Reverse Mutations in Pigmentation Induced by Sodium Azide in the IR64 Rice Variety

Pigmentation in rice is due mainly to the accumulation of anthocyanins. Five color mutant lines, AZ1701, AZ1702, AZ1711, AZ1714, and AZ1715, derived from the sodium azide mutagenesis on the non-pigmented IR64 variety, were applied to study inheritance modes and genes for pigmentation. The mutant line AZ1711, when crossed with IR64, displays pigmentation in various tissues, exhibiting a 3:1 pigmented to non-pigmented ratio in the F₂ progeny, indicating a single dominant locus controlling pigmentation. Eighty-four simple sequence repeat (SSR) markers were applied to map the pigment gene using 92 F₂ individuals. RM6773, RM5754, RM253, and RM2615 markers are found to be linked to the color phenotype. RM253 explains 78% of the phenotypic variation, implying linkage to the pigmentation gene(s). Three candidate genes, OsC1 (MYB), bHLH, and 3GT, as anthocyanin biosynthesis-related genes, were identified within a 0.83 Mb region tightly linked to RM253. PCR cloning and sequencing revealed 10 bp and 72 bp insertions in the OsC1 and 3GT genes, respectively, restoring pigmentation as in wild rice. The 72 bp insertion is highly homologous to a sequence of Ty1-Copia retrotransposon and shows a particular secondary structure, suggesting that it was derived from the transposition of Ty1-Copia in the IR64 genome.