Abstract
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.
Highlights
Glucosinolates (GS) are nitrogen- and sulfur-rich secondary metabolites characteristic of the order Capparales [1]
An in silico search of aliphatic GS genes from 1g and 2g chromosomes of Arabidopsis ([26] encompassing the syntenous region of Quantitative trait locus (QTL) J16Gsl4 (Fig 1) flanked by the markers At1g15370 and At2g21620 in B. juncea [21] and the corresponding regions in B. rapa
Emphasis was laid for uncovering B genome specific allelic polymorphism of the genes as the QTL J16Gsl4 regulating the sinigrin synthesis was located in the Linkage group (LG) B4 of B. juncea
Summary
Glucosinolates (GS) are nitrogen- and sulfur-rich secondary metabolites characteristic of the order Capparales [1]. Brassica crops primarily contain methionine derived aliphatic GS (up to 95% of the total GS). Brassica species consist of various combinations of the above three types of aliphatic GS namely, 2-propenyl (commonly known as sinigrin) of 3C GS, 3-butenyl (known as gluconapin) and 2-hydroxy-3-butenyl (known as progoitrin) of 4C GS and 4-pentenyl (known as glucobrassicanapin) of 5C GS. The GS profile of the three allotetraploid Brassica species, B. juncea (AABB, 2n = 36), B. napus (AACC, 2n = 38) and B. carinata (BBCC, 2n = 34) is a combination of the GS profiles of the progenitor diploid Brassica species [2]. In allotetraploid oilseed mustard (Brassica juncea; AABB), 3C and 4C constitute about 99% of the total aliphatic GS. Among the two gene pools in mustard—the east European and the Indian [3], cultivars belonging to the former primarily contain sinigrin of 3C GS whereas those belonging to the latter contain both sinigrin of 3C GS (~20%) and gluconapin of 4C GS (~80%) [4,5]
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