A naturally occurring functional allele of the rice waxy locus has a GT to TT mutation at the 5' splice site of the first intron

In cultivated rice two wild-type alleles, Wx^a and Wx^b, predominate at the waxy locus, which encodes granule-bound starch synthase. The activity of Wx^a is 10-fold higher than that of Wx^b at the level of both protein and mRNA. Wx^b has a +1G to T mutation at the 5' splice site of the first intron. Sequence analysis of Wx^b transcripts revealed that splicing occurs at the mutant AG/UU site and at two cryptic sites: the first is A/GUU, one base upstream of the original site and the second is AG/GU found approximately 100 bases upstream of the mutant splice site. We introduced single base mutations to the 5' splice sites of both Wx^a and Wx^b, fused with the gus reporter gene and introduced them into rice protoplasts. Analysis of GUS activities and transcripts indicated that a G to T mutation in Wx^a reduced GUS activity and the level of spliced RNA. Conversely, a T to G mutation of Wx^b restored GUS activity and the level of spliced RNA to that of wild-type Wx^a. These results demonstrated that the low level expression of Wx^b results from a single base mutation at the 5' splice site of the first intron. It is of interest that the Wx^b allele of rice carrying the G to T mutation of intron 1 has been conserved in the history of rice cultivation because there is a low amylose content of the seed caused by this mutation.