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Starch quality studies over the decades highlighted the immense health benefits of resistant starch (RS), but still much is needed to elucidate the science behind its inherent formation. Till date, there is no report to establish the relationship between inherent RS content and pullulanase (PUL) activity in any of the crops. In this study, we emphasize the novel role of debranching enzyme, PUL towards inherent RS formation, using rice as a model crop. Biochemical analysis of 51 rice genotypes for amylose content (AC) revealed a good amount of variation ranging from 7.03 to 37.33%. Further, hierarchical clustering which resulted in 11 genotypes of varying RS (0.33–2.7%), highlighted medium dependency towards amylose and low dependency towards amylopectin content. The discrete differences in microstructure, unimodal distribution and tight packing of starch granules observed in higher RS genotype indicated …