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

key messageWe identified digenic control of leaf variegation in a barley mutant. Candidate genes, identified through whole-genome resequencing, are consistent with possible roles in chloroplast proteostasis. Variegation mutants provide valuable insights into chloroplast biogenesis. We characterized a newly identified variegated barley mutant, in which the phenotype is controlled by two epistatic recessive genes. The causal loci, var4 and var5, were mapped on chromosomes 2H and 3H. We used genotyping-by-sequencing, linkage mapping, and PACE genotyping, followed by 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. While functional confirmation is still required, mild, independent defects in proteostasis from each mutation may act synergistically to surpass a functional threshold, impairing chloroplast development in early leaves while allowing partial recovery in later stages. These findings reveal a novel digenic basis for variegation in barley and identify candidate genes and pathways linked to chloroplast proteostasis.