Clones were sequenced with an ABI PRISM 3730 DNA Sequencer (ABI Big Dye Terminator Cycle Sequencing Kit, Perkin-Elmer). The obtained sequences were used in a BLAST search against the NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi) database with default blastn settings and assigned to specific taxa using MEtaGenome Analyzer (MEGAN) software (Huson et al. 2011). With MEGAN software, the lowest common ancestor (LCA) algorithm was used
for taxonomic classification, with the p38 MAPK signaling pathway required parameters of the LCA assignment set as minimum support = 1, minimum score = 500, top percentage = 1. Metagenomic barcoding of the fungal community in orchid roots Six DNA fragments derived from four DNA regions, namely, nrITS (ITS1/2 and ITS3/4), nrLSU (LR and U), mitochondrial large subunit rDNA (mtLSU), and mitochondrial ATPase subunit 6 (mtATP6), were PCR-amplified using genomic DNA isolated from roots of cultivated Phalaenopsis KC1111. PCR primers and annealing temperatures are listed in Table S1. Amplification was conducted
as described in the gene cloning section. All PCR products of ca. 250–300 bp were purified, pooled, and sequenced with Illumina GAIIx high-throughput paired-end sequencing to survey the composition of fungal community. Raw reads were sorted into six categories according to the primer sequences, and the GS-1101 mw reads with an N residue in the sequences were discarded. Sorted sequences were merged to haplotypes for computing the copy numbers, and single-copy haplotypes were removed to lessen the effect of sequencing errors. These haplotypes were further clustered into operational taxonomic units (OTUs) using the BLASTClust program in the standalone BLAST v2.2.26 package of the NCBI. Because the average minimal divergence between fungal species is around 2.5–3 % (Seena et al. 2010; Stockinger
et al. 2010), the stringency of clustering was set with two parameters at 97 % similarity and 80 % coverage between sequences and referred to as the average Reverse transcriptase minimal divergence of species between fungi. From reads sorting, singleton removal, to OTU generation, all steps were conducted with our own Perl scripts. BLAST analyses were performed on all reads against the NCBI nucleotide database, and the results were further processed for taxonomic assignations using MEGAN. An optional score adjustment was used when paired reads matched the same species. The required parameters of the LCA assignment were set as minimum support = 2, minimum score = 80, top percentage = 1 (Murray et al. 2011; Montaña et al. 2012). Classification results were manually checked to correct the Y-27632 cell line ambiguous assignation caused by synonyms for fungal species or an ambiguous annotation in the NCBI database. Evaluating biodiversity based on metagenomic data As recommended by Haegeman et al.