| 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.
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