A high-quality genome assembly of the tetraploid Teucrium chamaedrys unveils a recent whole-genome duplication and a large biosynthetic gene cluster for diterpenoid metabolism

Abstract

Teucrium chamaedrys, commonly known as wall germander, is a small woody shrub native to the Mediterranean region. Its name is derived from the Greek words meaning "ground oak," as its tiny leaves resemble those of an oak tree. Teucrium species are prolific producers of diterpenes, endowing them with valuable properties widely utilized in traditional and modern medicine. Sequencing and assembly of the 3-Gbp tetraploid T. chamaedrys genome revealed 74 diterpene synthase genes, with a substantial number of these genes clustered at four synteny genomic loci, each harboring a copy of a large diterpene biosynthetic gene cluster. Comparative genomics revealed that this cluster is conserved in the closely related species Teucrium marum. Along with the presence of several cytochrome p450 sequences, this region is among the largest biosynthetic gene clusters identified. Teucrium is well known for accumulating clerodane-type diterpenoids, which are produced from a kolavenyl diphosphate precursor. To elucidate the complex biosynthetic pathways of these medicinal compounds, we identified and functionally characterized several kolavenyl diphosphate synthases from T. chamaedrys. The remarkable chemical diversity and tetraploid nature of T. chamaedrys make it a valuable model for studying genomic evolution and adaptation in plants.

Keywords: BGC; Lamiaceae (mint); Teucrium; biosynthetic gene cluster; diterpenoid.

Figure 1

Clerodane skeleton and select clerodanes from T. chamaedrys. Teucrium, specifically T. chamaedrys, is rich in clerodane-type diterpenoids. Middle box features numbered carbons on a typical clerodane skeleton.

Figure 2

The tetraploid genome of T. chamaedrys. (A) Image of mature T. chamaedrys shrub. (B) A representative metaphase cell prepared from a root tip. (C) Smudgeplot analysis showing evidence for genome duplication, with k-mers present at 4n configurations AAAB and AABB. (D) Orthogroup proportions between T. chamaedrys and A. thaliana. Approximately 3000 orthogroups have four times as many orthologs in T. chamaedrys as in A. thaliana.

Figure 3

Phylogenetic analysis of the diterpene gene content in three Teucrium species. (A) This tree is rooted by the class II/class I bifunctional ent-kaurene synthase from Physcometrium patens. Genes from T. chamaedrys are in gold, T. marum in blue, and T. canadense in green. Those without highlights are previously characterized diTPSs from other Lamiaceae species and A. thaliana. Bolded genes were functionally characterized in this study. Red and pink rings denote physical clustering in the genome of Teucrium and C. americana, respectively. Clades are labeled according to Johnson et al. (2019). Figure was created with iTOL and BioRender.com. (B) Syntenic analysis between closely related T. marum (blue) and T. chamaedrys (gold) show a 1:4 syntenic relationship in a genomic region containing the majority of diTPSs genes. Inset shows the TPSs and CYPs (cytochromes P450) present in the T. marum cluster. Designated T. chamaedrys nomenclature (3A), Tca40(___), Tca12(___), Tca20(___), and Tca10(___), individually clustered TPS in four loci; (3B) Tc(_____), Tm10 corresponding to individual contigs. Figure was created with SynVisio and BioRender.com.

Figure 4

Extracted ion chromatogram (191 m/z) demonstrating iso-kolavenyl diphosphate synthase activity. (A) Extracted ion chromatograms were stacked and shifted to facilitate comparison of products. Tested enzymes TchaTPS1, TchaTPS2, TchaTPS3, and TcanTPS1 were compared to the known iso-KDP synthase, ArTPS2, and the negative control, DXS+GGDPS. DXS+GGDPS is present in all samples. The peak at ∼11.5 min corresponds to iso-kolavelool (1), and the peak at ∼13.5 min corresponds to iso-kolavenol (2). (B) Representative mass spectra of ArTPS2 corresponding to iso-kolavelool (1) and iso-kolavenol (2) peaks. Mass spectra of all relevant peaks are provided in Supplemental Figure 9. A representative chromatogram of three replicates is shown. Compound identification, level 1 (authentic standard, retention time, fragmentation pattern, m/z, high-resolution GC–MS data, Supplemental Table 4).