Microsynteny Analysis Of The BnMs-candidate-gene Region Among The Brassica A, C Genomes And Arabidopsis Genome

Brassica is one of the most important and largest genera of Cruciferae plants,consisting of more than 300 families. Many of them are vegetables, oil and feed cropscommonly consumed and used in the world. Brassica and Arabidopsis thalianan, whichstarted to diverge from a common ancestor during 14.5 to 20.4MYA, belong to the samefamily, Brassicaceae. Arabidopsis as a model plant of dicotyledon, its genetic map,physical map and the whole genome sequence have been done perfect researches. Manyimportant genes have been characterized functionally. In the Brassica, many geneticlinkage maps have been already contructed and many QTLs of main traits have beenmapped. So the comparative studies between Brasssica and Arabidopsis could emphasizethe developing of Brassica Genome Research.Currently, the discovery and study of some recessive genie male sterile (GMS) linesin Brassica napus plays an important role in the utilization of heterosis because of someadvantages, such as very stable and complete sterility, rich sources of cytoplasm, widelyspread of restorers and availability of 100ï¼…sterile population. A recessive genie malesterile (RGMS) system, S45AB, has been developed from spontaneous mutation inBrassica napus canola variety Oro. The male sterility of S45 was controlled by twoduplicated recessive genes, named as Bnms1 and Bnms2. Yi et al. (2006) mapped theBnMs1 locus to a region of 0.4 cM. A syntenic interval of Arabidopsis genome delimitedby the nearest flanking markers of BnMs1. Two candidates of which have been identifiedand genetic transformation is in progress. Then, using the same method, Lei et al. (2007)mapped the BnMs2 locus to a region of 0.075 cM and identified a collinear regioncontaining 68 Arabidopsis genes.Those genes include the two candidates of BnMs1gene.So,the region of Arabidopsis including the two candidate genes was calledBnMs-candidate-gene region in this study.In this study, we detected the distribution and copies of theBnMs1-syntenic-interval of Arabidopsis in Brassica A, C genomes by ACGMs(Amplified Consensus Genetic Marker), which were designed based on the sequence ofBnMs1-syntenic-interval of Arabidopsis. Furthermore, we analysed the gene conservationand microsynteny of the BnMs-candidate-gene region among the Brassica A, C genomesand Arabidopsis genome by sequenceing. This study could also provide the theory basisand help for the fine mapping and cloning of BnMs2 gene. The major results were asfollows:1. A total of 25 ACGM primer pairs were designed according to the different genes inBnMs1 syntenic interval of Arabidopsis. Of these, each of the majority (24)amplified a clear, strong and single band from the total genomic DNA of allparents and Arabidopsis when detected by 1ï¼…agarose gels. Using 6ï¼…PAGE or1ï¼…agarose gels analysis, a total of 9 ESTs (Expression Sequence Tags) or genesfrom B. napus showed polymorphisms between the parents of the mapping population, providing total 12 marker loci in the two mapping population. Thosemarkers, respectively, located in the linkage groups N2, N7, and N16 of B. napus.2. Two kinds of bulks constructed based on the male sterile and fertile plants from theNIL (near-isogenic line) population of S4516AB were screened using the 25ACGM primer pairs. Two ACGMs linked closely to the target gene were identified,one marker A2 is co-segregated with BnMs2, the other marker B4 is the closestflanking marker, which was mapped at 0.038cM away from the target gene.3. Two clones containing the BnMs2 gene were obtained, by screening the BAClibraries of B. oleracea and B. napus, respectively.4. Totals of 9.8kb and 9.6kb sequences containing the target candidate genes from thecandidate clones of B. oleracea and B. napus were sequenced, respectively.5. Using the Arabidopsis BnMs-candidate-gene region genome sequence as areference, we have analysed aspects of gene conservation and microsynteny amongthe four homologous regions of Brassica A, C genomes and Arabidopsis. Theresults indicated that the three genes of Arabidopsis in this region showed highlyconserved synteny with their orthologues across genomes. The sequences of theexons of those genes have little or no changes to the genes in Brassica. TheBnMs-candidate-gene region sequences of Brassica A, C genomes were allconsisting of five conservative segments, and these segments were the sameorientation and almost exactly the same sequence, but the difference is theseconservative segments of this region in the different genomes are separated bysome different large inserts.The BnMs-candidate-gene region sequences of Agenome in B.napus and B. rapa have the high sequence similarity, the same as Cgenome in B.napus and B. oleracea, the similarity of the former was higher thanthe latter.