Multispectral imaging and automated analysis for quantifying grain quality to reveal known and potential novel alleles affecting grain traits in wheat

To accelerate the pace of wheat ( Triticum aestivum L.) improvement worldwide, desired seed-level characteristics and seed quality receive a growing attention as they directly impact early seedling establishment, seed longevity, and grain quality. Nevertheless, the throughput and accuracy of seed-level phenotyping and analysis have become a key limiting factor in this research domain, requiring new solutions to relieve this bottleneck. In this study, we first combined automated multispectral seed imaging (MSI; i.e. the VideometerLab 4 and Autofeeder systems) with a variety of machine learning and computer vision techniques to establish a high-throughput pipeline to analyse wheat seeds. Then, using 493 lines selected from the Niab Diverse MAGIC (NDM) population, we applied the pipeline to segment individual seeds from MSI seed-lot images. This enabled us to perform seed-level measurement of sixteen morphological (e.g. seed size, length, width, and roundness) and spectral traits, ranging from ultraviolet (i.e. 375 nm, correlating with crude protein) to near-infrared (e.g. 975 nm, for assessing water content) wavelengths. After verifying these seed quality related traits (R2 ≥ 0.949; p < 0.001), we applied genome-wide association studies (GWAS) to link the computationally derived traits to genetic loci and identified eleven significant loci. Some of the loci were previously reported, with two unknown loci valuable for further assessment. Taken together, we believe this integrated MSI analysis pipeline provides a powerful solution for seed research and crop improvement in wheat, enabling us to bridge MSI, seed-level analysis, and genetic mapping to assess seed morphology, seed quality, and their underlying genetic architectures effectively.