Quantscript reverse transcriptase kit

Total RNA was extracted from freshly leaves of PN40024 with the RNAprep Pure Kit (Tiangen Biotech, Beijing, China). RNA samples were reverse-transcribed according to the instructions of Quantscript Reverse Transcriptase Kit (Tiangen Biotech, Beijing, China). Based on the sequence of VvIAA18 (Genbank accession No. XM_010656314), we designed one gene-specific primers (GC-F/R) of RT-PCR (Table S1) to obtain its full-length cDNA sequence. PCR was performed with an initial denaturation 94 °C for 3 min, followed by 35 cycles of 94 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min and final extension 72 °C for 10 min. PCR products were separated on a 1.0% (w/v) agarose gel. Target DNA bands were recovered by gel extraction, then cloned into PMD19-T (TaKaRa, Beijing, China), and finally transformed into competent cells of E. coli strain DH5α. Examine hite colonies were by PCR and sequence positive colonies (Invitrogen, Beijing, China).

According to the manufacturer’s protocol, TRIZOL reagent (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA from various tissues at different stages after the bacterial infection. The RNA samples were processed with DNase to remove potential genomic DNA contamination. Subsequently, agarose gel electrophoresis was performed and a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA) was used to evaluate the quality of the separated RNAs. Reverse transcription of RNA samples was conducted using Quantscript Reverse Transcriptase Kit (Tiangen Biotech, Beijing, China). The obtained cDNA solution was used as the template for PCRs.

Total RNA of Perilla frutescens leaves was reverse-transcribed according to the Quantscript Reverse Transcriptase Kit (TIANGEN Biotech, Beijing, China). cDNA was used as a template to measure gene expression. The specific primers involved in the anthocyanin biosynthesis genes and the Perilla frutescens actin gene (internal control) are listed in Table S5. Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted by a real-time PCR ABI Prism 7500 system (software for 7500 and 7500 Fast Real-Time PCR Systems, V2.0.1, Foster City, CA, USA) using SYBR Premix Ex Taq II (TaKaRa Code No. RR820A, https://www.takara biomed.com.cn). The comparative CT method (2^−ΔΔCT method) was used to quantify gene expression^{55 (link)}.

Total RNA was extracted from 0.5 g of fresh leaves of 4-week-old in vitro-grown plants of ND98 with the RNAprep Pure Plant Kit (Tiangen Biotech, Beijing, China). RNA samples were reverse-transcribed according to the instructions of Quantscript Reverse Transcriptase Kit (Tiangen Biotech, Beijing, China). A rapid amplification of cDNA ends (RACE) procedure was employed to amplify the 5′ and 3′ ends of the coding region using GeneRacer™ Kit (Invitrogen, Carlsbad, CA, USA). Based on the sequence of EST, primers were designed using the Primer 3 program (http://frodo.wi.mit.edu/primer3/) and listed in Table 1.
PCR amplifications were performed with an initial denaturation at 94°C for 3 min, followed by 35 cycles at 94°C for 30 s, 55°C for 30 s, 72°C for 1 min and final extension at 72°C for 10 min. PCR products were separated on a 1.0% (w/v) agarose gel. Target DNA bands were recovered by gel extraction, then cloned into PMD19-T (TaKaRa, Beijing, China), and finally transformed into competent cells of Escherichia coli strain DH5α. White colonies were checked by PCR and the positive colonies were sequenced (Invitrogen, Beijing, China).

The whole storage roots of the sweet potato cultivars were used for RNA extraction. The cultivars, Tengfei and JK20 were inoculated with stem nematodes (Gao et al., 2011 ) and the cultivars, Santiandao and JK142 were inoculated with C. fimbriata (Muramoto et al., 2012 (link)). Samples were collected at eight-time points (0 h, 6 h, 12 h, 1 day, 2 days, 4 days, and 6 days) after the inoculation. Root sample without inoculation was used as a control or mock. Total RNA of the samples was isolated using RNAprep Pure Plant Kit (Tiangen Biotech, Beijing, China) and first-strand cDNA was synthesized by Quantscript Reverse Transcriptase Kit (Tiangen Biotech, Beijing, China). The sweet potato β-actin gene (Genbank AY905538) was used as a control and was used to normalize the relative quantities of the three individual targeted DEGs based on its consistency across the different time points of each treatment of stem nematode and C. fimbriata (Liu et al., 2014 ). Three biological replicates were performed at each time point, and the gene expression changes were calculated using the 2^–ΔΔCt method for each sample (Schmittgen and Livak, 2008 (link)). The qRT-PCR was performed as described previously (Zhai et al., 2016 (link)) using the generated primers (Supplementary Table 1) through Primer-BLAST software (Ye et al., 2012 (link)).

Roots of plants treated for 0, 1, 6, 12, 24 or 48 h with 200 mM NaCl in Hoagland solution were used to extract total RNA using the RNAprep Pure Plant Kit (Tiangen Biotech, Beijing, China). RNA samples were reverse-transcribed using the Quantscript Reverse Transcriptase Kit (Tiangen Biotech, Beijing, China). The cDNA solution was used as the template for PCR amplification with specific primers (Supplementary Table S8). The sweet potato β-actin gene was used as an internal control (Supplementary Table S8). The amplifications were performed according to Zhai et al.50 (link).