Journal of Heredity Supplementary Material Carlos Driscoll, Nobuyuki Yamaguchi, Steven J. O’Brien and David W. Macdonald A Suite of Genetic Markers Useful in Assessing Wildcat (Felis silvestris ssp.) - Domestic Cat (Felis silvestris catus) Admixture Materials and Methods Oligonucleotide primers for mitochondrial amplification (Supplementary Table 4) were designed using PRIMER 3 (ROZEN 2000) and are conserved across all cats and the domestic dog (hence ‘CD’). ND5 and ND6 are not known to be incorporated as nuclear transpositions (numts) in cats of the domestic cat lineage (KIM 2006; LOPEZ et al. 1996; LOPEZ et al. 1994). Initial PCR of the 2.6 kb ND5 and ND6 segment was done using amplification primers CD-F1C (Forward) and CD-R1C (Reverse). Genomic DNA (50 ng) underwent amplification by PCR, performed using 200 nM final concentration of each oligonucleotide primer in 1.5 mM MgCl2, with AmpliTaq-GOLD DNA Polymerase (Applied Biosystems Inc. [ABI]) under the following conditions: initial 95°C denaturation for 9:45 and 20 sec denaturation cycles of 94°C, then annealing for 30 sec at 60°C (3 cycles), 58°C (5 cycles), 56°C (5 cycles), 54°C (5 cycles), 52°C (5 cycles), and 50°C (22 cycles), or with annealing for 30 sec at 60°C (3 cycles), 58°C (4 cycles), 56°C (11 cycles), 54°C (27 cycles). Final extension was 2.5 min at 72°C. After enzyme-purification (HANKE and WINK 1994), overlapping sequences were generated from the PCR products using forward sequencing primers CD-F2B, CD-F3B, CD-F4B, CD-F5B and reverse sequencing primers CD-R2A, CD-R3B, CD-R4, CD-R5B (Supplementary Table 4) in addition to amplification primers, with ABI Big Dye Terminator kits following manufacturer’s instructions. Fragments were analyzed on an ABI 3730. Sequences were concatenated, compared and aligned using SEQUENCHER 4.5 (Gene Codes Corporation). Sequences were re-aligned by eye using CLUSTALX (JEANMOUGIN 1998) and duplicate haplotypes were collapsed using MACCLADE (MADDISON 2005). Microsatellite loci initially developed in the domestic cat (MENOTTI-RAYMOND et al. 1999) were selected on the basis of their map locations (MENOTTI-RAYMOND et al. 2003a; MENOTTI-RAYMOND et al. 2003b; MENOTTI-RAYMOND et al. 1999; MURPHY et al. 1999) to represent both linked and unlinked loci (See Supplemental Table 6 for primer sequences and chromosome locations). Markers were amplified and genotyped as described previously (DRISCOLL 2002; MENOTTI-RAYMOND et al. 1999) and products were resolved on an ABI 3700. Binning was done visually with the assistance of ALLELOGRAM 1.2 (MANASTER 2009). Supplementary Tables can be found and downloaded from: http://dx.doi.org/10.5061/dryad.n40h2 Supplementary Table 1 Genotyped individuals Supplementary Table 2 Mitochondrial sequence alignment showing variable sites Supplementary Table 3 Synopsis alignment of Felis silvestris phylogenetically informative mitochondrial sites Supplementary Table 4 Primer sequences for mitochondrial amplification of F. silvestris ssp. informative sites Supplementary Table 5 STR genotypes ascertained in range-wide survey. Header rows indicate cat chromosome location of STR loci with distances given according to conventions used in PHASE (STEPHENS 2004). Alleles are indicated by base-pair length. Missing data is denoted by -9. Individual cats are listed by LGD code in column one. Refer to Supplemental Table 1 for additional information regarding provenance and taxonomic assignment. Data is provided for 77 loci, however only the 36 that amplified most completely across different runs were further characterized and used in published analysis (DRISCOLL et al. 2007). A limited examination of alternative locus combinations (data not shown) that include loci other than the published 36 indicates similar levels of performance. For future studies requiring the greatest genetic resolution these additional data could be considered. Supplementary Table 6 Locus by locus tabulation of allele frequencies broken down by results of categorical genetic assignments. References DRISCOLL, C. A., M. MENOTTI-RAYMOND, A. L. ROCA, K. HUPE, W. E. JOHNSON et al., 2007 The Near Eastern origin of cat domestication. Science 317: 519-523. DRISCOLL, C. A., MENOTTI-RAYMOND, M., NELSON, G., GOLDSTEIN, D., O'BRIEN, S.J., 2002 Genomic Microsatellites as Evolutionary Chronometers: A test in Wild Cats. Genome Res 12: 414-423. HANKE, M., and M. WINK, 1994 Direct DNA sequencing of PCR-amplified vector inserts following enzymatic degradation of primer and dNTPs. Biotechniques 17: 858, 860. JEANMOUGIN, F., THOMPSON,J.D., GOUY,M., HIGGINS,D.G. AND GIBSON,T.J., 1998 Multiple sequence alignment with Clustal X. Trends Biochem Sci 23: 403-405. KIM, J.-H., ANTUNES, A., LUO, S., MENNINGER, J., NASH, W.G., O’BRIEN, S.J., AND JOHNSON, W.E., 2006 Evolutionary analysis of a large mtDNA translocation (numt) into the nuclear genome of the Panthera genus fields. Gene 366: 292-302. LOPEZ, J. V., S. CEVARIO and S. J. O'BRIEN, 1996 Complete nucleotide sequences of the domestic cat (Felis catus) mitochondrial genome and a transposed mtDNA tandem repeat (Numt) in the nuclear genome. Genomics 33: 229-246. LOPEZ, J. V., N. YUHKI, R. MASUDA, W. MODI and S. J. O'BRIEN, 1994 Numt, a recent transfer and tandem amplification of mitochondrial DNA to the nuclear genome of the domestic cat. J Mol Evol 39: 174-190. MADDISON, D. R., AND MADDISON, W. P., 2005 MacClade 4: Analysis of phylogeny and character evolution. Version 4.08. Sinauer, Sunderland, MA. MANASTER, C., 2009 Allelogram 2.0, pp., http://code.google.com/p/allelogram/. MENOTTI-RAYMOND, M., V. A. DAVID, R. AGARWALA, A. A. SCHAFFER, R. STEPHENS et al., 2003a Radiation hybrid mapping of 304 novel microsatellites in the domestic cat genome. Cytogenet Genome Res 102: 272-276. MENOTTI-RAYMOND, M., V. A. DAVID, Z. Q. CHEN, K. A. MENOTTI, S. SUN et al., 2003b Second-Generation Integrated Genetic Linkage/Radiation Hybrid Maps of the Domestic Cat (Felis catus). J Hered 94: 95-106. MENOTTI-RAYMOND, M., V. A. DAVID, L. A. LYONS, A. A. SCHAFFER, J. F. TOMLIN et al., 1999 A genetic linkage map of microsatellites in the domestic cat (Felis catus). Genomics 57: 9-23. MURPHY, W. J., M. MENOTTI-RAYMOND, L. A. LYONS, M. A. THOMPSON and S. J. O'BRIEN, 1999 Development of a feline whole genome radiation hybrid panel and comparative mapping of human chromosome 12 and 22 loci. Genomics 57: 1-8. ROZEN, S., AND SKALETSKY, H., 2000 Primer3 on the WWW for general users and for biologist programmers., pp. pp 365-386 in Krawetz S, Misener S (eds) Bioinformatics Methods and Protocols: Methods in Molecular Biology. . Humana Press, Totowa, NJ. STEPHENS, M., SMITH, N.J. AND DONNELLY, P., 2004 Documentation for PHASE, version 2.1, pp., Seattle.