Rox reference dye

Expression levels of chitinase-like genes in B. tabaci were determined by qRT-PCR with gene-specific primers designed by Primer premier 5.0 (Table S1). ABI PRISM 7500 Real-time PCR System (Applied Biosystems, Foster City, CA, USA) was used for conduction of qRT-PCR with a 20-μL reaction system containing 0.4 μL of 50 × ROX reference dye (TIANGEN, Beijing, China), 0.6 μL of each specific primer, 1 μL of cDNA template, 7.4 μL of ddH2O, and 10 μL of 2 × SuperReal PreMix Plus (SYBR Green) (TIANGEN, Beijing, China). The qRT-PCR program was as follows: 95 °C for 10 min (initial denaturation), followed by 40 cycles of 95 °C for 5 s (denaturation), 60 °C for 15 s (annealing), and 72 °C for 35 s (elongation). qRT-PCR primers which meet the amplification efficiencies of 90%–110% were used and listed in Table S1. Relative expression levels were quantified using the 2^−ΔΔCt method [49 (link)]. Two reference genes 60S ribosomal protein L29 (RPL29) (GenBank accession no. EE596314) and elongation factor 1 alpha (EF1-α) (GenBank accession no. EE600682) were used for normalization of target genes expression [50 (link)]. For each sample, three biological replicates and four technical replicates were performed.

The specific primers for COEs of B. tabaci MED were designed using Primer Premier 5.0 and used to verify the expression levels of COEs by qRT-PCR (Table S2). qRT-PCR was performed using an ABI 7500 system (Applied Biosystems) with the 25-μL reaction containing 0.5 μL of each specific primer, 0.5 μL of 50 × ROX reference dye (TIANGEN, Beijing, China), 1 μL of cDNA template, 10 μL of ddH₂O, and 12.5 μL of 2 × SuperReal PreMix Plus (SYBR Green) (TIANGEN, Beijing, China). The qRT-PCR programme was as follows: 95 °C for 10 min (initial denaturation), followed by 40 cycles of 95 °C for 5 s (denaturation), 60 °C for 15 s (annealing), and 72 °C for 35 s (elongation). Only the qRT-PCR primers with 90–110% amplification efficiencies were used for the subsequent data analysis.
Relative expression levels were quantified using the 2^−ΔΔCt method [66 (link)]. The geometric mean of the reference genes 60S ribosomal protein L29 (RPL29) (GenBank accession no. EE596314) and elongation factor 1 alpha (EF1-α) (GenBank accession no. EE600682) was used to normalize the expression of target genes [67 (link),68 (link)]. Three biological replicates and four technical replicates were performed for each sample. One-way analysis of variance (ANOVA) (SPSS 23) was used to detect significant differences between samples.

The expression levels of target genes were quantified using the QuantStudio 3 Real‐Time PCR System (Applied Biosystems). The gene‐specific primers of BtFAD2 used for the real‐time quantitative PCR (qPCR) analysis were designed by the Primer Premier 5.0 software (Table S1, Supporting Information). The 25 µL PCR reactions included 0.5 µL of 50 × ROX Reference Dye (TIANGEN), 0.75 µL of each specific primer, 1 µL of cDNA template, 9.5 µL of ddH₂O, and 12.5 µL of 2 × SuperReal PreMix Plus (SYBR Green) (TIANGEN). The qPCR reactions were performed in an ABI 7500 system (Applied Biosystems) with the following protocol: initial denaturation of 94 °C for 3 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s. The amplification efficiencies were determined by dissociation curve analysis using five two‐fold serial dilutions of B. tabaci cDNA template. Only primers with 90%–110% amplification efficiencies were used for the subsequent studies.
Relative quantification was calculated according to the 2^−ΔΔCt method,^[47 (link)
^] to accurately analyze the expression of the target genes, the expression data were normalized to the internal gene elongation factor 1 alpha (EF1‐a) (GenBank accession number EE600682). Three independent biological replicates and four technical replicates were performed for each whitefly sample.

For real-time PCR (qPCR) experiments, the primer pair ga4F1/ga4R1 was used for specific amplification of C. cassiicola. qPCR was conducted in a 20-μL reaction volume containing 1 μL template DNA, 10 μL SuperReal PreMix Plus (Tiangen Biotech, Inc., Beijing, China), 0.4 μL of each 10 μmol/L primer, and 0.4 μL of 50 × ROX Reference Dye (Tiangen Biotech, Inc.). Amplification was performed in an Applied Biosystems 7500 Real-Time PCR System (ABI) with an initial denaturation step at 95°C for 15 min to activate the Taq polymerase, followed by 40 cycles of 95°C for 10 s and 60°C for 32 s. Cycle threshold (C_t) values were calculated automatically by ABI7500 software.
The standard curve for the quantification of C. cassiicola was generated by analyzing a 10-fold dilution series of spore suspensions from 5 × 10⁶ to 5 spores/mL (An et al., 2006 (link); Diguta et al., 2010 (link)). Each suspension of 1 mL was used for genomic DNA extraction by a Plant Genomic DNA Kit (Tiangen Biotech, Inc.) according to the manufacturer’s guidelines. The extractions were repeated three times for each spore concentration to create the DNA stock for the standard curve. All DNA was run in technical triplicates in 96-well reaction plates.