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Percorrer por autor "Soltani, Chiraz"

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  • Development of a cost-effective SNP tool to detect genetic pollution in honey bee spermatheca content
    Publication . Soltani, Chiraz; Henriques, Dora; Pinto, M. Alice
    The loss of genetic complexes adapted to local conditions through genetic introgression is one of the many threats affecting the honey bee (Apis mellifera). Large- scale displacement of honey bees has altered their native distribution. One extreme example is A. m. mellifera, a European subspecies that was once widespread but is now threatened with extinction in numerous countries due to introgression and replacement by the C lineage. As a result, conservation measures may be needed to preserve the genetic diversity of these subspecies. Acknowledging the significance of native genetic diversity, several conservation and breeding programs have been established. Their effectiveness hinges on the availability of accurate and cost-effective molecular tools for assessing subspecies introgression. Whole-genome data has offered valuable insights into honey bee evolution, yet its practical application is hampered by the need for specialized bioinformatics expertise and computational resources, often unavailable in conservation and breeding centers. To bridge this gap, a novel SNP (Single Nucleotide Polymorphism) tool, based on the NEBNext Direct Genotyping Solution, has been developed. This tool was designed from 228 whole-genome sequence data generated from 148 M-lineage drones and 80 C-lineage drones. From 5,007 highly differentiated SNPs, we selected 130 SNPs. After eliminating problematic SNPs, we retained 82 SNPs that demonstrated exceptional accuracy in estimating the degree of genetic introgression in known samples. This innovative tool represents a significant advancement in the genetic analysis of honey bee colonies, with applications spanning breeding and conservation efforts for A. m. mellifera, A. m. iberiensis, A. m. carnica and A. m. ligustica.
    2023Dissertação de mestrado Acesso aberto Ver mais
  • Genomic DNA isolation methods from honey bee (Apis mellifera L.) Spermatheca
    Publication . Ariel Yadró, Carlos; Lopes, Ana; Henriques, Dora; Soltani, Chiraz; Marques, Manoela; Wegener, Jakob; Musin, Eduard; Pinto, M. Alice
    The 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.
    2022Documento de conferência Acesso aberto Ver mais