Methods are described for the optimisation of the generation of radiation hybrids suitable for physical mapping of a plant (barley) genome. A combination of PCR-based technologies, involving the use of whole genome, mixed primer and hemi-nested primer amplifications, can greatly extend their utility for the physical mapping of expressed sequence tags (ESTs). Using panels of hybrids and ESTs, donor DNA retention and individual marker retention frequencies for the expressed portion of the barley genome in the hybrids were estimated.