SHORT COMMUNICATION doi:10.1111/j.1365-2052.2009.01997.x SNPs in the porcine GOT1 gene improve a QTL for serum aspartate aminotransferase activity on SSC14 G. Reiner*, N. Clemens*, E. Lohner† and H. Willems* *Department of Veterinary Clinical Sciences, Justus-Liebig-University, D-35392 Giessen, Germany. †Animal Health Service Baden Wurtemburg, D 70111 Fellbach, Germany Summary Clinical–chemical traits are essential parameters to quantify the health status of individuals and herds, but the knowledge about their genetic architecture is sparse, especially in swine. We have recently described three QTL for serum aspartate aminotransferase activity (sAST), and one of these maps to a region on SSC14 where the aspartate aminotransferase coding gene GOT1 is located. This QTL was only apparent under the acute burden of a model disease. The aim of the present study was to characterize GOT1 as a candidate gene and to test the effects of different GOT1 SNPs as potential quantitative trait nucleotides (QTNs) for sAST. Nine SNPs within GOT1 were identified, and SNP c.-793C>G significantly increased the QTL effects and narrowed the confidence interval from 90 to 15 cM. Additionally, we found a significant association of SNP c.-793C>G in a commercial outbred line, but with reversed phase. We conclude that GOT1 is a putative candidate gene for the sAST QTL on SSC14, and that SNP c.-793C>G is close to the responsible QTN. Keywords clinical–chemical traits, GOT1, quantitative trait loci, single nucleotide poly- morphism, swine. Cytosolic aspartate aminotransferase (EC. 2.6.1.1), formerly details on the parasite model are given in Reiner et al. 2002, known as glutamate oxalacetate transaminase (GOT), is 2007b). The aim of the present study was to increase the involved in several central metabolic pathways, and repre- significance and to narrow the confidence interval of the sents a link between amino acid, carbohydrate and fat QTL by consecutive inclusion of GOT1 SNPs into the QTL metabolism (McPhalen et al. 1992). Aspartate amino- analysis, thus identifying candidate QTN for the sAST QTL transferase activity in serum (sAST) is a common clinical– on SSC14. chemical marker for parenchyma damage, mainly of muscle Experiments were carried out with 126 F2 pigs of the and liver cells (Jackson 2007). Significant breed differences Pietrain/Meishan F2 family described by Reiner et al. in sAST have been described (Friendship & Henry 1992; (2007a, 2009). Existing genotypes and phenotypes (sAST Reiner et al. 2002), but the genetic background of this from the healthy pigs and during the acute phase of variation still remains unknown. Variants in the GOT1 gene Sarcocystosis as a model disease) were supplemented with might be relevant both for metabolism and diagnostics, but new SNP information. Additionally, association studies of until now, no GOT1-polymorphisms have been reported in the GOT1 SNPs were carried out with 153 unrelated, the literature. We have recently mapped three QTL for the healthy gilts (120 kg live weight; German Landrace · Large activity of sAST (Reiner et al. 2007a). One of these QTL was White) of one outbred population, derived from four mapped to a region on SSC14 where GOT1 is located, commercial production facilities. making it reasonable to search for the basic gene variant Genomic DNA was extracted from EDTA-stabilized blood. within the gene itself. The moderate QTL was significant on PCR was performed with the Qiagen Multiplex PCR Kit. PCR a genome-wide level only after the acute burden of a model primers were designed with OLIGO 4.0 (Eurofins MWG infection (by the metazoic parasite Sarcocystis miescheriana; operon). Primer design was based on the GOT1 mRNA sequence (acc. no. M24088), because the respective DNA Address for correspondence sequence was not available. Amplicons of the GOT1 pro- G. Reiner, Department of Veterinary Clinical Sciences, Justus-Liebig- moter were amplified with primers deduced from BAC clone University, D-35392 Giessen, Germany. CH242-276N12 (preEnsemble, http://pre.ensembl.org/). All E-mail: [email protected] primers, annealing temperatures and amplicon sizes are Accepted for publication 23 September 2009 listed in Table S1. PCR conditions were as follows: initial Ó 2009 The Authors, Journal compilation Ó 2009 Stichting International Foundation for Animal Genetics, Animal Genetics 1 2 Reiner et al. activation of the HotStarTaq DNA polymerase at 95 °C for consecutively into the microsatellite-based QTL analysis to 15 min, followed by 35 cycles of denaturation at 94 °C for compare the effects of each individual SNP with each other 30 s, annealing at the specific temperature given in Table 1 and with the situation without additional SNP information. for 1 min, extension at 72 °C for 1 min and a final exten- Nine SNPs were identified by comparative sequencing sion at 72 °C for 10 min. (Fig. S1, Table S3). Five of them were found within the DNA was prepared for comparative sequencing from each 5¢-flanking region of GOT1, including one within the 5¢UTR of the founder breeds (Pietrain boars, n = 4; Meishan sows, (c.-43T>C), and three were located within exon 2. All three n = 4) of the F2 family. Amplicons were sequenced on exonal SNPs were silent mutations. Based on the SNP 0.25 lm PAGE gels on a LI-COR DNA analyser 4200. information, GOT1 was mapped between Sw2519 and Sequences were aligned with CLUSTALW (http://www. Sw761 by linkage mapping. The distances between Sw2519 ebi.ac.uk/Tools/clustalW2) to detect SNPs. and GOT1 and between GOT1 and Sw761 were 14 and All nine detected SNPs were genotyped in the F2 family by 19 cM respectively. Predicted solely on the information of PyrosequencingTM on a Pyromark ID system (Biotage) flanking microsatellite markers, the sAST QTL was not according to the recommendations of the manufacturer. significant in the healthy pigs (before infection), but the Pyrosequencing primers were designed with the PSQ Assay F-value was 8.4 during the acute stage of Sarcocystosis. The design software (Biotage). PCR primers, sequencing primers QTL explained 12.1% (acute disease) of log sAST phenotypic and annealing temperatures are summarized in Table S2. variance (Table 1). The inclusion of SNPs c.-793C>G or PCR conditions were as described above. Pyrosequencing c.156A>G into QTL analysis improved F-values (Fig. 1) and was performed using a Pyromark Gold Q96 Reagent Kit explained phenotypic variance (Vp) significantly. The con- (Biotage). Pyrograms were analysed with the Pyromark ID fidence interval was narrowed by SNP c.-793C>G to 15 cM. software (Biotage). With one of these two SNPs included in the analysis, the GOT1 was mapped relative to the microsatellites geno- sAST QTL reached significance in healthy pigs also. Log typed on SSC14 with the software package CRIMAP, version sAST was decreased by Pietrain alleles and increased by 2.4 (Green et al. 1990), according to the guidelines of Keats Meishan alleles. Associations between SNPs and log sAST et al. (1991). QTL analysis was performed using the web- are given in Table 2. based application ÔQTL expressÕ (Seaton et al. 2002) as Associations between SNP c.-793C>G genotypes and log described by Reiner et al. (2007a). GOT1 SNPs were included sAST values of the commercial population are given at the Table 1 Effects of SNPs on SSC14-QTL for log sAST values in healthy pigs and during acute disease. Disease burden SNP cM FPVp x ±SD a ±SDa d ±SDd CI95l CI95u DCI Non Non1 49 4.5 6.8 1.250 0.071 )0.055 0.019 )0.032 0.031 0 90 90 c.-793C>G 49 10.6 *** 15.2 1.275 0.067 )0.0496 0.019 )0.0784 0.031 40 55 15 c.-744A>G 55 4.9 7.6 1.247 0.071 )0.0577 0.019 )0.0238 0.030 2 89 87 c.-605T>A 65 4.0 6.3 1.253 0.071 )0.046 0.017 )0.021 0.024 3 83 80 c.-483C>T 54 4.6 7.2 1.267 0.071 )0.045 0.024 )0.059 0.036 1 83 82 c.-470G>A 56 4.7 7.2 1.254 0.071 )0.056 0.019 )0.036 0.03 4 87 83 c.-43T>C 56 4.5 7.1 1.254 0.070 )0.075 0.025 )0.032 0.036 1 83 82 c.156A>G 51 9.1 *** 16.3 1.241 0.068 )0.090 0.024 0.011 0.037 11 63 52 c.255T>C/ 57 4.69 7.4 1.243 0.071 )0.061 0.021 )0.015 0.032 5 83 78 c.258T>C Acute Non 40 8.4 ** 12.1 0.924 0.081 )0.091 0.022 )0.0022 0.035 12 52 40 c.-793C>G 42 13.7 *** 18.5 0.960 0.077 )0.103 0.023 )0.035 0.037 21 55 34 c.-744A>G 37 8.0 ** 11.6 0.917 0.081 )0.083 0.021 0.0063 0.031 13 73 60 c.-605T>A 34 7.9 ** 11.4 0.926 0.081 )0.092 0.023 0.003 0.033 14 72 58 c.-483C>T 38 8.0 ** 11.7 0.922 0.081 )0.098 0.025 0.023 0.036 14 65 51 c.-470G>A 33 7.5 ** 11.1 0.923 0.081 )0.077 0.02 0.0034 0.003 14 70 56 c.-43T>C 40 9.7 *** 13.7 0.932 0.079 )0.122 0.029 0.013 0.042 13 56 43 c.156A>G 40 12.2 *** 16.7 0.941 0.090 )0.110 0.026 )0.022 0.041 20 57 37 c.255T>C/ 32 7.5 ** 11.1 0.925 0.081 )0.076 0.019 0.004 0.027 10 73 63 c.258T>C F, F-values; P, significance thresholds of F-values: 5.1 (*P < 0.05); 6.8 (**P < 0.01); 8.9 (***P < 0.001); Vp, percentage of phenotypic variance explained by the QTL; x, mean; ±SD, standard deviation of mean; a and d, additive and dominance effects of the QTL; ±SDa and ±SDd, standard deviations of the a and d values; CI95l and CI95u, lower and upper bound of 95% confidence interval; DCI, span of 95% confidence interval.