RU2790450C1 - Method for diagnosing the breeding value of duroc pigs using the developed test system for the mc4r gene - Google Patents

Abstract

FIELD: biotechnology and animal husbandry.

SUBSTANCE: invention relates to the agricultural field of biotechnology and animal husbandry, and in particular to methods for selecting animals in pig breeding for diagnosing the breeding value of Duroc pigs in the early stages of development. The method includes DNA isolation, gene fragment amplification using primers 5'-TACCCTGACCATCTTGATTG-3', 5'-ATAGCAACAGATGATCTCTTTG-3', restriction of the amplified MC4R gene fragment with TaqI restriction endonuclease, genotyping and selection of gilts with the AA/MC4R genotype, boars with the genotype GG. Animals with the AA/MC4R genotype have high rates of average daily gain, body length, and low rates of age to reach a live weight of 100 kg and bacon thickness. The presence of the GG genotype for the gene in boars is associated with the best indicators of average daily gain, body length and fat thickness.

EFFECT: invention makes it possible to more effectively diagnose the breeding value of Duroc pigs and to select animals with genetically confirmed breeding value at an early age, as well as to increase the debugged accuracy in breeding work and reliability in the genetic potential of breeding pigs.

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Description

The invention relates to agricultural biotechnology and animal husbandry, and in particular to methods for selecting animals in pig breeding for diagnosing the breeding value of Duroc pigs in the early stages of development.

Significant scientific and practical interest is given to the development of methods that allow assessing the productivity of animals not only by the phenotypic manifestation of traits, but also directly at the genetic level.

It is known that such methods include testing of single nucleotide substitutions (SNP testing), which are located in genes that have an impact on biochemical and physiological processes in the body. Associative links between SNP genotypes and productive qualities of pigs can be used as genetic markers in the selection and selection of animals. In the paternal breed of pigs used in the hybridization system in order to increase precocity, average daily growth, body length in commercial hybrids, it is important to assess the breeding value of producers. Therefore, in order to diagnose the breeding value of Duroc pigs, it is necessary to identify genetic factors that affect the fattening and meat qualities of animals.

A known method for assessing the fertility of Landrace and Large White pigs, including DNA isolation, amplification of a ZJF gene fragment using primers 5'-ATGTGGATGTGGCCTACGG-3' and 5'GGGAACAAGGTGGTGATGG-3', restriction of the amplified LIF gene fragment with DraIII restriction endonuclease, genotyping and selection animals with the AA/LIF genotype, with one fragment of 407 bp in size. corresponds to the AA genotype, two fragments 266 and 144 bp in size. - genotype BB, three fragments 407-, 266- and 144 bp - genotype AB (RF Patent No. 2634404, IPC A01K 67/02, C12Q 1/68, 10/26/2017, Bull. No. 30).

The disadvantage of this method is that it is designed to assess the reproductive qualities of pigs and is aimed at improving their reproductive functions. Also, in contrast to our proposed method, a marker gene of another direction for assessing productivity, reproductive, was used.

A known method of early selection of pigs for the thickness of the back fat based on the complex genotype by DNA markers, including determining the number of G alleles that cause an increase in the thickness of the back fat, and the assessment takes into account the complex genotype by DNA markers IGF2, MC4R, CCKAR. (RF patent No. 2744594, IPC A01K 67/02, publ. 03/11/2021, Bull. No. 8) - prototype.

The disadvantage of this method is that it allows the evaluation of the breeding value of young stock only on one indicator of meat productivity - the thickness of the fat, so it cannot be considered effective enough. This is not enough to obtain the most comprehensive results for the evaluation of the breeding value of pigs and the early selection of animals. Also, in contrast to our method, the studies were carried out on maternal breeds of pigs (Landrace and Large White breed), we conducted studies on the paternal breed of pigs (Duroc). This causes different inheritance of productivity traits and different transmission of their offspring. The main disadvantage of the prototype lies in the purposeful increase in the fat thickness indicator, while all pig breeding companies in the world strive to breed pigs with increased meatiness and reduced fat thickness. This is supported by numerous studies in the field of pig production. (Romantsova, S.S. The effectiveness of predicting productivity in pigs / S.S. Romantsova, N.S. Kryuchkova // Best research work 2021: collection of articles of the International Research Competition, Petrozavodsk, December 13, 2021. - Petrozavodsk: International center for scientific partnership "New Science" (IE Ivanovskaya I.I.), 2021. - P. 492-501. - EDN YVBNEU; Rud, A.I. Post-slaughter evaluation of the meat characteristics of pig carcasses / A.I. Rud, A. Tretyakova, O. L. Analysis of fattening and meat qualities of purebred young animals / O. L. Tretyakova, V. S. Bondarenko // Bulletin of the Don State Agrarian University. - 2018. - No. 1-1 (27. - P. 23-31. - EDN YOOHJH and others)

The objective of our invention is to create an effective method for diagnosing the breeding value of Duroc pigs genetically predisposed to higher rates of growth energy, feed conversion and yield of carcass meat.

The objective of the invention is achieved by using the developed test system for the MC4R gene based on SNP testing by determining the genetic potential of breeding value (expressed in the ability to transmit high indicators of precocity, average daily gain, body length and low fat thickness to offspring) of pigs based on testing SNP located in 7 exon of the MC4R gene (melanocortin receptor gene - 4), by PCR-RFLP.

The technical result of the invention is achieved by using the developed test system for the MC4R gene based on SNP testing and helps to determine the effect of the MC4R gene on the fattening and meat qualities of Duroc pigs, to determine and fix the "desirable" genotypes associated with high productivity, the method allows reduce the cost of growing breeding young pigs of the Duroc breed.

The method was developed to determine the alleles and genotypes of a gene using polymerase chain reaction based on specific primers. The proposed method allows selection of replacement livestock (replacement boars and gilts) at the stage from birth to obtaining an assessment of their own productivity, helping to optimize the cost of assessing replacement livestock for fattening and meat qualities, reducing the cost of expensive reagents and diagnostic time.

The MC4R - gene (melanocortin receptor gene - 4) is of particular interest among candidate signaling molecules involved in the regulation of energy homeostasis, acting as an endocrine organ. A point mutation in the seventh exon of the MC4R gene leads to a disruption in the conduction of the hormonal signal of leptin through MC4R and, thereby, affects the traits that determine the fattening and meat productivity of the animal. The gene is associated with the control of eating behavior (food intake), regulation of metabolism and body weight. Investigated for the diagnosis of obesity.

During the operation of the test system for the MC4R gene, the polymorphism of this gene is determined at position 1426 of chromosome No. 1 of the pig in the region (SSC1) q22-q27. It is known that, with the help of restriction enzyme TaqI, the replacement of one nucleotide G by A leads to a change in the amino acid composition of the MC4 receptor. As a result, there is a violation of the regulation of the secretion of adipose tissue cells and leads to a change in lipid metabolism, which directly affects the process of formation of traits that characterize the fattening and meat qualities of pigs. It is also known that in some populations of pigs, the single nucleotide polymorphism of this gene is significantly associated with fattening and meat qualities, in particular, with the thickness of the dorsal fat (fat thickness).

As a result of the research, it was found that the presence of the AA genotype in the MC4R gene in pigs is associated with better early maturity, average daily gains, and body length. In boars, high indicators of the productivity of fat thickness, body length and average daily weight gain are associated with the GG genotype for the MC4R gene. The results of this study show that a positive effect of the MC4R/TaqI gene on the productive qualities of pigs has been revealed, which makes it possible to use the developed method as a test system for diagnosing the breeding value of Duroc pigs.

A feature of the invention claimed by us is the use of the method directly for Duroc meat breeds by determining genotypes by SNPs that affect the meat and fattening qualities of animals.

Long-term selection of the Duroc breed to increase fattening and meat productivity has led to the formation of some morphophysiological features in them that distinguish them from other breeds. It has been established that the amount of energy in fat in Duroc pigs is less than in Large White pigs. The Danish Duroc breed has become widespread in many countries of the world. Animals are characterized by a compact body, a wide ham, medium-sized ears, normal hair. Animals of strong constitution and sufficiently resistant to various adverse environmental factors, well adapted to the conditions of detention. Pigs of this breed are used in hybridization systems at the final stage of crossing.

The method is carried out as follows. Based on the results of molecular genetic tests (SNP testing) performed by PCR followed by restriction analysis of amplification products (PCR-RFLP method), allelic variants and genotypes of the SNP of the MC4R gene are determined. To do this, DNA is isolated from a tissue sample from the animal's auricle using a DIAtom DNA Prep 100 reagent kit (NPF Genlab LLC) or any other standard method. It is possible to use other animal tissues or cells (blood, hair follicles, sperm, etc.). Amplification of the interesting fragment (226 bp) of the MC4R gene is carried out using specialized primers 5'-TACCCTGACCATCTTGATTG-3',5'-ATAGCAACAGATGATCTCTTTG-3' in the following mode: preliminary denaturation - 5 min at 94°C; 35 cycles, denaturation 94°С - 30 s, annealing 62°С - 30 s, elongation 72°С - 30 s, final elongation at 72°С - 7 min.

Restriction of the amplified fragment of the MC4R gene is carried out using restriction endonuclease TaqI. The size of the obtained restriction fragments is determined by electrophoresis in 2% agarose gel with the addition of ethidium bromide, and depending on the length of the fragments, the genotype is determined individually for each animal. One fragment of 226 bp, which corresponds to the AA genotype, two fragments of 156- and 70 bp to the GG genotype, three fragments of 226-, 156-, and 70 bp. - AG genotype. (drawing) The results of the SNP-MC4R test are entered into the electronic breeding database and, when assessing fertility, the genotype AA / MC4R is used as an additional criterion, the presence of which indicates the genetic predisposition of pigs to higher rates of precocity, average daily growth, body length. The presence of the GG genotype for the MC4R gene in boars is associated with the best indicators of the average daily gain, body length and fat thickness. According to the results of the test, it is possible to evaluate the fattening and meat qualities of Duroc pigs and select animals that are genetically predisposed to higher breeding values at an early age. This reduces economic costs.

Thus, the presented method of SNP-MC4R testing makes it possible to more effectively diagnose the breeding value of pigs, since takes into account genetic factors that affect the fattening and meat functions of the body. The invention is illustrated by the following example.

Example. The control use of the proposed method according to the developed test system for determining the polymorphism of the MC4R gene was carried out and tested in CJSC Plemzavod-Yubileiny, Ishimsky district, Tyumen region, on 55 Duroc pigs. To conduct molecular genetic studies in animals, tissue samples were taken from the auricle with an area of 1 cm ² (ear tweezers). The influence of genotypes on fattening and meat qualities was assessed in terms of early maturity (days), fat thickness (mm), body length (cm) and average daily gain (g) based on the results of control rearing to a live weight of 100 kg according to the "Instructions for grading" from 2009 .

DNA was isolated using a DIAtom DNA Prep 100 kit (NPF Genlab LLC). The analysis was carried out by PCR-RFLP (polymerase chain reaction - restriction fragment length polymorphism). PCR was carried out on a Tertsik amplifier.

Amplification of a fragment (226 bp, including 7 exons) of the MC4R gene was performed using forward and reverse primers 5'-TACCCTGACCATCTTGATTG-3', 5'-ATAGCAACAGATGATCTCTTTG-3', respectively, in the following mode: preliminary denaturation - 5 min at 94 °C; 35 cycles, denaturation 94°С - 30 s, annealing 62°С - 30 s, elongation 72°С - 30 s, final elongation at 72°С - 7 min.

The size of the obtained restriction fragments and genotypes were determined by electrophoresis in 2% agarose gel with the addition of ethidium bromide (drawing). Visualization of electrophoregrams was carried out on a transilluminator in UV light (drawing). Statistical data processing was carried out in M. Excel "Analysis Package" according to standard methods. The study revealed genotype frequencies in gilts of 51.1 (AG), 40.0 (AA) and 8.9 (GG)% and in boars 50, 40 and 10%, respectively.

As a result of our studies on the fattening and meat qualities of Duroc pigs, depending on the genotypes of the MC4R gene, a significant effect of MC4R on the inheritance of complex traits was established: precocity, fat thickness, body length, and average daily gain (Table 1).

Duroc gilts with the genotype AA/MC4R significantly differed in better performance compared to other genotypes GG and AG: by 1.5 mm in fat thickness, by 1 and 5.9 cm in body length, and by 50 cm in average daily gain. .8 and 37.2 g, respectively.

Duroc boars with the “desirable” genotype GG (Table 2) significantly differ in better indicators in terms of fat thickness from animals with AG and AA genotypes by 2.6 and 1.7 mm, in body length - by 4.2 and 1, 6 cm and for the average daily weight gain - by 92.2 and 100.4 g, respectively.

Thus, the developed method can be used to identify animals that are carriers of the desired AA genotype in gilts of the MC4R gene associated with the meat and fattening qualities of Duroc pigs. The “desirable” GG genotype was established in boars. The proposed method is applicable in the genetics of farm animals to identify the polymorphism of the MC4R gene in pigs, with the aim of subsequently using the results obtained in breeding and breeding pigs to improve fattening and meat qualities. The method allows to increase the debugged accuracy in breeding work and reliability in the genetic potential of breeding pigs.

The method of selecting pigs according to this test system was tested at the enterprise CJSC Plemzavod-Yubileiny.

The results obtained provide a basis for the use of the developed methods in breeding pig farms as test systems for diagnosing the breeding value of pigs. Selection pressure on the herd in breeding enterprises makes it possible to obtain a new generation of replacement boars with improved performance in terms of the main breeding traits: early maturity, average daily weight gain, body length and thinner fat. For the qualitative improvement of the herd of boars-producers, it is necessary to evaluate and select replacement young stock as early as possible.

Claims (1)

A method for diagnosing the breeding value of Duroc pigs using the developed test system for the MC4R marker gene, including the determination of alleles that affect the inheritance of breeding qualities, the selection of young pigs at an early stage, characterized in that the assessment is carried out on the paternal breed of Duroc pigs according to a set of breeding indicators, and when evaluating gilts, the genotype AA / MC4R is used, and boars - the genotype GG / MC4R.

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