The chromosome-level reference genome assembly for Panax notoginseng and insights into ginsenoside biosynthesis

Abstract

Panax notoginseng, a perennial herb of the genus Panax in the family Araliaceae, has played an important role in clinical treatment in China for thousands of years because of its extensive pharmacological effects. Here, we report a high-quality reference genome of P. notoginseng, with a genome size up to 2.66 Gb and a contig N50 of 1.12 Mb, produced with third-generation PacBio sequencing technology. This is the first chromosome-level genome assembly for the genus Panax. Through genome evolution analysis, we explored phylogenetic and whole-genome duplication events and examined their impact on saponin biosynthesis. We performed a detailed transcriptional analysis of P. notoginseng and explored gene-level mechanisms that regulate the formation of characteristic tubercles. Next, we studied the biosynthesis and regulation of saponins at temporal and spatial levels. We combined multi-omics data to identify genes that encode key enzymes in the P. notoginseng terpenoid biosynthetic pathway. Finally, we identified five glycosyltransferase genes whose products catalyzed the formation of different ginsenosides in P. notoginseng. The genetic information obtained in this study provides a resource for further exploration of the growth characteristics, cultivation, breeding, and saponin biosynthesis of P. notoginseng.

Keywords: P. notoginseng; chromosome-level; genome; ginsenoside; regulation; transcriptome.

Figure 1

Genome assembly characterization and chromosome locations of P. notoginseng. Landscape of the P. notoginseng genome: from outside to inside, chromosome number and length, coverage of second-generation data, density of repetitive sequences, gene density, GC content, noncoding RNA density, and genomic synteny.

Figure 2

Genome evolution and transcription factor regulation analysis of P. notoginseng. (A) Inferred phylogenetic tree with 458 single-copy genes from eight plant species. Gene family expansions are indicated in green, and gene family contractions are indicated in red. The timings of WGD and whole-genome triplications (WGT) are superimposed on the tree. Divergence times are estimated by maximum likelihood (PAML). (B) Distribution diagram of 4DTv values. The dark green-filled part indicates the 4DTv analysis inside P. notoginseng, and the peaks marked by the dotted line indicate where the two WGD events of P. notoginseng occurred. (C) Syntenic dot plots show a 2:1 chromosomal relationship between the P. notoginseng genome and the V. vinifera genome. The area in the pink box on each horizontal line represents the collinear block between the two genomes. (D) Correlation analysis of transcription factors with pathway genes. Pathway genes are represented by hexagons and transcription factors by circles. The line indicates the nature of the correlation: red for a positive correlation and blue for a negative correlation. The darker the color, the higher the correlation.

Figure 3

Temporal expression profile of key enzyme genes in the saponin biosynthesis pathway. (A) A brief view of the morphological changes in P. notoginseng as the years of growth increase during the cultivation process. (B) Temporal expression heatmap of terpenoid biosynthetic pathway genes in P. notoginseng. Taking the leaf's heatmap as an example, the Arabic numerals in the label indicate the years of growth; for example, 2-leaf indicates that the sample is the leaf of a 2-year-old P. notoginseng plant. Based on the gene expression levels, the pattern of expression change for any one gene can be observed after the data in each column are standardized. The area marked by the red box indicates high gene expression levels. Each heatmap has its own color scale: the higher the expression, the greener the color.

Figure 4

Screening for candidate UGT genes and functional verification of five UGT genes. (A) Phylogenetic analysis of UGT genes. The UGT gene families clustered into one clade are represented by different colors. The bootstrap value associated with each branch is represented by a light-purple circle: the larger the radius, the greater the bootstrap value. (B) UPLC/Q-TOF analysis of five functional UGT genes. In catalytic reactions, PnUGT3 uses PPT and F1 as substrates, PnUGT1 uses PPD, PPT, and Rh2 as substrates, PnUGT5 uses PPD as a substrate, and PnUGT2 and PnUGT4 use Rh2 as a substrate to generate corresponding ginsenoside compounds. The chemical structures and characteristic mass spectrum peaks of products from each reaction are displayed in the dashed box of each track. (C) WGCNA analysis of UGT genes.

Figure 5

Overview of the saponin biosynthetic pathway in P. notoginseng and expression profiles of key enzyme genes. The genes in the green box are the UGT genes identified in this study.