Screening of low-copy-number nuclear genes
Low-copy-nuclear (LCN) genes are ideal molecular markers for inferring genetic relationships between plant lineages and identifying potential mechanisms of selection driven by environmental factors (Du et al., 2020; Levin, Whelan, & Miller, 2009; Li et al., 2017). We screened LCN genes using the genome of S. baicalensis and its associated annotation file as a reference (Zhao et al., 2019). The genes were screened using two thresholds: (1) gene length > 500 bp and (2) physical distance > 100 kb. Primer3 (Untergasser et al., 2012) was used to design primers with a CG content of 40%–60%, length of 18–25 nt, and annealing temperature of 55–65 °C. A total of 132 primer pairs were designed and tested in polymerase chain reaction (PCR) analysis of 14 individuals representing seven Scutellariaspecies (Table S2). Amplicons that produced a single bright band were assumed to be LCN genes and were verified by Sanger sequencing. Finally, 52 LCN genes that were successfully amplified in all seven species were retained for subsequent sequencing (Table S6).