Pan-genomic analysis highlights genes associated with agronomic traits and enhances genomics-assisted breeding in alfalfa

Alfalfa (Medicago sativa L.), a globally important forage crop, is valued for its high nutritional quality and nitrogen-fixing capacity. Here, we present a high-quality pan-genome constructed from 24 diverse alfalfa accessions, encompassing a wide range of genetic backgrounds. This comprehensive analysis identified 433,765 structural variations and characterized 54,002 pan-gene families, highlighting the pivotal role of genomic diversity in alfalfa domestication and adaptation. Key structural variations associated with salt tolerance and quality traits were discovered, with functional analysis implicating genes such as MsMAP65 and MsGA3ox1. Notably, overexpression of MsGA3ox1 led to a reduced stem-leaf ratio and enhanced forage quality. The integration of genomic selection and marker-assisted breeding strategies improved genomic estimated breeding values across multiple traits, offering valuable genomic resources for advancing alfalfa breeding. These findings provide insights into the genetic basis of important agronomic traits and establish a solid foundation for future crop improvement.