Genetic analysis and fine mapping reveal that AhRt3, which encodes an anthocyanin reductase, is responsible for red testa in cultivated peanuts.

AhRt3, which governs the red testa of peanut, was narrowed down to a 125.30 kb region, and one gene encoding anthocyanin reductase was identified as the putative candidate gene. Testa color is a special characteristic of peanuts (Arachis hypogaea L.), and those with dark testa have been focused on recent years owing to their high-anthocyanin content and increased antioxidant nutritional value. However, the genetic mechanisms underlying this trait remain limited. To identify the gene responsible for the red testa color in peanuts, an F population was constructed by crossing YH91 (pink testa) with JHT1 (red testa). Genetic analysis revealed that the red testa was controlled by a single dominant gene named AhRt3 (Arachis hypogaea Red Testa 3). Through bulked segregant analysis sequencing, AhRt3 was preliminarily mapped to the chromosome Arahy.03 and subsequently narrowed to a 125.30 kb genomic region containing 12 potential candidate genes. RNA-seq analysis revealed that 4,880 genes were differentially expressed in the seed testa, with only the candidate gene Arahy.W8TDEC exhibiting higher expression levels in JHT1 than in YH91. Additionally, sequence variation, functional annotation, and expression profiling confirmed that Arahy.W8TDEC, which encodes an anthocyanin reductase, may be a candidate gene for AhRt3. The structural variation involving an inversion between the sixth exon and the 3'UTR of Arahy.W8TDEC resulted in altered amino acids closely associated with the red testa phenotype in peanuts. In conclusion, this study highlights the role of a novel gene in regulating red testa and contributes valuable insights into the genetic basis of seed testa in peanuts.