<div>Gene-specific primers were used in PCR reactions (20µl) containing 7µl
of ddH2O, 10µl of 2xSYBR Green MasterMix (Bio-Rad), 1µl of each specific
primer (10mM), and 1µl of first-strand cDNA template. The qPCR program
included an initial denaturation for 3 min at 95°C followed by 40 cycles
of denaturation at 95°C for 10s, annealing for 30s at 55°C, and
extension for 30s at 72°C. For melting curve analysis, a dissociation
step cycle (55°C for 10s, and then 0.5°C for 10s until 95°C) was added.
All primers were tested for efficiency prior to use, and only primer
pairs with the same efficiency as the primers for housekeeping genes
were used. Primers for 18S and 28S were obtained from Pan et al.
(Pan <i>et al.</i> , 2015). Candidate
gene-specific primers used were as follows (F, forward primer; R,
reverse primer): <i>DPOGS201379-F</i> , 5’- CTGACCAGCACGAAGAGAAA-3’;<i>DPOGS201379-R</i> , 5’- GACAATATCCCGGCGAATAGAA-3’;<i>DPOGS211203-F</i> , 5’-GATGCGATTGCTGCATTGAATA-3’;<i>DPOGS211203-R</i> , 5’- ATACCGCTGCCATCACTAAC-3’; <i>DPOGS202675</i> -F,
5’-CTCCCTTGTCGTGATGTTGT-3’; <i>DPOGS202675</i> -R,
5’-GTCGGCTCTCAATCCAGTAAA -3’; <i>DPOGS200868</i> -F,
5’-TCGGAACAGGAGGAGTATCT-3’; <i>DPOGS200868</i> -R,
5’-GCCTCTATGCCTCTCTTCTATG-3’; <i>DPOGS215054</i> -F,
5’-GTCGCTGACTTCTCCATCATAC-3’; <i>DPOGS215054</i> -R;
5’-GTTCTCGTTCAGAGGATCCATATT-3’; <i>DPOGS211604</i> -F,
5’-CAACGAGGAAGCCAGACTAAA-3’; <i>DPOGS211604</i> -R,
5’-TGTGGCATTGGTCTTCCATAA-3’.</div><div>Due to homogeneity-of-variance and error normality assumptions, we could
not use linear models to analyze gene expression. Instead, we used
one-tailed Mann-Whitney U tests in R 3.1.3
(R Development Core Team, 2012) to
determine whether gene expression was higher in eastern than western
monarchs.</div>