Detection of genome-specific ribosomal DNA sequences from bread wheat by a modified PCR-based method

Abstract

Although the nuclear ribosomal ITS region is the marker most frequently used for the molecular analysis of plant origin, little use has been made of this region to determine the origin of common wheat. The present work demonstrates that the nrITS region is suitable for research on the origin or evolution of wheat, not via direct PCR and sequencing, but by means of a novel PCR technique. This PCR analysis involved a combination of high denaturing temperature and high-fidelity Pfu polymerase, followed by product cloning and the sequencing. In this way sequences were revealed that remained undetected using the conventional technique and that bore traces of earlier hybridisations, allowing conclusions to be drawn on the original ITS sequences of the units involved in the hybridisation. It was demonstrated that the direct nrITS sequence of common wheat may be hybrid in nature, and that the results obtained by means of direct sequencing must be treated with caution in wheat and other allopolyploid organisms. With the help of the method described here, it should be possible to avoid such errors.