| Summary: | The <i>complementary sex determiner</i> (<i>csd</i>) gene plays an essential role in the sex determination of <i>Apis mellifera</i> L. Females develop only if fertilized eggs have functional heterozygous genotypes at this gene whereas males, being haploids, are hemizygous. Two identical <i>csd</i> alleles produce non vital males. In light of the recent decline in honey bee populations, it is therefore important to monitor the allele variability at this gene. In this study, we tested the application of next generation semiconductor-based sequencing technology (Ion Torrent) coupled with environmental honey DNA as a source of honey bee genome information to retrieve massive sequencing data for the analysis of variability at the hypervariable region (HVR) of the <i>csd</i> gene. DNA was extracted from 12 honey samples collected from honeycombs directly retrieved from 12 different colonies. A specifically designed bioinformatic pipeline, applied to analyze a total of about 1.5 million reads, identified a total of 160 different <i>csd</i> alleles, 55% of which were novel. The average number of alleles per sample was compatible with the number of expected patrilines per colony, according to the mating behavior of the queens. Allele diversity at the <i>csd</i> could also provide information useful to reconstruct the history of the honey.
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