Plant material
Mature seeds of 3-year-old A. sinensis (Mingui No. 1) (family Apiaceae, alt. Umbelliferae) were permitted to collect from the county-owned garden located in Minxian county (2,520 m asl; 34o28'33"N, 104o05'51"E) of Gansu province, P. R. China in July 2017. The species was identified by professor ling Jin (Gansu University of Chinese Medicine, Lanzhou, Gansu, China). A voucher specimen (No. 20200182) was deposited in the herbarium of College of Life Science and Technology, Gansu Agricultural University, Lanzhou, Gansu, China. Seeds were pre-treated in water (30°C) for 24 hrs. and sown at a soil depth of 0.5 cm located in Minxian county (2,730 m asl; 34o28'8"N, 104o36'22"E) in June 2018. Seedlings were dug up in October 2018, aired in the shade for approximately 15 days and then stored in a natural-rain-proof environment for the winter.
On April 3, 2019, the stored seedlings (root tip diameter 4.5-5.0 mm) were transplanted into pots (diameter 17 cm, depth 20 cm; one seedling per pot) with nutrition matrix and seedlings were greenhouse grown with controlling matrix volumetric moisture content of 60%-70%, light condition of 10-12 hrs. per day and air temperature 15-22°C. No additional fertilizer was applied after the transplant. On July 3, 2019, samples including the second-tip leaves and lateral roots (1:1, g/g fresh weight) from BP and UBP (Fig.S10) were collected (n = 20 plants) and then flash frozen in liquid nitrogen for transcriptomic analysis and GA metabolite analysis.
Total RNA isolation and Illumina sequencing
Total RNA samples were extracted using a Trizol reagent, enriched using Oligo (dT) beads (Invitrogen, CA, USA), fragmented into short mRNA segments (200-700 nt) using a fragmentation buffer and reverse transcribed into cDNA with random primers. Second-strand cDNA was synthesized via DNA polymerase I, RNase H, dNTP and buffer and cDNA fragments were purified using a QiAquick PCR extraction kit, successively repaired-end, added poly (A), and ligated to Illumina sequencing adapters. Finally, the ligation products were sequenced using an Illumina HiSeqTM 4000 platform by Gene Denovo Biotechnology Co., Ltd. (Guangzhou, China).
Sequence filtration, assembly and unigene expression analysis
Raw reads obtained from the Illumina sequencing were further filtered to get high quality clean reads by removing reads containing adapters, more than 10% unknown nucleotides as well as more than 40% low quality (Q-value≤10) bases. De novo assembly of clean reads was carried out using Trinity software [62] that combined three components: Inchworm, Chrysalis and Butterfly, respectively for assembling a collection of linear contigs, building graphs for each cluster of related contigs and outputting one linear sequence for each alternatively spliced isoform and transcripts. The expression level of each transcript was calculated and normalized to reads per kb per million reads (RPKM) [63]. In this study, the level of differential expression for each transcript with a criterion of |log2 (fold-change) ≥ 1 and p value ≤ 0.05 to identify DEGs between BP and UBP.
Basic annotation of DEGs and gene cluster analysis
Unigenes were annotated against the databases including: NCBI non-redundant protein (NR), Swiss-Prot protein, Kyoto Encyclopedia of Genes and Genomes (KEGG), euKaryotic orthologous groups of proteins (KOG), and gene ontology (GO) by using a BLASTx procedure with an e-value ≤ 10-5 [64]. Molecular Evolutionary Genetics Analysis (MEGA) 7.0 was used for the gene cluster analysis (Fig. S11).
qRT-PCR validation
Total RNA samples from samples of the BP and UBP plants were extracted using a plant RNA kit. Primer sequences of the 40 DEGs (Table 4) were designed with the tools for primer-blast in NCBI. First-strand cDNA was synthesized using a FastKing RT kit with one cycle at 42°C for 15 min and then 95°C for 3 min. PCR amplification was carried out using a SuperReal PreMix with one cycle at 95°C for 15 min, followed by 40 cycles at 95°C for 10 s, 60°C for 20 s and 72°C for 30 s. Melting curves were analyzed after an incubation at 72oC for 34 s. Actin was used as an internal standard, the relative expression level (REL) of gene was calculated based on a 2-△△Ct method [65].
GA quantification and identification
Five GAs including GA1, GA4, GA8, GA9 and GA20 were quantified and identified using a HPLC (Agilent1290, USA)-MS/MS (QTRAP 6500, AB SCIEX, USA) by Shanghai Biotree biotech Co., Ltd. (Shanghai, China). Representative chromatograms of reference standard of the 5 GAs are shown in Fig. S12, and representative chromatograms of the BP and UBP are shown in Fig. S13. The content of the 5 GAs was calculated based on calibration curves (Table S1).
Soluble sugar measurement
Soluble sugar was measured using a sulfuric acid-phenol protocol [66]. A dried powder (1.0 g) was soaked in 10% EtOH (25 mL) for 72 hrs. at 22oC and then centrifuged (4°C, 8000 r/min, 10 min). Extracts (30 µL) were added into 9% phenol reagent (1 mL), sulfuric acid (3 mL) was added after oscillation and then reacted at 22oC for 30 min. Absorbance was measured at 485 nm, soluble sugar content was evaluated based on mg of Suc.
Statistical analysis
All the measurements were performed using three replicates. A t-test for independent samples was performed and SPSS 22.0 was used, with p<0.05 as the basis for significant differences.