High Resolution Melting DNA analysis for the traceability of plants and juices of blond and pigmented sweet orange

The economic relevance of sweet orange [Citrus sinensis (L.) Osbeck] raised the interest for the set-up of robust and scalable methods to assess the origin of plants and derived products. Molecular markers, such as single-nucleotide polymorphisms (SNPs), represent a robust tool for the univocal identification of the genetic origin of a specific cultivar (or group of accessions) with relevant application for traceability. In this work, the whole-genome sequencing of 29 accessions representing the four varietal groups of sweet orange (i.e.: Common, Navel, Acidless and Pigmented) allowed the in silico detection of unique SNPs to discriminate each group (group-specific SNPs) and ten pigmented orange accessions (genotype-specific SNPs). A subset of the SNPs detected in silico was then validated through a high-resolution melting assay (HRM) on additional 81 genotypes held in three ex-situ collections in Italy and Spain and on several matrices: leaves, hand‐squeezed and commercial fresh-squeezed juices, processed orange juices, and beverages. The HRM assay allowed the identification of 13 group-specific and 30 genotype-specific SNPs showing consistent results across the whole germplasm and the different food matrices tested. The HRM assay proved its efficacy also for the identification of juice blending with other sweet orange varieties (with a detection limit of 5 %). The identified unique SNPs represent a valuable tool to trace the varietal correspondence of plants and fruit-derived products. Their implementation with a scalable and robust HRM assay could be readily employed by growers and the food processing industry to ensure traceability along the chain.