| Summary: | Sexual regulation in <i>Apis mellifera</i> is controlled by the <i>complementary sex-determiner (csd)</i> gene: females (queens and workers) are heterozygous at this locus and males (drones) are hemizygous. When homozygous diploid drones develop, they are eaten by worker bees. High <i>csd</i> allelic diversity in honeybee populations is a priority for colony survival. The focus of this study is to investigate <i>csd</i> variability in the genomic sequence of the hypervariable region (HVR) of the <i>csd</i> gene in honeybee subspecies sampled in Italy. During the summer of 2017 and 2018, worker bees belonging to 125 colonies were sampled. The honeybees belonged to seven different <i>A. mellifera</i> subspecies: <i>A. m. ligustica</i>, <i>A. m. sicula</i>, <i>A. m cecropia</i>, <i>A. m. carnica</i>, <i>A. m. mellifera</i>, Buckfast and hybrid Carnica. Illumina genomic resequencing of all samples was performed and used for the characterization of global variability among colonies. In this work, a pipeline using existing resequencing data to explore the <i>csd</i> gene allelic variants present in the subspecies collection, based on <i>de novo</i> assembly of sequences falling within the HVR region, is described. On the whole, 138 allelic sequences were successfully reconstructed. Among these, 88 different alleles were identified, 68 of which match with <i>csd</i> alleles present in the NCBI GenBank database.
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