This is the accepted version of the article: Mármol-Sánchez, Emilio; Luigi Sierra, Maria Gracia; Quintanilla Aguado, Raquel; [et al.]. «Detection of homozygous genotypes for a putatively lethal recessive mutation in the porcine argininosuccinate synthase 1 (ASS1) gene». Animal Genetics, Vol. 51, Issue 1 (February 2020), p. 106-110. DOI 10.1111/age.12877 This version is avaible at https://ddd.uab.cat/record/216805 under the terms of the license 1 RUNNING HEAD: Nonsense mutation in the pig ASS1 gene 2 3 Detection of homozygous genotypes for a putatively lethal 4 recessive mutation in the porcine argininosuccinate synthase 1 5 (ASS1) gene 6 7 Emilio Mármol-Sánchez1, María Gracia Luigi-Sierra1, Raquel Quintanilla2 and Marcel 8 Amills1,3 9 1 10 Department of Animal Genetics, Centre for Research in Agricultural Genomics, CSIC‐ 11 IRTA‐UAB‐UB, Universitat Autònoma de Barcelona, Bellaterra, 08193, Spain; 2 12 Animal Breeding and Genetics Program, Institute for Research and Technology in 13 Food and Agriculture (IRTA), Torre Marimon, Caldes de Montbui, 08140, Spain; 3 14 Departament de Ciència Animal i dels Aliments, Universitat Autònoma de Barcelona, 15 Bellaterra, 08193, Spain. 16 Corresponding author: marcel.amills@uab.cat 17 18 19 20 21 22 23 24 1 25 Summary 26 27 The sequencing of the pig genome revealed the existence of homozygous individuals 28 for a nonsense mutation in the argininosuccinate synthase 1 (ASS1) gene (rs81212146, 29 c.944T>A, L315X). Paradoxically, an AA homozygous genotype for this polymorphism 30 is expected to abolish the function of the ASS1 enzyme that participates in the urea 31 cycle, leading to citrullinemia, hyperammonemia, coma and death. Sequencing of five 32 Duroc boars that sired a population of 350 Duroc barrows revealed the segregation of 33 the c.944T>A polymorphism, so we aimed to investigate its phenotypic consequences. 34 Genotyping of this mutation in the 350 Duroc barrows revealed the existence of 7 35 individuals homozygous (AA) for the nonsense mutation. These AA pigs had a normal 36 weight despite the fact that mild citrullinemia often involves an impaired growth. 37 Sequencing of the region surrounding the mutation in TT, TA and AA individuals 38 revealed that the A substitution in the second position of the codon (c.944T>A) is in 39 complete linkage disequilibrium with a C replacement (c.943T>C) in the first position 40 of the codon. This second mutation would compensate the potentially damaging effect 41 of the c.944T>A replacement. In fact, this is the most probable reason why pigs with 42 homozygous AA genotypes at the 944 site of the ASS1 coding region are alive. Our 43 results illustrate the complexities of predicting the consequences of nonsense mutations 44 on gene function and phenotypes, not only because of annotation issues but also due to 45 the existence of genetic mechanisms that sometimes limit the penetrance of highly 46 harmful mutations. 47 48 Keywords: pig, single nucleotide polymorphism, nonsense mutation, citrullinemia, 49 premature stop codon. 2 50 The sequencing of the pig genome led to the discovery of 157 nonsense 51 mutations mapping to 142 genes, and 11 of them were reported to have pathological 52 effects in humans (Groenen et al. 2012). Although most of these 11 damaging nonsense 53 variants were found in a heterozygous state, two mutations mapping to the 54 argininosuccinate synthase 1 (ASS1, rs81212146, c.944T>A, L315X) and to the RB 55 binding protein 8 endonuclease (RBBP8) genes displayed homozygous genotypes. The 56 inactivation of the RBBP8 gene causes embryonic lethality (Polato et al. 2014), but it 57 should be noticed that the current release of the Ensembl database 58 (https://www.ensembl.org) does not report any stop gained mutation in the porcine 59 RBBP8 gene. With regard to the ASS1 enzyme, its inactivation leads to the disruption 60 of the urea cycle and to citrullinemia, a disease characterized by increased ammonia 61 levels in blood, stupor, convulsions, coma and death (Endo et al. 2004). Groenen et al. 62 (2012) argued that homozygosity for the nonsense ASS1 mutation might be associated 63 with a milder form of citrullinemia. However, the mild course of this disease is usually, 64 but not always, explained by mutations causing only a partial abolishment of the 65 function of the ASS1 enzyme (Häberle et al. 2003). In some cases, the mild form of the 66 disease involves the development of symptoms such as poor growth, liver failure, 67 cerebral infarction, or spasticity, while in other occasions patients remain asymptomatic 68 (Häberle et al. 2003). 69 Whole-genome sequencing (our unpublished data) of the five Duroc boars that 70 sired a purebred population of 350 Duroc barrows (Gallardo et al. 2008, 2009) revealed 71 the segregation of the rs81212146 ASS1 nonsense polymorphism, providing an 72 opportunity to investigate its phenotypic effects. Indeed, the consequences of this 73 polymorphism were just predicted by NCBI automated computational analysis, but no 74 experiment has been previously made to assess the accuracy of such prediction. By 3 75 using a QuantStudio 12K flex Real-Time PCR System available at the Servei Veterinari 76 de Genètica Molecular at the Universitat Autònoma de Barcelona 77 (http://sct.uab.cat/svgm/en), we genotyped the 350 offspring of the five boars with a 78 dedicated TaqMan Open Array multiplex assay. In total, 323 pigs were successfully 79 genotyped for the rs81212146 polymorphism, which led to the identification of 239 TT, 80 77 TA and 7 AA pigs, hence confirming the existence of homozygous individuals for 81 this mutation in the population under study. Since one of the potential symptoms of 82 mild citrullinemia is retarded growth, we inspected the final weight of the AA pigs 83 compared with their TT and TA counterparts. Live weights were measured before 84 slaughtering and carcass weights were also collected after evisceration at the abattoir. 85 The average live weights at 190 days of TT, TA and AA pigs were 122.55 ± 12.18 kg, 86 121.26 ± 16.66 kg and 119.92 ± 21.91 kg respectively. Moreover, carcass weights of 87 TT, TA and AA pigs were 94.47 ± 10.18 kg, 95.09 ± 11.78 kg and 93.67 ± 11.67 kg, 88 respectively (Supplementary Figure 1). An analysis of variance (ANOVA) performed 89 with the aov R function and contrasting ASS1 genotypic means for both live (P-value = 90 0.724) and carcass (P-value = 0.893) weights did not reveal any significant difference. 91 In summary, we did not find evidence of a significantly decreased weight, before or 92 after slaughter, in AA pigs. 93 In order to further investigate the potential consequences of the rs81212146 94 polymorphism, we sequenced the region of the ASS1 gene containing the putative 95 nonsense mutation by making use of both genomic DNA and complementary DNA 96 (cDNA) as templates. A total of sixteen liver samples belonging to each of five TT and 97 AA and six TA animals were selected at random. Genomic DNA extraction was 98 performed by digestion of 30 mg of liver tissue in 0.5 mL lysis buffer (50 mM Tris- 99 HCl, pH = 8; 20 mM EDTA, pH = 8; 2% SDS) plus 15μL 1 μg/μL proteinase K and 4 100 incubated overnight at 56 °C. Subsequently, 500 μL of the lysate were deproteinized 101 with 0.5 mL of a mixture of phenol: chloroform: isoamyl alcohol (25:24:1). The 102 resulting supernatant was mixed with 1 mL ice-cold pure ethanol plus 50 μL NaCl (2 103 M) and centrifuged for 30 minutes at maximum speed. The DNA pellets obtained in this 104 way were washed with 500 μL of ethanol 70% and resuspended in 50 μL of ultrapure 105 water. We also extracted RNA from the same selected liver samples corresponding to 106 TT, AA and TA pigs. In brief, liver samples were pulverized in liquid nitrogen with a 107 mortar and a pestle and subsequently submerged and homogenized in 1 mL of TRI 108 Reagent (Thermo Fisher Scientific, Barcelona, Spain). Total RNA was then purified 109 with the RiboPure kit (Ambion, Austin, TX) in accordance with the instructions of the 110 manufacturer. The concentration and purity of DNA and RNA samples were assessed 111 with a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Barcelona, 112 Spain). A Bioanalyzer-2100 equipment (Agilent Technologies Inc., Santa Clara, CA) 113 was employed for determining RNA integrity (RIN) with the Agilent RNA 6000 Nano 114 Kit (Agilent Technologies, Inc., Santa Clara, CA). All RNA samples had RIN values > 115 7. The average RIN values of RNA preparations corresponding to TT, TA and AA pigs 116 were 7.46, 7.24 and 7.52, respectively. Reverse transcription (RT) was carried out with 117 the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, 118 Barcelona, Spain). Each reverse transcription reaction contained 2 µL 10 × RT Buffer, 119 0.8 µL 25 × dNTP Mix (100 mM), 2 µL 10 × RT Random Primers, 1 µL MultiScribe 120 Reverse Transcriptase (50 U/µL) and 10 µL total RNA (~ 100 ng/ µL). Ultrapure water 121 was added until reaching a final volume of 20 µL. The RT thermal profile included an 122 incubation step at 25 ºC for 10 minutes, followed by 120 minutes at 37 ºC and an 123 inactivation step at 95 ºC for 5 minutes. 5 124 Genomic DNA and cDNA samples were then subjected to polymerase chain 125 reaction (PCR) amplification. Primers (Supplementary Table 1) were designed with 126 the Primer3 software (Untergasser et al. 2012) to span contiguous exon-intron and 127 exon-exon junctions for genomic DNA and cDNA amplicons, respectively. Expected 128 sizes were 278 bp and 221 bp for PCR products amplified from genomic DNA and 129 cDNA templates, respectively. The relative position of genotyped rs81212146 130 polymorphism in genomic and cDNA amplicons is depicted in Supplementary Figure 131 2. Amplification reactions contained 2 µL of 10 × PCR buffer, 0.2 µL dNTPs (25 mM), 132 0.6 µL of each primer (10 µM), 2 µL of MgCl2 (25 mM), 2.5 µL of genomic DNA (10 133 ng/µL) or 2.5 µL of a 5-fold dilution of the RT-reaction, and 0.2 µL AMPLITAQ 134 GOLD DNA Polymerase (5 U/µL) (Thermo Fisher Scientific, Barcelona, Spain) 135 Ultrapure water was added until reaching a 20 µL final volume. The thermal profile 136 included a denaturation step at 95 ºC for 10 minutes, followed by 35 cycles of 137 denaturation at 95 ºC for 1 minute, annealing at 60 ºC for 1 minute and extension at 72 138 °C for 1 minute, plus a final extension step at 72 °C for 7 minutes. Amplicons with the 139 expected size were purified with the ExoSAP-IT PCR Clean-up kit (Thermo Fisher 140 Scientific, Barcelona, Spain). They were subsequently sequenced with the BigDye 141 Terminator Cycle Sequencing Kit v1.1 (Applied Biosystems, Foster City, CA) and 142 primers listed in Supplementary Table 1. Sequencing reactions were electrophoresed 143 in an ABI 3730 DNA analyzer (Applied Biosystems, Foster City, CA). The Mega 144 software v.6.0 (Tamura et al. 2013) was employed to visualize the results of the 145 sequencing experiment. Partial ASS1 sequences obtained from genomic DNA (accession 146 numbers: MN296492-MN296493) and cDNA (accession numbers: MN296494- 147 MN296495) were submitted to Genbank database. 6 148 The predicted consequence of the replacement of T by A at the second position 149 of codon 315 would be the introduction of a premature stop codon (TTG>TAG) 150 completely abolishing the function of the ASS1 enzyme. However, sequencing of ASS1 151 DNA and cDNA amplicons revealed that the A allele in the second position of the 152 codon is linked to a C replacement (rs81212145, c.943T>C) in the first position of the 153 codon, both at genomic and transcriptomic level (Figure 2, Supplementary Figure 3), 154 leading to the generation of a benign missense mutation. As revealed by PolyPhen-2 155 algorithm (Adzhubei et al. 2010) the substitution of leucine (TTG) by glutamine (CAG) 156 is predicted to be tolerated (PolyPhen-2 score = 0.012). Moreover, this second mutation 157 is expected to compensate the potentially damaging effect of the c.944T>A 158 replacement. Indeed, homozygosity for the TAG codon at position 315 should be lethal 159 in pigs, and in consequence, it might have been strongly selected against. Interestingly, 160 all sequenced animals displaying an AA genotype for the second position of codon 315 161 were also homozygous CC for the first position (rs81212145), i.e. all of them were 162 CAG for codon 315, suggesting the existence of complete linkage disequilibrium 163 between both polymorphisms. By using a previously generated liver microarray data set 164 from the same Duroc population analyzed herewith (Manunza et al. 2014), we 165 compared the levels of ASS1 mRNA expression between two c.944T>A genotypes i.e. 166 TA (N = 18) vs TT (N = 67). A t-test analysis performed with t.test R function did not 167 reveal any significant difference between these two genotypes (P-value = 0.346), 168 suggesting that the c.944T>A polymorphism does not have any effect on the 169 transcriptional rate of the ASS1 gene. 170 In order to estimate the co-association between the two mutations in the first and 171 second positions of codon 315, 120 whole-genome sequences belonging to European 172 and Asian domestic pigs and wild boars were retrieved from the NCBI Sequence Read 7 173 Archive (SRA, https://www.ncbi.nlm.nih.gov/sra). Detailed information about these 174 whole-genome sequences is available in Supplementary Table 2. All raw SRA files 175 were converted into FASTQ format by using the fastq-dump 2.8.2 tool from the SRA- 176 toolkit package (https://www.ncbi.nlm.nih.gov/sra/docs/toolkitsoft). The FASTQ files 177 were subsequently filtered for any sequencing adaptors with the Trimmomatic v. 0.36 178 software (Bolger et al. 2014). Paired-end filtered sequences where then aligned to the 179 porcine reference genome (Sscrofa11.1, Warr et al. 2019) with the BWA MEM 180 algorithm (Li 2013). Alignment files were sorted and binarized and PCR duplicates 181 were marked and removed with the PICARD tool 182 (https://broadinstitute.github.io/picard). INDEL realignment and base recalibration were 183 performed and the HaplotypeCaller function from the GATK 3.8 tool (McKenna et al., 184 2010) with default parameters was used to generate variant call format (vcf) files and 185 hard filtering was applied according to GATK best practices 186 (https://software.broadinstitute.org/gatk/best-practices/). The rs81212145 and 187 rs81212146 contiguous polymorphisms were retrieved and their co-segregation in 188 European and Asian domestic pigs as well as in European and Asian wild boars was 189 investigated by estimating the r2 parameter which defines the amount of linkage 190 disequilibrium between two markers. (Hill & Robertson 1968). 191 This analysis supported the notion that rs81212145 and rs81212146 192 polymorphism are in complete linkage disequilibrium (Table 1), implying the existence 193 of two potential sequences CAG and TTG at codon 315. In contrast, the TAG sequence, 194 which would imply severe deleterious effects on ASS1 enzyme activity, was not 195 detected in our whole-genome sequence data set. The frequency of the CAG haplotype 196 was much higher in pigs and wild boars from Asia than in those with a European origin 8 197 (Table 1). This result is probably due to the high genetic divergence between Asian and 198 European pigs, which separated 1 million years ago (Frantz et al. 2015). 199 There is an increasing interest in characterizing nonsense mutations associated 200 with lethality because they can have a negative effect on the profitability of pig farms. 201 For instance, Derks et al. (2017) analyzed, with an 80K SNP array, 24,000 pigs from 202 commercial farms and found 35 haplotypes with complete absence or depletion of 203 homozygous genotypes and showing adverse effects on reproduction traits. Moreover, 204 Derks et al. (2019) detected five relatively frequent recessive lethal haplotypes in two 205 commercial Norwegian Landrace and Duroc purebred populations which cause 206 important reductions (15.1 to 21.6%) of litter size due to the embryonic death of 207 homozygous individuals. Interestingly, these recessive lethal haplotypes increase litter 208 size in crossbred individuals due to a positive heterotic effect on fertility. 209 The results of our study reflect the difficulties of predicting the outcome of 210 putative loss-of-function mutations, either because problems in their correct annotation 211 (Narasimhan et al. 2016) or due to the existence of mechanisms of genetic 212 compensation that prevent lethality. Indeed, the rs81212145 and rs81212146 SNPs are 213 annotated as synonymous and stop gained substitutions in the Sscrofa10.2 and 214 Sscrofa11.1 assemblies of the pig genome, respectively, but according to our analysis 215 they should be jointly considered as a dinucleotide polymorphism in codon 315 with a 216 missense effect. With regard to genetic compensation, an analysis of 589,306 human 217 genomes led to the identification of 13 individuals with homozygous (autosomal 218 recessive disease) or heterozygous (autosomal dominant disease) genotypes for eight 219 severe Mendelian childhood diseases (Chen et al. 2016). These individuals should have 220 manifested serious clinical symptoms before the age of 18 years but, apparently, they 221 were perfectly healthy (Chen et al. 2016). The only explanation for such paradoxical 9 222 result is that there are mechanisms at play that decrease the penetrance of nonsense 223 mutations, including suppressor mutations able to change the sequence of the affected 224 codon or to induce splicing events eliminating the exon carrying the nonsense mutation 225 (MacArthur et al. 2012). Alternatively, the readthrough of the premature stop codon 226 during ribosomal translation might also prevent its truncating effect on protein synthesis 227 (Rausell et al. 2014). In conclusion, our data indicate that the c.944T>A mutation 228 reported by Groenen et al. (2012) probably does not have pathological consequences on 229 pigs due to the existence of an adjacent mutation that prevents the formation of a 230 premature stop codon. The considerable amount of deleterious variation segregating in 231 domestic animals (Makino et al. 2018) offers an unparalleled opportunity to explore the 232 effects of loss-of-function mutations on phenotypes of economic interest, as well as to 233 elucidate the genetic mechanisms that, in some occasions, counteract their harmful 234 consequences. 235 236 Conflict of interest 237 The authors declare no conflict of interest. 238 239 Acknowledgements 240 The authors are indebted to Selección Batallé S.A. for providing the animal material and 241 IRTA collaborators for participating in experimental protocols and animal management. 242 Part of the research presented in this publication was funded by grants AGL2013– 243 48742-C2–1-R and AGL2013–48742-C2–2-R awarded by the Spanish Ministry of 244 Economy and Competitivity. We also acknowledge the support of the Spanish Ministry 245 of Economy and Competitivity for the Center of Excellence Severo Ochoa 2016–2019 246 (SEV-2015-0533) grant awarded to the Centre for Research in Agricultural Genomics. 10 247 Emilio Mármol-Sánchez was funded by an FPU Ph.D. grant from the Spanish Ministry 248 of Education (FPU15/01733). María Gracia Luigi-Sierra was funded with an FPI Ph.D. 249 grant (BES-C-2017-0024) awarded by the Spanish Ministry of Economy and 250 Competitivity. Thanks also to the CERCA Programme the Generalitat de Catalunya. 251 252 253 Data availability 254 Partial ASS1 sequences obtained from genomic DNA (accession numbers: MN296492- 255 MN296493) and cDNA (accession numbers: MN296494-MN296495) have been 256 submitted to Genbank. 257 258 References 259 260 Adzhubei, I. A., Schmidt, S., Peshkin, L., Ramensky, V. E., Gerasimova, A., Bork, P., 261 Kondrashov, A. S. & Sunyaev, S. R. (2010) A method and server for predicting 262 damaging missense mutations. 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Nucleic Acids 362 Research 40, e115–e115. 363 Warr, A., Affara, N., Aken, B., Beiki, H., Bickhart, D. M., Billis, K., Chow, W., Eory, 364 L., Finlayson H. A., Flicek, P., Girón, C. G., Griffin, D. K., Hall, R., Hannum, 365 G., Hourlier, T., Howe, K., Hume, D. A., Izuogu, O., Kim, K., Koren, S., Liu, 366 H., Manchanda, N., Martin, F. J., Nonneman, D. J., O’Connor, R. E., Phillippy, 367 A. M., Rohrer, G. A., Rosen, B. D., Rund, L. A., Sargent, C. A., Schook, L. B., 368 Schroeder, S. G., Schwartz, A. S., Skinner, B. M., Talbot, R., Tseng, E., Tuggle, 369 C. K., Watson, M., Smith, T. P. L. & Archibald A. L. (2019) An improved pig 370 reference genome sequence to enable pig genetics and genomics research. 15 371 bioRxiv 668921. 372 373 374 375 Table 1. Frequency of the CAG ASS1 haplotype in a total of 120 sequenced pigs and 376 wild boars and measurement of the r2 parameter between polymorphisms c.943T>C and 377 c.944T>A. European European wild Asian Asian wild Parameter domestic pigs boars domestic pigs boars (N = 40) (N = 20) (N = 40) (N = 20) Missinga 0.375 1 0.05 0.15 CAG frequencyb 0.08 - 0.89 0.47 r2 LDc 1 - 1 1 a 378 Missing: percentage of pigs with missing genotypes for codon 315 of the ASS1 gene; b 379 CAG Freq: CAG haplotype frequency; cr2 LD: magnitude of the linkage disequilibrium 380 between polymorphisms c.943T>C and c.944T>A expressed as r2 (Hill & Robertson 381 1968). 382 383 384 385 386 387 388 16 389 LEGENDS TO FIGURES 390 391 Figure 1: Sequencing of codon 315 of the porcine ASS1 gene and its surrounding 392 region using genomic DNA as a template. The upper (A), central (B) and lower (C) 393 electropherograms display the three codon 315 genotypes (CAG/CAG CAG/TTG and 394 TTG/TTG) detected by Sanger sequencing in a sample of sixteen pigs. The c.943T>C 395 and c.944T>A polymorphisms are indicated with the (Π) and (*) symbols, respectively. 396 17 397 Supplementary Table 1. Primers employed in the PCR amplification and partial 398 sequencing of the porcine argininosuccinate synthase 1 (ASS1) gene1. Template Primer ID Primer sequence Tm (ºC) GC (%) Primer-F 5’-GGA CGA TTC TTT ACC ACG CTC-3’ 57 52 gDNA Primer-R 5’-TTA TGA ACG AGG CAG GTC CC-3’ 59 55 Primer-F 5’-ATG AAG TCC CGA GGT ATC TAC GA-3’ 58 48 cDNA Primer-R 5’-ACC TTC CCT TCC ACA CGC-3’ 57 61 1 399 gDNA: genomic DNA template, cDNA: complementary DNA template, Primer-F: 400 forward primer, Primer-R: reverse primer, Tm (ºC): melting temperature, GC%: GC 401 content of the primers. 402 403 404 405 406 407 408 409 410 411 412 413 414 415 18 416 Supplementary Table 2. List of the porcine whole-genome sequences used in the current study and genotype of codon 315 in Asian domestic 417 (ADM), Asian wild boar (AWB), European domestic (EDM) and European wild boar (EWB) pigs. Run BioSample Sample SRA Study Experiment Group Breed/Location Center Name ReleaseDate Genotype ERR173202 SAMEA1557410 ERS177334 ERP001813 ERX149165 ADM Meishan WUR-PBR 2012-10-12 TTG SRR1172563 SAMN02646543 SRS559499 SRP038221 SRX473146 ADM Tongchen HUAZHONG AGRICULTURAL UNIVERSITY 2014-08-22 CAG SRR1172577 SAMN02646545 SRS559500 SRP038221 SRX473147 ADM Tongchen HUAZHONG AGRICULTURAL UNIVERSITY 2014-08-22 CAG SRR1216635 SAMN02646544 SRS559501 SRP038221 SRX473148 ADM Tongchen HUAZHONG AGRICULTURAL UNIVERSITY 2014-08-22 - SRR1216636 SAMN02646546 SRS559502 SRP038221 SRX473149 ADM Tongchen HUAZHONG AGRICULTURAL UNIVERSITY 2014-08-22 - SRR3123375 SAMN04440480 SRS1261811 SRP068560 SRX1544453 ADM Jinhua NOVOGENE 2016-07-06 CAG SRR3123391 SAMN04440482 SRS1261812 SRP068560 SRX1544455 ADM Rongchang NOVOGENE 2016-07-06 CAG SRR3123398 SAMN04440481 SRS1261813 SRP068560 SRX1544454 ADM Meishan NOVOGENE 2016-07-06 CAG SRR448588 SAMN02953814 SRS688476 SRP011937 SRX131601 ADM Wuzhishan BGI 2012-11-26 CAG SRR5088931 SAMN06115553 SRS1844647 SRP075519 SRX2405926 ADM Rongchang CHINA AGRICULTURAL UNIVERSTIY 2017-01-01 CAG SRR5088932 SAMN06115552 SRS1844648 SRP075519 SRX2405927 ADM Rongchang CHINA AGRICULTURAL UNIVERSTIY 2017-01-01 CAG SRR949626 SAMN02298073 SRS465708 SRP028348 SRX329710 ADM Wuzhishan BGI 2015-07-22 CAG SRR949628 SAMN02298074 SRS465709 SRP028348 SRX329712 ADM Wuzhishan BGI 2015-07-22 CAG SRR949630 SAMN02298075 SRS465710 SRP028348 SRX329714 ADM Wuzhishan BGI 2015-07-22 CAG SRR949678 SAMN02298096 SRS465731 SRP028348 SRX329762 ADM Tibetan BGI 2015-07-22 CAG SRR949680 SAMN02298097 SRS465732 SRP028348 SRX329764 ADM Tibetan BGI 2015-07-22 TTG/CAG SRR949681 SAMN02298098 SRS465733 SRP028348 SRX329765 ADM Tibetan BGI 2015-07-22 CAG SRR949697 SAMN02298106 SRS465741 SRP028348 SRX329781 ADM Sichuan BGI 2015-07-22 CAG SRR949702 SAMN02298108 SRS465743 SRP028348 SRX329786 ADM Sichuan BGI 2015-07-22 CAG SRR949703 SAMN02298109 SRS465744 SRP028348 SRX329787 ADM Sichuan BGI 2015-07-22 CAG SRR949715 SAMN02298115 SRS465750 SRP028348 SRX329799 ADM Hetao BGI 2015-07-22 CAG SRR949717 SAMN02298116 SRS465751 SRP028348 SRX329801 ADM Hetao BGI 2015-07-22 TTG/CAG 19 SRR949724 SAMN02298118 SRS465753 SRP028348 SRX329808 ADM Hetao BGI 2015-07-22 CAG SRR949726 SAMN02298119 SRS465754 SRP028348 SRX329810 ADM Hetao BGI 2015-07-22 CAG SRR949727 SAMN02298120 SRS465755 SRP028348 SRX329811 ADM Hetao BGI 2015-07-22 TTG SRR949732 SAMN02298122 SRS465757 SRP028348 SRX329816 ADM Minzhu BGI 2015-07-22 CAG SRR949733 SAMN02298123 SRS465758 SRP028348 SRX329817 ADM Minzhu BGI 2015-07-22 CAG SRR949736 SAMN02298124 SRS465759 SRP028348 SRX329820 ADM Minzhu BGI 2015-07-22 CAG SRR949738 SAMN02298125 SRS465760 SRP028348 SRX329822 ADM Minzhu BGI 2015-07-22 CAG SRR949740 SAMN02298126 SRS465761 SRP028348 SRX329824 ADM Minzhu BGI 2015-07-22 CAG SRR949742 SAMN02298127 SRS465762 SRP028348 SRX329826 ADM Bamaxiang BGI 2015-07-22 CAG SRR949743 SAMN02298128 SRS465763 SRP028348 SRX329827 ADM Bamaxiang BGI 2015-07-22 CAG SRR949746 SAMN02298129 SRS465764 SRP028348 SRX329830 ADM Bamaxiang BGI 2015-07-22 CAG SRR949747 SAMN02298130 SRS465765 SRP028348 SRX329831 ADM Bamaxiang BGI 2015-07-22 CAG SRR949749 SAMN02298131 SRS465766 SRP028348 SRX329833 ADM Bamaxiang BGI 2015-07-22 CAG SRR949753 SAMN02298133 SRS465768 SRP028348 SRX329837 ADM Laiwu BGI 2015-07-22 CAG SRR949756 SAMN02298134 SRS465769 SRP028348 SRX329840 ADM Laiwu BGI 2015-07-22 CAG SRR949757 SAMN02298135 SRS465770 SRP028348 SRX329841 ADM Laiwu BGI 2015-07-22 TTG SRR949760 SAMN02298136 SRS465771 SRP028348 SRX329844 ADM Laiwu BGI 2015-07-22 CAG SRR949762 SAMN02298137 SRS465772 SRP028348 SRX329846 ADM Laiwu BGI 2015-07-22 CAG ERR173212 SAMEA1557411 ERS177344 ERP001813 ERX149175 AWB Japan WUR-PBR 2012-10-11 TTG ERR173220 SAMEA1557396 ERS177352 ERP001813 ERX149183 AWB South China WUR-PBR 2012-10-11 TTG ERR173222 SAMEA1557421 ERS177354 ERP001813 ERX149185 AWB North China WUR-PBR 2012-10-11 TTG ERR977151 SAMEA3497815 ERS804964 ERP011076 ERX1054134 AWB WAGENINGEN UNIVERSITY 2015-08-28 CAG ERR977165 SAMEA3497818 ERS804967 ERP011076 ERX1054148 AWB WAGENINGEN UNIVERSITY 2015-08-28 CAG ERR977168 SAMEA3497819 ERS804968 ERP011076 ERX1054151 AWB WAGENINGEN UNIVERSITY 2015-08-28 CAG ERR977180 SAMEA3497822 ERS804971 ERP011076 ERX1054163 AWB WAGENINGEN UNIVERSITY 2015-08-28 TTG/CAG SRR1581061 SAMN03031172 SRS703308 SRP047260 SRX703567 AWB Korea SEOUL NATIONAL UNIVERSITY 2015-03-16 TTG SRR1581063 SAMN03031173 SRS703309 SRP047260 SRX703568 AWB Korea SEOUL NATIONAL UNIVERSITY 2015-03-16 - SRR1581067 SAMN03031174 SRS703310 SRP047260 SRX703569 AWB Korea SEOUL NATIONAL UNIVERSITY 2015-03-16 CAG 20 SRR1581070 SAMN03031175 SRS703311 SRP047260 SRX703570 AWB Korea SEOUL NATIONAL UNIVERSITY 2015-03-16 CAG SRR1581073 SAMN03031176 SRS703312 SRP047260 SRX703571 AWB Korea SEOUL NATIONAL UNIVERSITY 2015-03-16 TTG SRR1581075 SAMN03031177 SRS703313 SRP047260 SRX703572 AWB Korea SEOUL NATIONAL UNIVERSITY 2015-03-16 TTG SRR1581080 SAMN03031179 SRS703315 SRP047260 SRX703574 AWB Korea SEOUL NATIONAL UNIVERSITY 2015-03-16 TTG SRR3745076 SAMN05362551 SRS1541811 SRP074357 SRX1898989 AWB Russia CRAG 2016-09-05 - SRR949641 SAMN02298081 SRS465716 SRP028348 SRX329725 AWB South China BGI 2015-07-22 TTG/CAG SRR949643 SAMN02298082 SRS465717 SRP028348 SRX329727 AWB South China BGI 2015-07-22 CAG SRR949650 SAMN02298084 SRS465719 SRP028348 SRX329734 AWB South China BGI 2015-07-22 CAG SRR949653 SAMN02298085 SRS465720 SRP028348 SRX329737 AWB South China BGI 2015-07-22 TTG SRR949654 SAMN02298086 SRS465721 SRP028348 SRX329738 AWB South China BGI 2015-07-22 - ERR875316 SAMEA3376937 ERS718609 ERP010412 ERX954919 EDM Pietrain WAGENINGEN UNIVERSITY 2015-05-15 - ERR875318 SAMEA3376938 ERS718610 ERP010412 ERX954921 EDM Pietrain WAGENINGEN UNIVERSITY 2015-05-15 - ERR875320 SAMEA3376939 ERS718611 ERP010412 ERX954923 EDM Pietrain WAGENINGEN UNIVERSITY 2015-05-15 - SRR1178916 SAMN02665306 SRS562609 SRP039012 SRX476909 EDM Mangalitza AGRICULTURAL BIOTECHNOLOGY CENTER 2014-08-15 TTG SRR1178923 SAMN02665304 SRS562610 SRP039012 SRX476910 EDM Mangalitza AGRICULTURAL BIOTECHNOLOGY CENTER 2014-08-15 TTG SRR1178925 SAMN02665305 SRS562611 SRP039012 SRX476911 EDM Mangalitza AGRICULTURAL BIOTECHNOLOGY CENTER 2014-08-15 TTG SRR1513307 SAMN02904857 SRS655955 SRP044261 SRX648632 EDM Iberian CRAG 2014-08-08 - SRR1577877 SAMN03031142 SRS703276 SRP047260 SRX703536 EDM Duroc SEOUL NATIONAL UNIVERSITY 2015-03-16 - SRR1577880 SAMN03031145 SRS703279 SRP047260 SRX703539 EDM Duroc SEOUL NATIONAL UNIVERSITY 2015-03-16 - SRR1581045 SAMN03031159 SRS703294 SRP047260 SRX703553 EDM Yucatan SEOUL NATIONAL UNIVERSITY 2015-03-16 TTG SRR1581046 SAMN03031160 SRS703295 SRP047260 SRX703554 EDM Yucatan SEOUL NATIONAL UNIVERSITY 2015-03-16 TTG SRR1581047 SAMN03031161 SRS703296 SRP047260 SRX703555 EDM Yucatan SEOUL NATIONAL UNIVERSITY 2015-03-16 - SRR1581048 SAMN03031162 SRS703298 SRP047260 SRX703557 EDM Yucatan SEOUL NATIONAL UNIVERSITY 2015-03-16 TTG/CAG SRR1581052 SAMN03031166 SRS703302 SRP047260 SRX703561 EDM Yucatan SEOUL NATIONAL UNIVERSITY 2015-03-16 TTG SRR1581055 SAMN03031169 SRS703305 SRP047260 SRX703564 EDM Yucatan SEOUL NATIONAL UNIVERSITY 2015-03-16 TTG SRR1581056 SAMN03031170 SRS703306 SRP047260 SRX703565 EDM Yucatan SEOUL NATIONAL UNIVERSITY 2015-03-16 TTG SRR1581135 SAMN03031189 SRS703326 SRP047260 SRX703586 EDM Yorkshire SEOUL NATIONAL UNIVERSITY 2015-03-16 - SRR1581136 SAMN03031190 SRS703327 SRP047260 SRX703589 EDM Yorkshire SEOUL NATIONAL UNIVERSITY 2015-03-16 - 21 SRR1581138 SAMN03031193 SRS703332 SRP047260 SRX703596 EDM Yorkshire SEOUL NATIONAL UNIVERSITY 2015-07-22 - SRR1581139 SAMN03031192 SRS703333 SRP047260 SRX703595 EDM Yorkshire SEOUL NATIONAL UNIVERSITY 2015-03-16 - SRR1581140 SAMN03031194 SRS703334 SRP047260 SRX703597 EDM Yorkshire SEOUL NATIONAL UNIVERSITY 2015-07-22 - SRR1917381 SAMN03421607 SRS875335 SRP074357 SRX958019 EDM Iberian UNIVERSITAT AUTONOMA BARCELONA 2015-04-27 TTG SRR3118615 SAMN04440474 SRS1261795 SRP068560 SRX1544456 EDM Hampshire NOVOGENE 2016-07-05 TTG SRR3123333 SAMN04440477 SRS1261808 SRP068560 SRX1544450 EDM Pietrain NOVOGENE 2016-07-06 TTG/CAG SRR3123347 SAMN04440478 SRS1261809 SRP068560 SRX1544451 EDM Large-White NOVOGENE 2016-07-06 TTG SRR4341272 SAMN05791662 SRS1725713 SRP090776 SRX2208350 EDM Landrace USDA-ARS-USMARC 2016-11-01 TTG SRR4341276 SAMN05791666 SRS1725717 SRP090776 SRX2208354 EDM Duroc USDA-ARS-USMARC 2016-11-01 TTG SRR4341277 SAMN05791667 SRS1725718 SRP090776 SRX2208355 EDM Duroc USDA-ARS-USMARC 2016-11-01 TTG SRR4341312 SAMN05791653 SRS1725753 SRP090776 SRX2208390 EDM Landrace USDA-ARS-USMARC 2016-11-01 TTG SRR4341337 SAMN05791657 SRS1725778 SRP090776 SRX2208415 EDM Landrace USDA-ARS-USMARC 2016-11-01 TTG SRR5131497 SAMN06115550 SRS1879087 SRP075519 SRX2445320 EDM Duroc CHINA AGRICULTURAL UNIVERSTIY 2016-12-31 TTG SRR5513124 SAMN06895011 SRS2168992 SRP044261 SRX2787051 EDM Large-White CENTRE FOR RESEARCH IN AGRIGENOMICS 2017-05-10 - SRR5515065 SAMN06895012 SRS2170012 SRP044261 SRX2788443 EDM Iberian CENTRE FOR RESEARCH IN AGRIGENOMICS 2017-05-10 TTG SRR5518325 SAMN06917569 SRS2173160 SRP106658 SRX2791684 EDM Large-White GEO 2017-09-07 - SRR5818327 SAMN07344468 SRS2347775 SRP111615 SRX2996556 EDM Pietrain INRA 2017-07-12 - SRR6251675 SAMN05791648 SRS1725708 SRP090776 SRX2208345 EDM Landrace USDA-ARS-USMARC 2017-11-05 TTG SRR6252601 SAMN05791652 SRS1725743 SRP090776 SRX2208379 EDM Landrace USDA-ARS-USMARC 2017-11-08 TTG SRR6252608 SAMN05791660 SRS1725711 SRP090776 SRX2208348 EDM Landrace USDA-ARS-USMARC 2017-11-07 TTG SRR6261392 SAMN05791663 SRS1725714 SRP090776 SRX2208351 EDM Duroc USDA-ARS-USMARC 2017-11-10 TTG SRR6261493 SAMN05791665 SRS1725716 SRP090776 SRX2208353 EDM Duroc USDA-ARS-USMARC 2017-11-09 CAG ERR173214 SAMEA1557433 ERS177346 ERP001813 ERX149177 EWB Netherlands WUR-PBR 2012-10-11 - ERR173217 SAMEA1557401 ERS177349 ERP001813 ERX149180 EWB France WUR-PBR 2012-10-11 - ERR173218 SAMEA1557403 ERS177350 ERP001813 ERX149181 EWB Switzerland WUR-PBR 2012-10-11 - ERR977316 SAMEA3497864 ERS805013 ERP011076 ERX1054299 EWB Netherlands WAGENINGEN UNIVERSITY 2015-08-28 - ERR977317 SAMEA3497865 ERS805014 ERP011076 ERX1054300 EWB Netherlands WAGENINGEN UNIVERSITY 2015-08-28 - ERR977326 SAMEA3497867 ERS805016 ERP011076 ERX1054309 EWB Netherlands WAGENINGEN UNIVERSITY 2015-08-28 - 22 ERR977330 SAMEA3497869 ERS805018 ERP011076 ERX1054313 EWB Netherlands WAGENINGEN UNIVERSITY 2015-08-28 - ERR977340 SAMEA3497872 ERS805021 ERP011076 ERX1054323 EWB Netherlands WAGENINGEN UNIVERSITY 2015-08-28 - ERR977341 SAMEA3497873 ERS805022 ERP011076 ERX1054324 EWB Netherlands WAGENINGEN UNIVERSITY 2015-08-28 - ERR977342 SAMEA3497874 ERS805023 ERP011076 ERX1054325 EWB Netherlands WAGENINGEN UNIVERSITY 2015-08-28 - ERR977343 SAMEA3497875 ERS805024 ERP011076 ERX1054326 EWB Netherlands WAGENINGEN UNIVERSITY 2015-08-28 - ERR977352 SAMEA3497877 ERS805026 ERP011076 ERX1054335 EWB Switzerland WAGENINGEN UNIVERSITY 2015-08-28 - ERR977356 SAMEA3497879 ERS805028 ERP011076 ERX1054339 EWB Italy WAGENINGEN UNIVERSITY 2015-08-28 - ERR977359 SAMEA3497880 ERS805029 ERP011076 ERX1054342 EWB Greece WAGENINGEN UNIVERSITY 2015-08-28 - ERR977374 SAMEA3497885 ERS805034 ERP011076 ERX1054357 EWB Middle East WAGENINGEN UNIVERSITY 2015-08-28 - ERR977377 SAMEA3497886 ERS805035 ERP011076 ERX1054360 EWB Italy WAGENINGEN UNIVERSITY 2015-08-28 - ERR977380 SAMEA3497887 ERS805036 ERP011076 ERX1054363 EWB Italy WAGENINGEN UNIVERSITY 2015-08-28 - ERR977386 SAMEA3497890 ERS805039 ERP011076 ERX1054369 EWB Ukraine WAGENINGEN UNIVERSITY 2015-08-28 - SRR1513306 SAMN02904855 SRS655622 SRP044261 SRX648328 EWB Spain CRAG 2014-08-08 - SRR3745077 SAMN05362552 SRS1541812 SRP074357 SRX1898990 EWB Spain CRAG 2016-09-05 - 23 418 Supplementary Figure 1: Boxplots depicting the distribution of (A) live weight and 419 (B) carcass weight for pigs with TT (N = 239), TA (N = 77) and AA (N = 7) 420 rs81212146 genotypes. 421 422 423 424 425 24 426 Supplementary Figure 2. Primer binding regions (highlighted in bold) in the ASS1 427 amplicons generated from (A) genomic DNA and (B) cDNA. The position of the 428 rs81212146 polymorphism is depicted in red. Exon-intron structure of the two 429 amplicons is also indicated. 430 431 432 433 434 435 436 437 438 439 440 441 25 442 Supplementary Figure 3. Sequencing of codon 315 of the porcine ASS1 gene and its 443 surrounding region using cDNA as a template. The upper and lower electropherograms 444 display two codon 315 genotypes (CAG/CAG and TTG/TTG) detected by Sanger 445 sequencing in a sample of sixteen pigs. The c.943T>C and c.944T>A polymorphisms 446 are indicated with the (Π) and (*) symbols, respectively. 447 448 449 450 451 26