**AWARDS ISSUED PRIOR TO JANUARY 20, 2025, WERE FUNDED UNDER PREVIOUS ADMINISTRATIONS AND MAY NOT REFLECT THE PRIORITIES AND POLICIES OF THE CURRENT ADMINISTRATION.** BEFORE THE INTRODUCTION OF A DEVASTATING FUNGAL PATHOGEN, CHESTNUT (CASTANEA) SPECIES WERE ECOLOGICALLY KEYSTONE AND PROVIDED HIGH-VALUE TIMBER AND FOOD PRODUCTS IN EASTERN NORTH AMERICA. CONSIDERABLE ADVANCEMENTS IN GENETIC RESOURCES OVER THE LAST 15 YEARS HAVE BROUGHT US CLOSE TO THE BROAD REALIZATION OF CHESTNUT ACROSS THIS LANDSCAPE. AS IMPROVEMENT PROGRAMS CONTINUE ADVANCED BREEDING SELECTIONS AND POPULATIONS, INCREASED CAPACITY IS NEEDED TO REVEAL PREDICTIVE GENOMIC REGIONS AND MARKERS. CHESTNUT IMPROVEMENT, WHETHER FOR NUT PRODUCTION, RESTORATION, OR TIMBER, IS BASED ON MULTIVARIATE SELECTION CRITERIA AND ENVIRONMENTS, WHERE EXCEPTIONALLY LARGE BREEDING GENERATIONS ARE DEVELOPED AND DECENTRALIZED ACROSS REGIONS. ROBUST MARKER-ASSISTED SELECTION AND GENOMIC PREDICTION WILL ACCELERATE PROGRAM EFFICIENCY AND PRECISION.OUR OVERALL GOAL FOR THIS PROPOSAL IS TO ACCELERATE MARKER-TRAIT ASSOCIATION STUDIES IN CHESTNUT. WITH GROWING GENOMIC RESOURCES, AND DECLINING ILLUMINA SEQUENCING COSTS, THE BOTTLENECKS TO THESE STUDIES ARE NOW IN SAMPLE PREPARATION. ROBOTIC TECHNOLOGY ADAPTED TO CHESTNUTS CAN ACCELERATE THE RATE OF DNA EXTRACTION AND LIBRARY PREPARATION FOR SEQUENCING, ENABLING GREATER MARKER AND PUTATIVE GENE DISCOVERY. THIS PROJECT FOCUSES ON THREE OBJECTIVES: (1) IMPLEMENT ROBOTIC TECHNOLOGY TO ACCELERATE DNA EXTRACTION AND GENOTYPING-BY-SEQUENCING LIBRARY PREPARATION, 2) IDENTIFY QUANTITATIVE TRAIT LOCI AND DEVELOP GENOMIC PREDICTION IN CHINESE CHESTNUT AND HYBRID BI-PARENTAL FAMILIES FOR (A) PHENOLOGICAL TRAITS, AND (B) GALL WASP AND PHYTOPHTHORA ROOT ROT RESISTANCE, AND 3) TARGET CONSERVATION OF CLIMATE-ADAPTIVE GENETIC DIVERSITY BY COMPARING AMERICAN BACKCROSS POPULATIONS TO RANGE-WIDE WILD TYPE AMERICAN CHESTNUTS.THIS PROJECT SUPPORTS EXTENSIVE CHESTNUT BREEDING EFFORTS THAT SEEK TO RELEASE IMPROVED PLANT MATERIALS BROADLY THROUGHOUT THE EAST AND MIDWEST US. THESE EFFORTS ARE CHALLENGED BY COMPLEX SELECTION CRITERIA AND THE NEED FOR EXCEEDINGLY LARGE POPULATIONS WITH A LARGE PHYSICAL FOOTPRINT AND A LONG JUVENILE PERIOD. NEVERTHELESS, DEDICATED INSTITUTIONS AND STAKEHOLDERS HAVE PROGRESSED POPULATIONS THAT CAN NOW BE EXPLOITED BY MODERN TECHNOLOGY AND GENETICS. BROADER IMPACTS INCLUDE ENGAGING STUDENTS IN BREEDING AND GENETICS DISCIPLINES, SHARING DISCOVERIES WITH STAKEHOLDER GROUPS, CONNECTING RESEARCHERS FROM MULTIPLE BREEDING PROGRAMS, AND IMPLEMENTING SELECTION TOOLS THAT DIRECTLY LEAD TO THE ADOPTION OF IMPROVED GERMPLASM.