Mapping of two SHOOTLESS genes, SHL1 and SHL2, by RFLP analysis

13. Mapping of two SHOOTLESS genes, SHL1 and SHL2, by RFLP analysis M.ITO1, Y. SATO1, N. NAGASAWA’, H. Kitano2, Y. NAGATO3 and M. MATSUOKA 1) Bioscience Center, Nagoya University, Nagoya, 464-8601 Japan 2) Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, 464-8601 Japan 3) Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo, 113-8657 Japan In plants, most morphogenetic events occur in the post-embryonic phase, all of which develop from the shoot and root apical meristems produced in embryo. Thus the mechanism of initiation of the meristems during embryogenesis should be one of the most important subjects to be explored in developmental plant biology. To elucidate the mechanism of the shoot apical meristem (SAM) initiation, we are trying to isolate two SHOOTLESS genes, SHLJ and SHL2, by map-based cloning. Satoh et a!. (1999) have isolated and characterized nine shootless mutants derived from four independent loci (SHLJ - SHL4). These mutants fail to develop the SAM and the lateral organs such as leaf primordia, coleoptile and epiblast, while radicle and scutellum are normally produced. Through in situ hybridization analyses of the shi embryos, it has been postulated that SHL1 and SHL2 function upstream of a rice homeobox gene, OSHI, which is considered to be indispensable for the establishment and maintenance of the SAM throughout the life cycle (Sato eta!. 1996). Thus, the isolation of these genes is important not only for elucidating the molecular mechanism of the SAM initiation during embryogenesis but also for unraveling the molecular interaction between homeobox genes and the SHL genes. As a first step, we mapped SHL1 and SHL2 loci by the linkage analysis using RFLP markers. As shi homozygous mutants have no shoot, we can’t obtain enough genomic DNA for he DNA gel blot analysis from shi homozygous plants. Therefore, we isolated genomic DNA from heterozygous plants (SHL1/shl1 or SHL2/shl2). First, we crossed the japonica rice cv. Taichung 65 heterozygous for shl1 or shl2 with an indica rice, cv. Kasalath, homozygous for the wild type alleles (SHUSHL). Then, the F, plants were grown and self-pollinated for obtaining F2 seeds. The F2 seedlings (SHLISHL or SHL/shl) were used for the isolation of genomic DNA for DNA gel blot analysis. Genotypes of F2 plants were identified by screening the phenotypes of F3 embryos. We used one hundred F2 plants heterozygous for SHLI or SHL2 for the RFLP analysis. The SHL1 and SHL2 loci were roughly mapped on the chromosome 12 and 1, respectively. For further analysis, we have selected several markers located near SHL1 or SHL2 loci. Four markers, C1336, R2672, R3375 and R617 were selected for SHLI. Using these markers, SHLI was mapped between R3375 and R6l7 with a distance of 5.5 cM and 1.4 cM, respectively (Fig.l-A). Similarly, eight markers, R210, C585, C178, R2504, S10626, S765, R2159 and R1928, were selected for SHL2, and SHL2 was mapped between S 10626 and S765 with a distance of 4.2 cM and 9.9 cM, respectively (Fig.1-B). Since these RFLP markers are far away from SHL loci, we have already tested all the available markers identified by the Rice Genome Research Program, several new markers are required for further analysis. References Harushima, Y.,M. Yano, A. Shomura, M. Sato, T. Shimano, Y. Kuboki, T. Yamamoto, S.Y. Lin, B.A. Antonio, A. Parco, H. Kajiya, N. Huang, K. Yamamoto, Y. Nagamura, N. Kurata, G.S. Khush and T. Sasaki, 1998. A high-density rice genetic linkage map with 2275 markers using a single F2 population. Genetics 148: 479-494. Sato, Y., S.K. Hong, A. Tagiri, H. Kitano, N. Yamamoto, Y. Nagato and M. Matsuoka, 1996. A rice homeobox gene, OSHI, is expressed before organ differentiation in a specific region during early embryogenesis. Proc. Nati. Acad. Sci. USA 93: 8117-8122. Satoh, N., S.-K. Hong, A. Nishimura, M. Matsuoka, H. Kitano andY. Nagato, 1999. Initiation of shoot apical meristein in rice: characterization of four SHOOTLESS genes. Development 126: 3629-3636.

13. Mapping of two SHOOTLESS genes, SHL1 and SHL2, by RFLP analysis
M.ITO1, Y. SATO1, N. NAGASAWA’, H. Kitano2, Y. NAGATO3
and M. MATSUOKA

1) Bioscience Center, Nagoya University, Nagoya, 464-8601 Japan

2) Graduate School of Bioagricultural Sciences, Nagoya University,
Nagoya, 464-8601 Japan

3) Graduate School of Agricultural and Life Sciences, University of Tokyo,
Tokyo, 113-8657 Japan

In plants, most morphogenetic events occur in the post-embryonic phase, all of which develop from the shoot and root apical meristems produced in embryo. Thus the mechanism of initiation of the meristems during embryogenesis should be one of the most important subjects to be explored in developmental plant biology. To elucidate the mechanism of the shoot apical meristem (SAM) initiation, we are trying to isolate two SHOOTLESS genes, SHLJ and SHL2, by map-based cloning.

Satoh et a!. (1999) have isolated and characterized nine shootless mutants derived from four independent loci (SHLJ - SHL4). These mutants fail to develop the SAM and the lateral organs such as leaf primordia, coleoptile and epiblast, while radicle and scutellum are normally produced. Through in situ hybridization analyses of the shi embryos, it has been postulated that SHL1 and SHL2 function upstream of a rice homeobox gene, OSHI, which is considered to be indispensable for the establishment and maintenance of the SAM throughout the life cycle (Sato eta!. 1996). Thus, the isolation of these genes is important not only for elucidating the molecular mechanism of the SAM initiation during embryogenesis but also for unraveling the molecular interaction between homeobox genes and the SHL genes. As a first step, we mapped SHL1 and SHL2 loci by the linkage analysis using RFLP markers.

As shi homozygous mutants have no shoot, we can’t obtain enough genomic DNA for he DNA gel blot analysis from shi homozygous plants. Therefore, we isolated genomic DNA from heterozygous plants (SHL1/shl1 or SHL2/shl2). First, we crossed the japonica rice cv. Taichung 65 heterozygous for shl1 or shl2 with an indica rice, cv. Kasalath, homozygous for the wild type alleles (SHUSHL). Then, the F, plants were grown and self-pollinated for obtaining F2 seeds. The F2 seedlings (SHLISHL or SHL/shl) were used for the isolation of genomic DNA for DNA gel blot analysis. Genotypes of F2 plants were identified by screening the phenotypes of F3 embryos.

We used one hundred F2 plants heterozygous for SHLI or SHL2 for the RFLP analysis. The SHL1 and SHL2 loci were roughly mapped on the chromosome 12 and 1, respectively. For further analysis, we have selected several markers located near SHL1 or SHL2 loci. Four markers, C1336, R2672, R3375 and R617 were selected for SHLI. Using these markers, SHLI was mapped between R3375 and R6l7 with a distance of 5.5 cM and 1.4 cM, respectively (Fig.l-A). Similarly, eight markers, R210, C585, C178, R2504, S10626, S765, R2159 and R1928, were selected for SHL2, and SHL2 was mapped between S 10626 and S765 with a distance of 4.2 cM and 9.9 cM, respectively (Fig.1-B). Since these RFLP markers are far away from SHL loci, we have already tested all the available markers identified by the Rice Genome Research Program, several new markers are required for further analysis.

References

Harushima, Y.,M. Yano, A. Shomura, M. Sato, T. Shimano, Y. Kuboki, T. Yamamoto, S.Y. Lin, B.A. Antonio, A.

Parco, H. Kajiya, N. Huang, K. Yamamoto, Y. Nagamura, N. Kurata, G.S. Khush and T. Sasaki, 1998. A

high-density rice genetic linkage map with 2275 markers using a single F2 population. Genetics 148:

479-494.

Sato, Y., S.K. Hong, A. Tagiri, H. Kitano, N. Yamamoto, Y. Nagato and M. Matsuoka, 1996. A rice homeobox gene, OSHI, is expressed before organ differentiation in a specific region during early embryogenesis. Proc. Nati. Acad. Sci. USA 93: 8117-8122.

Satoh, N., S.-K. Hong, A. Nishimura, M. Matsuoka, H. Kitano andY. Nagato, 1999. Initiation of shoot apical meristein in rice: characterization of four SHOOTLESS genes. Development 126: 3629-3636.