** AWARDS ISSUED PRIOR TO JANUARY 20, 2025, WERE FUNDED UNDER PREVIOUS ADMINISTRATIONS AND MAY NOT REFLECT THE PRIORITIES AND POLICIES OF THE CURRENT ADMINISTRATION.** CATFISH IS THE MOST IMPORTANT AQUACULTURE SPECIES IN THE US. ALTHOUGH CHANNEL CATFISH IS MORE USED IN THE US AQUACULTURE THAN BLUE CATFISH, THE INTERSPECIFIC F1 HYBRID PRODUCED FROM FEMALE CHANNEL CATFISH AND MALE BLUE CATFISH IS CURRENTLY THE PREDOMINANT GENOTYPE OF THE US CATFISH INDUSTRY. HOWEVER, PRODUCTION OF THE INTERSPECIFIC HYBRIDS IS HINDERED BY REPRODUCTIVE ISOLATION. UNDERSTANDING REPRODUCTIVE ISOLATION AND MOLECULAR BASIS OF HYBRID INCOMPATIBILITY WILL ALLOW THE SCREENING OF CHANNEL CATFISH AND BLUE CATFISH FOR EFFICIENT AND RELIABLE PRODUCTION OF THE HYBRIDS. THROUGH THE SUPPORT OF OUR PREVIOUS USDA GRANT, WE FOUND THAT CHANNEL CATFISH HAS AN EXCEPTIONALLY UNIQUE SEX DETERMINATION MECHANISM, WHERE SEX IS DETERMINED BY EPIGENETICALLY REGULATED EXPRESSION OF THE Y CHROMOSOME-BORNE ALLELE OF HYDIN. IN THE PROPOSED PROJECT, WE WILL DETERMINE GENES INVOLVED IN REPRODUCTIVE ISOLATION AND HYBRID INCOMPATIBILITY WITH THREE SPECIFIC OBJECTIVES: 1) IDENTIFY THE SEX DETERMINATION LOCUS OF BLUE CATFISH, AND DEVELOP SEX MARKERS FOR BLUE CATFISH; 2) COMPARATIVE GENOMIC AND EPIGENOMIC ANALYSIS OF SEX DETERMINATION OF BLUE CATFISH AND CHANNEL CATFISH BY WHOLE GENOME METHYLATION AND RNA-SEQ ANALYSIS; AND 3) COMPARATIVE EPIGENOMIC AND EXPRESSION ANALYSES OF RECIPROCAL INTERSPECIFIC F1 HYBRIDS TO UNDERSTAND POSTZYGOTIC REPRODUCTIVE ISOLATION, PARTICULARLY ALLELIC METHYLATION AND EXPRESSION, IN RELATION TO EMBRYONIC DEVELOPMENT AND SEX DIFFERENTIATION. ACHIEVING THESE GOALS WILL ALLOW THE IDENTIFICATION OF SEX MARKERS FOR BLUE CATFISH, UNDERSTANDING OF SEX DETERMINATION MECHANISM IN BLUE CATFISH AND ITS DIFFERENCES FROM THAT OF CHANNEL CATFISH, ESPECIALLY IN EPIGENETICALLY REGULATED EXPRESSION OF GENES IN THE SEX DETERMINATION REGION (SDR). IN PARALLEL, HYBRID INCOMPATIBILITY GENES WILL BE IDENTIFIED, AND METHODS FOR SCREENING COMPATIBLE FISH WILL BE DEVELOPED. WE HAVE GENERATED SIGNIFICANT PRELIMINARY RESULTS AND ASSEMBLE A SYNERGISTIC RESEARCH TEAM, ASSURING SUCCESS OF THE PROJECT.