44. 
Organization of the 260 kb region in the vicinity of the waxy locus of rice
H. 
NAGANO’, S. KAWASAKI2, Y. KISHIMA’ and Y. SAN01
1) 
Faculty of Agriculture, Hokkaido University, Sapporo, 060-8589 Japan
2) 
National Institute of Agricultural Resources, Kan’non-dai 2-1-2, Ibaraki, 305-0856 Japan
     Recently an interest has been focused on the nature and structure of the repetitive sequences surrounding functional genes. Drastic changes in the repetitive sequences were demonstrated to correlate with genome size among maize, sorghum and rice (Chen et a!. 1998). However, the dynamic changes during the course of evolution have remained largely unknown. The sequence analyses in non-coding regions suggest that repetitive sequences tend to rapidly change during the course of evolution in contrast to extensive preservation of both gene content and gene order in the grasses. Although the importance of genome structure to gene regulation is not clear, we intend to compare homologous regions including repetitive sequences among related rice taxa. As the first step, the present study was carried out to characterize molecular organization of the 260 kb region in the vicinity of the waxy locus in rice. To grasp structural organization focused on single copy regions around the waxy locus, the 260 kb restriction fragments map constructed by using a bacterial artificial chromosome (BAC) library from the Shimokita, a Japonica cultiver (Nagano eta!. 1997, Nagano et a!. unpublished results) was used for this study.
     Overlapping 60 probes, which cover the entire 260 kb region, were prepared from HindIII fragments and/or EcoRI fragments. The probes ranging from 1.4 to 10 kb in fragment size were hybridized with HindIll-digested Shimokita total DNA. The hybridization patterns obtained were classified into four classes; the patterns indicating discrete band(s) (class 1), multiple discrete bands without smear background (class 2), discrete band(s) with smear background (class 3) and the patterns consisting of solely smear bands (class 4). The characteristics of their patterns and their distribution within the 260 kb are shown in Fig. 1. Each characteristic is interpreted as follows.
Class 1: Four segments were classified into this class. These sites showed a single or a very few bands in the Southern hybridization patterns. A distribution of the four sites appeared to be spread in the 260kb region. The length of the four segments was 17 kb in total, corresponding to 6.5% of the region. These segments should be solely present in the genome.
Class 2: About 55 kb in total length of ten segments was regarded as the class 2. Interestingly, the 15 kb Hindill fragment with the wx locus was observed as the class 2. Since this fragment contains known Tnrl elements (Bureau et a!. 1996), the smear background in the pattern of the class 2 might be caused by short repetitive elements. Alternatively, a single copy region might be flanked by repetitive sequences. The positions of the segment in this class, as well as the class 1, may contain genic regions which have sequences for functional genes.
Class 3: In this class, one site gave rise to multiple discrete bands without smear background. The patterns from the three probes showed multiple copy fragments which are dispersed in a genome. This sort of pattern was observed at a quite limited proportion (3.7%) of is genomic region.
Class 4: The class 4 is a major characteristic around the wx locus and possibly in other regions as well. About 70% of the 260 kb region showed to have strong smear background in the hybridization pattern. The segments classified into this class are considered to have two possible structures. One is a long repetitive sequence. Second is highly repetitive short sequence(s) with an unique sequence.

References

Bureau, T.E., P.C. Ronald and S.R. Wessler, 1996. A computer-based systematic survey reveals the predominance of small inverted-repeat elements in wild-type rice genes. Proc. Nati. Acad. Sci. USA 93:8524- 8529.
Chen, M. P. SanMiguel and J.L. Bennetzen, 1998. Sequence organization and conservation in sh2/al-homolo- gous regions and rice. Genetics 148: 435-443.
Nagano, H., 1. Wu, S. Kawasaki, Y.Kishima and Y. Sano, 1997. Construction of a 300-kb BAC contig containing waxy locus in Japonica rice. RON 14: 121-123.