Generation and Characterization of Novel Low-Amylose Rice Germplasm via Prime Editing of the Wx Promoter.

Amylose content (AC) is primarily regulated by the Wx gene and acts as a key determinant of rice eating and cooking quality. Natural low-AC alleles are limited in fine-tuning effects across genetic backgrounds, and studies exploiting natural allelic variation are nearing saturation. Here, we employed prime editing (PE) to generate precise promoter deletions near the transcription start site (TSS) of Wx in japonica rice Huaidao 5, obtaining two homozygous mutant lines: Wx5d harboring the designed 5 bp deletion and Wx32d carrying an unintended 32 bp promoter deletion. Compared with the wild-type, AC was significantly reduced by 3.95 and 2.77 percentage points, respectively, with no obvious changes in grain transparency or the eight major agronomic traits evaluated in this study. The edited lines showed higher taste values measured by a Satake cooked rice taste analyzer, with Wx32d achieving the highest score (76.6 versus 72.6 for the wild-type). Gel consistency was correspondingly longer in both edited lines, and Rapid Visco Analyser (RVA) pasting profiles showed higher breakdown viscosity and more negative setback, most prominently in Wx32d. Reduced AC was accompanied by decreased Wx transcript accumulation and lower granule-bound starch synthase I (OsGBSSI) protein abundance. These two alleles were absent in 3032 natural rice accessions. This study illustrates that PE-mediated promoter editing can efficiently generate valuable low-amylose rice germplasm with minimal agronomic trade-offs among the traits evaluated under the tested environment, providing valuable intermediate materials for rice quality breeding.