This is the accepted version of the article: Mármol-Sánchez, Emilio; Quintanilla Aguado, Raquel; Jordana i Vidal, Jordi; [et al.]. «An association analysis for 14 candidate genes mapping to meat quality quantitative trait loci in a Duroc pig population reveals that the ATP1A2 genotype is highly associated with muscle electric conductivity». Animal Genetics, Vol. 51, Issue 1 (February 2020), p. 95-100. DOI 10.1111/age.12864 This version is avaible at https://ddd.uab.cat/record/216804 under the terms of the license 1 RUNNING HEAD: Candidate gene analysis for pig meat quality 2 3 An association analysis for 14 candidate genes mapping to meat quality QTL in a 4 Duroc pig population reveals that the ATP1A2 genotype is highly associated with 5 muscle electric conductivity 6 7 Emilio Mármol-Sánchez1, Raquel Quintanilla2, Jordi Jordana3, Marcel Amills1,3 8 1 9 Department of Animal Genetics, Centre for Research in Agricultural Genomics (CRAG), 10 CSIC-IRTA-UAB-UB, Campus de la Universitat Autònoma de Barcelona, Bellaterra, 11 Spain. 2 12 Animal Breeding and Genetics Programme, Institute for Research and Technology in Food 13 and Agriculture (IRTA), Torre Marimon, Caldes de Montbui, Spain. 3 14 Departament de Ciència Animal i dels Aliments, Universitat Autònoma de Barcelona, 15 Bellaterra, Spain. 16 17 Address for correspondence: Marcel Amills, Department of Animal Genetics, Centre for 18 Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB, Campus de la 19 Universitat Autònoma de Barcelona, Bellaterra 08193, Spain. E-mail: 20 marcel.amills@uab.cat. Tel. +34 93 563 6600 21 22 23 24 1 25 Summary 26 27 In previous genome-wide association studies carried out in a Duroc commercial line 28 (Lipgen population), we detected on pig chromosomes 3, 4 and 14 several QTL for gluteus 29 medius muscle redness (GM a*), electric conductivity in the longissimus dorsi muscle (LD 30 CE) and vaccenic acid content in the LD muscle (LD C18:1 n-7), respectively. We have 31 genotyped, in the Lipgen population, 19 single nucleotide polymorphisms (SNP) mapping 32 to 14 genes located within these three QTL. Subsequently, association analyses have been 33 performed. After correction for multiple testing, two SNPs in the TGFBRAP1 34 (rs321173745) and SELENOI (rs330820437) genes were associated with GM a*, while 35 ACADSB (rs81449951) and GPR26 (rs343087568) genotypes displayed significant 36 associations with LD vaccenic content. Moreover, the polymorphism of the ATP1A2 37 (rs344748241), ATP8B2 (rs81382410) and CREB3L4 (rs321278469 and rs330133789) 38 genes showed significant associations with LD CE. We made a second round of association 39 analyses including the SNPs mentioned above as well as other SNPs located in the 40 chromosomes to which they map to. After performing a correction for multiple testing, the 41 only association that remained significant at the chromosome-wide level was that between 42 the ATP1A2 genotype and LD CE. From a functional point of view, this association is 43 meaningful because this locus encodes a subunit of the Na+/K+-ATPase responsible of 44 maintaining an electrochemical gradient across the plasma membrane. 45 46 Keywords: Pig, single nucleotide polymorphism, meat quality, Na+/K+-ATPase. 47 48 2 49 Meat quality traits are of paramount importance for the pig industry because they 50 determine, to a great extent, consumer acceptance and financial profit. Once pigs are 51 slaughtered, there is a decline of the pH of the skeletal muscle due to the production of 52 lactic acid through anaerobic glycolysis (Rosenvold & Andersen 2003). The rate of muscle 53 acidification has a strong effect on meat color and water-holding capacity. In this way, a 54 low ultimate pH (5.4-5.3) is associated with pale, soft and exudative (PSE) meat as well as 55 with an increased electrical conductivity (CE) and elevated drip and cooking losses (Lee et 56 al. 2000; Rosenvold & Andersen 2003). In contrast, a high ultimate pH (6.3 or higher) 57 results in dark, firm and dry (DFD) meat with a high water-holding capacity and a lower 58 CE (Lee et al. 2000; Kim et al. 2016). Adverse effects on meat quality are influenced by 59 both genetic and environmental factors. Recessive and dominant genotypes in the porcine 60 ryanodine receptor 1 (RYR1) and the protein kinase AMP-activated non-catalytic subunit γ3 61 (PRKAG3) genes, respectively, are strong predisposing factors to the occurrence of PSE 62 meats (Fujii et al. 1991; Milan et al. 2000). On the other hand, there are multiple factors 63 related with pig management and transportation (pre-slaughter stress), stunning method at 64 slaughter, carcass chilling and pelvic suspension of carcasses that influence pork quality 65 (Rosenvold & Andersen 2003). Another important parameter that determines meat quality 66 is intramuscular fat (IMF) composition. In this regard, it is well known that fatty acid 67 composition can have important consequences on the oxidative stability of meat during 68 processing and retail display as well as on fat firmness (Wood et al. 2008). 69 In previous genome-wide association studies, we identified several genomic regions 70 containing quantitative trait loci (QTL) for meat Minolta a* value (redness), CE (González- 71 Prendes et al. 2017) and IMF composition (González-Prendes et al. 2019) traits measured 72 in the longissimus dorsi (LD) and gluteus medius (GM) muscle samples of 350 Duroc 3 73 barrows (Lipgen population). Details about the rearing of the pigs can be found in Gallardo 74 et al. (2009), while a thorough description of QTL mapping methods is reported in 75 González-Prendes et al. (2017). The measurement of CE was done 24 hours after slaughter 76 by using a Pork Quality Meter (Intek GmbH), while Minolta a* value was determined with 77 a Minolta Chroma-Meter CR-200 (Konica Minolta) equipment at the same time point. 78 Muscle fatty acid composition was measured as previously described by Quintanilla et al. 79 (2011). In the current work, we have selected 14 candidate genes located within QTL 80 regions for GM a* on SSC3, LD CE on SSC4, and LD vaccenic content on SSC14 (Table 81 1). These genes were: phosphorylase kinase catalytic subunit γ 1 (PHKG1), transforming 82 growth factor ß receptor associated protein 1 (TGFBRAP1), selenoprotein I (SELENOI), 83 hydroxyacil-CoA dehydrogenase trifunctional multienzyme (HADHA), coatomer protein 84 complex subunit α (COPA), proliferation and apoptosis adaptor protein 15 (PEA15), 85 calsequestrin 1 (CASQ1), ATPase Na+/K+ transporting α2 subunit (ATP1A2), ATPase 86 phospholipid transporting 8B2 (ATP8B2), cAMP responsive element binding protein 3 like 87 4 (CREB3L4), CREB regulated transcription coactivator 2 (CRTC2), acyl-CoA 88 dehydrogenase short/branched chain (ACADSB), G protein-coupled receptor 26 (GPR26) 89 and C-terminal binding protein 2 (CTBP2). 90 Genes were selected based on bibliographic information about their biological 91 functions which suggested that they could be involved in the determinism of any of the 92 three traits under study (GM a*, LD CE and LD C18:1 n-7). Based on available RNA-Seq 93 (Cardoso et al. 2017) and whole-genome data (our unpublished results), we called 19 SNPs 94 mapping to these 14 genes by using the GATK Best Practices workflow for SNP calling 95 (https://software.broadinstitute.org/gatk/best-practices/workflow?id=11145) in accordance 96 with protocols reported by Mármol-Sánchez et al. (2019). Nineteen SNPs were finally 4 97 selected because the SnpEff software predicted that they might have functional effects 98 (Cingolani et al. 2012), as reported in Supplementary Table 1. The 19 selected SNPs 99 (Table 1) were genotyped at the Servei Veterinari de Genètica Molecular of the Universitat 100 Autònoma de Barcelona (http://sct.uab.cat/svgm/en) by using a QuantStudio 12K Flex 101 Real-Time PCR System (ThermoFisher Scientific). Association analyses between SNPs 102 and phenotypes were performed with the Genome-wide Efficient Mixed-Model Association 103 (GEMMA) software (Zhou & Stephens 2012). The following statistical model was used: 104 105 y = Wα + xδ + u + ε 106 107 where y is the vector of phenotypic observations for every individual; α corresponds 108 to a vector including the intercept plus the fixed effects, i.e. batch effect with 4 categories 109 (all traits), and farm origin effect with 3 categories (all traits). The α vector also contains 110 the regression coefficients of the following covariates: (1) Carcass weight at slaughterhouse 111 for meat quality traits, and (2) IMF content in the LD muscle for LD fatty acid 112 composition; W is the incidence matrix relating phenotypes with the corresponding effects; 113 x is the vector of the genotypes corresponding to the set of selected polymorphisms; δ is the 114 allele substitution effect for each polymorphism; u is a vector of random individual effects −1 115 with a n-dimensional multivariate normal distribution MVNn (0, λ τ K), where τ−1 is the 116 variance of the residual errors, λ is the ratio between the two variance components and K is 117 a known relatedness matrix derived from the SNPs; and ε is the vector of residual errors. 118 Results were corrected for multiple testing by using the false discovery rate (FDR) method 119 reported by Benjamini & Hochberg (1995). The correction for multiple testing took into 120 account the number of candidate SNPs (2nd column of Table 1) mapping to each one of the 5 121 SSC3 GM a* (5 SNPs), SSC4 LD CE (11 SNPs) and SSC14 LD (C18:1) n-7 (3 SNPs) 122 QTL. 123 Performance of association analyses with the methodology described above 124 revealed the existence of several associations that remained significant even after correction 125 for multiple testing. We have found, for instance, an association between GM Minolta a* 126 value and missense mutations in the TGFBRAP1 and SELENOI genes, which map to two 127 different GM a* QTL on SSC3 (Table 1). The inactivation of the TGFBRAP1 gene results 128 in the suppression of aerobic glycolysis and increased levels of mitochondrial respiration 129 and fatty acid oxidation (Yoshida et al. 2013), while SELENOI encodes a selenoprotein 130 fundamental for the synthesis of phosphatidylethanolamine, a molecule with important 131 effects on the oxidation of lipid membranes, oxidative phosphorylation and mitochondrial 132 morphology (Tasseva et al. 2013; Poyton et al. 2016). We have also detected significant 133 associations between LD CE and SNPs in the ATP1A2, ATP8B2 and CREB3L4 genes, 134 which map to SSC4 LD CE QTL covering two regions spanning from 85.6 to 91 Mb and 135 from 95.2 to 97.8 Mb. These findings are very suggestive because the ATP1A2 gene, the 136 one showing the most significant association, is preferentially expressed in the skeletal and 137 heart muscle and brain and it encodes the α2 subunit of the ion pump Na+/K+ ATPase 138 (Clausen et al. 2017). Noteworthy, Na+/K+-ATPases provide the energy necessary for the 139 maintenance of Na+ and K+ electrochemical gradients across the plasma membrane by 140 hydrolyzing ATP (Clausen et al. 2017; Sampedro et al. 2018). These gradients are essential 141 for the preservation of the resting membrane potential as well as for the generation of 142 electrical impulses in the skeletal muscle and nervous system (Clausen et al. 2017; 143 Sampedro et al. 2018). The ATP8B2 protein is also an ATPase with flippase activity 6 144 towards phosphatidyl choline, a key component of phospholipid membranes with important 145 effects on the functioning of the sarcoendoplasmic reticulum Ca2+ATPase pumps (Shin & 146 Takatsu 2018; Fajardo et al. 2018), while CREB3L4 is a transmembrane bZip transcription 147 factor involved in the modulation of endoplasmic reticulum stress (Kim et al. 2014). Our 148 association analysis has also revealed the existence of significant associations between the 149 phenotypic variation of LD vaccenic (C18:1 n-7) content and SSC14 SNPs located in the 150 ACADSB gene, which catalyzes the oxidation of branched-chain fatty acids (Porta et al. 151 2019) and the GPR26 gene, whose inactivation leads to hyperphagia, glucose intolerance, 152 hyperinsulinemia, dyslipidemia and obesity in mice (Chen et al. 2012). 153 We have made a second round of association analyses in which the SNPs that 154 previously showed evidence of statistical significance were compared against the whole 155 sets of the Porcine SNP60 BeadChip SNPs co-localizing to the same chromosome 156 (chromosome-wide analysis) i.e. 3,123 SNPs on SSC3, 3,899 SNPs on SSC4 and 4,203 157 SNPs on SSC14. These 11,225 SNPs were obtained from previously published porcine 158 SNP60 BeadChip data reported by González-Prendes et al. (2017). In this case, the 159 correction for multiple testing took into account the number of SNPs mentioned above for 160 each one of the three chromosomes under analysis, i.e. 3,128, 3,910 and 4,206 independent 161 tests were taken into consideration when performing association analyses for pig 162 chromosomes SSC3, SSC4 and SSC14. Interestingly, the rs344748241 SNP in the ATP1A2 163 gene was the only one that surpassed the chromosome-wide threshold of significance (q- 164 value < 0.05) (Table 1, Figure 1). Noteworthy, this SNP was not significant when we 165 made an association analysis at the genome-wide level (data not shown). Additionally, we 166 used the LD function of gaston R package (v1.5.5; Perdry & Dandine-Roulland 2019) to 167 evaluate the presence of linkage disequilibrium among the SNP markers that showed 7 168 significant associations with LD CE after correction for multiple testing at the 169 chromosome-wide level (Supplementary Figure 1). The amount of linkage disequilibrium 170 was expressed as r2 in accordance with the definition of Hill & Robertson (1968). As 171 shown in Supplementary Figure 1, we observed a high degree of linkage disequilibrium 172 between the rs344748241 (ATP1A2 gene) and the rs80782100 (IGSF8 gene) markers. 173 Noteworthy, the rs80782100 SNP, which maps to an intronic position within the 174 immunoglobulin superfamily member 8 gene, displays the highest association with the LD 175 CE phenotype, as described in González-Prendes et al. (2017). 176 As previously discussed, we consider that the ATP1A2 gene is a strong positional 177 and functional candidate to explain the CE QTL found on SSC4 because Na+, K+ ATPases 178 are fundamental to induce an electrochemical gradient across the plasma membrane of cells 179 (Suhail 2010), and their kinetics are modulated by the extracellular pH (Salonikidis et al. 180 2000), a parameter which also displays strong effects on muscle electrical conductivity. In 181 pigs, the ATP1A2 gene has been sequenced (Henriksen et al. 2013) and its polymorphisms 182 have been associated with fat cut percentage (Fontanesi et al. 2012). A next step would be 183 to re-sequence the whole gene in Lipgen pigs with alternative genotypes (QQ vs qq) for the 184 LD CE QTL on SSC4, to build a complete catalogue of SNPs with potential effects on 185 protein activity and expression and to investigate their association with CE in the Lipgen 186 population. Subsequently, functional tests should be applied to ascertain whether any of the 187 mutations in the pig ATP1A2 gene with highly significant q-values also have causal effects 188 on muscle conductivity. 189 190 Acknowledgments 191 8 192 The authors are indebted to Selecció Batallé S.A. for providing the animal material 193 used in this study. We gratefully acknowledge J. Reixach and J. Soler (IRTA) for their 194 collaboration in the experimental protocols. This research was partially funded with grants 195 AGL2013-48742-C2-1-R and AGL2013-48742-C2-2-R awarded by the Spanish Ministry 196 of Economy and Competitiveness and grant 2014 SGR 1528 from the Agency for 197 Management of University and Research Grants of the Generalitat de Catalunya. Emilio 198 Mármol-Sánchez was funded with a FPU Ph.D. grant from the Spanish Ministry of 199 Education (FPU15/01733). We also acknowledge the support of the Spanish Ministry of 200 Economy and Competitiveness for the Center of Excellence Severo Ochoa 2016-2019 201 (SEV-2015-0533) grant awarded to the Center for Research in Agricultural Genomics. 202 Thanks also to the CERCA Programme of the Generalitat de Catalunya. 203 204 Data availability 205 206 These 11,225 SNPs included in this study were obtained from published Porcine SNP60 207 BeadChip data reported by González-Prendes et al. 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(2012) Genome-wide efficient mixed-model analysis for 328 association studies. Nature Genetics 44, 821-824. 14 329 Table 1. An association analysis between 19 SNPs mapping to 14 candidate genes and meat quality traits recorded in a Duroc pig 330 population (significant associations are shown in bold)1. Gene SNP Type Trait P-value q-value P-value* q-value* δ ± SE A1 MAF rs697732005 Splice region -0.02 PHKG1 0.88661 0.88661 0.68325 0.96577 A 0.3443 (3:16.830 Mb) variant (G/A) (0.142) rs321173745 Missense variant 0.549 TGFBRAP1 0.00361 0.00902 0.03108 0.67220 G 0.1875 (3:49.516 Mb) (A/G) (0.186) rs330820437 Missense variant 0.643 SELENOI GM a* 0.00039 0.00196 0.01307 0.51778 G 0.1757 (3:112.635 Mb) (A/G) (0.181) rs81215086 Missense variant -0.102 0.53993 0.67491 0.62966 0.96577 A 0.2899 (3:112.794 Mb) (G/A) (0.169) HADHA rs344578723 Missense variant -0.104 0.53466 0.67491 0.67980 0.96577 A 0.2866 (3:112.796 Mb) (G/A) (0.169) rs340853721 Splice region 0.014 0.90735 0.95684 0.79005 0.99942 T 0.4351 (4:90.163 Mb) variant (T/C) (0.091) COPA rs333099339 Splice region 0.017 0.87813 0.95684 0.88586 0.99942 T 0.4381 (4:90.183 Mb) variant (T/C) (0.090) rs80949931 Missense variant -0.002 0.95684 0.95684 0.68990 0.99942 A 0.4335 (4:90.186 Mb) (A/G) (0.091) rs329681990 LD CE -0.014 PEA15 Splice region 0.85666 0.95684 0.58021 0.99942 G 0.433 (4:90.266 Mb) variant (G/A) (0.091) rs334946278 Splice region 0.005 CASQ1 0.95267 0.95684 0.92240 0.99942 A 0.1304 (4:90.280 Mb) variant (G/A) (0.104) rs344748241 Splice region 6.515E- 7.167E- -0.325 ATP1A2 0.00006 0.02518 G 0.497 (4:90.356 Mb) variant (G/A) 06 05 (0.066) ATP8B2 rs81382410 Splice region 0.00285 0.01565 0.00256 0.21113 -0.233 T 0.3345 15 (4:95.435 Mb) variant (T/C) (0.077) rs329686514 Missense variant -0.155 0.08043 0.17695 0.22592 0.97957 T 0.3063 (4:95.717 Mb) (C/T) (0.088) rs321278469 Missense variant -0.228 CREB3L4 0.00639 0.01757 0.00554 0.30475 C 0.3084 (4:95.717 Mb) (C/A) 0.083) rs330133789 Missense variant 0.254 0.00493 0.01757 0.01769 0.57188 A 0.3373 (4:95.721 Mb) (G/A) (0.075) rs330198768 -0.083 CRTC2 Intron variant (C/T) 0.32931 0.60373 0.56310 0.99942 T 0.3687 (4:95.740 Mb) (0.085) rs81449951 Missense variant 0.093 ACADSB 0.04036 0.08073 0.0424837 0.8322423 A 0.2109 (14:132.588 Mb) (C/A) (0.045) rs343087568 Splice region LD (C18:1) -0.096 GPR26 0.00333 0.01334 0.1269422 0.9956111 G 0.4632 (14:133.182 Mb) variant (A/G) n-7 (0.032) rs339956077 Splice region 0.007 CTBP2 0.88166 0.88166 0.1269422 0.9956111 A 0.2094 (14:134.334 Mb) variant (G/A) (0.046) 1 331 The P-value and the q-value terms define the statistical significance of the association analysis before and after correcting for 332 multiple testing with a fase discovery rate approach, respectively. The correction for multiple testing took into account the number of 333 candidate SNPs (2nd column of Table 1) mapping to each one of the SSC3 GM a* (5 SNPs), SSC4 CE (11 SNPs) and SSC14 LD 334 (C18:1) n-7 (3 SNPs) QTL. The P-value* and the q-value* terms define the statistical significance of the chromosome-wide 335 association analysis before and after correcting for multiple testing with a false discovery rate approach, respectively. In this case, the 336 correction for multiple testing took into account the number of markers in the Porcine SNP60 BeadChip mapping to pig chromosomes 337 SSC3 (3,123 SNPs), SSC4 (3,899 SNPs) and SSC14 (4,203 SNPs). Other terms that need to be defined are: δ, estimated allele 338 substitution effect and its standard error (SE); A1, minor allele; MAF, minor allele frequency; GM a*, Minolta a* value (redness) in the 16 339 gluteus medius muscle; LD CE, electric conductivity in the longissimus dorsi muscle; and LD (C18:1) n-7, vaccenic acid content in the 340 longissimus dorsi muscle. 17 341 LEGENDS TO FIGURES 342 Figure 1: Manhattan plot depicting associations between electrical conductivity in the 343 longissimus dorsi muscle and the genotypes of markers in the ATP1A2 (rs344748241), 344 ATP8B2 (rs81382410) and CREB3L4 (rs321278469 and rs330133789) loci plus 3,899 345 additional SNPs mapping to pig chromosome 4 (SSC4). The positions of these three genes 346 are SSC4: 90.292-90.371 Mb (ATP1A2), SSC4: 95.426-95.446 Mb (ATP8B2) and SSC4: 347 95.714-95.723 Mb (CREB3L4). The green line represents the nominal P-value of 348 significance, while the blue line indicates the P-value of significance after correcting for 349 multiple testing with a false discovery rate approach (q-value). The rs344748241 SNP in 350 the ATP1A2 gene is located 23 kb away from the peak of the LD CE QTL, i.e. 351 ALGA0026686 (rs80782100; 4:90.378 Mb) SNP, as reported by González-Prendes et al. 352 (2017). 353 354 355 356 357 358 359 360 18 361 362 363 364 365 366 367 19 368 SUPPLEMENTARY DATA 369 Supplementary Table 1: Additional information about selected SNP and their potential 370 impact and deleteriousness (SIFT). 371 Supplementary Figure 1: Graph depicting the magnitude of linkage disequilibrium among 372 SNPs that showed significant associations with longissimus dorsi electric conductivity after 373 correction for multiple testing at the chromosome-wide level. Here, the amount of linkage 374 disequilibrium is expressed as r2 as defined by Will & Robertson (1968) and such 375 parameter was calculated with the LD function of gaston R package. 376 377 378 379 380 381 382 383 384 385 20 386 SNP_ID Allele Consequence Impact Symbol SIFT rs697732005 A splice_region_variant,intron_variant LOW PHKG1 - rs321173745 G missense_variant MODERATE TGFBRAP1 tolerated(0.22) rs330820437 G missense_variant MODERATE SELENOI tolerated(1) rs81215086 A missense_variant MODERATE HADHA deleterious(0.04) rs344578723 A missense_variant MODERATE HADHA tolerated(0.81) rs340853721 C splice_region_variant,intron_variant LOW COPA - rs333099339 C splice_region_variant,intron_variant LOW COPA - rs80949931 G missense_variant MODERATE COPA tolerated(0.15) rs329681990 A splice_region_variant,intron_variant LOW PEA15 - rs334946278 A splice_region_variant,synonymous_variant LOW CASQ1 - rs344748241 A splice_region_variant,synonymous_variant LOW ATP1A2 - rs81382410 G splice_region_variant,intron_variant LOW ATP8B2 - rs329686514 T missense_variant MODERATE CREB3L4 tolerated_low_confidence(0.05) rs321278469 A missense_variant MODERATE CREB3L4 tolerated(0.24) rs330133789 A missense_variant MODERATE CREB3L4 tolerated(0.32) rs330198768 T intron_variant MODIFIER CRTC2 - rs81449951 A missense_variant MODERATE ACADSB tolerated(0.32) rs343087568 G splice_region_variant,intron_variant LOW GPR26 - rs339956077 T splice_region_variant,intron_variant LOW CTBP2 - 387 388 21 389 22