Characterizing the chromosomes of the platypus (Ornithorhynchus anatinus) Daniel McMillan 1L , Pat Miethke 1L , Amber E. Alsop 1L , Willem Rens 2 , Patricia O_Brien 2 , Vladimir Trifonov 2 , Frederic Veyrunes 1,2 , Kyriena Schatzkamer 3 , Colin L. Kremitzki 3 , Tina Graves 3 , Wesley Warren 3 , Frank Gru ¨tzner 4 , Malcolm A. Ferguson-Smith 2 & Jennifer A. Marshall Graves 1 * 1 Comparative Genomics Group, Research School of Biological Sciences, The Australian National University, Canberra, ACT 2601, Australia; Tel: 612 61252492; E-mail: jenny.graves@anu.edu.au; 2 Molecular Cytogenetics Laboratory, Centre for Veterinary Science, Department of Veterinary Medicine, University of Cambridge, Cambridge CB3 0ES, UK; 3 Genome Sequencing Center, Washington University School of Medicine, St. Louis, MO 63108, USA; 4 School of Molecular and Biomedical Sciences, University of Adelaide, South Australia, Australia L Daniel McMillan, Pat Miethke and Amber E. Alsop contributed equally to this work. * Correspondence Received 18 September 2007. Received in revised form and accepted for publication by Wendy Bickmore 20 October 2007 Key words: chromosome painting, karyology, Ornithorhynchus anatinus, platypus, sex chromosomes Abstract Like the unique platypus itself, the platypus genome is extraordinary because of its complex sex chromosome system, and is controversial because of difficulties in identification of small autosomes and sex chromosomes. A 6-fold shotgun sequence of the platypus genome is now available and is being assembled with the help of physical mapping. It is therefore essential to characterize the chromosomes and resolve the ambiguities and inconsistencies in identifying autosomes and sex chromosomes. We have used chromosome paints and DAPI banding to identify and classify pairs of autosomes and sex chromosomes. We have established an agreed nomenclature and identified anchor BAC clones for each chromosome that will ensure unambiguous gene localizations. Introduction The platypus (Ornithorhynchus anatinus) was first considered to be a hoax because of its combination of mammalian and reptilian features. Although it bears fur and feeds its young with milk, it shares some skeletal features with reptiles, and lays eggs like reptiles (Griffiths 1978). Platypus and its only close relatives (four species of echidna) are now classified as the Mammalian Subclass Prototheria (single order Monotremata), which diverged from Theria, which then diverged into marsupial and eutherian (Fpla- cental_) mammals (Figure 1). Divergence dates have been long debated; for consistency with the platypus sequence paper (Warren et al. 2008), we have used divergence dates of 166 million years (MYa) and 148 MYa respectively that were recently proposed (Bininda-Emonds et al. 2007). As a basal mammal and outgroup for all other mammals, the platypus has long been of unique value for comparisons of mammalian anatomy, biochemistry, physiology and genetics. Because of its particular value for comparative genomics, a 6-fold shotgun sequence of the entire female platypus genome was recently completed and assembled (Warren et al. 2008). Assembly onto a chromosome scaffold requires an Chromosome Research (2007) 15:961–974 # Springer 2007 DOI: 10.1007/s10577-007-1186-2