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.
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.