Figure 3.

Physical mapping of BAC and cDNA clones containing rspo1 gene in female Pogona vitticeps. a: merged image showing locations of BAC clones Pv03_L07 (green) and Pv100_O13 (orange) containing rspo1; highly amplified signals from the sex microchromosome BAC clone Pv03_L07 was observed on the W microchromosome, while clear signals from both rspo1 BAC clones were observed on a different pair of microchromosomes. Note that a yellow signal (overlap of orange and green fluorochromes) is observed on W chromosome, suggesting hybridization of shared repetitive sequences from both BAC clones containing sex chromosome sequences and rspo1 gene sequences. BAC clone Pv03_L07 hybridised onto P. vitticeps sex chromosomes and telomeric region of long arm of chromosome 2; b: merged image showing locations of BAC clones Pv03_L07 (green) and Pv215_D15 (orange) containing rspo1, which had the same pattern as the BAC clone Pv100_O13 (Figure  3a); c: merged image of two rspo1 BAC clones Pv215_D15 (green) and Pv100_O13 (orange), showing co-locations of both BAC clones on the same pair autosomal microchromosomes. Note the absence of hybridisation signal on the W chromosome (3a,b) which was suppressed by species specific c0t-1 DNA d: hybridization signal of rspo1cDNA clone in female metaphase chromosome spreads; e: two colour FISH on the same slide as panel d, showing mapping of P. vitticeps’ sex chromosome BAC clone Pv03_L07 (green) with BAC clone Pv100_O13 (orange) containing rspo1. Hybridisation signals of sex chromosome BAC clone and the rspo1 cDNA clone are not on the W chromosomes, suggesting that rspo1 is autosomal in P. vitticeps. The hybridization signal on the W chromosome from BAC clone FISH is a result of hybridization of shared repeat sequences contained in those BAC clones. G = green; O = Orange; r = rspo1; W = W chromosome; Z = Z chromosome. Scale bars represent 10 μm.