Genes can be mapped by recombination in E. coli. In ex-conjugants, selection is made, for merozygotes bearing the leu+ marker, which is donated late. The early markers (arg+ and met+) may or may not be inserted, depending on the site, where recombination between the Hfr fragment and the F- chromosome takes place. The frequencies of events are used to obtain the relative sizes of the leu-arg and arg-met regions. In all the region DNA is inserted into the F- chromosome survives the other fragment is lost.
Therefore, to produce a completely prototrophic ex-conjugant, two crossovers are required.