41

The allelic diversity which results from mutations at the coding and noncoding regions of a single locus has been proposed as an important source for phenotypic diversity

We intend to construct a fine map of the wx locus as a first step to compare the regulatory and structural regions. The wx locus on chromosome 6 specifies the synthesis of amylose in the triploid endosperm and the haploid pollen. The wild-type produces amylose and amylopectin whereas wx mutants lack amylose. The frequency of intragenic recombination can be examined in a large number of pollen grains stained with I₂-KI. This advantage makes it possible to detect recombination events within locus occurring at extremely low frequencies. Pollen analysis has been used to construct a fine structure genetic map in the wx locus of maize (Nelson 1962) and rice (Li et al. 1968). This report shows a preliminary result of the two mutation sites detected on the restriction map of the wx locus. We first constructed a fine map based on pollen analysis using spontaneous and induced wx mutants. The frequency of recombinants at wx locus was estimated in heteroallelic wx plants based on more than 10 x 10" pollen grains counted per crosscombination. Fig. 1A shows only a core of the map indicating a total length of 0.189cM within the locus.

To get information on the mutation sites on the physical map of the wx locus, we attempted to locate the mutation site of a -rays induced wx mutant (M-g A) which might have a detectable deletion. Total genomic DNAs from the mutant and the parent were analyzed by southernblotting by using a BamHI fragment of 0.75kb as probe which includes exon 6 to exon 9. Digestion with HindIII revealed that M-g A showed a hybridized

Research Notes 121

fragment of 11kb while a conserved length of 15kb fragment was detected in the parent. suggesting a deletion of about 4kb in M-g A. Comparisons of restriction sites (BamHI, ApaI and EcoRI) revealed that the upstream end of the deletion was present between BamHI site in exon 9 and ApaI site in exon 10 (Fig. 1B and C). Intragenic recombination was examined to locate the deletion on a fine map based on pollen analysis. The results revealed that the two EMS induced wx mutants (M-c and M-d) showed a close linkage with the deletion mutant since M-c showed linkage intensities of 0.005cM and 0.011 cM in crosses with M-g A and M-d, respectively.

The mutation sites in M-c is expected to be located upstream of the deletion because the downstream region from exon 10 was deleted in M-g A. Therefore, the region around exon 10 was sequenced and compared between the two mutants and the original. So far, a base pair substitution causing an amino acid alteration (alanine to valine) was detected within exon 9 of M-d although the mechanism causing inactivation of the gene product is uncertain. The distance on physical map might support a close linkage based on pollen analysis, suggesting that the mutation site could be estimated from pollen analysis to some extent.

The genetic distance based on recombination frequency greatly varies depending on chromosomal positions. The upstream sequence of about 3.5kb in the wx was sufficient to direct the gene expression in transgenic rice plants (Hirano et al. 1995), suggesting that the physical length of the wx locus might be less than 10kb. If the assumed physical length corresponded to 0.189cM observed in this study, lcM is expected to be less than 53kb. To determine the whole range of the wx locus, further studies are needed to identify the mutation sites of various wx alleles.

Hiranu, H.-Y. and Y. Sano, 1991. Molecular characterization of the waxy locus of rice (0ryzau sativa). Plant

Li. H.W., H.P. Wu, L. Wu and M.Y. Chu. 1968. Further studies of the interlocus recombination of the

Nelson, O.E.. 1962. The waxy locus in maize. I. Interlocus recombinations frequency estimated by pollen and