Sequencing detection system v1

RNA samples were treated with RNase-free DNase (New England Biolabs, Hitchin, UK) to remove remnant DNA, then underwent synthesis of first-strand cDNA by the use of the SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA). Gene-specific primers for PeTCP genes were designed with the use of Primer Express (Applied Biosystems, Foster City, CA, USA) and are listed in Supplementary Table S1 at JXB online. PeActin4 (5′-TTGTGAGCAACTGGGATG-3′ and 5′-GCCACGCGAAGTTCATTG-3′) and 18S rRNA (5′-TTAGGCCACGGAAGTTTGAG-3′ and 5′-ACACTTCACCG GACCATTCAA-3′) (Lin et al., 2014 (link)) were used as internal control. Each real-time RT-PCR contained 5ng of cDNA, 20mM primers and 12.5ml of SYBR GREEN PCR Master Mix (Applied Biosystems), and water was added to 25ml. Real-time PCR involved use of the ABI 7 500 Real-Time PCR Instrument (Applied Biosystems). For each real-time RT-PCR, each sample was analysed in triplicate. Data were analysed by the use of the Sequencing Detection System v1.2.3 (Applied Biosystems). The software MultiExperiment Viewer was used to construct heatmap representations for expression patterns.

After RNA extraction, total RNA was treated with RNase-free DNase (NEB, Hertfordshire, UK) following the manufacturer’s protocols to remove contaminating genomic DNA. Reverse transcription to cDNA was synthesized using the Superscript II kit (Invitrogen, Carlsbad, CA, USA). Real-time RT-PCR was performed on the ABI 7500, Applied Biosystems System using SYBR GREEN PCR Master Mix (Applied Biosystems, Warrington, UK). PCR was performed with the following reaction conditions: 95 °C for 10 min, 40 cycles of 95 °C for 15 s, and 60 °C for 1 min. All the raw data were analyzed with the Sequencing Detection System v1.2.3 (Applied Biosystems). PeActin4 (PACT4, AY134752) was used for normalization [34 (link)]. Mean and standard error were calculated from three biological and technical replicates. Primers used for real-time RT-PCR were designed by using Primer Express 3.0 (Thermo Fisher Scientific, Foster City, CA, USA) and listed in Supplementary Table S2.

RNA was used as a template for cDNA synthesis with reverse transcriptase and the SuperScript II kit (Invitrogen). Transcripts of PaMLS, PaICL, and 8 candidate TF genes including PaANT, PaHB5, PaMADS2, PaMYB4, PaPIF3, PaRAV1-1, PaWRKY18 and PaWRKY71 were detected by RT-PCR and real-time quantitative PCR. The primer pairs are in Supplementary Table S1.
The methods of RT-PCR and real-time quantitative RT-PCR were as described^{39 (link)} with modification. The RT-PCR program was 94 °C for 5 min for denaturation, then 94 °C for 30 s, 72 °C for 30 s, and extension at 72 °C for 30 min. Annealing temperature and number of amplified cycles varied with different primer pairs (PaICL and PaMLS: 58 °C/25 cycles, candidate TF genes: 62 °C/33 cycles). The amplified products were analyzed on agarose gel and photographed. Only one amplified band with expected size was detected for each of PaICL and PaMLS (Supplementary Fig. S1).
For real-time quantitative RT-PCR analysis, the PCR program was incubation at 50 °C for 2 min, then 95 °C for 10 min, and thermal-cycling for 40 cycles (95 °C for 15 s and 60 °C for 1 min) by using the ABI 7500 Real-Time PCR instrument^{39 (link)}. Triplicate experiments were performed for each sample. Sequencing Detection System v1.2.3 (Applied Biosystems) was adopted for data analysis.