** AWARDS ISSUED PRIOR TO JANUARY 20, 2025, WERE FUNDED UNDER PREVIOUS ADMINISTRATIONS AND MAY NOT REFLECT THE PRIORITIES AND POLICIES OF THE CURRENT ADMINISTRATION.** DECADES OF STUDYING PLANT GENE FUNCTION HAVE REVEALED THAT DNA THAT DOES NOT ENCODE PROTEINS IS FAR FROM JUNK DNA, RATHER, IT HAS A STRONG AND WIDESPREAD IMPACT ON PLANT TRAITS. IN CORN, FOR EXAMPLE, GENETIC VARIATION IN NONCODING REGIONS EXPLAINS ON AVERAGE 40% OF THE GENETIC VARIATION ACROSS AGRONOMIC TRAITS. ON A MOLECULAR LEVEL, WE KNOW THAT BINDING BETWEEN SHORT SEGMENTS OF NONCODING DNA AND CERTAIN PROTEINS DICTATES THE EXTENT, TIME AND SPACE AT WHICH A PARTICULAR GENE IS EXPRESSED (OR SILENCED). THERE ARE TENS OF THOUSANDS OF SUCH NONCODING REGULATORY DNA SEGMENTS IN A GENOME THAT POTENTIALLY OPERATE AND INTERACT TO CONTROL GENE EXPRESSION, AND ULTIMATELY, AGRONOMIC TRAITS. WE KNOW THAT VARIATION IN THESE NONCODING DNA ELEMENTS EXPLAINSAGRONOMIC PERFORMANCE, AND THAT TARGETED CHANGES TO SPECIFIC NONCODING SEQUENCES CAN ALTER AGRNOMIC TRAITS SUCH AS YIELD. HOWEVER, PREDICTING PHENTOYPIC OUTCOMES FROM CHANGES TO INDIVIDUAL REGULATORY DNA SEQUENCES REMAINS CHALLENGING. CONSEQUENTLY, OUR LIMITED UNDERSTANDING OF NONCODING DNA LIMITS CROP IMPROVEMENT.THE OVERARCHING GOAL IS TO UNCOVER PUTATIVE REGUALTORY DNA ELEMENTS AS CONSERVED NONCODING SEQUENCES WITHIN GRASS GENOMES, ESTABLISH CORRELATIONS BETWEEN CONSERVED NONCODING SEQUENCE VARIATION AND AWN TRAITS, AND DISSECT THE FUNCTION OF CNSS WITH SUSPECTED ROLES IN AWN TRAITS. TWO PRIMARY QUESTIONS WILL BE ADDRESSED: 1) HOW DOES VARIATION IN CONSERVED NONCODING SEQUENCES RELATE TO AWN TRAITS OVER DEEP EVOLUTIONARY TIME? 2) HOW DO SPECIFIC CONSERVED NONCODING SEQUENCES REGULATE AWN DEVELOPMENT? THE CENTRAL HYPOTHESIS IS THAT AWNS REPEATEDLY EVOLVED IN THE GRASSES VIA CIS-REGULATORY MODIFICATION TO A CONSERVED LEAF BLADE DEVELOPMENTAL PROGRAM. ULTIMATELY, THE PROPOSED WORK WILL UNVEIL PRINCIPLES AND STRATEGIES FOR PRECISELY ENHANCING CROP PLANTS THROUGH TARGETED EDITING OF REGULATORY DNA ELEMENTS.THIS PROJECT AIMS TO DISSECT THE FUNCTION OF SPECIFIC NONCODING DNA ELEMENTS. TO DO SO, I WILL ASK HOW VARIATION IN PUTATIVE REGULATORY DNA ELEMENTS IS CORRELATED WITH IMPORTANT AGRONOMIC TRAITS ACROSS GRASS SPECIES, USING THE GRASS AWN AS AN EXAMPLE. AWNS IMPACT AGRICULTURAL YIELD, PLAYING ROLES IN PHOTOSYNTHESIS, SEED DISPERSAL AND SPROUTING, AND DEFENSE. WHILE MANYGRASSES HAVE AWNS, THEY ARE NOT ESSENTIAL AND ARE THOUGHT TO HAVE ARISEN AT LEAST 12 TIMES INDEPENDENTLY. STRUCTURALLY, AWNS ARE A MODIFIED LEAF BLADE, WHICH ARE PRESENT IN ALL GRASSES, SUGGESTING THAT THE AWN ARISES THROUGH EXPRESSIONOF CONSERVED LEAF BLADE GENES IN A NEW TIME AND PLACE, POSSIBLY THROUGH MODIFICATION OF REGULATORY DNASEQUENCES NEAR GENES KNOWN TO BEREQUIRED TO FORMLEAF BLADES. DESCRIPTIONS OF AWN TRAITS ARE AVAILABLE FOR OVER 11,000 GRASS SPECIES AND THE GENOMES OF APPROXIMATELY 180 GRASS SPECIES HAVE BEEN SEQUENCED,PROVIDING A WEALTH OF PHENOTYPIC AND GENETIC DATA TO ASSOCIATE VARIATION IN NONCODING SEQUENCE WITH SPECIES-LEVEL TRAITS. FURTHERMORE, GENOME EDITING TOOLS ARE AVAILABLE FOR MULTIPLE GRASS SPECIES.FIRST, I WILL USE,CONSERVATORY, A RECENTLY DEVELOPED ALGORITHM, TO GENERATE A COMPREHENSIVE DATABASE OF PUTATIVE REGULATORY DNA ELEMENTS AMONG 180 SEQUENCED GENOMES REPRESENTING GRASS DIVERSITY. SECOND, I WILL ASSOCIATE REGULATORY DNA VARIATION WITH PUBLICLY AVAILABLE AWN PHENOTYPES, FOCUSING ON GENES EXPRESSED IN DEVELOPING AWNS. FINALLY, TO INVESTIGATE THE FUNCTION OF SPECIFIC NONCODING SEQUENCES, I WILL USE GENOME EDITING TO TEST THE FUNCTION OF INDIVIDUAL OR COMBINATIONS OF PUTATIVE REGULATORY DNA SEQUENCES. INITIAL EDITING WILL BE CARRIED OUT IN MAIZE AND BRACHYPODIUM DISTACHYON, WITH A FOCUS ON FIVE CANDIDATE REGULATORY SEQUENCES NEAR A GENE REQUIRED FOR AWN DEVELOPMENT KNOWN AS DROOPING LEAF (DL).