Soil DNA was extracted from 0.5 g fresh soil samples using the FastDNA SPIN Kit for soil (Aidlab Biotechnologies Co., Ltd., Beijing, China) according to the manufacturer’s instructions. The extracted soil DNA samples were diluted to 10 ng μL−1 with double-distilled H2O and stored at −20 °C for subsequent molecular analysis.
A nested polymerase chain reaction (PCR) was used to overcome the difficulty of amplification caused by the presence of interfering substances such as humic acid in soil. The AML1/AML2 primers (for detailed sequences 5′–3′, see [34 (link)]) were used in the first PCR reaction and the NS31/AM1 (for detailed sequences 5′–3′, see [35 (link),36 (link)]) were used in the second PCR. The PCR system and conditions were adapted from Xiang et al. [37 (link)], and the detailed conditions of the experiment are described by Zou et al. [29 (link)].
The sequences of quantified amplicons were determined on a 454-PLX+ system (Shanghai, China). Overall, the average number of sequencing data per sample was 10,000 and the average length of the sequence read length was 300−600 bp. The availed sequences (ambiguous nucleotides were discarded) were clustered into operational taxonomic units (OTUs) according to the 97% identity threshold, using the unsupervised Bayesian clustering algorithm CROP. Simultaneously, the most abundant sequence in each OTU was selected as the representative sequence. The sequences were clustered by using Usearch (version 7.1http://drive5.com/uparse/ , accessed on 27 November 2015). The fungal taxonomy was identified with UNITE (version 6.0 http://unite.ut.ee/index.php , accessed on 27 November 2015) [38 (link)]. The OTUs that could not be identified at the family or class taxon levels were verified with the National Center for Biotechnnology Information Genbank (http://www.ncbi.nlm.nih.gov/ , accessed on 27 November 2015).
A nested polymerase chain reaction (PCR) was used to overcome the difficulty of amplification caused by the presence of interfering substances such as humic acid in soil. The AML1/AML2 primers (for detailed sequences 5′–3′, see [34 (link)]) were used in the first PCR reaction and the NS31/AM1 (for detailed sequences 5′–3′, see [35 (link),36 (link)]) were used in the second PCR. The PCR system and conditions were adapted from Xiang et al. [37 (link)], and the detailed conditions of the experiment are described by Zou et al. [29 (link)].
The sequences of quantified amplicons were determined on a 454-PLX+ system (Shanghai, China). Overall, the average number of sequencing data per sample was 10,000 and the average length of the sequence read length was 300−600 bp. The availed sequences (ambiguous nucleotides were discarded) were clustered into operational taxonomic units (OTUs) according to the 97% identity threshold, using the unsupervised Bayesian clustering algorithm CROP. Simultaneously, the most abundant sequence in each OTU was selected as the representative sequence. The sequences were clustered by using Usearch (version 7.1
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