Fig. 1

Genetic variation in barley at the 4H B tolerance locus (HvBot1). a HvBot1 coding sequence alleles detected in a diverse set of barley genotypes. All SNPs relative to the Sahara allele are shown as lines, with those resulting in an amino acid change shown as circles. The positions of the xBot1 CAPS marker (blue) and the Sahara-specific wri57 KASP^TM assay (green) are also indicated. Allele nomenclature (Bot1.a to Bot1.g) is given according to published guidelines for barley [35]. We designated the allele from the sequencing reference genome of cv. Morex as Bot1.a. b Expression levels of HvBot1 determined by q-RT-PCR, in roots of seedlings of barley genotypes carrying different HvBot1 alleles. Refer to Additional file 8: Table S5 for a list of barley genotypes included in each HvBot1 allele class. Transcript levels were normalised using the RNA housekeeping genes for tubulin, GAPDH and HSP70. c Growth of yeast expressing the Sahara HvBot1 allele (Bot1.b), the non-functional Haruna Nijo HvBot1 allele containing two residue substitutions relative to Sahara (Bot1.g; Leu234His/Thr541Met), and Bot1.b with single residue mutations (Leu234His or Thr541Met) relative to an empty vector control, on a low B medium (left) or at high B (right). Clones are positioned vertically and spots across the plates represent 10 L of 10⁻⁰, 10⁻¹, 10⁻² and 10⁻³ dilutions from a starting culture containing approximately 3 × 10⁷ cells L⁻¹. Photographs were taken after 2 d (low B) or 4 d (20 mM B) incubation at 30 °C, and are representative of two experiments using independent clones