The club-shaped embryo (cle) mutant fails to develop most organs and tissues, and is characterized by the unique embryo shape (club-shape, see Fig. 1A-F), which is not observed in wild-type and the other organless-mutant embryos in rice (Hong et al. 1995). Three NMU-induced cle1 mutant alleles (cle1-1, cle1-2, and cle1-3) have been briefly reported previously (Hong et al. 1995).

Firstly, we studied a detailed histological analysis of the three cle1 alleles. It was revealed that these showed the same phenotype, which failed to develop shoot apical meristem (SAM) and root apical meristem (RAM), but developed the vascular-like tissue and the scutellar epitherium-like tissue (see Fig. 1B-D).

Next, we screened the other cle-like lines in a callus-culture induced mutant library. Of them, the embryos of the two lines (cle1-4, and cle1-5, see Fig. 1E and F respectively) also showed the cle phenotype, but the embryos were obviously larger (Fig. 1E and F compared with Fig. 1B-D). We carried out the allelism test, and the result showed that the two lines are allelic to cle1 (data not shown). The difference of the phenotype should depend on the each mutation, which causes the different activity of the gene product.

A rice homeobox gene OSH1, which is specifically expressed in the shoot meristem region before the morphological development in wild-type (Fig. 1G), was misexpressed in cle1-1 (the cle small-allele) embryo; the expression was observed in the basal region of the embryo on both the dorsal and the ventral sides (Fig. 1H). The same expression pattern was also observed in cle1-5 (the cle large-allele, Fig. 1I). These observations imply that the pattern formation is impaired both in the small- and the large-allele embryos. It should be noted that this pattern is obviously different from that of another organless-mutant, organless1 (orl1), in which the expression pattern of OSH1 is reported to be similar to that in the wild-type embryo (Kamiya et al. 2001).

In the aim of cle1 gene mapping, we used the genomic DNAs of F₂ heterozygous plants (CLE1/cle1-1), which were identified by the segregation of the cle phenotype in F₃ embryos. We used 46 F₂ heterozygous plants for the linkage analysis. The result demonstrated that CLE1 is located between K0432D01 and TG1020 on chromosome 3 (Fig. 2). According to the RGP (Rice Genome Project) map (Harushima et al. 1998), the genetic distance between them is 5.2 cM (Fig. 2). We are narrowing down of this region to determine the CLE1 gene.

References

Harushima Y., M. Yano, A. Shomura, M. Sato, T. Shimono, 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 makaers using a single F₂ population. Genetics 148: 479-494.

Hong S. K., T. Aoki, H. Kitano, H. Satoh and Y. Nagato, 1995. Phenotypic diversity of 188 rice embryo mutant. Dev. Genet. 16: 298-310.

Kamiya N., M. Ashikari, Y. Sato, Y. Nagato and H. Kitano and M. Matsuoka, 2001. Mapping of the ORGANLESS1 gene, ORL1. Rice Genet. Newsletter, 18: 33.