Lightcycler 480 2 real time system

Twelve unigenes related to anthocyanin biosythesis in the pink tea flower were selected for validation using quantitative real-time PCR (qRT-PCR). The specific qRT-PCR primers were designed (Supplementary Table S6). The qRT-PCR was conducted using the LightCycler® 480 II Real-Time System (LightCycler® 480 II cycler, Roche, Carlsbad, CA, USA) with a 96-well plate. The thermal profile for the PCR amplification was 95 °C for 5 min, followed by 40 cycles of 10 s at 95 °C and another 40 cycles at 60 °C for 30 s. The HieffTM qPCR SYBR Green Master Mix (No Rox) (Yeasen Biotech Co., Ltd., Shanghai, China) was used for all PCR reactions according to the instruction’s protocol. All qRT-RCRs analyses were conducted using three technical and three biological replicates. The reference gene (β-actin gene) was used as an internal expression control. The expression level of different genes to the control was calculated according to the 2−ΔΔCT method [67 (link)].

The quantitative real-time PCR (qRT-PCR) was performed as described before (Zhou et al., 2020 (link)). Briefly, total RNA was extracted from petals by using RNAqueous™ Total RNA Isolation Kit (Thermo, MA, United States). Primers of selected gene members were designed with the Primer Premier 5.0 software and listed in Supplementary Table 4. β-Actin was used as the reference gene. The fluorescence PCR reagent was the Hieff™ qPCR SYBR Green Master Mix (No Rox) (Yaesen Biotech Co., Ltd., Shanghai, China). The experiment and analysis were carried out on the LightCycler^® 480 II Real-Time System (Roche, CA, United States). Metabolome was carried out by using liquid chromatography–mass spectrometry.
The determination of the non-volatile metabolome of tea petals was described as earlier (Zhou et al., 2020 (link)). Briefly, 100 mg powder samples were extracted in 1.0 ml methanol (70%) at 4°C for 24 h, and 5 μl supernatant was injected into ultra-performance liquid chromatography (UPLC, Shimadzu Co., Kyoto, Japan) with a mass system (MS, Applied Biosystems 6500 Q TRAP, MA, United States). Metabolites were identified using MWDB (Metware Database, Metware Biotechnology Co., Ltd., Wuhan, China) and subject to the partial least squares (PLS) discriminant analysis. The significant dissimilarities of metabolites were set as the variable importance (VIP) ≥1 and the fold change ≥2 or ≤0.5.

Total RNA was extracted from whole bodies of the first- to fifth-instar nymphs and newly emerged brachypterous male and female adults. It was also extracted from salivary glands, guts, ovaries, fat bodies, and integuments dissected from brachypterous female adults. For each biological replicate, 50 nymphs, 20 adults, and various tissues from 200 brachypterous female adults were collected. Each experiment was repeated in triplicate. Total RNA was isolated with the SV Total RNA Isolation System (Promega, Madison, WI, United States) according to the manufacturer’s instructions. After DNase treatment and quantification, 1 μg total RNA sample was reverse-transcribed with a PrimeScript RT-PCR Kit (TaKaRa Bio Inc., Dalian, China). The quantitative polymerase chain reaction (qPCR) reactions were performed with a TB Green™ Premix Ex Taq™ Kit (TaKaRa Bio Inc.) according to the manufacturer’s protocol and run in a LightCycler^® 480 II Real Time System (Roche Diagnostics, Basel, Switzerland). Elongation factor 2α (ef2) served as the internal normalization controls. The qPCR primers are listed in Supplementary Table 1. The gene expression levels were calculated by normalizing the target gene mRNA level to ef2 mRNA abundance via the 2^–ΔCt method (Chyla et al., 2019 ; Tian et al., 2021 (link)).