Sybr green detection protocol

For temporal expression analysis, male and female flowers were sampled at different developmental stages according to the study by Bai et al. (2004) (link) at zeitgeber time (ZT) 4. The whole flower was sampled and dissected under a stereomicroscope (LEICA, S8APO, Germany) for receptacle and nectary samples. Three biological replicates were prepared. For gene expression analysis in different tissues, including root, stem, leaf, male and female flowers, and ovary/fruit, the samples were harvested at ZT4 of the 2-month-old cucumber plant at anthesis.
Total RNA from different tissues was extracted using the RNeasy Plant Kit (Huayueyang, Beijing, China) according to the protocol of the manufacturer. Reverse transcription was carried out using the FastQuant RT Kit (with gDNase; Tiangen, Beijing, China). Gene expression analyses were performed by reverse transcription-quantitative PCR (RT-qPCR) with the SYBR green detection protocol (TaKaRa, Japan) on an ABI 7500 Real-Time PCR Detection System (Bio-Rad, United States). The relative expression level was normalized to the housekeeping gene Tubulin using the 2^–ΔΔCT method (Livak and Schmittgen, 2001 (link)). Primers used in this study are as shown in Supplementary Table 1.

Total RNA was extracted from the qsk1 plants using the Plant RNA Mini Kit (peQlab, Germany) according to the instructions provided by the manufacturer. RNA was digested with DNaseI (Roche Diagnostics, Germany) to remove the genomic DNA. First-strand cDNA was synthesized using PrimeScript RT reagent kit (TaKaRa). Quantitative real-time PCR analysis was performed using the Bio-Rad CFX Connect real-time PCR system (BioRad Laboratories; Munich, Germany) with the SYBR green detection protocol (TaKaRa, Saint-Germain-en-Laye, France). The Actin and Tubulin genes were used as reference genes, and the relative expression of the gene of interest was calculated by the 2∧-ΔΔCq method. RT Primers for QSK1 was F 5-TGAGTCATGCCAATCTCGTGAC-3, R 5-GCAATATCGCAGACAAGCTTCC-3. Primers used for Actin and Tubulin were: Actin-F 5-ACTTTCATCAGCCGTTTTGA-3, Actin-R-5-ACGATTGGTTGAATATCATCAG-3 and Tubulin-F 5-ACCTACTGGTCTGAAGATGGCAT-3 and Tubulin-R5-TTTCTCCTGAACATAGCTGTGAAC-3.

Total RNA was extracted individually from root, stem, leaf, developing embryo, and endosperm as well as young tassel and young ear. Reverse transcription was conducted using PrimeScriptTM Ⅱ 1st strand cDNA Synthesis Kit (TaKaRa, Tokyo, Japan) with Oligo-T as primer. The full-length CDS was amplified by the TransStart FastPfu Fly DNA Polymerase kit (TransGen) using 1st strand cDNA as a template. Quantitative real-time PCR analyses were conducted through a 7500 Fast Real-Time PCR System (Applied Biosystem, Foster city, CA, USA) using SYBR green detection protocol (TaKaRa). Gene-specific primers and ubiquitin gene primers for the control standard were used in the RT-qPCR. The primer sequences used in cDNA synthesis and RT-qPCR are listed in Table S2.