Figure 1.
Putative pathway for scutellarin and chlorogenic acids biosynthesis in E. breviscapus.
The major products previously found in E. breviscapus are surrounded by shaded boxes. Enzymes found in this study are boxed; new putative enzyme (flavone 6-hydroase, F6H) was surrounded by dashed line box. Enzymes involved in the pathways are: 4CL, 4-coumarate: CoA ligase; C3H, p-coumarate 3-hydroxylase; C4H, cinnamate 4-hydroxylase; CHI, chalcone isomerase; CHS, chalcone synthase, DFR, dihydroflavonol 4-reductase; F3′5′H flavonoid 3′5′ hydroxylase; F3′H, flavonoid 3′ hydroxylase; F3H, flavanone 3-hydroxylase = flavonol synthase (FLS); FSII, flavone synthase II; HCGQT, hydroxycinnamoyl D-glucose: quinate hydroxycinnamoyl transferase; HCT, hydroxycinnamoyl CoA shikimate/quinate hydroxycinnamoyl transferase; HQT, hydroxycinnamoyl CoA quinate hydroxycinnamoyl transferase; LDOX, leucoanthocyanidin dioxygenase = anthocyanidin synthase (ANS); OMT, O-methyltransferase; PAL, phenylalanine ammonia lyase; RT, rhamnosyl transferase; UFGT, UDPG-flavonoid glucosyl transferase; UGCT, UDP glucose: cinnamate glucosyl transferase; F7GAT, flavonoid 7-O-glucuronosyltransferase.
Table 1.
Summary of Illumina Paired-end sequencing and assembly for E. breviscapus.
Figure 2.
Overview of E. breviscapus transcriptome assembly.
Figure 3.
The number of unigenes annotated by BLASTX with an E-value threshold of 10−5 against protein databases.
The numbers in the circles indicate the number of unigenes annotated by single or multiple databases.
Table 2.
Summary of the annotation percentage of E. breviscapus unigenes as compared to public databases.
Figure 4.
Gene Ontology classification of assembled unigenes.
Total 17,156 unigenes were categorized into three main categories: biological process, cellular component and molecular function.
Figure 5.
COG function classification of E. breviscapus unigenes.
Figure 6.
Pathway assignment based on the KEGG.
(A) Classification based on metabolism categories; (B) Classification based on biosynthesis of other secondary metabolites.
Table 3.
Transcripts involved in flavonoids and chlorogenic acids biosynthesis in E. breviscapus.
Figure 7.
Phylogenetic tree of the E. breviscapus CYPs.
Phylogenetic tree constructed based on the deduced amino acid sequences for the E. breviscapus CYPs (bold letters) and other plant CYPs. Protein sequences were retrieved from NCBI GenBank using the following accession numbers (source organism and proposed function, if any, are given in parentheses): CYP82D33, JX162212 (Ocimum basilicum, flavonoid 6-hydroxlase); CYP82D62, JX162214 (Mentha x piperita, flavonoid 6-hydroxylase); CYP82B1, AAC39454 (Eschscholzia californica, (S)-N-methylcoclaurine 3′-hydroxylase); CYP82N2v2, BAK20464 (Eschscholzia californica, protopine 6-hydroxylase); CYP82C2, O49394 (A. thaliana); CYP82H1, AAS90126 (Ammi majus); CYP82A2, CAA71515 (Glycine max); CYP82Q1, ABB20912 (Stevia rebaudiana); CYP82E4v1, ABA07805 (Nicotiana tabacum, nicotine demethylase); CYP82G1, NP_189154 (Arabidopsis thaliana, geranyllinalool catabolism); CYP75A1, CAA80266 (Petunia x hybrid, flavonoid 3′,5′-hydroxylase); CYP75B1, AAD56282 (Petunia x hybrida,flavonoid 3′-hydroxylase); CYP93A1, NP_001241186 (G. max, 3,9-dihydroxypterocarpan 6a-hydroxylase); CYP93B1, BAA22423 (Glycyrrhiza echinata, flavone synthase); CYP93B2, AAD39549 (Gerbera hybrid, flavone synthase); CYP93B6, BAB59004 (Perilla frutescens var. crispa, flavone synthase); CYP93B23, JX162213 (Ocimum basilicum, flavone synthase); CYP71AJ1, AAT06911 (Ammi majus, psoralen synthase); CYP71E7, AAP57704 (Manihot esculenta, cyanogenic glucoside oxim metabolism); CYP71D18, AAD44150 (Mentha spicata, limonene 6-hydroxylase), CYP71D9, CAA71514 (G. max, flavonoid 6-hydroxylase); CYP71D12, CAB56503 (Catharanthus roseus, tabersonine 16-hydroxylase); CYP71AV1, ABB82944 (Artemisia annua, amorpha-4,11-diene C-12 oxidase); CYP80A1, AAC48987 (Berberis stolonifera, berbamunine synthase); CYP80F1, ABD39696 (Hyoscyamus niger, littorine mutase); CYP73A1, CAA78982 (Helianthus tuberosus, trans-cinnamate 4-hydroxylase); CYP98A13, AAL99200 (Ocimum basilicum, p-coumaroyl shikimate 3′-hydroxylase); CYP79A1, AAA85440 (Sorghum bicolor, tyrosine N-hydroxylase); CYP703A1, BAA92894 (Petunia x hybrid, lauric acid hydroxylase); CYP701A1, AAG41776 (Cucurbita maxima, ent-kaurene oxidase); CYP719A1, BAB68769 (Coptis japonica, methylenedioxy bridgeforming enzyme); CYP72A1, AAA33106 (C. roseus, secologanin synthase); CYP86A1, P48422 (A. thaliana, fatty acid -hydroxylase); CYP88A1, AAC49067 (Zea mays, entkaurenoic acid oxidase); CYP90A1, Q42569 (A. thaliana, 6-oxocathasterone 23a-hydroxylase); CYP51G1, BAB61873 (A. thaliana, obtusifoliol 14-demethylase); CYP74A1, AAA03353 (Linum usitatissimum, allene oxide synthase); CYP74B1, AAA97465 (Capsicum annuum, fatty acid hydroperoxide lyase).
Figure 8.
Phylogenetic tree of the E. breviscapus R2R3-MYB transcription factors.
Phylogenetic tree constructed based on the deduced amino acid sequences for the E. breviscapus R2R3-MYB transcription factors (bold letters) and other plant R2R3-MYB transcription factors. Accession numbers in the NCBI GenBank database are as follows: AtMYB111, Arabidopsis thaliana (NM124310); GtMYBP3, Gentiana triflora (AB733016); VvMYBF1, Vitis vinifera (FJ948477); SlMYB12, Solanum lycopersicum (EU419748); AtMYB11, Arabidopsis thaliana (NM116126); AtMYB12, Arabidopsis thaliana (NM130314); GtMYBP4, Gentiana triflora (AB289446); GhMYB1, Gerbera hybrid (AJ554697); SbY1, Sorghum bicolor (AY860968); ZmP1, Zea mays (M73028); ZmP2, Zea mays (AF210616); ZmC1, Zea mays (MZEMYBAA); ZmPL, Zea mays (AF015268); AtMYB123, Arabidopsis thaliana (AJ299452); AtMYB75, Arabidopsis thaliana (AF325123); AtMYB114, Arabidopsis thaliana (AY519567); AtMYB114, Arabidopsis thaliana (AY519567); AtMYB90, Arabidopsis thaliana (AF325124); GtMYBP5,Gentiana triflora (AB733616); GtMYBP6, Gentiana triflora (AB733617); AtMYB113, Arabidopsis thaliana (NP176811); PhAN2, Petunia x hybrida (AF146702); AmVENOSA, Antirrhinum majus (DQ275531); AmROSEA1, Antirrhinum majus (DQ275529); AmROSEA2, Antirrhinum majus (DQ275530); AmMIXTA, Antirrhinum majus (X79108).
Table 4.
Summary of SSR searching results.
Table 5.
Distribution of identified SSRs using the MISA software.
Figure 9.
Dendrogram constructed with UPGMA clustering method among 13 different accessions of E. breviscapus.