Comparative Genome Analysis of Scutellaria baicalensis and Scutellaria barbata Reveals the Evolution of Active Flavonoid Biosynthesis

Abstract

Scutellaria baicalensis (S. baicalensis) and Scutellaria barbata (S. barbata) are common medicinal plants of the Lamiaceae family. Both produce specific flavonoid compounds, including baicalein, scutellarein, norwogonin, and wogonin, as well as their glycosides, which exhibit antioxidant and antitumor activities. Here, we report chromosome-level genome assemblies of S. baicalensis and S. barbata with quantitative chromosomal variation (2n = 18 and 2n = 26, respectively). The divergence of S. baicalensis and S. barbata occurred far earlier than previously reported, and a whole-genome duplication (WGD) event was identified. The insertion of long terminal repeat elements after speciation might be responsible for the observed chromosomal expansion and rearrangement. Comparative genome analysis of the congeneric species revealed the species-specific evolution of chrysin and apigenin biosynthetic genes, such as the S. baicalensis-specific tandem duplication of genes encoding phenylalanine ammonia lyase and chalcone synthase, and the S. barbata-specific duplication of genes encoding 4-CoA ligase. In addition, the paralogous duplication, colinearity, and expression diversity of CYP82D subfamily members revealed the functional divergence of genes encoding flavone hydroxylase between S. baicalensis and S. barbata. Analyzing these Scutellaria genomes reveals the common and species-specific evolution of flavone biosynthetic genes. Thus, these findings would facilitate the development of molecular breeding and studies of biosynthesis and regulation of bioactive compounds.

Keywords: Flavonoid biosynthesis; Scutellaria; Species-specific evolution; Tandem duplication; Whole-genome duplication.

Figure 1

Genome colinearity reveals the chromosome rearrangement in Scutellaria A. Morphological differences between the aerial parts of S. baicalensis and S. barbata. B. Comparison of nucleotide sequences of 9 S. baicalensis chromosomes (Sbai1–Sbai9; gray bars) and 13 S. barbata chromosomes (Sbar1–Sbar13; colored bars). Mapped regions with > 90% sequence similarity over 5 kb between the two species were linked. The red and black dots represent up-regulated genes (Log₂ FC > 1, FPKM > 10) in the root tissue compared to other tissues (stem, leaf, and flower) in S. baicalensis and S. barbata, respectively. C. Content distribution of flavone compounds in different tissues of S. baicalensis and S. barbata, including root, stem, leave, and flower, determined by UPLC. FC, fold change; FPKM, fragments per kilobase of exon model per million reads mapped; UPLC, ultraperformance liquid chromatography; R, root; S, stem; L, leaf; F, flower.

Figure 2

Shared WGD events of Lamiaceae and Pedaliaceae A. The phylogenetic tree based on the concatenated method using 235 single-copy orthologous genes from 11 angiosperms was constructed. The basal angiosperm Amborella trichopoda was chosen as the outgroup. The red branches represent two Scutellaria species, S. baicalensis and S. barbata. Speciation time was estimated based on the reported divergence time for A. trichopoda–Vitis vinifera (173–199 MYA) and Populus trichocarpa–Arabidopsis thaliana (98–117 MYA). The dashed line represents the divergence time between S. baicalensis and S. barbata. The orange ovals represent the reported WGT events. The purple and red ovals represent the reported WGD events and the newly identified WGD event in this study, respectively. The reported WGT/WGD events represent the WGT/WGD events identified in previous studies, including the WGT–γ event in core eudicots, WGT event in Solabun tuberosum, and WGD events in Sesamum indicum, A. thaliana, P. trichocarpa, Oryze sativa, and Brachypodium distachyon. B. Synonymous substitution rate (Ks) distributions of syntenic blocks for the paralogs and orthologs of S. baicalensis, S. barbata, S. miltiorrhiza, S. indicum, and V. vinifera. The gray box indicates the shared WGD event in S. baicalensis, S. barbata, S. miltiorrhiza, and S. indicum.C. Comparison with AEK chromosomes. The syntenic AEK blocks are painted onto S. baicalensis and S. barbata chromosomes separately. MYA, million years ago; WGT, whole-genome triplication; WGD, whole-genome duplication; AEK, ancestral eudicot karyotype.

Figure 3

Conserved flavonoid biosynthesis and species-specific gene expansion in Scutellaria A. Genes related to the biosynthesis of flavones and their glycosides. The red ovals represent the hydroxyl groups that can be glucosylated by UGT. The dashed box means the glycoside. B. The expression profile and orthologous gene pairs of flavone biosynthetic genes in S. baicalensis and S. barbata. C. Tandem duplication and phylogenetic analysis of CHS genes. The phylogenetic tree was constructed based on maximum likelihood method. PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; FNSII, flavone synthase II; F6H, flavone 6-hydroxylase; F8H, flavone 8-hydroxylase; UGT, UDP-glycosyltransferase; F8OMT, flavone 8-O-methyltransferase.

Figure 4

Tandem repeat of flavone hydroxylase genes revealed the divergent evolution A. Identification and expression of CYP82D subfamily genes, including F6H and F8H. B. Colinearity of CYP82D1 (F6H) regions between S. baicalensis and S. barbata. C. Colinearity of CYP82D2 (F8H) regions between S. baicalensis and S. barbata. The green triangles represent CYP450 genes. The orange triangle represents MYB transcription factor gene. The black triangles indicate neighboring genes of the selected CYP450 genes. D. Phylogenetic tree of F6H genes. E. Phylogenetic tree of F8H genes. Grape CYP82D (GSVIVT01009608001, http://plants.ensembl.org/Vitis_vinifera) was chosen as the outgroup to generate both trees. MYB, my elob lastosis.