Supplemental Material for Amini et al., 2019

Supplementary Materials.docx contains FileS1, Tables and Figures

File S1 contains Supplementary Materials and Method and Results and Discussion sections.

Table S1 contains The calibration curves for umbelliferone, luteolin, umbelliprenin.

Table S2 contains RNAseq data obtained from different organs of F. assafoetida.

Table S3 contains Summary statistics on de novo assembly of F. assafoetida.

Table S4 contains Summary evaluation of F. assafoetida transcriptome assemblies.

Table S5 contains Putative annotation and transcript identities of candidate genes of terpenoid biosynthesis.

Table S6 contains Putative annotation and transcript identities of candidate genes of coumarin-type phenylpropanoid biosynthesis.

Table S7 contains genes upregulated in sesqui- and tri-terpenoid biosynthesis with ko00909.

Table S8 contains Genes upregulated in flavonoid biosynthesis with ko00941.

Figure S1 contains Schematic overview of core pathway reactions in the biosynthesis of terpenoids (A) and phenylpropanoids (B) targeted in this study as relevant bioactive constituents in Ferula assafoetida. Key enzymes are highlighted in bold: TPS, terpene synthase; TTS, triterpene synthase; P450, cytochrome P450-dependent monooxygenase; PAL, phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate:CoA-ligase; C2?H, p?coumaroyl-CoA 2??hydroxylase; CHS, chalcone synthases; CHI, chalcone isomerases; FNS, flavone synthases; F3’H, flavanone 3-hydroxylase.

Figure S2 contains Length distribution of transcripts of assembled transcriptomes (A) and fraction of the RNAseq reads uniquely mapped to the assembled transcriptome by STAR 2.5.2b (B).

Figure S3 contains Distribution of assembled transcriptome best hits to different plants as a result of blastx against all RefSeq plant databases.

Figure S4 contains Gene ontology (GO) classification of the transcripts derived from F. assafoetida.

Figure S5 contains Determination of soft threshold (power) for network construction.

Figure S6 contains Relative terpenoid composition in the essential oils of different organs.

Figure S7 contains Quantification of the amount of umbelliferone (A) and luteolin (B) among different organs. Quantification of the amount of umbelliferone, umbelliprenin and luteolin (C) in oleo-gum-resin. The yield was microgram per gram dry weight (µg/g) of these compounds for each sample. Each value represents the average of two injections for each sample by LOD (limits of detection) at 10 ng/ml and LOD at 20 ng/ml.

Figure S8 contains Venn diagram of differentially expressed genes used in pairwise statistical tests and contrast matrix of different organs (Significance level: FDR <0.05).

Figure S9 contains Over-represented GO terms in differential expressed genes between pairwise comparison organs for three ontologies biological process (A), cellular component (B) and molecular function (C). “down” in Flower vs Root means higher expression in Root; “down” in Flower vs Stem means higher expression in Stem; “down” in Leaf vs Root means higher expression in Root.

“up” in Flower vs Root indicates higher expression in Flower; “up” in Flower vs Stem indicates higher expression in Flower; “up” in Leaf vs Root indicates higher expression in Leaf; “up” in Flower vs Leaf indicates higher expression in Flower; “up” in Leaf vs Root means higher expression in Leaf.

Figure S10 contains Pathway analysis result for finding over-represented KEGG orthology by using the KEGG database. “up” in Flower vs Leaf indicates higher expression in Flower; “up” in Flower vs Root indicates higher expression in Flower; “up” in Flower vs Stem indicates higher expression in Flower; “up” in Leaf vs Root indicates higher expression in Leaf.

Figure S11 contains Visualization of terpenoid pathway branches that were identified as significantly enriched in flowers versus stems of F. assafoetida. F. assafoetida transcripts significantly mapped to sesquiterpenoid biosynthesis are highlighted in red.

Figure S12 contains Visualization of phenylpropanoid pathway branches that were identified as significantly enriched in flowers versus stems of F. assafoetida. F. assafoetida transcripts significantly mapped to phenylpropanoid and flavonoid biosynthesis are highlighted in red.

Figure S13 contains Gene network of F. assafoetida was constructed using WGCNA by setting the soft power to 12, type to signed hybrid, minModuleSize to 30, dissimilarity threshold to 0.2, and deepslit to 2.

Figure S14 contains Over-representation analysis (ORA) of darkseagreen3 module (A). The eigengenes expression value of darkseagreen3 module across different organs (B).

Figure S15 contains The contribution of the darkseagreen3 and coral modules in sesquiterpene biosynthesis. The co-expression analysis indicates that the darksagreen3 module is negatively correlated with production of sesquiterpene in roots while the coral module is positively correlated with production of sesquiterpene.

Figure S16 contains The TPM (Transcripts Per Kilobase Million values) of terpene synthase candidate genes located in the coral module for different organs.