Plant materials and sampling
O. japonica is widely planted in China, and accordingly, it is not restricted by any relevant legislation or guidelines. Fruits of O. japonicas were collected from a germplasm garden situated at Zhejiang A&F University, Hangzhou, China (N 29◦56′ to 30◦23′, E 118◦51′ to 119◦52′), and the collection of plant material was allowed by college for experimental research. The fruits were collected evenly from the scape of fifteen to twenty plants located at the same area. Five growth stages for the collection were set based on the color and size of fruits. The first stage (S1) was arranged on September of 2020, followed by the second (S2) on October, the third (S3) on November, the fourth (S4) on December and the fifth (S5) on January, 2021. All the freshly picked fruits were frozen immediately in liquid nitrogen after sampling, and stored at − 80°C in Zhejiang Provincial Key Laboratory of Germplasm Innovation and Utilization for Garden Plants, Key Laboratory of National Forestry and Grassland Administration on Germplasm Innovation and Utilization for Southern Garden Plants, School of Landscape Architecture, Zhejiang Agriculture & Forestry University, Hangzhou 311300, China.
Determination on the Pigment content
200 mg fruit from the assigned growth stage was firstly ground into powder in 200 µl absolute ethyl alcohol (EA), then sealed and preserved in dark place. When the fruits turned colorless, the chlorophyll was considered to be dissolved in supernatant liquid. After filtrating the crushed fruits, the filter was collected and ready to be determined. The absorbance of extraction was measured at 470 nm, 649 nm, and 665 nm, respectively. The content of chlorophyll was computed according to the method described by Hu 43. The total anthocyanin was extracted at 4℃ in dark by a mixed solution which was formulated as EA: H2O:HCl = 80:20:0.3 (calculated as volume, mL) 32. The extracts were measured at the absorbance 530 nm and 600 nm. The relative content (amount) of total anthocyanin (TAC) was computed according to Liu's method 44.
Sample of metabonomics preparation and disposition
The freeze-dried sample was crushed with zirconia beads using a mixer mill (MM 400, Retsch) at 30 Hz for 1.5 min. 50 mg powder was vortexed in 0.5 ml methanol/water/hydrochloric acid (799:200:1,V/V/V) for 10 min and ultrasonically extracted for 10 min. The extracts were then centrifuged at 12, 000 rpm under 4°C for 3 min to allow phase separation. The extraction was repeated twice and the total supernatants were filtrated and collected (PTFE, 0.22 µm; Anpel) for LC-MS/MS analysis.
UPLC Conditions
The extracts were analyzed using UPLC-ESI-MS/MS system (UPLC, Shim-pack UFLC SHIMADZU CBM30A system, www.shimadzu.com.cn/; MS, Applied Biosystems 6500 Triple Quadrupole, www.appliedbiosystems.com.cn/), and the conditions were as follows. The WatersACQUITY BEHC18 (1.7 µm, 2.1 mm*100 mm) column and a mobile phase composed of water (0.1% formic acid) and methanol (0.1% formic acid) was used for separation under the following gradient conditions: 95:5 V/V initially, 50:50 V/V at 6min, 5:95 V/V at 12 min, holding for 2min, 95:5 V/V at 14 min, holding for 2min. The flow rate was 0.35 mL/min, with the column temperature at 40°C and injection volume of 2 µL. The effluent was alternatively connected to an ESI-triple quadrupole-linear ion trap (QTRAP)-MS.
ESI-Q TRAP-MS/MS
Linear ion trap and triple quadrupole (QQQ) scans were acquired by a triple quadrupole-linear ion trap mass spectrometer (Q TRAP). API 6500 Q TRAP UPLC/MS/MS System, equipped with an ESI Turbo Ion-Spray interface, was operated in positive and negative ion mode and controlled by Analyst 1.6.3 software (AB Sciex). The ESI source operation parameters were as follows: ion source with turbo spray, source temperature at 550℃; ion spray voltage (IS) at 5500 V (positive ion mode), curtain gas (CUR) at 35 psi, and the highly collision active dissociation (CAD). Each ion pair was scanned according to the optimized declustering potential (DP) and collision energy (CE). The metabolites eluted in each period were quantified using Multiple Reaction Monitoring (MRM).
Total RNA extraction and quality analysis
Total RNA was extracted by RNA extraction kit (TIANGEN RNAprep FFPE Kit) according to the corresponding specifications. The integrity of RNA was detected by Agilent 2100 Bioanalyzer (Agilent Technologies Inc., CA, USA) and agarose gel electrophoresis, while the purity and concentration of the RNA were analyzed by a NanoDrop™ One/OneC system (Thermo Fisher Scientific, MA, USA).
Iso-Seq library construction and Data Analysis
Total RNA of the five stages was maximized to acquire the full-length transcriptome. Iso-Seq library was prepared according to the Isoform Sequencing protocol (Iso-Seq) and using the Clontech SMARTer PCR cDNA Synthesis Kit protocol as described by Pacific Biosciences(www.pacb.com, PN 100-092-800-03). After PacBio system sequencing, 33.75 Gb subreads were obtained. SMRTlink 7.0 software was used to process these sequence data as follows. The circular consensus sequences (CCS) were generated from subreads of BAM files, and the resulted CCS contained full length non-chimeric (FLNC) sequences. The final isoforms were gained from high-quality consensus FLNC reads after removing the redundant FLNC through Quiver and CD-HIT-v4.6.754, respectively.
Construction of Illumina RNA-seq library and estimation on expression level
RNA sequences of fruits from five growth stages, with three replicates for each stage were concluded, which means a total number of fifteen libraries were established. Each sequencing library was prepared with 1 µg RNA and was generated from NEBNext® UltraTM RNA Library Prep Kit of Illumina® (NEB, USA). The quantitation and quality of library was detected by Qubit2.0 Fluorometer and Agilent 2100 bioanalyzer, respectively. When passing the quality inspection, the library preparations were sequenced on an Illumina Novaseq platform and 150 bp paired-end reads were generated.
The expression level of each gene was determined by the fragments-per-kilobase of transcript-per-million-mapped reads (FPKM), which was calculated by featureCounts v1.5.0-p3. Differential expression analysis between two stages was performed using the DESeq2 R package (1.16.1). The obtained P-values were then adjusted according to the Benjamini and Hochberg’s approach for controlling the false discovery rate. Genes with an adjusted P-value < 0.05 detected with DESeq2 were designated as differentially expressed genes (DEGs) (fold-change ≥ 2, false discovery rate < 0.01).
qRT-PCR analysis
The extraction of RNA was carried out according to the method mentioned above and PrimerScript™ RT Master Mix cDNA (TaKaRa, Japan) was used for cDNA synthesis. The primer pairs for each isoform/unigene were designed using primer 5. The obtained cDNA samples were analyzed by qRT-PCR in a 20 µl reacting solution formulated with TB Green™ Premix Ex Taq™ (TaKaRa, Japan), primer pairs and double distilled water. The qRT-PCR analysis was performed on a Light-Cycler 480II real-time PCR detection system (Roche, USA).
Statistical analysis
SIMCA 14 was applied to conduct the Principal Component Analysis (PCA) and Variable Importance in Projection (VIP). One-way ANOVA was prformed by means of Duncan’s multiple range test of the Statistical Product and Service Solutions program (version 19) (SPSS Inc.,Chicago, IL), and the graphs were plotted using Graphpad prism 8.0.