KR100774849B1 - Development of molecular marker of fatty acid-binding proteinFABP gene associated with longissimus muscle area and backfat thickness in Korean cattle - Google Patents

Abstract

    The present invention is to determine the carcass weight and carcass weight of individual cattle as the main breeding target traits of Korean cattle. As a molecular labeling selection method, an intracellular protein having a function of transporting fatty acids from a cell membrane to a fatty acid oxidation, phospholipid, or triacylglycerol synthesis site, a fatty acid binding protein having a function of intracellular fatty acid concentration and a specific fat metabolism ( Analysis of restriction fragment length polymorphism (RFLP) of the fatty acid-binding protein (FABP) gene and the development of RFLP molecular markers related to the dorsal root cross-sectional area and the backfat thickness of Hanwoo carcass Prediction and selection of Korean cattle with high carcass weight and carcass weight And it includes information for using the tool.

In the present invention, a primer comprising a specific region of Intron No. 1 of the Hanwoo Heart Fatty Acid Binding Protein (H-FABP) gene was amplified by PCR, cloned, and analyzed by sequencing. In this area, a single nucleotide polymorphism (SNP) site was found, and the association of these monobasic polymorphisms with meat quality and carcass traits of Hanwoo was analyzed.

That is, the single base polymorphism (SNP) site generated by cytosine (C) ↔ thymine (T) base substitution was discovered at the 64th base sequence of the total 302 bp sequence including the specific region of intron 1 of the Hanwoo H-FABP gene. It was. This monobasic polynucleotide sequence region was digested with TspRI restriction enzyme using PCR-RFLP technique and three genotypes (C / C, C / T and T / T) were detected. Among them, the C / C genotype was found to be significantly associated with the backfat thickness of Hanwoo and the T / T and C / T genotypes were significantly associated with the dorsum muscle cross-sectional area.

Therefore, the present invention is broad in the length of the dorsal muscle of the heart fatty acid binding protein (H-FABP) gene having a significant effect on the backfat thickness and Longgissimus muscle area of the Hanwoo This study aims to develop a DNA marker that uses a thin SNP genotype to improve the genetic ability of Korean cattle, regardless of their age and gender, and to predict and select Korean cattle with high carcass weight and carcass weight.

Hanwoo, cardiac fatty acid binding protein (H-FABP), sequencing, PCR-RFLP analysis, dorsal muscle cross-sectional area, dorsal fat thickness-related DNA markers    

Description

Development of molecular marker of fatty acid-binding protein (FABP) gene associated with longissimus muscle area and backfat thickness in Korean cattle}

1: Sequencing the SNP region of the Hanwoo heart fatty acid binding protein (H-FABP) gene in the present invention

Figure 2: PCR-RFLP analysis using TspR I restriction enzyme at the 64th monobasic polymorphism (SNP) site in the intron 1 region of the Hanwoo heart fatty acid binding protein (H-FABP) gene in the present invention

FIG. 3: Chromatogram of the 64th single nucleotide polymorphism (SNP) analysis in a specific amplification site of the Hanwoo heart fatty acid binding protein (H-FABP) gene intron 1 in the present invention

  The present invention is to determine the carcass weight and carcass weight of individual cattle as the main breeding target traits of Korean cattle. As a molecular labeling selection method, an intracellular protein having a function of transporting fatty acids from a cell membrane to a fatty acid oxidation, phospholipid, or triacylglycerol synthesis site, a fatty acid binding protein having a function of intracellular fatty acid concentration and a specific fat metabolism ( Analysis of restriction enzyme fragment polymorphism (RFLP) of fatty acid-binding protein (FABP) gene and the development of RFLP molecular markers related to dorsum muscle cross-sectional area and dorsal fat thickness among carcass traits of Hanwoo, improving heredity and carcass weight and carcass The purpose of this study is to use high-yield Hanwoo as a tool to predict and select early.

  In general, the improvement of capacity for beef meat and meat quality has been largely dependent on the statistic breeding approach based on quantitative genetics. The conventional Hanwoo breeding method requires a capacity test for large herds and selects individuals with excellent genetic ability through subsequent tests, and it takes a long time to improve the capacity and relies only on phenotypic measurements governed by genetics and the environment. As a result, the efficiency of selection is not high, and the improvement efficiency is greatly reduced. Carcass traits also have many difficulties in improving carcass traits that rely only on traditional breeding methods because phenotypic measurements are difficult in livestock and accurate measurements are possible after slaughter. As such, the traditional Hanwoo selection and breeding method requires a lot of cost, time and labor, which means that it is a high-cost, low-efficiency breeding system. Therefore, in order to maximize the efficiency and improve the performance of carcass quality, which is the most important improved quality of Hanwoo, it is necessary to actively develop new breeding techniques and breeding strategies.

  Recent advances in molecular genetics and biotechnology offer the possibility of developing more accurate and powerful new breeding models for animal genetic improvement. The approach using advanced molecular genetic techniques provides new insights into breeding and improvement projects by providing breakthrough information that can overcome the problems of traditional breeding methods in terms of breeding lead times and genetic capacity assessment accuracy. have. In particular, research on the development and use of DNA markers related to economic traits can directly identify genetic differences between animal individuals at the molecular level of DNA, enabling more accurate use of genetic information for estimating breeder value and assessing genetic capacity. Although DNA markers can be applied fundamentally to individual identification, paternity, breed identification, and household analysis, which are the basis for livestock improvement, their use as DNA markers associated with economically important traits in livestock has greater value in breeding improvement. do. The big advantage of using economically relevant DNA markers for animal breeding is that most DNA markers are co-dominant and follow the simple Mendelian genotype, so they can directly determine the genotype of an individual and are not affected by both sex and age. As a result, the genetic potential of an individual can be predicted and selected early. In the meantime, we have developed various DNA markers according to the remarkable progress of DNA marker analysis technology and analyzed the association between these markers and major economic traits to detect related DNA markers affecting quantitative traits, The development of molecular selection sarcoma by marker-assisted selection (MAS), which directly selects individuals with genotypes, is a direct selection sarcoma by genotype itself in a traditional selection sarcoma system that was dependent solely on conventional phenotypes. This realization made it a new era in animal breeding.

  Fatty Acid-Binding Protein (FABP) is one of the evolutionarily conserved groups of proteins involved in long chain fatty acid metabolism (Hirsch et al., 1998). Β-oxidation and triacylglycerols from plasma membranes (triacylglycerol) or phospholipids are small intracellular proteins that transport fatty acids to synthetic sites (Roy et al., 2003). In particular, fatty acid binding proteins are known to regulate intracellular fatty acid concentrations and various cellular and lipid metabolism (Veerkamp et al., 1995) and because of this physiological action are noted as candidate genes for intramuscular fat content in mammals. (Nechtelberger et al., 2001). The heart-type fatty acid binding protein (H-FABP) gene is mainly expressed in muscle cells, while the fat-type fatty acid binding protein (A-FABP) is almost entirely expressed in adipocytes. In particular, H-FABP protein, which is present in many tissues with high fatty acid demand, such as myocardium and skeletal muscle or metabolic mammary tissue (Roy et al., 2003), has been reported to be associated with intramuscular fat content, backfat thickness and growth rate in pigs. (Gerbens et al., 1998, 1999, 2000). However, in case of cattle, Roy et al. (2003) recently reported that the H-FABP3 gene was located on chromosome 2 by RH mapping technique. However, there have been no reports on the association between carcass trait and meat quality.

  The present invention utilizes a restriction enzyme fragment polymorphism (RFLP) molecular marker of the H-FABP gene, which has a function of fatty acid transport and fat metabolism in the cytoplasm of mammals, to determine the carcass weight and carcass weight of the individual. The large, backfat thickness provides advanced technology using DNA molecular markers for early selection of Hanwoo cattle.

The present invention is to find out the restriction enzyme fragment polymorphism (RFLP) marker of cardiac fatty acid binding protein (H-FABP) gene involved in fatty acid transport and lipid metabolism of higher animals in Hanwoo and PCR-RFLP (restriction fragment length Polymorphism) analysis method Development of carcass trait-related RFLP marker genotypes significantly affecting the dorsal root cross-sectional area and backfat thickness of Korean cattle, ultimately identifying DNA markers for early selection of Hanwoo cattle with high carcass weight and carcass weight It is used to actively utilize high-performance Hanwoo breeding and improvement.

The present invention will be described in detail according to the following examples.

Example 1 Detection of SNP Genotype of Hanwoo Heart Fatty Acid Binding Protein (H-FABP) Gene

1. Test material and DNA separation and purification

Hanwoo used in the present invention was registered in the National Hospitality Assurance Project, and a total of 500 candidate species of cattle breeds with lineage records and carcass scores were selected as a public axis through the Hospitality Test. Separation and purification of genomic DNA from each coaxial blood was separated and refined by some modifications of Miller et al. (1988). After measuring the DNA concentration using a spectrometer, TE buffer (10 mM Tris-HCl, pH 7.4; 1 mM EDTA), stored in -20 ℃ freezer and used as a test material.

2. Detection of single nucleotide polymorphism (SNP) of Hanwoo heart fatty acid binding protein (H-FABP) gene by PCR-RFLP technique

Primers for amplifying a 302bp fragment containing a specific region of intron 1 of the Hanwoo heart fatty acid binding protein (H-FABP) gene were designed based on the nucleotide sequence information registered in GenBank (DQ174319). The base sequences of the primers used in the present invention are as follows <Table 1>.

 Primer sequencing for PCR amplification of the Hanwoo heart fatty acid binding protein (H-FABP) gene gene Primer Sequence (5'-3 ') Amplified area Fragment size (bp) H-FABP (3460-3761) F: AACGTCTTTGAAGGTTCTCA R: CACCTTGACTTTCCTGTCAT Intron 1 302

The TspRI restriction enzyme recognition site (5'-NNCASTGNN ^▼ -3 ') is present at the 64th SNP region of the intron 1 region amplified from the 3460 to 3761 bases of the Hanwoo heart fatty acid binding protein (H-FABP) gene. Then, three types of SNP genotypes (C / C, C / T and T / T) of the heart fatty acid binding protein (H-FABP) gene were detected by PCR-RFLP technique (Fig. 2). PCR-RFLP analysis of the Hanwoo heart fatty acid binding protein (H-FABP) gene was performed by adding 5 units of TspRⅠ restriction enzyme to approximately 5µl of PCR amplification products and then reacting at 65 ° C incubator for 3 hours or more. DNA fragments obtained by cleaving PCR amplification products with restriction enzymes were electrophoresed for 3 hours using a 3% agarose gel using TBE buffer (90 mM tris-borate, 2 mM EDTA, pH 8.0) to observe the DNA band pattern and each assay. Individual SNP genotypes were determined. First, in the case of the C allele, one restriction enzyme recognition site exists, resulting in two bands having a size of 68 and 234 bp, respectively. In the T allele, there was no restriction enzyme recognition site, so the original amplification product size was 302bp. band was detected. In addition, the C / T heterozygote type detected all three of these bands and showed a banding pattern of 68, 234 and 302bp. In this figure, only 68234 bands with less than 100bp of low expression level are not shown, so only C / C type shows 234bp band, T / T type shows 302bp, and C / T type shows only 234bp and 302bp bands. >. The frequency of detected allele was 28.2% for the C allele and 71.8% for the T allele, with a significantly higher incidence of the T allele and 1.4% of the C / C genotype. , C / T type was about 53.7%, and T / T type was 44.9%, showing the highest frequency of C / T heterozygote type.

Example 2 Analysis of Association Between SNP Genotypes of Carcasses of Heart Fatty Acid Binding Protein (H-FABP) and Carcass and Meat Quality

To investigate the effect of the SNP genotype of the Hanwoo heart fatty acid binding protein (H-FABP) gene on the estimates of carcass and meat quality breeding values, the MTDFREML computer program (Boldman et al., 1993) was used. The statistical analysis of the association between the SNP genotype of FABP) gene and various carcass and meat traits showed that it had a significant effect on the backfat thickness and the dorsum muscle cross-sectional area (Table 2).

That is, in the present invention, the C / C genotype in the intron 1 region of the Hanwoo heart fatty acid binding protein (H-FABP) gene has a backfat thickness of 1.150 and is about 0.526 and 0.494, respectively, compared to the C / T and T / T types. It was higher (P <0.05), and the C / C type was 0.0792, which was significantly higher than the 0.135 and -0.048 of C / T and T / T type. 0.05). In addition, the C / C SNP genotype was -4.26 and significantly lower than that of C / T type 0.101 and T / T type 1.213 (4.361 and 5.473, respectively). <0.05).

 Effect of SNP Genotype on Intron 1 Region of Hanwoo Heart Fatty Acid Binding Protein (H-FABP) Gene Carcass and meat quality SNP genotype P-value C / C type C / T type T / T type Live weight / kg 585.000 ± 36.121 540.379 ± 5.747 548.939 ± 6.287 0.3253 Conductor weight / kg 328.500 ± 23.200 308.531 ± 3.691 315.015 ± 4.038 0.3825 Conductor Rate /% 56.200 ± 1.095 57.048 ± 0.174 57.333 ± 0.190 0.3698 Back fat thickness / cm 1.150 ± 0.189 ^a 0.624 ± 0.030 ^b 0.656 ± 0.033 ^b 0.0238 ^* Longest root cross-sectional area / ㎠ 69.000 ± 5.787 74.518 ± 0.920 76.045 ± 1.007 0.3060 Local muscle degree / 1 ~ 7 3.000 ± 0.875 2.177 ± 0.139 1.909 ± 0.152 0.2460 Carcass weight (breed price) / kg 5.705 ± 7.189 2.436 ± 1.143 5.078 ± 1.251 0.2882 Longest root cross-sectional area (breed price) / ㎠ -4.260 ± 2.405 ^b 0.101 ± 0.382 ^a 1.213 ± 0.418 ^a 0.0215 ^* Back fat thickness (breed price) / cm 0.792 ± 0.401 ^a 0.135 ± 0.063 ^ab -0.048 ± 0.069 ^b 0.0311 ^* Local muscle map (breed price) / 1 ~ 7 0.199 ± 0.090 -0.026 ± 0.014 -0.039 ± 0.015 0.0762

^* P <0.05

^{a, b} statistically significant difference (P <0.05)

Therefore, it was proved that a specific SNP genotype of the Hanwoo heart fatty acid binding protein (H-FABP) gene of the present invention had a high correlation with the dorsal fat thickness and the dorsum muscle cross-sectional area of Hanwoo. In other words, individuals with the C / T and T / T genotypes were found to have a smaller backfat thickness and broader dorsum muscle area than those with the C / C genotype, resulting in improved heredity and higher carcass weight and carcass volume. It can be used as a tool to predict and select Hanwoo early.

  The molecular marker of the fatty acid binding protein (H-FABP) gene of the present invention described in detail above directly affects carcass weight and carcass weight, which influence the breeding grade of the individual in relation to the selection breeding improvement project of high-capacity Hanwoo cattle at the national level. Its effect is to identify and select early breeders of long-eared and thin-backed cows with wide backbone area and back fat thickness, which can lead to improved selection efficiency and genetic improvement.

In addition, in the cattle breeding farming unit, when the grading grade is determined after slaughter, it is possible to reduce the feed cost significantly and reduce the management cost such as the specification management labor and labor force by early identifying and breeding high-quality calf or beef cattle that can cause large carcass weight and carcass weight. As a result, the rationalization of management through productivity improvement can greatly increase the economic income of farmers.

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Claims (3)

The Hanwoo shown in SEQ ID NO: 1 by PCR-RFLP analysis comprising the step of treating the TspR I restriction enzyme to the Hanwoo H-FABP gene amplified product amplified by the primers of SEQ ID NO: 2 and SEQ ID NO: 3 in the present invention H-FABP gene sequence The 64th C↔T detection of individual SNP markers according to base substitution and analysis of each genotype (CC, CT and TT type) predicted Hanwoo beef with thin back fat thickness and broad dorsum muscle area How to judge The method according to claim 1, Among the three SNP marker genotypes (CC, CT, and TT) of the Hanwoo H-FABP gene detected by the PCR-RFLP assay of the present invention, Hanwoo individuals exhibiting CT and TT genotype markers have thin back fat thickness and broad dorsum muscle cross-sectional area How to predict by judging delete

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