The hybrid test is based on 117 SNPs which are evenly distributed on all chromosomes of the honey bee. These SNPs were chosen because they show maximal differences between the Mellifera and Carnica groups. Therefore, it is possible to detect the hybridization between the M and C groups with high accuracy. The test was developed and validated in a scientific study with numerous samples from across Europe, including 87 samples from Switzerland.
Single drones, single bees and mixed drone or bee samples (30 antennas or larvae) can be tested. A single drone covers half of the queen's genetics, while a single bee additionally tests a patriline. A drone mix sample allows to test the purity of the whole queen. A bee mix sample on the other hand allows to test both the queen and possible paternal lines. In this case, unwanted alleles can be detected up to a proportion of 10%. However, if less than 1 out of 10 matings of the queen happen with another subspecies, it is difficult to detect it.
Sampling tubes with ethanol are available if ordered in advance.
DNA extraction and genotyping
DNA is extracted with QIAGEN's EZ1 DNA Tissue Kit (QIAGEN Redwood City). Genotyping of SNPs is performed on the MassARRAY® MALDI-TOF platform (Agena Biosciences).
Analysis and result
The calculation of the genetic descent is performed with ADMXITURE software and a reference set of A. m. ligustica, A. m. carnica and A. m. mellifera from all over Europe. The result is composed of a percentage which indicates the proportionate descent of the M or C group and a standard error that indicates the probability (accuracy) of the result.
It should be noted that a mixture of different honeybee subspecies also occurs naturally. Therefore, a possible selection based on the test results should not be carried out too strictly.
On demand. Note that in order to fill up the genotyping plates, 23 samples must be ordered and processed together.
Parejo, M., Wragg, D., Gauthier, L., Vignal, A., Neumann, P. & Neuditschko, M. (2016). Using Whole-Genome Sequence Information to Foster Conservation Efforts for the European Dark Honey Bee, Apis mellifera mellifera. Frontiers in Ecology and Evolution, 4, 140. Link
Henriques, D., Browne, K., Kryger, P., Muñoz, I., Parejo, M., Barnett, M., McCormack, G., Garnery, L. & Pinto, M.A. (2018). High sample throughput genotyping for estimating C-lineage introgression in the dark honeybee: an accurate and cost-effective SNP-based tool. Scientific Reports, 8 (8552). Link
Muñoz, I., Henriques, D., Johnston, J.S., Chávez-Galarza, J., Kryger, P. & Pinto, M.A. (2015). Reduced SNP panels for genetic identification and introgression analysis in the dark honey bee (Apis mellifera mellifera). PLoS ONE, 10. Link
Parejo, M., Henriques, D., Pinto, M.A., Soland-Reckeweg, G., & Neuditschko, M. (2018). Empirical comparison of microsatellite and SNP markers to estimate introgression in Apis mellifera mellifera. Journal of Apicultural Research, 57:4, 504-506, Link
Muñoz, I., Henriques, D., Jara, L., Johnston, J.S., Chávez-Galarza, J., De La Rúa, P. & Pinto, M.A. (2016). SNPs selected by information content outperform randomly selected microsatellite loci for delineating genetic identification and introgression in the endangered dark European honeybee (Apis mellifera mellifera). Molecular Ecology Resources. Link
Software Admixture : Alexander, D. H., Novembre, J., & Lange, K. (2009). Fast model-based estimation of ancestry in unrelated individuals. Genome Research, 19(9), 1655-1664. doi: 10.1101/gr.094052.109. Link