Percorrer por autor "Wegener, Jakob"
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- Genomic DNA extraction from honey bee (Apis mellifera) queen spermathecal contentPublication . Yadró, Carlos A.; Lopes, Ana Rita; Henriques, Dora; Musin, Eduard; Wegener, Jakob; Pinto, M. AliceGenetic analysis of the honey bee spermathecal content can be particularly useful to provide an estimate of the genetic diversity and purity of the surrounding populations. Here we compared the concentration and quality of DNA extracted from queen spermatheca using four commercial kits to determine the best method to obtain DNA suitable for single nucleotide polymorphism genotyping by next-generation sequencing. The four kits were tested with different adjustments in the lysis incubation time, use of RNA-carrier, elution conditions and number of re-elutions. Only the use of QIAamp DNA Microkit with 3 h of lysis incubation, the addition of RNA-carrier and multiple re-elutions produced a DNA concentration over the required threshold.
- Genomic DNA isolation methods from honey bee (Apis mellifera L.) SpermathecaPublication . Ariel Yadró, Carlos; Lopes, Ana; Henriques, Dora; Soltani, Chiraz; Marques, Manoela; Wegener, Jakob; Musin, Eduard; Pinto, M. AliceThe honey bee queen (Apis mellifera L.) has a polyandrous mating system, meaning that the queen mates on average with 17 drones from the surroundings in a congregation area. After the mating event, the spermatozoa of the drones are stored in an organ called spermatheca. Genetic analysis of the spermathecal content can provide an estimate of the genetic diversity and purity of the surrounding honey bee populations. This can be particularly useful for conservation and mating centers that need to monitor their populations’ genetic backgrounds. However, isolating enough DNA for genomic applications from such a small and complex matrix can be a challenge. Here, we compared the quantity and quality of DNA isolated using five methods: (i) phenol-chloroform-isopropanol, (ii) QIAamp DNA Minikit, (iii) QIAamp DNA Microkit, (iv) Macherey- Nagel Nucleospin Tissue, and (v) NEB Monarch Genomic DNA Purification Tissue. For each kit, when appropriate, variations including different isolation protocols, lysis incubation times, and the addition of RNA carrier were assayed. The quantity and quality of DNA extracted was assessed by spectrophotometric (SpectroStar®Nano LVis Plate) and fluorometric methods (Quantus ™ Fluorometer). Spectrophotometric quantification indicated nucleic acid concentrations ranging from 2.00 to 55.58 ng/μL, and in 91.43% of the cases, the A260/280 ratios were over 2.00, indicating an elevated presence of RNA. The fluorometric quantification, specific for double-stranded DNA, provided values ranging from 0.02 to 2.30 ng/μL. From the five methods, two alternative protocols of the commercial kit QIAamp DNA Microkit produced a sufficient DNA quantity (≥1.7 ng/μL measured by Quantus) for applications involving SNP genotyping, namely: the Tissue protocol with 6 hours of lysis incubation and the Tissue protocol with 3 hours of incubation, both with addition of RNA carrier. In contrast, overnight lysis decreased the DNA yield. The other methods generally produced low and/or inconsistent DNA recovery. According to our results, QIAamp DNA Microkit with the use of RNA carrier and lysis incubation times between 3 to 6 hours produce the required DNA quantities for SNP genotyping.
- A molecular tool to detect genetic introgression from Spermatheca contentPublication . Henriques, Dora; Smith, Stephen; Lopes, Ana; Wegener, Jakob; Musin, Eduard; Hoppe, Andreas; Parejo, Melanie; McCormack, Grace P.; Pinto, M. AliceThe genetic integrity of Apis mellifera mellifera is threatened by introgression in many places of its native distribution, after recurrent importations of commercial queens typically belonging to the divergent C-lineage. A growing interest in keeping and protecting A. m. mellifera has motivated the development of conservation programs in many places of Europe. As part of the conservation efforts, isolated mating stations are set to avoid unwanted crosses, but these are not always effective as matings with unwanted drones are frequently reported. An interesting method to monitor the degree of isolation of mating stations could be through genetic analysis of the queen spermatheca contents. While this method implies that queens selected for monitoring are sacrificed, it can be a powerful way of assessing the effectiveness of mating stations because it would allow easy detection of unwanted alleles. Here, we developed an SNP-based tool suited to the analysis of DNA extracted from spermatheca or from pooled DNA of varying sources. To that end, we first designed an SNP panel from whole-genome sequence data generated from 228 drones, of which 148 belonged to the M-lineage (117 A. m. iberiensis and 31 A. m. mellifera) and 80 to the C-lineage (46 A. m. carnica and 34 A. m. ligustica). A total of 5,007 highly differentiated SNPs was found. Based on different criteria, 130 SNPs were selected to be included in the genotyping tool. This tool is based on the NEBNext Direct Genotyping Solution that allows high-throughput, sequence-based target genotyping of single-individual or pooled DNA. To assess the tool’s sensitivity and accuracy, 142 samples (DNA extracted from spermatheca and tissue, as well as known DNA mixtures) were genotyped. After removing the problematic SNPs, 81 were retained and these were able to provide an estimate of the pool introgression level with great accuracy. This tool represents a significant advance in the genetic analysis of honey bee colonies with a variety of applications, including breeding and conservation of A. m. mellifera.