Analysis of Complementary Sex-Determiner (csd) Allele Diversity in Different Honeybee Subspecies from Italy Based on NGS Data

Abstract

Sexual regulation in Apis mellifera is controlled by the complementary sex-determiner (csd) 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 csd allelic diversity in honeybee populations is a priority for colony survival. The focus of this study is to investigate csd variability in the genomic sequence of the hypervariable region (HVR) of the csd 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 A. mellifera subspecies: A. m. ligustica, A. m. sicula, A. m cecropia, A. m. carnica, A. m. mellifera, 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 csd gene allelic variants present in the subspecies collection, based on de novo 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 csd alleles present in the NCBI GenBank database.

Variability and Number of Circulating Complementary Sex Determiner (Csd) Alleles in a Breeding Population of Italian Honeybees under Controlled Mating

“…Each sequence was assigned an Amelcsd-HVR ID, numbered sequentially from the most to the least common (from Amelcsd-HVR1 to Amelcsd-HVR225) [ 19 ]. Out of the 47 alleles found, 42 were reported and named by Bilodeau et al, 2 alleles (allele 7 and allele 14) were described only by Paolillo et al [ 20 ], while 3 (Allele 35, 39, 42) were novel alleles. In Supplementary Table S1 , allele sequences are reported together with their frequencies and the corresponding Amelcsd-HVR numbers or Paolillo Allele numbers [ 19 , 20 ].…”

“…Out of the 47 alleles found, 42 were reported and named by Bilodeau et al, 2 alleles (allele 7 and allele 14) were described only by Paolillo et al [ 20 ], while 3 (Allele 35, 39, 42) were novel alleles. In Supplementary Table S1 , allele sequences are reported together with their frequencies and the corresponding Amelcsd-HVR numbers or Paolillo Allele numbers [ 19 , 20 ]. Moreover, Figure 2 visually represents the aligned amino acid sequences of the 47 alleles, highlighting the notable differences in both amino acid composition and length.…”

“…A bioinformatic pipeline was used for this purpose, which included manual review to achieve a curated, reference-independent sequence assembly. The pipeline was tested and validated by GenBank data in a previous study [ 20 ].…”

“…The HVR region sequence of 51% of the total samples in the study was successfully reconstructed. The obtained sequences were validated by comparison with known alleles found in the literature [ 19 , 20 ]. As shown in Table S1 , 44 out of 47 alleles showed 100% identity with already-published sequences, while the remaining 3 are novel alleles.…”

“…The number of csd alleles was initially estimated to be about 10–13 [ 11 ], but further studies have identified a higher number of circulating alleles, ranging from 16 locally [ 12 , 13 , 14 , 15 , 16 ] to over 200 alleles worldwide [ 9 , 17 , 18 , 19 ]. In the Italian honeybee population, 88 different alleles have recently been identified by analyzing 125 colonies of different genetic types in 12 different Italian regions [ 20 ].…”