The conversion of RFLP markers to allele specific amplicons linked to QTLs governing malting quality in barley

Application of marker-assisted selection with RFLP based markers has been constrained by high cost and time requirements in situations involving a large number of plants. RFLP markers mapped on a Harrington/TR306 population have been identified elsewhere as linked to quantitative trait loci (QTL) governing malting quality. The probes ABG610, ABC622, as well as probes for the Nar1, Amy1 and Nar7 were sequenced and locus specific primers developed. These locus specific primers were applied to genomic DNA from both Harrington and TR306. Sequence analysis of the resultant monomorphic fragments revealed sequence divergence for the Xabg610, Xabc622, Amy1 and Nar1 loci, but not for the Nar7 locus. Application of a set of Hor2 primers to genomic DNA from the barley lines Harrington and TR306 led to the direct amplification of codominant alleles. Allele-specific primers were designed based on the sequence divergence identified among the Xabg610, Xabc622 and Nar1 alleles. Amplification conditions were optimized for each of these alleles such that only the favourable allele from Harrington was amplified. The usefulness of these primers for selecting Harrington alleles was demonstrated by their failure to amplify the corresponding alleles from the lines, Sterling, Stella and WM872. The Amy1 allele-specific amplicon was only capable of differentiating this locus between Harrington and TR306. The conversion of these markers into PCR amplifiable, allele-specific amplicons would greatly facilitate their application to barley breeding programs.