Comparative Analysis of Isatis indigotica Varieties Based on Integrated Metabolomic and Transcriptome Analyses

Objectives: Isatis indigotica is a core medicinal crop for producing Radix Isatidis and Folium Isatidis, although cultivar genotype severely affects medicinal quality. This study aimed to systematically dissect the phenotypic, physiological, metabolomic, and transcriptomic differences between two cultivars, Songming No. 1 (SL, new elite line) and Dinglan No.1 (DL, conventional cultivar), and uncover the molecular mechanisms underlying SL’s superior agronomic and medicinal traits. Methods: Agronomic phenotypes, photosynthetic pigments, antioxidant enzyme activities, and leaf indirubin content were measured across seven growth stages (A–G). Untargeted LC-MS/MS metabolomics analyses were conducted at stages A, B, and E, and full-stage transcriptome sequencing was performed followed by qRT-PCR validation. Multi-omics correlation was visualized via nine-quadrant analysis. Results: SL exhibited a significantly larger leaf size, higher leaf biomass, and persistently elevated indirubin concentration at all stages. Its chlorophyll, carotenoid, Calvin-cycle enzyme, and AsA-GSH antioxidant enzyme activities were mostly higher than those in DL. A total of 1217 metabolites and massive differentially expressed genes (DEGs) were identified; flavonoids, especially peonidin-3-glucoside, stably accumulated in SL and mapped to anthocyanin biosynthesis. Transcriptional variations between cultivars concentrated on chloroplast pathways, with two candidate genes, LOC18994026 and LOC104719239, potentially boosting SL photosynthesis. Seventeen differential metabolites correlated with 170 DEGs, while DL showed stronger cold tolerance linked to tryptophan-synthesis gene overexpression, and SL was inferred to be more sensitive to high light. Conclusions: In conclusion, SL possesses comprehensive advantages in yield, photosynthetic performance, and medicinal ingredient accumulation, serving as an excellent germplasm for large-scale cultivation. This multi-omics study clarifies genotypic divergence mechanisms and provides theoretical support for high-quality I. indigotica breeding.