The anther-specific CYP704B is potentially responsible for MSG26 male sterility in barley.

Plant male sterility is a valuable trait in breeding and hybrid seed production. The barley male-sterility gene msg26 was mapped to a 0.02-cM region that anchors to a 506-kb low-quality assembly between two cleaved amplified polymorphic sequence (CAPS) markers, SP1M14 and SP1M49. The barley gene HORVU4Hr1G074840, which encodes a putative cytochrome P450 CYP704B protein, appears to be a strong candidate for the MSG26 trait. Barley (Hordeum vulgare L.) is an important cereal crop worldwide. Traditional breeding in barley is time-consuming and labor-intensive. The use of male-sterile genotypes may significantly improve the efficacy of hybrid breeding and seed production. The barley accession 'GSHO745' is a spontaneous male-sterile mutant from the barley variety, 'Unitan'. The male sterility in 'GSHO745' is controlled by the recessive gene, msg26 (originally named as ms-u). We revealed that the barley plants homozygous for msg26 proceeded normally through Meiosis II until the tetrad stage, but became fully defective in the late uninucleate microspores and developed pollen-less anthers. Using seven barley F populations, we mapped MSG26 to a 0.02-cM region that anchored to a 506-kb low-quality assembly between two cleaved amplified polymorphic sequence markers, SP1M14 and SP1M49. The HORVU4Hr1G074840 gene that encodes a putative cytochrome P450 protein (CYP704B) was identified as the most plausible candidate for MSG26. First, HORVU4Hr1G074840 is located in a collinear region of the rice CYP704B2 and the maize CYP704B1. Both of these genes are essential for male gamete production. Second, the male-sterile allele of HORVU4Hr1G074840 in GSHO745 contained a 4-bp deletion in the last exon. The resulting frame shift causes a Gly436Gln substitution, scrambles the sequence of the remainder of the protein, and forms a new termination site at the 70th triplet of the shifted reading frame. We thus called the variant protein CYP704B:p.G436Qfs*70. Third, the barley HORVU4Hr1G074840 gene was specifically expressed in anthers. Altogether, HORVU4Hr1G074840 represents a strong candidate for MSG26 in barley.