Third generation DNA fingerprinting for food authenticity testing

DNA analysis against food fraud

Oct/Nov 2020. Due to the complexity of global food supply chains food fraud is becoming a growing problem. Fraudsters are getting increasingly inventive and authenticity testing methods have to keep pace with their creativity. In particular, DNA based methods like barcoding, sequencing and genetic fingerprinting are gaining increasing importance for the control of the authenticity of numerous food products, since these technologies are rapidly developing. Next-Generation Sequencing became a routine method not only in medicine and human genetics but also for food authenticity testing. Second-generation DNA fingerprinting based on Simple Sequence Repeats (SSRs) is routinely applied to control the authenticity of rice specialities like Basmati and Jasmine.

Basmati-Rice – commercial success by authenticity testing with the DNA fingerprint

Since the first application of DNA fingerprinting by Sir Alec Jeffreys to solve an immigration case in 1986 DNA fingerprinting revolutionized human forensics. Fourteen years later the pioneering work of Dr Frances Bligh (then a researcher at the University of Nottingham, England) on Basmati rice lead to the breakthrough of this technology also in food authenticity testing. Her DNA fingerprinting method of the second generation, after some refinements, became a routine test offered also by Eurofins to industry and trade.

The UK Code of Practice on Basmati Rice of the Rice Association, British Rice Millers Association and British Retail Consortium defines Basmati authenticity and determines DNA fingerprinting for its control. The code is accepted not only in the UK but also in most EU member states and other countries like Switzerland. It improved the quality of this rice on the market significantly and this was rewarded by the consumers with EU import volumes nearly doubling since 2010 (Nader et al., 2020 ^[2]). 

DNA fingerprinting of the second and third generation – a comparison

In 2017 the UK Code of Practice was revised and 26 Basmati varieties were added to the 15 in the first Code issued in 2005. These had been newly developed by Indian and Pakistani plant breeders to improve the quality, yield, tolerances against pests, salinity and drought. Due to this extension of the list of approved Basmati cultivars the authenticity testing method had to be adapted and Eurofins published a suitable procedure based on SSR markers (Nader et al., 2019 ^[3]), which can differentiate all varieties with the exception of 9 highly related varieties.

New approach with the KASP^TM genotyping assay

A different approach was followed by Dr Steele with a KASP^TM (competitive allele-specific PCR) genotyping assay based on SNP, insertion and deletion (InDel) markers (Steele et al., in press ^[1]). Advantages of SNPs over SSRs include their higher abundance within genomes, higher stability due to lower mutation rates and more homogenous distribution over the genome. Because of that Dr Steele could differentiate all 41 Basmati varieties with a selection from 364 KASP^TM designs. On the other hand, a major advantage of SSRs over SNPs is their polyallelic nature. By analysing only a small panel of SSR markers, crop varieties can be detected and differentiated in food in a quantitative manner. In contrast to the KASP^TM method, more than two varieties in mixtures can be differentiated in a quantitative manner. Accordingly, both methods will remain of importance for rice authenticity testing for the time being and will complement each other.

Potential of third-generation DNA fingerprinting for food analysis in general

Third generation DNA fingerprinting has a potential far beyond food testing of Basmati rice only. Fingerprinting based on SNPs became a routine tool to trace microbial and viral pathogens like SARS-CoV2 back to the source. As traceability is the most efficient antidote against food fraud, SNPs can be also used to trace food ingredients back to their origin. Plant and animal breeders use SNPs as selection markers and apply the KASP^TM genotyping method routinely. Similarly, high-value food ingredients with special characteristics like aroma and texture can be identified with these markers, as applied by Dr Steele et al. ^[1] for the fgr marker (aroma) and the waxy gene (amylose content) of Basmati rice.

GMOs (Genetically Modified Organisms) of the second generation are now engineered with the CRISPR-Cas9 technology and fall under EU regulations 1829/2003 and 1830/2003 (traceability). In particular targeted gene mutations (base pair modifications similar to SNPs and InDels) created with this technology by non-homologous end joining (NHEJ) are difficult to distinguish from those obtained by conventional breeding. If these mutations are in the public domain, e.g. in the GMO register of the EU, they can be detected and quantified with the  KASP^TM genotyping method in the same manner as described by Steele for Basmati rice.

Contacts für further information:

Dr Werner Nader, Senior Consultant, retired managing director of Eurofins Global Control GmbH, Hamburg, WernerNader@eurofins.de

Dr Katherine Steele, Senior Lecturer in Sustainable Crop Production, Bangor University, Wales, k.a.steele@bangor.ac.uk

References

[1] Steele, K., Tulloch, M.Q., Burns, M. and Nader, W.: Developing KASP markers for identification of Basmati rice varieties, Food Analytical Methods (2020). Available for download here.
[2] Nader, W., Maier, M., Miebach, M. and Linder, G.: Pesticide residue legislations challenge international trade of food and feed. Cereal Technology, 2: 84-99 (2020). Available for download here.
[3] Nader, W., Elsner, J., Brendel, T. and Schubbert, R.: The DNA fingerprint in food forensics – the Basmati rice case. Agro FOOD Industry Hi Tech, 30(6): 57-61, 2019. Available for download here.