Primer premier 6

Quantitative real-time PCR was used as proof that high-throughput sequencing was accurate. To detect the expression trend of the sequencing results, 12 genes were chosen at random. TRIzol reagent (Invitrogen Corporation, Carlsbad, CA, USA) was used to extract the cells’ total RNA, and the Primer-Script RT reagent Kit (TaKara, Tokyo, Japan) was used to reverse-transcribe the extracted total RNA into cDNA. The CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) was used for qRT-PCR analysis. Based on sequence data of the NCBI database (www.ncbi.nlm.nih.gov/genbank, accessed on 20 November 2022), the specific primers utilized for RT-PCR were designed or modified by Primer Premier 6.0 software (Premier Biosoft International, Palo Alto, CA, USA) [26 (link)] and are listed in Table 2. The thermal cycling program consisted of 95 °C for 30 s, followed by 40 cycles at 95 °C for 5 s and 60 °C for 30 s. The housekeeping gene glyceraldehyde-3-phophate dehydrogenase (GAPDH) was used to standardize the expression levels of the genes, and the changes in relative gene expression were computed by the 2^−ΔΔCt method. Significant differences were evaluated by Student’s t-test using SAS software (version 9.0), and a value of p < 0.05 was considered to be significant, which was calculated at least three times from independent biological replicates.

Following the three treatments (Tri, Aalm, and Tri + Aalm) at 28°C and 70% humidity, the treatment of samples was the same as that described for RNA-Seq, three plants (biological replicates) each treatment were set. Leaf samples were collected at 6 and 48 hpi, and untreated seedlings served as controls, leaf samples were collected at 0 hpi. Total RNA was extracted using the CTAB method, and reverse-transcribed into cDNA for quantitative real-time polymerase chain reaction (RT-qPCR). The gene-encoding MsActin (MZ605395) in M. sieversii was used as an internal reference. Three technical replicates were performed for each cDNA sample. Primers for RT-qPCR (Supplementary Table 1) were designed by using Primer Premier 6.0 software (PREMIER Biosoft, San Francisco, CA, United States).

Cultured KCs were treated with Trizol to extract total cellular RNA. Extraction of total RNA was performed according to the manual. Total RNA was used as the template for the synthesis of first-strand cDNA. The resulting cDNA was used as the template, Taq DNA polymerase and the downstream and upstream primers for the target genes were added, and PCR amplification was performed according to the manual. Primer design was carried out using Primer Premier 6.0 software (PREMIER Biosoft International), and the primer sequences and product length used in gene detection are shown in Table 1. The reaction conditions were as follows: denaturation at 94°C for 5 min; 32 cycles of 94°C for 1 min, 51°C for 45 s, and 72°C for 45 s; and extension at 72°C for 10 min. The resulting PCR product was electrophoresed in 1.5% agarose gel, and β-actin was used as the internal reference. Electrophoresis results were measured by BIO-RAD Quantity-One 4.7 image analysis software to calculate the relative gray scales of the detected genes as compared with that of β-actin.

Based on the sequencing information of the cucumber genome database website (http://cucurbitgenomics.org/), the physical location of the locus was determined, and the genes within the confidence interval were identified. Gene function was analyzed and predicted by BLAST in the CuGenDB, Swiss-Prot, TAIR and Gene Ontology (GO) databases to identify the candidate genes [16 (link)]. The candidate genes were selected based on the gene annotation and the function of its homolog in other plants. Primer Premier 6.0 software (Premier Biosoft, San Francisco, CA, USA) was used to design primers (Supplementary Materials Table S3). PCR amplification was performed with 2 × Phanta Max Master Mix (Vazyme, Nanjing, China), and the PCR products were sequenced by Sangon Biotech. The candidate genes in the parents CG104 and CG37 were amplified by PCR, and SnapGene 4.26 was used for sequence alignment and analysis.

In this study, an RNA-Seq experiment with 3 individual leaf samples was used to identify RNA editing events. The total RNA was extracted from mature foliage using an Extract kit (RP3301, BioTeke, China). The RNA-Seq library construction and sequencing were performed at Novogene Bioinformatics Technology Co., Ltd. (Beijing, China). The filtered paired-end reads obtained from an Illumina HiSeq 2000, were aligned to the B. platyphylla cp genome using HISAT2 (v2.1.0) software with strict comparison conditions. SAMtools (v1.9), bedtools (v2.25.0) and ChloroSeq were used to call and analyse precise RNA editing sites [28 ]. Because SNPs or mismatches may interfere with the results, we also mapped the set of PE 100 bp-long reads that was used to assemble the B. platyphylla cp genome back to the cp genome sequence using bowtie2 (v2.3.4.1) software and then checked the SNPs. Finally, we designed several pairs of primers using Primer Premier 6.0 software (PREMIER Biosoft International, Canada) and amplified the target sequence by PCR to form genomic DNA (gDNA) and complementary DNA (cDNA). The target representative editing sites were confirmed by Sanger sequencing. The relevant primer information is summarized in Additional file 1: Table S2.

Specific primers for amplification of the 3′ and 5′ ends were designed using Primer Premier 6.0 software (Premier Biosoft International, Palo Alto, CA, USA) (Table S2). The SMARTer^® RACE 5′/3′ Kit (Clontech, Mountain View, CA, USA) was used to perform 3′ and 5′ RACE reactions. First-strand cDNA synthesis and PCR amplification were performed according to the instructions of the SMARTer^® RACminE 5′/3′ Kit. The amplification reaction procedure was as follows: 25 cycles of 94 °C for 30 s, 72°C for 30 s, and 72 °C for 3 min. The PCR products were purified and cloned into linearized pRACE vectors, which were sequenced by Sangon Biotech (Shanghai, China). We assembled and analyzed the RACE sequences.