遗传连锁图谱进行图谱比较分析,在新型甘蓝型油菜基因组中发现了大量的染色体重排事件。大约三分之一的重排事件是由于白菜型油菜Ar基因组与甘蓝型油菜的An基因组自身存在的结构差异而导致的,而大部分重排事件是由于种间杂交诱发的新的基因组结构变异。对TH RIL群体中所检测到的各类序列变异以及染色体重排事件之间的总体关系进行分析发现,新的SSR序列变异的发生与重排事件的发生以及新的反转录转座子序列变异的发生均呈显著相关(P<0.01)。虽然没有检测到反转录转座子新的序列变异与重排事件之间存在相关性,但很多重排事件都涉及到了新的反转录转座子位点的出现。这暗示着基因组内的染色体重排以及反转录转座子的活动会加速SSR的变异,而反转录转座子的活动与染色体重排密切相关。进一步分析了TH RIL群体的3个产量性状以及6个产量相关性状的两年田间数据,结果表明新型甘蓝型油菜基因组中新的结构变异对植株的农艺性状影响很大,其中新的反转录转座子位点的出现对农艺性状的影响最大。根据本研究的分析结果,探讨了种间杂交导致各类基因组结构变异的可能机制及亚基因组间杂种优势产生的可能机制,提出了进一步进行新型甘蓝型油菜的遗传改良和开拓亚基因组间杂种优势的新途径。更多还原

【Abstract】 As a result of long-term reproductive isolation and artificial selection, the three basic genomes (A, B, C) in different species have undergone profound changes in Brassica crops. In order to distinguish this differentiation, our laboratory previously proposed the concept of "subgenome" to define the same genome in different species, and used the initials of the Latin names of different species as a superscript to distinguish different subgenomes. B. napus (AnAnCnCn) is one of the world’s most important oil crops, however, its limited history of cultivation and domestication, and the recent frequent intraspecific hybridization lead to an increasingly narrow genetic base and the stagnancy of heterosis utilization. To take full advantage of subgenomic differences for the genetic improvement of oilseed rape, we partially replaced the An and Cn genome of B. napus by the Ar genome of B. rapa (ArAr) and Cc genome of B. carinata (BcBcCcCc) resulting in the first generation of new type B. napus. Heterosis between "traditional" B. napus and the new type B. napus can therefore be defined as "intersubgenomic heterosis". Significant intersubgenomic heterosis was observed in the hybrids made with the first generation lines of new type B. napus over the past ten years of field trials, and positively related with the introgressed subgenomic components in the parental new type B. napus.Based on the previous research basis, a second generation of new type B. napus was developed with improved agronomic traits and higher introgressed subgenomic components in this project, by selecting the plants with higher proportion of exotic-subgenomic components from segregated populations of first generation of new type B. napus and screenging the agronomic traits under different environments. The intersubgenomic hybrids made with the second generation lines of new type B. napus exhibited stronger heterosis than the intersubgenomic hybrids made with the first generatin lines in two years on the traits of seed yield and biomass. The excellent lines of the second generation of new type B. napus were selected for breeding new type of male sterile lines and bridge materials for the Chinese breeders and international company abroad, and used as the recurrent parents for the construction of the gene resource pool of the third generation lines of new type B. napus for broadening the avenue of intersubgenomic heterosis in rapeseed. Genetic diversity analysis was conducted with 117 pairs of SSR markers distributed on published genetic map of B. napus, among 24 lines of the first generation of new type B. napus,79 lines of the second generation of new type B. napus,9 Brassica species as the original parents for the synthesis of new type B. napus, and 69 varieties of traditional B. napus. The results showed that there was significant genetic difference between new type B. napus and traditional B. napus, and bigger genetic distance was observed among the lines of the second generation of new type B. napus than the first generation of new type B. napus. Along with the increment of the genomic components of Ar/Cc in new type B. napus, the genetic difference between new type B. napus and traditional B. napus was enlarged. Introgressing of the genomic components from B. rapa and B. carinata synchronously into B. napus introduced more genetic variation than simply introgressing Ar component, which suggested that replacing the A and C genome of B. napus at the same time was an efficient way in broadening the genetic basis of B. napus. Compared with the traditional B. napus, the new type B. napus (especially the second generation lines) contained more alleles and group-special alleles per SSR locus, and constructed more new linkage disequilibrium loci pairs associated with the phenotypic variation. This implied the process of interspecific hybridization for the development of new type B. napus brought out profound genomic changes for new type B. napus.To further dissect the genomic changes of new type B. napus and their impacts on the phenotypes, three kinds of molecular markers based on the SSR, intron, and retrotransposon were used to evaluate the sequence variation in a recombination inbred line population (TH RIL population) derived from an interspecific hybridization between B. rapa (Tianmenyoucaibai) and B. napus (Huashuang3). The results showed that abundant novel sequence variation was observed in TH RIL population along with the introgression of all specific sequence variation between subgenomes. The majority of the novel sequence variation was inherited from the process of interspecific hybridization, most of which showed normal segregation, however, a considerable portion of the novel sequence variation exhibited with distorted segregation possibly because of competitive disadvantages or advantages for the survival of the population. Novel sequence variation in retrotransposon showed advantages was much more than that in SSR and intron. The results on sequencing the novel variation showed that the novel sequence variation for SSR were mainly resulted from the changes of motifs, which was possibly associated with gene function; the newly appeared retrotransposon variation were detected in new retrotransposon loci, which was possibly resulted from the insertion of new copies of retrotransposon because of reactivation.672 molecular markers were mapped on the genetic map of TH RIL population, representing 628 loci exhibited with various sequence variation. Three genetic maps of B. rapa and two genetic maps of B. napus was integrated, respectively. A lot of rearrangement events were detected in TH RIL population by the comparison among the two integrated maps and TH genetic map.Apporximately one third of the rearrangement events were resulted from the introgression of the pre-existed rearrangements between the genome of Ar and An, but most of the rearrangements were resulted from novel genomic structural variation induced by interspecific hybridization. The coefficient of correlation among various sequence variation and rearrangement events in TH RIL population was analyzed. The data showed that the novel sequence variation of SSR was positively and significantly (P<0.01) associated with the rearrangement events and new retrotransposon loci. Though no significant relativity was detected between the appearance of new retrotransposon loci and rearrangement events, considerable rearrangement events were involved with new retrotransposon loci. These resultes implied that the rearrangement evnents and reactivation of retrotransposon in new type B. napus prompted the mutation of SSR, and the reactivation of retrotransposon were intimately associated with rearrangement events. We further analyzed the two-year of field data about three yield traits and six yield related traits, which showed that the novel genomic structural variation had great impacts on the important agronomic traits of new type B. napus, and the novel retrotransposon loci showed the most significant impacts on the agronomic traits.Based on the results of this paper, the mechanism of various genomic structural variation and intersubgenomic heterosis was discussed, and new approaches in improving the new type B. napus and exploring intersubgenomic heterosis was put forward.更多还原