Creation of high‐resistant starch rice through systematic editing of amylopectin biosynthetic genes in rs4

Summary Resistant starch (RS) is a special kind of starch with beneficial effects on obesity, type 2 diabetes and other chronic complications. Breeding high‐RS rice varieties is considered a valuable way to improve public health. However, most rice cultivars only contain an RS level lower than 2% in cooked rice, and cloning of RS genes is critical to improve RS levels in rice. The loss of function of Starch Synthases IIIa ( SSIIIa ) and SSIIIb , two amylopectin biosynthetic genes, could elevate RS levels up to 10%. Here, we performed a systematic genetic study of 14 amylopectin biosynthetic genes in the ssIIIa ssIIIb double mutant via genome editing, and investigated their effects on RS formation, the eating quality and grain yield. The results showed that deficiency in SSIIa , SSIVb or ISA2 under the ssIIIa ssIIIb background could each elevate RS content to above 14%, and the quadruple mutants of sbeI sbeIIb ssIIIa ssIIIb and sbeI ssIVb ssIIIa ssIIIb could further increase RS levels to over 18%. Furthermore, the eating quality of cooked rice and grain yield decreased along with the elevated RS contents, showing a trade‐off among these traits. In these mutants, ssIIIa ssIIIb showed the balanced performance of RS and grain yield. This study provides insights into RS biosynthesis with a series of RS genes in the amylopectin biosynthesis pathway and practical strategy to breed high‐RS rice varieties with balanced performance.