Abstract

Drought stress in Mediterranean regions limits forage availability for livestock. Alfalfa (M. sativa L.) is an important drought resistant perennial forage crop. However, its genetic drought tolerance is not well understood. Current study used genome-wide association studies (GWAS) on 250 half-sib alfalfa populations containing different genetic makeups, evaluated over three years (2021–2023), to identify drought-tolerant populations with high forage yield (FY) under irrigated and rainfed conditions in Central Chile (Cauquenes). Trials followed an ?-lattice design with two replications. Phenotyping included FY, leaf/stem traits, and high-throughput data via thermal and RGB imaging. The plants were genotyped via genotyping by sequencing (GBS) method. GWAS identified 7 significant single nucleotide polymorphisms (SNPs) for total FY, 3 for winter FY, and 9 for spring FY under irrigation, and 3 SNPs for spring FY under rainfed conditions. Leaf dry weight and specific leaf area associated with 169 under irrigated and 33 SNPs under rainfed condition respectively. The stem-to-leaf ratio, stem diameter, and stem dry weight associated with 195, 6, and 167 SNPs under irrigation, and 139, 129, and 1 under rainfed, respectively. RGB derived indices intensity and u* showed 14 and 3 SNPs under irrigation, and 172 and 93 SNPs under rainfed conditions, respectively. Canopy temperature difference (CTD) was linked to 5 SNPs under rainfed conditions. Functional annotation of significant SNPs revealed novel SNP-trait associations and candidate genes to support alfalfa breeding for improved FY under drought conditions. This study establishes a strong foundation for the integration of beneficial genomic regions into future alfalfa drought tolerant varieties. © 2025