Engineering waxy wheat with customized starch properties by integrated breeding and identification of key residues governing Granule‐bound starch synthase I <scp>TaGBSSI</scp> function

Abstract BACKGROUND Waxy wheat ( Triticum aestivum L.) is valuable for tailoring starch functionality, but efficient strategies for developing elite waxy lines and an understanding of the Triticum aestivum granule‐bound starch synthase I (TaGBSSI) function at the residue level remain limited. This study combined accelerated breeding with structure‐guided mutagenesis to generate waxy wheat and identify residues affecting granule‐bound starch synthase I (GBSSI) activity. RESULTS Stable BC 3 F 5 waxy wheat lines carrying null alleles of the three Wx homeologs were developed through tissue‐culture‐assisted rapid generation advancement, pollen iodine staining, and kompetitive allele‐specific PCR (KASP) marker‐assisted selection. Amylose content decreased from 242.2 g kg −1 in SM133 to 26.2–55.1 g kg −1 in the waxy lines, whereas total starch reached 609.8–749.8 g kg −1 . Selected lines showed lower sedimentation values, shorter farinograph development time and stability, and distinct Rapid Visco Analyzer (RVA) pasting profiles. Starch structural analysis showed an increased degree of branching and moderately higher relative crystallinity, while retaining an A‐type crystalline pattern and similar amylopectin chain‐length distribution. Six residues near the predicted ADP‐glucose (ADPG)‐binding pocket of TaGBSSI were selected for mutagenesis. Among them, H465R caused the strongest reduction in starch‐binding capacity, ADPG‐binding capacity, and enzymatic activity. CONCLUSION The integrated strategy generated waxy wheat germplasm with weak‐gluten and modified starch‐quality traits. The mutagenesis results identified H465 as a key residue influencing TaGBSSI substrate binding and activity, providing molecular targets for future starch‐quality improvement and enzyme engineering. © 2026 Society of Chemical Industry.