Screening And Molecular Verification Of Uracil Auxotrophic Mutants Of Flammulina Velutipes And Pleurotus Eryngii

In recent years,molecular genetics and biotechnological approaches had been developed and applied in the breeding of edible fungi.Flammulina velutipes and Pleurotus eryngii are important cultivation mushrooms in China.Their genomes had been completed sequenced,and promote the rapid development of their molecular genetic research.However,the researches on their genetic transformation,hybrid breeding,identification and protection need the effective genetic markers.This study was mainly to screen uracil auxotrophic strains of F.velutipes and P.eryngii by the UV mutagenesis of protoplast,and to further identify the genotype of the mutants.The uracil auxotrophic dikaryontic strains were created by hybridization,and were also cultivated.It provides scientific basis for the application of the uracil auxotrophic strains of F.velutipes and P.eryngii in the future.This study mainly obtained the following results:1.The monokaryon strains ‘DG1-1’(A1B1)and ‘DG1-29’(A2B2)of F.velutipes are obtained by protoplast monokaryonization.Through the experiment of the sensitivity of5-FOA,the result showed that the strain‘DG1-1’had a higher sensitivity.The‘DG1-1’protoplast was irradiated by ultraviolet light to obtain 3 the mutant strains,‘NG1-65’and ‘NG1-92’,which have the point mutation of pyr F gene,and ‘NG1-95’,which has a point mutation of pyrG gene.2.The 38 uracil auxotrophic dikaryontic strains of F.velutipes were obtained by the hybridization.15 strains of uracil auxotrophic dikaryontic strains were random selected to measure the growth rate on MM,PDA and sawdust media containing different concentrations of uracil.The results showed that the mycelium growing was promoted when the concentration of uracil was low(0.05 mmol · L-1);the mycelial growing was inhibited,while concentration of uracil was high(0.15 mmol · L-1).The Cultivation test of 3 uracil auxotrophic dikaryontic strainsd was carried out.The results showed that the yield of the uracil auxotrophic dikaryontic strains was significantlyreduced,and was significant correlated with the content of uracil adding in the substrate.3.The monokaryon ‘DX8’(A1B1)and ‘DX25’(A2B2)of P.eryngii were obtained by protoplast monokaryonization.The‘DX8’and‘DX25’protoplast were irradiated by ultraviolet light,and one mutant strain‘NX1-265’was obtained from‘DX8’and three mutant strains(‘NX2-0’,‘NX2-11’and ‘NX2-23’)were derived from‘DX25’.All these mutants had the point mutation in pyrF gene.4.The 29 uracil auxotrophic dikaryontic strains of P.eryngii were obtained by the hybridization,from which,15 strains were randomly selected to measure the growth rate on MM,PDA and sawdust media containing different concentrations of uracil.The result showed that when the concentration of uracil was low(0.05 mmol · L-1),the mycelium growing was promoted,while the concentration of uracil was high(0.15 mmol · L-1),the mycelial growth was inhibited.The cultivated test of four uracil auxotrophic dikaryontic strains was done,and the results showed that the uracil auxotrophic dikaryontic strains were able to produce normal fruiting bodies but the yield was significantly correlated with the uracil concentration of the substrate.