SEED DORMANCY IS AN ADAPTIVE TRAIT OF GREAT ECOLOGICAL AND AGRICULTURAL IMPORTANCE. IN AGRICULTURE, STRONG DORMANCY IS THE BIOLOGICAL REASON FOR THE WEED PERSISTENCE, NON-UNIFORM GERMINATION OF CROP CULTIVARS, AND FERAL PLANTS, IN PARTICULAR THOSE FROM GENETICALLY ENGINEERED CROPS. ON THE OTHER HAND, NO/WEAK SEED DORMANCY OFTEN CAUSES PRE-HARVEST SPROUTING (PHS) IN CEREAL CROP PRODUCTION. THE GOAL OF THIS PROJECT IS TO ELUCIDATE GENETIC, EVOLUTIONARY, AND DEVELOPMENTAL MECHANISMS DIRECTLY REGULATING THE NATURAL VARIATION OF SEED DORMANCY IN THE GRASS FAMILY. THIS WILL PROVIDE FUNDAMENTAL KNOWLEDGE AND NOVEL GENES TO MANIPULATE GERMINATION ABILITY OF CULTIVARS AND TO DEVICE NEW STRATEGIES FOR WEED MANAGEMENT.OUR PREVIOUS RESEARCH ISOLATED OR CLONED A SET OF QUANTITATIVE TRAIT LOCI (QTL) FUNCTIONALLY DIFFERENTIATED FOR SEED DORMANCY BETWEEN WEEDY AND CULTIVATED RICE (ORYZA SATIVA). OF THE QTL, QSD12 HAD THE LARGEST EFFECT ON GERMINATION INHIBITION AND IS ASSOCIATED SPECIFICALLY WITH EMBRYO DORMANCY. MAP-BASED CLONING OF QSD12 IDENTIFIED THREE TIGHTLY LINKED GENES, DESIGNATED SD12A, B AND C. EACH OF THE THREE HAS AN INDEPENDENT EFFECT, AND TWO OR MORE OF THEM ACT CUMULATIVELY IN EFFECT ON THE DEGREE OF SEED DORMANCY. SD12A AND C ENCODE THE BHLH FAMILY TRANSCRIPTION FACTORS, WHILE SD12B HAS AN UNKNOWN MOLECULAR FUNCTION. IT IS KNOWN THAT A BHLH TRANSCRIPTION FACTOR WORKS AS A HOMODIMER BY A SELF-INTERACTION, OR A HETERODIMER BY AN INTERACTION WITH A PARTNER FROM THE SAME OR A DIFFERENT FAMILY, TO TURN ON/OFF THE EXPRESSION OF DOWNSTREAM TARGET GENES. THUS, THE OBJECTIVES OF THIS PROJECT ARE TO IDENTIFY INTERACTION PARTNERS AND DIRECT TARGETS OF SD12A AND C, AND TO EVALUATE GENETIC COMPONENT EFFECTS OF SD12A, B, AND C ON PHS RESISTANCE IN AN ISOGENIC BACKGROUND.THE INTERACTION PARTNERS WILL BE SCREENED AND VERIFIED USING A SERIES OF BIOCHEMICAL OR MOLECULAR BIOLOGICAL APPROACHES (E.G., BIFC, Y2H OR CO-IP ASSAYS). THE TARGET GENES WILL BE IDENTIFIED USING Y1H AND CHIP-SEQUENCING ANALYSIS. THE GENETIC EFFECTS ON PHS RESISTANCE WILL BE EVALUATED USING A SET OF ISOGENIC LINES IN A TEMPERATURE- AND HUMIDITY-CONTROLLED GREENHOUSE. EXPECTED OUTCOMES FROM THIS PROJECT INCLUDE: 1) GENES CAPABLE OF INTERACTING WITH SD12A OR C TO FORM HETERODIMERS TO REGULATE GENE EXPRESSION; 2) IMMEDIATE DOWNSTREAM GENES OF SD12A AND/OR C IN REGULATORY NETWORKS FOR THE DEVELOPMENT OF EMBRYO DORMANCY; 3) ISOGENIC LINES FOR SD12A, B AND C, AND THEIR COMBINATIONS; AND 4) INFORMATION ABOUT GENE MAIN (ADDITIVE) AND EPISTATIC EFFECTS ON PHS RESISTANCE.THE GENES IDENTIFIED, GENETIC MATERIALS DEVELOPED, AND GENIC EFFECTS EVALUATED IN THIS RESEARCH WILL PROVIDE BEST CANDIDATES FOR FUNCTIONAL ANALYSIS OF GENE REGULATORY NETWORKS BY GENOME EDITING AND OTHER TECHNIQUES. THIS INFORMATION WILL ALSO ASSIST CROP BREEDING EFFORTS TO IMPROVE THE RESISTANCE OF CONVENTIONAL AND HYBRID VARIETIES TO PHS. IN ADDITION, THE KNOWLEDGE FROM THIS RESEARCH, SUCH AS THE GENE REGULATORY NETWORKS AND THE GENETIC CORRELATION BETWEEN THE DEGREE OF SEED DORMANCY AND PHS RESISTANCE, WILL BE IMPORTANT INFORMATION FOR RESEARCH ON SIMILAR PROBLEMS IN THE OTHER CEREAL CROPS.