Promoter editing of starch branching enzyme IIb and granule-bound starch synthase I balances resistant starch content and amylose content in rice

Increasing attention is being focused on the glycemic index (GI) of daily food for humans, and the resistant starch content (RSC) is an important indicator of GI for starch-rich staple foods. In recent years, some studies revealed that the loss function of single or multiple key enzymes in the primary pathway of starch synthesis substantially increases RSC in rice, such as starch branching enzyme IIb (BEIIb) and soluble starch synthase IIIa (OsSSIIIa). However, a noteworthy negative characteristic of these high RSC mutants is the substantially increased amylose content (AC). AC as a major determinator of rice eating quality, must not be higher than an acceptable limit for most consumers. To solve this problem, in this study, we adopted two promoter editing (PE) editing strategies to develop rice germplasms with a better balance of RSC and AC: one is to edit the promoter of BEIIb in a low AC rice variety, another is to edit the promoter of Waxy (Wx) gene in a BEIIb loss of function mutant. Using AC[≤]20%, which is the range of premium quality rice in China as a criteria, we finally obtained 2 homozygous lines with significantly increased RSC ([≥]5%) in the NG46 background by promoter editing of BEIIb and 1 homozygous line in the YouTang2 (YT2, a BEIIb mutant) background by promoter editing of Wx gene. Further analysis revealed that AC and the amount of long-chain branches of amylopectin are positively correlated with RSC in the population of BEIIb PE lines. However, unexpectedly, the Wx PE-line with substantially decreased AC (17.7%) also showed significantly increased RSC (16.9%). Our study not only produces useful germplasms for the high RSC rice breeding in the future but also provides an insight into understanding the relationship between AC and RSC in defective BEIIb rice.