Beacon designer 7

The RNA sequencing samples that were isolated were also used to perform real-time quantitative (qPCR) analysis. Total RNA samples with 2 μg per reaction were reverse-transcribed into cDNA using M-MLV Reverse Transcriptase (Promega, Madison, WI, USA). qPCR-specific primers were designed based on the candidate gene sequences close to the 3’ end using Beacon Designer 7.0 software from Premier Biosoft International, Palo Alto, CA, USA. The Histone3 (GenBank NO: AF024716) was used as reference gene. The primers of histone3 and GarWRKY5 were forward primer 5’-CGGTGGTGTGAAGAAGCCTCAT-3’ and reverse primer 5’-AATTTCACGAACAAGCCTCTGGAA-3’ and forward primer 5’-GCCTTGTCATTTCATGGTGGA-3’ and reverse primer 5’-GGGTTGTCGTTGCCTTGC-3’, respectively. The light cycler carried out using IQ SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) based on the manufacturer’s instructions and the qPCR products were quantified using ABI 7500 Fast (Applied Biosystems, Foster City, CA, USA). The amplification reaction conditions for PCR were performed as follows: 94 °C for 3 min, followed by 40 cycles at 94 °C for 15 s, 60 °C for 15 s, and 72 °C for 30 s. The relative expression levels were calculated using the 2^−ΔΔCt method with three biological replicates and three experimental replicates [42 (link)].

The sequences of the primers and probes for expression analysis of the chosen genes Ccn1, Dusp1, Fos, Klf2, and Zfp36 and reference genes Psmd6 and Sars were designed using Beacon Designer 7.0 software (Premier Biosoft International).
Relative levels of the transcripts in the experimental groups were calculated as R = 2^(– ΔΔCt)^{78 (link)}. The levels of the transcripts studied in the control group were set as 1. The significance of group differences was evaluated using the nonparametric Kruskall–Wallis test in the R programming language.

Total RNA was extracted from the collected samples with a method modified from the previously described [48 ]. First strand cDNA synthesis was carried out using 1 μg total RNA and a PrimeScritpt RT reagent Kit (TaKaRa, Japan) following the manufacturer’s instructions. Reverse transcription - quantitative PCR (qRT-PCR) was performed as previously described [7 , 33 (link)]. The gene-specific primers used in the qRT-PCR analysis of MKK genes were designed using Beacon Designer 7.0 software (Premier Biosoft International, USA) and targeted the 3′ UTR of each of the genes and designed to generate approximately 200 bp products (Additional files 8: Table S5). Grapevine actin gene was selected as an internal control to normalize the total amounts of cDNA present in each reaction. The relative expression levels of the target genes were assessed using the 2^-ΔΔCt method, where ΔΔCt = (Ct_{target gene} - Ct_actin) _treatment - (Ct_{target gene} - Ct_actin) _control [7 ]. Each sample comprised three biological replicates and the analysis represented the results of three independent experiments.

Quantitative real-time PCR was carried out in 96-well plates with an ABI7500 Real-Time PCR Detection System (Applied Biosystems, USA) using SYBR Green Master ROX (Roche, Japan) following the manufacturer’s instructions. Primers were designed using Beacon Designer 7.0 (Premier Biosoft International, USA); Primer 5.0 (Premier Biosoft International, CA) and the reference genes ACT2/7 and UBQ10 were selected for use based on previous research (Additional file 8). Reaction mixtures contained 10 μL SYBR Green Mix, 0.2 μM of each primer and 1.5 μL of 1:10 diluted cDNA as template in a final volume of 20 μL. Reactions were subjected to the following conditions: 95 °C for 10 min, followed by 40 cycles of 95 °C for 30 s and 58 °C for 1 min [26 (link), 74 (link)]. After the reactions, a melting curve analysis was conducted to evaluate the primer specificity. The relative expression levels of the selected transcripts, normalized to ACT2/7 and UBQ10, were calculated using the 2^-∆∆Ct method. All reactions were performed with three independent biological replicates, and the expression levels calculated for each sample were based on three technical replicates. Data were analyzed using the ABI7500 manager software, and statistical analyses were conducted with SAS Version 9.0 software (SAS Institute, Cary, NC, USA) using Duncan’s multiple range test at the P < 0.05 level of significance.

Based on the known sequences of V. dahliae strain VdLs17 available from the genome database (http://genome.jgi.doe.gov/Verda1/Verda1.home.html), gene‐specific primers (Table S1) were designed using Primer 5.0 software (PREMIER Biosoft, Palo Alto, USA) to amplify the homologous genes of RGS with complete open‐reading frames in the Vd8 strains. High‐fidelity ExTaq DNA polymerase (Takara Biotechnology [Dalian] Co. Ltd., Dalian, China) was used in standard PCRs, and all the PCR products were cloned into pMD19‐T cloning vectors (Takara, Dalian, China) and transformed into E.coli DH5α bacterial strains obtained from our laboratory (State Key Laboratory of Crop Genetics & Germplasm Enhancement, Nanjing Agriculture University, Nanjing, China). At least five clones per gene were randomly selected and sequenced.
For the quantitative real‐time PCR (qPCR) analysis, gene‐specific primers (Table S1) were designed using Beacon Designer 7.0 (PREMIER Biosoft, Palo Alto, USA), and β‐tublin (KF555285.1) was used as a reference gene. Real‐time PCR amplification reactions were performed on an ABI 7500 Real Time PCR System (Applied Biosystems, Carlsbad, USA) using AceQ SYBR Green Master (Low Rox Premixed) (Vazyme) with three technical replicates for each biological sample. Expression data from three biologically independent experiments were analysed and presented as means ± SD.

Total RNA used for RT-qPCR analysis was prepared from the P. sinensis testes in April, July, and October with three biological replicates. The cDNA was synthesized using the SuperScript First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA). Primer sequences were designed by using Beacon Designer 7.0 (Premier Biosoft International, USA) software (Supplementary Table 1). The RT-qPCR analysis with three technological replications was performed on an iCycler Real-Time PCR Detection System (Bio-Rad, USA) according to a previous report (Kim et al., 2013 (link)). The β-Actin gene was used as the internal control. The relative expression levels of the genes were calculated using the delta-delta-CT method.

qRT-PCR was performed to determine the expression patterns of UGT genes in different strains and tissues using an Applied Biosystems™ QuantStudio™ 6 Flex Real-Time PCR System. Primers were designed by Beacon Designer 7.0 (Premier Biosoft International, Palo Alto, CA, USA) and are listed in Table S3 (Supplementary Materials). A ChamQ^TM SYBR^® qPCR Master Mix (Vazyme biotech Co., Ltd., Nanjing, China) was used according to the manufacturer’s protocol. Normalization of each experimental gene expression level was conducted based on the geometric mean of two selected reference genes, G3PDH and Actin A1 [8 (link)]. The expression stability of the two reference genes was analyzed with geNorm Software [35 (link)]. Data were calculated according to the 2^−ΔΔCt method.