A Single‐Nucleotide Substitution in the <i>Pullulanse</i> Gene Reduced Starch Digestibility in Low‐Amylose Rice due to a Linearized Chain of Amylopectin

ABSTRACT Processed rice, known for its high glycemic index (GI) value, is considered a risk component for non‐communicable diseases, like type 2 diabetes. People consuming milled rice in their staple diet, along with sedentary lifestyles, are more prone to modern lifestyle‐related diseases like obesity and type 2 diabetes. Rice with high resistant starch (RS) and low GI digests slowly, resulting in lower postprandial blood glucose levels. Apart from amylose content (AC) in rice grain starch, linear chains of amylopectin also impact the rate of starch digestibility. To understand the effect of amylose and amylopectin linear chains on the rate of starch digestibility, biochemical analysis was done to estimate the GI, RS, and AC in 110 milled rice genotypes. A significant variation was observed in GI (54.76–67.00), RS (0.40%–2.19%), and AC (4.82%–25.52%). Out of 110, three rice genotypes (IG 72, IG 23, and IG 40) were selected for the molecular analysis of two genes, granule bound starch synthase I ( GBSSI ) and pullulanase ( PUL ), based on their contrasting AC, RS, and GI values. A low GI (54.76) rice IG 23 was identified, where a single nucleotide substitution (“G” instead of “C”) in the endosperm‐specific motif (AACA) under the promoter sequence of PUL gene was found. This mutation most likely leads to overexpression of the PUL gene in IG 23, enhancing amylopectin branch trimming and accumulation of longer amylose‐like linear structures, thus lowering the GI value. However, the highest expression of GBSSI and PUL genes was observed in the mid stage of grain development in all the genotypes. These results highlight the importance of identifying rice genotypes with low starch digestibility, which may be suitable for consumption by diabetics or people prone to diabetes, and developing molecular markers for a breeding program to develop such rice cultivars.