Study On The Molecular Mechanism Of Multiple-Allele Inheritance Genetic Male Sterility In Chinese Cabbage

Chinese cabbasge is a typically allogamous plant with complete flowers and obvious heterosis. In the process of preparing hybrid, the utilization of male sterile material is a good way. The research on genetic mechanism and molecular mechanism of male sterility in Chinese cabbage has been the focus of attention. In the previous studies, our research group found Chinese cabbage multiple allele genetic male sterile materials, and the first to prepare a 100% sterile lines. Use this male-sterile line to construct separation group, and the sterile gene was located between the A07 chromosome 6700000 and 6800000. In order to further narrowed the positioning rang of male-sterile gene, this study used the BAC library constructed by the sterile homozygous sterile plant (MsMs) to screen the BAC clones in the location region, and to find out the differentially genes by sequencing, by high throughput sequencing analysis of fertile (Msf Ms) and sterile (MsMs) buds of male-sterile AB line, the male sterile related genes and miRNAs were studied at the transcriptional level. Found out the differentially expressed genes and analyze their functions and participate in the metabolic pathway, identified the differentially expressed miRNAs in the bud of Chinese cabbage, and predicted target genes for these miRNAs. These studies lay a foundation for screening and identification of Chinese cabbage male sterility alleles genes (Ms、Msf and ms), providing a database for the study of male sterility related genes and miRNAs, which will help to reveal the molecular mechanism of male sterility in Chinese cabbage. The main results are as follows:1. Though the observation of the morphological characteristics analysis of fertile and sterile buds of male-sterility AB line found that, in addition to sterile anthers do not produce pollen the other organs are nomal. Used paraffin sections to observe cell structure of sterile and fertile anthers. There was no significant difference in the early anther development stage, but after that sterile anther did not undergo noemal meiosis and the tapetum cells were abnormally expanded, eventually lead the pollen sacs of sterile anthers appeared shruke.2. Basis on the preliminary study of male-sterile gene Ms and restorer gene M/in Multiple-allele Genetic Male sterile Chinese cabbage, designed library screening primer,2 BAC clones corresponding to the positioning range were screened in BAC library, which are FH20-SRF1 and FH48-LCSK5. Using the third generation sequencing technology, the 2 BAC clones were sequenced. The length of FH20-SRF1 was 127658bp, and the length of FH48-LCSK5 was 130005bp. Two BAC sequences can be tiled, the length after stitching was 233305bp, the overlapping area was 24358bp. On the sequence after stitching, we can found the library screening primers, SKRF1 and LCSK5, and the positioning mark LZY6 was located in the splicing sequence region of 68462-68849bp. This indicated that these 2 BAC clones can completely cover the interval of chromosome A076700000-6800000. Compared with the sequence in the Brassica Database, in some region of BAC sequence took place inversion, and has a large fragment insertion. A total of 57 genes were predicted in the splicing sequence. These genes were cloned and sequenced in the meterial which genotype is Msf Msf, and 7 genes were found to be different from the BAC clones. And the 7 genes were all located in the region of the inserted fragment.3. Used high-throughput sequencing technology, fertile and sterile buds of Chinese cabbage male sterile AB line was sequenced. Unigenes were assembled according to no reference genome,32765 and 31955 unigenes obtained in fertile and sterile buds respectively. We compared 7 genes which have different sequences compared with ungenes. There are 5 genes can be compared, indicated that these 5 genes exist and can be normal expression.4. Gene expression analysis of fertile and sterile buds in Chinese cabbage male sterile AB line. There are 1013 differentially expressed genes between the fertile and sterile buds, including pollen development related AMS, MS2, VGD1 and pectinase gene etc.. Among these differentially expressed genes,481 were specific expressed in fertile buds and 6 were specific expressed in sterile buds. Among them,82 genes encoding unknown function proteins, most of which are expressed in pollen and pollen germination stages. We also performed GO analysis to reveal the main biological functions of the DEGs. Only 19 GO terms were significantly enriched. In the pathway enrichment analysis,5 KEGG pathways were significantly enriched, that is, Pentose and glucuronate interconversions, Alanine, aspartate and glutamate metabolism, Cysteine and methionine metabolism, Ascorbate and aldarate metabolism and Starch and sucrose metabolism. Finally, we used qRT-PCR to analyze the expression of 31 DEGs, the results were consisent with the sequencing results, which verified the accuracy of the sequencing results.5. Using the fertile and sterile buds of male-sterile AB line of Chinese cabbage as the test material to constructed small RNA libraries. Using high-throughput sequencing, we obtained 132 and 144 known miRNAs in two libraries respectively, and predicted 22 and 24 novel miRNAs, predicted 233 target genes of 35 miRNA families and identified 19 differentially expressed miRNAs. In these 19 differentially expressed miRNAs, most of them had high expression level in the sterile buds, only 5were expressed highly in fertile buds. These differentially expressed miRNAs target genes, which are related to the development of flower, stamens and pollen, such as AP2, pectin methylesterase and so on. There are 5 miRNAs’ target genes, which are differentially expressed genes in transcriptome sequencing. qRT-PCR verified that the expressions of all 19 differentially expressed miRNAs were basically consistent with the sequencing results.