Leaf variegation in a barley EMS mutant is controlled by two epistatic mutations

Abstract Variegation mutants provide valuable insights into chloroplast biogenesis. We characterized a newly identified variegated barley mutant, in which the phenotype is controlled by duplicate dominant epistasis—representing, to our knowledge, the first reported case of digenic control in chloroplast-deficient mutants. The causal loci, Var4 and Var5 , were mapped on chromosomes 2H and 3H. We used whole-genome resequencing to identify candidate genes. Our two top candidate genes are an NBR1-like selective autophagy receptor gene and a DNAJ-domain containing gene, respectively. Based on their homology-based functional annotation, both candidates could be implicated in chloroplast proteostasis, regulating protein import, folding, and/or degradation. We propose that mild, independent defects in proteostasis from each mutation act synergistically to surpass a functional threshold, impairing chloroplast development in early leaves while allowing partial recovery in later stages. These findings highlight a novel digenic mechanism underlying variegation and point to proteostasis as a central vulnerability in chloroplast biogenesis. Highlight We identified digenic control of leaf variegation in a barley mutant. Candidate genes, identified through whole-genome resequencing, suggest roles in chloroplast proteostasis.