| Brassica oleracea includes many important vegetable crops in the Brassicaceae family. It is widely cultivated in the world, and its heterosis is strong. Ogura cytoplasmic male sterility(Ogura CMS), with stable sterility and easy transfer, is an important way to utilize the heterosis in cruciferae crops. Nowadays, the Ogura CMS has been extensively used for hybrid seed production in B. oleracea vegetables. However, due to maternally inheritance, all the hybrids produced by CMS lines are male sterile and can not be self-pollinated. It hinders the germplasm maintenance and innovation. One effective way to overcome this problem is to identify and develop Ogu-CMS restorer line. In addition, the development of B. oleracea Ogu-CMS restorer materials is also of great importance to break the monopoly of unique germplasm resource owned by foreign companies. However, until now, Ogura CMS restorer material has not been found in B. oleracea crops. Thus, there is an urgent need to introduce the Rfo gene from other crops and develop B. oleracea Ogu-CMS restorer line.At present, the rapaseed Ogu-CMS restorer line has been successfully developed and used for hybrid seed production. In this study, in order to create the B. oleracea Ogu-CMS restorer material, the Rfo gene was transferred from the rapaseed restorer lines into Chinese kale by distant hybridization and embryo rescue. Screening with the foreground markers and background markers helped to select the fertile hybrids. Morever, the embryo rescue stage, morphological characteristics, ploidy identification of interspecific hybrids, and fertility performance were also investigated and analyzed in this research. The main results were as follows:1. Interspecific hybrids between Ogu-CMS Chinese kale and restorer rapaseed were obtained, and embryo rescue stage was confirmed. Nine F1 interspecfic hybrids between Ogu-CMS Chinese kale and rapaseed were obtained through hand pollination combined with embryo rescue technology. Six interspecific hybrids(2.5%) were obtained using embryo rescue, while three interspecific hybrids(0.05%) were obtained when only using hand pollination(without embryo rescue). This result suggested that the use of embryo rescue can overcome the reproductive barriers in distant hybridization and improved the seed-setting rate of interspecific hybrids. The hybrid plants were morphologically intermediate between the two parents and grew more vigorously than their parents. Ploidy identification showed that all hybrids were triploid plants and DNA relative content was close to the mean of the parents. Significant differences were observed in the rate of interspecific hybrids production at different stages of embryo rescue. In this study, the treatment(10DAP+10DAC) was the optimal stage for embryo rescue. Younger embryos were difficult to survive when pods removed from plants too early, in contrast, most of the embryos started to abort if it was removed too late.2. Seven Ogu-CMS fertility-restored interspecific hybrids between Ogu-CMS Chinese kale and restorer rapeseed were created in B. oleracea crops for the first time. In addition, the new Rfo specific molecular markers for foreground selection of interspecific hybrids were developed. Owing to the presence of non-specific amplification, the previous Rfo primers designed for rapaseed cannot be used as a specific marker to differentiate B. oleracea restorer line from non-restorer line. The Rfo homologous sequences in B.oleracea, B.alboglabra and B.rapa, obtained through BRAD database or re-sequencing data, shared over 89% identity to the Rfo sequence. Based on the cloning and sequencing results, a novel Rfo specifc primer pair(Bn RFO-AS2F/Bn RFO-NEW-R) was designed, and it could be used for the early selection of restorer individuals in interspecific hybrids of B. oleracea. Screened with the novel Rfo markers showed seven of the nine hybrids carried the Rfo gene. Further studies indicated that all the seven hybrids with Rfo gene were fertile, but no pollen was observed in the hybrids without Rfo gene. It suggested that the novel designed primers were proved useful for the selection of fertility restored progenies in B. oleracea. At anthesis, there was a significant difference in fertility performance among seven progenies containing the Rfo gene. Two progenies, YL2 and YL6, showed a better fertility performance than other hybrid plants at the whole flowering stage. Furthermore, there was significant variation in fertility performance at different DAFs(days after flowering). Generally, all restored hybrids showed better fertility performance before 12 DAF, while pollen quantity and pollen viability became lower after 12 DAF.3. The BC1 interspecific hybrids, containing Rfo and closer to the genetic background of Chinese kale parent, were obtained by foreground selection and background selection. In order to eliminate rapeseed background, the individuals YL2 and YL6, with better fertility restoration, were selected to conduct self-pollination and backcross, respectively. No F2 seed was obtained from self-pollination of triploid plants(YL2). One hexaploid individual(YL2-3), obtained after colchicine treating, showed better fertility performance and could be self-pollinated. The F2 population was originated from self-pollination of YL2-3. The morphological characteristics of F2 population were more similar to rapaseed parent and showed obvious morphological separation. All F2 plants harbored Rfo gene showed fertility-restored. 35 BC1 interspecific hybrids were obtained through the backcross of CMS Chinese kale and YL2-1, YL2-3, YL6, respectively. Screening with the novel specific Rfo marker showed 29 BC1 progenies harbored Rfo gene and showed fertility-restored, although the fertility performance were different among these BC1 Rfo progenies. A total of 33 pairs of SSR primers were selected to analyze the genetic background of three F1 plants and 35 BC1 plants. The result showed that the genetic background of F1 plants was closer to rapeseed than to Chinese kale, while the BC1 plants had a big genetic background difference among BC1 plants. The morphological characteristics of BC1 plants showed obvious morphological segregation. Screening with foreground marker and background marker showed the genetic background of fertility-restored individual 14Y1 was the closest to Chinese kale, which was further confirmed by morphological observation. Thus, this individual could be use as the key material for continuous backcross. |
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