Integrated GWAS and transcriptomic analyses reveal genetic variation in stomatal density contributing to water-use efficiency in strawberry
Water availability is a major constraint for intensive crops such as strawberry (Fragaria × ananassa), yet the genetic, physiological and molecular basis of water use efficiency remains poorly understood. Here, we evaluated a diverse collection of 132 accessions under well-watered (WW) and water-limited (WL) conditions (40% reduced irrigation) to dissect variation in leaf-level water use efficiency (WUEL), agronomic performance, and fruit quality. Substantial phenotypic variation was observed across 25 traits. Under WW conditions, lower stomatal density was associated with higher WUEL, yield, fruit weight, and firmness, suggesting a favorable carbon–water balance. Population structure analysis revealed that modern and exotic cultivars combine higher WUEL and agronomic performance with reduced stomatal density, suggesting an indirect selection during breeding. Genome-wide association studies detected 25 marker–trait associations for 11 traits, with all but one specific to water regime, highlighting a complex genetic architecture and important environmental effects. Transcriptomic analysis of accessions contrasting in stomatal density and WUEL identified 4837 differentially expressed genes and revealed two distinct strategies underlying water management: (1) an energy-conservative strategy characterized by reduced stomatal density and higher WUEL, enhanced ABA signaling and optimized photosynthetic electron transport; and (2) an energy-demanding strategy associated with higher stomatal density and lower WUEL, increased autophagy, activated stress-mitigating pathways and higher investment in secondary metabolism. Together, these results provide the first integrated phenotypic, genetic and molecular characterization of WUEL in octoploid strawberry and identify candidate loci and pathways, thereby establishing a foundation for improving WUEL-related traits.