Loss of function of SSIIIa and SSIIIb coordinately confers high RS content in cooked rice.

The sedentary lifestyle and refined food consumption significantly lead to obesity, type 2 diabetes, and related complications, which have become one of the major threats to global health. This incidence could be potentially reduced by daily foods rich in resistant starch (RS). However, it remains a challenge to breed high-RS rice varieties. Here, we reported a high-RS mutant rs4 with an RS content of ~10.8% in cooked rice. The genetic study revealed that the loss-of-function SSIIIb and SSIIIa together with a strong Wx allele in the background collaboratively contributed to the high-RS phenotype of the rs4 mutant. The increased RS contents in ssIIIa and ssIIIa ssIIIb mutants were associated with the increased amylose and lipid contents. SSIIIb and SSIIIa proteins were functionally redundant, whereas SSIIIb mainly functioned in leaves and SSIIIa largely in endosperm owing to their divergent tissue-specific expression patterns. Furthermore, we found that SSIII experienced duplication in different cereals, of which one SSIII paralog was mainly expressed in leaves and another in the endosperm. SSII but not SSIV showed a similar evolutionary pattern to SSIII. The copies of endosperm-expressed SSIII and SSII were associated with high total starch contents and low RS levels in the seeds of tested cereals, compared with low starch contents and high RS levels in tested dicots. These results provided critical genetic resources for breeding high-RS rice cultivars, and the evolutionary features of these genes may facilitate to generate high-RS varieties in different cereals.