Abstract

Food authentication and traceability is a complex problem, strictly correlated to fraud andadulteration detections that dramatically affect the consumer protection. Analysis of protein,metabolite and DNA represents robust tools for food authentication. In particular, DNA-basedmethods are more reliable, thanks to the stability of DNA under production and processing techniquesapplied along the food-chain. Therefore, DNA markers offer a powerful tool to address the validationof food authenticity and traceability of primary products. Single nucleotide polymorphism (SNP)markers have become the most used markers in genetic characterization studies as well as intranslational genomic even in plants. SNP are, in fact, the most abundant forms of genetic variationamong individuals of a species. In particular, SNP analysis by next generation sequencing (NGS)(e.g.genotyping by sequencing (GBS) and double-digest restriction site-associated DNA sequencing(ddRAD-Seq) or by high resolution melting analysis (HRM), e.g. single-base variants and smallinsertions or deletions, have rapidly become popular due to their flexibility and relatively low cost.The ddRAD-Seq technology has the advantage over GBS of high accuracy read mapping by paired-endsequencing of identical loci. Progress in NGS technology has led to the availability of several plantgenomes. This situation makes it possible to simulate ddRAD-Seqin silico, allowing prediction of thenumbers, sizes, and genome positions of digested fragments. However, few reports have evaluatedthe in silico predictions by comparative experiments using several combinations of restriction enzymesand multiple samples with different SNP density. HRM analysis has several advantages over traditionalmethods for gene scanning and genotyping, making it faster, less laborious and more suitable for highsample throughput. In this study, two approaches are proposed for the authentication of the Italianrice cultivars Carnaroli and Roma: in silico and empirical ddRAD-Seq analysis and HRM analysistargeting an A/C SNP in exon 6, responsible for the Wxin allele. The ddRAD-Seq approach consisted ofa workflow, as follows:(i) in silico prediction of optimum restriction enzymes from the reference ricegenome,(ii) verification of the prediction by ddRAD-Seq data of Carnaroli and Roma genomes (iii)establishment of a computational data processing pipeline for high confidence SNP calling, and (iv)validation of SNP accuracy. In silico prediction prior to sequencing analysis will contribute tooptimization of the experimental conditions for ddRAD-Seq and could help to accelerate the detectionof DNA markers useful for the authentication of rice cultivars Carnaroli and Roma. Preliminary resultsof HRM analysis show potential for rice cultivar differentiation since Carnaroli was distinguished fromRoma, among others (Carnise/Karnak, Gladio, Sant'Andrea and others) with high level of confidence(>98%). Acknowledgments: This work has

Autore Pugliese

• Mule' Giuseppina , Logrieco Antonio Francesco , Ferrara Massimo , De Paolis Angelo

Tutti gli autori

• M. Ferrara; L. Grazina; A. De Paolis; A. F. Logrieco; I. Mafra; J. S. Amaral; G. Mulè

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Anno di pubblicazione

2017

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