Chromosome-Linked Gene Mapping in Pogona

We mapped Pogona vitticeps derived 8 BAC clones containing 8 chromosome-linked genes (WAC; KLF6; APTX; CHD1; CTNNB1; TAX1BP1; OPRD1/RCC1 and NR5A1)^{12 (link),19 (link),52 ,53 (link)} to B. constrictor male and female metaphase chromosomes. The clones were selected from Pogona vitticeps genomic BAC library as previously described in Ezaz et al.^{12 (link),52} , Young et al.^{53 (link)} and Deakin et al.^{19 (link)}. All 8 BACs were anchored to P. vitticeps metaphase chromosomes as control (data not shown). BAC DNA was extracted using the Promega Wizard Plus SV Minipreps DNA Purification System following the manufacturer’s protocol, with volumes scaled up for 15 ml cultures. The BACs were labeled with SpectrumOrange-dUTP or SpectrumGreen-dUTP (Abbott, North Chicago, Ill., USA) and hybridized for 2 days. The slides were then washed twice in 0.4 × SSC, 0.3% IGEPAL (Sigma-Aldrich) at 55 °C for 5 min each and after air-dried, counterstained using DAPI (1.2 µg/ml) and mounted in an antifade solution (Vector, Burlingame, CA, USA).