RADseq of Etheostoma caeruleum cross used in generating a linkage map. Samples include a male and female pair and their 85 offsping from a single clutch.
We created an F1 mapping cross with a single male and female rainbow darter pair. Both parents were collected via kick seine from the Salt Fork of the Vermillion River Drainage (Champaign County, Illinois) in April 2018. The pair were allowed to spawn in the laboratory and eggs were collected. A total of 85 fry survived to two months post-hatching. At this time, the parents and fry were euthanized and DNA was extracted using the Puregene protocol (Qiagen; www.qiagen.com). A RAD library was constructed using 1000 ng of DNA from each of the parents and offspring. We used the restriction enzyme SbfI for RAD library construction at the University of Illinois at Urbana-Champaign, following the methodology of Baird et al. (2008). We ensured a higher depth of coverage for the parents by including 2X the amount of DNA for both parents compared to the fry in the RADseq library. This library was sequenced as 1x100 bp reads on two rapid-run lanes on an Illumina HiSeq2500 machine at the Biotechnology Center at the University of Illinois at Urbana-Champaign.