Chromosome-level genome and multi-omics analyses provide insights into the geo-herbalism properties of Alpinia oxyphylla

Abstract

Introduction: Alpinia oxyphylla Miquel (A. oxyphylla), one of the "Four Famous South Medicines" in China, is an essential understory cash crop that is planted widely in the Hainan, Guangdong, Guangxi, and Fujian provinces. Particularly, A. oxyphylla from Hainan province is highly valued as the best national product for geo-herbalism and is an important indicator of traditional Chinese medicine efficacy. However, the molecular mechanism underlying the formation of its quality remains unspecified.

Methods: To this end, we employed a multi-omics approach to investigate the authentic quality formation of A. oxyphylla.

Results: In this study, we present a high-quality chromosome-level genome assembly of A. oxyphylla, with contig N50 of 76.96 Mb and a size of approximately 2.08Gb. A total of 38,178 genes were annotated, and the long terminal repeats were found to have a high frequency of 61.70%. Phylogenetic analysis demonstrated a recent whole-genome duplication event (WGD), which occurred before A. oxyphylla's divergence from W. villosa (~14 Mya) and is shared by other species from the Zingiberaceae family (Ks, ~0.3; 4DTv, ~0.125). Further, 17 regions from four provinces were comprehensively assessed for their metabolite content, and the quality of these four regions varied significantly. Finally, genomic, metabolic, and transcriptomic analyses undertaken on these regions revealed that the content of nootkatone in Hainan was significantly different from that in other provinces.

Discussion: Overall, our findings provide novel insights into germplasm conservation, geo-herbalism evaluation, and functional genomic research for the medicinal plant A. oxyphylla.

Keywords: Alpinia oxyphylla; genome; geo-herbalism; metabolomics; nootkatone; transcriptomics; valenene synthase.

Figure 1

Morphology of A. oxyphylla and overview of A. oxyphylla gnome. (A) Morphological characteristics of A. oxyphylla. (a), Plant; (b), inflorescence; (c), fruits; (d), dried fruit; (e) seed and pericarp. (B) Circos plot of A. oxyphylla genome assembly. Elements are arranged in the following scheme (from inner to outer): (a) GC content; (b) gene density shown as the distribution densities from high (red) to low (green); (c) repeat sequence densities; (d) non-coding RNA (ncRNA) density; (e) tandem repeat density; (f) the density of other repeats except tandem repeats. The window size is 1Mb.

Figure 2

Transcriptomic and metabolic analysis and identification of differentially expressed genes. (A) Venn diagram shows the number of differentially expressed genes in four regions. (B) KEGG enrichment analysis of differentially expressed genes. (C) Heatmap showing the expression level of top 30 genes associated with pharmacodynamic component kaempferol. (D) Venn diagram shows the number of differentially expressed metabolites in four regions. (E) KEGG enrichment analysis of differentially metabolites. (F) Heatmap showing the expression level of top 30 genes associated with pharmacodynamic component nootkatone. The metabolisms KEGG annotation and correlation coefficient between genes and metabolites of this figure are from Supplementary Data 2, 3 . Sample class: H1 (A1-A3): Danzhou; H2 (A4-A6): Baoting; L1 (B1-B3): Nanning; L2 (B4-B6): Zhangpu. The fpkm values are log2-based. Red and blue indicate high and low expression levels, respectively.

Figure 3

Genome and gene family evolution of A. oxyphylla. (A) Phylogenetic tree of 10 species A. thaliana, S. bicolor, O.sativa, A. comosus, M. balbisiana, M. acuminata, Z. officinale, C. alismatifolia, W. villosa, and A. oxyphylla. Gene family expansions/contractions are indicated in green/pink. Inferred divergence times (MYA, million years ago) are denoted at each node. (B) Gene categories used from all the species (the resource of this figure is from Supplementary Data 1 ). (C) Syntenic blocks between A. oxyphylla and Z. officinale. (D, E) Distribution of 4DTV values and Ks values of syntenic orthologous genes in the genomes of five species (M. acuminata, Z. officinale, C. alismatifolia, W. villosa, and A. oxyphylla. (F) Insertion time of LTRs in M. acuminata, C. alismatifolia, W. villosa, and A. oxyphylla.

Figure 4

Gene family and functional enrichment analysis of A. oxyphylla. (A) The shared and unique gene families among five species (M. acuminata, Z. officinale, C. alismatifolia, W. villosa and A. oxyphylla). (B–D) KEGG enrichment analysis of expanded gene families, contracted gene families, and positively selected gene families in A. oxyphylla.

Figure 5

Terpenoid and flavonoid metabolic pathways involved in the biosynthesis of nootkatone and kaempferol in A. oxyphylla. Heatmap showing the expression level of candidate genes involved in nootkatone (A) and kaempferol (B) synthesis in different regions. Based on the million mapped fragments (FPKM) value, genes with the identity>70% were selected on the nootkatone and kaempferol biosynthesis pathway; for functional annotation of related genes, see Supplementary Data 4, 5 . The FPKM values are log2-based. Red and blue indicate high and low expression levels, respectively. Enzyme abbreviations: MVA pathway: AACT (acetyl-CoA acetyltransferase); HMGS (3-hydroxyl-3-methylglutaryl-CoA synthase); HMGR (3-hydroxy-3-methylglutaryl-CoA reductase); MK (mevalonate kinase); PMK (phosphomevalonate kinase); MPDC (pyrophosphomevalonate diphosphate decarboxylase); IPPI (IDP isomerase); MEP pathway: DXS (1-deoxy-D-xylulose-5-phosphate synthase); DXR (1-deoxy-D-xylulose-5-phosphate reductoisomerase); MCT (2-C-methyl-D-erythritol-4-phosphate cytidylyltransferase); CMK (4-(cytidine 5-diphospho)-2-C-methylerythritol kinase); MDS (2-C-methul-D-erytyritol 2,4-cyclodiphosphate synthase); HDS (4-hydroxy-3-methylbut-2-enyl-diphosphate synthase); HDR (4-hydroxy-3-methylbut-2-enyl-diphosphate reductase); PAL (phenylalanine ammonia-lyase); C4H (cinnamate-4-hydroxylase); 4CL (4-coumarate CoA ligase); CHS (chalcone synthase); CHI (chalcone isomerase); F3H (flavanone 3-hydroxylase); FLS (flavonol synthase). Compound abbreviations: HMGCoA (3-hydroxyl-3-methyglutaryl-CoA); MVA (mevalonate); MVAP (mevalonate-5-phosphate); MVAPP (mevalonate-5-diphosphate); IPP (isopentenyl diphosphate); DMAPP (dimethylallyl diphosphate); GA-3P (D-Glyceraldehyde 3-phosphate); DXP (1-Deoxy-D-xylulose 5-phosphate); MEP (2-C-Methyl-D-erythritol 4-phosphate); HMBPP (4-(Cytidine 5’-diphospho)-2-C-methyl-D-erythritol); CDP-ME (2-Phospho-4-(Cytidine 5’-diphospho)-2-C-methyl-D-erythritol); MECPP (2-C-Methyl-D-erythritol 2,4-cyclodiphosphate); HMBDP (4-Hydroxy-3-methylbut-2-enyl-diphosphate); FPP (farnesyl diphosphate).

Figure 6

Analysis of TPS gene family in A. oxyphylla. (A) Phylogenetic tree of TPS genes from A. oxyphylla (59 genes), O. sativa (32 genes), A. thaliana (31 genes), and O. sanctum (47 genes). The outer circle and branch colors represent different TPS gene subfamilies. (B) Schematic map presentation of the genomic localization of 59 AoxTPSs. Blue represents tandem duplicates, and purple-linked genes represent genome-wide replication. (C) Heatmap (row scale) showing the differentially expressed of AoxTPSs according to the transcriptome data in four regions. Uppercase AOX represents AoxTPSs from a or b subfamily, and lowercase aox represents AoxTPSs from other subfamilies. Underlined AoxTPSs represents highly expressed in A1-A6 areas. Sample class: H1 (A1-A3): Danzhou; H2(A4-A6): Baoting; L1 (B1-B3): Nanning; L2(B4-B6): Zhangpu.