An alternative method to identify CSD alleles of the honey bees based on NGS data

Focus of this study is the allelic variability of the complementary sex determination (CSD) gene, which regulates sex determination in honey bees: males are hemizygous for CSD, while females are heterozygous. Diploid males develop from fertilized eggs, which are homozygous for CSD; diploid males are eaten by worker bees in the larval stage. Therefore, there is a strong advantage for heterozygotes leading to a high number of distinct csd alleles. A hypervariable region (HVR), located in exons 6 and 7, is responsible for csd allelic variability. One hundred twenty-five worker colonies were sampled within summer 2017 and summer 2018 in twelve different Italian regions: Abruzzo, Emilia Romagna, Liguria, Lombardy, Marche, Piedmont, Sicily, Trentino, Tuscany, Apulia, Umbria and Veneto. In each region, beekeepers were chosen among those belonging to the ‘Associazione Italiana Allevatori Api Regine (AIAAR)’ and the ‘Albo Nazionale Allevatori Api Italiane’ associations. Genomes of the 125 worker colonies were sequenced through Illumina NextSeq with a 150 bp paired end module. Sequences were mapped to the HAv3.1 Apis mellifera genome and the HVR of the CSD gene was manually inspected with the Integrative Genomics Viewer. The nucleotide sequences of both strands of this region were successfully reconstructed for 65 samples, and each strand was translated to obtain protein sequences for each allele of the heterozygous sequenced individuals. The resulting 130 sequences include variants of the HVR region which have not been described in literature, yet, contributing to characterize CSD allelic variability in Italian honey bee colonies.