Computational protocol: Communities of Arbuscular Mycorrhizal Fungi in Pyrus pyrifolia var. culta (Japanese pear) and an Understory Herbaceous Plant Plantago asiatica

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Protocol publication

[…] Fresh fine roots, around 10–100 mg from Py. pyrifolia and around 50–800 mg from Pl. asiatica were used to analyze the AMF communities. DNA was extracted using a DNeasy Plant Mini Kit (Qiagen, Tokyo, Japan). The AMF communities were examined on the basis of the partial fungal DNA sequences (approximately 750 bp) of the nuclear small subunit ribosomal RNA gene (SSU rDNA), which was amplified by nested polymerase chain reaction (PCR) as follows. The universal primer set NS1 () and NS4 () was used in the first PCR. The reaction mixture contained 1.0 μL of extracted DNA, 0.15 μL of Taq DNA polymerase, 3 μL of PCR buffer (100 mM Tris-HCl [pH 8.3], 500 mM KCl, and 15 mM MgCl2), 2.4 μL of each deoxynucleotide triphosphate, and 0.15 μM of each primer in a total volume of 30 μL. The PCR program was as follows: an initial denaturation step at 94°C for 2 min, followed by 30 cycles of 94°C for 30 s, 40°C for 1 min, and 72°C for 1 min, and a final elongation step at 72°C for 10 min (PC-818S Program Temp Control System; Astec, Fukuoka, Japan). In the second PCR, 1 μL of a 1:10 dilution of the first PCR product was used with the AMF-specific primers AML1 and AML2 () in the same reaction mixture. The PCR program was as follows: an initial denaturation step at 94°C for 2 min, followed by 35 cycles at 94°C for 30 s, 50°C for 30 s, and 72°C for 1 min, and a final elongation step at 72°C for 10 min. The PCR products were purified using a Gel/PCR DNA Fragment Extraction Kit (RBC Bioscience, Taipei, Taiwan) and cloned using a pGEM-T Easy Vector System I (Promega, Madison, WI, USA), according to the manufacturer’s instructions. The clones were analyzed by colony PCR, which was followed by RFLP using HinfI to exclude plant sequences from further analysis. The DNA inserts were sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) with the promoter primer T7 on a 3130 Genetic Analyzer (Hitachi, Tokyo, Japan). Multiple sequence alignments were performed using ClustalX version 2.0.12 for all sequenced data (). The aligned sequences were analyzed using the neighbor-joining method () with 1,000 bootstrap analysis (), and the phylogenetic trees were drawn using TreeView (). The AMF phylotypes were defined on the basis of tree topology and sequence similarity computed using ClustalX. A rarefaction curve was computed for each sample by plotting the number of AMF phylotypes detected against the number of sequences using Analytic Rarefaction version 1.3 (https://www.uga.edu/_strata/software/AnRare/Readme.html). After selecting representative sequences of each phylotype, BLAST searches were performed to download similar sequences. Multiple sequence alignments, neighbor-joining analysis, and phylogenetic tree drawing were performed as described above for the sequenced and downloaded data. Furthermore, the pairwise sequence similarities between AMF from Py. Pyrifolia and Pl. asiatica in the same soil cores were computed using ClustalX. […]

Pipeline specifications

Software tools Clustal W, TreeViewX
Application Phylogenetics
Organisms Pyrus pyrifolia