adaptor sequences for 12 cycles using Platinium PCR SuperMix High Fidelity (Invitrogen) standard PCR conditions. PCR products were then enriched using a custom SureSelect 120 bp probe set in solution (Agilent). Probes were designed using eArray web-based software (Agilent) to cover a 1.74 Mb region from 55,853,187 to 57,599,455 bp which includes the homozy- gosity region of BTA 17 and three additional segments of the bovine genome with a total size of 2.46 Mb as control. Repetitive elements were excluded from the probe design. A total of 56,923 probes were created for the target regions. For hybridization, we followed the manufacturer’s directions with the following modi- fications. The library was hybridized at 65uC for 24 h, and then purified using streptavidin M280 beads (Dynal, Invitrogen). Eluted DNA was amplified using adaptor-specific primers for 12 cycles as before hybridization. Enriched products were analyzed using an Agilent Bioanalyzer to determine their size and concentration. High Throughput Sequencing, Read Mapping and Variant Calling The paired-end library was sequenced on each end for 50 and 35 cycles, respectively, on one quarter of a slide on a SOLiD4 sequencer (Life Technologies). We collected 383 million reads (191.5 million pairs) and aligned them to the bovine genome (UMD3.1) using BioScope (Life Technologies) with default settings. Alignment data were stored in BAM format and visualized using IGV software [20]. Duplicate pairs were removed, and only reads with reliable alignment quality .20 were used for the variant (SNP and small indel) detection. Sequence variants were detected using BioScope software. High-confidence variants were supported by 8 or more high-quality aligned reads on both strands. Lower confidence variants for low coverage regions were required to be supported by at least 4 reads on each strand. Sanger Re-sequencing Two variants were genotyped by re-sequencing of targeted PCR products using Sanger sequencing technology. PCR products were amplified using AmpliTaqGold360Mastermix (Life Technologies). PCR products were directly sequenced on an ABI 3730 capillary sequencer (Life Technologies) after treatment with exonuclease I and shrimp alkaline phosphatase. Sequence data were analyzed with Sequencher 4.9 (GeneCodes). References 1. Testoni S, Militerno G, Rossi M, Gentile A (2009) Congenital facial deformities, ascites and hepatic fibrosis in Romagnola calves. Vet Rec 164: 693–694. 2. Charlier C, Coppieters W, Rollin F, Desmecht D, Agerholm JS, et al. (2008) Highly effective SNP-based association mapping and management of recessive defects in livestock. Nat Genet 40: 449–454. 3. Thompson PN, Heesterbeek JA, van Arendonk JA (2006) Changes in disease gene frequency over time with differential genotypic fitness and various control strategies. J Anim Sci 84: 2629–2635. 4. The Bovine Genome Sequencing and Analysis Consortium, Elsik CG, Tellam RL, Worley KC (2009) The genome sequence of taurine cattle: A window to ruminant biology and evolution. Science 324: 522–528. 5. Zimin AV, Delcher AL, Florea L, Kelley DR, Schatz MC, et al. (2009) A whole- genome assembly of the domestic cow, Bos taurus. Genome Biol 10: R42. 6. Dro¨gemu¨ ller C, Tetens J, Sigurdsson S, Gentile A, Testoni S, et al. (2010) Identification of the bovine arachnomelia mutation by massively parallel sequencing implicates sulfite oxidase (SUOX) in bone development. PLoS Genet 6: e1001079. 7. Dro¨gemu¨ ller C, Reichart U, Seuberlich T, Oevermann A, Baumgartner M, et al. (2011) An unusual splice defect in the bovine mitofusin 2 gene (MFN2) is associated with degenerative axonopathy in Tyrolean Grey cattle. PLoS ONE 6: e18931. 8. Homepage Bos taurus UMD3.1 assembly (2012). Available: http://www.cbcb. umd.edu/research/bos_taurus_assembly.shtml#12.Accessed 2012 March 22. 9. He J, Kallin EM, Tsukada Y, Zhang Y (2008) The H3K36 demethylase Jhdm1b/Kdm2b regulates cell proliferation and senescence through p15(Ink4b). Nat Struct Mol Biol 15: 1169–1175. KDM2B Mutation in Cattle Supporting Information Figure S1 Pedigree tree of 46 paunch calf syndrome cases with available family records. Filled symbols represent paunch calf syndrome affected calves, open symbols represent normal cattle. DNA samples were available only for the numbered individuals. Animals genotyped as carriers of the KDM2B c.2503G.A mutation are shown with half-filled symbols. The mutation probably occurred in sire A and was spread into the population by his son B, grandson C, and great-grandson D. Most of the mothers of affected calves are related to these bulls. (EPS) Figure S2 KDM2B mutation. (A) Visualization in the IGV browser: Individual reads overlapping with the mutation are displayed. Seventy of eighty-one reads show the homozygous mutation at genomic position BTA 17:5690109031. (B) Sanger sequencing electropherograms of a wildtype control animal, a heterozygous carrier and a PCS affected calf are shown. The protein translation of the wild-type and mutant sequence is shown above the electropherograms. (PDF) Table S1 Sequence variants in reference cattle genome sequence. (XLSX) Acknowledgments comparison to the We would like to thank the Functional Genomics Center Z u¨rich and the Next Generation Sequencing Platform of the University of Bern for their technical support. The authors would also like to thank all Romagnola cattle breeders and the ANABIC for donating samples and sharing pedigree data. Author Contributions Conceived and designed the experiments: ST AG CD. Performed the experiments: ST EB MR AP RB PL AG CD. Analyzed the data: EB RB JT CD. Contributed reagents/materials/analysis tools: ST AP RB PL JT AG CD. Wrote the paper: CD. 10. Tsukada Y, Fang J, Erdjument-Bromage H, Warren ME, Borchers CH, et al. 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