FUSARIUM WILT, CAUSED BY [FUSARIUM OXYSPORUM F. SP. VASINFECTUM (FOV)], IS A SERIOUS ECONOMIC DISEASE OF COTTON (GOSSYPIUM HIRSUTUM) IN THE U.S. AND MANY OTHER COUNTRIES. FOV IS A SOIL AND SEED BORNE PATHOGEN SURVIVING FOR LONG PERIODS WITHOUT A HOST AND CANNOT BE ERADICATED ONCE ESTABLISHED. RECENTLY, NEW EXTRAORDINARILY PATHOGENIC FOV ISOLATES HAVE APPEARED ON COTTON IN THE U.S. FOV RACE 4 (FOV4) HAS BEEN IDENTIFIED IN CALIFORNIA SOILS IN 2003 AND SINCE THEN, HAS SPREAD THROUGHOUT CALIFORNIA'S SAN JOAQUIN VALLEY. FOV4 NOW HAS BEEN FOUND AROUND EL PASO, TEXAS NOW THREATENING UPLAND COTTON PRODUCTION IN TEXAS, THE LARGEST COTTON-PRODUCING STATE, AND ACROSS THE CONTIGUOUS U.S. BELT. FURTHERMORE, GREENHOUSE PATHOGENICITY TESTS SHOW FOV4 SEVERITY INCREASES IN THE PRESENCE OF ROOT KNOT NEMATODE (RKN) (MELOIDOGYNE INCOGNITA) AND FOV RACE 1 (FOV1) PATHOGENS. THE RAPID TRANSMISSION OF FOV4 ACROSS THE U.S. MAKES THE PROPOSED WORK OF UTMOST URGENCY, LEADING TO SHORT- TO MEDIUM-TERM APPLICATION OF RESULTS PROVIDING SOLUTIONS THAT WILL BE IMPLEMENTED WITHIN TWO YEARS OF THE PROJECT CONCLUSION THAT AIDS STAKEHOLDERS. HOST-PLANT RESISTANCE IS THE ONLY COST-EFFECTIVE SOLUTION, SERIOUSLY IMPEDED BY UPLAND COTTON'S NARROW GENETIC BASE. CONSEQUENTLY, CONVENTIONAL BREEDING HAS INTROGRESSED GENES FOR TOLERANCE/RESISTANCE TO FOV4, PROVIDING A DIAGNOSTIC BASIS TO UNDERSTAND THE GENETIC AND GENOMIC BASIS FOR FOV4 TOLERANCE/RESISTANCE MECHANISMS IN COTTON. THE HYPOTHESIS IS THAT THE COTTON RESISTANCE MECHANISM TO FOV4 IS GENETICALLY BASED AND WILL BE DETERMINED BY IDENTIFYING GENETIC POLYMORPHISMS AND GENE EXPRESSION CHARACTERISTICS RELATING TO DEFENSE THROUGH THE WELL DOCUMENTED CAPABILITY OF RNA SEQUENCING (RNA SEQ), EXPEDITING BREEDING FOR FOV4 RESISTANCE IN UPLAND COTTON FROM A DIVERSITY OF GOSSYPIUM SOURCES. EXPERIMENTAL REACTIONS ARE ANALYZED IN THE PROJECT, SPANNING EIGHT COTTON GENOTYPES, SELECTED BASED ON THEIR KNOWN FOV4, FOV1 AND RKN RESISTANCE/TOLERANCE/SUSCEPTIBLE PROFILES, THEIR CURRENT USE AS WHOLE GENOME REFERENCE SEQUENCE GENOTYPES, AND AS PARENTS OF RECOMBINANT INBRED LINE (RIL) GENETIC MAPPING POPULATIONS. THESE GENOTYPES INCLUDE BARBREN-713-32-38 (FOV4 SUSCEPTIBLE) AND A BACKCROSSED-DERIVED ISOLINE (BC5S2, FOV4 RESISTANT INTROGRESSED FROM G. ARBOREUM), BOTH FOV1 AND RKN RESISTANT; PIMA S-7 AND PIMA 379 (PIMA TYPES HIGHLY SUSCEPTIBLE TO FOV4, TOLERANT TO FOV1); ACALA NEMX AND TM1 AND UH001 (TOLERANT/RESISTANT TO FOV4, SUSCEPTIBLE TO FOV1 AND FOR NEMX RKN RESISTANT); PIMA S-6 (TOLERANT TO FOV4, SUSCEPTIBLE TO FOV1 AND RKN). PIMA S-7, PIMA 379, TM1 AND NEMX ARE RIL POPULATION PARENTS. THE PROPOSED PROJECT IS FACILITATED BY THE TM1 PRIMARY G. HIRSUTUM GENOME REFERENCE. OTHER EMPLOYED GENOMES INCLUDE G. BARBADENSE, 243 ADDITIONAL RE-SEQUENCED COTTON GENOMES, FOV1 AND RKN. THE IN-HOUSE RNA-SEQ PIPELINE ANALYZES COTTON RESPONSES TO SOIL-INFESTATION IN SERIES OF INOCULATIONS AS THE PROJECT NARRATIVE OUTLINES. AS DEMONSTRATED IN RELATED STUDIES RNA-SEQ ANALYSES CLARIFY, GENETICALLY, KNOWLEDGE OF DEFENSE (AND SUSCEPTIBILITY) OCCURRING IN A DISEASE MATRIX MADE THROUGH MORPHOLOGICAL STUDIES. EXPRESSED FOV AND RKN GENES IN THE DIFFERENT PATHOGEN INFECTED REACTIONS ARE ALSO IDENTIFIED, LEADING TO THE IDENTIFICATION OF PATHOGEN EFFECTORS THAT CAN BE USED IN COTTON FOV DEFENSE RECEPTOR IDENTIFICATION. IN CONJUNCTION WITH SNP MAP DATA, KEY DEFENSE/TOLERANCE/SUSCEPTIBILITY GENES ARE IDENTIFIED, SUBJECTED TO FUNCTIONAL VALIDATION USING IN-HOUSE TRANSGENIC PROCEDURES (OVEREXPRESSION, RNA INTERFERENCE (RNAI), VIRUS INDUCED GENE SILENCING (VIGS) AND CRISPR/CAS9). VALIDATED DEFENSE GENES ARE USED IN RESPONSE TO DEVELOP RNA BASED MARKERS TO AID FOV4 RESISTANCE BREEDING, MAKING TOOLS AVAILABLE TO SCIENTISTS, EXTENSION, STAKEHOLDERS AND PUBLIC.