| Jinhua-fungi are the dominant microorganisms in dark tea,which have huge application potential in tea deep processing and the development of fungal active ingredients.However,the research relevant to the germplasm resources of Jinhua-fungi is insufficient.The genetic diversity of Jinhua fungal strains from different regions is still unclear,and the techniques used to identify their intraspecific units have rarely been reported.Moreover,Jinhua-fungi and their related species,Aspergillus pseudoglaucus and Aspergillus chevalieri,have not been systematically classified,and their nomenclature is also controversial.For the above problems,in this study,Jinhua-fungi isolated from samples in different dark tea producing areas were selected as test strains,and the genetic differentiation characteristics and germplasm resources of the strains were examined from different angles such as physiological traits,mitochondrial genes,expression sequences,and microsatellite markers,thus providing a basis for the systematic classification and nomenclature of Jinhua-fungi.On this basis,molecular markers suitable for the identification of intraspecific and interspecific units of Jinhua-fungi were developed,in a bid to provide technical support for the selection of germplasm resources and standardized production of excellent strains for dark tea fermentation.The main research contents are as follows:1.Analysis of morphological diversity of Jinhua-fungi from different sourcesA total of 70 strains of Jinhua-fungi were isolated from dark tea samples collected in 6 provinces.The genetic diversity of the isolated strains was analyzed using morphological markers.The results showed significant differences in colony characteristics among the 70 strains.The strains were divided into 4 groups with significant differences according to their morphological traits.Additionally,correlations could be found between strain diversity and geographical distribution.Most of the strains from the same region clustered on the same branch,which preliminarily confirms the existence of genetic diversity of Jinhua-fungi in dark tea.2.Differential analysis of physiological traits of representative Jinhua fungal strains and related speciesBased on the results of morphological analysis,the physiological traits of Jinhua fungal strains JH1805,A1,A11,B1,and D1,as well as their related species A.pseudoglaucus and A.chevalieri which greatly differ from each other were further investigated.The results are specified as follows:(1)Ability of raw tea fermentation:All the 7 test strains grew well in raw dark tea and formed golden cleistothecia with high similarity;(2)Colony morphology: The colonies of Jinhua-fungi and their related species were similar on the plates dominated by teleomorphs,while the anamorphic colonies formed on the CZ60 plates with a high osmotic pressure were greatly different;(3)Microstructure characteristics: Some differences were found in the spore structures among Jinhua-fungi,A.pseudoglaucus,and A.chevalieri,which can be used as a supporting method to differentiate the 3 fungi types;(4)Extracellular enzyme activity: The activities of 4 extracellular enzymes showed significant differences among the liquid media of the 7 test strains for the fermentation of raw dark tea powder,and the activities of Jinhua-fungi were generally higher than those of the related species.JH1805 and B1 had high enzyme activities,which can be used as the excellent strains for further breeding for dark tea fermentation.3.Phylogenetic analysis and barcode screening of Jinhua-fungi and related species based on mitochondrial genomeThe whole mitochondrial genome sequences of Jinhua-fungi JH1805 and A.chevalieri XW1803 were determined and annotated,and compared with the mitochondrial DNA sequences of related species.The phylogenetic tree based on protein-coding genes of mitochondrial genome demonstrated the closest genetic relationship between Jinhua-fungi and A.pseudoglaucus,followed by A.chevalieri.All the 3 species and Aspergillus were classified into one cluster,and were in accordance with the characteristics of Aspergillus species in gene arrangement and characteristics of amino acid codons.Therefore,this study supports the classification of Jinhua-fungi into Aspergillus and the nomenclature as A.cristatus.The potential of 6 mitochondrial genes(cob,cox1,cox2,cox3,nad2,and nad6)with the length of 600-1800 bp to develop molecular markers was evaluated.cox1 sequence presented the highest resolution,but it was not effective in differentiating the species such as A.flavus and A.ustus.Based on this,molecular markers with combined nad6 + cox1 sequences were developed in this paper,which could more effectively distinguish some species that fail to be differentiated by monogenes.The topological structure of the constructed phylogenetic tree was basically the same as that of the phylogenetic tree of complete mitochondrial protein-coding genes,and the confidence level of branching was high.Therefore,the proposed molecular markers are suitable for the differentiation of Aspergillus and Penicillium.4.Differential analysis of fungal transcriptome between Jinhua-fungi with two reproduction modesRNA sequencing was performed on the teleomorphic/anamorphic strains of Jinhua-fungi to analyze the genes related to the two reproduction modes and their expression mechanisms.A total of 3,810 DEGs were found,which were mainly enriched in secondary metabolites,bio-membrane,amino acid biosynthesis,and other related metabolic pathways,and were responsible for the regulation of redox reaction,catalytic activity,metal ion binding,and transmembrane transport,among others.The functional analysis of the differentially expressed genes showed that the DEGs regulating spore wall biosynthesis,sexual differentiation,and calcium metabolic pathways were significantly different between the fungi with two reproduction modes.These genes provide resources for the reproductive regulation and efficient sporulation-growth and fermentation process of Jinhua-fungi.5.Development of EST-SSR markers and analysis of genetic diversity in Jinhua-fungiA total of 2,183 EST-SSR loci were found in 10,542 unigenes of the transcriptome sequences of teleomorphic/anamorphic Jinhua-fungi.30 SSR designed primers were selected for PCR amplification using the DNA of 15 strains of Jinhua-fungi and A.chevalieri as templates.According to the phylogenetic tree constructed by the results of electrophoretic bands,A.chevalieri clustered into a single branch as an outgroup,the 15 strains could be divided into 5 groups at the genetic similarity coefficient of 0.983,and had correlations with the differential groups divided by morphology,which confirms the different groups in physiological differentiation and genetic diversity of Jinhua-fungi. |
PDF Full Text Request