不同扩增引物对高通量测序分析盐角草内生真菌多样性的影响

【背景】高通量测序分析作为深入了解环境微生物群落组成的重要方法,已成为植物内生真菌多样性研究的有效手段,然而由于引物的扩增差异,采用不同引物可对实验结果分析造成影响。同时,盐角草作为世界上最耐盐的植物之一,存在着多种功能性的内生真菌,而较为全面介绍其内生真菌组成和多样性的报道鲜见。【目的】为了揭示盐角草内生真菌的多样性,解析不同扩增引物对内生菌多样性分析的影响。【方法】分别采用真菌高通量测序常用引物对ITS1-5F、ITS1-1F、ITS2对采自乌鲁木齐达坂城盐湖的盐角草内生真菌进行扩增,开展其内生真菌OTU的分析。【结果】通过不同引物对扩增并测序共获得102个盐角草内生真菌OTU,涉及真菌界8个门和未分类菌群,其中子囊菌门(Ascomycota)占绝对优势,其次为担子菌门(Basidiomycota);在属层次上,盐角草内生真菌共涉及64个属及20个未分类属,其中Alternaria、Cladosporium、Podospora等3个属为盐角草内生真菌优势菌群。对不同引物对扩增测序结果分析表明,不同引物对扩增对分析内生真菌OTU数量和种类具有明显的影响,在全部所得的102个OTU中,...

Effects of different amplification primers on diversity analysis of endophytic fungi in Salicornia europaea by the high-throughput sequencing

[Background] High-throughput sequencing methods are widely used in analyses of environmental microbial communities and diversities, and make it easy to investigate endophytic fungi in plants. However, choices of primers often lead to difference in results because fungal species detected depend on fungal coverage rate of primers. Plants in the genus Salicornia are one kind of the most salt-tolerant plants. Some endophytic fungi isolated from the plants produced important functional metabolites. But few investigations on the diversity of endophytic fungi were reported. [Objective] To investigate the endophytic fungi community in Salicornia europaea and reveal effects of different PCR primers on high-throughput sequencing analysis. [Methods] Plants of Salicornia europaea were sampled from Guhya Salt Lake of Urumqi county in Xinjiang, and ITS DNA of endophytic fungi was amplified with primer pairs of ITS1-5F, ITS1-1F and ITS2. Then OTUs based on high-throughput sequencing were analyzed. [Results] In total 102 OTUs were obtained involving 8 phyla and unclassified flora in fungal domain. Among them, Ascomycota was dominant phylum followed by Basidiomycetes. A total of 64 genera and 20 unclassified genera were discovered, in which Alternaria, Cladosporium and Podospora were major groups. Effects of different PCR primers on high-throughput sequencing were measured. In total of the 102 OTUs, 44 OTUs were obtained from the ITS1-5F primer pair, 55 OTUs from the ITS1-1F primer pair and 25 OTUs from the ITS2 primer pair, whereas only 5 OTUs were detected at the same time by all three primers pairs. Additionally, ITS1-1F primer pair was recommended with ITS1-5F primer pair as the supplementary strategy in investigation of the diversity of endophytic fungi. [Conclusion] There are abundant resources of endophytic fungi in Salicornia europaea and obvious effect on analyzing the composition and distribution of endophytic fungi in high-throughput sequencing.