Differentiation of homokaryons and heterokaryons of Agaricus bisporus with inter-simple sequence repeat markers.

Morphologically different fifteen slow growing single spore isolates (SSIs) were screened from germinated basidiospores of Agaricus bisporus; assumed to be homokaryons, and subjected to growth rate, spawn run, fruiting and inter-simple sequence repeat (ISSR) analysis for confirmation of true homokaryons. The present studies are the first report of fingerprinting on differentiating homo- and hetero-karyotic SSIs using ISSR markers. The patterns were highly polymorphic and very reproducible. Among 40 primers tested, 7 ISSR primers were selected for the analysis of genomic DNA and generated a total of 54 ISSR fragments, sufficient to differentiate the 15 isolates from each other. ISSR fingerprinting detected 46.30% polymorphic loci. All appressed homokaryons carried a subset of ISSR markers found in the heterokaryons, and these were not able to produce a fruiting body. A test of cross-fertility and the following fruiting trial proved that 3 of the 15 SSIs with different ISSR fingerprints were homokaryons. None of the discarded SSIs have any missing bands present in the parental heterokaryotic control; these are heterokaryons derived from non-sister nuclear pairs. It is revealed that homokaryons are probably restricted to the assumed morphological classes of SSIs. These results demonstrate that ISSR markers provide an efficient alternate for identification of homokaryons and suggest these markers be considered as new tools for the survey of Agaricus species.