THIS PROJECT WILL GENETICALLY IMPROVE SEED SIZE AND SEED OIL CONTENT OF FIELD PENNYCRESS (THLASPI ARVENSEL.; PENNYCRESS) FOR ITS USE AS A NEW WINTER ANNUAL CASH COVER CROP FOR THE U.S. MIDWEST. PENNYCRESS IS A WINTER HARDY COVER CROP THAT PROVIDES ECOSYSTEM SERVICES SUCH AS REDUCED SOIL EROSION AND NUTRIENT LOSS IN BETWEEN FALL CORN HARVEST AND SPRING SOYBEAN PLANTING. UNLIKE TRADITIONAL COVER CROPS, FIELD PENNYCRESS PRODUCES A MATURE OILSEED IN LATE SPRING, ALLOWING FARMERS TO HARVEST TWO CASH CROPS IN ONE YEAR. WILD-DERIVED PENNYCRESS LINES HAVE BEEN SHOWN TO YIELD 1,500 KG/HA ON AVERAGE. PENNYCRESS SEEDS CONTAIN APPROXIMATELY 33% OIL BY WEIGHT, AND THE OIL IS AN EXCELLENT BIOFUELS FEEDSTOCK. AFTER PRESSING, THE REMAINING PENNYCRESS MEAL COULD BE USED AS ANIMAL FEED. HOWEVER, DESPITE THESE ENVIRONMENTAL AND ECONOMIC BENEFITS, PENNYCRESS IS CURRENTLY LIMITED BY ITS SMALL SEED SIZE (1 MG/SEED), WHICH CAN COMPLICATE PLANTING, HARVESTING, AND HANDLING THE SEED. INCREASING PENNYCRESS SEED SIZE WOULD ALSO INCREASE THE EFFICIENCY OF OIL EXTRACTION. IN ADDITION TO IMPROVING THE SEED SIZE, INCREASED OIL CONTENT IN THE SEED WOULD ALSO IMPROVE THE ECONOMICS OF GROWING AND PROCESSING OF PENNYCRESS AS A BIOFUELS FEEDSTOCK.WITH PREVIOUS USDA NIFA FUNDING, WE DEVELOPED SEVERAL EMS-INDUCED PENNYCRESS MUTANT LINES EXHIBITING KEY DOMESTICATION TRAITS SUCH AS REDUCED SEED POD SHATTER, EARLIER FLOWERING, AND IMPROVED FATTY ACID PROFILES. WE HAVE ALSO DEVELOPED AND DEMONSTRATED THE UTILITY OF PENNYCRESS AGROBACTERIUM-MEDIATED PLANT TRANSFORMATION AND CRISPR-CAS9 GENOME EDITING, GENERATING PENNYCRESS LINES WITH UNDETECTABLE LEVELS OF ERUCIC ACID IN SEED OIL. WITH PENNYCRESS DOMESTICATION WELL UNDER WAY, OUR NEW GOAL IS TO IDENTIFY, CHARACTERIZE, AND INTROGRESS INTO BREEDING LINES TRAITS THAT WILL IMPROVE PENNYCRESS EFFICIENCY AND UTILITY AS A BIOFUEL FEEDSTOCK SPECIES AND MAKE THE SEED EASIER FOR PRODUCERS TO HANDLE, NAMELY: 1) INCREASED SEED SIZE AND 2) INCREASED SEED OIL CONTENT. THESE TWO TRAITS WILL BE KEY FOR RAPID COMMERCIALIZATION OF THE SPECIES. IN ADDITION TO IDENTIFYING AND CHARACTERIZING EMS-INDUCED MUTANT LINES WITH INCREASED SEED SIZE AND OIL CONTENT, WE WILL ALSO EVALUATE UNTAPPED GENETIC VARIATION FOR THESE TRAITS IN WILD-DERIVED PENNYCRESS LINES USING QUANTITATIVE TRAIT LOCI (QTL) MAPPING. FINALLY, WE WILL INTROGRESS IDENTIFIED QTLS AND BENEFICIAL MUTATIONS INTO ELITE PENNYCRESS LINES USING MARKER ASSISTED SELECTION. THIS RESEARCH WILL AID IN THE GENERATION OF HIGH-YIELDING, ELITE PENNYCRESS VARIETIES WITH LARGER SEED SIZE AND HIGHER OIL CONTENT. PRODUCERS HAVE EXPRESSED INTEREST IN PLANTING PENNYCRESS AS A CASH COVER CROP, HOWEVER THEY HAVE BEEN CONCERNED ABOUT THE SMALL SEED SIZE. LARGER SEEDED PENNYCRESS VARIETIES WILL BE EASIER TO HANDLE WITH CONVENTIONAL EQUIPMENT AND WILL LEAD TO INCREASED ADOPTION OF PENNYCRESS AS A WINTER OILSEED COVER CROP FOR THE U.S. MIDWEST. INCREASING THE SEED OIL CONTENT WILL GREATLY IMPROVE THE ECONOMICS OF GROWING AND PROCESSING PENNYCRESS. BY INTEGRATING NEW PENNYCRESS VARIETIES INTO THE CORN-SOYBEAN ROTATION SYSTEM, U.S. PRODUCERS CAN INCREASE SUSTAINABILITY BY REDUCING SOIL EROSION, NUTRIENT LOSS, AND HERBICIDE APPLICATIONS. ESTABLISHING PENNYCRESS AS AN OFF-SEASON CROPPING SYSTEM WILL IMPROVE RURAL ECONOMIES BY CREATING NEW JOBS AND DEVELOPING NEW PRODUCTS THAT REPLACE FOSSIL FUELS WITH LOCALLY-PRODUCED BIOFUELS.