Cloning And Functional Analysis Of The Genetic Male Sterility Gene BnMS5 In Brassica Napus L.

Rs1046A and FM195 A are dominant genetic male-sterility lines derived from a spontaneous mutation in Yi3 A. Genetic analysis indicated that the fertility is controlled by a monogenically multiallelic locus with three different alleles, BnMS5a(restorer type), BnMS5b(male-sterile type) and BnMS5c(normal male-fertile type). The dominance relationship of three alleles is BnMS5 a > BnMS5 b > BnMS5 c. In order to understand the sterile molecular mechanism of these lines, we cloned this novel meiotic gene BnMS5 by a map-based cloning approach. The evolution dynamics of BnMS5 in the Brassica and the functional divergence of different alleles were characterized. Combining with different cytological approaches, we demonstrated that BnMS5 likely determines the proper meiotic chromosome structure by regulating chromosome condensation during early prophase I. The results were described as follows:1. The cloning of BnMS5aThe physical map of BnMS5 locus was achieved through screening the BAC library HZBnB which contains BnMS5 a and BnMS5 b locus. The BACs containing the BnMS5 a, BnMS5 b and previously selected BnMS5 c were annotated according to the new sequenced B. napus reference genome(Darmor-bzh). Sequences restricted by BE10 and SCD8 in three locus were compared and only one unique genes, were likely candidates for BnMS5. The genetic complementation result suggested that BnaA08g25920 D from restorer line can rescue the sterile phenotype and corresponds to BnMS5 a.2. Sequence analysis of the three BnMS5 allelesAlignment of the full-length cDNA fragments and the corresponding genomic DNA sequences showed that the Bn MS5 a and Bn MS5 c displayed high CDS sequence identity with only 18 amino acid changes. In addition, we found that they showed a relatively low sequence similarity(56.1%) in the 670-bp region upstream of the transcription start site. BnMS5 b is a loss of function mutaion because of a Mutator-like transposable element(MULE) insertion in the second intron of BnMS5 a. Sequence analysis revealed that BnMS5 encodes functon unknown protein which contains a coiled-coil domain and a Brassicaceae-specific DUF626 domain.3. The phenotypic characterization of FM195 A by cytological analysesMale meiotic chromosome spreads at different stages showed that chromosomes did not get any further condensation, and aggregated into a disordered group after leptotene. Transition from leptotene to zygotene is arrested in FM195 A male meiocytes.4. The function of BnMS5 in meiosisFISH analysis using 45 S, 5SrDNA and pAtT4 as a probe showed that homologous chromosomes do not pair and telomere bouquet formation is defective in BnMS5bBnMS5 b. Immunostaining assay(γH2AX) demonstrated that DSB formation occurs normally in BnMS5bBnMS5b; ASY1 localizes to chromosomes normally during early prophase I in BnMS5bBnMS5 b, suggesting that the installation of chromosomal axes occurs during early prophase I in BnMS5 bBn MS5b meiocytes. No detectable SYN1, ZYP1 and HEI10 signals were observed in any BnMS5bBnMS5b(FM195A) meiocytes, suggesting that MS5 is important for SC installation and homologous recombination.5. The expression patterns of BnMS5qRT-PCR results showed that BnMS5 expressed in various tissues especially in reproductive organs. RNA in situ hybridization and GUS detection were performed in BnMS5aBnMS5 a anthers and Arabidopsis respectively to determine precisely the spatial and temporal expression patterns of BnMS5. Strong hybridization signals of BnMS5 a were observed in early meiocytes and tapetum. The GUS activity of ProBnMS5a-GUS was detected in all organs and similar with the RNA in situ hybridization results. Because of the reduced expression level and abnormal mRNA splicing of Bn MS5 b due to the MULE insertion in the second intron of BnMS5 a, BnMS5 b is a non-function mutation of BnMS5 a. The expression and protein accumulation of BnMS5 in MS5aMS5 a were far higher than MS5cMS5 c and MS5bMS5 b, with the lowest amount of MS5 in MS5bMS5 b.6. The origin and evolution of BnMS5BnMS5 homologs were isolated from B. rapa, B. oleracea and B. napus. Two BnMS5 homologs named BnMS5-COPY2 and BnMS5-COPY3 were found in B. napus. Sequence and phylogenetic analysis indicated that BnMS5 displays the closest relationship with its B. rapa orthologue(Bra018456) and two homologs are derived from B. oleracea. BnMS5 is a Brassica-specific novel gene which was an orphan gene evolved recently.