## 3 Results and Discussion
### Expression Level of GpOSCs Among Different Populations  
Numerous studies have demonstrated that the highest content of gprenosides in _G. pentaphyllum_ is predominantly found in the leaves [9]. Additionally, key enzyme genes involved in the gprenoside biosynthesis pathway exhibit relatively high expression levels in both stems and leaves [30]. Therefore, in our study, we collected _G. pentaphyllum_ plants from various regions and cultivated them in a common garden setting. Twelve young leaves from four distinct populations were collected to analyze the expression levels of _GpOSC_ genes based on RNA sequencing (Tables S3 and S4), and experimentally validated using qRT-PCR. A heatmap illustrating the expression patterns of 11 _GpOSC_ genes across different populations was generated (Figure S6). The results indicated that five \(\beta\)-AS genes (_GpOSC2_, _GpOSC3_, _GpOSC8_, _GpOSC9_, and _GpOSC11_) along with one \(\beta\)-AS-like gene (_GpOSC10_) exhibited consistently low expression levels across all populations. Notably, _GpOSC1_, a _CAS_ gene, showed the highest expression level among populations except for the QC population. The expression levels of _GpOSC4_, _GpOSC5_, and _GpOSC6_ showed significant variation among different populations. From a population perspective, _GpOSC1_ was highly expressed within the DH population, followed by elevated expressions of both _GpOSC4_ and _GpOSC5_. In contrast, high expressions of both _GpOSC1_ and _GpOSC6_ were observed in XA and ZJ populations; however, the expression levels of all _GpOSC_ genes were relatively low in the QC population (Figure S6). Correspondingly, the results of qRT-PCR showed a consistent result that the DH population had the highest expression level across _GpOSC1_, _GpOSC4_, and _GpOSC5_, while the _GpOSC6_ gene was highly expressed in XA and ZJ populations (Figure S7).  
A study on _Citrus grandis_ integrated transcriptome and metabolome data to identify key medicinal components that significantly varied across different growing regions while conducting association analyses with differentially expressed functional genes. This approach provided valuable insights into quality differences observed in _C. grandis_ sourced from diverse regions [58]. Similarly, analyses of RNA-seq data from _Citrus_ reticulata cultivated in various regions revealed that transcripts associated with monoterpene biosynthesis were more prevalent in the core areas. Furthermore, it was also demonstrated that genes responsive to biotic and abiotic stress exhibited a correlation with monoterpene production in these regions, suggesting that unique soil properties and microbial environments may affect monoterpene accumulation [59]. Consequently, it was speculated that _G. pentaphyllum_ distributed across different regions might exhibit significant variations in the expression of members of the _OSC_ gene family due to genetic differences or environmental factors such as soil conditions. This could lead to the synthesis of diverse types of gprenosides in _G. pentaphyllum_ from various regions, which underpins the authenticity of _G. pentaphyllum_ as a traditional Chinese medicinal herb. Therefore, investigating the expression patterns of _OSC_ gene family members in _G. pentaphyllum_ from different regions could elucidate the underlying mechanisms responsible for quality discrepancies and provide a foundation for cultivating, producing, and breeding high-quality varieties of _G. pentaphyllum_. This would contribute to the sustainable development of high-quality traditional Chinese medicine. However, further studies on gprenoside content and genetic evidence remain necessary through biochemical molecular genetics and multi-omics approaches, including metabolomic analyses in the future.