PCR programs and ITS2 sequencing

ISSR and SCoT PCR reactions were conducted in 20 μL volume containing 10 μL 2 × Taq Mix (CWBIO, China), 8 μL ddH₂O, 1 μL primer (1 mM), 1 μL template DNA (20 ng/μL). 100 ISSR and 85 SCoT primers were based on University of British Columbia and Collard and Mackill (Collard and Mackill 2009), respectively. All the primers were synthesized by Sangon Biological Engineering Technology & Services Company (Shanghai, China). ISSR-PCR reaction started at 94 °C for 5 min, followed by 35 cycles of 94 °C for 45 s, 55 °C for 40 s and 72 °C for 1.5 min, the final extension step at 72 °C was held for 7 min. SCoT-PCR reaction started at 94 °C for 5 min, followed by 35 cycles of 30 s at 94 °C, 1 min at 50 °C and then 1.5 min at 72 °C, with the extension at 72 °C for 7 min.

The PCR reaction for ITS2 was conducted in 50 μL volume containing 25 μL 2 × Taq Mix, 19 μL ddH₂O, 2 μL primer forward (1 mM), 5′-ATGCGATACTTGGTGTGAAT-3′, 2 μL primer reverse (1 mM), 5′-GACGCTTCTCCAGACTACAAT-3′ and 2 μL template DNA (20 ng/μL). ITS2 PCR reaction started at 94 °C for 5 min, followed by 40 cycles of 94 °C for 30 s, 56 °C for 30 s and 72 °C for 45 s, the final extension step at 72 °C was held for 10 min (Chen et al. 2010). PCR reactions were performed in a Veriti 96 Thermal Cycler (Applied Biosystems, USA). PCR productions were visualized by agarose gel electrophoresis and sequenced by AuGCT Technology & Services Company (Beijing, China).