WSF: A Transformer-Based Framework for Microphenotyping and Genetic Analyzing of Wheat Stomatal Traits

Stomata on the leaves of wheat serve as important gateways for gas exchange with the external environment. Their morphological characteristics, such as size and density, are closely related to physiological processes like photosynthesis and transpiration. However, due to the limitations of existing analysis methods, the efficiency of analyzing and mining stomatal phenotypes and their associated genes still requires improvement. To enhance the accuracy and efficiency of stomatal phenotype traits analysis and to uncover the related key genes, this study selected 210 wheat varieties. A novel semantic segmentation model based on transformer for wheat stomata, called Wheat Stoma Former (WSF), was proposed. This model enables fully automated and highly efficient stomatal mask extraction and accurately analyzes phenotypic traits such as the length, width, area, and number of stomata on both the adaxial (Ad) and abaxial (Ab) surfaces of wheat leaves based on the mask images. The model evaluation results indicate that coefficients of determination (R²) between the predicted values and the actual measurements for stomatal length, width, area, and number were 0.88, 0.86, 0.81, and 0.93, respectively, demonstrating the model's high precision and effectiveness in stomatal phenotypic trait analysis. The phenotypic data were combined with sequencing data from the wheat 660 K SNP chip and subjected to a genome-wide association study (GWAS) to analyze the genetic basis of stomatal traits, including length, width, and number, on both adaxial and abaxial surfaces. A total of 36 SNP peak loci significantly associated with stomatal traits were identified. Through candidate gene identification and functional analysis, two genes-TraesCS2B02G178000 (on chromosome 2B, related to stomatal number on the abaxial surface) and TraesCS6A02G290600 (on chromosome 6A, related to stomatal length on the adaxial surface)-were found to be associated with stomatal traits involved in regulating stomatal movement and closure, respectively. In conclusion, our WSF model demonstrates valuable advances in accurate and efficient stomatal phenotyping for locating genes related to stomatal traits in wheat and provides breeders with accurate phenotypic data for the selection and breeding of water-efficient wheat varieties.