Chloroplast and Nuclear DNA Extraction and Sequencing

Genomic DNA was extracted using the Favorprep^TM Plant Genomic DNA Extraction Mini Kit (Favorgen, Taiwan) according the manufacturer’s suggested protocol. The isolated DNA was quantified using a Nanophotometer (Implen, Germany). A total of five chloroplast DNA (cpDNA) regions—two coding cpDNA loci, matK and rbcL; the trnL intron; and two non-coding cpDNA intergenic spacer loci, trnL-trnF and psbC-trnS—and an nrDNA ITS region were amplified (Table 2). PCR was conducted in a final reaction volume of 25 μL, containing 12.5 μL of 2x PCRBIO Taq Mix Red (PCRBiosystems, United Kingdom), 10 mM of each primer, and 20 ng of genomic DNA as a template. PCR amplification was conducted in a MyCycler^TM thermal cycler system (Bio-Rad, United States). PCR conditions (Lee et al., 2016 (link)) and annealing temperatures for each primer set are shown in Supplementary Table S1. PCR products were visualized in 1% agarose gel prior to direct DNA sequencing (ABI PRISM 3730xl Genetic Analyzer, Applied Biosystems, United States), performed by the First Base Laboratory Sdn. Bhd., Malaysia. In the case where targeted sequences were available in the GenBank database through previous studies of the same plant specimen, the records were included in this study without performing additional PCR and sequencing (Table 3).