Hybrid sterility is the main barrier in utilizing the heterosis of subspecies in rice. A knowledge of the underlying molecular mechanism of the hybrid sterility will be useful for overcoming the barrier. In this research, the F 1 pollen sterility locus, S-b, was mapped between SSR markers PSM8 and PSM202. To fine map the locus, one F 2 mapping population of 3910 plants was developed using the near-isogenic lines of the locus. Ninety-seven recombinants between two markers were selected. Moreover, a series of markers, including two SSR markers, two InDel markers and four CAPS markers, were developed on the region. Linkage analysis showed that marker W4 was co-segregated with locus S-b, while makers A8 and A14 were located on the two sides of the locus with a distance of 0.026 and 0.038 cM, respectively. The markers were then integrated with the sequences of the clones of the region. Results showed that all the polymorphic markers were anchored on the three end-to-end jointed clones AC093089, AC079021 and AC134931. According to the physical information of the markers, locus S-b was finally delimited to a region of 27 kb between A8 and A14. Seven ORFs were identified on the region based on the annotation results of RiceGAAS system. These results laid the foundation for further cloning the gene.Firstly, the recombinants were selected from the large population by assaying the genotypes of the tightly linked markers. Then the selected recombinants were subjected to pollen fertility examination which was confirmed again in the F Keywords: hybrid sterility, molecular marker, fine mapping, rice.Strong heterosis existed in the hybrid of the subspecies in rice [1,2] . However, the partial sterility of the hybrid hinders the utilization of the heterosis [3,4] . Ikehashi et al. [5,6] considered the female gamete as the main sterility form and proposed single locus S-5 controlling the hybrid fertility of the subspecies. Subsequently a series of female and male sterility loci were identified in different researches [7][8][9][10][11][12][13][14][15][16][17] . Zhang and Lu [18] found three F 1 pollen sterility loci by diallel analysis of Taichung 65 and its near-isogenic lines. Thereafter other three F 1 pollen sterility loci were identified by testcross between the near-isogenic lines of Taichung 65 and a large number of varieties [19] . Of the six F 1 pollen sterility loci identified so far, three loci, namely S-a [20] , S-b [21] and S-c [22,23] , have been mapped and one locus, S-c, has been fine mapped [24] .Locus S-b was primary mapped on the short arm of chromosome 5 and tightly linked with markers RM13 and R830STS by screening the published microsatellite markers and developing cleaved amplified polymorphic sequence (CAPS) markers [25] . Rice genome sequencing project [26][27][28][29] provided abundant sequences for developing sequence-based microsatellite markers [30,31] and Insertion-Deletion (InDel) markers [32] . To map-based clone locus S-b, a large mapping population was developed and several putative ORFs were identi...