Plant dna extraction kit

To validate the SSR markers, a total of 100 primer pairs were randomly selected and synthesized. Wild almond germplasm from six species (A. communis, A. ledebouriana, A. mongolica, A. pedunculata, A. tangutica, and A. triloba) were used to validate the SSR markers. The total genomic DNA was extracted from fresh leaves using a plant DNA extraction kit (TIANGEN^®, China). The DNA quality and concentration were tested with a NanoDrop ND 1000 spectrophotometer (Thermo Scientific, USA). PCR amplification reactions were performed in a 25 μL volume, containing 40 ng of DNA, 0.2 mM dNTPs, 1.5 pM aliquots of forward and reverse primers, 2.5 mM Mg²⁺, 1 U Taq DNA polymerase (TaKaRa Biotechnology Dalian Co., Ltd., China), and 1× Taq Buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl). PCR amplification was performed with the following conditions: initial denaturation at 94 °C for 5 min; 30 cycles at 94 °C for 30 s, a primer-specific annealing temperature for 60 s, and 72 °C for 90 s; and 72 °C for 7 min. PCR products were separated by electrophoresis on denaturing 6% polyacrylamide gels and visualized using silver staining. The molecular size of the amplified fragments was estimated using a 10-bp DNA ladder (TransGen Biotech, China).

Total DNA of fresh young leaves was extracted using a plant DNA extraction kit (TIANGEN Biotech, Beijing, China). Based on the quality, integrity, and concentration of the extracted DNA, the Illumina HiSeq PE150 double-end sequencing strategy was used to build the library. Then FastQC was used to evaluate raw read quality and then raw reads were filtered by removing low-quality reads at the cutoff of Q20 using Trimmomatic (Bolger et al., 2014 (link)) to obtain clean reads. GetOrganelle¹ was used to assemble the plastid genome sequence by selecting 15 million reads from the dataset of clean reads. Both our newly acquired plastid genomes and the downloaded plastid genomes from NCBI website were annotated using the online annotation tool GeSeq (Tillich et al., 2017 (link)). All the annotations were manually curated. In addition, we used HMMER (Wheeler and Eddy, 2013 (link)) and ARAGORN Version 1.2.38 (Laslett and Canback, 2004 (link)) to ensure the prediction accuracy of the encoded protein and RNA genes, respectively. Finally, the resulting plastid genome maps were drawn with Chloroplot (Zheng et al., 2020 (link)).

Total genomic DNA was extracted from 24 Uncaria samples (∼100 mg) according to the instructions of the plant DNA extraction kit (Tiangen Biotech Co., Beijing, China). All the DNA samples were stored at −20 °C before analysis. The ITS2 regions of nuclear DNA were amplified using the universal primers (ITS2F/3R) according to previously described methods [20 ]. The PCR mixture (30 µL) contained template DNA 2 µL, forward primer (10 µM) 1.2 µL, reverse primer (10 µM) 1.2 µL, Mix-Taq enzyme 15 µL and ddH2O 10.6 µL. The PCR conditions were 94 °C for 2 min, followed by 30 cycles at 94 °C for 30 s, 55 °C for 30 s and 72 °C for 1 min, with a final incubation at 72 °C for 10 min. PCR-amplified products were examined by 1.0% agarose gel electrophoresis before sequenced by the Sino GenoMax Co., Ltd (Beijing, China). To ensure the accuracy, samples were all sequenced in two-way mode.