Computational protocol: Effect of Rice Cultivation Systems on Indigenous Arbuscular Mycorrhizal Fungal Community Structure

Similar protocols

Protocol publication

[…] A clone library was generated using pooled amplicons from three replicates of the same plot (3 plot replicates were carried out for a sample). Pooled PCR products were gel-purified to improve the efficiency of the ligation reaction (QIAquick gel purification kit; Qiagen). Purified products were ligated into the pGEM-T-Easy vector system cloning kit according to the manufacturer’s instructions (Promega) and transformed into Escherichia coli (JM109). Ninety-six positive transformants were selected randomly. Amplified inserts were then digested by the restriction enzyme HinfI. Distinct RFLP profiles containing one or more clones were considered to be a distinct species. Each pattern of RFLP was screened and sequenced to ensure sequence identity with both directions of vector primers SP6 and T7 on ABI PRISM 3730 (Applied Biosystems) DNA Analyzer System at Macrogen (Seoul, South Korea). All sequences were compared in the public database by BLASTN searches and fifteen AMF sequences (phylotypes) were deposited in GenBank (accession numbers: JF906731–JF906744, JF906749). To separate AMF sequences from non-target sequences, the BLAST () was used. The resulting sequences (GenBank) were subjected to phylogenetic analysis using CLUSTALX version 1.83.1 (), together with 46 reference sequences, and 3 sequences of other fungi were used as the outgroup. AMF sequences were automatically aligned, their closest BLAST matches, and additional AMF sequences from a phylogenetically representative set of Glomeromycota downloaded from GenBank. A neighbor-joining distances tree was constructed using MEGA version 4 () with 1,000 bootstrap replicates. All sequences were subjected to in silico restriction digestion with the program BioEdit 7.0 () to predict the fragment sizes of each double-labeled sequence fragment. Using this procedure, predicted fragment lengths were calculated, taking the differences in migration caused by dye properties (HEX vs FAM) into account. These in silico RFLP analyses were performed to putatively assign the most common T-RF pairs to specific sequences (AMF types). This procedure was only performed for T-RF pairs that were specific to a certain sequence. Related sequences having approx. 2 bp differences in T-RF sizes were considered to be the same, and this criterion was also used to match the observed T-RFs to sequences. […]

Pipeline specifications

Software tools BLASTN, Clustal W, MEGA, BioEdit
Application Phylogenetics
Organisms Oryza sativa