The barley ear row-number allele ancestral to the six-row allele vrs1.a1 is found in wild barley from the Fertile Crescent

Barley ( Hordeum vulgare L.) exhibits two main inflorescence phenotypes: the wild-type ‘two-row’ form in which only the central spikelet at each rachis node is fertile, and the ‘six-row’ form, where mutations in the homeobox gene Six-rowed spike 1 ( VRS1 ) confer fertility to all three spikelets. While the six-row alleles vrs1.a2 and vrs1.a3 arose independently from point mutations in the ancestral Vrs1.b2 and Vrs1.b3 alleles, the origin of the hypothesised wild-type Vrs1.b1 allele ancestral to vrs1.a1 is unclear. To explore the origin and exploitation of VRS1 alleles, we re-sequenced VRS1 in 98 cultivars, 170 landraces and 69 wild barley accessions, identifying 39 haplotypes. Sequence analysis confirmed vrs1.a1 as the most commonly used six-row allele in European cultivars. Subsequent analysis of the landrace and wild barley data identified three occurrences of the haplotype consistent with a vrs1.b1 allele, all from wild barley. These wild barley accessions originated from the eastern Fertile Crescent (Iran) and the Caspian Sea region, suggesting vrs1.a1 arose via mutation of Vrs1.b1 within barley’s domestication centre - unlike vrs1.a2 and vrs1.a3 , which originated in the Western Mediterranean and East Asia cultivated barley genepools, respectively. In addition, we identified ten novel VRS1 amino acid changes in wild barley accessions, which given the pleiotropic effects of VRS1 on traits such as leaf size, vein number and tiller number, may be of interest for future functional investigation. Overall, this study provides insights into the evolution, domestication and utilisation of genetic variation at VRS1 , a key gene influencing barley architecture and agricultural performance.