Primer premier v5

The mRNA expression of autophagy marker LC3 was quantified using real-time quantitative polymerase chain reaction (RT-qPCR). Total RNA was isolated from rat RV and LV free wall tissues using TRIzol (Invitrogen, Carlsbad, CA, USA), and then subjected to reverse transcription into cDNA using a Reverse Transcription Kit (Takara, Dalian, China). The purity of RNA (260/280 nm ratio) was determined spectrophotometrically using the NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific Inc., Rockford, IL, USA). Two-step qRT-PCR was used to quantify relative LC3 mRNA by the ABI PRISM® 7500 qPCR System (Applied Biosystems, Foster City, CA, USA) using SYBR Green. The relative LC3 mRNA expression was normalized to the level of glyceraldehyde-3 phosphate dehydrogenase (GAPDH) using ΔΔC_T method. Each reaction was carried out in triplicate. Using Primer Premier v5.0 software (PREMIER Biosoft International, Palo Alto, CA, USA), primers for LC3 and GAPDH were designed as follows:

rat LC3 (NCBI accession number: NM_199500.2): 5′-CGAGAGCGAGAGAGATGAAGACGG-3′ (sense) and 5′-GGTAACGTCCCTTTTTGCCTTGGTA-3′ (antisense);

rat GAPDH (NCBI accession number: NM_017008): 5′-TCCATGACAACTTTGGCATCGTGG-3′ (sense) and 5′-GTTGCTGTTGAAGTCACAGGAGAC-3′ (antisense).

The specific primers used to isolate the 3’-UTR sequences of the Large White pig BMP7 and BMP15 genes were designed using Primer Premier v5.0 software (PREMIER Biosoft, Palo Alto, CA, USA), according to the sequences of the porcine BMP7 and BMP15 genes provided by the NCBI database (https://www.ncbi.nlm.nih.gov/). The primers (listed in Table 1) were synthesized by Tsingke Biological Technology (Tsingke, Beijing, China).

The highly repeated ITS region of P. beihaiensis (GenBank accession no. JN054741) was selected as a target sequence. Candidate RPA primers were first sought using Primer Premier v.5.0 software (Premier Biosoft, Palo Alto, CA, USA) following TwistAmp® reaction kit (TwistDx) guidelines (the best primers are 30–35 bases in length and have 30–70% GC content). No suitable RPA primers were screened, as gel-electrophoresis results lacked bands or produced unequally sized bands for the target fragment. Of 10 pairs of candidate RPA primers, one pair of efficient RPA primers (Pits6-F and Pits6-R, amplicon size 186 bp) was subsequently screened using primers of normal length (20–25 bases) (Table 1). For adaptation to the lateral flow detection system, the 5′ end of the reverse primer (Pits6-R) was labelled with a biotin (Table 1). Of 5 candidate RPA probes, one probe was screened out and its 5′ end labelled with a carboxyfluorescein (FAM) group, a C3 spacer (SpC3) on the 3′ end, and a tetrahydrofuran (THF) residue to replace an internal base (Table 1).

To validate the reliability of the RNA-Seq analysis, we performed quantitative real-time PCR (qRT-PCR) using ten randomly selected genes. The primers were designed using Primer Premier v.5.0 software (Premier Biosoft International, Palo Alto, CA, United States). We synthesized the first-strand cDNA using a TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix Kit (TransGen Biotech, Beijing, China). We conducted the qRT-PCR on an ABI StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, United States) using SYBR Premix Ex Taq (TaKaRa, Dalian, China). We used Actin1 (Os03g0718100) as an internal standard to calculate the relative expression level and determined expression values based on the relative quantitative method (Livak and Schmittgen, 2001 (link)).

The first important step in RT-qPCR reference gene selection is to select an initial set of candidate reference genes. Seven genes that were commonly used as stable reference genes in abiotic stresses were chosen [2 , 19 (link), 23 (link)] (Table 1). Primers were designed using Primer Premier v5.0 software (Premier Biosoft International) with melting temperatures (T_m) of 83.3–90.5°C, primer lengths of 20–22 bp, and amplicon lengths of approximately 110–224 bp (Table 2). Specificity of the amplification product was tested by qPCR. The expected size of the primer amplicons was further verified by agarose gel electrophoresis. Amplicon purity was assumed where a single melting peak was produced.

For qPCR analysis, cDNA was synthesized using the PrimeScript^TM RT reagent kit (Perfect Real time; Takara, Japan) according to the manufacturer’s protocol with random hexamer primers. The primer pairs for qPCR were designed using the Primer Premier v5.0 software (Premier Biosoft, USA) and listed in Supplementary Table S3. Melting curve analysis was performed for each primer pair prior to further analyses. qPCR reactions were carried out in 96-well plates with CFX96^TM Real-Time PCR Detection System (Bio-Rad, USA) using SYBR Premix ExTaq^TM (Takara, Japan) quantitation PCR kit. cDNA was diluted five times for qPCR. The reaction mixture consisted of 2 μl diluted cDNA samples, 0.4 μl each of the forward and reverse primers (10 μM), 10 μl real-time master mix and 7.2 μl PCR-grade water in a final volume of 20 μl. The target gene expression levels were normalized using actin gene (TR77358|c0_g1_i4) as internal reference. The relative abundance of transcript levels was calculated relative to the internal reference gene according to the method proposed by Paffl^{73 (link)}. Three biological replicates and triplicates of each biological replicate were performed for each qPCR analysis and a NTC (non-transcribed control) was also included to confirm correct DNase digestion.

Plants of the S and R lines were grown together in a growth chamber at 25/15°C day/night with a 12-h photoperiod (200 μmol m⁻² s⁻¹ photosynthetic photon flux density, with ~75% relative humidity). Weed seedlings at the three- to four-leaves stage were treated with fenoxaprop-P-ethyl at the field-recommended rate (62.1 g ai ha⁻¹). Fresh shoots of the S and R plants were sampled at the same time at 0 (untreated), 6, 12, and 24 HAT, frozen in liquid N₂, and stored at −80°C. RNA was extracted from each sample using the TRIzol Reagent (Invitrogen) and assessed as described above.
Primers were designed using Primer Premier v.5.0 software (PREMIER Biosoft International) and summarized in Supplementary Table S3. For the genes encoded by multiple alleles, primers were designed based on the conserved regions of different alleles. RT-qPCR was performed on the CFX96 Touch Real-Time PCR Detection System (Bio-Rad). cDNA was prepared as described above from 0, 6, 12, and 24-h RNAs extracted from the S and R plants. The expression of each putative P450 gene was normalized relative to the mean of ACT, 18S, and RUBP, and the results were analyzed by the 2^−ΔΔCq method. Two threshold values including the fold change (twofold) and t-test (p < 0.05) were used to determine the up- or down-regulation of a target gene by fenoxaprop-P-ethyl treatment.