A novel enzyme-linked immunosorbent assay for the detection of Wx-B1 null wheat lines using a monoclonal antibody and its application for the detection of marker heterogeneity within commercial cultivars

A monoclonal antibody was raised with specificity for the granule bound starch synthase (GBSS1) Wx-B1 homeoallele of wheat (Triticum aestivum L.) using a synthetic peptide immunogen. This was used to develop a simplified enzyme-linked immunosorbent assay (ELISA) for the discrimination of Wx-B1a and Wx-B1b alleles which differ in the starch properties they confer. Discrimination of these alleles is important for the selection of wheat lines for Udon-style noodle production. The simplified ELISA worked in a single antibody (indirect) format and gave improved ease of use, discrimination of alleles and resolution relative to a previously developed 2-antibody (sandwich) ELISA. When the test was validated using breeders seed of a panel of commercial cultivars, heterogeneity of Wx-B1 alleles was observed for a significant proportion of the cultivars tested and this was confirmed using PCR analysis of the Wx-B1 and Wx-A1 genes. This observation has implications for cultivar wheat quality assessment, the application of molecular markers for variety identification purposes and the establishment of mapping populations from commercial cultivars.