Analysis of the Coptis chinensis genome reveals the diversification of protoberberine-type alkaloids

Abstract

Chinese goldthread (Coptis chinensis Franch.), a member of the Ranunculales, represents an important early-diverging eudicot lineage with diverse medicinal applications. Here, we present a high-quality chromosome-scale genome assembly and annotation of C. chinensis. Phylogenetic and comparative genomic analyses reveal the phylogenetic placement of this species and identify a single round of ancient whole-genome duplication (WGD) shared by the Ranunculaceae. We characterize genes involved in the biosynthesis of protoberberine-type alkaloids in C. chinensis. In particular, local genomic tandem duplications contribute to member amplification of a Ranunculales clade-specific gene family of the cytochrome P450 (CYP) 719. The functional versatility of a key CYP719 gene that encodes the (S)-canadine synthase enzyme involved in the berberine biosynthesis pathway may play critical roles in the diversification of other berberine-related alkaloids in C. chinensis. Our study provides insights into the genomic landscape of early-diverging eudicots and provides a valuable model genome for genetic and applied studies of Ranunculales.

Fig. 1. Landscape of genome assembly and annotation of Coptis chinensis.

a Chromosome-scale pseudomolecules (chr1–chr7). b Density of transcripts. c Gene density. d Density of long retrotransposon terminal repeats. e Density of DNA transposable elements. f Density of biallelic heterozygous single-nucleotide polymorphisms. g Intragenomic synteny information.

Fig. 2. Species tree comprising 12 angiosperm species, including Coptis chinensis.

Expansion and contraction of gene families are denoted as numbers with plus and minus signs, respectively. The green numbers in the brackets indicate the estimated divergence time of each node, and the green bars show the 95% confidence interval of divergence time, in millions of years. All the nodes are 100% bootstrap support. A Venn diagram at the side of the tree shows the sharing and specificity of gene families presented between four Ranunculales species.

Fig. 3. One-round ancient whole-genome duplication in Coptis chinensis.

a Histogram distribution of synonymous divergence (K_s) for orthologue duplicates identified within C. chinensis (both transcriptome and genome data were used), A. coerulea and between C. chinensis and A. coerulea. b–e The syntenic depth ratios between different species genome pairs. f Dot plots of orthologues between C. chinensis and grape. The red circles highlight examples of major duplication events which suggesting a 2:3 syntenic relationship. g The estimated whole-genome duplication (WGD) events within the Ranunculales clade and the episodic burst of gene duplications which are statistically consistent with the WGD shared by the three core eudicot species in the Multi-tAxon Paleopolyploidy Search (MAPS) analysis (marked as an asterisk in MAPS3). The taxa included in each MAPS analysis were denoted in round-corner boxes and the ingroup taxa of each analysis were linked as a solid line.

Fig. 4. Evolution of CYP719 members in Coptis chinensis.

a A gene cluster in relation to tandem duplication of CYP719 genes on chromosome 3. b Tissue-specific expression of CYP719 genes identified in C. chinensis. The expression values were log2 transformed. c The Ranunculaceae subclade of the CYP719 gene tree. See Supplementary Fig. 27 for a full version of the CYP719 gene tree. Gene sequences from the present study are marked in green. Aco: Aquilegia coerulea, Tfl: Thalictrum flavum, Cch: C. chinensis, Cja: C. japonica. d Putative branch pathways for the biosynthesis of berberine, coptisine and epiberberine. The box with the green dashed line borders indicates pathways proposed from the results of other plant species (coptisine) or suspected from the chemical structure per se (epiberberine). See Supplementary Data 5 for the abbreviations of the enzymes. The known CYP719 enzymes are denoted with a prefix of species name. Cj: C. japonica, Ec: Eschscholzia californica, Am: Argemone mexicana. e–g Extracted ion chromatograms showing the in vitro catalytic activity of Cch00017825 with three different substrates. In each case, enzyme-mediated activity is indicated by the green chromatograms, while those for the empty vector (EV) negative control assays are in black. The speculated product of nandinine was marked in gray. Source data underlying Fig. 4b are provided as a Source Data file.