Diploid chromosome-level reference genome and population genomic analyses provide insights into Gypenoside biosynthesis and demographic evolution of Gynostemma pentaphyllum (Cucurbitaceae)

Abstract

Gynostemma pentaphyllum (Thunb.) Makino is a perennial creeping herbaceous plant in the family Cucurbitaceae, which has great medicinal value and commercial potential, but urgent conservation efforts are needed due to the gradual decreases and fragmented distribution of its wild populations. Here, we report the high-quality diploid chromosome-level genome of G. pentaphyllum obtained using a combination of next-generation sequencing short reads, Nanopore long reads, and Hi-C sequencing technologies. The genome is anchored to 11 pseudo-chromosomes with a total size of 608.95 Mb and 26 588 predicted genes. Comparative genomic analyses indicate that G. pentaphyllum is estimated to have diverged from Momordica charantia 60.7 million years ago, with no recent whole-genome duplication event. Genomic population analyses based on genotyping-by-sequencing and ecological niche analyses indicated low genetic diversity but a strong population structure within the species, which could classify 32 G. pentaphyllum populations into three geographical groups shaped jointly by geographic and climate factors. Furthermore, comparative transcriptome analyses showed that the genes encoding enzyme involved in gypenoside biosynthesis had higher expression levels in the leaves and tendrils. Overall, the findings obtained in this study provide an effective molecular basis for further studies of demographic genetics, ecological adaption, and systematic evolution in Cucurbitaceae species, as well as contributing to molecular breeding, and the biosynthesis and biotransformation of gypenoside.

Figure 1

Plant morphology, karyotypes, assembled genome information, and evolutionary analyses of G. pentaphyllum. (a) Wild plant and habitat of G. pentaphyllum. (b) Venn diagram for the genome annotated based on five databases. (c) Circos graph showing the genome characteristics from the outer circle to the inner ring (A–G): (A) circular representation of the 11 pseudochromosomes featured in 10-Mb intervals across the genome; (B–D) distributions of (B) Ty3-gypsy long terminal repeat retrotransposons (LTR-RTs), (C) Ty1-copia LTR-RTs, and (D) all transposable elements; (E) gene density, (F) GC distribution, and (G) intraspecific collinearity. (d) Flow cytometry analysis. (e) Anatomical sections of G. pentaphyllum chromosomes. (f) Hi–C chromatin interaction heatmap for 11 pseudochromosomes in the G. pentaphyllum genome. (g) Inferred phylogenetic tree, divergence time, and gene family expansions and contractions. The numbers at the node positions represent the divergence time of each species in millions of years. The numbers in parentheses indicate the confidence interval for the divergence time, which can be used to estimate the divergence time of target species and other species. The yellow points are the calibration times used to correct the time of species divergence. The green, red, and blue sections in the pie graph indicate the expanded, contracted, and other gene families, including extinctions and no change gene families, respectively. The red and green numbers represent the exact precise quantities of expanded and contracted gene families, respectively. (h) Clusters of orthologous and paralogous gene families in G. pentaphyllum and 11 other sequenced plant genomes. (i) Whole-genome duplication (WGD) analysis of G. pentaphyllum and genomes of related species.

Figure 2

Comparative transcriptome analysis of genes involved in the gypenoside biosynthetic pathway. The expression value of each identified candidate gene is colored in log₁₀(FPKM) in five tissues: fruit, flower, stem, leaf, and tendril. Low to high expression is indicated by the change in color from blue to red.

Figure 3

Genetic structure, demographic history, and global climatic space constructed over all background areas and realized niches of G. pentaphyllum. (a) Spatial genetic structure of G. pentaphyllum based on 32 populations within the natural species range in China. The pie chart colors indicate the probability of sample assignment based on SNPs analyzed using ADMIXTURE at the most likely K = 3. Standard maps produced by the Ministry of Natural Resources were used as the base maps with drawing approval numbers GS(2016)1554 and GS(2016)1606. (b) Results of ADMIXTURE analyses for 107 G. pentaphyllum individuals at K = 2 and K = 3. (c) Non-rooted maximum-likelihood phylogenetic tree based on SNPs in 107 G. pentaphyllum individuals. Color coding of the branches reflects the structure of genetic groups at K = 3. (d) Past effective population size history of G. pentaphyllum assessed by PSMC. (e) Demographic changes on recent timescales established for G. pentaphyllum by using SMC++. (f) Plot of cross-validation errors for ADMIXTURE runs with K values ranging from 1 to 8. (g) Three-dimensional principal component analysis (PCA) results for G. pentaphyllum. (h) The 20% occurrence density includes the contribution and direction of each variable to the first two components of PCA-env. (i) An occurrence density of 100% is denoted by the thin line and 100% of the available climatic background by the thick line. (j–o) Kernel density plots for six environmental parameters for G. pentaphyllum showing niche differences among the three groups.