Genome-wide association studies (GWAS) identified novel QTLs for forage yield, stem-leaf and canopy traits of 250 half-sib alfalfa populations grown under Mediterranean conditions

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 α-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. • GWAS revealed SNPs linked to forage yield under irrigated and rainfed conditions. • Leaf and stem traits showed strong genetic associations with drought adaptation. • RGB indices and canopy temperature identified genomic regions for stress tolerance. • Candidate genes support breeding of drought-tolerant alfalfa for Mediterranean environments.