| Summary: | Seed size is the major yield component and a key target trait that is selected during peanut breeding. However, the mechanisms that regulate peanut seed size are unknown. Two peanut mutants with bigger seed size were isolated in this study by <sup>60</sup>Co treatment of a common peanut landrace, Huayu 22, and were designated as the “big seed” mutant lines (<i>hybs</i>). The length and weight of the seed in <i>hybs</i> were about 118% and 170% of those in wild-type (WT), respectively. We adopted a multi-omics approach to identify the genomic locus underlying the <i>hybs</i> mutants. We performed whole genome sequencing (WGS) of WT and <i>hybs</i> mutants and identified thousands of large-effect variants (SNPs and indels) that occurred in about four hundred genes in <i>hybs</i> mutants. Seeds from both WT and <i>hybs</i> lines were sampled 20 days after flowering (DAF) and were used for RNA-Seq analysis; the results revealed about one thousand highly differentially expressed genes (DEGs) in <i>hybs</i> compared to WT. Using a method that combined large-effect variants with DEGs, we identified 45 potential candidate genes that shared gene product mutations and expression level changes in <i>hybs</i> compared to WT. Among the genes, two candidate genes encoding cytochrome P450 superfamily protein and NAC transcription factors may be associated with the increased seed size in <i>hybs</i>. The present findings provide new information on the identification and functional research into candidate genes responsible for the seed size phenotype in peanut.
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