Development of a ddRAD library preparation and HRM approaches for SNP discovery and genotyping of Italian rice cultivars Carnaroli and Roma

Food authentication and traceability is a complex problem, strictly correlated to fraud and adulteration detections that dramatically affect the consumer protection. Analysis of protein, metabolite and DNA represents robust tools for food authentication. In particular, DNA-based methods are more reliable, thanks to the stability of DNA under production and processing techniques applied along the food-chain. Therefore, DNA markers offer a powerful tool to address the validation of 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 in translational genomic even in plants. SNP are, in fact, the most abundant forms of genetic variation among 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 small insertions 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-end sequencing of identical loci. Progress in NGS technology has led to the availability of several plant genomes. This situation makes it possible to simulate ddRAD-Seqin silico, allowing prediction of the numbers, sizes, and genome positions of digested fragments. However, few reports have evaluated the in silico predictions by comparative experiments using several combinations of restriction enzymes and multiple samples with different SNP density. HRM analysis has several advantages over traditional methods for gene scanning and genotyping, making it faster, less laborious and more suitable for high sample throughput. In this study, two approaches are proposed for the authentication of the Italian rice cultivars Carnaroli and Roma: in silico and empirical ddRAD-Seq analysis and HRM analysis targeting an A/C SNP in exon 6, responsible for the Wxin allele. The ddRAD-Seq approach consisted of a workflow, as follows:(i) in silico prediction of optimum restriction enzymes from the reference rice genome,(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 to optimization of the experimental conditions for ddRAD-Seq and could help to accelerate the detection of DNA markers useful for the authentication of rice cultivars Carnaroli and Roma. Preliminary results of HRM analysis show potential for rice cultivar differentiation since Carnaroli was distinguished from Roma, among others (Carnise/Karnak, Gladio, Sant'Andrea and others) with high level of confidence (>98%). Acknowledgments: This work has been supported by the European project FOODINTEGRITY (FP7-KBBE-2013-single-stage, No 613688).