Cutting corners: The impact of storage and DNA extraction on quality and quantity of DNA in honeybee (Apis mellifera) spermatheca

The purpose of our study was to investigate methods of short-term storage that allow preservation, transport and retrieval of genetic information contained in honeybee queen's spermatheca. Genotyping of the honeybee colony requires well ahead planned sample collection, depending on the type of data to be acquired. Sampling and genotyping of spermatheca's content instead of individual offspring is timesaving, allowing answers to the questions related to patriline composition immediately after mating. Such procedure is also cheaper and less error prone. For preservation either Allprotect Tissue Reagent (Qiagen) or absolute ethanol were used. Conditions during transportation were simulated by keeping samples 6-8 days at room temperature. Six different storing conditions of spermathecas were tested, complemented with two DNA extraction methods. We have analysed the concentration of DNA, RNA, and proteins in DNA extracts. We also analysed how strongly the DNA is subjected to fragmentation (through amplification of genetic markers ANT2 and tRNA(leu)-COX2) and whether the quality of the extracted DNA is suitable for microsatellite (MS) analysis. Then, we tested the usage of spermatheca as a source of patriline composition in an experiment with three instrumentally inseminated virgin queens and performed MS analysis of the extracted DNA from each spermatheca, as well as queens' and drones' tissue. Our results show that median DNA concentration from spermathecas excised prior the storage, regardless of the storing condition and DNA extraction method, were generally lower than median DNA concentration obtained from spermathecas dissected from the whole queens after the storage. Despite the differences in DNA yield from the samples subjected to different storing conditions there was no significant effect of storage method or the DNA extraction method on the amplification success, although fewer samples stored in EtOH amplified successfully in comparison to ATR storing reagent. However, we recommend EtOH as a storing reagent due to its availability, low price, simplicity in usage in the field and in the laboratory, and capability of good preservation of the samples for DNA analysis during transport at room temperature

The Carniolan Honeybee from Slovenia-A Complete and Annotated Mitochondrial Genome with Comparisons to Closely Related Apis mellifera Subspecies

The complete mitochondrial genome of the Carniolan honeybee (Apis mellifera carnica) from Slovenia, a homeland of this subspecies, was acquired in two contigs from WGS data and annotated. The newly obtained mitochondrial genome is a circular closed loop of 16,447 bp. It comprises 37 genes (13 protein coding genes, 22 tRNA genes, and 2 rRNA genes) and an AT-rich control region. The order of the tRNA genes resembles the order characteristic of A. mellifera. The mitogenomic sequence of A. m. carnica from Slovenia contains 44 uniquely coded sites in comparison to the closely related subspecies A. m. ligustica and to A. m. carnica from Austria. Furthermore, 24 differences were recognised in comparison between A. m. carnica and A. m. ligustica subspecies. Among them, there are three SNPs that affect translation in the nd2, nd4, and cox2 genes, respectively. The phylogenetic placement of A. m. carnica from Slovenia within C lineage deviates from the expected position and changes the perspective on relationship between C and O lineages. The results of this study represent a valuable addition to the information available in the phylogenomic studies of A. mellifera-a pollinator species of worldwide importance. Such genomic information is essential for this local subspecies' conservation and preservation as well as its breeding and selection