Determination of Starch Size Distribution in Single Grains (SSDSG): A Novel High-Efficiency Method for Characterizing Starch Granule Morphology in Wheat

Wheat starch granule size distribution is a critical determinant of processing quality and end-use functionality. However, conventional methods for characterizing starch granules are labor-intensive, require relatively large sample quantities, and may preferentially lose small B-type granules during starch extraction. To address these limitations, we developed SSDSG (Determination of Starch Size Distribution in Single Grains), a novel, high-throughput, image-based method for in situ and non-destructive phenotyping of starch granule size distribution at the single grain level. A morphological correction factor (K~=0.4113) enabled statistical conversion to volume equivalent measurements, and the method showed a significant positive correlation with the Coulter counter reference method (R2 = 0.55) across 20 wheat varieties. SSDSG characterized the temporal dynamics during grain filling and intra-spike spatial heterogeneity, providing technical support for dissecting grain development and analyzing superior and inferior grains. High-throughput screening of 1667 EMS-mutagenized lines revealed broad variation in B-type starch proportion in total starch (84.01–98.69%), which is significant for further genetics and phenotypic analysis. Moreover, the non-destructive workflow allows a single grain being used for both phenotyping and propagation. The top 10% of the 204 F2 breeding lines, with highest B-type starch proportion, were selected and propagated for further genetic analysis. Finally, characterization of barley and 24 diverse-ploidy wheat accessions revealed that tetraploid wheat had relatively lower B-type starch proportions. This is significant for understanding starch traits evolution during wheat polyploidization. Overall, SSDSG provides a practical high-throughput platform for single-grain starch phenotyping and quality-oriented wheat breeding.