Pre-breeding in alfalfa germplasm develops highly differentiated populations, as revealed by genome-wide microhaplotype markers.

Plant genebanks contain large numbers of germplasm accessions that likely harbor useful alleles or genes absent in commercial plant breeding programs. Broadening the genetic base of commercial alfalfa germplasm with these valuable genetic variations can be achieved by screening the extensive genetic diversity in germplasm collections and enabling maximal recombination among selected genotypes. In this study, we assessed the genetic diversity and differentiation of germplasm pools selected in northern U.S. latitudes (USDA Plant Hardiness Zone 7 or below) originating from Eurasian germplasm. The germplasm evaluated included four BASE populations (C0) from different geographical origins (Central Asia, Northeastern Europe, Balkans-Turkey-Black Sea, and Siberia/Mongolia), 20 cycle-one populations (C1) derived from each of the four BASE populations selected across five locations in the U.S. and Canada, and four commercial cultivars. Using a panel of 3,000 Diversity Array Technologies (DArTag) marker loci, we retrieved 2,994 target SNPs and approximately 12,000 microhaplotypes. Microhaplotypes exhibited higher genetic diversity values than target SNPs. Principal component analysis and discriminant analysis of principal components revealed significant population structure among the alfalfa populations based on geographical origin, while the check cultivars formed a central cluster. Inbreeding coefficients (F) ranged from - 0.1 to 0.006, with 27 out of 28 populations showing negative F values, indicating an excess of heterozygotes. Interpopulation genetic distances were calculated using Rho pairwise distances (F adapted for autotetraploid species) and analysis of molecular variance (AMOVA) parameters. All BASE populations showed lower Rho values compared to C1 populations and check cultivars. AMOVA revealed that most of the genetic diversity was among individuals within populations, especially in BASE populations (92.7%). This study demonstrates that individual plants in BASE populations possess high genetic diversity, low interpopulation distances, and minimal inbreeding, characteristics that are essential for base-broadening selection. The populations developed in this project serve as valuable sources of novel alleles for North American alfalfa breeding programs, offering breeders access to diverse, regionally adapted pools for improving various alfalfa traits.