Comparative Analysis Of The Genomic Region Containing Recessive Genic Male Sterility Gene In Rapeseed And Development Of Sequence-specific Markers

Brassica napus is a polyploid resulting from the natural hybridization of B. rapa (containing the Brassica A genome) and B. oleracea (containing the Brassica C genome). The A and C genome differentiated from a common ancestor Brassica about 400 million years ago.They have highly conserve genome structure and high micro-collinearity (Rana et al., 2004), which may hamper the genetic mapping in Brassica napus.The research of the variance between A and C genomes on DNA sequence level can be used to distinguish homologous genomes between A and C genomes which have high microcollinearity, and therefore may provide some assistance of the research of Brassica napus.9012AB is a novel genic male sterility (GMS) material which has great potential in three line seed production, because of it's a series of advantages, such as complete sterility, high stability, sufficient restore lines, and so on. Genetic analysis indicated that the male sterility of 9012AB was controlled by two pairs of recessive duplicate sterile genes (ms3ms3 and ms4ms4) interacting with one pair of a recessive epistatic inhibitor gene (rfrf) (Chen et al., 1998). The previous study ( He et al., 2008) shown that the ms3 gene was fine mapped to a region of 0.034cM which including at least six BACs and the BAC clone which contained the target gene was identified by subclone sequencing. In order to develop ms3 mocular makers, confirm the results of BAC subclone and compare the micro-collinearity between B. oleracea and B. rapa, we randomly selected 20kb sequences on the basis of the subclone library for comparative sequencing and analyzing from differenent matirials, which can provide the theory basis and help for the mapping and cloning of ms4 gene. The major results were as follows:1. A total of 18 primer pairs which were designed according to the 20kb sequences were used in 9012A,9012B,B. rapa and B. oleracea. The amplification products from these primer pairs could constitute a contig that can coverage this region completely in theory. Splicing the sequence of these amplification products, we obtained the the sequence information of these materials.2. Based on the section sequence, we analyzed the microcollinearity of Tapidor,9012A,9012B,B. rapa and B. oleracea in the selective region. The results indicated that 9012A and B. rapa had the high sequence similarity, so as the 9012B,Tapidor and B. oleracea. Gene prediction of this region refer to the Arabidopsis genome sequence shown that this region included three highly conserved genes with the same direction. The sequences of the exons of those genes had little or no changes, however, the sequences of the introns of those genes had mutation, insertion or deletion. According to these significant differences, we developed six markers as to distinct 9012A and 9012B,markers named DM1 and HWY to distinct B. rapa and B. oleracea.3. Five markers developed from 9012A and 9012B showed co-segregated with the target gene ms3 by tested recombination plants of 20,391 individuals. As a result, we did not reduce the physical region of the gene ms3, although five new markers were developed.4. According to the different sequences between B. rapa and B. oleracea, Two codominant markers, which showed stable polymorphism in forty nine B. rapa and eight B. oleracea materials were developed. Then, we used the markers to identity the Brassica napus BAC library, which contained the target gene ms3. The amplified products with the same size and sequence information of B. rapa genome,were obtained, however, clone with the same size in B. oleracea genome was not found. According to the previous studies, ms3 gene located in the N19 linkage groups, which corresponding to B. oleracea chromosome of C9. It was speculated that the BAC clones we selected could be the homology region of ms3 in B. rapa.5. We used the SSR markers from BAC clones and the A, C genome-specific markers to detect the polymorphism in ms4 mapping population. All markers were not linked with ms4 gene. It is suggested that the ms3 and ms4 were different homologous genes, so the statement about ms3 and ms4 were two pairs of recessive duplicate sterile genes needed to be further confirmed.