高通量测序技术分析进境高粱种子的真菌多样性
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摘要
[目的]利用高通量测序技术对进口美国和澳大利亚的高粱种子真菌多样性进行分析,以期为筛查进口高粱携带真菌种类及检疫风险的评估提供科学依据。[方法]收集美国和澳大利亚的高粱种子24份,提取DNA,对ITS1区进行Illumina Hiseq高通量测序,通过真菌通用数据库(UNITE database)及自行构建的高粱病原真菌核心数据库,应用USEARCH 10.0软件对高通量测序数据进行生物信息学分析。[结果]质量控制后得到1 794 895条优化序列,聚类得到901个OTU(operational taxonomic units),测序已覆盖样品中绝大部分的真菌类群。多样性分析表明:2个国家的高粱种子携带的真菌数量相对稳定,但2个国家的高粱种子真菌群落结构差异显著,美国高粱主要真菌群落分布比澳大利亚均匀。2个国家进口高粱种子携带的真菌种类(属水平上)有:链格孢属、曲霉属、隐球菌属、附球菌属、镰刀菌属、戴维氏属、汉纳酵母属、球梗孢属、茎点霉属、耐干霉菌属、尾孢菌属、枝孢属、麦角菌属、旋孢腔菌属、刺盘孢属、弯孢霉属等。采用通用数据库有29个OTU比对到种水平;而采用高粱病原真菌核心数据库有47个OTU比对到种水平。[结论]通过高通量测序技术全面筛查进境高粱携带的病原真菌种类,一次性检出了多种重要的高粱病原真菌;并且构建高粱核心病原真菌数据库,可以显著提高鉴定到种的准确率,为快速筛查高粱病原真菌提供科学依据,为口岸科学评估进口高粱检疫风险提供技术支持。
[Objectives]In order to provide the scientific data for the classification of fungi in imported sorghum and the risk analyzed of biological safety,the diversity of fungal communities in sorghum imported from America and Australia was analyszed by high-throughput sequencing. [Methods]24 sorghum seeds were collected from America and Australia and their DNA were extracted. Illumina Hiseq high-throughput sequencing was used to measure the sequence of ITS1 region. USEARCH 10.0 software program was employed to analyze bioinformatics by the UNITE database and the core database of sorghum pathogenic fungi. [Results]Totally of 1 794 895 sequences were obtained by controlling quality and 901 OTU(operational taxonomic units)were obtained by clustering. The communities of fungi in the samples were obtained. Diversity analysis showed that the number of fungi in sorghum seeds from two countries was relatively stable. But there were significant differences in the community structure of fungi between two countries. The distribution of major fungal communities in sorghum from the United States was relatively uniform compared with that from Australia. At the same time,the main population of fungi in sorghum seeds from two countries(at genus level)were identified. The major genus included Alternaria,Aspergillus,Cryptococcus,Davidiella,Epicoccum,Fusarium,Hannaella,Kabatiella,Phoma,Xeromyces,Cercospora,Cladosporium,Claviceps,Cochliobolus,Colletotrichum and Curvularia. Annotations with UNITE database of phytopathogenic fungi on sorghum,29 OTU were assigned to species. Annotations with the core database of sorghum pathogenic fungi,47 OTU were assigned to species. [Conclusions]The pathogenic fungi in sorghum were screened by high throughput sequencing,and most important pathogenic fungi were detected simultaneously. The construction of the core database of sorghum pathogenic fungi can significantly improve the identification accuracy of species. High-throughput sequencing provides scientific basis for screening pathogenic fungi in sorghum rapidly and risk assessment of imported sorghum.
[Objectives]In order to provide the scientific data for the classification of fungi in imported sorghum and the risk analyzed of biological safety,the diversity of fungal communities in sorghum imported from America and Australia was analyszed by high-throughput sequencing. [Methods]24 sorghum seeds were collected from America and Australia and their DNA were extracted. Illumina Hiseq high-throughput sequencing was used to measure the sequence of ITS1 region. USEARCH 10.0 software program was employed to analyze bioinformatics by the UNITE database and the core database of sorghum pathogenic fungi. [Results]Totally of 1 794 895 sequences were obtained by controlling quality and 901 OTU(operational taxonomic units)were obtained by clustering. The communities of fungi in the samples were obtained. Diversity analysis showed that the number of fungi in sorghum seeds from two countries was relatively stable. But there were significant differences in the community structure of fungi between two countries. The distribution of major fungal communities in sorghum from the United States was relatively uniform compared with that from Australia. At the same time,the main population of fungi in sorghum seeds from two countries(at genus level)were identified. The major genus included Alternaria,Aspergillus,Cryptococcus,Davidiella,Epicoccum,Fusarium,Hannaella,Kabatiella,Phoma,Xeromyces,Cercospora,Cladosporium,Claviceps,Cochliobolus,Colletotrichum and Curvularia. Annotations with UNITE database of phytopathogenic fungi on sorghum,29 OTU were assigned to species. Annotations with the core database of sorghum pathogenic fungi,47 OTU were assigned to species. [Conclusions]The pathogenic fungi in sorghum were screened by high throughput sequencing,and most important pathogenic fungi were detected simultaneously. The construction of the core database of sorghum pathogenic fungi can significantly improve the identification accuracy of species. High-throughput sequencing provides scientific basis for screening pathogenic fungi in sorghum rapidly and risk assessment of imported sorghum.
引文
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[2] da Silva Barreto S,de Rezende D V,Blum L E B.Fungi on seeds of ornamentals[J].Revista Brasileira de Sementes,2011,33(3):561-573.
[3] Buyer J S,Roberts D P,Russek-Cohen E.Microbial community structure and function in the spermosphere as affected by soil and seed type[J].Canadian Journal of Microbiology,1999,45(2):138-144.
[4] Khan Z,Doty S L.Characterization of bacterial endophytes of sweet potato plants[J].Plant and Soil,2009,322(1/2):197-207.
[5] Illeghems K,de Vuyst L,Papalexandratou Z,et al.Phylogenetical analysis of a spontaneous cocoa bean fermentation metagenome reveals new insights into its bacterial and fungal community diversity[J].PLoS One,2012,7(5):e38040.
[6] Hardoim C C P,Esteves A I S,Pires F R,et al.Phylogenetically and spatially close marine sponges harbour divergent bacterial communities[J].PLoS One,2012,7(12):e53029.
[7] Nicolaisen M,Justesen A F,Knorr K,et al.Fungal communities in wheat grain show significant co-existence patterns among species[J].Fungal Ecology,2014,11:145-153.
[8] Mancini V,Murolo S,Romanazzi G,et al.Diagnostic methods for detecting fungal pathogens on vegetable seeds[J].Plant Pathology,2016,65(5):691-703.
[9] Links M G,Demeke T,Gr?fenhan T,et al.Simultaneous profiling of seed-associated bacteria and fungi reveals antagonistic interactions between microorganisms within a shared epiphytic microbiome on Triticum and Brassica seeds[J].New Phytologist,2014,202(2):542-553.
[10] Chen W,Turkington T K,Levesque C A,et al.Geography and agronomical practices drive diversification of the epiphytic mycoflora associated with barley and its malt end product in western Canada[J].Agriculture,Ecosystems and Environment,2016,226:43-55.
[11] Edgar R C.UPARSE:highly accurate OTU sequences from microbial amplicon reads[J].Nature Methods,2013,10(10):996-998.
[12] Nilsson R H,Tedersoo L,Ryberg M,et al.A comprehensive,automatically updated fungal ITS sequence dataset for reference-based chimera control in environmental sequencing efforts[J].Microbes and Environments,2015,30(2):145-150.
[13] Lozupone C,Knight R.UniFrac:a new phylogenetic method for comparing microbial communities[J].Applied and Environmental Microbiology,2005,71:8228-8235.
[14] Robinson M D,McCarthy D J,Smyth G K.EdgeR:a bioconductor package for differential expression analysis of digital gene expression data[J].Bioinformatics,2010,26(1):139-140.