分子系統樹解析(分類解析)の例
Molecular Phylogenetic Analysis―example of Entorhoma mushrooms

 To build a robust identification method, collecting many sequences from Database and your samples is extremely important to avoid sequence variation. Sequences in wild mushrooms and plants usually include some SNP and InDels (insertion/deletion). Entorhoma mushroom in eastern Japan are partly different from Entorhoma mushroom in western Japan, for example.

How to classify your mushroom/plant of interest.

1) Morphological observation/microscopic analysis to find unique characteristics of your samples
(carefully check colors of cap, stipe, spores, basidia, cystidia and pileipellis)
Ref. きのこの基礎実験技術(第5回)(実験講座) in Japanese
Ref2. MushroomExpert.Com
There are many Web sites describing how to observe mushroom.

2) Sequence analysis to identify the genus and species of your sample
(ITS, RPB2, CO1 for mushroom, matK, rbcL, trnH-psbA for plant)
The following Web site can help you analyze sequences- which gene should I use?
DNA barcoding - International Barcode of Life – includes public data portal and primer DB
(https://ibol.org/about/dna-barcoding/)

Note: If you find a novel species, you register it at NCBI (DDBJ) and Mycobank, and keep the novel species at your Institute or National Museum of Nature and Science in an appropriate way.

Procedure in detail (example of Entoloma mushrooms)

Fungal sampling, DNA isolation, PCR, sequencing and dataset assembly.

 To examine genetic variations, we collected E.rhodopolium, E.sinuatum and E.sarcopum mushrooms from various regions in Japan, from Hokkaido (northern) to Shimane (southwestern) to cover genetic variations among locations. Entoloma rhodopolium were morphologically identified based on the comparison with the neotype reported by Kokkonen13 and our observations. Genomic DNA was extracted using DNeasy Plant Mini Kit (Qiagen) or by the CTAB method following grinding in liquid nitrogen. PCR and alignment. Two markers, internal-transcribed spacers (ITS) and RNA polymerase subunit II (RPB2), were used. The sequencing primers for ITS were ITS-1F and ITS-4. The primers for RPB2 were RPB2-6F and RPB2-7R for the first PCR, and RPB2-i6F and RPB2-i7R for the second nested PCR8. PCR amplification was performed in a 50 μL reaction volume: 1 μL primers (final 0.5–1 μM), 10 μL 10 × PCR buffer, 4 μL dNTP (200–400 μM), 5 μL DNA template (50 μg), 1 μL KOD-FX or KOD-FX neo (1.0U, Toyobo, Japan), and sterile MilliQ water. The PCR reaction conditions used for ITS were: 3 min initial incubation at 95 °C, followed by 45 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min. The first PCR conditions for RPB2 were: 3 min initial incubation at 95 °C, followed by 30 cycles of 95 °C for 30 s, 61 °C for 30 s, and 72 °C for 1 min. The second nested PCR conditions for RPB2 were 3 min initial incubation at 95 °C, followed by 30 cycles of 95 °C for 30 s, 51 °C for 30 s, and 72 °C for 45 s. Sequences obtained were aligned using the MUSCLE25,26 multiple sequence alignment programs in CLC Genomics Workbench as described in the Molecular phylogeny section below. For alignment of these Entoloma species, datasets from our study and sequences from the NCBI database were used, and the sequences were adjusted to the same sequence length. ITS and RPB2 yielded alignment lengths of 926 and 621 bp, respectively. We collected 48 samples of E.rhodopolium, E.sinuatum, and E.sarcopum. From those samples, 37 ITS and 15 RPB2 sequences were obtained.

Molecular phylogenetic analysis.

 Phylogenetic analyses were performed by the maximum likelihood method using a general-time-reversible model with a gamma distribution rate of variable sites in the CLC Genomics Workbench. Bootstrap values (%) were calculated with 1,000 replicates, and a phylogenic tree was constructed. For the out-groups, two taxa were used; sequences of Clitocybe dealbata and Lyophyllum leucophaeatum were downloaded from NCBI. Phylogenetic trees were rooted by outgroups. For the in-group, a total of 37 Entoloma spp. were sequenced for ITS. Sequences of other Entoloma species were obtained from NCBI. All reference datasets are summarized. When two sequences were obtained from a single mushroom, the sequences were designated as -seq 1 and -seq 2 (e.g., KUB-127-seq 1 and KUB-127-seq 2) in phylogenetic trees. KUB-127-seq 1 and -seq 2 were the same sequence except for a nine-nucleotide insertion or deletion (InDel). Other KUB samples, such as KUB-3-seq 1 and–seq 2 also had the same sequence except for a one-nucleotide indel. Two sequences from a single sample were categorized within the same clade. MEGA software was also used to compare our data with the previous report. Phylogenetic analysis was performed by the maximum likelihood method using a general-time-reversible model with a gamma distribution rate of invariable sites. Gap/missing sites were used. Bootstrap values (%) were calculated with 500 replicates.

See Scientific Reports | 7: 14942 | DOI:10.1038/s41598-017-14466-x (2017)—free access

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