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Jilin ginseng, Panax ginseng , is a valuable medicinal herb whose ginsenosides are its major bioactive components. The ginseng oxidosqualene cyclase (PgOSC) gene family is known to play important roles in ginsenoside biosynthesis, but few members of the gene family have been functionally studied. The PgOSC gene family has been studied by an integrated analysis of gene expression-ginsenoside content correlation, gene mutation-ginsenoside content association and gene co-expression network, followed by functional analysis through gene regulation. We found that five of the genes in the PgOSC gene family, including two published ginsenoside biosynthesis genes and three new genes, were involved in ginsenoside biosynthesis. Not only were the expressions of these genes significantly correlated with ginsenoside contents, but also their nucleotide mutations significantly influenced ginsenoside contents. These results were further verified by regulation analysis of the genes by methyl jasmonate (MeJA) in ginseng hairy roots. Four of these five PgOSC genes were mapped to the ginsenoside biosynthesis pathway. These PgOSC genes expressed differently across tissues, but relatively consistent across developmental stages. These PgOSC genes formed a single co-expression network with those published ginsenoside biosynthesis genes, further confirming their roles in ginsenoside biosynthesis. When the network varied, ginsenoside biosynthesis was significantly influenced, thus revealing the molecular mechanism of ginsenoside biosynthesis. At least five of the PgOSC genes, including the three newly identified and two published PgOSC genes, are involved in ginsenoside biosynthesis. These results provide gene resources and knowledge essential for enhanced research and applications of ginsenoside biosynthesis in ginseng. • Three new PgOSC genes involved in ginseng ginsenoside biosynthesis were identified. • Analyses of gene expression, mutation and network identify strong candidate genes. • Genes involved in ginsenoside biosynthesis are interacted in a form of network. • Variation of the gene network interactions greatly affects ginsenoside biosynthesis. [ABSTRACT FROM AUTHOR]