** AWARDS ISSUED PRIOR TO JANUARY 20, 2025, WERE FUNDED UNDER PREVIOUS ADMINISTRATIONS AND MAY NOT REFLECT THE PRIORITIES AND POLICIES OF THE CURRENT ADMINISTRATION.** THE OVERARCHING GOAL OF THIS PARTNERSHIP PROPOSAL IS TO USE NEW CRISPR/CAS9 MUTANTS OF SORGHUM [SORGHUM BICOLOR (L.) MOENCH] TO IDENTIFY WHICH GENE AT THE INHIBITION OF ALIEN POLLEN (IAP) LOCUS CONTROLS THE ABILITY OF ALIEN POLLEN TUBES TO REACH THE OVARY DURING INTERSPECIFIC CROSS-POLLINATION. IAP IS THE ONLY KNOWN REPRODUCTIVE BARRIER LOCUS IN SORGHUM. WE WILL CHARACTERIZE GENERAL PLANT MORPHOLOGY, REPRODUCTIVE BIOLOGY, OFF-TARGET COPY NUMBER, AND TARGET GENE EXPRESSION IN THREE MUTANT LINES HAVING LESIONS IN THREE CANDIDATE GENES AT IAP (ONE PUTATIVELY KNOCKED-OUT GENE PER LINE). THE SPECIFIC IAP ALLELE (OR COMBINATION THEREOF) THAT REDUCES/KNOCKS OUT THE EXPRESSION OF THE INHIBITED ALIEN POLLEN PHENOTYPE WILL BE IDENTIFIED. OUR SPECIFIC AIMS ARE TO 1) IDENTIFY WHICH IAP ALLELE (OR COMBINATION OF ALLELES) ENABLES ALIEN POLLEN TUBES TO REACH THE SORGHUM OVARY, 2) DIFFERENTIATE TISSUE-SPECIFIC GENE EXPRESSION PROFILES DURING CROSS-POLLINATION EVENTS, AND 3) IDENTIFY KEY GENES INVOLVED IN A COMPATIBLE VERSUS INCOMPATIBLE CROSS-POLLINATION EVENT USING A CO-EXPRESSION ANALYSIS ACROSS DIFFERENT TIMEPOINTS. OUR LONG-TERM VISION IS TO USE INFORMATION GAINED FROM THE NEW CRISPR MUTANTS TO DEVELOP METHODS TO EITHER PREVENT OR ENCOURAGE OUTCROSSING AMONG SORGHUM AND ITS WILD RELATIVES, DEPENDING ON CONTEXT. FOR EXAMPLE, OUTCROSSING BETWEEN THE CROP AND CLOSELY-RELATED WEEDS IS UNDESIRABLE, BUT ENHANCING THE ABIOTIC STRESS TOLERANCE OF THE CROP THROUGH BREEDING WITH MORE DISTANT RELATIVES IS DESIRABLE.