Epistatic interaction of the genes Raw1 and Raw7 controls barb size and frequency in barley awn roughness

Abstract Awns of wild barley ( Hordeum vulgare ssp. spontaneum L.) are rough by default due to silicified upward-oriented trichomes on the awn’s epidermis, forming a ratcheted surface, which is advantageous for seed dispersal and burial. Cultivated barley, however, may carry smooth awns covered by smaller barbs or lacking barbs completely. The gene Raw1 on chromosome 5H is a major factor controlling barley awn roughness and was shown to encode a LONG AND BARBED AWN1 (LABA1) homolog. Here we report, by using quantitative analysis of the barb trait, map-based cloning and Cas9-mediated gene knock-out, a second gene Raw7 , located on barley chromosome 7H, encoding a putative two-component response regulator. We propose that Raw7 acts downstream of Raw1 in a cytokinin signaling pathway underlying cell cycle control in epidermal barb primordia cells. Raw1 and Raw7 show epistatic interaction, suggesting that Raw1 acts as the primary driver of barb initiation, while Raw7 modulates barb size and frequency. Our findings provide the foundation to study the selection and domestication history of the awn roughness trait in barley, and thus to dissect if awn roughness is providing an advantage in cultivated barley or if the trait persisted after domestication due to linkage drag. Summary The presence of silicified upward-oriented trichomes or barbs arising from the epidermis of barley awns is a prominent trait. They form a ratcheted surface which is advantageous for seed dispersal and burial and defense against herbivory. Previous work identified Raw1 on chromosome 5H as a major determinant of awn roughness in barley. Here, we identify a second awn roughness gene, Raw7 on chromosome 7H, combining quantitative phenotyping of barb traits, map-based cloning and Cas9-mediated targeted mutagenesis for functional analyses. Genetic and functional evidence suggests a complex epistatic interaction in which Raw1 primarily drives barb formation and Raw7 fine-tunes endoreduplication-dependent epidermal cell expansion and patterning in barb primordia cells. Raw1 and Raw7 likely act in a cytokinin-dependent two-component signaling pathway, where Raw1 promotes local cytokinin activation and Raw7 , a type-B response regulator, mediates downstream transcriptional responses. The proposed pathway suggests additional undetected loci may contribute to awn roughness, and emerging barley pangenome and pan-transcriptome resources provide a framework to identify and functionally validate new candidates.