DNA Extraction and Sequencing Protocol for Plant Barcodes

Leaf tissues were first dried in silica gel. Ten milligrams of each of the dried tissues was rubbed for one minute at a frequency of 30 times/second in a FastPrep bead mill (Retsch MM400, Germany). DNA extractions were performed using the Plant Genomic DNA Kit (Tiangen Biotech Co., China) according to the manufacturer's instructions. The sequences of the universal primers for the DNA barcode to be tested, including those for psbA-trnH, matK, rbcL, rpoC1, ycf5 and ITS, and general PCR reaction conditions were obtained from previous studies [9] (link), [17] (link), [18] (link), [21] (link). Based on the conserved regions of 18S and 5.8S, we designed four pairs of primers for ITS1. Similarly, according to a previous study [25] (link) and the conserved regions of 5.8S and 26S, we also designed four pairs of primers for ITS2. PCR amplification was performed in 25-µl reaction mixtures containing approximately 30 ng of genomic DNA template, 1 X PCR buffer without MgCl₂, 2.0 mM MgCl₂, 0.2 mM of each dNTP, 0.1 µM of each primer (synthesized by Sangon Co., China) and 1.0 U Taq DNA Polymerase (Biocolor BioScience & Technology Co., China), with a Peltier Thermal Cycler PTC0200 (BioRad Lab, Inc., USA). Purified PCR products were sequenced in both directions with the primers used for PCR amplification on a 3730XL sequencer (Applied Biosystems, USA). To estimate the quality of the generated sequence traces, the original forward and reverse sequences were assembled using CodonCode Aligner 3.0 (CodonCode Co., USA). Base calling was carried out using the Phred program (version no. 0.020425.c). The quality values were defined for three levels: low quality (0 to 19 QV), medium quality (20 to 30 QV) and high quality (higher than 30 QV). The sequences showing >2 bases with a quality value below 20 QV in a 20-base window were trimmed. The forward and reverse reads have a minimum length of 100 bp, a minimum average QV of 30, and the post-trim lengths should be >50% of the original read length. In addition, the assembled contig should have a minimum average QV score of 40 and >50% overlap in the alignment of the forward and reverse reads. All sequences of the second set of plant samples containing the “internal transcribed spacer 2”or “psbA-trnH” were retrieved according to Keller et al. [42] (link) and GenBank annotations. Subsequences marked as ITS2 or psbA-trnH intergenic spacer were recovered after deleting sequences with ambiguous nucleotides and those shorter than 100 bp.