K. R. Gale

Two PCR-based assays were examined for tracing the presence of a Thinopyrum chromosome segment (Tc6 or Tc14) conferring barley yellow dwarf virus (BYDV) resistance in wheat breeding lines. The microsatellite gwm37 was used to assay the Thinopyrum chromosome segment or its wheat, Group 7, homoeologous segment, and was effective in characterising breeders material since heterozygous lines could be identified. A new set of primers derived from a Thinopyrum -specific DNA segment (csTiB1) provided a dominant marker that was readily scored by agarose gel electrophoresis. It was also demonstrated that the csTiB1 primers could be used to establish a solid phase PCR assay that avoided the requirement for gel electrophoresis and was amenable to use in a high-throughput, microtitre plate format. Depending on the number of DNA samples to be assayed, both primer pairs appear to have a place in breeding programs.

An enzyme-linked immunosorbent assay (ELISA) for the discrimination of Wx-B1a and Wx-B1b genotypes at the granule-bound starch synthase I (GBSSI) or waxy locus of hexaploid wheat ( Triticum aestivum L.) was adapted to a high-throughput, 96-well microtitre plate format. This test is applicable to the direct analysis of starch, flour, or crushed grain and requires less than 1 grain to perform. Several hundred samples may be routinely analysed in one day. The assay was validated using quantitative trait locus (QTL) analysis of a doubled haploid mapping population of the cross Cranbrook ( Wx-B1a )/Halberd ( Wx-B1b). This demonstrated that the assay unambiguously identified 153 of 161 lines analysed, with a highly significant QTL (LRS value 270) accounting for 83% of ELISA variation, at the Wx-B1 locus on chromosome 4AL. In addition, measurement of total GBSSI variation using a non-isoform-specific GBSSI detection monoclonal antibody also gave a significant QTL (LRS of 84, accounting for 42% of ELISA variation) at the Wx-B1 locus. Application of the assay to crude flour extracts of 8 grains for each of 1093 progeny from 4 crosses segregating at the Wx-B1 locus permitted the unambiguous scoring of lines as pure Wx-B1a or pure Wx-B1b . The scoring by ELISA was strongly related to the flour swelling volume of the lines, thus demonstrating the utility of this high-throughput screening method for the faster, more efficient development of Australian noodle wheats.