Polymorphisms in Several Porcine Genes are Associated with Growth Traits

Corresponding Author: Getmantseva Lyubov Head of the laboratory of molecular diagnostics and biotechnology of farm animals, Persianovsky, Rostov region, Russia Email: ilonaluba@mail.ru Abstract: The purpose of this study was to confirm the relation between polymorphism of POU1F1, GH, PRLR and MC4R genes and economically important traits of the Duroc pigs bred in Russia. Studies were carried out on purebred Duroc breed pigs (male n = 360). All pigs were kept under identical and standard conditions. The traits of analysis: The number of Days to 100-kg (Days to 100 kg), Average Daily Gain (ADG), Length of Body (LB) and Backfat Thickness (BF). The additive and dominance effects of genes were calculated. The signification effects were found for POU1F1 on LB (a = 0.75); GH on Days of 100 kg (a = 2.43), LB (d = 0.71); PRLR on Day of 100 kg (d = 1.11) and ADG (d = -29.73); MC4R on Days of 100 kg (a = 2.58; d = 2.49) and LD (d = -0.83). Our result showed the influence of POU1F1, GH, PRLR and MC4R on the growth trait and perceptivity of their use in breeding programs.


Introduction
The intensification of the livestock industry demands introduction of new effective methods of evaluating animals. Molecular and genetic analysis techniques based on the polymorphic nature of DNA allow rapid identification of genes controlling the formation of various features of animals as well as their productivity (Fontanesi et al., 2012;Ma et al., 2014;Rostellato et al., 2014).
Transcription factors represent a group of proteins capable to interact with the DNA characteristic sectors located in the regulatory pieces of gene and initiating programs of increasing or decreasing transcription. The main function of transcription factors is to read and interpret the genetic information that allows providing each gene with a unique regulation method in the process of an organism's development (Fan et al., 2012). The specific transcription factor POU1F1 (also known as Pit-1 or GHF-1) effectively stimulates the expression of growth hormone genes (GH), of prolactin (PRL) and Thyroid Stimulating Hormone (TSH) in the pituitary gland. The growth hormone and prolactin refer to the family of prolactin-like proteins and effect the growth, anabolic, hyperglycemic, lipolytic and lactogenic activity. The growth hormone has the highest anabolic and growth activity. Te Pas et al. (2005) reported that on injecting the growth hormone to swine the growth rate and muscle hypertrophy increased, while the growth of muscle hypertrophy, fat storage and the number and size of fat cells decreased. Growth hormone introduction reduces deposition of lipids, regardless of gender, breed and age (Louveau and Gondret, 2004). At the same time Weber et al. (2002) notes that the long-term excess of the growth hormone leads to a disease associated with myopathy which induce muscle hypertrophy and feebleness.
Prolactin affects the reproductive quality, development of mammary glands and lactation. The prolactin receptor which refers to membrane receptors associated with cytoplasmic protein kinases is a hormonal signal conductor for both the prolactin and the growth hormone. Molecular studies in the sectors of the hypothalamus periventricular nucleus have revealed the expression of gene melanocortin-4 receptor (MC4R) encoding the second type of neuronal melanocortin receptors. These studies have led to the assumption of melanocortin-4 receptor's participation in the regulation of the hypothalamo-pituitary system. In the research of mice with a knockout MC4R gene (Huzar et al., 1997) the impact of this gene on obesity was experimentally proved.
Biological features genes of the transcription pituitary factor (POU1F1), the growth hormone (GH), the prolactin receptor (PRLR) and the melanocortin receptor -4 (MC4R) served as an occasion for revealing association between their polymorphism and growthweight characteristics. In studies Yu et al. (1995) found the relationship of POU1F1 gene's polymorphism with fattening and meat qualities of pigs. Similar results were obtained in the further studies when the effect of POU1F1gene's polymorphism on the growth signs was considered (Piórkowska et al., 2013;Kim et al., 2014). POU1F1 is located in chromosome 13 and consists of six exons and five introns. Song et al. (2007) reported that genetic variation in the intron 1 (insertions or deletions from 313 pairs of the bases) of the POU1F1 was connected with young pigs' growth, genotypes frequencies with intron 1 varying according to a breed. Yu et al. (1995) presented POU1F1/MspI polymorphisms in the intron 1 and POU1F1/RsaI in the intron 4 in Large White pigs and in the Large White x Landrace cross. In the subsequent researches of Large White, Landrace, Duroc pigs of the Polish selection the influence of POU1F1/RsaI (dbSNP rs80904061) on selection signs of pigs was established (Piórkowska et al., 2013).
The GH is located in swine chromosome 13 and consists of five exons and four introns. A number of investigations of pigs breeds such as Large White, Landrace, Duroc, Pietrain ones demonstrated a significant effect of the GH gene's polymorphism on the growth signs, but the effects of GH genotypes are not universal and depend on the pigs' breed, line or cross (Pierzchala et al., 2003;Faria et al., 2006). Polymorphism of PRLR/AluI gene (dbSNP rs45435440) is considered mainly for the effect in terms of reproductive indexes. However, the studies of Alonso et al. (2003;Do et al., 2012) demonstrated the effect of PRLR gene on fattening and meat quality of pigs. In our research of hybrids the influence of PRLR gene on meat quality was also found (Mihailov et al., 2014). The available literature data show that polymorphism of the MC4R in pigs is associated with the growth rate, back fat thickness and feeding (Dvorakova et al., 2011;Munoz et al., 2011). Our results obtained previously from researches on pigs of Danish Landrace, Canadian Landrace and Commercial Crossbreds breeds showed the effect of MC4R gene genotypes on Days to 100 kg, ADG and BF (Klimenko et al., 2014). Recent studies of Van den Broeke et al. (2015) demonstrated the possibility of using MC4R in selection against boar taint, as well as for lower feed intake and ADG and consequently for a better carcass quality.
The purpose of this study was to confirm or disprove the relation between polymorphisms of POU1F1, GH, PRLR and MC4R genes and economically important traits of the Duroc pigs bred in Russia.

Materials and Methods
Animals Studies were carried out on purebred Duroc breed pigs (male n = 360) developed to Breeding Farm «Yubileiny» in Russia. The farm specializes in breeding purebred pigs Landrace, Large White and Duroc. The Landrace and Large White breeding are aimed at improving the reproductive traits and Duroc is on growth and meat (Leonova et al., 2015). All pigs were kept under identical and standard conditions.

Studied Traits
The productivity of pigs takes account the following traits: The number of Days to 100-kg (Days to 100 kg), Average Daily Gain (ADG), Length of Body (LB) and Backfat Thickness (BF). All traits were obtained according to the results of growing up to 100 kg.

Genotyping
Extraction, manipulation and subsequent analysis of porcine genomic DNA were performed in the Laboratory of molecular diagnostics and biotechnology Don State Agrarian University. DNA was isolated from blood leukocytes using a kit Diatom DNA Prep100 (Isogene Lab.Ltd.Russia). Specific oligonucleotide primers for the PCR were constructed on base of literature data (Table  1). The PCR amplification (25 µL final volume) was performed using 20 ng of genomic porcine DNA, 1×PCR buffer (Evrogene, Russia), 100 µM each dNTP, 10 pmol each primer and 2 U Taq polymerase (Evrogene. Russia).
Conditions were: for POU1F1 -94°C for 4 min, followed by 35 cycles of 94°C for 60 s, 61°C for 60 s and 72°C for 180 s, the final cycle 72°C for 7 min; for GH -94°C for 4 min, followed by 35 cycles of 94°C for 60 s, 64°C for 60 s and 72°C for 60 s, the final cycle 72°C for 5 min; for PRLR 94°C for 4 min, followed by 35 cycles of 94°C for 30 s, 55°C for 60 s and 72°C for 30 s, the final cycle 72°C for 5 min; for MC4R 94°C for 4 min, followed by 35 cycles of 94°C for 30 s, 62°C for 30 s and 72°C for 30 s, the final cycle 72°C for 5 min.

Statistical Analysis
Analyses of gene effect in the observed traits were examined using a linear model:

Results
Restriction analysis of the POU1F1 in Duroc pig breed defined the presence of three genotypes, which were presented as three monomorphic (774-, 153-and 108 bp) and three polymorphic (710 bp -allele E, 388-and 322 bp -allele F) fragments. In our group the genotypes EE, EF and FF are distributed with the same frequency ( Table 2). The GH is presented by polymorphic fragments of 604 bp -allele A and 345and 259 bp -allele G. The three genotypes AA, AG and GG were identified. The highest frequency is specific for the heterozygous AG genotype. The polymorphic fragments (104 bp -allele A and 85-, 59-, 19 bp -allele B) of the PRLR were observed in the group under study. The highest frequency presented allele A and genotype AA.  The analysis of pigs' production traits (Table 3) showed significant effect of POU1F1 polymorphism on the LB. The EE homozygous had a higher LD than the FF homozygous in Duroc pigs (a = 0.75). The significant effect of POU1F1 polymorphism on the Day of 100 kg (a = 6.6), ADG (a = 73.5), LB (a = 1.2) and BF (a = 0.15) has not been defined.
The GH polymorphism exhibited an effect of genotypes on the growth traits of Duroc pigs. The significant additive effect on Days of 100 kg (a = 2.43) and dominant effect on LB (d = 0.71) were found. The impact of the genotypes GH on ADG and BF in the study population of Duroc pigs was not ascertained.
Our results showed that the genotype GG/GH was associated with best Days of 100 kg and the AG/GH with the smallest LB in pigs. Bižienė et al. (2011) also detected an association between genotype GG/GH and Days of 100 kg as well lowest feed consumption of 1 kg in various breed pigs. Result in commercial sows (Faria et al., 2006) detected that homozygous genotype GG/GH was responsible for greater carcass length means, lower drip loss and higher mean pH 24 h after slaughtering. Table 3. Additive and dominance effects for the genotypes of POU1F1, GH, PRLR, MC4R genes in Russian Duroc pigs Polymorphism of PRLR the influence on growth trait was found in our study. The lowest growth rates exhibited pigs of heterozygous genotype AB/PRLR. The significant effect of PRLR polymorphism on the Day of 100 kg (d = 1.11) and ADG (d = -29.73) were found. The best performance of Day of 100 kg observed in pigs homozygous genotype BB/PRLR, but these were not significant. Different results for the Day of 100 kg were obtained by us a previous study (Mihailov et al., 2014) that with the best genotype AB/PRLR was determined in Landrace pigs.
Influence of genotype MC4R on the growth traits of Duroc pigs identified in this study. The result showed the significant effect of genotype GG/MC4R on Days of 100 kg (a = 2.58). The heterozygous AG/MC4R had a lower Days of 100 kg (d = 2.49), ADG (d = -2.58) and LD (d = -0.83) than the homozygous in Duroc pigs. In general, our research defined the best indicators of growth traits of Duroc pigs associated with homozygous genotype GG/MC4R. Nevertheless, the low frequency of GG/MC4R genotype can be noted in the population under study probably due to the fact that in the recent selection of pigs special importance was given to reproductive indicators. Works of Leonova and Svyatogorova (2014) showed impact of AA/MC4R genotype on reproductive traits of pigs. Perhaps this influence has fixed the high frequency of genotype AA/MC4R in our population. As far as the main objective in the Duroc pigs selection is to improve the growth and meat traits so polymorphism MC4R can be used as a genetic marker. Our result has shown importance of increasing GG/MC4R genotype in the population of Duroc pigs.

Conclusion
Research of purebred Russian Duroc pigs (n = 360) showed the presence of polymorphisms in genes POU1F1, GH, PRLR and MC4R. Our findings demonstrate the influence of POU1F1, GH and MC4R on the growth traits and perceptivity of their use in breeding programs. Stable effect of the PRLR gene on growth characteristics was not established and we suppose that this gene is better to use in the programs aimed at improving reproductive traits of pigs.

Funding Information
The studies were performed using the funds of the grant of President of Russian Federation for state support of young Russian scientists MK-7781.2016.11 (contract № 14.W01.16.7781-MK).
Siroj Bakoev and Anatoly Kolosov: Developed analytical tools and analysed data.
Vyacheslav Vasilenko and Aleksander Klimenko: Designed and performed experiments.
Anastasia Radyuk: Collected and analyzed data.

Ethics
This article is original and contains unpublished materials. The corresponding author confirms that all of the other authors have read and approved the manuscript and no ethical issues involved.