17. Fine map of d-I gene in rice 

M. ashikari, 0. ideta, a. yoshimura, and N.iwata

Plant Breeding Laboratory, Faculty of Agriculture, Kyushu University, Hakozaki 6-10-1, Higashi-ku, Fukuoka 812-81,Japan

 A chimeric rice plant maintained in our laboratory shows normal and dwarf morphology in many parts of the plant body. Allelic and progeny test revealed that the chimerism appeared to occur at the locus of recessive gene d-l located on chromosome 5. To elucidate the mechanism to generate the chimerism, cloning of d-l gene is essential. In this study, fine RFLP linkage map around d-l locus was carried out as an initial step for map-based cloning of the gene.

A small mapping population of 100 F2 plants derived from the cross between Indica variety IR24 and Japonica marker line FL423 with d-l was used for linkage analysis with 20 RFLP markers on chromosome 5 (Saito et al. 1991; Kurata et al. 1994). The d-l is located at a distance of 7.8 ± 2.0 cM between RFLP markers C309 and XNpb251 (Fig. I ).

To obtain a large mapping population, d-l plants in the Fz population from the cross between IR24 and FL423 (d-l) was backcrossed to IR24 and the resultant BC3F2 population was used for fine mapping of d-l. Out of about 2600 seedlings of BC3F2 population, 640 dwarf plants (homozygous recessive) were selected at the seedling stage for pooled-sample mapping (Churchill et al. 1993). Two mature leaves of each individual were collected. Leaves of five plants were bulked for 1 pool and DNA of the 128 pools were extracted. RFLP analysis were performed with some RFLP markers around d-l. Out of 128 pools, 10,10 and 41 recombinant pools were detected with RFLP markers C309, S2351 and G1458, respectively. RFLP marker G1458 instead of XNpb251 was used for pooled-sample mapping, because Southern hybridization image of G1458 was clearer than XNpb251. Furthermore, out of 97 pools, 32 and 42 recombinant

Research Notes 77
Fig. 1. Linkage map of d-l with RFLP markers on chromosome 5. Linkage map of d-l constructed by using F2 is shown under the bold bar. Fine linkage map constructed by using BC3F2 population is shown above the bold bar. Map distance in pooled-sample mapping was calculated based on the follwoing formula; r=(l/k)ln[1-(yA/n)] (Churchill et al. 1993), r: the approximate maximum likelihood estimator of genetic distance from the target locus, k: the number of individuals per pool, yA: total number of recombinant pools, n: the number of pools.


pools were detected with C282 and R569. In this pooled-sample mapping, the exact number of recombinants in a pool is difficult to determine. Therefore, to determine the exact number of recombinants for the closest RFLP marker, DNA of 640 dwarf plants was individually extracted. For C309 and S2351, 10 and 10 recombinants were detected, respectively. RFLP markers C309 was tightly linked to S2351 at 0 cM. The genetic distance between d-l and C309 (S2351)was 0.8 cM. And the genetic distance between d-l and G1458 was 3.9 cM. The order of d-l and RFLP markers in the vicinity of the d-l was C309(S2351) - d-l- G1458. (Fig. 1).

Since the genetic distance d-l and the flanking RFLP marker C309 (S2351) in one side was less than lcM, C309 (S2351) appears to be a starting point for chromosome walk to d-l. However, flanking marker G1458 in the other side was far from d-l. We need markers located closely around d-l for cloning. The 9 RFLP and 6 RAPD markers are located between C309 and G1458 (Kurata et al. 1994). These markers could not be used in the present mapping, because there is no polymorphism for these markers between Japonica marker line FL423 (d-l) and Indica variety IR24. We detected polymorphisms for these markers between of FL423 (d-l) and an Indica variety Kasalath. So we are generating Fine mapping population derived from cross between FL423 (d-l) and Kasalath. (Gene symbol: Old system)

 References

Churchill, G.A., J.J. Giovannoni and S.D. Tanksley, 1993. Pooled-sampling makes high-resolution mapping practical with DNA markers. Proc. Natl. Acad. Sci. USA. 90: 16-20.

Kurata, N., Y. Nagamura, K. Yamamoto, Y. Harushima, N. Sue, J. Wu, B. A. Antonio, A. Shomura, T. Shimizu, S-Y. Lin, T.Inoue, A. Fukuda, T. Shimano, Y. Kuboki, T. Toyama, Y. Miyamoto, T. Kirihara, K. Hayasaka, A. Miyao,
    L. Monna, H. S. Zhong, Y. Tamura, Z-X. Wang, T. Momma, Y. Umehara, M. Yano, T. Sasaki and Y. Minobe, 1994. A 300 kilobase interval genetic map of rice including 883 expressed sequences. Nature Genetics 8: 365-372.

Saito, A., M. Yano. N. Kishimoto , M. Nakagahra, A. Yoshimura, K. Saito. S. Kuhara. Y. Ukai, M. Kawase, T. Nagamine. S. Yoshimura, 0. Ideta. R. Ohsawa, Y. Hayano, N. Iwata and M. Sugiura, 1991. Linkage map of restriction fragment      length polymorphism loci in rice. Japan. J. Breed. 41: 665-670.