Genetic Mapping Of A Dominant Male Sterility Gene In Brassica Oleracea And Transcriptional Analysis Of The Male Sterility

Cabbage (Brassica oleracea var. capitata) is a worldwide vegetable crop belonging to the family Cruciferae. Breeding and utilization of male sterile lines is important for hybrid seed production. A dominant male sterility gene (Ms) identified from a nature population of a cabbage line has been well utilized in commercial cabbage hybrid seed production. However, molecular genetics and gene expression of the male sterility has not been characterized. The present research aimed to map the gene and characterize it at transcriptional level. Genetic Mapping of the Ms GeneAmplified Fragment Length Polymorphism (AFLP) was adopted to identify DNA markers linked to the Ms gene in a 3-generation backcross of Brassica oleracea var. alboglabra into a male sterile Brassica oleracea var italica line harboring the gene. Four markers, M3247, M356(K M3658 and M3461, linked to the Ms gene were identified using Bulked Segregant Analysis (BSA) from 2 male sterile DNA pools and 2 male fertile ones. The genetic distances, determined by Joinmap 3.0, between the markers and the Ms gene were 7cM, 7cM, lOcM and 12cM respectively.A high-density genetic linkage map of Brassica Oleracea was constructed with AFLP markers using a Brassica oleracea var. alboglabra Brassica oleracea var italica F1-derived doubled-haploid (DH) population of 46 individuals. The total map length of nine main linkage groups containing 337 markers was 801.5 cM with an average marker interval of 3.6 cM.Basing on the chromosomes characterized using a B. oleracea var. alboglabra line A12DH by Howell, we used chromosome specific SSR markers to assign two linkage groups of the genetic map to chromosomes 4 and 9. Fortunately, one of the markers, MS3247, linked to the Ms gene was segregating in the mapping population, which allowed us to locate it on a linkage group assigned to chromosome 9 by its specific SSR marker MB4.Transcriptional AnalysiscDNA-AFLP was used to detect differentially expressed genes in buds of male sterile and fertile lines of cabbage and broccoli. One hundred and twenty eight primer combinations generated proximately 26,000 cDNA fragments, of which 24 fragments were differential ones. Among the 24 fragments, 11 were expressed only in fertile buds, while the other 13 were much more abundant in fertile buds than in sterile ones.We isolated and sequenced 11 differential cDNA-AFLP fragments by extended NNN primer method, which simplified the band patterns by further selective amplification. BLAST analysis found that 7 fragments out of 11 were highly homologous or similar to known genes, while the other 4 shared no significant homology to any sequence in TAIR (The Arabidopsis Information Resources) DNA sequence database. MS1124110 and MS1620200 were two fragments sharing very high homology respectively with pectin methylesterase (PME) and pectase lyase, two enzymes playing important roles in the maturation of microspore by releasing them from the tetrad. MS1615300 revealed 89% similarity to the gene thioredoxin, which is related to S -locus protein in the Brassica family; MS1620200 was homologous to a peptide signal rapid alkalinization factor (RALF), which is a conserve domain in Brassica. MS1324200 is similar to anther-specific proline-rich protein APG precursor.In summary, informative markers linked to the Ms gene were generated. These markers will be useful not only for marker assisted breeding of the male sterility gene, but also for identifying in arabidopsis chromosomal micro-colinearity, which will lead to a possible way to map based cloning of the Ms gene. Findings of Ms related genes provided information improving our understanding of the molecular and cellular process involved in microspore development in Brassica crops. These genes can be further explored in the construction of controllable artificial male sterility genes.