Key message The pepper mutants (‘221-2-1a’ and ‘1559-1-2h’) with very low pungency were genetically characterized. The Pun4 locus, responsible for the reduced pungency of the mutant fruits, was localized to a 208 Mb region on chromosome 6. DEMF06G16460, encoding 3-ketoacyl-CoA synthase, was proposed as a strong candidate gene based on the genetic analyses of bulked segregants, DEG, and expression analyses. Abstract Capsaicinoi...