Identification of glucosinolate profile in Brassica oleracea for quantitative trait locus mapping

Abstract

Glucosinolates are a group of secondary plant metabolites, which have been shown to play important roles in human health and nutrition. Identification of novel genes or regulators of expression are important for optimising the glucosinolate composition of Brassica crops. This project aimed to develop a HPLC based methodology for quantifying these compounds within Brassica leaf material and to use this to map Quantitative Trait Loci for individual glucosinolates within Brassica oleracea mapping populations. Glucosinolates were analysed using an optimized HPLC-UV method developed in this study for complete separation of desulfated glucosinolates with high resolution for quantification measurements. The reproducibility of the desulfation reaction was improved for robust enzymatic reaction of sulfatase. A data dependent MS and MS/MS methodology was developed to confidently identify seven glucosinolates in the 89 AGDH plant lines distributed between aliphatic and indolic glucosinolate, with different combinations from the parental plants A12DHd and GDDH33. For the quantitative measurements of glucosinolates, an optimized level of glucotropaeolin was used as an internal standard (IS1). In addition, we have demonstrated the first use of a second internal standard (IS2) to significantly improve the reproducibility of the quantitative measurements. Aliphatic glucosinolates were predominant over indolic glucosinolates, where progoitrin has the highest abundance. This methodology was then used to identify Quantitative Trait Loci for individual glucosinolates and for key points in their biosynthesis. A major gene effect was found near the top of B. oleracea LG9 associated with aliphatic glucosinolate synthesis. In addition other Quantitative Trait Loci were identified which corresponded with previous work by other groups and to which individual gene function could be attributed. A number of novel Quantitative Trait Loci were also found which control the synthesis of glucosinolates distributed on the nine chromosomes of C genome. A combination of the quantitative data and genetic analysis of glucosinolate profiles was used to infer the existence of factors at distinct loci and associated these with specific steps in the biosynthesis pathway of glucosinolates in B. oleracea. The assignment of genes or gene regulator functions to Quantitative Trait Loci identified in this study was consistent with known positions of Brassica candidate genes and collinear regions of the Arabidopsis genome. Consequently, this information can be applied to other Brassica species for breeding vegetable crops with modified glucosinolate profiles