The genetic integrity of Apis mellifera mellifera is threatened by introgression in many
places of its native distribution, after recurrent importations of commercial queens
typically belonging to the divergent C-lineage. A growing interest in keeping and protecting
A. m. mellifera has motivated the development of conservation programs in many places
of Europe. As part of the conservation efforts, isolated mating stations are set to avoid
unwanted crosses, but these are not always effective as matings with unwanted drones are
frequently reported. An interesting method to monitor the degree of isolation of mating
stations could be through genetic analysis of the queen spermatheca contents. While this
method implies that queens selected for monitoring are sacrificed, it can be a powerful way
of assessing the effectiveness of mating stations because it would allow easy detection of
unwanted alleles. Here, we developed an SNP-based tool suited to the analysis of DNA
extracted from spermatheca or from pooled DNA of varying sources. To that end, we first
designed an SNP panel from whole-genome sequence data generated from 228 drones, of
which 148 belonged to the M-lineage (117 A. m. iberiensis and 31 A. m. mellifera) and 80 to
the C-lineage (46 A. m. carnica and 34 A. m. ligustica). A total of 5,007 highly differentiated
SNPs was found. Based on different criteria, 130 SNPs were selected to be included in
the genotyping tool. This tool is based on the NEBNext Direct Genotyping Solution that
allows high-throughput, sequence-based target genotyping of single-individual or pooled
DNA. To assess the tool’s sensitivity and accuracy, 142 samples (DNA extracted from
spermatheca and tissue, as well as known DNA mixtures) were genotyped. After removing
the problematic SNPs, 81 were retained and these were able to provide an estimate of the
pool introgression level with great accuracy. This tool represents a significant advance in
the genetic analysis of honey bee colonies with a variety of applications, including breeding
and conservation of A. m. mellifera.info:eu-repo/semantics/publishedVersio
