2.3 DNA Extraction and PCR Amplification
The total genomic DNA was extracted from dried leaves of 1340 samples
using the Plant genomic DNA kit (Biomed DL114-01, China) following the
CTAB protocol (Saghai-Maroof et al., 1984) with minor
modifications. The DNA amplification was carried out using a 2,720
thermal cycler (Applied Biosystems, USA) in a 15 μl reaction mixture.
Each reaction contained 5 μl buffer (ddH2O), 7 μl 2 × Taq PCR MasterMix
(0.1 U Taq Polymerase/μl, 500 μM dNTP each, 20 mM Tris–HCl, 100 mM KCl,
3 mM MgCl2) (TIANGEN KT201, TIANGEN Biotech (Beijing) Co., Ltd), 1 μl
each primer (forward and reverse) (10 μM), and 1 μl template DNA. The
PCR cycling was performed at 94 °C, 3 min; 35 cycles of 94 °C, 30 s; 55
°C, 30 s; and 72 °C, 1 min; and 72 °C, 10 min for the final extension.
The PCR products (2 μl) were diluted with 8 μl of ultrapure water and
scoured with 100% alcohol for 15 min. The diluted DNA was then dried
and mixed with highly deionized formamide (Applied Biosystems, USA) and
submitted to fragment analysis by capillary electrophoresis using an
Applied Biosystems 3130 xl DNA Analyzer (Applied Biosystems, USA). The
analysis of DNA fragment size and allele calling was performed using
GeneScan and GeneMapper software (Applied Biosystems, USA), followed by
manual allele binning. To analyze the FAM-, TAMRA-, and HEX-labelled DNA
fragments with the ABI3130 series systems (Applied Biosystems, USA), the
matrix standard kit was used to generate the ”multi-component matrix”.
The Data Collection Software (Applied Biosystems, USA) was used to
record the data of the multi-component matrix. The instrument
automatically analyzed three different coloured fluorescent dye-labelled
samples in a single capillary, and reference lanes were further verified
by polyacrylamide gel electrophoresis. In the case of non‐amplification,
the PCR was repeated to exclude technical failure, and a null allele was
recorded if both PCRs failed.