An Integrated Framework Coupling Hierarchical Hyperspectral i-trait Screening, Gene Identification, and Functional...

Cotton is an economically pivotal fiber crop worldwide, but its growth and yield are critically limited by soil salinity. Salt tolerance is a complex quantitative trait, and conventional destructive phenotyping methods have long hindered the efficiency and accuracy of gene discovery and genetic dissection. To overcome this phenotyping bottleneck and establish a streamlined workflow from phenotype to gene, we developed an integrated framework coupling dynamic hyperspectral phenomics, hierarchical trait selection, genome-wide association study (GWAS), and functional validation, and applied it to dissect the genetic basis of salt tolerance using a natural population of 419 upland cotton accessions. We first confirmed abundant salt tolerance phenotypic diversity in the population via four core physiological traits and their phenotypic plasticity indices. Using time-series hyperspectral imaging (HSI), we extracted large-scale image-derived traits (i-traits) capturing dynamic physiological responses to salt stress. A three-tiered screening strategy integrating t-test, broad-sense heritability (H²) estimation, and multi-algorithm machine learning consensus was applied to identify core salt-responsive i-traits with biological relevance, genetic stability, and low redundancy. Using these core i-traits as phenotypic inputs for GWAS, we successfully mapped GhST7 as a key candidate gene. Functional validation via CRISPR/Cas9-mediated knockout and haplotype-specific overexpression demonstrated that GhST7 acts as a key regulator of cotton salt stress responses, and natural variation in its promoter region confers differential salt tolerance by modulating gene expression levels. Collectively, this study not only identifies a functionally verified salt tolerance gene with breeding application value, but also establishes a coherent phenomics-genomics-functional validation framework, providing an efficient paradigm for high-precision genetic dissection of complex stress-tolerance traits in crops.