KR20100070511A - Dna marker and distinction method associated with the intramuscular fat content in pig - Google Patents

Abstract

PURPOSE: A marker regarding pig intramuscular fat content is provided to ensure exact heredity measuring ability and contributes to the national development of breeding pig. CONSTITUTION: A marker taking part in the pig intramuscular fat content comprises a standard (+1)-196 loci transcription initiation based on adenylate kinase gene transcription regulation region within pig 6 chromosome. The -196 loci is a TT genotype.

Description

Labeling Factors Involved in Pig Muscle Content and Methods for Selecting Piglets Using the Same {DNA marker and distinction method associated with the intramuscular fat content in pig}

The present invention analyzes genes and gene mutations that regulate quantitative trait loci (QTL) associated with intramuscular fat content of porcine chromosome 6 to develop biomarkers associated with intramuscular fat content. Adenylate kinase (AK4) gene present in the locus was selected as a candidate gene, DNA single sequence polymorphism (SNP) was found by comparing DNA sequencing corresponding to the transcriptional regulatory region of the gene, and the found single nucleotide polymorphism was found in muscle fat content. The purpose of this study is to identify significant monobasic polymorphisms by analyzing whether they are related to traits and to develop molecular markers required for pig marker selection help (MAS) technique.

The annual income of sows was 3,113 in 1997, 1,867 in 1999, 1,599 in 2000, 1414 in 2001, 1,274 in 2002, 760 in 2003, 1,361 in 2004, 1,836 in 2005, 1,850 in 2006, In 2007, 1,832 heads of pigs imported more than 1,000 heads of pigs every year, and foreign currency was spent at $ 2,474,000 for the annual purchase of pigs.

In developed countries, the genetic improvement program for sows and the search for excellent sows are carried out in the practical stages of breeding molecular breeding by using DNA markers to improve meat quality, breeding traits, growth traits, genetic diseases and breed characteristics.

This information at the DNA level gives producers as well as breeders important information in selecting specific key variants. DNA information can be used to select quantitative traits called marker factor help selection (MAS). Molecular markers can be used to increase the accuracy of selection, to select sex-limited traits, and to be useful for carcass traits such as meat quality. To date, several genes or markers have been used in the pig industry.

When selecting pig breeders, the introduction of DNA markers in the existing method based on phenotype can improve the accuracy of selection, thereby maximizing genetic capacity improvement.

Therefore, the present invention is to provide a marker factor involved in the intramuscular fat content of pigs that can be used in the development of marker-assisted selection (MAS) technique and a method of selecting sows using the same.

In order to solve the above problems, the present invention relates to the intramuscular fat content of the pig including a -196 locus on the basis of the transcription initiation base of the adenylate kinase gene transcription control region in the swine chromosome 6 (+1). It provides a labeling factor.

The present invention also provides a method for determining (1) monobasic polymorphisms (SNPs) on specific loci of genes related to intramuscular fat content in pigs;

(2) analyzing the intramuscular fat content trait of pigs based on the above analysis results; Provides a method for selecting sows consisting of a process.

Existing methods for selecting longitudinal axes have been selected only by selection indexes based on the phenotype. However, simulations show that marker selection help (MAS) can be effectively used not only for traits controlled by quantitative locus, but also for selection or selection of variants using molecular markers. It is confirmed. Therefore, the development of a molecular marker that is statistically significant is an essential technique for the development of marker selection help. As a result, the monobasic polymorphism associated with the intramuscular fat content identified in the present invention can be used for the selection of sows. It can greatly contribute to upbringing.

In order to amplify and confirm the transcriptional regulatory region of the adenylate kinase (AK4) gene, two pairs of primers were prepared and used as shown in [Table 1]. PCR reaction buffer (500mM KCl; 100mM Tris-Cl, pH 8.2; 15mM MgCl ₂ ; 0.1% Triton X-100, TaKaRa Co, Japan) 2.5 μl, 2.5 μm dNTPs 2 μl, 1 pm primer of 10 pmole , 25-50ng genomic DNA, 1U Taq DNA polymerase (CoreBio) was added to the final volume of 25μ was filled with sterile distilled water. PCR amplification reaction was 94 ℃ 30 minutes, after the denaturation reaction 94 ℃ 30 seconds, 53 ℃ 30 seconds, 72 ℃ 1 minute 10 seconds amplification for 35 revolutions and then performed the final extension reaction at 72 ℃ 10 minutes. After PCR, 5 μl of PCR product was amplified in 1.2% agarose gel. On the other hand, after determining the base sequence to determine whether the adenylate kinase (AK4) gene is correct for the amplification product, the DNA sequence was compared to confirm that the desired adenylate kinase (AK4) gene [Fig. 1] .

In order to discover a single base polymorphism in the transcription control region of the adenylate kinase (AK4) gene, the product amplified by PCR reaction was purified using a PCR amplification product purification kit (PCR Purification Kit, CoreBio PPHTS-30). Direct sequencing was performed using BigDye Terminator ver3.1 Cycle Sequencing Kit (Applied Biosystems) and ABI 3730XL DNA sequencer (Applied Biosystems).

      The nucleotide sequence determined by individual was searched for the single nucleotide polymorphism in the DNA nucleotide sequence through multi-alignment using SeqMan program (DNASTAR).

As a result of the correlation analysis between the single nucleotide polymorphism and the trait found in the transcription control region of the adenylate kinase (AK4) gene, as shown in Table 2, the locus significantly associated with the intramuscular fat content was based on the transcription start base. When set to (+1), the polymorphism of -196 C / T showed statistical significance of 5%. This polymorphism showed an additive effect as shown in Table 2, where the TT genotype had the highest intramuscular fat content, the CT genotype was the next highest, and the CC genotype had the lowest intramuscular fat content. Therefore, it would be advantageous to select individuals with TT genotype to select individuals with high intramuscular fat content.

TABLE 1 Primers for monobasic polymorphism analysis in the transcriptional regulatory region of adenylate kinase (AK4) gene

Primer set Primer Sequence Amplification Product Size (bp) Annealing Temperature (℃) AK4_pro_1 Fw: 5-TAAGTACAGTGCCAACATCG-3 '
Rv: 5-CCTCCCATCTCTATCCCTTA- ' 1096 55

Table 2: Association of -196 C / T polymorphisms in the electronic regulatory region of adenylate kinase (AK4) genes with intramuscular fat content in pigs

   Figure 1 shows the nucleotide sequence of the transcriptional regulatory region of the adenylate kinase (AK4) gene.

Figure 2 shows the expression pattern of -196 C / T polymorphism of the electronic regulatory region of the adenylate kinase (AK4) gene.

Claims (5)

A marker factor involved in the intramuscular fat content of the pig including a -196 locus on the basis of the transcription initiation base of the Adenylate kinase gene transcription control region in the swine chromosome 6 (+1). The method of claim 1, The -196 locus is a TT genotype, characterized in that the marker factor involved in the fat content of pig muscle. (1) determine monobasic polymorphisms (SNPs) on specific loci of genes related to pig intramuscular fat content; (2) analyzing the intramuscular fat content trait of pigs based on the above analysis results; Selection method of sows consisting of the process. The method of claim 3, wherein Piglet intramuscular fat content-related gene in the step (1) is characterized in that the transcriptional regulatory region (promoter region) of the adenylate kinase gene in pig chromosome 6. The method of claim 4, wherein Single base polymorphisms (SNPs) in the process (1) is -196 selection method, characterized in that the phase shift of -196 locus based on the transcription start base.

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