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

Abstract:

[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.