M. longissimus thoracis et lumborum (LTL) and M. semimembranosus (SM) tissues and blood were collected from two animal resources (86 crossbred and 295 purebred pigs) in which meat quality phenotypic data was available for a number of traits as previously described [43, 45, 46, 49]. The cross-bred resource comprised of 31 Large White-sired, 23 Duroc-sired and 32 Pietrain-sired F1 female offspring with a common Large White × Landrace background. The purebred animals were females sampled from each of three closed populations (breeding lines) based on Large White (n = 98), Duroc (n = 99) and Pietrain (n = 98) and all reared in the same production system The purebred animals were reared in the same conditions and fed the same diet and were slaughtered at 140 days with a live weight of 109.56 ± 7.81 kg. . The Pietrain line was homozygous dominant, NN (“Normal”) for the RYR1 [45, 46].
PRKAG3 gene expression quantification
PRKAG3 gene expression was quantified in the LTL tissue samples which were collected from the crossbred F1 animal resource for transcriptomic analysis. All tissue was taken from approximately the same posterior location, in RNAse free conditions and preserved in RNALater® (Ambion Ltd., Cambridge, UK) within 10 min post-exsanguination, snap-frozen in dry ice, kept overnight at 4°C, and then stored at −20°C. RNA was extracted using the Qiagen RNeasy® Fibrous Tissue Mini Kit (Qiagen, Hilden, Germany Ltd, West Sussex, UK) according to the manufacturer’s instructions, together with a DNase treatment. RNA integrity was assessed using the Bioanalyser 2100 RNA nano chip (Agilent Technologies, Santa Clara, California, USA) and quantified with the NanoDrop 1000 Spectrophotometer (Thermo Scientific, Waltham, MA).
Reverse transcription was carried out using total RNA to generate a cDNA template for use with the QuantiTect SYBR Green PCR Kit (Qiagen, Hilden, Germany). For the reverse transcription 2.5 μg of total RNA, 1 μl oligo (dT)12-18 (500 μg/ml), 1 μl of a 10 mM dNTP mix were combined together in a final volume of 12 μl, heated at 65°C for 5 min and then placed immediately on ice. The contents were collected by a brief centrifugation before adding 4 μl 5X first strand buffer, 2 μl 0.1 M DTT, 1 μl SUPERase-In (Ambion, Foster City) and 1 μl of Superscript III RNase H reverse transcriptase (200 u/μl) (Invitrogen, Carlsbad, CA). The reverse transcription was carried out at 50°C for 1 h followed by an enzyme inactivation step of 70°C for 15 min. The cDNA was diluted to 10 ng/μl for use as a template for quantitative PCR (qPCR).
All qPCR was performed on the Mx3000P™ Real-Time PCR System (Stratagene, La Jolla, California, USA) using the QuantiTect™ SYBR Green Kit (Qiagen, Hilden, Germany). qPCR reactions were carried out using 2X QuantiTect SYBR Green PCR Master mix, 1 μl of 10 mM sense and anti-sense gene specific primers, (final conc. 0.5 mM), 3 μl dH2O and 5 μl of total cDNA template (10 ng/ml) in a total volume of 20 μl. The thermal profile was 95°C for 15 min, followed by 40 cycles of 94°C for 15 s, 60°C for 30 s and 72°C for 30 s.
PRKAG3 mRNA expression levels were determined for both splice variants using gene specific primers [Genbank:NM_2140777]:
Forward: 5' CTCCGACTCCAACACAGACCATCT 3',
Reverse: 5' TTCTGCAGCTCATCATCCCAGC 3'. PRKAG3 gene expression was normalised using the geometric mean of reference genes: Ribosomal protein L4 (RPL4): [Genbank: DQ845176]:
Forward: 5' AGAGATCCAAAGAGCCCTCCGC 3' and
Reverse 5' GCCTGGCGAAGAATGGTGTTTC 3' and TATA box binding protein (TBP): [Genbank: DQ845178]: Forward: 5' TTAATGGTGGTGTTGTGGACGGC 3', Reverse: 5'CCAAATAGCAGCACAGTACGAGCAA 3'. These reference genes were previously found to be stable for gene expression analysis in LTL muscle . Stability was re-confirmed on these samples using Genorm . PCR efficiencies (E) was calculated for each target PRKAG3 (99%), RPL4 (102%) and TBP (99.9%) and hence were suitable for comparison. The relative expression was calculated according to an established protocol [52, 53].
DNA preparation, promoter SNP discovery and SNP genotyping
Genomic DNA was extracted from LTL muscle tissue samples using DNeasy kit from Qiagen (Qiagen, Hilden, Germany) or from whole blood using the Wizard Genomic DNA Purification Kit (Promega, Madison, USA). DNA quantity and purity (A260/A280 ratio) for each sample was assessed using the NanoDrop™ 1000 Spectrometer (Thermo Scientific, Waltam, MA, USA).
The promoter region of the PRKAG3 gene (1322 bp long) was sequenced in the crossbred F1 population.
Primers were designed from draft sequence of a BAC clone (GenBank Ref. AY263454) which contained sequence flanking the PRKAG3 gene . Primers were designed using the web based application Primer 3 .
A 1322 bp fragment located in the promoter region was amplified using 20 pmol of primer: Forward: 5′ AGGGATGCTGCAGAAGAAGA '3 and Reverse: 5′ CACACAGAACCGCACAGACT '3, 20 ng genomic DNA using Qiagen PCR Master Mix (Qiagen, Hilden, Germany) in a 50 μl volume. The PCR conditions for the touchdown PCR reaction are as follows: 95°C for 2 min, and for 14 cycles; 95°C for 30 s, 62.3°C (decrease 0.5°C per cycle) for 30 s, 72°C for 2 min 20 s. Followed by 19 cycles; 95°C for 30 s, 53.3°C for 30 s and 72°C for 2 min, 20 s.
PCR products were analysed by agarose gel electrophoresis (2%) and ethidium bromide staining and visualised on MultiDoc Imaging System (UVP, Upland, CA, USA). Products were purified prior to sequencing using GenElute™ Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich Corp., St. Louis, MO, USA) and quantified on a NanoDrop™ 1000 Spectrometer (Thermo Scientific, Waltam, MA, USA). Sequencing of the purified PCR product was carried out in both directions by Eurofins MWG-Biotech (Ebersberg, Germany). Sequences were aligned and data analysed using MEGA® (Molecular Evolutionary Genetic Analysis) v 4.0 software . The I199V locus was genotyped using nested PCR followed by restriction digest with 10 U of Hga1 (Fermentas, Vilnius, Lithuania) at 37°C for 6 hrs .
Tagging SNP analysis
Based on patterns of linkage disequilibrium in crossbred animals, minor allele frequencies and the ability to characterise haplotype blocks, two SNPs (g.-995A>G, g. SNP -311A>G) in the promoter region and one in the coding region I199V were selected for association analysis in the three purebred populations. Genotyping of SNPs g.-311A>G and I199V was performed using the Sequenom iPLEX assay (Sequenom, Hamburg, Germany). SNP g.-995A>G was genotyped using a custom TaqMan assay, Assay ID: AHN1HPD (Applied Biosystems, Warrington, UK).
Meat quality phenotypic information for LTL and SM were available for cross-bred samples  and purebred samples  as previously described. pH and temperature at 45 min and 24 hr were measured in cross-bred samples as detailed in  and in pure-bred animals according to . For both sets of LTL samples, drip loss (driploss %) was determined after 3 days according to the method of Honikel (1998) and expressed as a percentage of the initial weight . Percentage cooking loss (cookloss %) was measured in cross-bred samples only and was determined by weighing transverse sections of the LTL before and after they were heated to a core temperature of 75°C in a circulating water bath held at 77°C. Electrical conductivity for both sets of samples was measured at a frequency of 1 KHz using a Pork Quality Meter (PQM, INTEK, Aichach, Germany) in accordance with manufacturer’s instructions. Bloomed CIE L* (lightness), a* (redness) and b* (yellowness) values were determined in the LTL at 7 days post mortem in the cross-bred animals as detailed in  and for pure-bred pigs at the last rib at 1 day post mortem using a Minolta C2002 Spectrophotometer (Minolta, Japan) as described in . For cross-bred animals, intramuscular fat (IMF%) concentrations were determined in thawed minced LTL samples using the Smart System 5 microwave moisture drying oven and NMR Smart Trac Rapid Fat Analyser (CEM Corporation USA) using AOAC Official Methods 985.14 & 985.26, 1990. In purebred samples, IMF% levels in LTL and SM were assessed using a Near Infrared Spectroscopy apparatus .
The primers used to amplify the promoter region were modified (modification highlighted in bold) at the 5' end to contain HIND III and BGL II restriction sites in the Forward:
5' CCTTAGATCTGGGATGCTGCAGAAGAAGAG 3’ and Reverse: 5' GGATAAGCTTAGGAGTGCGCAACACTGTATC 3’ primers, respectively.
The promoter region was amplified using 80 ng DNA, 45 μl Platinum High Fidelity Master Mix (Invitrogen Carlsbad, California, USA) and 20 ng primer in a final volume of 50 μl. The PCR conditions were as follows 95°C, 5 min, and for 30 cycles; 95°C for 1 min, 60°C for 1 min and 72°C for 2 min. This was followed by a single step of 72°C for 5 min. Samples were purified and quantified.
The PCR products for each haplotype (1 μg) and (4 μg) reporter vector pGL 4.17 (Promega, Corp., Madison, WI, USA) were then digested using HIND III (Promega, Corp., Madison, WI, USA) at 37°C for 3 hr and purified using GenElute PCR Purification Kit (Sigma-Aldrich Corp., St. Louis, MO, USA) and then digested with BGL II (Promega, Corp., Madison, WI, USA) at 37°C for 3 hr. 100 ng of vector and 70 ng of digested product were then ligated using T4 DNA Ligase (Promega, Corp., Madison, WI, USA) at 4°C.
Chemically competent E. Coli XL1 Blue host cells were transformed with the ligated product 5 μl and selected on LB medium containing ampicillin (100 μg/ml). The genotypes of the positive clones were verified with DNA sequencing (Eurofins MWG-Biotech, Ebersberg, Germany).
Cell culture and transient transfection assay
Mouse 3T3-L1 pre-adipocytes were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in a dulbecco modified eagle’s medium (DMEM, Gibco, Invitrogen Corp., San Diego, CA, USA) containing 10% fetal calf serum (Gibco, Invitrogen Corp., San Diego, CA, USA) and 1% penicillin-streptomycin (Sigma-Aldrich Corp., St. Louis, MO, USA) in a 37°C humidified incubator with 5% CO2. Medium was replaced every alternate day.
The day prior to transfection, 3T3-L1 cells were cultured 3 ×104 per ml cells in DMEM containing 10% fetal calf serum in a 24 well cell culture plate (Greiner Bio-One, Gmbh, Germany) in a 37°C humidified incubator with 5% CO2. The transfection cocktail (for each well) contained 25 μl DMEM basal media, 0.8 μl FuGENE HD Transfection Reagent (Roche Diagnostics GmbH, Mannheim, Germany) and 200 ng of endotoxin free PRKAG3 promoter construct. Following incubation at room temperature for 15 min, this cocktail was introduced drop wise onto the cells. Cells were then grown on a DMEM containing 10% fetal calf serum for 24 hr.
After 24 hr the media was removed and the cells washed with 500 μl of sterile phosphate buffer saline. Lysis of the cells was performed by adding 200 μl of passive lysis buffer (Promega Corp., Madison, WI, USA) followed by incubation at 37°C in a shaking incubator for 30 min at 800 rpm. Luciferase activity was measured in 20 μl of cell lysate after adding 100 μl luciferase assay reagent in a luminometer GLOMAX™, (Promega Corp., Madison, WI, USA). All measurements were normalised to the total protein.
Identification of putative regulatory SNP’s in the PRKAG3 promoter region
To identify SNPs that putatively affect promoter elements, the upstream sequence containing the SNPs was screened for the presence of putative selective transcription factor binding sites in silico using five prediction tools including;TFSEARCH , TESS , MatInspector , AliBaba2  and MATCH .
Particular attention was paid to binding sites which changed on substitution to the minor allele as well as sites known to be of relevance to mammalian muscle with high similarity scores and matrix similarities.
Estimation of SNP frequencies and tests for departure from Hardy Weinberg equilibrium were carried out using the Excel Microsatellite Toolkit  at each locus for each breed. Linkage disequilibrium and haplotype blocks were identified using Haploview . Following haplotype inference the ELB algorithm was used to assign the most likely haplotype combination to each individual animal as implemented using Arlequin .
The relationship between normalised expression of PRKAG3 and meat quality traits was calculated using Pearson correlation in PASW Statistics 18.0 software (SPSS, Inc., Somers, NY, USA). Association analysis was carried between genotyped SNPs/haplotypes and values of meat and carcass quality traits in each breed using the least square means method of GLM (General Linear Model) procedure in SAS (version 9.1; SAS Institute, Cary, NC, USA). Slaughter date was included in the model as a covariate. Each analysis tested for a difference in least square means of meat and carcass quality traits where 0, 1, or 2 copies of each haplotype were present.
The significance of the in vitro reporter assay across the three haplotypes and the association between genotypes and PRKAG3 expression were established using One-Way ANOVA, in PASW Statistics 18.0 software (SPSS, Inc., Somers, NY, USA).