GENETIC CHARACTERIZATION OF THE MIRGOROD PIG BREED, OBTAINED BY ANALYSIS OF SINGLE NUCLEOTIDE POLYMORPHISMS OF GENES

47 Introduction. One of negative consequences, resulting from the distribution of industrial technology of animal production, has become the decrease in genetic diversity of agricultural breeds. In pig breeding, the main bulk of the global livestock is presented with three breeds – Large White (Yorkshire), Landrace and Duroc. In general, these breeds are more productive and better adapted to the conditions of industrial technology compared to local ones. However, local breeds, most of which are at the verge of extinction, have some unique traits, which could be useful in case of changes in economic conditions and the loss of which could result in depauperation of the global gene fund of animals. Among these traits of local breed pigs, one can distinguish high adaptation and resistance abilities, conformational and performance stability, high viability, plasticity, plain feeding, excellent reproduction and maternal characteristics, high product quality [1–3].


Introduction.
One of negative consequences, resulting from the distribution of industrial technology of animal production, has become the decrease in genetic diversity of agricultural breeds. In pig breeding, the main bulk of the global livestock is presented with three breeds -Large White (Yorkshire), Landrace and Duroc. In general, these breeds are more productive and better adapted to the conditions of industrial technology compared to local ones. However, local breeds, most of which are at the verge of extinction, have some unique traits, which could be useful in case of changes in economic conditions and the loss of which could result in depauperation of the global gene fund of animals. Among these traits of local breed pigs, one can distinguish high adaptation and resistance abilities, conformational and performance stability, high viability, plasticity, plain feeding, excellent reproduction and maternal characteristics, high product quality [1][2][3].
Mirgorod pig breed is one of such local breeds, which has come to the verge of extinction, regardless of unique useful characteristics. This breed was approved in 1940. It was obtained via complicated reproductive crossing of local short-eared pigs with English breeds -Berkshire, Tamworth, Middle Large, and Large White. Long-term mass selection while breeding local pigs of VASHCHENKO et al. Poltava region «within themselves» played a great role in the process of establishment [4,5].
It is well known that the content of intramuscular fat is one of the main factors, impacting the tenderness and energy value of meat. According to the data, presented in the articles [6][7][8], muscular tissue of purebred Mirgorod pigs contains extra-high level of intramuscular fat (6.56 %) and is remarkable for a low content of proteins (20.33 %). Along with low content of moisture in the meat of this breed, this trait conditions its excellent taste qualities. In addition to the abovementioned, another specifi c feature of this breed is its ability to use pastures and a considerable percentage of non-concentrated fodder in the ratio [9]. This is conditioned by the fact that Mirgorod breed was established on the basis of local pigs, which had been better adapted to digesting fi bers.
Until recently, the main problem in preserving the gene fund of Mirgorod breed was its low number of livestock and, as a result, forced use of in-breeding in its selection.
However, the situation was not critical. The studies, conducted in 2010-2014, noted a low degree of inbreeding, and thus, a possibility of purebred selection without the risk of inbred depression [9]. This conclusion is also confi rmed by the analysis of microsatellite loci of Mirgorod breed, which determined that its genetic variability was high, but lower (Na = 2.92; H o = = 0.382; FIS = 0.178), compared to three other Ukrainian local pig breeds -Ukrainian Meat, Ukrainian White Steppe and Ukrainian Spotted Steppe (mean Na = 5.00-8.42; H o = 0.549-0.668; FIS = 0.027-0.066) [10].
However, the spreading of African swine fever (ASF) in Ukraine has become the highest threat for breed preservation. According to the data of the World Organization for Animal Health [11], since the beginning of 2016, 123 thousand pigs were eliminated in Ukraine due to the outbreak of ASF, not counting the ones, eliminated in private households.
In August 2018, the only purebred herd of Mirgorod pig breed in the world, which belonged to SI "EF named after Decemberists" was eliminated too, due to the outbreak of ASF. As of December 2018, only 9 animals of Mirgorod breed remained alive in the experimental station of the Institute of Swine Production and Agroindustrial Production, NAAS, and 14 pigsin private households. In addition, the genetic material bank of the Institute of Animal Breeding and Genetics, NAAS, keeps the sperm of 5 Mirgorod breed boars, obtained in 2016. Therefore, there are some prerequisites for restoration of Mirgorod breed. Here, one of possible constituents of solving this task is using the genetic material of Belorussian Black-and-Spotted pig breed, the selection scheme of establishing of which was similar to the scheme of establishing the Mirgorod breed, and which is close to the latter by the phenotype features. Another possibility is the involvement of Poltava Meat breed, which was created using Mirgorod breed sows and inherited specifi c mitochondrial haplotype from the latter. It is important for the population of the restored breed to be maximally genetically similar to the initial Mirgorod breed, which is practically nonexistent at present.
Taking this fact into consideration, it is relevant to obtain genetic characteristics of the Mirgorod breed using the information about single nucleotide polymorphisms. Genetic characterization is supplemented with the associative analysis of the relationship between some SNPs and the manifestation of productive features of purebred pigs.

MATERIALS AND METHODS
The population studies involved the use of blood samples of pedigree Mirgorod pigs, bred at SI «Experimental farm named after Decemberists», the Institute of Swine Production and the Agroindustrial Production of NAAS in Poltava region, which were obtained in 2015-2017. The number of pigs, genotyped by each gene under investigation, is presented in Table 1.
The association of genetic markers with productive traits was determined for the pigs of Mirgorod breed (the breeding farm of SI «Experimental farm named after Decemberists»),yusing the following indices: age of reaching the bodyweight of 100 kg (days), thickness of backfat, measured at the level of vertebrae 6-7 (mm) at the bodyweight of 100 kg, average daily gain (g) during the feeding period, length of a semi-carcass (cm) and Eye Muscle Area (sq.cm.). DNA from blood samples was extracted using Chelex 100 reagent [12].
DNA amplifi cation with PCR was conducted using a programmed thermostat TERTSIK-2 (DNA-Technologies, Russian Federation) according to the instructions of DNA-polymerase manufacturer (Thermo Fisher Scientifi c). The restriction analysis using relevant endonucleases was conducted according to the manufacturer's instructions (Thermo Fisher Scientifi c). PCR products were separated using 2 % agarose gel-electrophoresis in 1 × Tris-borate electrode buffer (TBE) for 2 h at the current of 50 mA in the electrophoretic chamber (Cleaver Scientifi c Ltd, UK). DNA of plasmid pUC19, hydrolyzed by endonuclease Msp I (Thermo Fisher Scientifi c), was used as a marker of molecular mass. After electrophoresis, the gel was stained with the solution of ethidium bromide (10 mg/cc) and the electrophoresis results were documented with the digital camera using the transilluminator [29].
DNA-typing of animals by gene CTSK (SNP g.15G>A) was conducted by the method of allele discrimination (Taq Man -analysis), and by SNP c.1987C>T of gene LEPR -by HRM (High Resolution Melting) using CFX96 TouchTM Real Time PCR Detection System (Bio-Rad, Laboratories Inc., USA) [30].  68  50  28  61  37  17  20  43  20  50  50  38  14  36  50  50  50  50  49  50  50  50  50  50 50 GENALEX 6 program was used to estimate population characteristics [31]. The analysis of associations between genotypes and indices of productive traits of pigs was conducted using single-factor disperse analysis (ANOVA, analysis of variance) in Excel 2007. Table 1 presents the frequencies of alleles of 22 genes of quantitative trait loci, determined both by the results of our own genotyping of pedigree stock of Mirgorod pig breed, and in the investigations of other authors, the works of which have been referenced.

RESULTS AND DISCUSSION
The analysis of allele frequencies demonstrated some specifi cities of the Mirgorod breed in comparison with local and transborder breeds of different performance lines [34].
For instance, by ryanodine receptor 1 gene (RYRI), the frequency of minor allele g.1843Т, associated with the hereditary drawback of pigs -enhanced stress sensitivity, the critical manifestation of which is malignant hyperthermia [14], was 0.04. However, the phenotypic manifestation of the mentioned allele was not registered in the Mirgorod breed herd. At the same time, allele g.1843T is absent in local breeds -Ukrainian White Steppe and Ukrainian Spotted Steppe, and in meat breeds it occurs with the frequency from 0.1 to 1.00.
Estrogen receptor 1 gene (ESR1), traditionally viewed as a genetic marker of reproductive qualities of sows (PvuII-polymorphism of 3rd intron of the gene) [35][36][37] in the Mirgorod breed, was remarkable for polymorphism similar to most other domestic and foreign breeds. However, according to our data, contrary to the Large White breed, the «desired» allele ESR1 was not associated with multiple pregnancy of sows in the Mirgorod breed.
Enhancing reproductive traits in pig breeding involves the use of genetic markers, based on polymorphism of prolactin receptor gene (PRLR, AluI-polymorphism, SNP c.1789 G˃A) [38] and follicle-stimulating hormone beta-subunit gene, HaeIII-polymorphism) [39]. According to our data and the results, obtained in the work [40], the mentioned genes are notable for polymorphism in the Mirgorod breed; no specifi cities in terms of allele frequencies and differences from other breeds were determined.
According to the data, presented in [40], secreted phosphoprotein 1 gene (OPN or SPP1) [41], which has registered impact on such reproductive traits of pigs as a number of piglets at birth and motility of spermia in boars, was notable for not a high level of polymorphism (indel in SINE element) at the frequency of a minor allele А -0.136. The distribution of allele frequencies in the population of the Mirgorod breed had slight differences from that for Large White, Large Black, Poltava Meat and Landrace breeds.
As for the growth hormone gene (GH), involved in the control of meat and fi nishing qualities of pigs [42], as of 2017, both alleles of the gene (BsuRI-polymorphism) were found in the population of the Mirgorod breed, whereas our previous studies of this breed did not determine allele B of this marker. Allele B was absent in local breeds of Ukrainian White Steppe and Ukrainian Spotted Steppe, and in a number of other breeds, produced in Ukraine, this gene occurred with different frequencies of alternative alleles.
In terms of a physiological function of encoded proteins and localization of some QTLs in regions, cathepsin genes are referred to the candidate genes, involved in the control of meat productivity traits, meat quality and carcass structure [19][20][21][43][44][45][46]. As for allele frequencies of cathepsin genes S, F, and B, the Mirgorod breed does not have any signifi cant differences from other domestic and foreign breeds, for which relevant population studies were conducted [47]. At the same time, its specifi city was a relatively high frequency of the minor allele A by SNP of gene CTSK g.15G>A (q = 0.16) and practically no polymorphism of gene CTSL by SNP g.143C>T (q = 0.01).
Leptin genes (LEP) and leptin receptor genes (LEPR) are also candidate genes to QTL, as they play an important role in regulating lipid exchange [48,49]. In the Mirgorod breed, allele frequencies by genetic markers LEPR (SNP c.1987C>T) and LEPR (SNP c.232Т>А), associated with the features of fi nishing and meat traits, had no signifi cant differences from the frequencies of these alleles in the populations of other breeds, in particular, in Large White, according to the data, obtained in [32]. Among genetic markers of leptin gene SNP g.2845 A>T, SNP g.3469 T>C and SNP g.3996 T>C, the Mirgorod breed differed from other breeds in terms of allele frequency only by the latter. For instance, Yorkshire and Large White breed of Ukrainian selection had only one allele (LEP g.3996 С) [23], whereas the Mirgorod breed demonstrated the prevalence of allele LEP g.3996 T.
Melanocortin 4 receptor gene (MC4R) (SNP c.1426 G>A)) and growth hormone-releasing hormone gene (GHRH) ( Alu1-polymorphism), impacting the fi nishing traits of pigs and fat deposition [50,51] in the Mir-gorod breed were characterized by a considerable level of polymorphism by the corresponding genetic markers. However, these genes were also polymorphic in a number of other breeds, bred in Ukraine [52,30].
Genes, impacting the processes of fat deposition, include perilipin gene (PLIN) [53]. According to [40] polymorphisms SNP g.4119A > G and SNP g.7966T > > C occurred in the Mirgorod breed with rather high frequencies of alternative alleles (Table 1), which were close to the frequencies of these alleles in the Large White breed.
Actinin alpha 1 gene (ACTN1), associated with the development of muscle tissue, meat quality and reproductive traits, was represented in the population of Mirgorod breed by two alleles G and A (BstE II -polymorphism) with the prevalence of the former [40], which did not distinguish it from other breeds, investigated by this locus [54].
The mutant allele of calpastatin 2 gene (KPL2/m), which differs from the normal («wild» type), by the insertion of transposon in the area of intron 30, and is known to be responsible for immotile, short-tail sperm defect (ISTS) in boars, was not found in the population of the Mirgorod breed [55]. This defect is manifested in animals, homozygous by the mutant allele, and in Finnish Yorkshire, mainly. It has recently been demonstrated that the carriers of this allele may be pigs of other breeds [56].
Mucin 4 gene (MUC4) encodes mucin 4 -glycoprotein, playing a relevant role in protecting intestinal epithelium from pathogenic microorganisms, in particular, from adhesive strains of Escherichia coli. A point replacement of g.1849 G > С in intron 7 of the gene conditions the existence of two alleles, including g.1849 G, associated with the resistance of pigs to Escherichia coli bacteria, causing colibacteriosis [57]. One of the specifi cities of the Mirgorod pig breed was found to be high frequency of allele g.1849 G of mucin 4 gene. Contrary to a number of other breeds, it was 0.8, whereas, for instance, in the populations of the Large White the allele g.1849 G frequency was in the range of 0.16-0.67 [58].
Fucosyltransferase 1 gene (FUT1), encoding α-fucosyltransferase 1, is also associated with the resistance of pigs to diseases, caused by coliform bacilli. The animals, homozygous by recessive allele A of gene FUT1 (SNP g.307 G>A), are notable for resistance to colibacteriosis, the mutation of gene G→A in position 307 b.p. is «desired» in pigs. In the Mirgorod breed, the frequency of allele g.307 A was at the level, close to the frequency of this allele in Duroc breed (0.21 and 0.28, respectively), and in different populations of the Large White it was in the range of 0.06-0.43 [27]. Thus, no specifi c features in terms of the distribution of gene FUT1 alleles were found in the Mirgorod breed.
In general, the analysis of 25 SNPs of 22 genes demonstrated a high level of genetic variability in the population of pigs of Mirgorod breed. Only three out of all the investigated genes were found to be monomorphic (the accepted criterion of polymorphism was the frequency of a minor allele ≥ 0.05). Therefore, the percentage of polymorphic loci was 88.0 % with the consideration of several polymorphisms, present in one gene. To compare the Mirgorod breed against other breeds (in particular, Large White of different selection, Large Black, Landrace, Ukrainian White Steppe, Ukrainian Spotted Steppe, and Poltava Meat), the percentage of polymorphic loci was calculated separately for 12 genes of QTL from the investigated ones, namely, the ones, previously used by us for genotyping in subpopulations of these breeds (RYRI, ESR1, PRLR, GH, IGF2, CTSB, CTSK, CTSS, CTSL, MC4R, GHRH, LEPR) [47,30]. In this case, the percentage of polymorphic loci in the Mirgorod breed was 83.3 %, whereas, for instance, in different subpopulations of Large White (of English selection and intrabreed types of ULW-1 and ULW-3) it fl uctuated in the range of 58.3-66.7 %, in the population of Large Black -75.0 %. The percentage of polymorphic loci was also lower (75 % and 50 %, respectively) in the local breeds, Ukrainian White Steppe and Ukrainian Spotted Steppe, which are «closed» and in which the selection is conducted similar to that for the Mirgorod breed, mostly without the involvement of genetic material of other breeds.
Noteworthy is the fact that the average expected heterozygosity H e (Nei's genetic diversity) was at a relatively high level -0.326, which is another feature of high genetic variability in the Mirgorod breed. The latter is also confi rmed by the average number of alleles per locus -1.96 and the effective number of alleles -1.48. In addition, a high level of QTL gene polymorphism in the population of the Mirgorod breed, as one of the factors of genetic variability, is indicated by the indices of PIC, estimated for each genetic marker, used for typing. For instance, PIC values of 17 out of 25 genetic markers in the investigated population were at the level, exceeding 0.250, which corresponded to their high informative value, suffi cient to conduct associative studies in marker-associated selection [59]. VASHCHENKO et al. The abovementioned data demonstrate the absence of genetic erosion and contraction of genetic variability in the population of pigs of Mirgorod breed. This is in good agreement with the conclusions, obtained via the analysis of its genetic structure by immunological [60] and ISSR [61] markers and microsatellite loci [10]. This also corresponds to the conclusion, made by the results of the analysis of genealogy of intrabreed structures about the possibility of avoiding affi nity breeding and preserving the gene fund [62].
More detailed genetic characterization of the Mirgorod breed involved the analysis of the association of specifi c genetic markers with the main productive traits of the pigs of Mirgorod breed ( Table 2). The animals of the experimental group under investigation were estimated by fi ve parameters of the productive traits and genotypes by genetic markers IGF2/NciI (BcnI), RYRI (SNP g.1843C > T), MC4R (SNP c.1426 G > A), LEP (SNP g.2845 А ˃ Т), GH/BsuRI, CTSF (SNP g. 22 G > C). By the fi rst two markers, all the animals in the experimental group had similar genotypes, IGF2 АА and RYRI g.1843CС, respectively. As for the rest of genetic markers, pigs were divided into groups in accordance to the genotypes, among which the groups of animals with genotypes MC4R c.1426 GG and GH BB were absent. Unfortunately, the latter is explained by a small sampling of the animals for associative analysis.
It was determined that there were statistically signifi cant associations of the genetic marker MC4R SNP c.1426 G>A with such indices as the age of gaining 100 kg, the thickness of backfat and the Eye Muscle Area. We also determined the associations between the growth hormone gene polymorphisms (GH/BsuRI) and the age of gaining 100 kg and cathepsin gene F (CTSF SNP g. 22 G > C) with the Eye Muscle Area. There was a tendency, observed for statistically signifi cant differences between the groups of pigs with different genotypes by leptin gene (SNP g.2845 А ˃ Т) by thickness of backfat (p = 0.09). For comparison, there was no association between the genetic marker LEP SNP g.2845 А ˃ Т and thickness of backfat in the Large White.
The results regarding the association of SNP and the productive traits for Mirgorod breed with the consider- ation of a small sampling of the experimental animals may be viewed as preliminary data only and should have been validated using a larger number of animals. However, the elimination of the purebred herd and a limited number of remaining Mirgorod breed pigs exclude this possibility. However, the associations, determined in our study, give some ideas about the search for genetic markers of productive traits in pigs of the restored population of the Mirgorod breed for markerassociated selection.
Therefore, the results of the studies on genetic structure of the Mirgorod breed by a number of SNPs and associative analysis demonstrate its specifi cities and differences from other breeds, bred in Ukraine. First of all, these specifi cities refer to the general high level of genetic variability, determined by the results of our analysis of SNPs. This is in agreement with the statements of other authors about the Mirgorod breed, whose genetic structure was studied using other classes of genetic markers. The abovementioned proves the common thesis about local breeds, preserving higher level of genetic variability compared to the commercial breeds. The latter are subject to considerable breeding impact, aimed at the maximal manifestation of productive traits, which may result in some contraction of allelic diversity in their populations [63].
It should also be noted that the Mirgorod breed is remarkable for the absence of the mutant allele KPL2/m, conditioning genetic anomaly of ISTS, and the recessive allele RYR1 g.1843T, associated with stress sensitivity of animals, is present with very low frequency (0.04). The latter is the consequence of «new incoming blood» of Pietren -the breed, which was not included into the breeds-founders of the Mirgorod breed. It should be considered that the specifi city of the breed is rather a high frequency of the minor allele CTSК g.15A (q = 0.16) and, thus, rather a high level of informative value of the genetic marker CTSK (SNP g.15G > > A) (PIC = 0.233). Noteworthy is the polymorphism of gene LEP in the Mirgorod breed by the genetic marker of SNP g.3996 T > C, which distinguishes it from the Large White.

CONCLUSIONS
The associations between some genetic markers and productive traits have been determined in the herd of the Mirgorod breed. On the one hand, these associations may result from the immediate impact of polymorphisms of quantitative trait nucleotides (QTN) on their manifestation. However, they may be considered as a specifi c case of population dynamics process, due to which there are changes in the frequencies of alleles in the sampling not only by the candidate genes of QTL, but also by a number of others, functionally related or linked to them. The latter is relevant in the context of preserving genetic structure and variability of breeds to prevent the loss of their breed specifi city, which is especially urgent for numerically insignifi cant local breeds. In both cases, in our opinion, it is reasonable to check the presence of the very associations, determined in the original breed, during the breeding process using genetic markers in the restored population of the Mirgorod breed.
It is reasonable to carry out the restoration of the gene pool of the Mirgorod pig breed, taking into account the results regarding the SNPs of the studied genes and their associations with the productive traits, presented in this article. The selection of animals for restoration should be carried out after typing the young animals, giving preference to pigs with genotypes с.1426 MC4R GA , MC4R AA , g. 22  Ключевые слова: миргородская порода свиней, генетическая характеристика, QTL, SNP.