Genotyping of native and immunologically castrated gilts by QTL-marker of RYR1 and CTSD

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Genotyping of native and immunologically castrated gilts by QTL-marker of RYR1 and CTSD

Yelyzaveta Budakva

postgraduate student 204 «Technologies of production and processing of livestock products» specialization,

Junior researcher of the Laboratory of Genetics

Institute of Pig Breeding and Agricultural Production NAAS, Ukraine

Summary

In the study conducted, the allele effect of the SNP studied was determined (Single Nucleotide Polymorphism) ryanodine receptor gene 1 RYR1 (g.1843 C>T) and cathepsin gene D CTSD (g.70 G>A) for fattening performance of the control (native) and experimental (immunologically castrated) groups of gilts the final hybrid (Large White * Landrace) * Maxgro. It was held a population analysis of polymorphism using software «GenAlEX6» to find out if the allele effect of the investigated is different (SNP) on the productivity of hybrid gilts - of the native in the number of samples (n=38) and immunologically castrated (n=30) grown in conditions of LLC SPE "Globinsky Pig Farm". Laboratory tests are carried out in the laboratory of genetics the basis of the Institute of Pig Breeding and Agricultural Production NAAS of Ukraine. Due to the fact that information on polymorphism according to QTL markers of ryanodine receptor 1 and cathepsin D in the population of immunologically castrated pigs that are bred in Ukraine is absent requires an experiment to determine which alleles will be determined as desirable in marker breeding of pigs under conditions of immunological castration. Polymorphism of the studied gene RYR1 (g.1843 C>T) and CTSD (g. 70 G>A) was determined by the method of PCR-RFLP (Polymerase chain reaction- restriction fragment length polymorphism) analysis. Researched by QTL (quantitative trait loci) ryanodine receptor gene 1 RYR1 (g. 1843 C>T) - is a marker gene whose polymorphism is associated with the quality of meat, and is responsible for the developed muscles of the carcass and limbs as well as pigs' tendency to stress (PSS) - RYR1TT. In the SNP, the RYR1 allele T frequency was 0.941 and is higher than the allele frequency C of 0.059. The genotype RYR1CC was found in only one native pig with (ADG=0.677g/163day). The genotype RYR1TT with a frequency of 0.94 genotypes prevails RYR1CC =0.06. Gilts with genotype RYR1TT are stress-sensitive. Indicator (ADG) for immunologically castrated gilts is (ADG=0.875g/149day) with a significant difference with uncastrated pigs in indicator of - average growth rate 156days. The PIC level for locus RYR1 is low at 0.10, which limits its use for associated studies. No pigs with the RYR1CC genotype were found among the research team. However, it was found only in 1 head of female pigs - the control group as a result of a single case in this population. Animals with the genotype RYR1TT 7 days earlier reached a live weight of 100 kg from the control group. The gene of cathepsin D CTSD (g.70 G>A) - is associated with precocity and efficiency of feed use. The breed character of a frequency distribution of gene alleles was established CTSD (g.70 G>A) was 0.595 by G allele frequency significantly prevailing allele A 0.405 with the index of the information content of PIC loci at the optimal level of 0.37. A research group of pigs with monomorphicgenotype CTSDGG prevailed the control group (ADG=0.770g/159day) by age reaching a live weight of 100 kg in 151 days with an average daily increase of 0.808g. A similar situation was formed as a result of the average daily increase in experimental pigs with genotype CTSDGA 0.879 g. with the same age, reaching a live weight of 100 kg in 147 days (ADG=0.858g; 0.879/147day) and the average growth rate other than uncastrated gilts. Hybrid pigs with genotype CTSDGА=0.85 genotypes prevail CTSDGG with a frequency of 0.19.

Keywords: hybrid gilts, (Large White * Landrace) * Maxgro, native, immunologically castrated, QTL marker, RYR1 (g. 1843 C>T), CTSD (g.70 G>A), PCR-RFLP analysis, ADG.

Introduction

With an effective management strategy, the pig industry is a promising area of animal husbandry. However, for the last 24 years, the problem of the pig industry has been the manifestation of the specific smell of boar in the meat and fat of uncastrated gilts. Boar smell - this is a "defect" in the biological organism of uncastrated gilts which is expressed by a "specific" smell and aroma already present in ready-made meat for consumption in the diet of people [1]. This physiological defect manifests itself in pigs with the onset of puberty. The manifestation of a boar smell contributes to the level of accumulation of androstenone and scatole. One of the good reasons for producing the smell of boar in the meat and fat of female pigs is the conversion of androgens into estrogens. In the ovaries and fatty tissues of uncastrated gilts, the conversion of androstenone into testosterone is possible which is one of the negative reasons for the results of fattening performance indicators. According to domestic sources, the influence of the gene RYR1 (g.1843 C>T) and CTSD (g.70 G>A) for fattening productivity immunologically castrated female pigs in comparison with uncastrated were not carried out which became the interest of our research. Determination of polymorphism of the QTL-marker studied for labeling certain quantitative features in (Marker Assisted Selection) will allow you to identify gene complexes that affect the formation of the desired fattening signs of productivity. Stress syndrome in pigs (PSS) characterizes pathology encoded by the halothane gene - ryanodine receptor 1 and affects the well-being of pigs and the quality of the meat product [2]. Animals carrying one mutant allele in nucleotide 1843 (g.1843C>T) - replace arginine with cysteine in position 615 in the amino acid sequence (p.Arg615Cys) this leads to the formation of an abnormal protein [2,3]. PSS - affects the quality of meat, forming a pale, soft, and exudative product for fresh consumption as well as dry and hard meat. Consequently, the maintenance of pigs with signs of PSS leads to large economic losses in the production of pork products all over the world. Cathepsin D gene localized at the p-telomere end of SSC2 [4]. CTSD gene captures about 9 thousand par nucleotides and has 9 exons and 9 introns - GT/AG [5]. CTSD gene encodes lysosomal aspartyl protease. Transcription of the gene with sites that serve as a start site for estrogen-regulating transcript [4]. Pigs with genotype AG had a lower average daily increase and a greater number of days in reaching a live weight of 100 kg unlike pigs with homozygous genotype. Pigs with the AA genotype had higher levels of meat qualities and lower spitz thickness compared to pigs with the GG genotype [6]. Only by genotyping an experimental sample of uncastrated and immunologically castrated gilts, it will be possible to determine the feasibility of associative investigation of QTL markers polymorphism RYR1 (g.1843 C>T) and CTSD (g.70 G>A).

The purpose and objectives of the research. To investigate and evaluate the prospects of the study of the genetic marker ryanodine receptor 1 and cathepsin D on the indicators of fattening productivity of native (uncastrated) and immunologically castrated gilts based on population-genetic studies on gene polymorphism RYR1 (g.1843 C>T) and CTSD (g.70 G>A).

Materials and methods of research. Genomic DNA extracted from samples of bristles from pig auricle - of the native (n=38) and immunologically castrated gilts (n=30) by method (Korinnyi S.M., Pochernyaev K. F., Balatsky, V. M., 2005) was carried out using Chelex-100 ion exchange resin [7]. The obtained samples were subjected to PCR amplification using primers (Table 1).

Table 1

Structure of genetic marker RYR1 (g.1843 C>T) and CTSD (g.70 G>A)

for PCR amplification

Amplification of DNA fragments confirmed by electrophoresis in 2% agarose gel staining with bromide ethidium and visualization in UV light. For the gene of RYR 1 (g.1843 C>T) the size of the base pairs was obtained - 137, and for the CTSD (g.70 G>A) - 104. Fragmentary splitting of amplified samples was carried out using endonuclease for RYR 1 (g.1843 C>T) - Hha\ and CTSD (g.70 G>A) - Msc\ by the (Thermo Fisher Scientific™). Electrophoretic separation of DNA fragments was carried out in 8% polyacrylamide gel in 1xTBE buffer, at current strength (5V/cm) gel length. Visualization of restriction products was carried out by dyeing bromide ethidium and viewing on the transilluminator in UV light.

Results of the study and their discussion. DNA typing of the studied groups was carried out on the native (n=38) and immunological castrated gilts (n=26) for SNP RYR 1 (g.1843 C>T) and CTSD (g.70 G>A). DNA typing involves identifying allele gene variants alleles of which are characterized by restriction fragments the size of base pairs (bp). Fragments of the resulting electrophoregram (Fig. 1.): Track 1-5 with genotype GA (184, 117, 67 bp.) CTSD (g. 70 G>A). Track 6-12 with genotype NN (84, 53 bp) RYR1 (g. 1843 C>T).

Fig.1. Electrophoregram of restriction products MscI Locus DNA CTSD (g. 70 G>A), HhaI - RYR1 (g. 1843 C>T) у 8% PAAG. Molecular mass marker pBR322/MspI.

* For genotype CTSDGG (150 bp.); RYR1CC (134bp).

The study group (immunological castrated) with the genotype RYR1TT (ADG=0.875g/149day) on 7 days earlier reached a live weight of 100 kg from the control group (uncastrated) (ADG=0.811g/156day), with an advantage of 64g of average daily growth. With the genotype RYR1CC, there were only uncastrated gilts (1 head) with an average daily increase of 677g at the age of 163 days. No RYR1CC genotype was found among immunologically castrated gilts. This genotype in the study population among the uncastrated gilts - 38 heads and immunologically castrated - 30 heads is an isolated case. However, animals with the polymorphic genotype RYR1TC have also not been detected. Progenotyped by a DNA marker CTSDgg immunologically castrated gilts predominate uncastrated in terms of average daily growth of 0.808g.per 38g. and the age of reaching a live weight of 100 kg. 151 days 8 days earlier than uncastrated (ADG=0.770g/159day). With the CTSDga genotype, the research team prevails only by an average daily increase of 0.879 g. for 21 g. with the same indicator of reaching the age of live weight of 100 kg (ADG=0.858g/147day) (Table 2).

Table 2

Results of fattening indicators of uncastrated and immunologically castrated gilts (Large White x Landrace) x Maxgro progenotyped by locus RYR1 (g. 1843 C>T) and CTSD (g. 70 G>A)

For SNP behind locus RYR1 (g. 1843 C>T) found both homozygous alleles. Аіієіє T (0.941) higher in frequency allele C (0.059). For SNP behind locus CTSD (g.70 G>A) in the studied breed found both heterozygous alleles. Allele frequency G (0.595) in SNP CTSD was higher than allele A frequency (0.405). According to the SNP, there was no reliable deviation of frequencies with DNA typed Genotypes under the Hardy- Weinberg Equilibrium Law. At the same time, the frequency of the CTSDGA genotype

85 over the genotype CTSDGG 0.19 of the gene prevailed. Reliable deviations of the observed dispersion of genotypes from the expected ones were recorded in the SNP in frequency at x2 =25.789* and 17.000*. The fixed index also showed an excess of heterozygous genotypes at the level of F=-0.682 in the investigated micro population. A positive value of the fixed index F=1.000 shows the overwhelming number of homozygous genotypes (Table 3.).

Table 3

Distribution of frequencies of alleles and genotypes of DNA marker RYR1 (g.1843 C>T) and CTSD (g.70 G>A) in gilts (Large White x Landrace) x Maxgro

We have calculated the indicator of the information content of loci (PIC) (Polymorphism Information Content) by identifying the levels of locus polymorphism required for associative research by any genetic marker. Optimal indicators for associative research which provide the necessary variety of genotypes to establish their relationship with performance indicators are from 0.25 to 0.75. Thus, the SNP CTSD gene has an optimal value of 0.37 of the corresponding (PIC) level for associated studies. For the RYR1 locus alone, the (PIC) level is low at 0.10, which limits its use for associated studies (Table 4).

Table 4

Actual and theoretically expected heterozygousness and indicator РІС of gene CTSD (g.70 G>A) in female pigs (Large White x Landrace) x Maxgro

genetic marker heterozygousness

Of the sample of 17 samples studied, only one of the gilts with the genotype RYR1CC. 16 gilts were found to have the genotype RYR1TT - this indicates a tendency to stress, accompanied by aggressive behavior in the herd, and resistance to diseases. As a result of genotyping by DNA marker of gene performance traits, cathepsin D experimentally selected examined samples of control (uncastrated) (n=38) and experimental (immunologically castrated) (n=30) groups gilts with polymorphic genotype were identified by marker CTSDGA this result shows that the studied groups (uncastrated and immunologically castrated) effectively absorb feed and are characterized by precocity and high daily growth.

Conclusions

Assessed DNA marker cathepsin D demonstrated the presence of polymorphism. It is promising to continue research in the uncastrated and immunologically castrated gilts (Large White x Landrace) x Maxgro of LLC SPE "Globinsky Pig Farm".

It was clarified that immunologically castrated gilts with genotype CTSDGG were characterized by a higher indicator of (ADG=0.808g/151 day) and the average growth rate other than uncastrated gilts (ADG=0.770g/159day).

Gilts of control and research groups with genotype CTSDGA differ only in indicator (ADG) - (ADG=0.858g; 0.879/147day).

Hybrid pigs with genotype CTSDGA=0.85 genotypes prevail CTSDGG with a frequency of 0.19.

Using the cathepsin D gene in the MAS with other DNA markers of fattening performance is possible.

Gilts with genotype RYR1n are stress-sensitive. Indicator (ADG) for immunologically castrated gilts is (ADG=0.875g/149day) with a significant difference with uncastrated pigs in indicator of - average growth rate 156days.

The genotype RYR1CC was found in only one native pig with (ADG=0.677g/163day). The genotype RYR1TT with a frequency of 0.94 genotypes prevails RYR1CC=0.06.

To determine the prospect of further marker selection by gene RYR1 in the herd of hybrid gilts there is a need for more complete genotyping. Find out how often alleles are found RYR1CC - stress resistance. Perhaps in this population of alleles RYR1CC is a rare, isolated case.

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