Study of the variability of Csd alleles based on NGS data in Italian honeybees

Sex determination in Apis mellifera is regulated by the complementary sex determiner (csd) gene. Drones are hemizygous at this locus and females are heterozygous; diploid drones, containing homozygous genotypes for csd, are eaten by worker bees during the larval stage. Therefore, the prevention of allelic variability in this locus is essential to prevent colony losses and for conservation strategies. Focus of this study is to investigate the genomic variability of the csd gene on NGS data and to explore the variability of alleles circulating in Italy in different honeybee subspecies. During summer 2017 and 2018, 125 colonies of seven different subspecies were sampled in Italy. Whole Genome sequences of the 125 worker bees were obtained through Illumina NextSeq with a 150 bp paired-end module. Sequences were mapped to the HAv3.1 A. mellifera genome and the hypervariable region of the csd gene was manually examined with the Integrative Genomics Viewer (IGV). Each strand was translated to obtain protein sequences for each allele of the heterozygous sequenced individuals. Nucleotide sequences have been constructed for 65 samples, belonging to 33 Ligustica, 20 Buckfast, 5 Carnica, 4 Sicula, 1 hybrid Carnica, 1 Cecropia and 1 Mellifera honeybees. In total 107 sequences were identified using identity as a criterion. For each subspecies, 58 sequences were identified in the Ligustica honeybees, 35 in Buckfast, 10 in Carnica, 8 in Sicula and 2 in hybrid Carnica, Cecropia and Mellifera. The most frequent sequence identified occurs in the 12% of the population and is present in four different subspecies (Ligustica, Buckfast, Carnica and hybrid carnica). Reconstructed sequences will be validated through Sanger sequencing in the near future. This study contributes to the characterization of the csd allelic variability in Italian honeybee colonies.