Construction of Kongyu 131 mutant library provides genetic resources for rice functional genomics and germplasm...

Kongyu 131 (Oryza sativa ssp. japonica) is an elite rice cultivar widely planted in Heilongjiang Province, China. Here, we subjected Kongyu 131 to ethyl methanesulfonate (EMS)-induced mutagenesis to establish a genome-wide mutant library. On this basis, we generated 8770 M2 lines, and systematically screened 2215 lines with 42 distinct phenotypic variations across all developmental stages. Then, we employed a three-dimensional (3D) pooling strategy to combine 6144 M3 DNA samples into 224 multiplexed pools. By using the TILLING-seq (Targeting Induced Local Lesions in Genomes sequencing) approach, we identified 84 mutants across five key genes (GS3, Pi21, CSA, OsRR22, and OsaTRZ2), and validated their allelic variations. Phenotypic screening generated 18 lines with superior plant architecture, including a novel OsSPL14 allele identified via MutMap+ analysis. Whole-genome sequencing (WGS) of three M2 lines and TILLING-seq of mutants of the five key genes demonstrated a high mutation density (1 mutation per 121-288 kb). This 3D-pooled and genome-saturated EMS mutant library represents a robust resource for advancing functional genomic studies and precision breeding in rice.