SSR and AFLP analysis

Total genomic DNA for each sampled individual was extracted from the silica gel-dried leaf tissue using the modified CTAB method (Huang et al., 2000). The DNA quality was checked by loading DNA on a 1.0% agarose gel, and the DNA concentration of each sample was measured using a TBS-380 Fluorometer (Turner BioSystems Inc., Sunnyvale, CA). The samples were screened for 11 SSR markers. Of these loci, Qk15874, Qk17139, Qk17611, and Qk20944 have been developed for Q. kerrii from transcriptome data (An et al., 2016), QmC00693, QpZAG9, QpZAG16, QpZAG36, QpZAG110, CG371 were from the non-coding region (Steinkellner et al., 1997; Ueno et al., 2008; Tong et al., 2012), and {"type":"entrez-nucleotide","attrs":{"text":"CR627959","term_id":"75975942","term_text":"CR627959"}}CR627959 was predicted in the coding region of Cys-3-His zinc finger protein in nuclear genome (Ueno and Tsumura, 2008). All of these loci show a relatively high degree of transferability within the genus Quercus and an adequate degree of polymorphism of the studied taxa. For the 11 SSR primer pairs, 5′ end of forward primers were labeled with fluorescent dye tags (6FAM or HEX or ROX) (Sangon, Shanghai, China). The PCR reactions were performed in 20 μl reaction volume containing 10 μl TIANGEN PCR Master Mix (TIANGEN, Beijing, China), 0.3 μl/L of each primer (10 mM), and 20 ng genomic DNA; PCR reactions were performed as follows: 5 min initial denaturation at 94°C, 35 cycles of 40 s at 94°C, 30 s at 55°C, 1 min elongation at 72°C, and 7 min extension at 72°C. Finally, Gene-Scan-500 LIZ size standard (Applied Biosystem™) was added to all the samples before loading on an automated sequencer ABI 3730 (Applied Biosystem™). The final step was performed by a private professional commercial lab (Shanghai Majorbio Bio-pharm Technology Co., Ltd, Shanghai, China).

The AFLP method was performed following the protocol by Vos et al. (1995), with minor modifications. Briefly, 500 ng of genomic DNA were double-digested using 5 U of EcoRI and 2 U of MseI (New England BioLabs). The digestion mixtures were incubated at 37°C for 3 h and the digested mixture was then incubated at 70°C for 10 min to denature the enzymes. Subsequently, 4 μL of digested DNA were added to 16 μL of ligation mix containing 2 U T4 DNA ligase (New England BioLabs), 5 pmol EcoRI, and 50 pmol MseI adaptor. The mixture was incubated at 10°C for 14 h and then denatured at 70°C for 10 min. The ligated DNA samples were diluted 5-fold with double sterile water. Pre-selective amplification reactions were carried out using EcoRI-A (5′-GACTGCGTACCAATTCA-3′) and MseI-C (5′-GATGAGTCCTGAGTAAC-3′) in a 50 μL volume containing 1.5 mmol/L MgCl₂, 200 μmol/L of each dNTP, 1.25 μmol/L of each primer, and 0.6 U rTaq DNA polymerase (Takara Biotechnology, Dalian, China) under the following cycle: 3 min at 72°C, 30 cycles of 30 s denaturing at 94°C, 30 s annealing at 56°C, 1 min extension at 72°C, and a final extension for 5 min at 72°C. After a 1:20 dilution of pre-selective PCR products, seven primer combinations were performed in selective amplification (EcoRI-ACG/MseI-CAC, EcoRI-AGG/MseI-CAA, EcoRI-AGG/MseI-CAC, EcoRI-AGG/MseI-CAG, EcoRI-AGG/MseI-CTA, EcoRI-AGG/MseI-CTC, EcoRI-AGG/MseI-CTT). The EcoRI primers were fluorescently labeled with 6-FAM. These primer pairs were chosen because they generated clear and fewer bands (thus decreasing the risk of fragment non-homology) with sufficient variability in preliminary tests. Selective PCRs were carried out in a 20 μL volume containing 2.5 mmol/L MgCl₂, 200 μmol/L of each dNTP, 1.25 μmol/L of each primer, and 0.2 U of rTaq DNA polymerase (Takara Biotechnology, Dalian, China) and under the following cycle: 3 min at 94°C, 9 cycles of 40 s at 94°C, 30 s at 65–57°C touchdown (reducing the temperature at 1°C per cycle), 15 min at 72°C, 20 cycles of 40 s at 94°C, 30 s at 56°C, 1.5 min at 72°C, and a final extension for 7 min at 60°C. The PCR products were 10-fold diluted and mixed with Gene-Scan-500 LIZ size standard (Applied Biosystem™); products from each primer combination were loaded separately on an automated sequencer ABI 3730 (Applied Biosystem™) by the same commercial service provider mentioned above.

Raw data of SSR and AFLP samples were collected and analyzed using GeneMarker®v2.2.0. The samples with low quality of size calibrations or peaks were excluded from allele calling. The allele sizes of SSR were read manually and MicroChecker v 2.2.3 (Van Oosterhout et al., 2004) was used to check for potential errors. For AFLP, the variable fragments in the size range 50–500 base pairs (bp) were manually scored as present (1) or absent (0). We only considered fragments with similar fluorescence profile and intensities across the samples to maximize the probability of homology.