Biochemical analysis of a new sugary-type rice mutant, Hemisugary1, carrying a novel allele of the sugary-1 gene.

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CONCLUSION: A novel allele of the sugary-1 rice mutant was isolated. The single amino acid change led to isoamylase activity reduction and accumulation of high-molecular-weight phytoglycogen in seeds. A new sugary rice variety with an improved seed appearance has been isolated and designated Hemisugary1. This mutant, which was derived from Japonica-type cultivar Tsugaruroman treated with sodium azide, has about half the isoamylase activity of seeds in the original Tsugaruroman. The mutant also accumulates significant phytoglycogen, albeit approximately 40% of the total phytoglycogen in the existing sugary cultivar Ayunohikari which is defective in its most isoamylase activity. The site of mutation was identified using a re-sequence of the whole genome and a cleaved amplified polymorphic sequence (CAPS) marker. The hemisugary phenotypes of the F2 progeny were entirely consistent with the results of genotyping using the CAPS marker. Segregation analysis of the F2 population showed that the hemisugary phenotype was controlled by a single recessive gene, which was produced by a G → A single nucleotide polymorphism in the sugary-1 gene, resulting in a missense mutation from glycine to aspartic acid at amino acid position 333. Zymogram showed that this amino acid replacement resulted in a decrease in isoamylase activity with a concomitant reduction in the formation of isoamylase complexes. Phytoglycogen molecules from Hemisugary1 seeds were 3.5 times larger and contained more short glucan chains than did Ayunohikari seeds. Our data provide new insights into the relationship between isoamylase structure and phytoglycogen formation.