An Alternative, High Throughput Method to Identify Csd Alleles of the Honey Bee

Abstract

Applying instrumental insemination in closely related honey bee colonies often leads to frequent lethality of offspring causing colony collapse. This is due to the peculiarities of honey bee reproductive biology, where the complementary sex determination (csd) gene drives sex determination within a haplodiploid system. Diploid drones containing homozygous genotypes are lethal. Tracking of csd alleles using molecular markers prevents this unwanted event in closed breeding programs. Our approach described here is based on high throughput sequencing (HTS) that provides more data than traditional molecular techniques and is capable of analysing sources containing multiple alleles, including diploid individuals as the bee queen. The approach combines HTS technique and clipping wings as a minimally invasive method to detect the complementary sex determiner (csd) alleles directly from honey bee queens. Furthermore, it might also be suitable for screening alleles of honey harvested from hives of a closed breeding facility. Data on alleles of the csd gene from different honey bee subspecies are provided. It might contribute to future databases that could potentially be used to track the origin of honey. With the help of tracking csd alleles, more focused crossings will be possible, which could in turn accelerate honey bee breeding programmes targeting increase tolerance against varroosis as well.

Application of Next Generation Semiconductor-Based Sequencing for the Identification of Apis mellifera Complementary Sex Determiner (csd) Alleles from Honey DNA

“…It has also been suggested that csd may play a role in controlling the balance between inbreeding and outbreeding in honey bee reproduction [11,17,23]. Considering that heterozygosity at this gene is essential to generate vital females, several studies have investigated csd sequence diversity, demonstrating that a large number of functional alleles exist in all honey bee populations [21,[23][24][25][26][27][28][29]. These studies have mainly been carried out by Sanger sequencing the HVR from drones of different colonies, in order to maximize the probability of detecting the two alleles of the queen bees [23,24,28] or from workers after cloning the amplicons [25].…”

“…The level of diversity of this highly repetitive gene region has been investigated in different honey bee populations by several studies that mostly used Sanger sequencing of the HVR amplified from drones [21,[23][24][25][26]28,29]. These studies identified a high number of csd protein allele sequences that have been recently compiled in a scientific note [27].…”

“…Therefore, honey can be used to obtain a quite extensive population-wide picture of the presence of honey bee genetic features including information on variability at the csd gene. Kolics et al [29] already tested the possibility of obtaining sequence information of the csd HVR using amplicons produced from honey DNA and Illumina next generation sequencing.…”

Non-Destructive Genotyping of Honeybee Queens to Support Selection and Breeding

“…For example, using an appropriate set of primers, the entomological origin of honey can be determined to a certain degree, like distinguishing between species or, in some cases, even between some of the subspecies of honeybees [ 59 , 60 ]. While these sources do not seem directly applicable to queen breeding, genotyping of csd alleles using NGS technology yielded a diversity of these alleles within the colony [ 18 ]. Even if it may be difficult to envision such an approach in practice, it surely has a practical value in research.…”

“…The successful extraction of DNA from clipped wings and subsequent genotyping were reported in the past [ 13 , 14 , 15 , 16 , 17 ]. Recent research demonstrated the efficient high throughput method to identify csd alleles of the honeybee queens using wing clippings coupled with a next-generation sequencing approach [ 18 ]. Though successful, there is still a requirement to wait with wing clipping until mating flights are completed.…”