Cfx96 touch deep well real time pcr system

We genotyped all individuals with a diagnostic SNP developed by Souissi et al. (2022 (link)) in order to detect hybrids since there is a high level of genetic admixture between L. fluviatilis and L. planeri in the Oir population (Rougemont et al., 2015 (link)). L. fluviatilis and L. planeri individuals are homozygous at this marker called diagLpf (genotypes ff or pp, respectively) whereas hybrids are heterozygous (pf). We kept only homozygous individuals for competitive fertilization experiments in order to determine the paternity of offspring with the same marker. We used the kit NucleoSpin® 96 Tissue (Macherey‐Nagel, Düren, Germany) to extract DNA from fin clips according to the manufacturer's instructions. Then, we genotyped all individuals at the diagLpf locus with quantitative polymerase chain reactions (qPCR) by using the protocol of Souissi et al. (2022 (link)). The PCR products were run on a CFX96 Touch Deep Well Real‐Time PCR System (Bio‐Rad) and analyzed with CFX Maestro™ Software (version 4.1.2433.1219). We analyzed sperm traits for 26 males homozygous at the diagLpf locus: 13 L. fluviatilis and 13 L. planeri.

Total RNA was extracted using SteadyPure Plant RNA Extraction Kit [Accurate Biotechnology (Changsha, Hunan) Co., Ltd.]. The RNA was reverse-transcribed (Evo M-MLV One Step RT-PCR Kit) into cDNA and quantitative RT-PCR (qPCR) analysis performed using gene-specific primers (Supplementary Table 8). 18s gene was used as an internal control (Accession Number: EU747115). The qPCR was conducted using SYBR^® Green Premix Pro Taq HS qPCR Kit II (Accurate Biotechnology (Changsha, Hunan) Co., Ltd.) on CFX96 Touch Deep Well Real-Time PCR System (Bio-Rad) according to standard procedure. Relative transcript levels were calculated using the 2^–ΔΔCt formula and the results displayed using histograms drawn with GraphPad Prism 8 software. All histograms were merged using Adobe Photoshop (2020) software.

The real-time PCR assays were all performed on the CFX96 Touch Deep Well Real-Time PCR System (Bio-Rad, Hercules, CA, USA) in a final volume of 20 μL. The reaction mixture of SYBR Green real-time PCR contained 10 μL of 2× iTaqTM Universal SYBR Green Supermix (Bio-rad), 0.8 μL of each primer (10 μM), 1 μL of template DNA, and a final addition of ddH₂O to 20 μL. The PCR program followed these steps: 95 °C for 15 min denaturation; 50 cycles of 95 °C for 10 s, 60 °C for 30 s, 72 °C for 30 s; then a melting curve ranging from 65 °C to 95 °C at 0.5 °C/s. The reaction mixture of TaqMan real-time PCR included 10 μL of 2× Heiff UNICON^® qPCR TaqMan Probe Master Mix (Yeason, Shanghai, China), 0.8 μL of each primer (10 μM), 0.4 μL of TaqMan Probe (10 μM), 1 μL template DNA, and a final addition of ddH₂O to 20 μL. The TaqMan real-time PCR was performed following this program: 95 °C for 1 min, 45 cycles of 95 °C for 10 s, 60 °C for 60 s. The temperature of annealing and extension was increased to 66.8 °C when the LNA-containing probe was used for real-time PCR. The final data were collected using Bio-Rad CFX Manager software (v3.1.1517.0823).

Total RNA from leaves was extracted using the RNeasy Plant Mini Kit (Qiagen, Germany). RNA quality and quantity were assessed using a previously described method (Song et al., 2022 (link)). cDNA was synthesized using M-MLV reverse transcriptase (Promega, USA) following the manufacturer’s protocol. RT-qPCR was performed in a CFX96 touch deep well real time PCR System (Bio-Rad, USA) with the program in a previous study (Liu et al., 2020 (link)). RT-qPCR was used to examine the expression of the stomatal regulating genes SPCH, MUTE and FAMA from Saccharum spontaneum, Sorghum bicolor and Oryza sativa for qPCR. β-TUBLIN genes were used as an internal control to normalize the relative expression of genes. A standard cycling program was used for qPCR: initial denaturation at 94°C for 2 min, followed by 40x cycles of denaturation at 94°C for 15s and annealing at 65°C for 1 min. The primers used for Q-PCR are listed in Supplementary Table 2.

Total RNA of cell samples was extracted using the Monarch Total RNA kit (NEB, T2010S) and then reverse transcribed with iScript cDNA Synthesis kit (Biorad, 1708890). cDNA was measured using a CFX96 Touch Deep Well Real-Time PCR system (Biorad) with the probe-based PrimeTime Gene Expression Master Mix (IDT, 1055770). Fold changes in cDNA were determined using the ΔΔC_t method normalized to PPIA cDNA. Primer pairs were purchased from IDT's predesigned PrimeTime Standard qPCR assay; sequences are as follows:

For qRT-PCR analysis, two weeks-old Arabidopsis plants were grown under the same conditions as described for growth measurements. Total RNA was isolated as described for RNAseq analysis. One microgram of DNase-treated RNA was used for cDNA synthesis using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). qRT-PCR was performed on 3 μl of 20× diluted cDNA templates using 5× HOT FIREPol EvaGreen qPCR Mix Plus (Solis Biodyne) in a final volume of 8 μl, and Bio-Rad CFX96 Touch Deep Well Real-Time PCR System. Mean values of polyubiquitin 10 (UBQ10, AT4G05320) and glyceraldehyde-3-phosphate dehydrogenase C2 (GAPC2, AT1G13440) Ct were used as internal reference. Normalized relative transcript levels were obtained by the 2^−ΔΔCt method (Livak and Schmittgen, 2001 (link)). Experiments were repeated with three biological replicates. Oligonucleotides used in this study are listed in Supplementary Table 12.