Limitations of in situ hybridization with total genomic DNA in routine screening for alien introgressions in wheat

In situ hybridization with total genomic DNA (GISH) has become a powerful tool in characterization of alien introgressions in wheat. With recent simplification it can now be used in large scale screening for new chromosome constructs. Its level of resolution in routine applications was tested on sets of recombined wheat-rye chromosomes with genetically determined positions of the translocation breakpoints. The resolution level of GISH visualized by an enzymatic color reaction was much lower than that of GISH with fluorescent probes but both techniques failed to reveal the presence of some distally located breakpoints. The limits of resolution for the two methods were at least 9.8 and 3.5 cM of the relative genetic lengths of chromosome arms, respectively, in configurations with proximal rye and terminal wheat segments when rye DNA was used as a probe. When wheat DNA was used as a probe, a terminal wheat segment estimated to be ca. 1.6 cM in length could not be visualized. An example of induced recombination between a chromosome of Agropyron elongatum and wheat illustrates that these resolution limits of GISH may hamper isolation of critical translocation breakpoints in a chromosome engineering effort.