Turtle Metaphase Chromosome Preparation

All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Iowa State University. Metaphase chromosome preparations were obtained from C. picta and T. scripta cell cultures established previously using our standard protocols [12 (link),21 (link),26 (link)]. Briefly, turtle tissues were digested with collagenase (GIBCO, Thermo Fisher Scientific, Waltham, MA, USA) and cultured using a medium composed of 1:1 RPMI 1640:Leibowitz media (GIBCO) supplemented with 15% fetal bovine serum (One Shot, GIBCO), 2 mM L-glutamine (GIBCO), and 1% antibiotic–antimycotic solution (GIBCO). Cell cultures were incubated at 30 °C without CO₂ supplementation, and 10 µg/mL colcemid (KaryoMAX^®, GIBCO) was added four hours prior to harvesting. Metaphase chromosomes were harvested after hypotonic exposure and fixed in 3:1 methanol:acetic acid. Cell suspensions were dropped onto glass slides and air-dried. The slides were digested with pepsin and fixed with formaldehyde. High quality metaphase chromosomes were imaged and used for FISH. Because the subspecies of the slider turtle individual used for cytogenetics was not confirmed, we will use T. scripta or TSC to refer to cytogenetic data related to this individual, and T. s. elegans or TSE to refer to data from the recently published genome assembly [13 (link)].