The ITS has also recently been suggested for use as a suitable marker for fungal barcode recognition of species (Seifert, 2009). There are two common approaches to sequence PCR products – direct sequencing and sequencing after cloning (Gyllensten, 1989; Rao, 1994). Direct
sequencing of PCR products is likely to represent DNA that is accurately replicated (Gyllensten & Erlich, 1988). Also, it is a quicker and less expensive MK-1775 in vitro option than sequencing after cloning multiple copies of the product. However, it is not always the most successful method. Many studies have failed in direct sequencing of partial ITS PCR products for reasons other than DNA contamination (Vollmer & Palumbi, 2004; Mondiet et al., 2007; Lindner & Banik, 2009). Sequencing after cloning of PCR products is now a widely used
method. Misincorporation by Taq DNA polymerase can give rise to individual clones SB431542 with varying sequences (Tindall & Kunkel, 1988), and the PCR error rate may be higher than 10% (Kobayashi et al., 1999). At least three clones of each PCR product were sequenced to obtain a consensus sequence. Sequencing after cloning is expensive, time-consuming, and labor-intensive for larger scale studies. In our previous study, we obtained a success rate of about 50% with direct sequencing of PCR products of the ITS in 300 wild Pleurotus nebrodensis isolations. As a dikaryon, P. nebrodensis contains two genetically distinct nuclei. We suspected that there were differences in ITS in the two nuclei. Here, we sequenced amplified regions of the ITS of protoplast-derived monokaryons and clones of PCR products derived from dikaryons of P. nebrodensis. Two
dikaryotic Casein kinase 1 isolates of P. nebrodensis (00489 and 00491) from the China Center for Mushroom Spawn Standards and Control (CCMSSC) and their two protoplast-derived monokaryons, respectively, were used in this study (Table 1). All strains were maintained on potato dextrose agar (PDA) slants at 4 °C. All strains were cultured (7 days at 26 °C) on sterilized cellophane overlaid on PDA contained in Petri dishes. Mycelia were collected and suspended in lytic enzyme solution containing 1.5% lytic enzyme (Guangdong Institute of Microbiology, China), 0.6 mol L−1 mannitol, and incubated at 32 °C for 4 h. A 1-mL aliquot of lytic enzyme solution was used for each 100 mg of fresh mycelium. After incubation, the suspension was filtered through a syringe (50 mL) packed with 4-mm-thick cotton to remove mycelial debris. The filtrate was centrifuged at 800 g for 10 min at 4 °C and the supernatant discarded. Residues were dissolved with 1 mL 0.6 mol L−1 mannitol. The number of protoplasts in the filtrate was counted using a hemocytometer. A protoplast suspension (0.1 mL) containing 100–200 protoplasts was spread on regeneration medium (0.6 mol L−1 mannitol, 1.5% maltose, 1% glucose, 0.5% yeast extract, and 1.5% agar) contained in Petri dishes. Incubation was carried out at 25 °C.