Studies On The Homokaryon Differentiation And Hybrids Identification In Agaricus Bisporus

Button mushroom (Agaricus bisporus (Lange) Imbach) is one of the most popular nutritious edible mushrooms in the world for its unique taste and medicinal properties. In recent years, the production of edible mushrooms has been rising due to the rapid increase in international market share. Despite its value and importance, progress in direct improvement has been limited by genetic systems for mushrooms. The uninucleate homokaryotic materials are prerequisite and very essential for the genetic analysis and selective breeding of button mushroom, but differentiation and identification of these uncommon homokaryons is an extremely laborious task. In this research the ways of improvement of the homokaryons identification techniques to accelerate the button mushroom breeding by employing phenomenon of colony morphology along with usefulness of molecular markers viz., RAPD and ISSR, was investigated. Two cultivated domestic strains Ag1023 and As2796 of A. bisporus were used for isolation of single spores and protoplasts (protoclones), respectively.In 600 single spores isolates (SSIs) from strain Ag1023 fruit-body,60 showed differences in colony morphology and the size of colony no more than half those of both parental heterokaryotic control and fast growing heterokaryotic SSIs were selected.15 of 60 SSIs which exhibited similar morphology in the sub-cultures were assumed to be homokaryons. The other 30 SSIs, i. e.,15 of the discarded slow growing and 15 of the discarded faster growing, were randomly selected to perform the ISSR analysis.In 500 regenerated protoplasts (PRs) (from strain As2796 mycelia),45 colonies showed different morphology with colony sizes no more than half those of both parental heterokaryotic control and fast growing heterokaryotic protoplasts (protoclones) were selected for further studies. The 9 colonies of slow-growing protoclones showed unchanged morphology (either appressed or strandy) in each of 5 sub-cultures, were screened as putative homokaryotic protoclones.In the above mentioned 15 SSIs and 9 protoclones treated as putative homokaryons were subjected to growth rate, spawn run and fruiting trials for confirmation of true homokaryons. The variation in terms of growth rate and type of colony on PDA medium and spawn run was carefully investigated. Cross fertility and subsequent fruiting test was conducted to the isolated confluent zones.The use of inter-simple sequence repeats (ISSR) markers are first time report to differentiate homokaryons and heterokaryons in A. bisporus. The patterns were highly polymorphic between homokaryons and heterokaryons 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 putative homokaryotic isolates from each other. ISSR fingerprinting detected 46.30% polymorphic loci. All appressed single spore homokaryons carried a subset of ISSR markers found in the heterokaryons, and these were not able to produce any fruiting body. A test of cross-fertility and the following fruiting trial proved that 3 of the 15 SSIs were homokaryons. This study revealed that homokaryond and hetero-karyons of A. bisporus could be differentiated by ISSR technique. The SSIs identified as homokaryons showed an inability to produce fruiting bodies. These also exhibited a slower spawn run process than that of the self-fertile strandy colonized SSIs. None of the discarded SSIs have any missing bands present compared with the parental heterokaryotic control. 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 could be considered as new tools for the survey of A. bisporus.In this project, protoplast technology was used for obtaining putative homokaryotic protoclones of strain As2796. The present investigations is the first report of fingerprinting on differentiating homokaryotic and heterokaryotic protoclones using random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) markers. Among 80 primers tested,7 of each marker, which could generate excellent results were selected for DNA analysis, generated a total of 94 ISSR and 52 RAPD fragments. ISSR fingerprinting detected more polymorphic loci (38.29%) than RAPD fingerprinting (34.61%). PCA analysis was employed to evaluate the resolving power of the markers to differentiate protoclones. Mean PIC (polymorphism information content) for each of these marker systems (0.787 for RAPD and 0.916 for ISSR) suggest that the ISSR marker system was more effective in determining polymorphisms. The dendrograms constructed using RAPD, ISSR and integrated RAPD and ISSR marker systems were highly correlated with each other as revealed by high Mantel correlation (r=0.98). Pairwise similarity index values ranged from 0.64 to 0.95 (RAPD),0.67 to 0.98 (ISSR) and.67 to 0.98 (RAPD and ISSR) and mean similarity index values of 0.82,0.81 and 0.84 for RAPD, ISSR and combined data, respectively. As there was a good correspondence between RAPD and ISSR similarity matrices, ISSR may be used as an alternative to replace RAPD in the genetic diversity assessment and accurate differentiation of homokaryotic and heterokaryotic protoclones of A. bisporus. A test of cross-fertility and the following fruiting trial proved that 7 of the 9 protoclones which exhibited appressed colony with different ISSR fingerprints were homokaryons. The selected homokaryons (3 from SSIs and 7 from regenerated protoplasts) were used in crossbreeding to produce hybrids. A total of two novel hybrids were produced in 21 attempted crosses between paired homokaryons. One hybrid performed significantly better both in PDYA culture medium and in the wheat spawn. Recovery of both homokaryons and hybrid was verified by analysis of random amplified polymorphic DNA (RAPD) markers. According to present and absence of bands in the RAPD markers were described in such as-shared bands common in hybrids and both of its homokaryon parents, shared bands unique to specific homokaryons or lost bands common to the paired homokaryons. These are the good markers types to confirm the hybrids of its component homokaryon parents. One of two hybrids fruited in small-scale tests; further trial is needed on a larger scale as to know more details about hybrids. One non parental band expressed uniquely in hybrid has direct implication in identification of true hybrid.The results demonstrated that the ISSR analysis is very useful for the differentiation of homokaryons from heterokaryons comparing with the traditional methods. From the crosses of protoplast regeneration homokaryon and single spore isolate homokaryons could get heterokaryotic hybrids, but not all pairs of homokaryons. Only very few pairs could generate the hybrids. Single hybrid have the new fingerprints of RAPD, not the complement fingerprints of the two parental homokaryons, which possibly due to the interaction and recombination between two homokaryotic nuclei. It is need to be more deeply research. Based on the results of dissertation, it is possible that a series of ISSR primers could be selected to differentiate homokaryons of every commercial cultivated strain of A. bisporus.