Genotyping-by-sequencing of honey derived environmental DNA can retrieve information on the Apis mellifera subspecie.

Honey contains environmental DNA (eDNA) traces derived from all organisms that directly or indirectly contributed to its production or that have been part of the production niche and environment from which this matrix is obtained. We recently demonstrated that honey constitutes an easily accessible source of Apis mellifera DNA useful to retrieve population genetic information. We also recently demonstrated that honey bee mitochondrial DNA (mtDNA) specific lineages detected in the honey can be used to authenticate the entomological origin of the honey. In this study we analysed honey DNA and integrated honey bee mtDNA information with nuclear genome polymorphisms to set up an improved tool that can detect the honey bee subspecies using these two genome levels. To this aim, we designed and tested a genotyping by sequencing (GBS) assay to analyse 121 single nucleotide polymorphisms (SNPs) of A. mellifera nuclear genome using eDNA extracted from honey. Results were integrated with information derived from previous studies and whole genome resequencing datasets. Genomic analyses were obtained for 61 specimens (honey samples and honey bees) collected in a few Italian regions (Emilia-Romagna, Liguria and Sicily) and that included: (i) individual honey bees of the subspecies A. m. ligustica, A. m. mellifera and A. m. siciliana; (ii) groups of pooled DNA samples from more than 30 A. m. ligustica workers belonging to the same colonies from which honey samples (see below) have been collected; (iii) honey samples obtained from 32 single hives; (iv) undifferentiated honey samples produced from A. m. ligustica and A. m. siciliana. The GBS runs produced more than 53 million reads that were used to obtain genotype information of the selected bi-allelic SNPs. Allele frequency estimation combined with several multidimensional scaling approaches were able to 25th Congress of Animal Science and Production Associationcorrectly assign the honey to the honey bee subspecies that produced it with high correlations between samples and runs. Overall, results obtained from GBS demonstrated the possibility to use A. mellifera nuclear genome variability to authenticate the entomological origin of the honey by detecting the honey bee subspecies. Acknowledgements This study was supported by Regione Emilia Romagna – BEE-RER3 project