Chromosome-level Alstonia scholaris genome unveils evolutionary insights into biosynthesis of monoterpenoid indole alkaloids

Abstract

Alstonia scholaris of the Apocynaceae family is a medicinal plant with a rich source of bioactive monoterpenoid indole alkaloids (MIAs), which possess anti-cancer activity like vinca alkaloids. To gain genomic insights into MIA biosynthesis, we assembled a high-quality chromosome-level genome for A. scholaris using nanopore and Hi-C data. The 444.95 Mb genome contained 35,488 protein-coding genes. A total of 20 chromosomes were assembled with a scaffold N50 of 21.75 Mb. The genome contained a cluster of strictosidine synthases and tryptophan decarboxylases with synteny to other species and a saccharide-terpene cluster involved in the monoterpenoid biosynthesis pathway of the MIA upstream pathway. The multi-omics data of A. scholaris provide a valuable resource for understanding the evolutionary origins of MIAs and for discovering biosynthetic pathways and synthetic biology efforts for producing pharmaceutically useful alkaloids.

Keywords: Biological sciences; Genomics; Natural sciences; Omics; Plant Genetics; Plant biology.

Graphical abstract

Figure 1

Genome information and morphological features of A. scholaris (A) Characteristics of the 20 chromosomes of A. scholaris. The tracks from the outer to the inner regions of the circle individually represent the length of chromosomes (pink), gene numbers (dark green), the content of GC (black line), repeat sequences (blue), LTRs (green), LTR Copia (yellow), and LTR Gypsy (gray), and the links inside the circle show syntenic collinearity. (B) Hi-C plot of the pseudochromosome-level assembly of A. scholaris genome. The axis refers to the genome size, and each blue box represents one chromosome.

Figure 2

Gene family analysis and phylogenetic tree construction (A) Bar chart of the ortholog numbers in these 16 species. (B) Phylogenetic tree showing the sizes of significantly expanded and contracted gene families. The branch labels in yellow and blue represent the significantly expanded and contracted gene families (p value <0.05), respectively, of each node. The right column shows significantly expanded and contracted gene families of individual species. Furthermore, the statistical method of enrichment analysis is χ2 test. AdjustedPv is a corrected p value that is obtained by performing false discovery rate (FDR) testing on p values. AdjustedPv obtained from significance tests are generally considered to have significant statistical differences when AdjustedPv <0.05.

Figure 3

The analysis of whole-genome duplication in A. scholaris (A) The distribution of synonymous substitution rate (ks) distances observed for paralogs from A. scholaris, C. canephora, C. roseus, O. pumila, and V. vinifera. (B) The distribution of ks values of orthologs between A. scholaris and the previously mentioned species. (C) Synteny between genomic regions in A. scholaris, C. roseus, and O. pumila. The gray lines highlight major syntenic blocks spanning the genomes. The colored lines represent examples of syntenic genes found in two species that correspond to one copy in A. scolaris, and two in C. roseus and O. pumila.

Figure 4

Key genes involved in strictosidine biosynthesis (A and B) Maximum likelihood phylogenetic tree based on candidate STR and TDC gene families from these 18 species. “∗” represents functionally characterized genes. Each species is represented by a different color. (C) A gene cluster located on the third chromosome of the A. scholaris genome. The blue and orange lines show syntenic blocks of AsTDCs and AsSTRs with TDC and STR from C. roseus and O. pumlia, respectively. (D) The saccharide-terpene cluster. The bolded gene IDs represent genes that are significantly highly expressed in petioles and trunk barks and are also genes that are enriched in the monoterpene biosynthesis pathway.

Figure 5

Biosynthesis pathway of akuammiline alkaloid and the expression levels of candidate enzymes in the pathway (A) The akuammiline biosynthesis pathway. (B) The expression levels of candidate genes in the A. sholaris genome. The abscissa of each heatmap indicates the different tissues of A. scholaris. B: branch, P: petiole, T: trunk bark, L: leaf. The number represents duplication. Log2GeneCount refers to the normalization of FPKM (fragments per kilobase million) values by the log method of each row. Different expressed genes match adjusted p value <0.05 and a |log2FoldChange| ≥ 2. Dark-blue color indicates a high expression level, and blue indicates a low expression level.