*4.9. RNA Extraction and qRT-PCR Analysis*

Total-RNA was extracted from different samples using Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the instruction of manufacturer's protocol. Further treatment of RNA was achieved with RNase-free DNaseI to eliminate DNA contamination. The cDNA was synthesis by using the Prime-Script TM RT Reagent Kit (TaKaRa, Dalian, China). The quality of cDNA was checked by nanodrop (Thermo Scientific NanoDrop 2000C, Wilmington, DE, USA) and the required volume was calculated and adjusted the concentration up to 50 ng/µL. For qRT-PCR analysis, the gene-specific primers (Table S5) were designed by using Primer Premier 6.0 software package (Available online: http://www.premierbiosoft.com/primerdesign/index.html). The specificities of the primers were further confirmed through NCBI Primer BLAST (Available online: https://www.ncbi.nlm.nih.gov/ tools/primer-blast/). The pepper ubiquitin-conjugating protein gene (*CaUbi3*) was used as internal control [91], with a little modification of annealing temperature (60 ◦C for 30 s). The relative expression levels of all the CaChi genes were calculated using the 2−∆∆Ct method [92].
