A Genetic Framework for Enhancing Resistant Starch in Cooked and Retrograded Rice via Combined Effect of Amylose and Lipid Biosynthesis

Resistant starch (RS) is a functional polysaccharide of interest for its potential physiological effects, including glycemic control. However, the genetic regulation of its RS5 subtype—an amylose–lipid complex prevalent in cooked rice—remains incompletely understood. In this study, 101 rice accessions with varying apparent amylose content (AAC) were evaluated. A targeted-gene association study was performed to systematically dissect the genetic effects of starch and lipid biosynthesis-related genes on RS content in cooked rice (RSc) and retrograded rice (RSr). The associations of crude fat content (CFC) and eating and cooking qualities with RSc and RSr were also explored. The results demonstrated that Wx, PAL6, SSIVb, and SSIIa were all involved in regulating different RS types. Among these, Wx and PAL6, along with their statistical interaction, played dominant roles in the genetic regulation of RSc and RSr. The Wxa, Wx-T, and PAL6-2 alleles had significant positive effects on high RS accumulation. Besides that, Hap1 (Wxa–Wx-T–PAL6-1), Hap2 (Wxa–Wx-T–PAL6-2), and Hap4 (Wxa–Wx-C–PAL6-2) were significantly associated with elevated RSc and RSr. Hap1 and Hap4 demonstrated promising potential for quality breeding, being associated with high gel consistency and low pasting viscosity, respectively. In addition, RSc and RSr were significantly positively correlated with AAC but negatively correlated with CFC. Notably, we propose a plausible hypothesis that RS5 formation may exert an inhibitory effect on RS3 accumulation, and that this effect may be relieved by defatting treatment. Collectively, these findings provide a theoretical framework and valuable allelic resources for designing rice varieties with improved RSc and RSr.