| Plant male sterility genes are important genetic resources, also which play the vital role in crop breeding work, especially in the heterosis utilization. Gene mapping is an important work of heterosis and genetic research, also is a key foundation of breeding work. Therefore, clear the molecule markers, and its position on chromosomes will support for the further research and application in male sterility.In this study, male fertile and sterile Salvia miltiorrhiza Bunge were used as research materials, though the field observation and marked plants in tags. Based on previous work and result of our laboratory, combined the AFLP molecular marker and BSA technology to screen the molecule markers, and constructed the primary gene localization and constructed linkage map about the male sterile gene initially. The main results and conclusions were listed as follows:1. Based on the filed flowering stage observation and the sterile stability and abortive degree were investigated continuously, we found such a conclusion: according to the filaments length and the amount of pollen, the Salvia miltiorrhiza Bunge male sterile plants was subdivided into two categories: fertile type and sterility type, we marked them F and S. And in this study, sample was in strict accordance with the flower pollen, selected the plants that sterile plants and the fertile one. Based on the changed traditional protocol, an improved method for good quality leaf DNA isolation was established, this method could efficiently get rid of polysaccharides from Genomic DNA.2. In this study, the AFLP molecular marker and the BSA method were used to identify the polymorphisms and differences between FB and SB. A total number of 378 pairs of random AFLP primer combination(21×18) were used to screened, as a result, 80 pairs of them products were showed bright and different between the two gene pools.Through AFLP method eight materials were analyzed by selected 80 pairs of primer combination, only 26 pairs of primer combination were amplified clear and repeating bands, totally got 141 bands, and the average ratio was 18.4%. The above primer combinations were subjected to population test among all the plants, it was found that only seven primer combinations was tightly related to the target gene locus, named marker E01/M01,E01/M09,E03/M03,E03/M10,E05/M10,E05/M13 and E05/M04.3. Constructed by Mapmaker(3.0), a total of 26 markers meet this linkage, constructed two linkage groups, named EM1 and EM2. The group EM2 has 16 markers, the total genetic distance was 272.7 c M and the average lotus interval was 17.04 c M, the largest lotus interval was 40.2 c M.The group EM1 has 10 markers, the total genetic distance was 52.9 c M and the average lotus interval was 4.41 c M, and the largest lotus interval was 8.6 c M. Aong them, marker E01/M01 and marker E01/M09 have smaller lotus interval with the sterile gene, t he genetic distances were 2.1 c M.4. The target segment were sequenced, searched EST database, we found marker E01/M09 and marker E05/M04 were highly homologous with model plant Arabidopsis thaliana Male Sterile2(MS2) gene, the MS2 gene in Arabidopsis thaliana plays a key role in pollen wall development. Furthermore, marker E01/M09 and marker E05/M04 can be located on the third chromosomes of Arabidopsis thaliana, so they were greater likelihood with our study genes. |
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