Quantitative Color Analysis of Sesame Seed Coat

We also sampled parental seeds at 10, 15, 20, 25, and 30 DPA in Yangling, Shaanxi Province (N34°27′, E108°07′), in 2022. Quantitative color analysis of the seed coat was performed with a CIE-Lab color scale (Colorimeter, CS-820, Hangzhou, China) with a 6 mm aperture due to the small sample size (Dong et al., 2022 (link)). All samples were flash frozen in liquid nitrogen and stored at -80°C in the refrigerator until needed. Total seed RNA was extracted using a kit (DP441, TIANGEN, China) and first-strand cDNA was synthesized by the PrimeScript RT reagent kit (#6210A, Takara, Kusatsu, Japan). Three independent biological replicates of the qRT−PCR (#RR820A, Takara, Kusatsu, Japan) protocol were tested using cDNA as the template for each experiment. The sesame actin gene (SIN_1006268) was used as the internal reference gene (Wei et al., 2015 (link)), and relative gene expression was calculated using the 2^{− ΔΔCT} method (Livak and Schmittgen, 2001 (link)).