2.2 DNA extraction and AFLP genotyping
We extracted total DNA from each sample according to a modification of
the CTAB procedure (Doyle & Doyle, 1987) and accessed DNA quality using
1.0% agarose gel electrophoresis and A260/A280 ratio determined on a
Nanodrop 2000c. Our procedure to obtain AFLPs was based on a
modification of the method in Vos et al. (1995). First, we digested the
genomic DNA with the restriction enzymes EcoR I and MseIfor 5 h at 37°C, and ligated the adaptors of EcoR I andMse I to the digestion products over night at 4°C (Beijing Dingguo
Biotechnology Co., Ltd). Using the digested products, we performed a
two-stage PCR amplification comprising pre-amplification and selective
amplification. The selective amplification was conducted in 25 μl volume
of reaction mixture containing of 1.0 μl EcoR I/Mse I primer
combinations (AAC/CAA, AAG/CAC, ACA/CAG, ACT/CAT, ACC/CTA, ACG/CTC,
AGC/CTG, AGG/CTT; Table 1). Subsequently, we separated the
fluorescently-labeled fragments on an ABI PRISM 377 DNA Calibrator
(Applied Biosystems) using GeneScan ROX-500 with an internal size
standard, allowing visual inspection of all individual sites (Liu,
Harris, Gao, Su, & Ren, 2019). We recorded the presence or absence of
AFLP amplification bands (Figure S2) in a binary matrix as 1 or 0,
respectively, based on interpretations from GeneScan 3.1 (Applied
Biosystems). In total, we assessed 1728 AFLP markers for the 210
individuals, and all interpretations were performed randomly.