Fig. 6

a Partial sequence profiles of amplicon from Hfr-2 with primer pair, Hfr-2_F2b and Hfr-2_R2b, for one R cultivar, CASW02GH00010S, and one S cultivar Cham6. Seventeen SNPs were observed in this segment (Additional file 1: Table S1). Many of these SNPs occur in both cultivars (e.g. SNP labelled ‘1’ to ‘5’) as well as in all the cultivars sequenced (Additional file 1: Table S1). The SNP labelled ‘*’ occurs in the 3rd nucleotide of the codon for glycine and is redundant. The indicated SNP, SNP1294, ‘A/G’, is located at nt position 1294 of the Hfr-2 mRNA sequence, GenBank AY587018.1 (Fig. 2), and results in an amino acid substitution of isoleucine to valine. This SNP is located in the lectin domain of the gene. A SNP assay, SNP1294_Hfr-2, has been developed for high throughput genotyping (Fig. 6b). b The endpoint fluorescence in the genotyping of SNP 1294 in exon II of Hfr-2 using the SNP_1294_Hfr-2 assay (Table 2) on two different 96-well sample plates. The assay has been designed in the complement orientation and hence the nucleotide called was complementary to the corresponding nt in the gene. The SNP at nt 1294 (AY587018.1) segregates between the S cultivars with ‘T’ (CalFluor, green triangles on y-axis), and the R cultivars with either the ‘C’ (FAM, blue triangles on x-axis) or the heterozygotes (red triangles). This SNP in the coding sequence is ‘G/A’ and results in an amino acid change from isoleucine to valine in the alleles of R cultivars with the gene. No S cultivars have the ‘G’ allele. Twelve of the 39 R cultivars have the ‘G’ allele (Table 3). Refer to section under ‘Results’ for explanation on why the S cultivars are in a tight cluster while the R genotypes are in a loose cluster