DNA Extraction and Amplification for nrDNA-ITS

The seeds were germinated in a light environment at room temperature until the seedlings reached a height of 3 cm–4 cm. Total genomic DNA was then extracted from the seedlings using the modified CTAB (cetyltrimethyl ammonium bromide) method [18 ]. The upstream primer (ITS-P17 5′-CTACCGATTGAATGGTCCGGTGAA-3′) and downstream primer (ITS-26S-82R 5′-TCCCGGTTCGCTCGCCGTTACTA-3′) [19 (link)] used for amplifying nrDNA-ITS sequences were synthesized by Shanghai Sangon Biological Engineering Technology and Service Co. (Shanghai, China). Amplifications were performed in a Mastercycler Gradient PCR Machine (Eppendorf, Germany) using the following program: initial denaturation at 95 °C for 5 min; followed by 34 cycles of 94 °C for 30 s, 56 °C for 45 s, and 72 °C for 45 s; and a final extension at 72 °C for 10 min. The sequencing of the nrDNA-ITS region was performed by GENEWIZ CO., Ltd. (Suzhou, China).