Study Of Breeding Quality Strains Of Agaricus Bisporus Portobello By Genome Shuffling

Agaricus bisporus?J.E.Lange?Imbach Portobello mushroom,shortened to Portobello,was cultivated and consumed all over the world,and it had great contribution to economic value and friendly attitude to ecological environment.For the complex genetic background of A.bisporus and the conventional breeding methods limitation,A.bisporus breeding work developed slowly.Currently,A.bisporus strains used in industry were those derived from U or AS2796 series.In our research,we modified A.bisporus strains by genome shuffling technology,breeding superior strains for industry cultivation,which could improve the mushroom yields and had good mushroom qualities.The results were shown as below:1)33 couples of SSR primers wereused to amplify 7 kinds of Portobello strains genomic DNA,and 62 alleles were detected.The number of alleles in different locus was2⁴,the average number was 2.48.25 pairs of primers showed obvious polymorphismin 7Portobello genotype,which occupied 75.76%of the detected primers numbers.7 kinds of A.bisporus strains were classified into 4 genetic groups by SSR primers.The genetic similarity coefficient was 1 for ZONGXIU NO.1,LY and LC,the genetic similarity coefficient was 0.6452 compared with ZONGXIU NO.1 and MG.The results showed that Portobello strains in China were different from the foreign strains,it had practical significance in Portobello strains breeding.2)In order to improve the yield and regeneration rate of Portabello protoplast,we established response surface model for optimizing Portobello mushroom protoplast preparation and regeneration conditions.Mushroom strain was ZONGXIU No.1,and the parameters were designed by Design-Expert 8.0.6 software,which including mycelial age,lywallzyme enzyme concentration,snailase enzyme concentration,enzymolysis time and enzymolysis temperature.The results show that the yield of protoplast arrived at the highest level of 4.39×10⁷/mLin the condition of lywallzyme concentration 1.35%and snailase concentration 7.01%,enzymolysis time 3.00 h,mycelium age 8.36 days and enzymolysis temperature 31.4?.Protoplast regeneration rate with highest level of 15.1%was found in the condition of lywallzyme concentration 1.32%and snailase concentration 6.96%,enzymolysis time 2.97 h,mycelium age 9.00 days and enzymolysis temperature 31.0?.The ultraviolet mutagenesis and chemical mutagenesis were used to revealthe growth performance of modified protoplast,and eighteen improved growth performance strains were obtained.The establishing optimized protoplast preparation and regeneration system of Portobello mushroom Zongxiu NO.1 could provide the technical support of protoplasts transformation,genome shuffling and genome targeted editing for Portobello.3)Zongxiu NO.1 strains were processed by EMS,UV,60Co-?and electron beam irradiation to reveal their fatality rate.The suitable EMS concentration was 1.25%,UV irradiation time was 45s,⁶⁰Co-?irradiation dose was 600GY,electron beam irradiation dose was 500GY.33 kinds of mutant strains were obtained with great growth rate and stability.9mutants strains were obtained by EMS,5 mutants strains were obtained by UV,8 mutants strains were obtained by ⁶⁰Co-?,and 11 mutants strains were obtained by electron beam.The construction of mutants strains library could providea foundation for genome shuffling.4)The Portabello protoplast fatality rate was studied by different inactivated methods.The protoplasts could lose its ability at the condition of UV irradiation 100s,heat temperature 50?with 3min.3 strains with high growth ability were selected,named as GS4016?GS4017 and GS5005 respectively,processed at the fusion conditon of PEG concentration 35%,pH8,fusion time 30 min,fusion temperature 25?.The fusion system was carried out 4-5 times.3 kinds of strains had different effects on mycelium growth rate,primordia induction and industrial cultivation.Compared with Zongxiu NO.1,GS5005mycelium forming time was ahead of 1.5 days,primordia numbers were improved by28.66%,the yield of unit area was 26.3 kg/m²,which increased 32.83%compared the original protoplats.5)The genetic diversities among GS4016,GS4017,GS5005 and Zongxiu NO.1were compared,and the similarity coefficients were 0.5102⁰.8204.The similarity coefficient was 0.8204 between GS4017 and Zongxiu NO.1,while similarity coefficients were 0.5102 and 0.5306 among GS4016,GS5005 and Zongxiu NO.1.The genome shuffling could lead to various genetic variations and broaden genetic bases.6)In order to identify the characteristic taste components of Portobello mushroom,taste components in the stipe and pileus of Portobello mushroom harvested at different growth stages were extracted and identified,and principal component analysis?PCA?and taste active value?TAV?were used to reveal the characteristic taste components during the each of the growth stages of Portobello mushroom.In the stipe and pileus,20 and 14different principal taste components were identified,respectively,and they were considered as the principal taste components of Portobello mushroom fruit bodies,which included most amino acids and 5'-nucleotides.Some taste components that were found at high levels,such as lactic acid and citric acid,were not detected as Portobello mushroom principal taste components through PCA.However,due to their high content,Portobello mushroom could be used as a source of organic acids.The PCA and TAV results revealed that 5'-GMP,glutamic acid,malic acid,alanine,proline,leucine and aspartic acid were the characteristic taste components of Portobello mushroom fruit bodies.Portobello mushroom was also found to be rich in protein and amino acids,so it might also be useful in the formulation of nutraceuticals and functional foods.