RADseq of Etheostoma spectabile cross used in generating a linkage map. Samples include a male and female pair and their 145 offsping from a single clutch.
We used a single male and female pair of orangethroat darters to create an F1 mapping cross. Both orangethroat darter parents were collected via kick seine from the Salt Fork of the Vermillion River Drainage (Champaign County, Illinois) in May 2016. The male parent used in this cross was the same individual whose DNA was used to sequence all genomic libraries (i.e. Illumina shotgun, Illumina mate-pair, and PacBio) used in the orangethroat genome assebly UIUC_Espe_1.0; PRJNA556880 : Etheostoma spectabile genome sequencing; SAMN12373147 : Espe_m_wgs). We isolated DNA from the white muscle tissue of both parents and the entire body of each fry. DNA was extracted using the Puregene (Qiagen; www.qiagen.com) protocol and treated with RNase A. Samples were checked for purity on a Nanodrop 1000 machine and quantified on a Qubit fluorometer. Restriction site associated DNA sequencing (RADseq) libraries were constructed at Floragenex (Portland, OR) following Baird et al. (2008). The restriction enzyme SbfI was used to digest 750 ng of DNA from each of the progeny. To ensure that diploid genotypes were accurately called at each RAD locus for the two parents, 1.5 ug of DNA was included for both parents in the RADseq libraries. Libraries were sequenced as 1x100 bp reads on two lanes on an Illumina HiSeq4000 machine at the University of Oregon (Eugene, OR).