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Spermateka pri matici medonosne čebele (Apis mellifera L.) kot vir genetske informacije o očetovstvu v čebelji družini
ID Pavlin, Anja (Author), ID Prešern, Janez (Mentor) More about this mentor... This link opens in a new window, ID Moškrič, Ajda (Comentor)

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Abstract
Uporaba vsebine spermateke matice medonosne čebele namesto posameznih potomcev (čebel delavk) za genotipizacijo prihrani čas in omogoča vpogled v sestavo očetovskih linij takoj po parjenju. Raziskali smo načine kratkotrajnega shranjevanja in metode izolacije DNA, ki omogočajo ustrezno ohranjanje spermateke med transportom za pridobivanje genetskih informacij iz spermatozoidov. Testirali smo šest različnih načinov shranjevanja spermatek, ki smo jih dopolnili z dvema metodama izolacije DNA. Uspešnost le-teh smo preverjali z analizo koncentracij DNA, RNA in proteinov v izolatih DNA ter stopnje fragmentacije DNA s pomnoževanjem markerja ANT2 in primernost kakovosti DNA za mikrosatelitno (MS) analizo. Raziskali smo tudi uporabo MS in dela zaporedja gena csd za določevanje očetovskih linij v čebelji družini. Za poskus uporabe spermateke smo za analizo MS uporabili instrumentalno osemenjene deviške matice in naravno osemenjene matice s pripadajočimi troti oziroma zalego. Za analizo dela zaporedja gena csd smo uporabili le instrumentalno osemenjene matice s pripadajočimi troti. Naši rezultati kažejo, da je razpon mediana koncentracij DNA iz spermatek, izoliranih pred shranjevanjem, ne glede na način shranjevanja in metodo izolacije DNA, na splošno nižji od razpona median koncentraciji DNA iz spermatek, izoliranih iz matic po shranjevanju. Kljub razlikam v izplenu DNA iz vzorcev ni bilo pomembnega vpliva načina shranjevanja ali metode izolacije DNA na uspeh pomnoževanja MS. Za transport pri sobni temperaturi kot medij za shranjevanje priporočamo etanol zaradi njegove dostopnosti, nizke cene, enostavnosti uporabe na terenu in v laboratoriju ter zmožnosti dobrega ohranjanja vzorcev za analizo DNA. Rezultati za določanje očetovskih linij z uporabo MS kažejo, da je analiza na vsebini spermateke enakovredna analizi na zalegi, zato menimo, da je uporaba spermateke primernejša od zalege. Rezultati preliminarnega testiranja metode za določanje očetovskih linij na vsebini spermateke z uporabo gena csd nakazujejo na potencialno uporabnost le-te.

Language:Slovenian
Keywords:spermateka, medonosna čebela, Apis mellifera, način shranjevanja, metoda izolacije DNA, rejski program, očetovske linije, mikrosateliti, sekvenciranje naslednje generacije
Work type:Master's thesis/paper
Typology:2.09 - Master's Thesis
Organization:BF - Biotechnical Faculty
Year:2023
PID:20.500.12556/RUL-148487 This link opens in a new window
COBISS.SI-ID:162043651 This link opens in a new window
Publication date in RUL:25.08.2023
Views:1108
Downloads:96
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Secondary language

Language:English
Title:Queen’s spermatheca as a source of patrilineal genetic information in the colony of Apis mellifera L.
Abstract:
In this study, we explored a more efficient method for genotyping honeybee colonies using the contents of the queen bee's spermatheca instead of individual offspring (worker bees). This method saves time and provides insight into the composition of paternal lines immediately after mating. To obtain genetic information from spermatozoa in the spermatheca, we investigated different methods for short-term storage of spermatheca and DNA isolation. We tested six different storage methods coupled with two DNA isolation methods. Their success was verified by analyzing DNA, RNA, and protein concentrations, as well as DNA fragmentation and suitability of DNA quality for microsatellite (MS) analysis. We also investigated the use of MS analysis and the csd gene sequence to determine paternal lines in the bee colony. Our results showed that the range of medians of DNA concentrations from the spermathecae isolated before storage was generally lower than the range of medians of DNA concentrations from those isolated after storage. However, the storage method or DNA isolation method did not significantly affect the success of MS amplification. For transport at room temperature, we recommend ethanol as a storage medium due to its availability, low cost, ease of use in the field and laboratory, and the ability to preserve samples well for DNA analysis. The results for determining paternal lines with analysis of the contents of the spermatheca were found to be equivalent to te results with analysis ot the brood, indicating that the use of spermatheca is more suitable than brood. Preliminary testing of the method for determining paternal lines using the csd gene sequence in the spermatheca showed potential usefulness.

Keywords:spermatheca, honeybee, Apis mellifera, preservation method, DNA extraction, breeding, patriline, microsatellite, next generation sequencing

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