Deciphering mycorrhizal fungi in cultivated Phalaenopsis microbiome with next-generation sequencing of multiple barcodes

Identifying the species composition of a microbial ecosystem is often hampered by difficulties in culturing the organisms and in the low sequencing depth of traditional DNA barcoding. Metagenomic analysis, a huge-scale nucleotide-sequence-based tool, can overcome such difficulties. In this study, Sanger sequencing of 500 nrITS clones uncovered 29 taxa of 19 fungal genera, whereas metagenomics with next-generation sequencing identified 512 operational taxonomic units (OTUs) for ITS1/2 and 364 for ITS3/4. Nevertheless, high throughput sequencing of PCR amplicons of ITS1/2, ITS3/4, nrLSU-LR, nrLSU-U, mtLSU, and mtATP6, all with at least 1,300x coverage and about 21 million reads in total, yielded a very diverse fungal composition. The fact that 74 % of the OTUs were exclusively uncovered with single barcodes indicated that each marker provided its own insights into the fungal flora. To deal with the high heterogeneity in the data and to integrate the information on species composition across barcodes, a rank-scoring strategy was developed. Accordingly, 205 genera among 64 orders of fungi were identified in healthy Phalaenopsis roots. Of the barcodes utilized, ITS1/2, ITS3/4, and nrLSU-U were the most competent in uncovering the fungal diversity. These barcodes, though detecting different compositions likely due to primer preference, provided complementary and comprehensive power in deciphering the microbial diversity, especially in revealing rare species.