IMPROVING WHEAT YIELD POTENTIAL TO MEET FUTURE DEMANDS FOR FOOD WHILE REDUCING LAND USE AND PROMOTING SUSTAINABLE FARMING REQUIRES CONCERTED EFFORTS OF RESEARCH AND BREEDING COMMUNITY AIMED AT 1) DISCOVERING GENETIC VARIANTS CONTROLLING PHYSIOLOGICAL AND DEVELOPMENTAL PROCESSES THAT CONTRIBUTE TO TRAITS OF AGRONOMIC IMPORTANCE, ESPECIALLY PRODUCTIVITY AND NUTRITIONAL VALUE TRAITS, 2) EVALUATING THE PHENOTYPIC EFFECTS OF INDIVIDUAL AND STACKED BENEFICIAL VARIANTS IN ADAPTED GERMPLASM UNDER REALISTIC AGRICULTURAL SETTINGS, AND THEN 3) USING THIS KNOWLEDGE TO REDESIGN WHEAT PLANT BY STRATEGICALLY COMBINING THOSE ALLELES WHOSE INTERACTION GREATLY ENHANCES TRAIT EXPRESSION. WHILE PROGRESS WAS MADE TOWARDS IDENTIFYING THE GENETIC BASIS OF MANY AGRONOMIC TRAITS, WE STILL NEED TO EXPAND GENETIC DIVERSITY ACCESSIBLE FOR BREEDING BY EITHER INTRODUCING NEW DIVERSITY FROM THE ANCESTRAL POPULATIONS OF WILD RELATIVES OR LANDRACES, OR BY CREATING NOVEL VARIANTS BY MUTAGENESIS OR GENE EDITING. WE STILL HAVE LIMITED UNDERSTANDING OF HOW DISCOVERED GENES AND THEIR ALLELES WILL PERFORM IN DIVERSE GENETIC BACKGROUNDS OR ENVIRONMENTS, OR HOW TRAITS AFFECTING DIFFERENT ASPECTS OF WHEAT BIOLOGY WILL INTERACT WITH EACH OTHER ONCE COMBINED. TO ADDRESS THESE CHALLENGES, URGENT INTEGRATION OF INNOVATIVE TECHNOLOGIES AND BREEDING STRATEGIES INTO THE TRANSLATIONAL ACTIVITIES IS REQUIRED.OVER THE LAST DECADE, BY THE EFFORTS OF NATIONAL AND INTERNATIONAL WHEAT RESEARCH PROGRAMS, THE GENETIC BASIS OF MANY AGRONOMIC TRAITS HAS BEEN ESTABLISHED. THE RELEASE OF THE ANNOTATED WHEAT GENOME SEQUENCE AND DEVELOPMENT OF COMPARATIVE GENOMICS TOOLS AND RESOURCES PROVIDED A POWERFUL FRAMEWORK FOR EXTRAPOLATING GENE MAPPING INFORMATION FROM OTHER CROPS INTO WHEAT. THESE DISCOVERIES NOW PROVIDE UNIQUE OPPORTUNITY TO REDESIGN BIOLOGICAL PATHWAYS UNDERLYING MAJOR AGRONOMIC TRAITS IN WHEAT BY INTRODUCING THE FAVOURABLE ALLELES OF CAUSAL GENES INTO THE BREEDING PIPELINES.MANY STUDIES DEMONSTRATED THAT THE CRISPR-CAS SYSTEM CAN TAKE FULL ADVANTAGE OF THESE NEW GENOMIC RESOURCES AND FACILITATE THE CHARACTERIZATION AND DEPLOYMENT OF THE NOVEL GENE VARIANTS. OUR PROJECT WILL ESTABLISH PLANT BREEDING PARTNERSHIP INCLUDING KANSAS STATE UNIVERSITY (KSU) AND THE UNIVERSITY OF SASKATCHEWAN (CANADA) WITH THE AIM TO INTEGRATE THE CRISPR-CAS-BASED TECHNOLOGY INTO THE WHEAT PRE-BREEDING PIPELINES AND IMPROVE WHEAT PRODUCTIVITY AND NUTRITIONAL QUALITY TRAITS IN ADAPTED GERMPLASM. THIS PROJECT WILL BUILD ON THE RESOURCES GENERATED IN THE ON-GOING GENE EDITING PROJECTS CONDUCTED BY THE PROJECT DIRECTORS AND COLLABORATORS. THE PROJECT ACTIVITIES WILL BE INTEGRATED WITH THE WHEAT CAP AND IWYP PROJECTS, AND THE FUTURE NIFA IWYP WINTER WHEAT BREEDING INNOVATION HUB IN MANHATTAN (KS).HERE, WE WILL CREATE VARIATION IN THE PROTEIN CODING OR REGULATORY REGIONS OF GENES THAT CAN INFLUENCE PLANT GROWTH, SPIKE AND GRAIN DEVELOPMENT, AND NUTRIENT ACCUMULATION IN GRAIN. WE WILL MAKE USE OF CRISPR-EDITED WHEAT LINES GENERATED BY OUR TEAM IN A PREVIOUS PROJECT WITH MUTATIONS IN THE GENES AFFECTING GRAIN SIZE AND WEIGHT, AND NUMBER OF GRAINS PER HEAD.WE WILL TEST A NEW STRATEGY TO CREATE NOVEL PHENOTYPIC VARIATION BY MUTATING THE REGULATORY REGIONS OF GENES CONTROLLING NITROGEN UPTAKE, CARBON FIXATION, GROWTH AND NUTRIENT REMOBILIZATION. CURRENTLY, WE HAVE LIMITED UNDERSTANDING OF THE RANGE OF POSSIBLE QUANTITATIVE VARIATION FOR THESE TRAITS THAT CAN BE GENERATED BY EDITING THE REGULATORY REGIONS OF GENES. IN THIS PROJECT, WE WILL INVESTIGATE PHENOTYPES PRODUCED BY THE CRISPR-CAS9-EDITED PROMOTER VARIANTS AND ASSESS THEIR UTILITY FOR WHEAT IMPROVEMENT. BY COMBINING AND TESTING DIFFERENT REGULATORY VARIANTS OF GENES SHOWING DIFFERENT LEVELS OF EXPRESSION, WE WILL IDENTIFY OPTIMAL COMBINATIONS OF VARIANTS SUPPORTING EFFECTIVE NITROGEN UPTAKE AND PLANT GROWTH.THE IDENTIFICATION AND ASSEMBLY OF POSITIVELY INTERACTING COMBINATIONS OF GENES AFFECTING WHEAT PRODUCTIVITY TRAITS HAS A POTENTIAL TO SUBSTANTIALLY INCREASE YIELD. ONE OF THE PRE-REQUISITES FOR THE IMPLEMENTATION OF THIS STRATEGY IS THE IDENTIFICATION OF ALLELES THAT DO NOT SHOW NEGATIVE INTERACTION. BY TRANSFERRING THE CRISPR-EDITED GENE VARIANTS INTO THE ADAPTED GERMPLASM ALREADY SELECTED FOR OTHER BENEFICIAL AND COMPLEMENTARY TRAITS (E.G. HIGH BIOMASS, IMPROVED PLANT ARCHITECTURE, ETC.), THIS PROJECT WILL INVESTIGATE THE UTILITY OF QUANTITATIVE TRAIT VARIATION INDUCED BY GENE EDITING FOR WHEAT IMPROVEMENT, AND IDENTIFY THE COMBINATIONS OF GERMPLASM AND CRISPR-CAS INDUCED ALLELES CAPABLE OF SUPPRESSING NEGATIVE INTERACTION AMONG THE WHEAT PRODUCTION TRAITS.WITH GENE EDITING BECOMING ONE OF THE VALUABLE ASSETS IN THE BREEDER'S CROP IMPROVEMENT TOOLBOX, TRAINING OPPORTUNITIES FOR GRADUATE STUDENTS AND POSTDOCTORAL RESEARCHERS THAT INTEGRATE GENE EDITING SYSTEM WITH BREEDING METHODOLOGIES, BIOINFORMATICS, COMPARATIVE GENOMICS, AND MOLECULAR GENETICS ARE REQUIRED.IN OUR PROJECT, WE WILL DEVELOP EDUCATIONAL MODULES FOR POSTDOCTORAL RESEARCHERS AND PHD STUDENTS TO PROVIDE TRAINING IN APPLICATIONS OF GENE EDITING TECHNOLOGY FOR CROP IMPROVEMENT, REGULATORY ASPECTS OF CROP BIOTECHNOLOGY AND SCIENCE COMMUNICATION. THESE TRAINING IS INTENDED TO PREPARE THE NEW GENERATION OF CROP SCIENTISTS TO FACE THE CHALLENGES OF COMMUNICATING THEIR DISCOVERIES TO THE PUBLIC, CONSUMER GROUPS AND POLICYMAKERS TO ENSURE THAT THE SCIENCE-BASED POLICIES PREVAIL.