SHORT COMMUNICATION doi: 10.1111/age.12117 Genome-wide association analysis identifies quantitative trait loci for growth in a Landrace purebred population

† † † † ‡ ‡ § E. J. Jung* 1, H. B. Park* 1, J. B. Lee* , C. K. Yoo* , B. M. Kim , H. I. Kim , B. W. Kim and † H. T. Lim* † *Department of Animal Science, College of and Life Sciences, Gyeongsang National University, Jinju, 660-701, Korea. Institute ‡ of Agriculture and Life Science, Gyeongsang National University, Jinju, 660-701, Korea. Division of Applied Life Science, Gyeongsang § National University, Jinju, 660-701, Korea. Department of Animal Science, Pusan National University, Miryang, 627-706, Korea.

Summary Growth-related traits are complex and economically important in the livestock industry. The aim of this study was to identify quantitative trait loci (QTL) and the associated positional candidate affecting growth in pigs. A genome-wide association study (GWAS) was performed using the porcine single-nucleotide polymorphism (SNP) 60K bead chip. A mixed-effects model and linear regression approach were used for the GWAS. The data used in the study included 490 purebred Landrace pigs. All experimental animals were genotyped with 39 438 SNPs located throughout the pig autosomes. We identified a strong association between a SNP marker on 16 and body weight at 71 days of age (ALGA0092396, P = 5.35 9 10À9, Bonferroni adjusted P < 0.05). The SNP marker was located near the genomic region containing IRX4, which encodes iroquois 4. This SNP marker could be useful in the selective breeding program after validating its effect on other populations.

Keywords body weight, GWAS, purebred pigs, QTL

The advent of DNA technologies and statistical genetic date genes affecting growth-related traits using a GWAS in methodologies has facilitated the identification of individual a Landrace population. genetic factors at the quantitative trait (QTL) or A total of 490 (50 males and 440 females) purebred quantitative trait nucleotide (QTN) level affecting growth- Landrace pigs under the same controlled conditions from a related traits in livestock (Andersson & Georges 2004). breeding stock farm were used for this study. The pheno- Genome-wide association studies (GWASs), using high- typic data were measured within the farm (Table S1). density single nucleotide polymorphism (SNP) arrays Genetic and phenotypic correlations between pairwise covering entire genomes and historic recombinations accu- combinations of traits were computed in this study popu- mulated in a population for many generations, have been lation (Table S2). Blood samples were collected from the recently used to identify QTL or QTN (Balding 2006). jugular vein for genomic DNA isolation. The Several GWASs have revealed economically important trait was performed using the Porcine SNP 60K bead chip loci in chickens (e.g., Gu et al. 2011), pigs (e.g., Fan et al. (Illumina). After the filtering procedure, a total of 39 438 2011) and (e.g., Kim et al. 2011). The aim of this SNP markers on autosomes remained for GWAS. More study was to identify QTL and associated positional candi- detailed methods are described in Appendix S1. The GWAS identified only one significant SNP marker affecting the starting body weight (P < 0.05). The Address for correspondence Bonferroni SNP marker was identified as ALGA0092396 (rs81464168, B. W. Kim, Department of Animal Science, College of Natural P = 5.35 9 10À9), located on SSC16 (Fig. 1, Table 1). The Resources & Life Science, Pusan National University, Miryang 627-706, and frequencies at ALGA0092396 are Korea. E-mail: [email protected] shown in Table S3 together with the result of testing for and Hardy–Weinberg equilibrium. In addition, the genotypic H. T. Lim, Department of Animal Science, College of Agriculture & Life effect of ALGA0092396 on starting body weight is described Sciences, Gyeongsang National University, Jinju 660-701, Korea. in Fig. S2. Interestingly, the same SNP marker showed a E-mail: [email protected] suggestive association with both ending body weight 1These authors equally contributed. (P = 1.58 9 10À5) and age at reaching 90 kg (P = 2.53 9 À Accepted for publication 07 November 2013 10 5; Table 1). The nearest annotated of

© 2014 Stichting International Foundation for Animal 1 2 Jung et al.

Table 1 Single nucleotide polymorphism markers with significant and suggestive thresholds identified by the genome-wide association study.

Trait SSC1 SNP ID Location (bp) Allele2 MAF3 Model4 P-value Closest gene

À START_WT 16 ALGA0092396 85 541 183 C/T 0.1776 Add. 5.35 9 10 9* 17 831 bp at 5’side: IRX4 À † LMA 16 DRGA0016148 41 222 726 C/T 0.4816 Add. 6.63 9 10 6 41 163 bp at 3’side: LOC100738184 À † LOIN 16 DRGA0016148 41 222 726 C/T 0.4816 Add. 7.19 9 10 6 41 163 bp at 3’side: LOC100738184 À † END_WT 16 ALGA0092396 85 541 183 C/T 0.1776 Add. 1.58 9 10 5 17 831 bp at 5’side: IRX4 À † DAY90KG 16 ALGA0092396 85 541 183 C/T 0.1776 Add. 2.53 9 10 5 17 831 bp at 5’side: IRX4

SNP, single nucleotide polymorphism; START_WT, starting body weight; LMA, loin muscle area; LOIN, loin depth; END_WT, ending body weight; DAY90KG, age at reaching 90 kg; IRX4, iroquois homeobox 4. 1Sus scrofa chromosome. 2Major/minor allele. 3Minor allele frequency. 4Additive model. *P-value with genome-wide significance. † P-value with genome-wide suggestive significance.

(a) (b)

Figure 1 Results of the genome-wide association analysis for starting body weight (START_WT) in a purebred Landrace pig population. (a) Association between START_WT and 39 438 mapped single nucleotide polymorphism (SNP) markers in 18 pig autosomes using an additive model. The y-axis shows the Àlog10 (P-values), and x-axis shows the physical positions of the SNPs on the pig chromosome. (b) The corresponding quantile–quantile (QQ) plots for the association of 39 438 SNPs with START_WT. No indication of excess signal was observed except for the SNP marker ALGA0092396 on SSC16.

ALGA0092396 was iroquois homeobox 4 (IRX4). The IRX4 Tajima’s D-value was significantly positive (D = 2.68, gene influences ventricular differentiation during heart P < 0.05), representing a balancing selection at this locus. development, and IRX4 transcripts are present in the This result supports the hypothesis that the genomic region developing central nervous system, skin and vibrissae containing ALGA0092396 is an important locus. However, (Bruneau et al. 2000). This locus overlapped with the QTL we cannot exclude the possibility that the positive D-value region affecting the average daily weight gain (10– could reflect population bottlenecks (Biswas & Akey 2006). 22 weeks; Edwards et al. 2008). Therefore, we focused on We also further analyzed the haplotype structure surround- IRX4, the only gene annotated in the region containing ing ALGA0092396 and the neighboring SNP markers, and ALGA0092396 and developed additional SNP markers in eight major haplotypes with frequencies greater than 1% this region using massively parallel (Appendix were identified (Fig. S1). S2, Fig. S1). For further analysis of the newly developed The P-values for the associations between the neighbor- markers, we performed a genetic diversity and haplotype ing SNP markers of ALGA0092396 and growth-related analysis in a 184-kb region around ALGA0092396 includ- traits are summarized in Table S4. The results shown in ing the IRX4 gene, using the DNASP program (Librado & Table S4 demonstrate only a single SNP marker (i.e., Rozas 2009). The nucleotide diversity (p) was 0.326, and ALGA0092396) mapped 17.8 kb from IRX4 with a

© 2014 Stichting International Foundation for Animal Genetics, doi: 10.1111/age.12117 GWAS for growth in pigs 3 genome-wide level of significance for START_WT. Consis- an F2 Duroc 9 Pietrain resource population: I. Growth traits. tent with recent findings in human diseases, such as type 2 Journal of Animal Science 86, 241–53. (Diabetes Genetics Initiative 2007), the results Fan B., Onteru S.K., Du Z.Q., Garrick D.J., Stalder K.J. & Rothschild obtained in this study indicate that SNP markers mapping M.F. (2011) Genome-wide association study identifies loci for far from the adjacent coding sequences of a gene might body composition and structural soundness traits in pigs. PLoS ONE 6, e14726. affect the of interest, likely through remote Gu X., Feng C., Ma L. et al. (2011) Genome-wide association study effects on transcription or translation. of body weight in chicken F2 resource population. PLoS ONE 6, In summary, we performed a GWAS to identify QTL for e21872. growth in a purebred Landrace population. The results Kim Y., Ryu J., Woo J., Kim J.B., Kim C.Y. & Lee C. (2011) Genome- suggested that a SNP marker (i.e., ALGA0092396) was wide association study reveals five nucleotide sequence variants strongly associated with growth traits. Thus, this SNP for carcass traits in beef cattle. Animal Genetics 42, 361–5. marker could be useful in selective breeding programs after Librado P. & Rozas J. (2009) DNASP v5: a software for comprehen- validating its effect in other independent populations. sive analysis of DNA polymorphism data. Bioinformatics 25, 1451–2.

Acknowledgements Supporting information This work was supported by a grant from the IPET Program (No. 309022-5), RDA, Republic of Korea. Additional supporting information may be found in the online version of this article. Appendix S1. GWAS. References Appendix S2. Massively parallel sequencing. Andersson L. & Georges M. (2004) Domestic-animal : Figure S1 Genomic organization around ALGA0092396 deciphering the genetics of . Nature Reviews and the results of the haplotype analysis using the Genetics 5, 202–12. Haploview program. Balding D.J. (2006) A tutorial on statistical methods for population Figure S2 The genotypic effect of ALGA0092396 on association studies. Nature Reviews Genetics 7, 781–91. START_WT described using the least squares mean and Biswas S. & Akey J.M. (2006) Genomic insights into positive standard error. selection. Trends in Genetics 22, 437–46. Table S1 Summary of phenotypic data from a Landrace Bruneau B.G., Bao Z.Z., Tanaka M., Schott J.J., Izumo S., Cepko C.L., purebred population. Seidman J.G. & Seidman C.E. (2000) Cardiac expression of the Table S2 Phenotypic (below diagonal) and genetic correla- ventricle-specific homeobox gene Irx4 is modulated by Nkx2-5 tions (above diagonal) among growth traits measured from and dHand. 217, 266–77. Diabetes Genetics Initiative of Broad Institute of Harvard and MIT, a purebred Landrace pig population. Lund University & Novartis Institutes of BioMedical Research. Table S3 Genotype/allele frequencies and the result of (2007) Genome-wide association analysis identifies loci for type 2 Hardy–Weinberg equilibrium of ALGA0092396 in a pure- diabetes and triglyceride levels. Science 316, 1331–6. bred Landrace pig population. Edwards D.B., Ernst C.W., Tempelman R.J., Rosa G.J., Raney N.E., Table S4 Summary of SNP markers around ALGA0092396 Hoge M.D. & Bates R.O. (2008) Quantitative trait loci mapping in and association with growth traits.

© 2014 Stichting International Foundation for Animal Genetics, doi: 10.1111/age.12117