The SNP locations in each gene, Genbank accession number and the positions (intron/exon etc) are listed in Table 3. In brief the genes were CAPN1 (2 SNP), CAST (1 SNP), leptin (3 SNP), GHR (1 SNP), and acylCoA:diacylg- lycerol acyltransferase 1 (DGAT1) (1 dinucleotide substi- tution). All animals with phenotypes were genotyped, as were all available sires. Multiplex PCR of loci for SNP genotyping A single PCR was used to generate 12 amplicons. Each 10 μL reaction volume contained 5 μL DNA, 100 μM of each dNTP, 1 × QIAGEN PCR buffer which contained 1.5 mM MgCl2, 1× primer mix, 1 U Hot Start Taq (QIAGEN, UK) and 0.1 μg/μL BSA. PCR conditions consisted of 15 min at 95°C followed by 32 cycles of 30 s as 94°C, 1 min at 67°C and 1 min at 72°C. Following amplification, 4 μL of a solution containing 4 U Exonuclease I (NEB, UK), 1.4 μL of 10× Antarctic Phosphatase buffer (NEB, UK) and 2 U Antarctic Phosphatase (NEB, UK) was added to the PCR product. Samples were then incubated for 60 min at 37°C followed by 15 min at 72°C. Single base extension SNaPshot® (Applied Biosystems, UK) reactions included 5 μL of Antarctic Phosphatase/Exonuclease I treated PCR product, 2 μL of SNaPshot Multiplex Ready Reaction Mix (Applied Biosystems, UK), 2 μL H2O and 1 μL probe mix. Thermocycling conditions consisted of 30 s at 94°C and 20 s at 67°C. Following single base extension, 4 μL of CIP solution containing 0.4 μL of 10× NEBuffer 3 (NEB, UK), and 2 U CIP (NEB, UK) was added to each sample. The samples were then incubated at 37°C for 60 min followed by 85°C for 20 min. Table 3: SNP name and location Gene CAPN11 CAST2 LEP3 GHR4 DGAT15 Electrophoresis and scoring Five μL of the SNaPshot/CIP product was added to 10 μL of Hi-Di™ formamide (Applied Biosystems, UK) and the samples were electrophoresed on a 3100 Genetic Analyzer (Applied Biosystems, UK). Genemapper® v4.0 was used to interpret the genetic profiles. Data analysis SNP frequencies and linkage disequilibrium Genotype frequencies of each polymorphism were tested for deviations from Hardy-Weinberg equilibrium by χ2 tests [22] (significance based on P < 0.05). Pairwise geno- type combinations of the SNP were also tested for linkage disequilibrium (LD), the degree of non-random associa- tion of alleles at two or more loci, using the Haploview program, version 4 [23]. The Haploview program uses a two-marker EM to estimate the maximum-likelihood val- ues of the four gamete frequencies as well as D' and r 2 val- ues and LOD scores. Mixed model association analysis The relationship between the different genotypes of each SNP and the various traits recorded was evaluated using a single-marker mixed-model association analysis. Data were analyzed by fitting a linear mixed model using the restricted maximum likelihood method (REML) provided in Genstat, release 10 [24]. The statistical model included fixed effects of farm, genotype, sex and the genotype-sex interaction, and random effects of sire, slaughter date (panel date for the taste panel traits), interaction of sire and slaughter date (panel date for the taste panel traits) and interactions of sire and slaughter date (panel date for the taste panel traits) with the genotype/sex groups. These latter interactions took into account the possibility of gen- otype/sex effects varying with sire or slaughter date (panel date for the taste panel traits) or both. An additional term Genetics Selection Evolution 2009, 41:36 http://www.gsejournal.org/content/41/1/36 1 Calpain was mapped by Smith et al., (2000) [32], CAPN316 SNP reported by Page et al., (2002) [35] and CAPN4751 SNP reported by White et al., (2005) [13] 2 Calpastatin was mapped by Bishop et al., (1993) [36] and the SNP was reported by Schenkel et al., (2006) [11] 3 Leptin was mapped by Stone et al., (1996) [37], UASMS1 and UASMS2 SNP reported by Nkrumah et al., (2005) [9], Exon2FB SNP reported by Buchanan et al., (2002) [5] 4 Growth hormone receptor was mapped by Moody et al., (1995) [43], SNP reported by Blott et al., (2003) [42] 5 acylCoA:diacylglycerol acyltransferase 1 was mapped by Cases et al., (1998) [39], dinucleotide substitution reported by Grisart et al., (2002) [40] Page 4 of 12 (page number not for citation purposes) BTA SNP name Location GenBank Accession number and base position 29 7 4 20 14 CAPN316 CAPN 4751 UoGCAST UASMS1 UASMS2 Exon2FB F279Y K232A Exon 9 Intron 17 between exon 17 and 18 Intron 5 between exon 5 and 6 Leptin promoter region Leptin promoter region Exon 2 Exon 8 Exon 8 AF252504-5709 AF248054-6545 AY008267-282 AB070368-207 AB070368-528 AY138588-305 AY748827-914 AY065621-6829 AY065621-6830 SNP G/C C/T G/C C/T C/T C/T A/T A/G A/C