Cyclocarya paliurus is a relict plant species that survived the last glacial period and shows a population expansion recently. Its leaves have been traditionally used to treat obesity and diabetes with the well-known active ingredient cyclocaric acid B. Here, we presented three C. paliurus genomes from two diploids with different flower morphs and one haplotype-resolved tetraploid assembly. Comparative genomic analysis revealed two rounds of recent whole-genome duplication events and identified 691 genes with dosage effects that likely contribute to adaptive evolution through enhanced photosynthesis and increased accumulation of triterpenoids. Re sequencing analysis of 45 C. paliurus individuals uncovered two bottlenecks, consistent with the known events of environmental changes, and many selectively swept genes involved in critical biological functions, including plant defense and secondary metabolite biosynthesis. We also proposed the biosynthesis pathway of cyclocaric acid B based on multi-omics data and identified key genes, in particular gibberellin-related genes, associated with the heterodichogamy in C. paliurus species. Our study sheds light on evolutionary history of C. paliurus and provides genomic resources to study the medicinal herbs.

Cyclocarya paliurus (Batal) Iljinskaja is mainly used for harvesting leaves as materials for tea production and ingredients for the food industry. As its most important component, the contents of its total or specific flavonoids are supposed to vary at different ploidy levels. In the present study, two ploidy levels of C. paliurus are used to study their metabolome and transcriptome profiles. Though the total content of the flavonoids in leaves that were collected in September (the main harvesting season) presented insignificant differences between the two ploidies, flavonoid glucuronides were significantly accumulated in the tetraploid C. paliurus. Several structural genes related to the biosynthesis of these flavonoid glucuronides were expressed differentially, including PAL, 4CL, CHS, and CpUGTs. A weighted gene co-expression network analysis (WGCNA) revealed that nine genes were highly correlated with the flavonoid glucuronide contents. Furthermore, 3 CpMYB39 and 3 CpUGT71 were highly associated with this accumulation of flavonoid glucuronides in tetraploid C. paliurus. These results can provide a new perspective on how different polyploid levels alter the quantitative and qualitative patterns of the secondary metabolite production in C. paliurus.

The GRAS gene family, derived from GAI, RGA, and SCR, plays a crucial role in plant growth and development. In the diploid Cyclocarya paliurus (2n = 2x = 32) with heterodichogamous characteristics, 51 CpGRAS genes were identified and phylogenetically classified into 10 subfamilies. Structural analysis revealed that CpGRAS genes possessed a canonical GRAS domain, but 70% lacked introns. WGD/segmental duplication was the major driver in the expansion of the CpGRAS family. In addition, a Ka/Ks ratio below 1 for these genes implied functional constraints and evolutionary conservation. Transcriptional profiling revealed significant differential expressions of CpGRAS genes between male and female flowers from two mating types, protogyny (PG) and protandry (PA). Notably, members of the DELLA subfamily exhibited significant upregulation in female flowers at the inflorescence elongation (S3) stage. The expression level of CpSCL6-2 in late-flowering samples (PA-F and PG-M) was higher than in early-flowering ones (PA-M and PG-F). Co-expression analysis identified that CpRGL1 and CpGAI-2 of the DELLA subfamily, along with CpSCL6-2, acted as hub genes, implying their crucial roles in floral development and potential involvement in the heterodichogamous flowering mechanism in C. paliurus. These findings broaden our understanding of CpGRAS genes and provide new insights into the molecular basis of heterodichogamy.

Cyclocarya paliurus is a relict plant species that survived the last Glacial period and shows a population expansion recently. Its leaves have been traditionally used to treat obesity and diabetes with the well-known active ingredient cyclocaric acid B. Here, we present three C. paliurus genomes from two diploids with different flower morphs and one haplotype-resolved tetraploid assembly. Comparative genomic analysis revealed two rounds of recent whole-genome duplication events and identified 691 genes with dosage-effect that likely contribute to adaptive evolution through enhanced photosynthesis and increased accumulation of triterpenoids. Re-sequencing analysis of 45 accessions uncovered two bottlenecks, consistent with the known events of environmental changes, and many selectively swept genes involved in critical biological functions, including plant defense and secondary metabolism biosynthesis. We also proposed the biosynthesis pathway of cyclocaric acid B based on multi-omics data and identified key genes, in particular gibberellin related genes, associated with heterodichogamy in the species. Our research sheds light on evolutionary history and provides genomics resources to study the medicinal herb.

Cyclocarya paliurus (Batal.) Iljinskaja, a monotypic species in Cyclocarya of Juglandaceae, is regarded as one of important medical plants in China. In order to reveal the alterations in chloroplast (cp) genome with nuclear genome duplication, we presented the complete cp genomes of C. paliurus, and firstly analyzed on the basis of ploidy type (tetraploid and diploid C. paliurus). The total length of the cp genome of tetraploid and diploid C. paliurus is 160,938 and 161,105 bp, respectively. Both type genome consist of a large single-copy (LSC) region (90,221 and 90,391 bp), a small single-copy (SSC) region (18,593 and 18,590 bp), and an pair of invert repeats (IRs) regions (26,062 and 26,062 bp). Tetraploid and diploid plastid genome contain 132 and 137 genes, 87 and 88 protein-coding genes, 37 and 39 tRNA genes, and both eight rRNA genes, respectively. Closely phylogenetic relationship by analyzing 23 cp genomes suggests that tetraploid C. paliurus probably originated from diploid C. paliurus.