42. Construction of a 300-kb BAC contig containing waxy locus in Japonica rice H. nagano1 ,L. Wu1, 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

 A gene designated as S10 is involved in gamete elimination. It was detected in the backcross line when Wx from PTB10 (Indica) was introduced into the genetic background of T65Hwx (Japonica) (Sano et al. 1994). At the third backcross (BC3), pollen with Wx from the PTB10 was found to be lacking as determined through iodine staining. The

122 Rice Genetics Newsletter Vol. 14

Fig. 1. Construction of a 300-kb BAC contig around the rice Wx gene. The BAC library was prepared from cv. Shimokita (Nakamura et al. 1997). The contig contains 9 independent clones that were screened by the three probes, wx, 4104 and 6189. Each BAC clone has a partial HindIII fragment ranging from 70-kb to 120-kb. The clone number is indicated at the right side of the bar. elimination of the female gametes carrying PTB10 allele was also observed, since seeds from self pollination showed waxy phenotypes. Based on these results, it was proposed that S10 allele in T65wx eliminated the gametes carrying S10-a allele of PTB10. A recombination frequency between wx and S10 was estimated to be 0.15%. According to this value, S10 is expected to be about 50-kb apart from Wx locus, as lcM in rice genome is assumed to be 300-kb length. We are now attempting an isolation of S10 gene by map based cloning.

A sophisticated cloning method using bacterial artificial chromosome (BAC) provides us a tool to undertake molecular analysis of relatively large size (more than 100kb) of genomic DNA (Zhang et al. 1996: Nakamura et al. 1997). To isolate S10, we constructed 300-kb contig with Wx gene from rice BAC library developed by Nakamura et al. ( 1997). Initially, wx gene was used as a probe, and 7 independent clones were identified (Fig. 1 ). The relative locations of these clones were determined by either the outermost end probes produced by TAIL-PCR or the comparisons of HindIII restriction fragments in each clone. Two secondary probes, the 4104 and the 6189 fragments, were selected from single-copy regions near both the ends of the primary contig. These probes added new fragment in each side of the primary contig. The resultant contig covers a range of about 300-kb including the Wx region. It is large enough to start the effort for the isolation of S10 gene. We are constructing its subclones to make a detailed map for cDNAs prepared from developing anthers in T65wx. Furthermore, we are trying to find recombinants between wx and S10 in a large number of the F2 population from BC3F1 in order to obtain the precise location of 570 gene.

References

Nakamura, S., S. Asakawa, N. Ohmido, K. Fukui, N. Shimizu and S. Kawasaki, 1997. Construction of an 800-kb contig in the near-centromeric region of the rice blast resistance gene Pi-ta2 using a highly representative rice BAC library. Mol. Gen. Genet. 254: 611-620. 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.

Research Notes 123

Zhang. H.B., S. Choi, S.S. Woo, Z. Li and R.A. Wing, 1996. Construction and characterization of two rice bacterial artificial chromosome libraries from the parents of a permanent recombinant inbred mapping population. Mol. Breed. 2: 11-24.