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).