** AWARDS ISSUED PRIOR TO JANUARY 20, 2025, WERE FUNDED UNDER PREVIOUS ADMINISTRATIONS AND MAY NOT REFLECT THE PRIORITIES AND POLICIES OF THE CURRENT ADMINISTRATION.** ALFALFA, MEDICAGO SATIVA L., IS THE MOST IMPORTANT FORAGE CROP IN THE USA. HOWEVER, BIOMASS YIELD IMPROVEMENT HAS BEEN SLOW. WE PROPOSE TO INVESTIGATE DIPLOID ALFALFA FROM THREE INTERRELATED ANGLES TO BETTER UNDERSTAND THE GENETIC BASIS OF BIOMASS YIELD AND IDENTIFY POSSIBLE GENETIC APPROACHES TO FUTURE YIELD IMPROVEMENT. FIRST, WE WILL DEVELOP HIGHLY ACCURATE, HAPLOTYPE RESOLVED GENOME SEQUENCES FOR EIGHT DIPLOID GENOTYPES FROM BOTH ALFALFA SUBSPECIES AND RESEQUENCE AN ADDITIONAL EIGHT GENOTYPES TO GAIN FURTHER INFORMATION ON STRUCTURAL VARIATION AMONG THESE INDIVIDUALS. OUR SEQUENCES WILL INCLUDE THE FIRST WHOLE GENOME SEQUENCE OF THE YELLOW-FLOWERED SUBSPECIES FALCATA. SECOND, WE WILL INVESTIGATE THE BIOLOGICALLY INTERESTING PHENOMENON OF SEGREGATION DISTORTION TOWARD EXCESS HETEROZYGOSITY. WE WILL SURVEY MORE THAN 20 SEGREGATING POPULATIONS FOR EXCESS HETEROZYGOSITY, DESCRIBING SIMILARITIES AND DIFFERENCES BETWEEN THE REGIONS WITH DISTORTION. THIRD, WE WILL DEVELOP SEVERAL LARGE SEGREGATING POPULATIONS THAT EXPRESS HETEROZYGOTE EXCESS IN SOME GENOMIC REGIONS AND USE THEM TO GENETICALLY MAP QUANTITATIVE TRAIT LOCI (QTL) FOR YIELD AND YIELD COMPONENTS. FINALLY, WE WILL LOOK FOR COINCIDENCE OF QTL, LOCI WITH DISTORTED SEGREGATION, AND STRUCTURAL VARIANTS IN THE PARENTAL GENOMES TO HELP EXPLAIN VARIATIONS IN YIELD AND PROPOSE MECHANISMS FOR HOW THESE LOCI CONTROL YIELD. THESE RESULTS WILL LEAD TO GENETIC MARKERS TO ASSIST SELECTION FOR YIELD AND POTENTIALLY STRATEGIES TO MORE EFFECTIVELY SELECT FOR HIGHER YIELD.