KitBase Expanded: An Integrated Genomic and Phenotypic Resource for 3,268 Fast-Neutron-Irradiated Rice Mutants

Fast-neutron mutagenesis creates diverse genome-wide mutations, providing a powerful tool for crop functional genomics. Here, we present an expanded genomic and phenotypic analysis of 3268 fast-neutron (FN)-induced mutant rice lines (Oryza sativa L. cv. Kitaake). All FN lines were whole-genome sequenced, and mutations were identified by alignment in the Nipponbare and KitaakeX reference genomes. We cataloged over 428,000 mutations affecting 78.49% of Nipponbare genes and 70.38% of KitaakeX genes. In silico expression analysis indicates that 575 non-mutated Nipponbare genes are highly expressed and likely essential for viability. Each mutant carries, on average, 68.5 mutations in the Nipponbare alignments or 63.2 mutations for KitaakeX alignments, distributed randomly across all 12 chromosomes with no evident hotspots. FN lines have approximately 8.5% fewer mutations when using the KitaakeX alignment, underscoring the unique contributions of each reference genome and the importance of utilizing both for comprehensive mutation discovery. The majority of mutations are small deletions and single-base substitutions, with deletions predominating in their effect on genes. We found that 74.4% of all transcription factor Nipponbare genes were mutated at least once. Phenotypic characterization of over 2700 lines revealed a broad spectrum of variation in core agronomic traits (heading date, tiller number, plant height, panicle weight, seed yield components) and other morphological variants of interest. The integration of genomic and phenotypic data through the KitBase platform enabled the identification of candidate genes for several traits of interest. The KitBase website (https://kitbase.ucdavis.edu) has been updated to provide open access to all mutation data and seed stocks, as well as an intuitive query interface, facilitating forward and reverse genetic analyses in rice. This expanded resource enriches the rice functional genomics toolkit and highlights the value of coupling high-density mutation mapping with phenotypic data for rapid gene discovery and crop improvement.