RGB-imaged GWAS reveals the genetic basis of salt tolerance in chrysanthemum across salinity gradients

Soil salinization is a severe abiotic stressor that limits plant growth and productivity worldwide; however, the genetic basis of plant responses to salinity stress remains elusive. Here, we combined RGB image-based phenotyping with genome-wide association studies (GWAS) to dissect the phenotypic and genetic architecture of salt tolerance in a diverse panel of chrysanthemums. An RGB image-based high-throughput phenotyping framework was first established to discriminate natural variation in chrysanthemum salt tolerance. Based on the salt-tolerance index (STI) of 27 core image-derived traits (i-traits), GWAS detected 1,892 (52.4%) trait-specific and 1,716 (47.6%) multiple-trait signals under low salinity (LS; 100 mM NaCl), and 1,283 (23.3%) trait-specific and 4,215 (76.7%) multiple-trait signals under high salinity (HS; 200 mM NaCl). Cross-environment comparison identified 88 co-localized loci between LS and HS conditions, with 2,383 LS-specific and 2,163 HS-specific loci, suggesting distinct genetic basis across salinity gradients. Notably, the marked increase in multi-trait loci under HS suggested a substantial shift in the genetic architecture underlying salt-responsive phenotypes, a pattern further supported by the enrichment of HS-specific genes in endomembrane organization, cell-cycle regulation, and defense-related pathways. Given their strong pleiotropy, haplotype effects, salt-induced expression patterns, and transient functional validation, CmPME34 and CmEGR2 were identified as promising candidate targets associated with salt tolerance. This study established an integrated phenomics-to-genomics framework to dissect complex salt-stress-adaptive traits and provides valuable genetic resources for improving salt tolerance in chrysanthemum.