Guardians of quality: advancing Castanea sativa traceability using DNA analysis from seed to processed food

Castanea sativa is one of the most appreciated species in Italy, due to timber and fruit quality; nuts are usually intended for fresh consumption, but also for the production of industrial foods. The demand for an efficient traceability system to identify Castanea species and cultivars along the supply chain, from the chestnut orchard to the processed product, is crucial to protect consumers and producers from frauds. Here we present a traceability protocol to identify the species of the Castanea genus and trace the cultivars of Castanea sativa, based on DNA markers (SSRs and SNPs) analyses. Microsatellites were applied to identify the cultivar in plant material (leaves, seed, and episperm) and processed food (creams, beverages, flour, and cookies). Although SSRs made it possible to trace cultivar starting from somatic tissues (e.g.: leaves), the presence of foreign paternal DNA from pollenizer plants in seed and processed food, interferes with the identification of the maternal one. To overcome this issue, we Illumina sequenced six C. sativa cultivars to highlight maternal-inherited markers. A set of SNP/INDEL markers in two chloroplastic regions were identified, able to effectively distinguish and trace C. sativa from C. mollissima and C. crenata. The High Resolution Melting (HRM) analysis was performed as a practical and efficient method for SNP/INDELvalidation and to develop a fast, reliable, and reproducible protocol for chestnut traceability in food.