Cloning The Dominant Genic Male Sterile Gene Ms-cd1 In Brassica Oleracea And Identifing The Tapetum Specific Expressed Genes

Cabbage (Brassica oleracea var. capitata) is an imporant leafy vegetable crop which takes the second place of growth area in China. Utilization of male sterile lines is becoming the trends for cabbage heterosis breeding. Tapetum is the main organ of anther to provide nutrients and materials for pollen development. Therefore, obtaining the tapetum specific expressed genes and clarifing their functions become the focus of anther development researchThis study ideitifed the candidate gene of Ms-cdl by fine-map cloning and we also analysed the expression levels of two parapogues of this candidate gene in the male sterile and fertile B. oleracea. At genome level, this study compared the transcriptome profiles of the male sterile and fertile materials which lay a solid foundation for studying Ms-cdl affected genes. In addition, this study idendified a few candidate genes which specifically expressed in tapetum through screening four types of male sterile B. oleracea. In summary, the main results of this study were as following:1. We resequenced fertile and male sterile Brassica oleracea var. alboglabra materials and then confirmed the fine mapping results of Ms-cdl by using the "sliding window method" of each chromosome heterozygosity area and localization hybrid sites. We developed new Indel markers which was much closer to Ms-cdl for screening recombinant individuals. At last, the location of Ms-cdl was mapped on 39.4 kb physical region of chromosome C09. We predicted four genes in this region, namely Bol035717, Bol0357N2, Bol0357N3 and Bol035718. There is no difference among the nucletide sequences of these four genes. However, we identified Bol0357N3 significantly expressed in flower buds in male sterile B. olerace. Thus, this gene was considered as the candidate gene of Ms-cdl.2. We identified 1,396 differentially expressed genes by comparing the transcriptome of flower buds in fertile and male sterile Brassica oleracea var. alboglabra through sequencing mRNA (RNA-seq). we found that Ms-cdl also influenced the gene expression at early stage in addition to late stage of anther development. It was also the first time we identified that large number of up-regulated genes was related with callose degradation and failure of microspores release during tetrad stage. Gene function annotation showed large number of up-regulated genes were categrized in metabolic pathway, secondary metabolites synthesis pathway, carbon metabolic pathways, amino acid synthesis pathway. The down-regulated genes were significantly enriched in callose esterase activity, pectin lytic enzyme activity, galacturonic acid enzyme activity which are vital important for pollen mother cell wall degradation during tetrad stage. The decreased expression level of these genes were in accordance with the failure of pollen mother cell wall degradation.3. According to the analysis of DEGs in the three sub-genomes in B. oleracea, we found that 75% corresponding paralgous genes had the same direction of expression change, which means that functin of downstream genes which were affected by Ms-cdl are redundant in B. oleracea. The number of DEGs in LF is not significantly higher than that in MFs, indicating partial retention of DEGs in LF does not play amore significant role in the anther development than the other two sub-genomes.4. By comparing the cytological abortive time sequence and characteristics of the four types of male sterile B. oleracea (NiCMS, RGMS, OguCMS, DGMS), we proposed an anther sequential development interruption model. We detected 1,005 differentially expressed genes closely related to the anther development by comparing the RNA transcriptiom of the four types of male sterile B. oleracea and their corresponding fertile materials hybridized with the Arabidopsis microarray. Considering the cytological tapetum abortive features, we identified 105 candidate genes specifically expressed in tapetum by comparing our results with previous transcriptome of vegetative organs and pollen. According to anther sequential development interruption model, we clarified the expression sequence of certain tapetum specific candidate genes.5. According to the network of anther development and summarized the key genes differentially expressed in four types of male sterile materials, we determined the main factors of tapetum abortion in four type male sterilities. The results showed that most key genes invovling in anther development were down regulated in NiCMS; In RGMS and OguCMS, the expression levels of TDF1 and MYB103 (80) increased significantly although a large number of key genes were expressed decreasly; DGMS mainly affected the late pollen development genes. These results were highly consistent with the cytological results we observed.