Amomi Fructus (Sharen, AF) is a traditional Chinese medicine (TCM) from three source species (or varieties), including Wurfbainia villosa var. villosa (WVV), W. villosa var. xanthioides (WVX), or W. longiligularis (WL). Among them, WVV has been transplanted from its top-geoherb region, Guangdong, to its current main production area, Yunnan, for >50 years in China. However, the genetic and transcriptomic differentiation among multiple AF source species (or varieties) and between the origin and transplanted populations of WVV is unknown. In our study, the observed overall higher expression of terpenoid biosynthesis genes in WVV than in WVX provided possible evidence for the better pharmacological effect of WVV. We also screened six candidate borneol dehydrogenases (BDHs) that potentially catalyzed borneol into camphor in WVV and functionally verified them. Highly expressed genes at the P2 stage of WVV, Wv05G1424 and Wv05G1438, were capable of catalyzing the formation of camphor from (+)-borneol, (−)-borneol and DL-isoborneol. Moreover, the BDH genes may experience independent evolution after acquiring the ancestral copies, and the following tandem duplications might account for the abundant camphor content in WVV. Furthermore, four populations of WVV, WVX, and WL are genetically differentiated, and the gene flow from WVX to WVV in Yunnan contributed to the greater genetic diversity in the introduced population (WVV-JH) than in its top-geoherb region (WVV-YC), which showed the lowest genetic diversity and might undergo genetic degradation. In addition, terpene synthesis (TPS) and BDH genes were selected among populations of multiple AF source species (or varieties) and between the top- and non-top-geoherb regions, which might explain the difference in metabolites between these populations. Our findings provide important guidance for the conservation, genetic improvement, and industrial development of the three source species (or varieties) and for identifying top-geoherbalism with molecular markers, and proper clinical application of AF.

Amomum villosum Lour. is a perennial herb of the Zingiberaceae family, which is widely distributed in Xishuangbanna, Yunnan Province in Southwest China. Large amounts of volatile components contained in this plant enter the surrounding atmosphere and soil through volatilization, foliar leaching, root exudation, and residue decomposition. However, the ecological role of these compounds is currently unclear. The aim of this study was to compare the differences in the composition of volatile oils from stems, leaves, and young fruits of A. villosum, identify chemicals that had allelopathic effects, and explore the effects of the oil on the diversity and composition of soil microbiomes. Volatile oils were obtained by steam distillation and characterized by gas chromatography–mass spectrometry, and then were tested for allelopathic activity using seedlings of Lactuca sativa L. and Lolium perenne L. as test species. The results showed that the oils from stems and leaves were rich in monoterpene hydrocarbons, unlike the oxygenated monoterpenes which dominated oils from young fruits. Leaves > stems > young fruits: this was the order of the allelopathic effects of volatile oils from various A. villosum organs. Among the four main chemical components in the oils, only α-pinene, which is abundant in leaves, had a stronger allelopathic action than the crude oils, implying that it might be a potential allelochemical. Experiments on soil microorganisms indicated that 3.0 mg/mL oil had the greatest effect on the structure of the soil fungal community. It can be concluded that A. villosum is capable of releasing allelochemicals which affect the growth of other plant species and the diversity and community structure of soil microorganisms.

Amomum villosum Lour., a perennial medicinal plant in the Zingiber genus, usually requires approximately 3–4 years of vegetative growth from seed germination to first fruiting, resulting in high initial investment costs and a prolonged revenue cycle, which pose significant challenges to the industry’s sustainable development. Our research team observed a distinct premature fruiting phenomenon in A. villosum. We investigated the regulatory mechanisms underlying premature fruiting in A. villosum by identifying the key differentially expressed genes (DEGs) and metabolic pathways governing the premature fruiting (Precocious) and typical plants (CK) of the ‘Yunsha No.8’ cultivar. Transcriptomic sequencing (RNA-seq) and bioinformatic analyses were performed using the DNBSEQ^TM platform. The sequencing generated 29.0 gigabases (Gb) of clean data, and 115,965 unigenes were identified, with an average length of 1368 bp. Based on the sequencing results, 1545 DEGs were identified. Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were annotated for these DEGs. This study identifies phytohormone signaling, carbohydrate and lipid metabolism, and polysaccharide degradation as critical pathways controlling premature fruiting in A. villosum. Six randomly selected DEGs were validated using real-time fluorescence quantitative polymerase chain reaction (RT-qPCR), and the results corroborated the transcriptome data, confirming their reliability. This study lays the foundation for the elucidation of the molecular mechanisms and metabolic pathways driving premature fruiting in A. villosum.

As a perennial medicinal plant in the Zingiber genus, Amomum villosum Lour. faces agricultural challenges due to its prolonged vegetative and reproductive growth phases, which hinder efficient pollination and delay fruiting. To address this limitation, the present study aimed to identify the FD gene involved in regulating flowering in A. villosum to provide a basis for research on the molecular mechanisms of early fruiting cultivars. Based on the differentially expressed gene AvFD1 obtained from the transcriptome database of early fruiting plants and controls, specific primers were designed for PCR to clone the full-length sequence of AvFD1. The characteristics of the cloned AvFD1 gene were analyzed using online bioinformatics software. The expression profiles of AvFD1 in various tissues and in 1- and 2-year bearing A. villosum varieties were investigated by quantitative real-time PCR. This study successfully cloned the FD1 gene sequence of A. villosum, marking the first reported characterization of this gene in the species. Tissue-specific expression analysis revealed significantly elevated AvFD1 expression levels in stolon tips and flower buds compared to tender leaves, suggesting its potential role as a positive regulator of flowering initiation. The obtained sequence establishes essential molecular data for subsequent functional validation of AvFD1 in A. villosum.

Precocious-fruiting Amomum villosum Lour. is characterized by early fruit set, rapid yield formation, and shortened economic return cycles, indicating strong cultivation potential. However, the molecular mechanisms underlying its flowering transition remain unclear. To elucidate the flowering mechanism of A. villosum, we used the Illumina NovaSeq X Plus platform to compare gene expression profiles in three tissues (Rhizomes, R; Stems, S; Leaves, L) during the vegetative stage and three tissues (Rhizomes and Inflorescences, R&I; Stems, S; Leaves, L) during the flowering stage of individual plants: VS-R vs. FS-R&I, VS-S vs. FS-S, and VS-L vs. FS-L. We obtained 52.5 Gb clean data and 789 million reads, and identified 2963 novel genes. The 3061 differentially expressed genes (DEGs, FDR ≤ 0.05 and |log2FC| ≥ 1) identified in the three comparison groups included six overlapping genes. The DEGs were enriched primarily in GO terms related to cellular process, metabolic process, binding, catalytic activity, and cellular anatomical entity, as well as multiple terms associated with development and reproduction. KEGG enrichment analysis revealed enrichment primarily in metabolic pathways, including global and overview maps, energy metabolism, and carbohydrate metabolism. Moreover, the most significantly enriched core pathways included metabolic pathways, photosynthesis, and carbon assimilation. Among all alternative splicing (AS) events, skipped exons (SEs) accounted for the largest proportion (59.5%), followed by retained introns (RI, 19.4%), alternative 3′ splice sites (A3SS, 10.7%), alternative 5′ splice sites (A5SS, 6.8%), and mutually exclusive exons (MXE, 3.6%). A preliminary set of 43 key DEGs was predicted, displaying spatiotemporal expression specificity and strong interactions among certain genes. Nine genes were further selected for RT-qPCR validation to confirm the reliability of the RNA-seq results. This study established a foundational framework for elucidating the flowering mechanism of precocious-fruiting A. villosum.