Plant total rna extraction kit

Total RNA was isolated from S. miltiorrhiza tissues using the plant total RNA extraction kit (Aidlab, China). The isolation was carried out following the manufacturer’s instructions. RNA integrity was analyzed on an agarose gel. RNA quantity was determined using a NanoDrop 2000C spectrophotometer (Thermo Scientific, USA). Reverse transcription was conducted using PrimeScript™ RT reagent kit (TaKaRa, Japan). Gene specific primers were designed using Primer Premier 6 (PREMIER Biosoft Int, USA) based on SmDML coding sequences. SmUBQ10 was used as an internal control as described previously^{2 (link)}. The expression of Smi-miR7972a and Smi-miR7972b was analyzed using Mir-X miRNA qRT-PCR SYBR Kit (TaKaRa, Japan). The primers were listed in Table S2. qRT-PCR was performed in triplicate for each tissue sample using the SYBR premix Ex Taq™ kit (TaKaRa, China) on a CFX96 Touch™ real-time PCR system (Bio-Rad, USA). Three independent biological replicates were performed. Gene relative expression levels were calculated for Ct values using the 2^−ΔΔCq method^{75 (link)}. Differential expression among tissues and treatments was determined by one-way ANOVA using IBM SPSS 20 software (IBM Corporation, USA).

Roots, stems, leaves and flowers of whole genome-decoded S. miltiorrhiza Bunge (line 993) were collected and stored in liquid nitrogen until use. Total RNA was extracted using the plant total RNA extraction kit (Aidlab, China). Genomic DNA contamination was eliminated by pre-treating total RNA with RNase-Free DNase (Promega, USA). RNA integrity was analyzed on a 1.2% agarose gel. RNA quantity was determined using a NanoDrop 2000C Spectrophotometer (Thermo Scientific, USA). Total RNA was reverse-transcribed by Superscript III Reverse Transcriptase (Invitrogen, USA). The full-length CDSs of 110 SmMYBs were amplified by PCR using the primers listed in Additional file 5: Table S3. PCR products were gel-purified, cloned, and then sequenced. qRT-PCRs were performed as previously described by Ma et al. using gene-specific primers (Additional file 6: Table S4) [7 (link)]. The length of amplicons was between 80 bp and 250 bp. SmUBQ10 was used as a reference gene. The data was analyzed as described previously [7 (link)].

Two wounds (1 × 2 mm) were created uniformly at the equator of the citrus fruit with a 0.5 mm diameter needle. A 10 μL conidia suspension of P. italicum (1 × 10⁶ spores mL⁻¹) was injected into each wound. The inoculated fruits were placed in transparent sealed boxes and stored at 26 °C. The tissue samples (about 3 g) were collected from the wound after 72 hpi. They were immediately frozen with liquid nitrogen and stored at −80 °C for subsequent analysis. Three replicates were prepared for the sample. RNA was extracted using a plant total RNA extraction kit (Aidlab Biotechnologies Co., Ltd., Beijing, China), according to the manufacturer’s instructions (with three biological replicates). The Illumina HiSeq 2000 sequencing platform was used for sequencing total RNA, which was conducted by the BGI Company (Shenzhen, China). The raw RNA-Seq data files were submitted to the NCBI database under SRA accession number SRP362092.

The suspected cysteine protease gene members were obtained by searching and screening methods according to functional domain division in the populus tomentosa genome database website (https://genome.jgi.doe.gov/portal/, accessed on 13 October 2021). Total RNA was extracted from the leaves of P. trichocarpa, according to the instructions of the plant total RNA extraction kit (Aidlab, Beijing, China). First-strand cDNA synthesis was performed using M-MLV Reverse Transcriptase and an oligo (dT) primer (Promega, Madison, WI, USA). The PtCP5 cDNA sequence was amplified by PCR using the primers PtCP5-F and PtCP5-R.

Salvia miltiorrhiza tissues were used for total RNA extraction using the Plant Total RNA Extraction kit (Aidlab, China) according to the user manual. Total RNA was transcribed into cDNA using the TransScript-Uni One-Step gDNA Removal and cDNA Synthesis SuperMix kit (TransGen Biotech). Reverse transcription of Smi-miR858 was carried out with specific stem-loop primers. RT–qPCR was performed using the TransStart Tip Green qPCR SuperMix (TransGen Biotech) and analyzed using the Bio-Rad CFX96 detection system. Three biological replicates and four technical replicates for each biological replicate were performed. S. miltiorrhiza 5.8S rRNA and SmUbiquitin were used as internal controls for the analysis of miR858s and protein-coding genes, respectively. The primers used for RT–PCR analysis are listed in Supplementary Data Table S2.