Researches on polymorphisms in the porcine LEPR gene and their association with economic traits were widely performed in the past. Manny polymorphisms in different part of the LEPR gene were described and majority of them was associated with economic traits such as growth and fatness. In present study, LEPR gene in 68 Crna Slavonska pigs was genotyped for HinfI polymorphism in the 3.8kb part of LEPR promoter, for HpaII and RsaI polymorphisms in the intron 4 and for ApeKI polymorphism in the exon 14. Allelic and genotype frequencies on polymorphic sites were calculated. Restriction of the 3.8 kb of the promoter region with HinfI revealed presence of two distinct restriction patterns, which haven’t been described so far. Their exact location and also their potential role in LEPR expression, as well as their impact on important economic traits should be explored in the future. Allelic and genotypic frequencies for other three polymorphic sites studied were more or less comparable with previous findings in the literature

Goran Kušec

Ivona Djurkin Kušec

Kristina Gvozdanović

Polonca Margeta

Vladimir Margeta

Žarko Radišić

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299Agriculturae Conspectus Scientifi cus . Vol. 82 (2017) No. 3 (299-302)ORIGINAL SCIENTIFIC PAPERSummaryResearches on polymorphisms in the porcine LEPR gene and their association with economic traits were widely performed in the past. Manny polymorphisms in diff erent part of the LEPR gene were described and majority of them was associated with economic traits such as growth and fatness. In present study, LEPR gene in 68 Crna Slavonska pigs was genotyped for Hinf I polymorphism in the 3.8kb part of LEPR promoter, for HpaII and RsaI polymorphisms in the intron 4 and for ApeKI polymorphism in the exon 14. Allelic and genotype frequencies on polymorphic sites were calculated. Restriction of the 3.8 kb of the promoter region with Hinf I revealed presence of two distinct restriction patterns, which haven’t been described so far. Th eir exact location and also their potential role in LEPR expression, as well as their impact on important economic traits should be explored in the future. Allelic and genotypic frequencies for other three polymorphic sites studied were more or less comparable with previous fi ndings in the literature.Key wordsCrna Slavonska pig, LEPR polymorphismsGenotyping of the Leptin Receptor Gene in Crna Slavonska Pig – Preliminary Results Suggests New Variants of the Promoter Polonca MARGETAKristina GVOZDANOVIĆIvona DJURKIN KUŠECŽarko RADIŠIĆGoran KUŠECVladimir MARGETA ( )Department of Special Zootechnic, Faculty of Agriculture in Osijek, J.J. Strossmayer University of Osijek, Vladimira Preloga 1, 31000 Osijek, Croatia e-mail: vmargeta@pfos.hrReceived: May 16, 2017 | Accepted: August 18, 2017ACKNOWLEDGEMENTS This work has been fully supported by Croatian Science Foundation under the Project number 3396.Agric. conspec. sci. Vol. 82 (2017) No. 3300 Polonca MARGETA, Kristina GVOZDANOVIĆ, Ivona DJURKIN KUŠEC, Žarko RADIŠIĆ, Goran KUŠEC, Vladimir MARGETAIntroductionTh e leptin receptor (LEPR) belongs to the type I cytokine receptor. At least 6 isoforms arising from alternative splicing are found in the LEPR family, including a long form, 4 short forms, which are distinct due to the length of the cytoplasmic region and a soluble circulating form (Tartaglia et al., 1995). In mammals, leptin receptor (LEPR) gene plays an important role in the control of feed intake, energy homeostasis, body weight regulation and fat mobilization. It modulates feed intake and GH secretion (Barb et al., 2006).Due to its relevance on important economic traits such as growth and fatness, investigation on polymorphisms in the por-cine LEPR gene and their association with economic traits were widely performed during the last decade. Studies on Iberian x Landrace experimental cross report-ed signifi cant eff ects of SNPs located in LEPR on pig produc-tive traits (Ovilo et al., 2005). A signifi cant eff ect on fatness and growth has been reported for LEPR c.1987 C>T polymorphism in this population. Th e eff ect of this SNP on growth and fatness has been confi rmed also for other pig breed crosses (crossbred Iberian x Meishan, Duroc x Iberian and Duroc x Landrace/Large White pigs). Diff erential LEPRb (long form) expression connected to this SNP was found in hypothalamus (Ovilo et al., 2010). Moreover, Mackowski et al. (2005) identifi ed a signifi cant association between a Tsp509I RFLP LEPR genotype and back-fat over shoulder. Hirose et al. (2014) genotyped the LEPR poly-morphism c.2002C>T in exon14 (ApeKI restriction site) with an impact on average daily gain and backfat thickness.  Biallelic polymorphisms in the intron 4 were found with HpaII and RsaI restriction enzymes by Stratil et al. (1998) and co-dominant in-heritance of both polymorphisms was confi rmed. LEPR-HpaII polymorphism in Slovak Large White pigs showed signifi cant impact on backfat thickness and lean meat percentage (Bauer et al., 2009), which was also confi rmed on Large White x Landrace crossbreeds (Trakovická et al, 2016). Th e frequencies 0.214 and 0.786 were detected for LEPRHpaII alleles A and B in Slovak Large White pigs, while the frequency of the LEPR-RsaI allele A was only 0.00357 (Bauer et al., 2009). For the LEPR Hinf I poly-morphism, alleles A and B were described (Vincent et al., 1997). Th e frequencies for the A allele were lower than in Hampshire, Landrace, Duroc and Large White (from 0.9 to 0.18) and much higher than in Meishan (0.75).Th erefore, the aim of the present study was to genotype an-imals originating from Crna Slavonska pig at Leptin receptor (LEPR) gene and to estimate allele frequencies in investigated polymorphismsMaterials and methodsGenomic DNA was isolated from muscle samples of 68 Black Slavonian pigs using GeneJET™ Genomic DNA Purifi cation Kit (Th ermo Scientifi cTM) following the manufacturer’s instructions. HinfI polymorphism:Polymerase Chain Reaction (PCR) was carried out in a fi nal volume of 25 μl which contained 0.5 μM concentration of cor-responding forward and reverse primers (Vincent et al., 1997), 12.5 μl of Maxima Hot Start Green PCR Master Mix (2X) (Th ermo Scientifi cTM), 50-100 ng of template DNA and nuclease free water to the fi nal volume. Amplifi cation conditions were 950C for 7 min, followed by 35 cycles of 950C (30s), 57.50C (30s) and 720C (3min 30s), with a fi nal extension step of 10 min at 720C. Th e 3.8kb PCR product was then digested overnight with Hinf I re-striction endonuclease at 370C and the products were checked on 3% agarose gel.HpaII and RsaI polymorphisms.Th e PCR reactions were performed in a volume of 25 μl containing 50 - 100 ng genomic DNA, standard PCR buff er, 1.5 mM MgCl2, 200 mM each dNTP, 5 pmol each primer (Stratil et al., 1998) and 1,0 U Taq polymerase (Th ermo Scientifi cTM). Amplifi cation conditions were 950C for 7 min, followed by 35 cycles of 950C (50s), 650C (50s) and 720C (2min), with a fi nal ex-tension step of 10 min at 720C. PCR products were visualized on 2% agarose gel and the rest of products were divided into two restriction reactions, one containing HpaII and the other RsaI restriction endonucleases. Both reactions were incubated at 370C and the products were visualized on 4% agarose gel.ApeKI polymorphismTh e PCR reactions were performed in a volume of 15 μl con-taining 50 ng genomic DNA, standard PCR buff er, 1.5 mM MgCl2, 200 mM each dNTP, 5 pmol each primer (Hirose et al., 2014) and 1,0 U Taq polymerase (Th ermo Scientifi cTM). Amplifi cation conditions were 950C for 7 min, followed by 35 cycles of 950C (450s), 640C (45s) and 720C (45s), with a fi nal extension step of 10 min at 720C. PCR products were checked on 3% agarose gel. Th e restriction reaction with ApeKI was run overnight at 750C. Products of restriction reaction were checked on 5% agarose gel.Calculating genotypic and allelic frequenciesFor all analyzed polymorphisms, except of Hinf I, as it was found to be monoallelic, genotypic and allelic frequencies were calculated. Genotypic frequencies were obtained by dividing the number of each genotype by the whole number of samples. Allelic frequencies were determined in compliance with Hardy-Weinberg basic formulas, p2 + 2pq + q2 = 1 and p + q = 1. ResultsRestriction of the 3.8 kb leptin receptor promoter with HinfI revealed that majority of the genotyped samples possessed BB genotype (58 from 68 analyzed, i.e. 85.3%) with bands of ap-proximate length of 2100, 700, 395, 240, 140, 110, 50 and 40 bp, previously described by Vincent et al. (1997). Among analyzed samples, we didn’t fi nd any of AA or AB genotype, as stated in above mentioned research. Nevertheless, two new genotypes were discovered. Th e fi rst one, named N1, was found in 2 samples (3%) and is characterized by two additional bands of approxi-mate lengths 300 and 160 bp.  Th e second one, N2, was present in 8 samples (12%) and is characterized by two additional bands of approximate lengths 500 and 200 bp (fi gure 1).Restrictions of 2kb part of the LEPR intron 4 with HpaII and RsaI revealed biallelic polymorphisms in both cases. Fragment lengths aft er restriction with HpaII were for allele A 2kb (not cut) and for allele B 1450 and 550 bp. For RsaI, fragment lengths of the A allele were 1kb, 349, 334 and 300bp, and for the B allele 750, 349, 334, 300 and 250bp. Genotypic and allelic frequencies Agric. conspec. sci. Vol. 82 (2017) No. 3301Genotyping of the Leptin Receptor Gene in Crna Slavonska Pig – Preliminary Results Suggests New Variants of the Promoterof both polymorphisms are shown in the table 1. For both poly-morphisms, frequencies of the A allele were low, 0.125 for the HpaII and 0.04 for the RsaI, in which genotype AA was com-pletely absent. Digestion of the 133bp part of LEPR exon 14 with ApeKI also revealed a biallelic polymorphism with uncut allele A (133bp) and cut allele B (107 and 26 bp). Genotypic and allelic frequen-cies are shown in the table 1. Allelic frequencies were 0.38 for allele A and 0.62 for allele B and all three genotypes (AA, AB and BB) were found.DiscussionDiscovered new restriction patterns of the LEPR promot-er region suggest presence of new SNPs in that region in Crna Slavonska pigs. It is well known that polymorphisms in the pro-moter region can aff ect transcription factors binding sites, which in turn infl uences transcription and expression of genes (van ‘t Hooft  et al., 1999). Also, promoters have role in splicing of in-trons and some polymorphisms in the promoter region can lead to alternative splicing (Cramer et al., 1999). So polymorphisms in LEPR promoter could impact not only quantity of LEPR in diff erent tissues, but also its form, especially when taking into account that at least six diff erent splicing forms of LEPR exist (Tartaglia et al., 1995). Th e quantity of LEPR expression could have an impact on diff erent performance (average daily gain) and meat quality traits (backfat thickness, intramuscular fat). Considering above mentioned, new polymorphisms in the pro-moter region certainly deserve further attention. Regarding allelic frequencies for HpaII and RsaI polymor-phisms in the intron 4, allelic frequency of the A allele on LEPR- HpaII locus was 0.12, while in literature diff erent values for diff erent breeds were described: 0.214 for Slovak Large White and 0.0484 for Landrace pigs (Bauer et al., 2009), while Stratil et al. (1998) observed frequencies ranging from 0.07 in Meishan, between 0.17 and 0.29 for Landrace, Czech Meat Pig, Pietrain and Black Pied Přestice, to 0.50 for Large white and 0.75 for Hampshire. However, small number of animals per breed were included in the research, ranging from 6 to 15. Allelic frequen-cies for the LEPR- RsaI locus A allele, reported by Bauer et al. (2009), were 0.00357 in Slovak large white and 0.008 for Landrace pigs, which is comparable with our results, where the A allele frequency was 0.04. In Crna Slavonska pig, genotypic frequen-cies for HpaII polymorphism were between values, reported by Bauer et al. (2009) for the Landrace and Slovak large white. Also, on LEPR- RsaI locus the AA genotype was completely absent in both breeds and the frequency of AB genotype was low, which coincides with our results of the present study.Allelic and genotypic frequencies on LEPR- ApeKI locus in Crna Slavonska pigs were similar with that reported by Hirose et al. (2014) for Duroc pig breed.ConclusionsGenotyping four polymorphic sites in diff erent part of the LEPR gene revealed two new restriction patterns of the LEPR promoter region, suggesting a presence of new SNPs in that region in Crna Slavonska pigs. Th eir exact location and also their potential role in LEPR expression, as well as their impact on important economic traits should be explored in the future. Allelic and genotypic frequencies for other three polymorphic sites included in our study were more or less comparable with previous fi ndings in the literature. Nevertheless, correlation of all investigated LEPR polymorphic sites with important perfor-mance and meat quality traits in Crna Slavonska pigs is planned for the future.ReferencesBarb C.R., Kraeling R.R., Rampacek G.B., Hausman G.J. (2006). Th e role of neuropeptide Y and  interaction with leptin in regulat-ing feed intake and luteinizing hormone and growth hormone secretion in the pig. Reproduction 131(6): 1127-35.Bauer M., Bábelová A., Omelka R., Bahelka I., Bauerová M. (2009). Eff ect of leptin and leptin receptor genes on meat production traits of Slovak Large White and Landrace pigs. Slovak J. Anim. Sci. 42 (2): 49–53.Cramer P., Cáceres J.F., Cazalla D., Kadener S., Muro A.F., Baralle F.E., Kornblihtt A.R. (1999). Coupling of transcription with alternative splicing: RNA pol II promoters modulate SF2/ASF and 9G8 eff ects on an exonic splicing enhancer. Mol Cell. 4(2): 251-8.Hirose K., Ito T., Fukawa K., Arakawa A., Mikawa S., Hayashi Y., Tanaka K. (2014). Evaluation of eff ects of multiple candidate genes (LEP, LEPR, MC4R, PIK3C3, and VRTN) on produc-tion traits in Duroc pigs. Anim Sci J 85(3)198-206. doi: 10.1111/asj.12134.Figure 1. Restriction of the 3.8 kb leptin receptor promoter with Hinf I revealed two new genotypes, N1 and N2 in the pictureTable 1. Genotypic and allelic frequencies of analyzed LEPR polymorphisms with indicated location in the LEPR genePolymorphism Location Genotypic frequencies Allelic frequencies   AA AB BB A B HpaII intron 4 0.04 0.16 0.80 0.12 0.88 RsaI intron 4 0 0.09 0.91 0.04 0.96 ApeKI exon 14 0.12 0.54 0.34 0.38 0.62 Agric. conspec. sci. Vol. 82 (2017) No. 3302 Polonca MARGETA, Kristina GVOZDANOVIĆ, Ivona DJURKIN KUŠEC, Žarko RADIŠIĆ, Goran KUŠEC, Vladimir MARGETAMackowski M., Szymoniak K., Szydlowski M., Kamyczek M., Eckert R., Rozycki M., Switonski M. (2005). Missense mutations in exon 4 of the porcine LEPR gene encoding extracellular domain and their association with fatness traits. Anim Genet. 36(2): 135-7.Ovilo C., Fernández A., Noguera J.L., Barragán C., Letón R., Rodríguez C., Mercadé A., Alves E., Folch J.M., Varona L., Toro M. (2005). Fine mapping of porcine chromosome 6 QTL and LEPR eff ects on body composition in multiple generations of an Iberian by Landrace intercross. Genet Res 85(1): 57-67Ovilo C., Fernández A., Fernández A.I., Folch J.M., Varona L., Benítez R., Nuñez Y., Rodríguez C., Silió L. (2010). Hypothalamic expression of porcine leptin receptor (LEPR), neuropeptide Y (NPY), and cocaine- and amphetamine-reg-ulated transcript (CART) genes is infl uenced by LEPR gen-otype. Mamm Genome 21(11-12): 583-91. doi: 10.1007/s00335-010-9307-1.Stratil A., Kopecný M., Moser G., Schröff el J. Jr, Cepica S. (1998). HpaII and RsaI PCR-RFLPs within an intron of the porcine lep-tin receptor gene (LEPR) and its linkage mapping. Anim Genet 29(5): 405-6.Tartaglia L.A., Dembski M., Weng X., Deng N., Culpepper J., Devos R., Richards G.J., Campfi eld L.A., Clark F.T., Deeds J., Muir C., Sanker S., Moriarty A., Moore K.J., Smutko J.S., Mays G.G., Wool E.A., Monroe C.A., Tepper R.I. (1995). Identifi cation and expression cloning of a leptin receptor, OB-R. Cell 83(7): 1263-71.Trakovická A., Moravčíková N., Kukučková V., Nádaský R., Kasarda R. (2016). Th e associations of LEPR and H-FABP gene poly-morphisms with carcass traits in pigs. 24th Int. Symp. “Animal Science Days”, Acta argiculturae Slovenica, Supplement 5, pp 189–194. Van ‘t Hooft  F.M., von Bahr S.J., Silveira A., Iliadou A., Eriksson P., Hamsten A. (1999). Two common, functional polymorphisms in the promoter region of the beta-fi brinogen gene contribute to regulation of plasma fi brinogen concentration. Arterioscler Th romb Vasc Biol 19(12): 3063-70.Vincent A.L., Wang L., Rothschild M.F. (1997). Rapid communi-cation: restriction fragment length polymorphism in the por-cine leptin receptor (LEPR) gene. Journal of Animal Science, 75, 2287.acs82_59

Genotyping of the Leptin Receptor Gene in Crna Slavonska Pig – Preliminary Results Suggests New Variants of the Promoter

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Genotyping of the Leptin Receptor Gene in Crna Slavonska Pig – Preliminary Results Suggests New Variants of the Promoter

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