Objective To screen stable reference genes in Gastrodia elata and analyze the expression patterns of key enzyme genes in the gastrodin biosynthesis pathway, providing reliable normalization tools for molecular studies of G. elata. Methods Two transcriptome datasets, encompassing various tissues (tubers, stems, and flowers) at the jian-ma stage and four developmental stages post-inoculation, were integrated. Expression matrices were constructed using Transcripts Per Million (TPM) and normalized via the TMM method. Candidate reference genes were screened based on a coefficient of variation (CV) threshold of < 0.05, and specific primers were designed using the PrimerServer2 package. The expression levels of these candidates across different tissues and developmental stages were validated by qRT-PCR. Stability was comprehensively evaluated using delta-Ct, geNorm, NormFinder, BestKeeper, and RefFinder. Furthermore, the identified optimal reference gene was used to normalize the expression of six key enzyme genes in the gastrodin biosynthesis pathway. Results Gelref1 exhibited the highest stability across all samples, serving as a universal reference gene for various tissues and developmental stages of G. elata. Expression analysis revealed distinct patterns for the six key enzyme genes, with most showing high expression levels during the mid-to-late stages of tuber development. Conclusion This study establishes a systematic workflow for reference gene screening in G. elata based on RNA-Seq data, providing a reliable foundation for further research into the molecular mechanisms and functional validation of genes in gastrodin biosynthesis.

Gastrodia elata Blume  /  RNA-Seq  /  reference genes  /  gastrodin  /  gene expression