Full-length transcriptome atlas of Panax vietnamensis var. fuscidiscus reveals novel genes and alternative splicing in tissue-specific biosynthesis of ocotillol-type saponins

Background Panax vietnamensis var. fuscidiscus is the only known Panax species that accumulates exceptionally high levels of ocotillol (OCT)-type ginsenosides, particularly majonoside R2. However, the molecular mechanisms underlying OCT-type ginsenoside biosynthesis remain largely uncharacterized, due in part to the absence of comprehensive full-length transcriptome data and complete gene annotations.

We combined PacBio single-molecule real-time (SMRT) and Illumina RNA sequencing (RNA-Seq) technologies to construct the first tissue-specific, full-length transcriptome atlas of P. vietnamensis var. fuscidiscus. A total of 281,468 non-redundant transcripts were assembled, including 8,089 novel genes not present in existing annotations, substantially expanding the documented gene repertoire of this species. We identified 21 candidate genes putatively involved in triterpenoid saponin biosynthesis and uncovered distinct tissue-specific expression patterns across the oleanane (OA)-, protopanaxadiol (PPD)-, protopanaxatriol (PPT)-, and OCT-type biosynthetic pathways. Notably, we detected 46,917 alternative splicing (AS) events, with over 45% of expressed genes producing multiple isoforms. Several key biosynthetic genes—including dammarenediol synthase (DDS), cytochrome P450 716A53v2 (CYP716A53v2), and mevalonate diphosphate decarboxylase (MVD)—exhibited tissue-specific splicing patterns, such as retained introns and exon skipping, which may contribute to functional diversification of isoforms involved in OCT-type ginsenoside biosynthesis. These results highlight the importance of post-transcriptional regulation and transcript diversity in the evolution and fine-tuning of specialized metabolic pathways.

This study establishes a high-quality, full-length transcriptomic resource for P. vietnamensis var. fuscidiscus, revealing previously undescribed genes and extensive alternative splicing events related to ginsenoside biosynthesis. Our findings provide novel molecular insights into the mechanisms regulating OCT-type saponin accumulation and offer a foundation for future research into the functional characterization, regulatory mechanisms, and synthetic biological production of rare triterpenoid saponins. Competing Interest Statement The authors have declared no competing interest. Footnotes Figure 2 has been corrected to replace an image that was mistakenly duplicated from Figure 1, and Figure 5 has been corrected to replace an image that was mistakenly duplicated from Figure 4. The updated figures present the correct data and do not affect the results or conclusions of the manuscript. Abbreviations - AACT - Acetyl-CoA acetyltransferase - AF - Alternative first exon - AL - Alternative last exon - AS - Alternative splicing - A3 - Alternative 3′ splice site - A5 - Alternative 5′ splice site - CCS - Circular consensus sequence - CD-HIT - Cluster Database at High Identity with Tolerance - COG/KOG - Clusters of Orthologous Groups / Eukaryotic Orthologous Groups - CYP - Cytochrome P450 - DDS - Dammarenediol synthase - DEG - Differentially expressed gene - eggNOG - Evolutionary genealogy of genes: Non-supervised Orthologous Groups - FDR - False discovery rate - FL - Full-length - FLNC - Full-length non-chimeric - FPS - Farnesyl diphosphate synthase - FSM - Full splice match - GO - Gene Ontology - HMGR - 3-hydroxy-3-methylglutaryl-coenzyme A reductase - HMGS - 3-hydroxy-3-methylglutaryl coenzyme A synthase - HQ - High-quality - ICE - Iterative Clustering for Error Correction - ISM - Incomplete Splice Match - Iso-Seq - Isoform sequencing - KEGG - Kyoto Encyclopedia of Genes and Genomes - LQ - Low-quality - MVA - Mevalonate pathway - MVD - Mevalonate diphosphate decarboxylase - MVK - Mevalonate kinase - MX - Mutually exclusive exons - NIC - Novel in catalog - NNC - Novel not in catalog OCT Ocotillol - OSC - Oxidosqualene cyclase - PPD - Protopanaxadiol - PPT - Protopanaxatriol - RI - Retained intron - RNA-Seq - RNA sequencing - ROIs - Reads of insert - SE - Skipped exon - SE - Squalene epoxidase - SGS - Second-generation sequencing - SMRT - Single-Molecule Real-Time sequencing - SS - Squalene synthase - SSR - Simple sequence repeat - TSS/TTS - Transcription start site / transcription termination site - UGT - UDP-glycosyltransferase