| Hypsizygus marmoreus can be divided into two type according to the color of fruit-body:the brown and the white. The white H. marmoreus, the mutatied strain of the brown one, isnamed White Jade Mushroom. Recently, H. marmoreus has become one of the most popularedible and medicinal mushrooms in East Asia, for its pleasant flavor, and nutritional benefits.H. marmorues is an edible mushroom of commercial importance with medicinal propertieswhich can be produce in large-scale factory production.32strains of H. marmoreus werecollected from the following institutions:9strains from the Agricultural Culture Collection ofChina (ACCC), Beijing,7strains from Provincial Institutes of Agricultural Science (2fromFujian,2from Hunan,2from Sichuuan,1from Zhejiang);1strain from Huazhongagriculture university;4strains from Institutes of Edible Fungi and Mushroom (2fromGaoyou,1fromTianda,1from Mianyang);1strain from Changbaishan, Jinlin; and5strainsfrom commercial mushroom enterprises of mushroom (three from Shanghai,1fromShandong,1from Guangdong). The cultivation and genetic diversity were studied withcollected strains of H. marmoreus as material of research.The strains of H. marmoreus were identified for reconfirmation with the internaltranscribed spacer of fungus. The result of identification showed that the strain ofACCC50515was Coprinus comatus, strains of GDGM25484and GDGM26168were thesame strain.To cultivate each strain, the mycelia from a liquid culture were inoculated onto solidsubstrate (a commercial formulation used at the Starway Bio-Technology Co., LTD facility)in a wide-mouth polypropylene bottle, and grown for approximately80d at20°C. Fruitingwas induced by reducing the temperature to15.6°C in an incubation chamber with1,700—3,000ppm CO2and96%relative humidity. Each of the strains had16replications.Three strains of H. marmoreus did not survive because of pollution or other reasonsduring mycelial growth period. The remaining strains grew on solid substrate, produced whitemycelia, and fruit-bodies. The time for hyphae to cover the fully substrate (HCFS) was from34to50days in common, HCFS of some strain was longer more than50days, after thedecreasing temperature to induce fruiting,27strains produced fruiting bodies. The H. marmoreus strains could be divided into several types according to the fruiting bodymorphology (color, shape, uniformity). The color of the fruiting bodies varied among thestrains: dark grey, dark stipe, grey brown with water spots, grey, white, or ivory white. Thecommon diameter of pileus ranged from15to25mm, while the common length of stiperanged from25to50mm. The fresh weight of white strains ranged52.4to188.1g per bottle,the mean weight was112.37g per bottle. The fresh weight of dark strains ranged from0to227g per bottle, the average weight was93.51g per bottle. The factors of H. marmoreus’cultivation phenotype were analyzed by statistical package for the social sciences (SPSS). Theresult of principal components analysis (PCA) showed that the yields of H. marmoreus wasaffected by fresh-weight of mushroom per bottle, the diameter of pileus and the height of stipe;the quality was mainly affected by the diameter of pileus and the height of stipe. The result ofcluster analysis showed that the strains with lower fresh-weight, the diameter of pileus and theheight of stipe were clustered into one group, and the strain with the higher fresh-weight, thediameter of pileus and height of stipe were clustered into the other group.The genomic DNA extracted from H. marmoreus was amplified with18inter-simplesequence repeat (ISSR) primers, which generated199bands. The average number ofamplified bands per primer was11.10. There were173polymorphism bands, with a mean of9.61bands per primer. The mean percentage of polymorphism was87.46. The55primercombinations generated a total of533scorable bands, with an average of9.69bands perprimer combination of sequence-related amplified polymorphism (SRAP). The polymorphicbands were453. The percentage of polymorphic bands ranged between50.0%for Me4-Em2combination and100%for primers of Me1-EM4, Me1-EM2, and so on, with an average of84.67%. The polymorphism information content (PIC) value for ISSRs ranged from0.39to0.50(mean PIC value per ISSR was0.48). The PIC value for SRAPs ranged from0.37to0.50,(average PIC value per primer-combination was0.47). The strains of H. marmoreus had amedium level of polymorphism based on the data of ISSR and SRAP. There were plentifulpolymorphism among the H. marmoreus based on the conbination of cultivation and data ofmolecular makers analysis. The combination of molecular markers and cultivated traits wereone of effective methods in breeding and varietal improvement research for H. marmoreus.Some primers of target region amplified polymorphism were designed by the software of primer3(on line) based on sequence of H. marmoreus from GenBank. After screening,6fixed primers,20anchor primers, and28primer combinations, which could produce clearbands with good polymorphisms and reproducibility, were selected. The28primercombinations generated287scorable bands, including202polymorphic bands. The totalnumber of bands amplified by a primer-combination ranged from4to17with an average of10.27per primer combination. The proportion of polymorphic bands ranged from33.33%(JN00L1-ME11) to100%(JN00R1-ME6) with a mean of72.12%. The20strains of H.marmoreus were clustered into2groups, which one had2strain based on the data of TRAP.The cluster result of TRAP revealed that the cluster of strains were no association with thecolor, yield and stored type of strains.Recycling the bands that were produced in all strains of H. marmoreus and not incontrols, then they were cloned into Ecoli DH5α by vector pMD1-T. The clone productionswere sequenced by Beijing Genomics Institute (BGI) after PCR amplification checked usinguniversal primers (GV-M,5’-GAG CGG ATA ACA ATT TCA CAC AGG-3′; andM13-47,5′-CGC CAG GGT TTT CCC AGT CAC GAC-3′). There were36sequences of H.marmoreus after assembling sequences with the software of DNAstar package. There were6specific sequences which had no significant similarity sequences found in GenBank data, and5sequences that were the part of heat shock protein70(HSP70) of H. marmoreus by BLASTwith GenBank data online. Online analysis in Softberry site showed that18sequences hadone or more open reading frames (ORF). Six pairs of specific primer were designed withsoftware of primer6.0based on specific sequences which were no significant similaritysequences with GenBank data. The result of a serial check experiments show that one pairspecific primers of H. marmoreus was found. A specific sequence of H. marmoreus with120bp was found.There were plentiful genetic diversity among the strains of H. marmoreus based on thestudy results of cultivated phenotype, ISSR, SRAP and TRAP. The results of this study mightstrengthen genetic research foundation and the cultivation of new vaarieties of H. marmoreus.The study revealed the molecular markers of ISSR, SRAP and TRAP were useful tools for evaluating genetic diversity and germplasm resource from different aspects, selectingpotential strains and for predicting hybrid performance in the outbreeding of mushrooms. |
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