24th Int. Symp. “Animal Science Days”, Ptuj, Slovenia, Sept. 21st−23rd, 2016. Acta argiculturae Slovenica, Supplement 5, 189–194, Ljubljana 2016 COBISS: 1.08 Agris category code: L10, Q04 THE ASSOCIATIONS OF LEPR AND H-FABP GENE POLYMORPHISMS WITH CARCASS TRAITS IN PIGS Anna TRAKOVICKÁ 1, Nina MORAVČÍKOVÁ 2, Veronika KUKUČKOVÁ 3, Rudolf NÁDASKÝ 4, Radovan KASARDA 5 The associations of LEPR and H-FABP gene polymorphisms with carcass traits in pigs 1 Slovak University of Agriculture in Nitra, Faculty of Agrobiology and Food Resources, Department of Animal Genetics and Breeding Biology, Tr. A. Hlinku 2, 94976 Nitra, Slovakia, e-mail: anna.trakovicka@uniag.sk 2 Same address as 1, e-mail: nina.moravcikova@uniag.sk 3 Same address as 1, e-mail: veron.sidlova@gmail.com 4 Same address as 1, e-mail: qnadasky@is.uniag.sk 5 Same address as 1, e-mail: radovan.kasarda@uniag.sk ABSTRACT The aim of this study was to assess the impact of polymorphisms in porcine LEPR and H-FABP genes on carcass and meat quality traits in population of Large White x Landrace crossbreeds. The genomic DNA samples obtained from in total of 180 animals (86 boars and 94 sows) were genotyped using PCR-RFLP methods. The association analyses were carried out in relation to evaluate back-fat thickness, proportion of valuable meat parts, MLT area and propor- tion of thigh using One-way ANOVA procedure. The allele frequencies were as follows: H-FABP/HinfI H 0.31 and h 0.69, H-FABP/HaeII 0.65 and 0.35, and LEPR/HpaII 0.61 and 0.39. The average values of heterozygosity (0.37) and FIS index (0.17) indicated in population the prevalence of homozygous genotype across all evaluated loci. The statistical analyses showed significant effects on selected production traits only for H-FABP/HinfI (p < 0.001) and LEPR/HpaII (p < 0.0001) loci. The obtained results indicated the positive effect of H-FABP/HinfIH and LEPR/HpaIIA alleles in order to improvement of the economically important parameters for breeders. Key words: pigs, breeds, Landrace, Large White, genetic diversity, SNP genotyping, production traits, meat, car- cass, quality 1 INTRODUCTION For many years, a major objective of the swine in- dustry has been to increase the carcass meat percent- age. Carcass traits of pigs – such as back-fat thickness, fat meat weight, skin weight, loin eye area, lean meat weight, ratio of lean meat to fat meat, and ratio of leg and butt to carcass – are very important economic traits for pork production (Zhang et al., 2007). Genetic selec- tion has enabled dramatic improvements in the carcass composition of pigs (Jiang et al., 2012). Modern inten- sive swine production uses improved breeds with a high percentage of lean meat in the carcass. However, the complex conditions of handling and feeding lead to an appreciable decrease of the meat quality mainly due to the improvement of growth rate and lean percentage and reduction of intramuscular fat content values (Han et al., 2012; Temperan et al., 2014). Recently, the molecular technology advances have allowed the determination of many autosomal regions containing SNPs that affected pork meat traits (Dekkers et al., 2011). The analysis of structural genetic changes in candidate genes can make possible the selection footprint and deepen the under- standing of the genetic basis of complex traits (Cuong et al., 2012). The estimation of the frequency of genetic polymorphisms in different pig breeds allows identifica- tion of both males and females carrying beneficial alleles early in life, thereby improving accuracy of selection, re- ducing the generation interval and accelerating the rate Acta agriculturae Slovenica, Supplement 5 – 2016190 A. TRAKOVICKÁ et al. of genetic improvement of the trait (Pérez-Montarelo et al., 2015). The H-FABP gene, a member of the fatty acid bind- ing protein family that plays a critical role in intracellu- lar fatty acid transport by binding lipids and regulating metabolic homeostasis, has been mapped to the QTL region on SSC 6 (Gerbens et al. 1997; Grindflek et al., 2002). Moreover, the H-FABP gene may also regulate the concentration and lipid metabolism, as well as other pro- cesses connected with cell metabolism (Jankowiak et al., 2010). The impact of this gene on IMF has been analysed by many association studies (Gerbens et al., 1999; Siecz- kowska et al., 2006; Pang et al., 2006). Currently, 3 types of H-FABP restriction fragment length polymorphisms defined as HinfI, MspI and HaeIII loci have been deter- mined for many pig populations (Chen et al., 2014). The porcine leptin and its receptor genes are in- volved in food intake and energy homeostasis, and polymorphisms associated to growth and fatness traits have been detected in both genes (Pérez-Montarelo et al. 2015). The LEPR (leptin receptor) gene is considered a candidate gene for fatness traits. It is located on SSC 6 in a region in which quantitative trait loci for back-fat thickness, fat area ratios, and serum leptin concentra- tion have previously been detected (Uemoto et al., 2012). Stratil et al. (1998) identified the HpaII polymorphism located in the four intron of the LEPR gene. Several stud- ies suggested that the polymorphisms of LEPR gene are significantly associated with production traits including feed efficiency and back-fat thickness. The aim of present study was to analyse the effect of polymorphisms in H-FABP and LEPR genes on carcass traits in population of Large White x Landrace cross- breeds. Economically important parameters, including back-fat thickness, proportion of valuable meat parts, MLT area and proportion of thigh, have been evaluated to assess the importance of selected loci for the poten- tially improving accuracy of selection in analysed popu- lation. 2 MATERIAL AND METHODS Totally, 180 crossbreeds of Large White x Landrace (86 boars and 94 sows) from the Experimental Centre of Farm Animals (Department of Animal Husbandry, Slo- vak University of Agriculture in Nitra) have been includ- ed in present study. All selected animals were farmed in the same conditions, fed with standard feed mixtures and selected based on stratification method. The genomic DNA samples were extracted from blood samples according to Miller et al. (1988). Subse- quently, the concentration of DNA were tested by spec- trophotometry by the optical density at wave length of 260 nm. The genotyping of animals was performed using PCR-RFLP methods. The genotypes of HinfI and HaeIII polymorphisms in H-FABP gene have been identified according to the protocols described by Gerbens et al. (1997). The genotyping of HpaII loci located in the LEPR gene has been performed according to method by Stra- til et al. (1998). The PCR products and restriction frag- ments were separated and visualised by horizontal elec- trophoresis in 2.5 % agarose gels in 0.5 x TBE (130 V for 50 min) stained with dye GelRed. The allele and genotype frequencies were estimated in order to determine the population structure by using PowerMarker v3.25 software (Liu and Muse, 2005). The significance of differences between observed and expect- ed genotype frequencies were tested based on Chi-square (χ2) analysis. To estimate the state of genetic diversity in population the basic parameters, including observed (Ho) and expected heterozygosity (He), effective allele numbers (Ne), polymorphic information content (PIC), and FIS index has been calculated. The selected carcass and meat quality traits – back fat thickness (BFT), lean meat percentage (LM), thigh percentage (TP) and MLT area were measured by stand- ard technical norm STN 466164. The One-way ANOVA procedure adopted in SAS software (SAS Institute Inc., 2009) has been used to assess the impact of loci in H- FABP (HinfI, HaeIII), and LEPR (HpaII) genes on the selected production traits. 3 RESULTS AND DISCUSSION Across all analysed loci the prevalence of homozy- gous genotypes was found (Table 1). For the H-FABP gene the lowest proportion was observed for homozy- gous HH (HinfI) and dd (HaeIII) animals. In case of LEPR/HpaII loci the lowest proportion has been found for animals with BB genotype. The high distribution of homozygous animals was reflected in the increase of fre- quency of alleles h (H-FABP/HinfI), D (H-FABP/HaeIII) and A (LEPR/HpaII). The non-significant differences (p > 0.05) between observed and expected genotype fre- quencies were found only for the H-FABP/HaeIII poly- morphisms. In two other cases the differences caused the bias from the Hardy-Weinberg equilibrium. In accordance with observed alleles frequency sev- eral studies revealed the prevalence of h (HinfI) and D (HaeIII) H-FABP allele in different pig breeds (Gerbens et al., 1997; Kováčik et al. 2011; Chalupová et al., 2012). On the contrary, the obtained frequency of LEPR/HpaII allele was much smaller compared for example to Stra- til et al. (1998) and Mindeková et al. (2006). This stud- Acta agriculturae Slovenica, Supplement 5 – 2016 191 THE ASSOCIATIONS OF LEPR AND H-FABP GENE POLYMORPHISMS WITH CARCASS TRAITS IN PIGS ies showed that in many pigs breeds including Landrace, Meishan, Large White and Czech meat pig can be ex- pected the prevalence of B allele. However, Cuong et al. (2012) revealed that in spite of the similar genetic back- ground for IMF content of several pig breeds the selec- tion pressure on 1 locus should not influence the allelic frequencies of the other loci. Table 1 shows the summary of basic genetic diver- sity parameters observed in analysed population. The values of observed heterozygosity and also Wright’s FIS fixation index across all loci indicated the deficiency of heterozygous animals with comparison to the Hardy- Weinberg expectations. The FIS index is also considered as molecular equivalent of individual inbreeding coeffi- cient with respect to the population, thus its value re- flected in population the increase of homozygosity re- sulting from the high animal’s relatedness. The analysed loci showed median level of polymorphisms according to the classification of Botstein et al. (1980). The effec- tiveness of alleles was tested also by effective allele num- bers and in loci comparison the highest Ne was found for the LEPR/HpaII polymorphism. Generally, the value of Ne indicated relative good level of genetic variability in analysed population. The statistical analysis of H-FABP/HinfI locus ef- fect on selected production traits showed the signifi- cant impact of its genotypes on back-fat thickness and MLT area (p < 0.01), with H as desirable allele. In case of lean meat and thigh percentage based on different H-FABP/HinfI only slight tendency to improvement of this traits was found (Table 2). Similarly the genotypes of H-FABP/HaeIII showed only non-significant associa- tions (p > 0.05) with all analysed traits. The evaluation of LEPR/HpaII genotype effect on carcass and meat qual- ity traits showed its significant influence on all evaluated production traits. The results indicated that the animals with A allele in genotype can be perspective for the in- crease of production parameters in selected group of animals. In the production of pork there are many genes in- volved in the metabolic processes that control growth, fat reduction, back-fat thickness, intramuscular fat con- tent, stress resistance and other meat quality parameters (Kováčik et al., 2013). The intramuscular fat content (IMF) as a major determinant of pork quality is close- ly related with the tenderness, palatability, juiciness, and special flavor of meat (Wang et al., 2015). The IMF deposition is affected by genetic, physiological and bio- chemical factors. With respect to genetic factors, 4 to 13 quantitative trait loci (QTLs) for IMF content have been identified in different pig populations. These QTLs for IMF were found on 11 autosomes (Gao and Zhao, 2009; Cuong et al., 2012). In addition, many SNPs of candidate genes have been analysed in order to identify their asso- ciations with pork quality (Dekkers et al., 2011; Kováčik et al., 2013; Tempfli et al., 2015). Among them, a con- firmed effect on carcass and meat quality has been found for well-known polymorphisms of the H-FABP (heart fatty acid-binding protein) and LEPR (leptin receptor) genes (Cuong et al., 2012; Pérez-Montarelo et al. 2012). The results of association analysis of the H-FABP gene polymorphisms impact on pig production traits remained inconsistent. The H-FABP gene is mostly de- scribed as candidate gene for IMF content and its sig- nificant effect has been found in many studies (Árnyasi et al., 2006; Jankowiak et al., 2010; Han et al., 2012). These studies indicated that the ordering of IMF H-FABP genotypes is HH > Hh > hh (HinfI) and DD < Dd < dd (HaeIII) and that porcine meat quality might be im- proved by increasing the frequency of genotype dd-HH in pig breeds (Chen et al., 2014). But other studies have found no significant associations with IMF content (Ne- chtelberger et al., 2001; Chmurzyńska, 2006; Chalupová et al., 2012). On the other hand Wei-Jun et al. (2006) found the associations between the H-FABP gene pol- ymorphisms and fat deposition, when the HH and dd genotypes showed significantly higher deposition of fat in adipocites. Similarly Hanna et al. (2010) found that Locus Genotypes frequency Alleles frequency χ2 test Ho He Ne PIC FIS H-FABP/HinfI HH Hh hh H h ** 0.33 0.43 1.75 0.35 0.22 0.14 0.33 0.53 0.31 0.69 H-FABP/HaeIII DD Dd dd D d ns 0.41 0.46 1.84 0.29 0.11 0.44 0.41 0.15 0.65 0.35 LEPR/HpaII AA AB BB A B * 0.39 0.48 1.91 0.31 0.18 0.42 0.39 0.19 0.61 0.39 Table 1: Summary of basic genetic diversity parameters in analysed population Ho – observed homozygosity, He – observed heterozygosity, Ne – effective allele number, PIC – polymorphic information content, FIS – fixation index; ns – not significant, * p < 0.05, ** p < 0.01 Acta agriculturae Slovenica, Supplement 5 – 2016192 A. TRAKOVICKÁ et al. in Polish native Złotnicka Spotted pigs the meat with genotype hh was marked by a higher intramuscular fat content compared with the HH group. 4 CONCLUSION The statistical analyses showed significant effects of H-FABP/HinfI and LEPR/HpaII loci on selected produc- tion traits. The obtained results indicated the positive ef- fect of H-FABP/HinfIH and LEPR/HpaIIA alleles in order to improvement of the economically important param- eters for breeders. It is generally accepted that both of the presented genes has important role in intracellular fatty acid transport, fat deposition and are involved in food intake and energy homeostasis. Because of this they can be considered as candidate genes mainly for economi- cally important traits in different pig breeds. 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