Application of next generation semiconductor-based sequencing for the identification of Apis mellifera complementary sex determiner (csd) alleles from honey DNA

The complementary sex determiner (csd) gene plays an essential role in the sex determination of Apis mellifera L. Females develop only if fertilized eggs have functional heterozygous genotypes at this gene whereas males, being haploids, are hemizygous. Two identical csd alleles produce not vital males. In light of the recent decline of the honey bee populations, it is therefore im-portant to monitor the allele variability at this gene. In this study we tested the application of the next generation semiconductor-based sequencing technology (Ion Torrent) coupled with en-vironmental honey DNA as source of honey bee genomic information to retrieve massive se-quencing data for the analysis of variability at the hypervariable region (HVR) of the csd gene. DNA was extracted from 12 honey samples collected from honeycombs directly retrieved from 12 different colonies. A specifically designed bioinformatic pipeline, applied to analyse a total of about 1.5 million reads, identified a total of 160 different csd alleles, 55% of which were novel. The average number of alleles per samples was compatible with number of expected patrilines per colony, according to the mating behaviour of the queens. Allele diversity at the csd could al-so provide information useful to reconstruct the history of the honey.