Comparisons of wild and cultivated American ginseng (Panax quinquefolius L.) genomes provide insights into changes in root growth and metabolism during domestication

American ginseng (Panax quinquefolius L.) originated in the forests of North America and was introduced to China over 40 years ago. Cultivated P. quinquefolius in China had fewer lateral roots and significantly decreased ginsenoside Rg1 content compared to wild American varieties, both of which determines quality and yield of P. quinquefolius (Figure S1). To explore genetic basis to alterations in both root morphology and specialized metabolite production, we firstly sequenced and assembled the chromosome-level P. quinquefolius genome of wild American varieties by PacBio SMRT sequencing, 10× Genomics sequencing and chromatin conformation capture technology (Appendix S1; Appendix S2), and further analysed gene family expansion and contraction in P. quinquefolius and P. ginseng (fewer lateral roots) genomes compared to P. notoginseng (more lateral roots). As a result, there were 84 expanded gene families (1159 genes) shared by P. quinquefolius and P. ginseng compared to P. notoginseng (Figure S2; Table S1). Compared to P. notoginseng, there were significant expansions of the gene families incorporating 1,3-β-glucan synthase, cellulose synthase, expansin (EXP) and shikimate O-hydroxycinnamoyl transferase in P. quinquefolius, with increases of 28, 29, 14 and nine members, respectively, which are thought to participate cell wall synthesis (Fang et al., 2023), formation of cell wall skeleton (Lampugnani et al., 2019), cell extension (Samalova et al., 2023), hypothesizing that regulation of the cell wall composition and structure contribute to specific root morphology formation during P. quinquefolius speciation. To identify genes that played key roles in altering P. quinquefolius root morphology after its introduction to China, whole-genome resequencing was performed in 23 wild and 30 cultivated samples. Principal component analysis (PCA) and population structure analysis at K = 2 clustered the analysed accessions into two main groups (16 wild individuals; 30 cultivated and seven wild individuals) (Figure 1b,c; Figure S3), and linkage disequilibrium analysis showed that the cultivated population decayed more slowly than the wild population (Figure S3). To identify key selective signatures contributing to genetic changes in cultivated P. quinquefolius, we analysed selective sweep regions using the top 5% of the pairwise Wright's F statistics and genetic diversities. This yielded 263 and 397 positively selected genes (PSGs) in the wild and cultivated populations, respectively (Table S2; Table S3). Through comparative transcriptomic analysis of P. quinquefolius roots and leaves, we further analysed expression levels of PSGs to identify potentially functionally important variations during P. quinquefolius domestication. Twenty-one of the 397 PSGs in the cultivated population shown in Figure S3 were expressed at significantly higher levels in the roots compared to leaves (Table S4). Two positively selected differential expressed genes were related to cell wall development: an EXP gene (Pq17G51776; log2(FC) = 5.23, p.adj = 3.16e−09) and a beta-galactosidase gene (Pq06G18190; log2(FC) = 3.45, p.adj = 2.97e−10) (Figure S4; Table S4; Figure S6), which were involved in cell wall extension (Samalova et al., 2023) and cell wall degradation (Chen et al., 2021), respectively. These results supported the hypothesis that cell wall regulation was at the core of variations in P. quinquefolius root morphology. We also found that two peroxidase genes (Pq17G50742 and Pq17G50721), two isoflavone reductase genes (Pq23G66278 and Pq23G66277) and a β-galactosidase gene (Pq16G47641) were positively selected in wild P. quinquefolius and highly expressed in the root (Figure S4; Table S5). It is reported that isoflavone reductase and peroxidase enhance plant resistance to biotic and osmotic stress (Tenhaken, 2015), implying their importance for wild P. quinquefolius adaption to environmental stress. Peroxidases strengthen cell wall mechanical properties (Tenhaken, 2015), and β-galactosidase degrade cell wall polysaccharides (Chen et al., 2021), both of which counteract EXP activities. This could explain the key role of EXPs in cultivated populations but the importance of peroxidase and β-galactosidase in wild populations as determined from the selective sweep analysis. EXPs serve to relax stress generated in the cell wall and improve extensibility, contributing to root growth (Samalova et al., 2023). We identified a total of 65 EXP genes in the newly assembled genome and classified them into four subfamilies: the 44 α-expansins (EXPAs), the 14 β-expansins (EXPBs), the two expansin-like As (EXLAs) and the five expansin-like Bs (EXLBs) (Figure S5; Table S6). Positive selection of PqEXLB5 (Pq17G51776) in cultivated populations may indicate that the EXL gene subfamilies significantly contributed to genetic changes that led to differences in P. quinquefolius root morphology after introduction (Figure S3). To further determine the function of EXLA and EXLB gene subfamilies, we constructed overexpression (OE) and RNA interference (RNAi) knockdown vectors for seven EXL genes: PqEXLA1, PqEXLA2 and PqEXLB1–PqEXLB5 (Figure S7). Compared with wild P. quinquefolius calli, PqEXLA1-OE showed a significant increase in cell volume (73.7%) and rate of tissue growth (38.7%), while which significantly decreased 31.3% and 26.7% in PqEXLA1-RNAi calli (Figure 1d,e; Figure S8). Interestingly, PqEXLB1-OE calli significantly promote cell volume (111.9%) and cell wall thickness (120.2%), while which were significant reduced in PqEXLB1-RNAi calli, compared to wild-type (Figure 1d,f; Figure S9). These results indicated that PqEXLA1 (Pq21G63567) promoted tissue growth by increasing cell volume and PqEXLB1 (Pq23G66132) mediated regulation of cell wall thickness. Compared to wild calli, Rg1 levels were significantly decreased in PqEXLA1-OE and PqEXLB1-OE calli but increased in PqEXLA1-RNAi and PqEXLB1-RNAi calli (Figure S5), and the PqEXLA1-RNAi calli showed significant up-regulation of seven Rg1 biosynthesis genes (Figure S5). These results showed that PqEXLA1 and PqEXLB1 affected Rg1 accumulation in P. quinquefolius. Rg1 has autotoxicity in Panax roots because it causes overaccumulation of reactive oxygen species (ROS) and cell wall degradation (Yang et al., 2018). In wild P. quinquefolius, the high Rg1 contents are expected to cause ROS overaccumulation, which would require alleviation by antioxidants. This is consistent with our finding that two peroxidase genes (Pq17G50742 and Pq17G50721) were under positive selection in the wild population. Previous studies have revealed that ginsenosides may function as defensive compounds (Deng et al., 2023), which explains the retention of high Rg1 levels in wild P. quinquefolius despite the autotoxicity. Our results thus position PqEXLA1 and PqEXLB1 as hub genes that affects both cell growth and Rg1 accumulation, ultimately balancing the plant's requirements for growth and defence (Figure 1g). This work was supported by the National NSFC (82173926/81891013/82325049), STIP-CACMS (CI2023E002-04) and KP-VCMR (2060302). The authors declare no conflict of interest. Y.Y. and L.H. designed the project. Y.L., Z.W., H.J., J.H., J.S., Y.J., T.N. and Y.Z. prepared materials and analysed the data, T.W. and J.H. performed gene functional validation. Y.Y., Z.W. and H.J. wrote the manuscript. All authors read and approved the paper. The genome assembly and annotations (GWHDOGX00000000), raw sequence data of resequencing and transcriptome (GSA: CRA011933; CRA011888) were deposited in National Genomics Data Center. Figure S1–S9 Supplementary Figures. Table S1–S19 Supplementary Tables. Appendix S1 Methods. Appendix S2 Genome assembly and annotation. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.