GENETIC MODIFICATION (GM) TECHNOLOGY SHOWS PROMISING RESULTS FOR INCREASING NUTRITIONAL QUALITY AND YIELD OF CROPS. MORE RECENTLY, GENOME EDITING (GE) TECHNOLOGY USING CRISPR (CLUSTERED REGULARLY INTERSPERSED SHORT PALINDROMIC REPEATS) ASSOCIATED CAS9/SGRNA SYSTEM SHOWS GREAT POTENTIAL FOR CROP IMPROVEMENT. HOWEVER, CONSUMERS HARBOR NEGATIVE VIEWS TOWARD GM AND GE FOOD PRODUCTS. CONSUMER CONCERNS INCLUDE THE POTENTIAL FOR GENOMIC VARIANTS AND UNPREDICTABLE PHENOTYPIC CONSEQUENCES DUE TO THE INSERTION OF A TRANSGENE OR OFF-TARGET MUTATIONS GENERATED BY CRISPR/CAS9. MODERN NON-TRANSGENIC PLANT BREEDING TECHNIQUES (E.G., IRRADIATION OR CHEMICAL MUTAGENESIS, TISSUE CULTURE, SOMATIC HYBRIDIZATION, AND WIDE CROSSING) ARE USED ON A WIDE SCALE, AND MORE THAN 2,000 CROP VARIETIES DERIVED FROM MODERN NON-TRANSGENIC PLANT BREEDING TECHNIQUES HAVE ENTERED THE FOOD SUPPLY WITHOUT SPECIFIC SAFETY REGULATIONS. HOWEVER, THESE TECHNIQUES ALSO CREATE A VARIETY OF PLANT GENOME MUTATIONS THAT ARE COMPARABLE TO THOSE CREATED BY TRANSGENIC AND GENE EDITING PLANT BREEDING, AND IT IS STILL QUESTIONABLE WHETHER THESE MUTATIONS ARE OF A SIMILAR MAGNITUDE AND TYPE TO THOSE INTRODUCED BY GM AND GE PLANT BREEDING METHODS.USING TOMATO, WHICH IS ONE OF THE HIGHEST PRODUCED EDIBLE CROPS IN THE WORLD AND IS AN EXCELLENT MODEL PLANT FOR THE STUDY OF COMPARATIVE GENOMICS AND PHENOMICS, THE GOAL OF THIS STUDY IS TO COMPARE AND CONTRAST THE TYPES AND FREQUENCIES OF GENOMIC CHANGES ASSOCIATED WITH UNINTENDED PHENOTYPIC VARIATIONS RESULTING FROM CRISPR/CAS9-MEDIATED GENOME-EDITED TOMATO VERSUS EMS-MUTAGENIZED TOMATO, AS WELL AS TRANSGENIC TOMATO VERSUS TISSUE CULTURE-DERIVED NON-TRANSGENIC TOMATO. THE SPECIFIC OBJECTIVES ARE PROPOSED: WE WILL 1) GENERATE CRISPR/CAS9-MEDIATED PURPLE-KNOCKOUT TOMATO LINES AND EMS-MUTAGENIZED PURPLE-LOSS TOMATO LINES; 2) PERFORM WHOLE GENOME SEQUENCING IN THE CRISPR/CAS9-MEDIATED PURPLE-KNOCKOUT LINES AND EMS-MUTAGENIZED PURPLE-LOSS LINES, ALONG WITH TRANSGENIC PURPLE-MONITORING TOMATO LINES EXPRESSING DEL/ROS1 TRANSGENE AND TISSUE CULTURE-DERIVED NON-TRANSGENIC LINES WHICH ARE NOT ASSOCIATED WITH INSERTION OF THE TRANSGENE; 3) PERFORM COMPARATIVE TRANSCRIPTOMIC PROFILING AND WHOLE-GENOME DNA METHYLATION PROFILING IN THE SET OF THOSE LINES; AND 4) INVESTIGATE PHENOTYPIC VARIATIONS AND POTENTIAL CONSEQUENCES IN THE LINES. THE RESULTS OF A WHOLE GENOME SEQUENCING, COMPARATIVE TRANSCRIPTOME PROFILING, DNA METHYLATION PROFILING, AND OTHER PHENOTYPING APPROACHES WILL PROVIDE FEDERAL REGULATORY AGENCIES WITH RELEVANT NEW GENOMIC AND UNINTENDED PHENOTYPIC INFORMATION OF TRANSGENIC AND GENE EDITING CROPS. THIS PROJECT ALIGNS WITH USDA BRAG PROGRAM AREA #5: OTHER RESEARCH TOPICS DESIGNED TO FURTHER THE PURPOSES OF THIS PROGRAM: COMPARISON OF THE TYPES AND FREQUENCIES OF NUCLEIC ACID CHANGES INTRODUCED INTO IMPORTANT CROP GENOMES, VIA GENETIC ENGINEERING TECHNIQUES VERSUS OTHER PLANT BREEDING TECHNIQUES.