E coli dh5α competent cells

The SiFBA5 gene sequence was obtained from SaussureaKBase (http://www.shengtingbiology.com/SaussureaKBase/index.jsp.). Putative full-length SiFBA5 cDNA and genomic DNA were amplified using gene-specific primers SiFBA5-F and -R (Supplementary Table S1). SiFBA5 cDNA was ligated into the pMD®19-T simple vector (TaKaRa, China) and E. coli DH5α competent cells (TransGen, China); successful cloning was verified via Sanger sequencing (BGI, Beijing, China). All subsequent constructs were made using this clone as a template. SiFBA5 gene homologs were identified using BlastP in NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Multiple sequence alignments were performed in ClustalX2 with default parameters and DNAMAN. Alignments were then adjusted for constructing the phylogenetic tree in MEGA 5.0.

For confirmation, primers were designed to amplify the CDS sequence of netB gene. The primer sequence was NetB F: 5′- TTGAAAAGATTAAAAATTAT-3′ and NetB R: 5′- GTAAGAAATCAAATCATATTG-3′. The netB gene was amplified using T100 thermocycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA) in a reaction containing positive PCR product (1 µL), Premix Taq (LA Taq Version 2.0) 12.5 µL, primers (forward 0.5 µL), (reverse 0.5 µL), ddH2O (10.5 µL). The reaction conditions were 94 °C for 2 min, followed by 35 cycles of 98 °C for 10 sec, 47 °C for 30 s, and 68 °C for 1 min and final extension 72 °C for 10 min. The amplified product was electrophoresed on 2% agarose gel and observed under UV trans-illuminator. The purification of PCR product was performed through Gene Jet purification kit (OMEGA). After purification, Ligation was performed with T4 DNA Ligase, and recombinant plasmid (pMD18-T vector (Takara) containing netB gene sequence) was inserted into E. coli DH5α competent cells (Transgen Biotech, Beijing, China). Ligation was performed at 4 °C for overnight incubation. Transformation was confirmed by performing colony PCR of 4 single colonies by mixing with 10 µL ddH2O as a template. The cloned bacteria identified as positive by PCR were sent to Sangon Bioengineering (Shanghai) Co., Ltd. for sequencing.

Total RNA was extracted from mature-stage fruits, using the TaKaRa MiniBEST Plant RNA Extraction Kit (TaKaRa, Japan), following the manufacturer’s instructions. cDNA was synthesized using a TransScript^® Reverse Transcriptase Kit (TransGen Biotech, Beijing, China) for real-time RT-PCR (TaKaRa, Japan) following the manufacturer’s protocol. Specific primers were designed to amplify the ORF based on the complete sequence of coding sequence (cds) from the Prunus persica (http://www.ncbi.nlm.nih.gov; accession umber ABV32551.1); primers were synthesized by BGI (Beijing, China). Forward and reverse primer sequences were: ChSPS1-F (5′-ATGGCGAGCAACGATTGGATA-3′) and ChSPS1-R (5′-CTACGTCTTGACAACTCCGA-3′) respectively. PCR was carried out as follows: initial denaturation at 94°C for 1 min; followed by 36 cycles of denaturation at 94°C for 30 s, annealing 59.2°C for 30 s, and elongation at 72°C for 1 min, with a final 10min extension step at 72°C. PCR products were analyzed by electrophoresis on 1% (w/v) agarose gels. PCR products were ligated into the pMD18-T cloning vector (TaKaRa, Japan), and the pMD18-ChSPS1 was then transformed into E. coli DH5α competent cells (TransGen Biotech, Beijing, China) for sequencing by BGI.