Genescan 500 250

We selected 13 unlinked microsatellite markers previously characterized in domestic cats (Menotti-Raymond et al. 1999 (link)). Forward primers for all loci were fluorescently labeled and PCRs optimized into three multiplexes (see Table S2 for details). Briefly, each PCR contained about 20 ng of genomic DNA, 0.2 U Taq DNA polymerase (Kapa Biosystems, supplied by Lasec, Cape Town, South Africa), 1 X PCR reaction buffer, 0.5 mm MgCl₂, primers at specific concentrations (Table S2), with the final reaction volume adjusted to 10 μL with distilled water. All multiplex reactions were amplified using the following thermal cycle: an initial denaturation at 95°C for 3 min, followed by 30 cycles of initial denaturation at 95°C for 15 s, annealing at 60°C for 30 s, and elongation at 72°C for 30 s. A final extension was carried out at 72°C for 15 min. Successful amplification was verified using agarose gel electrophoresis. Purified PCR fragments were separated on an ABI Prism 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA), using GENESCAN™-500 (-250) as an internal size standard (Appl-ied Biosystems). Allele sizes were visualized and scored using GENEMARKER v1.95 (SoftGenetics LLC, State College, PA).

Multiplex ligation-dependent probe amplification (MLPA) was performed using the MRC Holland (Amsterdam, The Netherlands) SALSA MLPA probe mix P383-A1 TALL with three additional probes for the MAP3K7 gene according to the manufacturer’s instructions. The MAP3K7 probe sequences were:

Exon 1: 5´ GGGTTCCCTAAGGGTTGGACATGTCTACAGCCTCTGCCGCCTCCTCCTCCTCCTCGTCTTC/ GGCCGGTGAGATGATCGAAGCCCCTTCCCAGGTCCTCAACTCTAGATTGGATCTTGCTGGCAC 3´

Exon 5: 5´ GGGTTCCCTAAGGGTTGGACCACGCAATGAGTTGGTGTTTACAGTG/ TTCCCAAGGAGTGGCTTATCTTCACAGCATGCAACCCAAAGCGTCTAGATTGGATCTTGCTGGCAC 3´

Exon 7: 5´ GGGTTCCCTAAGGGTTGGACGTCTTCAGCTGGGGTATTATTCTTTGGGAAGTGATAACGCG/ TCGGAAACCCTTTGATGAGATTGGTGGCCCAGCTTCTAGATTGGATCTTGCTGGCAC 3´

Polymerase chain reaction (PCR) products were separated by capillary electrophoresis on an ABI-3130XL device. The size standard was GeneScan 500–250 (both Applied Biosystems).