Molecular Ecology (2008) 17, 964–980 doi: 10.1111/j.1365-294X.2007.03551.x © 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd Blackwell Publishing Ltd Genomics of natural bird populations: a gene-based set of reference markers evenly spread across the avian genome NICLAS BACKSTRÖM, SOFIE FAGERBERG and HANS ELLEGREN Department of Evolutionary Biology, Evolutionary Biology Centre, Uppsala University, Norbyvägen 18D, SE-752 36 Uppsala, Sweden Abstract Although there is growing interest to take genomics into the complex realms of natural populations, there is a general shortage of genomic resources and tools available for wild species. This applies not at least to birds, for which genomic approaches should be helpful to questions such as adaptation, speciation and population genetics. In this study, we describe a genome-wide reference set of conserved avian gene markers, broadly applicable across birds. By aligning protein-coding sequences from the recently assembled chicken genome with orthologous sequences in zebra finch, we identified particularly conserved exonic regions flanking introns of suitable size for subsequent amplification and sequencing. Primers were designed for 242 gene markers evenly distributed across the chicken genome, with a mean inter-marker interval of 4.2 Mb. Between 78% and 93% of the markers amplified a specific product in five species tested (chicken, peregrine falcon, collared flycatcher, great reed warbler and blue tit). Two hundred markers were sequenced in collared flycatcher, yielding a total of 122.41 kb of genomic DNA sequence (12096 bp coding sequence and 110 314 bp noncoding). Intron size of collared flycatcher and chicken was highly correlated, as was GC content. A polymorphism screening using these markers in a panel of 10 unrelated collared flycatchers identified 871 single nucleotide polymorphisms (π = 0.0029) and 33 indels (mainly very short). Avian genome characteristics such as uniform genome size and low rate of syntenic rearrangements suggest that this marker set will find broad utility as a genome-wide reference resource for molecular ecological and population genomic analysis of birds. We envision that it will be particularly useful for obtaining large-scale orthologous targets in different species — important in, for instance, phylogenetics — and for large-scale identification of evenly distributed single nucleotide polymorphisms needed in linkage mapping or in studies of gene flow and hybridization. Keywords: chicken, collared flycatcher, comparative anchored tagged sequences, linkage mapping, QTL analysis, single nucleotide polymorphism Received 22 May 2007; revision accepted 15 August 2007 Introduction There is a growing interest in exploring the possibility to merge knowledge, hypotheses and technologies from the fields of genomics and evolutionary biology (Feder & Mitchell-Olds 2003). Impetus for this would be, for instance, the long-term goals to understand and trace the genetic background to phenotypic traits under selection in natural populations and, subsequently, to study adaptation using the combined information from genotypes and phenotypes. Other motivations could be to reveal the genetic basis of reproductive isolation and to scale up analyses of popula- tion processes using population genomics approaches. However, although there is rapid progress in the generation and establishment of genomic resources in model species, we still lack resources and tools for the vast majority of wild species, and this is so even for initial and small-scale attempts to access the genome. Before the era of eukaryotic whole-genome sequences, O’Brien et al. (1993) and Lyons et al. (1997) introduced the concept of ‘comparative anchor tagged sequences’ (CATS). CATS are anchored reference loci with known location in a model genome that, based on evolutionary conservation, Correspondence: Hans Ellegren, Fax: +46-18-4716310; E-mail: hans.ellegren@ebc.uu.se