43. Identification of the DNA fragments located in the vicinity of waxy locus responsible for gamete elimination

L. WU1, H. NAGANO1, S. KAWASAKI2, Y. KISHIMA1 and Y. SANO1

1) Faculty of Agriculture, Hokkaido University, Sapporo, 060 Japan
2) National Institute of Agrobiological Resources, Kannon-dai 2-1-2, Ibaraki, 305 Japan

As described by Nagano et al. (in the previous paper) in this volume, the present study is now focused on the isolation of S10 gene from T65wx (Japonica) that acts to exclusively eliminate the gametes carrying S10-a allele from PTB10 (Indica) in the backcross line. This gene is very closely linked with the wx locus, according to the recombination value (0.15%) estimated between wx and S10 by means of pollen analysis (Sano et a1.1994).

In this study, we have subcloned the 300-kb BAC contig, which was constructed by Nagano et al. into the lambda Zap vector. Among randomly selected 300 subclones. 150 clones carrying inserts larger than 4-kb were employed in the following analysis, preceded by a conversion into pBluescript form. S10 gene is expected to express during the gametogenesis, since it acts gametophytically. Subsequently, crude cDNAs were synthesized with RNA prepared from the developing anthers of T65wx. The cDNAs thus obtained were used for probing the filter loading the representative subclones of the 300-kb BAC contig (Fig. 1A). As shown in Fig. 1B, there were several fragments that hybridized with the probe consisting of crude cDNAs from the anther RNA population. These results imply that the 300-kb contig contains transcribing fragments in the developing anther. It should be certified whether the hybridized segments correspond with certain cDNAs. To do this, we are in the process of constructing a detailed map using the subclones of the 300-kb contig, and at the same time we are isolating cDNA clones from its library by probing the hybridized fragments in the subclones.
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References 

Sano, Y., R. Sano, M. Eiguchi, and H.-Y. Hirano, 1994. Gamete eliminator adjacent to the wx locus as revealed by pollen analysis in rice. J. Hered. 85: 310-312.

Nagano, H., L.Wu, S. Kawasaki, Y. Kishima and Y. Sano, 1997. Construction of a 300-kb BAC contig containing waxy locus in Japonica type. RGN 14: 121-123.