Determining the Genetic Architecture and Breeding Potential of Quality Traits in Alfalfa (Medicago sativa L.) Through Genome-Wide Association Study and Genomic Prediction

Alfalfa (Medicago sativa L.) is a high-nutritive-value forage crop that provides livestock with abundant protein and essential nutrients. Breeding elite cultivars with superior quality has become a major goal in modern alfalfa improvement. This study systematically evaluated 12 quality-related traits under field conditions using a diverse panel of 176 alfalfa accessions and investigated the genetic basis underlying these traits. Phenotypic analysis revealed variability across all traits, with coefficients of variation ranging from 2.56% to 15.72%. Based on multi-trait clustering analysis, 16 accessions with overall superior quality were identified. Genome-wide association studies (GWAS) detected 45 significant single nucleotide polymorphisms (SNPs) and 12 structural variants (SVs). Within the associated genomic regions, eight candidate genes were prioritized. RT-qPCR validation indicated that three of these genes (Msa.H.0301430, Msa.H.0290550, and Msa.H.0313490) negatively regulate quality traits, while one gene (Msa.H.0479570) acts as a positive regulator. Haplotype analysis further revealed a positive correlation between the number of favorable haplotypes and phenotypic performance. Genomic prediction (GP) achieved accuracies ranging from 0.71 to 0.86 for the traits when incorporating the top 5000 SNPs identified from GWAS. This study provides valuable insights into the genetic architecture of quality-related traits in alfalfa and lays a solid foundation for future molecular design breeding.