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 primerLocus / Primer / Sequence (5’-3’) / Taxon / Reference
RPB1 / RPB1B-F / AACCGGTATATCACGTYGGTAT / Elata Clade / Du et al. (2012)
RPB1B-R / GCCTCRAATTCGTTGACRACGT / Du et al. (2012)
RPB1Y-F / CGATCTATTAGAACATGGGGCTTC / Esculenta Clade / Du et al. (2012)
RPB1Y-R / GTTGACAACGTGAGCTGGAGA / Du et al. (2012)
RPB2 / RPB2B-F / TAGGTAGGTCCCAAGAACACC / Elata Clade / Du et al. (2012)
RPB2B-R / GATACCATGGCGAACATTCTG / Du et al. (2012)
RPB2Y-F / CTTGCCACTACGCGGTCTAT / Esculenta Clade / Du et al. (2012)
RPB2Y-R / CACGGCTCTGGTATCCATTC / Du et al. (2012)
EF1-α / EF-595F / CGTGACTTCATCAAGAACATG / Morchella / Kauserud & Schumacher (2001)
EF-1R / GGARGGAAYCATCTTGACGA / Du et al. (2012)
ITS rDNA / ITS1 / TCCGTAGGTGAACCTGCGG / Morchella / White et al. (1990)
ITS4 / TCCTCCGCTTATTGATATGC / White et al. (1990)
LSU rDNA / NL1 / GCATATCAATAAGCGGAGG / Morchella / O’Donnell et al. (1997)
NL4 / GGTCCGTGTTTCAAGACGG / O’Donnell et al. (1997)
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.
Du XH, Zhao Q, O'Donnell K, Rooney AP, Yang ZL (2012) Multigene molecular phylogenetics reveals true morels (Morchella) are especially species-rich in China. Fungal Genet Biol 49:455–469
Kauserud H, Schumacher T (2001) Outcrossing or inbreeding: DNA markers provide evidence for type of reproductive mode in Phellinus nigrolimitatus (Basidiomycota). Mycol Res 105:676–683
O’Donnell K, Cigelnik E, WeberNS, Trappe JM(1997) Phylogenetic relationships among ascomycetous truffles and the true and false morels inferred from 18S and 28S ribosomal DNA sequence analysis. Mycologia 89:48–65.
Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Molecular Biology and Evolution 30:2725–2729.
Taylor JW, Jacobson DJ, Kroken S, Kasuga T, Geiser DM, Hibbett DS, Fisher MC(2000) Phylogenetic species recognition and species concepts in fungi. Fungal Genet Biol 31:21–32
White TJ, Bruns T, Lee S, Taylor J(1990) Amplification and direct sequencing of fungal ribosomal RNA for phylogenetics. PCR Protocols. Academic Press, San Diego. 315–322