Combining RGB imaging with a two-stage deep learning method to reveal genetic variation of wheat sprouting traits

Wheat emergence rate and emergence uniformity are key indicators for evaluating seed vigor and sowing quality, and they play an important role in wheat growth and yield formation. Traditional methods for measuring emergence rate and evaluating emergence uniformity rely on manual assessment, which is inefficient, highly subjective, and unable to meet the demand for large scale, high efficiency, and precise acquisition of wheat emergence data. In this study, RGB images and a two-stage deep learning algorithm were used to extract and analyze seedling traits of 420 wheat varieties under two nitrogen levels, and the results were applied to genome wide association studies to elucidate the genetic basis. The two-stage algorithm integrates a Bidirectional Feature Pyramid Network, small object detection layer, large size image input, and FasterNet to improve detection and instance segmentation speed and accuracy. The proposed method achieved an emergence rate accuracy of 0.929, with R² = 0.914 and RMSE = 2.448 compared to manual measurements, and required less than 0.2 s per image for analysis. By employing this two-stage algorithm for processing and analysis, varieties (e.g., Gao8901 and ShiYou20) that consistently exhibited high emergence rates and uniformity under multiple nitrogen treatments were identified. Furthermore, genome-wide association study identified the major loci qEmergence rate-3A and qUniformity-6B governing seedling emergence rate and uniformity, which likely enhance wheat seedling traits by modulating energy supply or related signaling molecules. The emergence-rate and uniformity data generated by the two-stage algorithm significantly accelerated the discovery of relevant genes and enabled the identification of wheat varieties with high emergence rate and uniformity, providing valuable insights and practical references for high-quality breeding and gene mining.