Identification of the Morels in This Study

Identification of the morels in this study

1. DNA isolation

DNA isolation was conducted by using the E.Z.N.A. Fungal DNA Kit (Omega Bio-Tek, USA). Extracted DNA was diluted to 10 ng/μL and stored at -40°C.

2. PCR amplification and sequencing

PCR and DNA sequencing primers are listed in Table 1. Each PCR reaction contained 2×Taq PCR Master Mix 25 μL, 2 μL of each primer (5 μM), 2 μLof 10 ng/μL genomic DNA, and the final volume was adjusted to 25 μLwith sterile distilled H2O. The PCR amplification program for LSU, EF1-α, RPB1 and RPB2 included initial denaturation at 94°C for 3 min, followed by 35 cycles of 94°C for 1 min, 50°C for 30 sec, and 72°C for 1 min, and a final extension at 72°C for 10 min.For ITS sequences, PCR included initial denaturation at 94°C for 4 min, followed by 35 cycles of 94°C for 1 min, 57 °C for 1 min, and 72 °C for 1 min, and a final extension at 72°C for 10 min. The PCR products were subjected to electrophoresis using 1.0% agarose gel (Fig. 1of this online resource).

3. Identification of the morels

The GCPSR(genealogical concordance phylogenetic speciesrecognition) method was used to identify the morelsin this study (Taylor, 2000). The concatenated data set (ITS, LSU, EF1-α, RPB1 and RPB2)were assessed for congruenceusing the Partition homogeneity test as implemented in Clustal X ver. 2.1 ( The maximum parsimony (MP) criterion was used in phylogenetic analyses with Mega 6.0 (Tamura et al. 2013).The study followed the sequence identification program based on multilocus sequence typing(MLST) ( and adopted sequences of reference materials implemented therein. The phylogenetic tree was constructed with reliable sequences obtained from BLAST one-by-one comparisons.Correlating sequences were submitted to GenBank (accession numbersMG121861–121865).The isolated strain Cyl-158 was phylogeneticallyidentified as Morchella importunaM. Kuo, O'Donnell & T.J. Volk.

Table 1 PCR and sequencing primer

Fig. 1. Electrophoretogram showing amplified PCR products and their size measured in basepairs (bp).

Fig. 2 Most parsimonious tree inferred from analyses of concatenated ITS, LSU, EF1-α, RPB1 and RPB2sequences.The phylogenetically-based identification of Cyl-158 is Morchella importuna.

References

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