Detection of almonds (Prunus dulcis) adulteration by genotyping of sweet and bitter almonds with double-mismatch allele-specific qPCR (DMAS-qPCR)

Abstract

The main ingredient in marzipan is sweet almonds (Prunus dulcis var. dulcis). Bitter almonds (Prunus dulcis var. amara) are also added to create a more intense flavour. In Germany the proportion of bitter almonds in marzipan is limited to a maximum of 12%. The use of debittered bitter almonds is prohibited. However, currently no analytical technique can reliably monitor compliance with this legal requirement. In this study we used next-generation sequencing technology to sequence the plastid genomes of six sweet cultivars (Larguetta, Ferragnes, Nonpareil, Carmel, Monterey, and Marcona) and six bitter almond batches of different origin (Iran, Morocco, Kyrgyzstan, Spain, Syria, and Turkey) to locate single nucleotide variants (SNVs). Sweet and bitter almonds share the same maternal lineage, but sequence variants on the plastid genome were present in most bitter almond populations. To exploit these differences for detection of bitter almonds in processed products, we used the double-mismatch allele-specific qPCR (DMAS-qPCR) for two polymorphic loci (rpoB, rps4). We provide evidence that the rps4 variant was detectable in almond raw pastes containing (debittered) bitter almonds, showing that the DNA sequence was sufficiently stable throughout food processing. A clear distinction from sweet almonds was observed for samples that contained at least 8% of bitter almonds. Our results present a promising approach to detect adulterations with bitter almonds in marzipan.