Breeding Of A Superior Strain Of Hypsizugus Marmoreus By Atmospheric Room Temperature Plasma Mutagenesis

In this study,ARTP mutagenesis technology was used to mutagenate the protoplast and fruiting body monosporin suspension of the strain O-T of Hypsizugus marmoreu.All the dikaryotic hyphae obtained in the experiment were screened,and the dominant strains were further screened out by analyzing the cultivation data and SSR molecular marker results.The test results are as follows:(1)In this experiment,the effects of different factors on the protoplast yield were studied by single factor experiment.The optimum conditions for protoplasm preparation were enzymatic hydrolysis temperature 32℃,enzymatic hydrolysis time 3.5 h,osmotic stabilizer0.6 mol/L mannitol and lysozyme concentration 20 mg/m L.Then ARTP mutagenesis technology was used to mutagenize protoplasts.When the mutagenesis time was 100 s,the fatality rate of protoplasts could reach 90%.A total of 290 mutagenesis strains were obtained by this step,among which 288 strains were dikaryotic strains and 2 strains were mononuclear strains.The results of dikaryotic strains screening showed that the growth rate of 30 strains was higher than that of the original strain O-T.Seven strains were with high cellulase production capacity(M-105,M-50,M-65,M-212,M-56,M-53,M-94)and four strains were with slightly high laccase production capacity(M-50,M-48,M-96,M-133)were obtained by plate screening.The antagonism test results showed that the original strain O-T had no antagonism with the strains M-133,M-96,M-53 and M-94,but had antagonism with other strains,but the degree of antagonism was weak.The dominant strain was screened out from 10 mutagenesis strains was M-56,which showed strong laccase production ability during the cultivation process.Compared with the original strain O-T,the enzyme activity increased by 1.69.The average fruiting body yield per vial of strain M-56 was 197.33 g/vial,which was 6.19% higher than that of the original strain O-T.The color of the cap is white with marble-like markings.In addition,the protein content in the fruiting body of this strain was significantly higher than that of the original strain O-T,and the protein content was increased by 27.26%.(2)In this study,single spore suspension of parent O-T was mutated,and the mutagenesis time was determined to be 120 s.A total of 51 strains of mononuclear hyphae were obtained after mutagenesis.In this study,mononuclear hyphae of strain O-T(including 2mononuclear hyphae obtained by protoplast mutagenesis)and mononuclear hyphae of strain B-4 were hybridized.A total of 53 hybrid combinations were obtained by random hybridization,among which 33 hybrid strains were obtained.In this experiment,22 strains with excellent growth condition were obtained from all the hybrid strains by plate screening.The antagonism test results showed that the 12 strains were all new strains different from 2 parents.The antagonism between the two parents was obvious.Cultivation experiment results showed that the hybrid strains of H-9 showed strong ability of enzyme production,which were significantly higher than that of two parents(strains O-T and strain B-4),and increased by 1.39 and 0.98 respectively.In addition,the fruiting body good agronomic traits(the cap is white),basic no marbled cover surface markings.The production(232 g/bottle)of fruiting body was extremely significant different with two parents(strain O-T and B-4),which was increased by 24.85% and 9.85% respectively.In addition,compared with parent O-T,the protein content was increased by 17.29%.(3)In this study,1893 SSR loci were obtained in genomes of Hypsizugus marmoreus which was searched by Hunter 1.3 software.Among them,double-base repeat and tri-base repeat SSRs were 993(accounted for 52.44%)and 710(accounted for 37.49%)respectively,In terms of the number of repeats,the maximum number of SSRs with 5 repeats was 1269,accounted for 67.04% of the total number of SSRs.On the part of repeat types,the number of repeat type in three,four and six bases was 28,28 and 24 respectively,and the repeat frequency was lower than that in two base repeated SSR..Primers were designed according to SSR loci information,20 primers were used to SSR-PCR.The electrophoresis results showed that 4 pairs of primers could amplify more bands.The results of this experiment further indicated that the strains M-56 was different with strain O-T,and H-9 was different with strain O-T and B-4.