**AWARDS ISSUED PRIOR TO JANUARY 20, 2025, WERE FUNDED UNDER PREVIOUS ADMINISTRATIONS AND MAY NOT REFLECT THE PRIORITIES AND POLICIES OF THE CURRENT ADMINISTRATION.** WINTER SQUASH AND PUMPKIN ARE AMONG THE RICHEST SOURCES OF CAROTENOIDS IN OUR DIET. UNDERSTANDING THE GENETIC AND MOLECULAR BASIS UNDERLYING CAROTENOID ACCUMULATION IN THESE CROPS, WHICH PROVIDE UP TO 250% RDA PER SERVING, IS IMPORTANT FOR IMPROVING HUMAN HEALTH AND NUTRITION. ONE GENE PAIR IN PARTICULAR, B AND L-2, HAVE LONG BEEN KNOWN TO GREATLY BOOST SQUASH CAROTENOID CONTENT. HOWEVER, THEIR IDENTITY AND MECHANISMS REMAIN UNKNOWN. GENOMIC RESOURCES HAVE EMERGED IN SQUASH ALONG WITH BREAKTHROUGHS IN UNDERSTANDING OF CHROMOPLAST DEVELOPMENT AND CAROTENOID ACCUMULATION. IT'S IMPORTANT TO CHARACTERIZE ESTABLISHED GENETIC SYSTEMS THAT UNDERLIE CAROTENOID ACCUMULATION BEYOND MODEL SPECIES, TO UNCOVER NEW PATHS TO IMPROVE THE QUALITY OF OUR FOOD. OUR OBJECTIVES ARE TO 1) UNDERSTAND THE MOLECULAR MECHANISMS OF THE B GENE IN SQUASH THAT INDUCES PRECOCIOUS CHROMOPLAST DEVELOPMENT, 2) UNRAVEL THE IDENTITY OF L-2 WHICH INTERACTS WITH B TO GREATLY INCREASE CAROTENOID ACCUMULATION BY AN ORDER OF MAGNITUDE, AND 3) CHARACTERIZE A SECOND B LOCUS IN ANOTHER SQUASH SPECIES WITH A MORE STABLE EXPRESSION. OUTCOMES AND IMPACTS WILL BE THE ELUCIDATION AND INFORMED DEPLOYMENT OF THE B AND L-2 INTERACTING GENE PAIR. THIS KNOWLEDGE WILL APPLY DIRECTLY TO INCREASE NUTRIENT DENSITY WITHIN SQUASH. IT WILL ALSO INFORM APPROACHES AND FUTURE HYPOTHESES FOR CAROTENOID BIOFORTIFICATION IN OTHER CROPS.