Rapid Identification of Chromosome-Specific Sequence-Tagged-Sites in Hexaploid Wheat, using Selective PCR from Nullisomic-Tetrasomic Lines

Abstract

RFLP markers flanking the R loci for red grain colour in hexaploid wheat were identified previously using barley cDNAs to probe Southern blots. Here we report a novel approach for isolating and identifying individual wheat homoeologues of the barley sequences. End sequences from the barley clones were used to design multiple sets of compatible forward and reverse primers which amplify from wheat genomic DNA in PCR. Amplification products from nullisomic-tetrasomic lines of Chinese Spring were obtained from some primer/line combinations but not from others. Appropriate combinations of primer pairs and nullisomic templates were used to clone products specific to each of the wheat chromosomes 3A, 3B and 3D. End sequences of these products were then used to design new primer combinations which specifically amplify products from single wheat chromosomes. This technique reduces the number of clones which must be sequenced in order to obtain all three homoeologues of each target marker, by allowing selective amplification from each homoeolocus in turn. We believe that the method has considerable potential for the development of sequence tagged sites in wheat from heterologous RFLP mapping probes.