【作者】 田恩堂；

【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】 异源多倍体在植物进化过程中扮演着重要的角色,在生长活力及环境适应性方面比低倍性物种具有更强的优势。但是,由于生值隔离和染色体稳定性的原因,像普通小麦和燕麦等高倍性作物在自然界中并不常见。芸苔属在自然界中包含有三个二倍体物种(基因组为：AA,BB和CC)及三个四倍体体物种(基因组为：AACC, AABB和BBCC),其中每一个四倍体物种均来源于两个对应二倍体物种的结合。而包含有芸苔属三个基本基因组的六倍体物种(AABBCC)在自然界中并不存在,因此人工合成芸苔属异源六倍体材料对于芸苔属生物学及作物育种均具有重要的意义。人工合成芸苔属异源六倍体的途径有很多,其中以芸苔属异源四倍体物种与对应的芸苔属二倍体物种进行种间杂交(AACC×BB, BBCC×AA, AABB×CC)及随后染色体加倍的方法最为可行与高效。本实验室先后对三种人工合成六倍体的途径进行了尝试,结果显示途径"BBCC×AA"最容易获得六倍体后代,其次是"AABB×CC",最难的途径是"AACC×BB"。因此,本实验室选取‘"BBCC×AA"途径作为人工合成芸苔属异源六倍体材料的主要途径。2004年,利用110份埃芥材料与29份白菜型油菜进行广泛种间杂交,总共完成405个杂交组合共计40154个杂交花朵,平均杂交亲和性为0.33,最终得到13235粒三倍体(ABC)杂交种子。利用表型鉴定、染色体计数、分子标记筛选等技术对三倍体杂种后代的假杂种进行了排除,总共获得了5111株三倍体真杂种幼苗。利用秋水仙素滴定茎尖加倍技术,有23.3%的三倍体真杂种幼苗的整株或分支得到了加倍,总共收获包含192个亲本杂交组合的1190个六倍体第一代(H1)单株。六倍体H1代单株的角果育性在株系间及株系内均表现出较大变异,因此选取育性较好的来源于411个杂交组合的822个H1代株系作为进一步研究用材料。在H1代株系的后代中,进一步进行了表型和育性筛选并选取出1753个H2代单株进行染色体数目鉴定工作,结果显示六倍体H2代染色体表现出极大的不稳定性。95%左右的H2代单株变成了非整倍体,非整倍体染色体数目在2n=26与2n=53之间变化。仅有80个(5%)H2代单株仍然是六倍体单株(2n=54),这些整倍体单株来源于35个亲本杂交组合。35个杂交组合中,71.4%的组合只产生1个整倍体单株,约有22.9%的组合产生了2到6个整倍体单株。而组合C21及C28中分别得到了17及11个六倍体单株,明显多于其他组合,这可能是两个亲本特异结合的结果。六倍体H2代单株的染色体稳定性很低,但随着世代的增加有了很大程度的提高：在H3代和H4代中分别为23.0%和26.3%。在同一个世代的不同组合中,六倍体单株的比率表现出较大的变异,这有利于通过不同六倍体组合之间的杂交来筛选染色体稳定性更高的六倍体材料。伴随着六倍体染色体稳定性的不断提高,其H4代的花粉育性及角果育性普遍高于H3代并表现出更大程度的变异。此外,H3代中六倍体单株的比率与H3代及H4代的花粉育性、H4代的六倍体单株的比率均表现出了显著的相关性,表明了六倍体单株比率向后代的传递性及进一步提升六倍体育性的可能性。六倍体早期世代中,不仅染色体稳定性和育性有了很大程度的提高,性状表现也明显不同于埃芥和白菜亲本。在06-07年及07-08年,分别对15个六倍体组合的埃芥与白菜型油菜亲本及H2代、H3代及H4代的17个田间性状进行了考察。结果显示两年的数据重复性很好：总共17个性状中,只有06-07年株高、干物重、全株角果数性状显著高于07-08年,表明六倍体性状的总体稳定性。而六倍体性状在同一年不同世代之间则未发现显著差异。与双亲性状相比较,六倍体后代的高亲性状比例高达35.3%,类似埃芥亲本性状比率为32.4%,低亲性状比率为20.6%,类似白菜亲本和中亲性状比率均为5.9%。此外,我们在08-09年进一步考察了六倍体H5代的小区产量及干物重性状,六倍体材料的小区平均产量显著高于其五倍体后代,低于双亲及甘蓝型油菜对照；六倍体材料的干物重显著高于双亲及甘蓝型油菜,仅略低于五倍体材料。为了解六倍体染色体稳定性的调控因素并为六倍体的稳定性的快速提高寻找可行的途径,本实验利用细胞学与分子标记技术对引起六倍体后代染色体不稳定性机制进行了解析。细胞学结果显示,六倍体的染色体在H2代和H3代配对不正常,能够产生大量的落后染色体及染色体提前分离现象,并且能够出现大量的多价体及单价体现象。在H2代的减数分裂中期,二价体的平均数目仅为22.30,并伴随有大量的多价体和单价体,而六倍体的染色体配对构型在H3代有了一定的改善,例如二价体的数目平均提高了1.12,单价体与多价体的数目也有了不同程度的降低。因此六倍体大量多价体和单价体的出现可能是导致其染色体不稳定的主要因素。此外利用129对SSR引物分析芸苔属异源六倍体A、B和C三个基因组的稳定性,发现A和C基因组稳定性远差于B基因组,据此可推断A和C基因组的高同源性可能是导致六倍体大量多价体及单价体出现的主要原因。此外,而分子标记结果则表明H4代的基因组比H2代和H3代更稳定。因此细胞学和分子标记结果不仅揭示了六倍体不稳定的机理,同时进一步证实了六倍体染色体稳定性不断提高的可行性。更多还原

【Abstract】 Allopolyploidy plays an important role in plant evolution, and confers obvious advantages on crop growth and breeding, compared to low ploidy levels. However, only a few plants, such as wheat and oat, are cultivated as crops at 6x level due to problems of reproductive isolation and chromosomal stability. In Brassica, three diploid species (the genomes of which are assigned as AA, BB, and CC, respectively) and three tetraploid species (AACC, AABB, and BBCC, respectively) exist in nature. Each of the latter three arose from the combination of two of the three diploid species. The synthesis of a new species of Brassica hexaploid with the genome AABBCC would be a significant step in Brassica biology and valuable for crop breeding.Many ways could be used to synthesize the Brassica hexaploid, the cross between tetraploid Brassica species and diploid Brassica species (AACCxBB, BBCCxAA, AABBxCC) and subsequent chromosome doubling would be the best way. We once tried all the three ways to synthesize the Brassica hexaploid, the "BBCC×AA" was more easier than "AABB×CC", and "AACC×BB" was the most difficult. So, we chosen "BBCC×AA" as the main way to synthesize Brassica hexaploid. In 2004 season,110 accessions of B. carinata and 29 accessions of B. rapa were used to widely hybridization, and 40154 hybrid flowers from 405 hybrid combinations were finished, the average was 0.33, and 13235 triploid seeds were finally obtained by us. A total of 5111 true hybrid seedlings were identified after screening them morphologically, and by chromosome counting and marker identification. After treatment with colchicine to induce allopolyploidy, chromosome doubling occurred in approximately 23% of the hybrid plants, and 1190 H1 (the first generations of hexaploid) plants from 192 hybrid combinations were gotten at last.The fertility of H1 generation changed greatly within the same lines or among different lines, and 822 H1 lines from 411 hybrid combinations were chosen to do further study. In H2 generation,1753 plants were further selected through morphological and fertility methods, which showed a great chromosomal instability. About 95% of H2 plants became aneuploids, whose chromosome number changing from 2n=26 to 2n=53, and only 80 plants (5%) from 35 hybrid combinations still had 54 chromosomes. In the 35 combinations, about 71.4% only had one hexaploid plants,22.9% had two to six hexaploid plants. The combinations C21 and C28 respectively had 17 and 11 plants, significantly higher than other combinations, which might be due to the special combination of their B. carinata and B. rapa parents.Since the chromosomal stability of H2 generation was lower, the ratio of hexaploid respectively increased to 23.0% in H3 and 26.3% in H4 with the increase of hexaploid generation. The hexaploid ratio changed greatly between different combinations within the same generation, which would be very useful to increase the chromosomal stability through the hybridization between different combinations. The pollen fertility and seed fertility of H4 exhibited a higher improvement and wide variation than H3 generation, although the change didn’t reached significant level. Besides, the ratio of hexaploid plant in H3 generation were significantly correlated with the pollen fertility in H3 and H4, the ratio of hexaploid plants in H4, showing the possibility that the ratio of hexaploid plants could inherit to their progenies.Not only the increase of chromosomal stability and fertility with the generation increase of Brassica hexaploid, but also the trait variation in hexaploid was great comparing with B. carinata and B. rapa parents. In 06-07 season and 07-08 season, the 17 traits in B. carinata and B. rapa parents, H2, H3, H4 from each 15 combinations were investigated. The results showed that most of the investigated traits marched well except for plant height, dry matter weight and number of siliques in whole plant. No significant difference was found between different hexaploid generations for each trait under the same season. Comparing with the B. carinata and B. rapa parents, the ratio of transgressive traits was about 35.3%, the B. carinata-like traits was 32.4%, the lower traits was 20.6%, B. rapa-like and intermediate traits were all about 5.9%. Besides, the seed product and dry matter weight in H5 were also investigated in 08-09 season. Detaily, the seed product of Brassica hexaploid was lower than two parents and B. napus control, significantly higher than the petaploid materials, while the dry matter weight was only a little lower than pentaploid materials and significantly higher than two parents and B. napus.To further improve the chromosomal stability of Brassica hexaploid, the cytological and molecular markers were used to study the mechanism of hexaploid stability. The cytological results showed that the chromosome pairing in H2 and H3 were abnormal and could produce many laggards, precocious segregation, multivalent and univalents. In H2, the average number of bivalents was 22.30 accompanying with higher ratio of multivalent and univalents, which was improved a lot in H3 generation with the increase of the number of bivalents and the decrease of multivalent and univalents. So, the appearance of large amount of multivalent and univalents might be the mainly reason of the chromosomal instability. Besides,129 pairs of SSR markers were further used to analysize the genomic stability of A, B and C genomes, and found that the genome stability in A and C genomes were lower than that of B genome, then the higher homologue between A and C genomes might be the main reason resulting in lots of multivalent and univalents in Brassica hexaploid. Besides, the molecular results showed that the higher hexaploid generation were more stable than lower generations of Brassica hexaploid. The cytological and molecular results not only interpreted the mechanism of chromosomal instability of Brassica hexaploid, but also approved the possibility that the hexaploid stability could increase.更多还原