Qiaamp tissue extraction kit

Maternity was determined by observation of suckling since 1971, but fathers were unknown until DNA analyses began in 1988. Hair, blood, or ear tissue was collected from all sheep at first capture from 1988 to 1993 and from 1997 to 2018. About 20–30 hairs including follicles and around 5 mg of ear tissue were used to extract DNA with the QIAamp tissue extraction kit (Qiagen Inc., Mississauga, Ontario). DNA was extracted from blood using a standard phenol–chloroform method. The genotyping protocol is detailed in Coltman et al. (2003) and Poissant et al. (2013). Paternity was assigned using the likelihood‐based approach from Marshall et al. (1998), and the software CERVUS was used to estimate the critical difference in log‐likelihood score for paternity assignment under a statistical confidence of 95% (Coltman et al., 2002). In 1988–2018, 770 lambs were seen, 721 were sampled for DNA and 380 were assigned to a known father. Only lambs that survived to be captured and genotyped could be assigned a father. Lambs that died before they were sampled, or were sired by immigrant males of unknown identity, were not included in subsequent analyses.

Genomic DNA was extracted individually from the half-part of each tick and from the I. hookeri specimens. In order to minimize PCR inhibitors from tick samples, we followed the extraction protocol described by Halos et al., 2004 [31 (link)]. Briefly, a bead-based physical disruption of the tick body within the Tissue-Lyser apparatus (Qiagen, Hilden, Germany), and 24 h of enzymatic digestion at 56 °C using buffer G2 supplemented with 25% of proteinase K were performed prior DNA extraction. Meanwhile, specimen I. hookeri subjected to enzymatic lysis with proteinase K followed by incubation at 56 °C overnight prior to DNA extraction. The extraction was performed using the QIAamp Tissue Extraction Kit (Qiagen, Courtaboeuf, France), in the QiagenEZ1 automated system, following the manufacturer’s instructions. DNA was eluted in 100 μL and stored at −20 °C until further use.