Non-lethal genotyping of honey bee queens and drones prior to artificial insemination for targeted mating

Abstract Honey bee ( Apis mellifera ) breeding and experimental studies often require precise control of parental genotypes. To produce diploid drones, homozygosity at the complementary sex determiner ( csd ) gene is essential. Here, we report a non-lethal, high-throughput workflow for csd genotyping of queens and drones prior to instrumental insemination, enabling targeted matings that yield diploid drones and controlled inbreeding while preserving queen vitality. For queens, DNA was extracted from exuviae and for drones, two middle-leg segments were clipped from newly emerged males. Exon 7 of csd was PCR-amplified, drone amplicons underwent direct Sanger sequencing, whereas queen amplicons were cloned to resolve heterozygous alleles. Translated sequences were aligned to assign each individual’s alleles, and brother-drones were grouped by homozygosity for one of two matching sister-queen alleles. Instrumental insemination with these cohorts produced seven queens that started oviposition and exhibited the scattered brood pattern characteristic of diploiddrone production. Our approach combines the precision of single-drone insemination with the fecundity of multiple-drone insemination, offering a rapid, reliable tool for breeding programs and experimental studies of diploid drones and csd -mediated sex determination.