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4.3. DNA Extraction, PCR Amplification and Sequencing
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Rooibos genomic DNA was extracted using a modified protocol adapted from [45] and [46]. Young rooibos leaves were ground in liquid nitrogen using mortar and pestle to fine powder. For each gram of leaf tissue, 4ml of heated (65 °C) CTAB extraction solution (100 mM Tris-Cl pH 8, 20 mM EDTA pH 8, 1.4M NaCl, 2% w/v CTAB, 1% w/v PVP, 2% v/v 2-ME) and 0.5 mL CTAB/NaCl solution (10% w/v CTAB, 7M NaCl) was added. After vigorous mixing, the homogenate was transferred into a 15 mL falcon tube. An equal volume of CTAB extraction solution was added, the solution was mixed thoroughly by inversion and then incubated at 65 °C for 1 h. To maximize yields, the solution was vortexed every 15 min. Thereafter, an equal volume of Chloroform: Isoamyl alcohol (24:1) was added and mixed by inversion. The top aqueous phase was recovered after centrifugation for 5 min at 7500× g. A 1/10th volume of 65 °C CTAB/NaCl solution was added to the supernatant and mixed well by inversion. The top aqueous phase was recovered after centrifugation for 5 min at 7500× g. An equal volume of CTAB precipitation solution (50 mM Tris-Cl pH 8, 10 mM EDTA pH 8, 1% w/v CTAB) was then added and the solution was incubated at 37 °C overnight. After centrifugation for 5 min at 500× g and 4 °C, the supernatant was recovered. DNA was precipitated by adding 0.6 volumes of isopropanol to the supernatant, mixed well and centrifuged for 15 min at 7500× g at 4 °C. The pellet was then washed with 80% ethanol, dried, re-suspended in 200 μL high salt TE buffer (10 mM Tris-Cl pH 8, 0.1 mM EDTA pH 8, 1M NaCl) per gram starting material and stored at −20 °C.
The ITS region was PCR amplified using the 17SE and 26SE primers published by Sun et al. [47]. Each reaction tube contained 30–50 ng of template DNA, 10 µL of 5X High-Fidelity Phusion® reaction buffer (Biolabs® Inc, New England), 5 µL of dNTP stock solution (containing 2 mM of each dNTP), 2.5 µL of each primer (10 µM; Inqaba Biotec, Pretoria, South Africa), 0.5 µL of 2 u/mL Phusion® High-Fidelity DNA polymerase (Biolabs® Inc, France SASU, New England) and nuclease-free water (final volume 50µL). The amplification was conducted in a T100 thermal cycler (Bio-Rad, USA) under the following conditions: initial denaturation at 98 °C for 3 min, 34 cycles of denaturation at 98 °C for 10 s, annealing at 72 °C for 30 s, and extension at 72 °C for 30 s; and a final extension at 72 °C for 10 min. To verify the size and concentration of the PCR products, 8 µl of the PCR reactions were analyzed on a 1.2% (w/v) agarose gel using PstI as a molecular weight marker. For samples that showed no bands or weak bands on the agarose gel, the PCR reactions were repeated. The PCR products were purified using the QIAquick PCR Purification Kit (Qiagen, Germantown, Maryland, USA) and stored at −20 °C. Amplicons were sequenced bidirectionally (with 17SE and 26SE primers) by Macrogen Inc. (Netherlands, Europe) using a 96-capillary ABI 3730xl DNA analyzer (Applied Biosystems, Foster City, Carlifonia, USA) following standard procedures. Sequencing reads were quality trimmed using CLC Genomics Workbench version 7.0.1 (QIAGEN-Bioinformatics, Germantown, Maryland, USA) using a quality limit of 0.01, removing all bases below a quality score of 20. Quality processed reads (Appendix A) were aligned with published ITS sequences from the Aspalathus genus obtained from NCBI (AJ74495, EU347722, EU347738, EU347723, EU347736, EU347729, EU347726, EU347732, EU347734, EU347731, EU347728, EU347733, EU347727, EU347725, EU347730) using MAFFT [48] with default settings, and the alignments were manually edited using MEGA X [49].
DNA library preparation for genome sequencing was performed at the UKHC Genomics Core Laboratory (UK Chandler Hospital Lexington, KY 40536, USA). One paired-end 300 bp insert size library was prepared using the Nextera DNA Library Preparation kit (Illumina, San Diego, CA, USA), following the manufacturer’s instructions. Sequencing was performed using two flow cells (1 lane each) on the Illumina MiSeq platform (Illumina, San Diego, CA, USA) and one flow cell (six lanes) on the Illumina HiSeq 2500 platform (Illumina, San Diego, CA, USA), generating in total 2.5 billion read pairs of 120 bp length according to the manufacturers protocols. Adapter trimming and removal of the PhiX spike was performed by the service provider using Illumina’s bcl2fastq2 Conversion Software v2.20 (Illumina, San Diego, CA, USA). Subsequently, quality trimming was performed using Trimmomatic (v0.38) [50] to remove remaining adapter sequences, leading and trailing nucleotides with a Phred score below 20, and reads shorter than 50 bp (ILLUMINACLIP:NexteraPE-PE.fa:2:30:10 LEADING:20 TRAILING:20 MINLEN:50). Error correction was performed on filtered reads using Lighter (v1.1.1) [51] with k set to 31, α set to 0.1, and specifying a 2x genome coverage of 2.5 Gbp (the use of a 2x genome coverage was suggested by the author of the program due to high heterozygosity predicted for the rooibos genome, personal communications). Quality filtering was performed for each lane, and subsequently assessed using FastQC (v0.11.7) [52] and MultiQC (v1.7) [53].
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