DNA-Based Food Authentication Techniques: Differentiation of Tetraploid and Hexaploid Wheat

Abstract

Authenticating food constituents is a major challenge to regulators and to food manufacturers and processors. Fraud in this area is considerable. A major concern is with wheat. Unlike bread and biscuit wheat, which are hexaploid, pasta wheat is tetraploid. The phenotypic consequence of the tetraploid nature is that this ‘durum’ wheat is particularly suitable for pasta usage and it grows less readily (grows in dry midoterranean climates) than the other wheat types and therefore commands a high price accordingly. The consequence of this, however, is that the durum wheat tends to be ‘diluted’ with other cheap, non-durum wheat whereupon an inferior product is produced. This project was directed towards developing a DNA-based quantitative real-time PCR (qPCR) assay to detect the bread wheat quantity in durum wheat. The currently available methods are either time consuming or labour intensive or less reliable especially in a variety of situations. In this research, several potential targets were investigated as suitable diagnostic markers including the D-genome specific Xwye838 sequences coding for ADPglucose pyrophosphorylase. Newly designed and established primers were tested for their suitability in terms of specificity and sensitivity with the cultivar varieties used is this research that distinguishing the D-genome alleles from the A and B alleles and were shown to be specific in end-point PCR. After establishing their suitability in end point PCR, attempts were made to develop both SYBR greenbased and TaqMan probe-based qPCR. To facilitate this, a fragment of the Dgenome gene was sequenced in a number of wheat cultivars. Some success was achieved with both qPCR methods but not at the level of sensitivity that was aimed for. The problems encountered while developing qPCRs and the troubleshooting protocols followed are discussed. Possible reasons for failure of the methods under challenging conditions are suggested. Alternative approaches, alternative targets and the potential of completion of wheat genome sequencing are discussed with respect to developing a successful assay able to detect very small quantities of bread wheat in durum wheat materials.

Date of Award	2010
Original language	English
Awarding Institution	Glasgow Caledonian University
Supervisor	John Craft (Supervisor) & Richard Tester (Supervisor)