Molecular Mechanism Of Dominant Self-Compatibility And Its Application In Brassica Napusl

Self-incompatibility (SI) is a kind of genetic mechanism that evolved by the plants to avoid inbreeding depression, promote outcrossing, which is necessary for adaptation. The research on SI has great significance not only for the basic research but also for the applied research. The cultivated Brassica napus (AACC) is self-compatible, although the resynthesized B. napus and its ancestor species, B. oleracea (CC) and B. rapa (AA) are self-incompatible. In the present experimentations and observations, three plant materials, including SI line ’S-1300’, self-compatible (SC) lines’10-9-8400’and’Westar’ were selected for studying the SI/SC underlying molecular mechanism using the research means such as genetic analysis, gene cloning, expression analysis, promoter analysis, genetic transformation, transcriptome sequencing and so on. This research will pave the way for further research on pollen-stigma interactions in rapeseed. Additionally, the molecular marker system was established to identify the S haplotypes which could be used for SI hybrid breeding, to improve the breeding efficiency and to fasten the breeding process. The main results were listed as follows:1. Genetic analysis of the self-incompatibility in’S-1300’By identifying the compositions of S haplotypes in SI line’S-1300’and the SC line ’10-9-8400’, we found that they shared the same S haplotypes BnS-6on C genome. While on the A genome’S-1300’contains the S haplotypes BnS-1300,’10-9-8400’contains the S haplotypes BnS-1. By analyzing the phenotype and S locus genotype of the plants in the segregation population’S-1300X10-9-8400’, we proved that the S haplotypes BnS-1300located on A genome determined the self-incompatibility in’S-1300’.2. Cloning the S locus genes on A genome in’S-1300’ with homology based candidate gene methodologyBnSP11-1300gene shows a length of378bp, it contains two exons and an intron, showing100%sequence similarity with BrSP11-60gene. BnSRK-1300gene has a length of7967bp, it contains7exons and6introns,100%sequence similarity with the CDS (coding sequence) of BrSP11-60gene. However, there were several nucleotides variance in intron1,3and5. Both BnSP11-1300and BnSRK-1300gene could expressed only in mature buds, but not in root, stem, leaf and siliques, conforming the expression character of SI genes in Brassica. 3. Set up of the SI molecular marker systemWe cloned the CDS of BnSRK-1gene and the genomic sequence of BnSP11-1gene with homology based candidate gene methodology. Based on the sequences of S locus genes in SI line ’S-1300’, restorer line ’10-9-8400’ and maintainer line’Bing409’, a SI molecular marker system was set up. Molecular marker SRK1-1and SCR1-1could be amplified only in restorer line’10-9-8400’, molecular marker SRK-1300could be amplified onlyin SI line’S-1300’and molecular marker SCR7-2could be amplified only in maintainer line’Bing409’. By modifying the PCR conditions, two multiplex PCR markers SRK-1300/SRK1-1and SRK-1300/SCR7-2were developed. By applying91SC lines and their hybrids crossed with’S-1300’, we observed that SI molecular marker system could be used effectively in SI hybrid breeding.4. Identification of Helitron transposon in the promoter of BnSP11-1gene and evolution analysis of B. napus.By analyzing the inserted3.6kb fragment in the promoter region of BnSP11-1gene, a non-autonomous Helitron transposable element which contains some truncated pseudogenes but no transposase gene was identified. Furthermore, by blasting the truncated pseudogenes an another non-autonomous Helitron transposable element downstream of SP11/SCR gene with a length of10393bp was identified in BrS-47in B. rapa. S haplotype BnS-1in B. napus was derived from BrS-47in B. rapa. As BnS-1was derived from BrS-47, both the Helitron type transposable elements showed sequence similarity at the termini and even they contain a similar small fragment of truncated pseudogene, they also shared the same predicted small hairpin structure near the3’end. Therefore, it was suggested that the movement of the Helitron type transposable element was responsible for the transition of the pollination system (from cross pollination to often cross-pollination) in B. napus, which can make the species survival and have a significant impact on the evolution of B. napus.5. Cis-regulatory element identification in the promoter of BnSPll-1geneBy promoter analysis, we found that there are several cis-regulatory elements responsible for the spatial and temporal expression patterns of BnSP11-1gene and a main cis-regulatory element was identified to be located in a10bp region nearby-227bp to-217bp.6. Functional complementary analysis of BnSP11-1geneThe self-incompatible B. napus plants were obtained by genetic transformation of functional BrSP11-47gene into’Westar’. The stigma of the wild type’Westar’could accept the self-pollen but rejects the pollens of transgenic plants. It established that nonfunctional SP11gene caused the self-compatible phenotype of B.napus.7. RNA-seq analysis of the compatible/incompatible reactions in stigmasTranscriptome analysis was conducted to explore the underlying molecular mechanisms for self-incompatible (SI) and self-compatible (SC) reactions in stigmas. The differentially expressed genes (DEGs) annotations showed that SC reactions might be more complex than the SI reaction. The SI and SC reactions shared several common signal transduction pathways. The composition and the change of expression of the transcription factors, protein kinase and protein ubiquitination related genes were also analyzed in both the SC and SI reactions. Also some potential signal transduction pathways which might function in the pollination reaction were discussed.