Elsevier

Food Chemistry

Volume 131, Issue 4, 15 April 2012, Pages 1493-1498
Food Chemistry

Short communication
Detection and quantification of tissue of origin in salmon and veal products using methylation sensitive AFLPs

https://doi.org/10.1016/j.foodchem.2011.09.120Get rights and content

Abstract

Food quality depends largely on the source of origin of the products used. Here we use methylation sensitive amplified polymorphisms (MSAP) analysis to diagnose and quantify the tissue of origin in salmon and veal products. We exploited the variability of MSAP profiles between tissues of an organism in order to characterise the origin of the food product. MSAP profiles provided robust discrimination of tissues from salmon (brain, eye, liver, and muscle) and veal (heart, kidney, liver, and muscle). Isolation of the diagnostic markers revealed by MSAP may ultimately allow for the development of a new STS-based epigenetic profiling technique for the detection and quantification of mixture of tissues in the same sample; offering not only rapid determination of the tissue of origin but also potential quantification of the components in processed food products.

Highlights

► We compared DNA methylation profiles of four tissues in salmon and veal using MSAPs. ► We test DNA methylation as a molecular marker (epiallele) for tissue identification. ► We generated qualitative and quantitative epialleles for each tissue and species. ► Epialleles can be used to determine and quantify tissue of origin in processed foods. ► Epialleles sequencing will permit designing quick and reliable food analysis assays.

Introduction

Certifying the authenticity of food products is crucial for labelling and assessment of value and is invaluable to avoid unfair competition and assure consumers protection against fraudulent practices (Reid, O’Donell, & Downell, 2006). Due to their market value, fish and meat products are often the target of fraudulent labelling, by replacement of more expensive products with others of inferior quality and commercial value. Examples of this practice are the substitution of expensive species by similar ones of lower value or the mixture of different animal organs in processed foods.

A large number of techniques have been developed over the recent years for the identification and authentication of food products including immunological (Asensio, González, García, & Martín, 2008), genetic (Koppel et al., 2008, Meyer and Candrian, 1996, Spaniolas et al., 2010), and chromatographic and electrophoretic (Czerwenka, Muller, & Lindner, 2010) based techniques (for a recent review see Sentandreu & Sentandreu, 2011). In general, all such techniques are focussed on enabling inspection programs to determine the species identity of, otherwise, undistinguishable products. Genetic methods, particularly those based on DNA sequencing, have proved the most taxon-specific and sensitive for food components authentication (Asensio et al., 2008) and are routinely used to detect the substitution of species even in highly processed food, such as cans, frozen fillets, or minced meat. Still, the identification of different tissues from a single species remains challenging, even for modern sequencing based techniques, because different tissues within an organism are, generally, genetically identical. Yet, the use of differences in organ-specific epigenetic markers as a tool for tissue identification remains unexplored.

Living organisms’ development depends upon the regulation of both spatial and temporal tissue-specific gene regulation programs. The execution and maintenance of such transcriptional programs rely on dynamic epigenetic changes during development (Morgan, Santos, Green, Dean, & Reik, 2005). DNA methylation, which usually involves the incorporation of a methyl group to carbon 5 of the cytosine pyrimidine ring to form 5-methylcytosine, plays a key role in establishing different developmental epigenetic states (Bird, 1996). DNA methylation is widespread across many distinct eukaryotic phyla, including plants, mammals, birds, fish, and invertebrates (Su, Han, & Zhao, 2011). C-methylation has attracted particular interest amongst molecular biologists because of its confirmed role in gene regulation, control of cellular differentiation in many animals, including humans (Eckhardt et al., 2006) and fish species (Rai et al., 2006), and in normal embryonic development, genomic imprinting, and X-chromosome inactivation (Jones & Laird, 1999). A series of locus-specific studies have implicated C-methylation in the targeted silencing of genes involved in developmental progression or tissue differentiation (Messeguer, Ganal, Steffens, & Tanksley, 1991).

Different tissues within an organism are, generally, genetically identical but present widespread differences in their DNA methylation profiles (Eckhardt et al., 2006). Characterising such differences may prove useful in identifying and quantifying the origin of tissues used as raw materials in many processed foods. Several approaches have been developed for the detection and localisation of DNA methylation, but all have limitations. More recent DNA methylation detection techniques are based on the prior application of sodium bisulphite (Wang, Gehrke, & Ehrlich, 1980) to the genomic DNA, followed by bisulphite genomic DNA sequencing (BGS) (Han, Cauchi, Herman, & Spivack, 2006) and methylation-specific PCR (MSP) (Herman, Graff, Myohanen, Nelkin, & Baylin, 1996). Both techniques require prior knowledge of the target sequence and genomic methylation screening to direct primer design to appropriate specific markers for analysis. In food analysis, problems may arise due to the limited number or, more commonly, the absence of known tissue-specific methylation markers, particularly in non-model species. Genome-wide analysis, using the differential ability of methylation sensitive restriction enzymes to digest methylated and nonmethylated DNA templates could be a better suited approach. Methylation-sensitive amplified polymorphism (MSAP) is a genome wide anonymous marker system, commonly used to investigate global changes to the distribution of C-methylation (Reyna-López, Simpson, & Ruiz-Herrera, 1997) and has proved useful to uncover epigenetic variability in a wide taxonomic spread of plant species (Rodríguez López, Wetten, & Wilkinson, 2010). Crucially, like AFLP, the technique holds open the capacity to isolate and convert the useful anonymous markers it generates into locus-specific markers (Massicotte, Whitelaw, & Angers, 2011).

We used MSAP in a range of tissues of fish (Atlantic salmon, Salmo salar) and veal (Bos taurus) in order to test the efficiency of the method for the identification of specific signatures of methylation amongst tissues.

Section snippets

Sample selection and DNA isolation

Seven Atlantic salmon and veal specimens were obtained from a local non-commercial hatchery and a local slaughterhouse in Verín, Spain. Fresh samples were dissected from several tissues (brain, muscle, eye, and liver from salmons and heart, kidney, liver, and muscle from veals) of all selected specimens. Genomic DNA was extracted using the NucleoSpin® Tissue Kit (BD Biosciences, Macery-Nagel, Germany), following the manufacturer’s recommendations and quantified using the Nanodrop 2000

Estimation of qualitative tissue dependent epigenetic variability

The two primer combinations, per species used, in the MSAP analysis generated 453 and 672 loci for the 28 samples of salmon and veal respectively. Of these, 14.1% (salmon) and 11.5% (veal) were considered to be partially polymorphic between tissues (i.e., always present or absent in one tissue type but not in all tissues). Finally, 12.1% of the loci in salmon and 8.6% in veal were deemed tissue-specific (i.e., present in one tissue and absent in the rest or absent in a tissue and present in the

Discussion

In order to protect consumers from falsely labelled fish and meat products, many molecular techniques have been developed for food authentication. Amongst these, DNA-based approaches offer the advantage of being the most specific and sensitive methods for food components authentication (Asensio et al., 2008). One of the limitations of the genetic methodologies based on DNA sequence analysis is that they cannot detect different organs of an animal in a meat mixture. MSAP has proven useful to

Conclusions

In conclusion, our results show that genetically identical tissues from salmon and veal present DNA methylation differences at a genome-wide scale and demonstrate that MSAP is sensitive enough to detect these changes. These results also reveal that even when there is a significant level of variation between individuals, MSAP analysis generates enough epigenetic markers to separate the different tissues. These findings open the way for STS-based epigenetic profiling techniques that could be the

Acknowledgements

We would like to thank Professor Mike J. Wilkinson for critical reading and improving the manuscript. This work was funded in part by Grants from Ministerio de Ciencia y Tecnología (CGL2010-14964) and Fondos FEDER.

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