Identification of SNPs in the waxy gene among glutinous rice cultivars and their evolutionary significance during the domestication process of rice

Common non-waxy (Wx) rice cultivars contain two different alleles at the waxy locus, designated Wx(a) and Wx(b), which encode different levels of granule-bound starch synthases and are hence involved in the control of endosperm amylose content. The Wx(a) allele was predominant in non-waxy indica cultivars, whereas the Wx(b) allele was common to the non-waxy japonica variety. Recently, some of the molecular mechanisms underlying the differentiation of Wx(a) from Wx(b) have been characterized. One structural difference between these two alleles was shown to be due to alternative splicing caused by a single-base substitution (AGGT to AGTT) at a donor site of the first intron within the Wx gene. In the case of waxy (wx) rice, it was not possible to distinguish whether the each wx allele was derived from Wx(a) or Wx(b) alleles by phenotypic analysis. However, we succeeded in developing a derived cleaved amplified polymorphic sequence (dCAPS) marker for the detection of the one-base splicing mutation without the need for sequencing. A mismatch primer was used to generate a restriction site in the Wx(a) allele (AGGT) but not in the Wx(b) allele (AGTT). Three hundred fifty-three waxy rice strains that are widely found in Asia were then employed for analysis using this dCAPS marker. Our findings suggested that waxy rice strains have both Wx(a)- and Wx(b)-derived alleles, but that the Wx(b)-derived allele was predominant, and its distribution was independent of indica-japonica differentiation. The wild relatives of cultivated rice all possessed the AGGT allele. It was concluded that the waxy mutations, and the corresponding rice cultivation, originated from japonica during the evolution and domestication process of rice and was preferentially selected by most Asian peoples.