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.
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