Genes are the keys to deciphering the molecular mechanism underlying a biological trait and designing approaches desirable for plant genetic improvement. Ginseng is an important medicinal herb in which ginsenosides have been shown to be the major bioactive component; however, only a few genes involved in ginsenoside biosynthesis have been cloned through orthologue analysis. Here, we report the identification of 21 genes controlling Rb1 biosynthesis by stepwise ginseng transcriptome and Rb1 content integrated analysis. We first identified the candidate genes for Rb1 biosynthesis by integrated analysis of genes with the trait from four aspects, including gene transcript differential expression between highest- and lowest-Rb1 content cultivars, gene transcript expression–Rb1 content correlation, and biological impacts of gene mutations on Rb1 content, followed by the gene transcript co-expression network. Twenty-two candidate genes were identified, of which 21 were functionally validated for Rb1 biosynthesis by gene regulation, genetic transformation, and mutation analysis. These genes were strongly correlated in expression with the previously cloned genes encoding key enzymes for Rb1 biosynthesis. Based on the correlations, a pathway for Rb1 biosynthesis was deduced to indicate the roles of the genes in Rb1 biosynthesis. Moreover, the genes formed a strong co-expression network with the previously cloned Rb1 biosynthesis genes, and the variation in the network was associated with the variation in the Rb1 content. These results indicate that Rb1 biosynthesis is a process of correlative interactions among Rb1 biosynthesis genes. Therefore, this study provides new knowledge, 21 new genes, and 96 biomarkers for Rb1 biosynthesis useful for enhanced research and breeding in ginseng.

GRAS transcription factors are a kind of plant-specific transcription factor that have been found in a variety of plants. According to previous studies, GRAS proteins are widely involved in the physiological processes of plant signal transduction, stress, growth and development. The Jilin ginseng (Panax ginseng C.A. Meyer) is a heterogeneous tetraploid perennial herb of the Araliaceae family, ginseng genus. Important information regarding the GRAS transcription factors has not been reported in ginseng. In this study, 59 Panax ginseng GRAS (PgGRAS) genes were obtained from the Jilin ginseng transcriptome data and divided into 13 sub-families according to the classification of Arabidopsis thaliana. Through systematic evolution, structural variation, function and gene expression analysis, we further reveal GRAS’s potential function in plant growth processes and its stress response. The expression of PgGRAS genes responding to gibberellin acids (GAs) suggests that these genes could be activated after application concentration of GA. The qPCR analysis result shows that four PgGRAS genes belonging to the DELLA sub-family potentially have important roles in the GA stress response of ginseng hairy roots. This study provides not only a preliminary exploration of the potential functions of the GRAS genes in ginseng, but also valuable data for further exploration of the candidate PgGRAS genes of GA signaling in Jilin ginseng, especially their roles in ginseng hairy root development and GA stress response.

Ginseng, an important medicinal plant, is characterized by its main active component, ginsenosides. Among more than 40 ginsenosides, Rg1 is one of the ginsenosides used for measuring the quality of ginseng. Therefore, the identification and characterization of genes for Rg1 biosynthesis are important to elucidate the molecular basis of Rg1 biosynthesis. In this study, we utilized 39,327 SNPs and the corresponding Rg1 content from 344 core ginseng cultivars from Jilin Province. We conducted a genome-wide association study (GWAS) combining weighted gene co-expression network analysis (WGCNA), SNP-Rg1 content association analysis, and gene co-expression network analysis; three candidate Rg1 genes (PgRg1-1, PgRg1-2, and PgRg1-3) and one crucial candidate gene (PgRg1-3) were identified. Functional validation of PgRg1-3 was performed using methyl jasmonate (MeJA) regulation and RNAi, confirming that this gene regulates Rg1 biosynthesis. The spatial–temporal expression patterns of the PgRg1-3 gene and known key enzyme genes involved in ginsenoside biosynthesis differ. Furthermore, variations in their networks have a significant impact on Rg1 biosynthesis. This study established an accurate and efficient method for identifying candidate genes, cloned a novel gene controlling Rg1 biosynthesis, and identified 73 SNPs significantly associated with Rg1 content. This provides genetic resources and effective tools for further exploring the molecular mechanisms of Rg1 biosynthesis and molecular breeding.

Ginseng, a traditional precious Chinese herb of high medicinal value, has ginsenoside as its main active medicinal ingredient. Modern pharmacological studi

The HD-Zip gene family plays a vital regulatory role in plant growth and development, but its function in plant secondary metabolism remains to be further elucidated. This study therefore investigated the involvement of the HD-Zip gene family in ginsenoside biosynthesis and identified the genes that were highly correlated. Through correlation analysis between gene expression and ginsenoside content, heatmap and co-expression network analyses, and association analysis between Single Nucleotide Polymorphism (SNP) variations and ginsenoside content, the PgHDZ01 gene was identified as the core candidate gene highly associated with ginsenoside biosynthesis. Subsequently, this gene was shown to significantly respond to Methyl Jasmonate (MeJA) treatment and correlated with changes in ginsenoside content. Further functional verification using Virus-Induced Gene Silencing (VIGS) in Panax ginseng (Ginseng) seedlings and gene overexpression in ginseng hairy roots confirmed the positive regulatory role of PgHDZ01 in ginsenoside biosynthesis. Moreover, its overexpression led to a significant increase in the content of ginsenoside Rb1, and its expression was positively correlated with the change in Rb1 content. The results of this study provide a reference for the comprehensive analysis of the functions of the HD-Zip gene family and the regulatory mechanisms of ginsenoside synthesis. The VIGS method used in this study also provides a new approach for the verification of functional genes in ginseng. The results demonstrate the critical role of PgHDZ01 in regulating ginsenoside biosynthesis, deepening our understanding of the underlying mechanisms of ginsenoside biosynthesis and offering a potential candidate gene for molecular breeding applications.