20.500.12556/RUL-155267 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. spermatheca honeybee Apis mellifera breeding preservation method microsatellite DNA extraction patriline true false true Angleški jezik Ni določen Članek v reviji 2024-03-22 13:56:20 2024-03-22 13:56:21 2024-03-24 03:14:07 0000-00-00 00:00:00 2023 0 0 10 str. art. 1139269 Vol. 14 2023 0000-00-00 Zaloznikova Objavljeno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 638 1664-042X 10.3389/fphys.2023.1139269 143854595 fphys-14-1139269.pdf fphys-14-1139269.pdf 1 D479B70D1AAB479E8D34671CE4A890AD e9971e7d8537491092e01e0917b5850b4f38c4d474629b96fc3304a1f8b1b59b ff984efa-e84b-11ee-b815-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=182085 https://www.frontiersin.org/journals/physiology/articles/10.3389/fphys.2023.1139269/full 0 f57b4e49-e84b-11ee-b815-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=182084 Biotehniška fakulteta 0 0 0