In order to understand the mechanisms regulating the seed length in rice, we are carrying out the identification of the causal genes for small and round seed mutants by the map-based cloning. To date, we have revealed that a dwarf rice mutant, d1, which showed pleiotropic phenotypes, such as the dwarfism, dark-green leaves and small, round seeds, had impairment in the gene encoding the heterotrimeric G protein α subunit (Ashikari et al. 1999, Fujisawa et al. 1999). Physiological experiments using d1mutants suggested that the heterotrimeric G protein was involved in the part of GA signaling pathway (Ueguchi-Tanaka et al. 2000). We also identified the mutated gene of the rice dwarf mutant, d11, which set small and round seeds. The D11 gene encoded a novel cytochrome P450 taking part in BR biosynthesis (Tanabe et al. 2005). In this paper, we report the mapping position of the mutated gene in a new small and round seed mutant of rice.

The mutant was obtained from Taichung 65 (T65) mutagenized with γ ray irradiation with 200 gray and was named small and round seed 1, srs1. The gross morphologies of srs1 mutant were compared with d1, d11 and T65 (Fig. 1). The srs1 mutant was not dwarf, but formed erect leaves at maturation stages (Fig. 1A) and set small and round seeds (Fig. 1B).

For mapping, the F₂ plants from a cross between srs1 (japonica) and Kasalath (indica) were used in this study. Genomic DNA was extracted from fresh leaf tissues of 46 F2 plants that set small and round seeds by CTAB method. The genetic linkage between the srs1 locus and molecular markers was determined using the sequence tagged site (STS) markers and the cleaved amplified polymorphic sequence (CAPS) markers reported by the Rice Genome Program and the microsatellite markers (McCouch et al. 2002a, 2002b). In addition to those markers, the ten primers for the five STS markers on rice chromosome 7, C1008F (5'-GAAGATGAAAATTGGCGTGTGAAG-3') and C1008R (5'-GCTAGTTGGTCAGGCAATAG-3'), L585F (5'-GGCAATATCTTGAATCACCTAG-3') and L585R (5'-GTTTTACTAGTTAAGACAAACGTG-3'), 231AF (5'-AACCAGGCCGCAATGCAATG-3') and 231AR (5'-ACTAGCTGCATGCATGCATG-3'), 7-240F (5'-CTGATTTCTCTTTCCTCCAC-3') and 7-240R (5'-CGTTGTCACCAGTGCTTTCG-3'), 253BF (5'-TTTGTGCGTTGTCAGCTGTC-3') and 253BR (5'-CCTAGTGGCGGTAGAGGTAC-3') were designed.

Next, 800 F₃ plants setting small and round seeds were selected and were used for semi-fine mapping of the SRS1 locus. We found that the srs1 mutation was located on chromosome 7 between the two STS markers 7-240 (81.4 cM) and R10253 (94 cM), and co-segregated with STS marker, 253B (91.7 cM) (Fig. 2). We are constructing more precise linkage map of SRS1 locus using STS markers between 7-240 and R10253.

References

Ashikari M., J. Wu, M. Yano, T. Sasaki and A. Yoshimura, 1999. Rice gibberellin-insensitive dwarf mutant gene Dwarf 1 encodes the a-subunit of GTP-binding protein. Proc. Natl. Acad. Sci. USA 96: 10284-10289.

Fujisawa Y., T. Kato, S. Ohki, A. Ishikawa, H. Kitano, T. Sasaki, T. Asahi and Y. Iwasaki, 1999. Suppression of the heterotrimeric G protein causes abnormal morphology, including dwarfism, in rice. Proc. Natl. Acad. Sci. USA 96: 7575-7580.

McCouch S. R., L. Teytelman, Y. Xu, K. B. Lobos, K. Clare, M. Walton, B. Fu, R. Maghirang, Z. Li, Y. Xing, Q. Zhang, I. Kono, M. Yano, R. Fjellstrom, G. G. DeClerck, D. Schneider, S. Cartinhour, D. Ware and L. Stein, 2002a. Development and mapping of 2240 new SSR markers for rice (Oryza sativa L.). DNA Res. 9: 199-207.

McCouch S. R., L. Teytelman, Y. Xu, K. B. Lobos, K. Clare, M. Walton, B. Fu, R. Maghirang, Z. Li, Y. Xing, Q. Zhang, I. Kono, M. Yano, R. Fjellstrom, G. DeClerck, D. Schneider, S. Cartinhour, D. Ware and L. Stein, 2002b. Development and mapping of 2240 new SSR markers for rice (Oryza sativa L.) (supplement). DNA Res. 9: 257-279.

Tanabe S., M. Ashikari, S. Fujioka, S. Taketsuto, S. Yoshida, M. Yano, A. Yoshimura, H. Kitano, M. Matsuoka, Y. Fujisawa, H. Kato and Y. Iwasaki, 2005. A novel cytochrome P450 is implicated in brassinosteroid biosynthesis via the characterization of a rice dwarf mutant, dwarf11, with reduced seed length. Plant Cell 17: 776-790.

Ueguchi-Tanaka M., Y. Fujisawa, M. Kobayashi, M. Ashikari, Y. Iwasaki, H. Kitano and M. Matsuoka, 2000. Rice dwarf mutant d1, which is defective in the a subunit of the heterotrimeric G protein, affects gibberellin signal transduction. Proc. Natl. Acad. Sci. USA 97: 11638-11643.