BackgroundSpike is the grain-bearing organ in cereal crops, which is a key proxy indicator determining the grain yield and quality. Machine learning methods for image analysis of spike-related phenotypic traits not only hold the promise for high-throughput estimating grain production and quality, but also lay the foundation for better dissection of the genetic basis for spike development. Barley (Hordeum vulgare L.) is one of the most important crops globally, ranking as the fourth largest cereal crop in terms of cultivated area and total yield. However, image analysis of spike-related traits in barley, especially based on CT-scanning, remains elusive at present.ResultsIn this study, we developed a non-invasive, high-throughput approach to quantitatively measuring the multitude of spike architectural traits in barley through combining X-ray computed tomography (CT) and a deep learning model (UNet). Firstly, the spikes of 11 barley accessions, including 2 wild barley, 3 landraces and 6 cultivars were used for X-ray CT scanning to obtain the tomographic images. And then, an optimized 3D image processing method was used to point cloud data to generate the 3D point cloud images of spike, namely ‘virtual’ spike, which is then used to investigate internal structures and morphological traits of barley spikes. Furthermore, the virtual spike-related traits, such as spike length, grain number per spike, grain volume, grain surface area, grain length and grain width as well as grain thickness were efficiently and non-destructively quantified. The virtual values of these traits were highly consistent with the actual value using manual measurement, demonstrating the accuracy and reliability of the developed model. The reconstruction process took 15 min approximately, 10 min for CT scanning and 5 min for imaging and features extraction, respectively.ConclusionsThis study provides an efficient, non-invasive and useful tool for dissecting barley spike architecture, which will contribute to high-throughput phenotyping and breeding for high yield in barley and other crops.