Bupleurum falcatum L. is frequently used in traditional herbal medicine in Asia. Saikosaponins (SSs) are the main bioactive ingredients of B. falcatum, but the biosynthetic pathway of SSs is unclear, and the biosynthesis of species-specific phytometabolites is little known. Here we resolved the transcriptome profiles of B. falcatum to identify candidate genes that might be involved in the biosynthesis of SSs. By isoform sequencing (Iso-Seq) analyses of the whole plant, a total of 26.98 Gb of nucleotides were obtained and 124,188 unigenes were identified, and 81,594 unigenes were successfully annotated. A total of 1033 unigenes of 20 families related to the mevalonate (MVA) pathway and methylerythritol phosphate (MEP) pathway of the SS biosynthetic pathway were identified. The WGCNA (weighted gene co-expression network analysis) of these unigenes revealed that only the co-expression module of MEmagenta, which contained 343 unigenes, was highly correlated with the biosynthesis of SSs. Comparing differentially expressed gene analysis and the WGCNA indicated that 130 out of 343 genes of the MEmagenta module exhibited differential expression levels, and genes with the most “hubness” within this module were predicted. Manipulation of these genes might improve the biosynthesis of SSs.

WRKY transcription factors play critical roles in plant growth, development, metabolism, and stress responses. In this study, we performed the first genome-wide characterization of the WRKY gene family in Bupleurum chinense, using a T2T-level assembly of the autotetraploid genome. A total of 303 BcWRKY genes were identified and found to be unevenly distributed across four subgenomes. Phylogenetic and structural analyses revealed that segmental duplications after polyploidization drove lineage-specific expansion of the family. Meta-transcriptome analysis demonstrated that BcWRKY genes exhibited tissue-specific expression patterns and dynamic responses to stress, suggesting functional diversification. Under drought, waterlogging, methyl jasmonate, and ABA treatments, the contents of saikosaponins A and D significantly increased. This increase was accompanied by transcriptional activation of multiple BcWRKY genes. Correlation analysis between ten BcWRKYs and ten saikosaponins biosynthetic associated genes (BcBASs, BcCYPs, and BcUGTs) identified BcWRKY22, BcWRKY33, and BcWRKY46 as potential regulators of saikosaponin metabolism under stress conditions. Our study provided a comprehensive framework for understanding BcWRKY gene evolution and secondary metabolic regulation in polyploid medicinal plants. It also offered candidate genes for breeding B. chinense cultivars with high saikosaponin content.

Accurate identification of Fritillaria taxa is essential for quality control of Fritillariae Cirrhosae Bulbus. This study evaluated morphological analysis, chloroplast DNA barcoding, and steroidal alkaloid profiling across 40 individuals from four taxa (F. unibracteata, F. unibracteata var. wabuensis, F. cirrhosa, and F. taipaiensis) from Sichuan province, China. Among five chloroplast markers, ycf1 and rps4 were the most polymorphic, with nucleotide diversity of 0.065 to 0.116 and 0.035 to 0.120, respectively, and generally distinguished the four taxa into well-supported clades, though two F. unibracteata nested within the F. cirrhosa group. Morphologically, F. unibracteata var. wabuensis produced the largest bulbs, with a mean fresh weight of 26.99 g, approximately 24-fold greater than that of F. unibracteata at 1.12 g, whereas F. unibracteata and F. cirrhosa overlapped substantially in all measured traits. PCA revealed that biomass traits were slightly more discriminative, while central bud height contributed the least. Among the samples analyzed, alkaloid profiles provided the most distinct diagnostic signals: F. unibracteata var. wabuensis accumulated the highest sipeimine at 298.40 μg/g and sipeimine-3-β-D-glucoside at 156.30 μg/g; F. taipaiensis was richest in peimine and peiminine; F. unibracteata uniquely contained sipeimine-3-β-D-glucoside without detectable sipeimine; and F. cirrhosa showed the lowest levels across all five compounds. The integration of chloroplast markers with alkaloid profiling improved species discrimination for the samples examined. Chloroplast markers for initial screening followed by the alkaloid profiling of the ambiguous sample might offer a practical framework for the authentication and quality control of Fritillariae Cirrhosae Bulbus, but this strategy requires validation across broader geographic scales and with larger sample sizes.