Strobl Frederic, Ross J Alexander, Stelzer Ernst H K
Physical Biology / Physikalische Biologie (IZN, FB 15), Buchmann Institute for Molecular Life Sciences (BMLS), Cluster of Excellence Frankfurt-Macromolecular Complexes (CEF-MC), Goethe Universität-Frankfurt am Main (Campus Riedberg), Max-von-Laue-Straße 15, Frankfurt am Main-Germany.
PLoS One. 2017 Aug 11;12(8):e0182564. doi: 10.1371/journal.pone.0182564. eCollection 2017.
The red flour beetle Tribolium castaneum has become the second most important insect model organism and is frequently used in developmental biology, genetics and pest-associated research. Consequently, the methodological arsenal increases continuously, but many routinely applied techniques for Drosophila melanogaster and other insect species are still unavailable. For example, a protocol for non-lethal genotyping has not yet been adapted but is particularly useful when individuals with known genotypes are required for downstream experiments. In this study, we present a workflow for non-lethal genotyping of T. castaneum adults based on extracting genomic DNA from wing tissue. In detail, we describe a convenient procedure for wing dissection and a custom method for wing digestion that allows PCR-based genotyping of up to fifty adults in less than an afternoon with a success rate of about 86%. The amount of template is sufficient for up to ten reactions while viability and fertility of the beetles are preserved. We prove the applicability of our protocol by genotyping the white / scarlet gene pair alleles from the black-eyed San Bernadino wild-type and white-eyed Pearl recessive mutant strains spanning four generations. Non-lethal genotyping has the potential to improve and accelerate many workflows: Firstly, during the establishment process of homozygous cultures or during stock keeping of cultures that carry recessively lethal alleles, laborious test crossing is replaced by non-lethal genotyping. Secondly, in genome engineering assays, non-lethal genotyping allows the identification of appropriate founders before they are crossed against wild-types, narrowing the efforts down to only the relevant individuals. Thirdly, non-lethal genotyping simplifies experimental strategies, in which genotype and behavior should be correlated, since the genetic configuration of potential individuals can be determined before the actual behavior assays is performed.
赤拟谷盗已成为第二重要的昆虫模式生物，常用于发育生物学、遗传学以及与害虫相关的研究。因此，相关的方法库在不断扩充，但许多常用于黑腹果蝇和其他昆虫物种的常规技术仍然无法应用于赤拟谷盗。例如，一种非致死基因分型方案尚未被适配，而当下游实验需要已知基因型的个体时，该方案会特别有用。在本研究中，我们提出了一种基于从翅组织中提取基因组DNA对赤拟谷盗成虫进行非致死基因分型的工作流程。详细地说，我们描述了一种便捷的翅解剖程序以及一种定制的翅消化方法，该方法能够在不到一个下午的时间内对多达五十只成虫进行基于PCR的基因分型，成功率约为86%。模板量足以进行多达十次反应，同时甲虫的活力和繁殖力得以保留。我们通过对跨越四代的黑眼圣贝纳迪诺野生型和白眼珍珠隐性突变株的白色/猩红色基因对等位基因进行基因分型，证明了我们方案的适用性。非致死基因分型有潜力改进和加速许多工作流程：首先，在纯合培养物的建立过程中或在携带隐性致死等位基因的培养物保种期间，费力的测交被非致死基因分型所取代。其次，在基因组工程分析中，非致死基因分型允许在将合适的奠基者与野生型杂交之前对其进行鉴定，从而将工作范围缩小到仅相关个体。第三，非致死基因分型简化了实验策略，在这些策略中基因型和行为应相互关联，因为可以在实际行为测定之前确定潜在个体的遗传构型。
