83 and 0.76), nrLSU-LR (1.47 and 0.68), mtLSU (1.09 and 0.58), and mtATP6 (0.18 and 0.07). Both indices Selleckchem GDC 0068 showed that the nrITS regions had better resolution in width and depth in uncovering the biodiversity than nrLSU and mitochondrial regions (Table 4). Fig. 3 OTU accumulation curves of multiple rarefactions with six markers sequenced with Illumina GAIIx Table 4 Indices of alpha diversity across markers Diversity index ITS1/2 ITS3/4 nrLSU-LR nrLSU-U mtLSU mtATP6 Shannon 2.49 2.02 1.47 1.83 1.09 0.18 Gini-Simpson 0.85 0.78 0.68 0.76 0.55 0.07 Data analysis using rank scoring to evaluate fungal AG-881 diversity The taxonomic assignment for the ten most abundant OTUs for each marker is shown in Table S4.
Unexpectedly, different dominant species were identified among markers. The most abundant OTUs were assigned as Alternaria, Penicillium, Trechispora, Trechispora, Serpula, and Ceratobasidium detected with ITS1/2, ITS3/4, nrLSU-LR, nrLSU-U, mtLSU and mtATP6, respectively. As each marker only represented AZD5363 clinical trial a part of the fungal community, the data across these markers must be combined to get an overview of the microbiome. Here, a rank-scoring strategy
was developed for integrating the information on species composition obtained from multiple markers. Value 0 suggests no reads detected. Abundance of each genus in the community was calculated by summing the rank scores for the five barcodes used; results for mtATP6 were excluded due to its biased detection toward Agaricomycetes. In the rank-scoring, the top 15 genera were Penicillium (including teleomorph Talaromyces), Sporothrix (including teleomorph Ophiostoma),
Trechispora, RG7420 order Fusarium (including teleomorph Gibberella), Candida, Cladosporium, Mortierella, Exophiala, Meira, Aspergillus, Devriesia, Leucocoprinus, Mycospharella, Trichoderma (including teleomorph Hypocrea), and Cladophialophora, all having rank scores between 40.34 and 84.21 (Fig. 4, Table S5). Fig. 4 Bar plot of rank scores at the genus level. Rank scores obtained from five markers are represented in different grayscale colors Discussion DNA barcoding for species identification Although molecular techniques using cloning and Sanger sequencing largely avoid the difficulties of microbial culture or morphotype identification, in the present study, sequencing the ITS1/4 region to investigate the fungal species diversity in orchid roots only identified 29 taxa from 500 clones. Even so, of the top 10 abundant genera (Table 1), nine were also recognized as the dominant genera in the metagenomic analyses (Table S5): Penicillium (20.0 %; meta-rank 2 in the NGS approach), Trechispora (17.6 %; meta-rank 3), Exophiala (6.6 %; meta-rank 8), Fusarium (4.8 %; meta-rank 4), Cladosporium (3.6 %; meta-rank 6), Alternaria (2.0 %; meta-rank 17), Leucocoprinus (2.0 %; meta-rank 12), Sporothrix (1.2 %; meta-rank 1), and Trichoderma (0.4 %; meta-rank 14). High repeatability in both methods reflects that Sanger sequencing may be capable of detecting common taxa.