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Among the different types of methionine-derived aliphatic glucosinolates (GS), sinigrin (2-propenyl), the final product in 3C GS biosynthetic pathway is considered very important as it has many pharmacological and therapeutic properties. In Brassica species, the candidate gene regulating synthesis of 3C GS remains ambiguous. Earlier reports of GSL-PRO, an ortholog of Arabidopsis thaliana gene At1g18500 as a probable candidate gene responsible for 3C GS biosynthesis in B. napus and B. oleracea could not be validated in B. juncea through genetic analysis. In this communication, we report the isolation and characterization of the gene CYP79F1, an ortholog of A. thaliana gene At1g16410 that is involved in the first step of core GS biosynthesis. The gene CYP79F1 in B. juncea showed presence-absence polymorphism between lines Varuna that synthesizes sinigrin and Heera virtually free from sinigrin. Using this presence-absence polymorphism, CYP79F1 was mapped to the previously mapped 3C GS QTL region (J16Gsl4) in the LG B4 of B. juncea. In Heera, the gene was observed to be truncated due to an insertion of a ~4.7 kb TE like element leading to the loss of function of the gene. Functional validation of the gene was carried out through both genetic and transgenic approaches. An F2 population segregating only for the gene CYP79F1 and the sinigrin phenotype showed perfect co-segregation. Finally, genetic transformation of a B. juncea line (QTL-NIL J16Gsl4) having high seed GS but lacking sinigrin with the wild type CYP79F1 showed the synthesis of sinigrin validating the role of CYP79F1 in regulating the synthesis of 3C GS in B. juncea. [ABSTRACT FROM AUTHOR]