Medicago super-pangenome reveals adaptive advantages and evolutionary constraints in autotetraploid alfalfa.

The genetic basis for the adaptive advantages of polyploids over their diploid relatives remains poorly understood. To address this knowledge gap, we generate a haplotype-resolved autotetraploid alfalfa (Medicago sativa subsp. sativa) genome and construct a super-pangenome from 13 genomes across seven Medicago taxa. We discover substantial gene content variation in alfalfa, with only 20.1% of genes present on all four haplotypes. Within this group, 53.3% are core genes conserved across the Medicago genus, which we term 'tetra-copy core genes'. We find these genes are significantly enriched in climate-adaptation-associated genes (1.60-fold) and stress-responsive differentially expressed genes (1.61-fold). Paradoxically, they also carry a high genetic burden, with 80.1% of deleterious variants located in coding regions. Indeed, overexpressing a representative tetra-copy core gene, the glycine decarboxylase (MsGDC), improves both biomass and nitrogen use efficiency, despite its high genetic burden. Our study reveals the trade-off between adaptation and evolutionary constraints mediated by tetra-copy core genes, facilitating polyploid genetics and alfalfa breeding.