Realplex4 system

Plant material was collected as previously described [39 (link)]. Briefly, the fruits of the ‘Furongli’ plum were harvested from 6-year-old field-grown trees in Fuda Village, Fujian Province, China. Fruit samples were picked 23, 43, 70, 98, 112, 127, and 157 days after flowering (DAF), respectively. All the trees received the standard horticultural practices and insect prevention. The fruit samples were stored at −80 °C until use. After that, they were peeled and sliced into appropriate pieces and frozen in liquid nitrogen immediately. For qRT-PCR analysis, the total RNA was isolated using the RNA prep Pure Plant Total RNA Extraction Kit (Tiangen, Beijing, China) following the manufacturer’s instructions. First-strand cDNA was synthesized for qPCR using the PrimeScript RT reagent kit with gDNA Eraser (Takara, Dalian, China). The qRT-PCR reactions were conducted in 20 µL volumes, utilizing 1 µL of cDNA and 2 × SYBR Premix Ex TaqTM II (Tli RNaseH Plus, TaKaRa), based on the Eppendorf RealPlex4 system (Hamburg, Germany). The experiment procedures of qRT-PCR were 40 cycles of 95 °C for 15 s and then 68 °C for 30 s. The actin gene was utilized as the control standard, and the 2^−ΔΔCT method was employed to analyze the relative expression levels of the genes [40 (link)]. Three biological and three technical replicates were performed. The primer sequences are provided in Table S1.