KR20160053670A - Novel snp marker for discriminating level of muscle fiber type i within porcine muscle and use thereof - Google Patents

Abstract

The present invention relates to: a SNP marker for determining a level of muscular fiber type I in swine muscle; a composition for determining a level of muscular fiber type I in swine muscle, comprising the SNP marker; a kit for determining a level of muscular fiber type I in swine muscle, comprising the composition; a microarray; a method for determining a level of muscular fiber type I in swine muscle; and a method for manufacturing swine at the increased level of muscular fiber type I in muscle. The composition for determining a level of muscular fiber type I in swine muscle comprises a formulation, capable of detecting or amplifying a polynucleotide constituted of 5 to 1000 continuous bases having single nucleotide polymorphism (SNP) or a complementary polynucleotide thereof. The SNP marker of the present invention can be used as a means for objectively evaluating a level of muscular fiber type I, which influences the meat quality in swine.

Description

SNP markers for judging the level of muscle fiber type I in pigs and their uses [

The present invention relates to a composition for determining the level of muscle fiber type I in a pig comprising the SNP marker, a SNP marker for determining the level of muscle fiber type I in the muscle of a pig, a level of muscle fiber type I in a pig containing the composition A judging kit, a microarray, a method for judging the level of muscular fiber type I in a muscle of a pig, and a method for producing a pig with an increased level of intramuscular muscle fiber type I.

Since breeding in the eastern part of India about 9,000 years ago, pigs have been raised as the most basic animal for the worldwide consumption of protein that people need, according to their age, situation and people's preferences.

In addition, pigs have been raised for a long period of time to increase feed efficiency and growth rate. As a result, stress-sensitive pigs have been produced and meat quality has been lowered.

The meat quality of pigs has been reported to be influenced by muscle fiber composition rather than number and size of muscle fibers. Muscle fiber in pigs is divided into three types depending on the type of myosin-heavy chain responsible for ATPase activity. Because muscle fiber type I slows the post-metabolism rate through slow-oxidative action, high content of muscle fiber type I has a positive effect on meat quality, while muscle fiber type IIb is fast-glycolytic ), So that the post-metabolism is performed rapidly. Therefore, when the content of muscle fiber type IIb is high, the color of meat is pale, and the possibility that the tissue is disturbed is judged to be abnormal. The remaining muscle fiber type IIa exhibits a slow-glycolytic property as an intermediate property. In order to improve the meat quality, studies are needed to increase the proportion of muscle fiber type I in the muscle fiber composition in the muscle.

The myocardial characteristic is a characteristic that can be measured only at the rosaceus muscle of the conductor, and measurement in the living body is impossible. Therefore, there is a need for a method for predicting the myocardial characteristics of an individual without slaughtering, and development of DNA marker and SNP markers is required in this respect. As a part of this research, various DNA analysis techniques such as random amplified polymorphic DNA (RAPD) and single strand conformation polymorphism (SSCP) using PCR techniques have been used as methods for analyzing genes. For example, Korean Patent Publication No. 2007-0113336 discloses a DNA marker for confirming the increase in the number of pig muscle cells using a mutation (SNP) due to a single nucleotide sequence difference in the 5 'promoter region of the Myogenin gene known to be involved in the myocardial differentiation of pigs Korean Patent Laid-Open Publication No. 2011-0139011 discloses a method for evaluating meat quality using a single-trait polymorphism biomarker for diagnosing fat content in pigs, Korean Patent Laid-Open Publication No. 2012-0052796 discloses a method for evaluating meat quality (SNP) site of the PPARGC1A gene involved in the characteristics of the pigs. However, no method has yet been developed for judging muscle fiber types affecting the meat quality of pigs, or for improving meat quality by controlling the level of certain types of muscle fibers.

Under these circumstances, the inventors of the present invention have made extensive efforts to develop a method for manufacturing pigs having improved meat quality by determining the level of muscle fiber type I and increasing the level of muscle fiber type I. As a result, The present inventors completed the present invention by confirming that the level of myofibrillar type I in the genetically determined pigs can be determined and increased by using SNPs.

It is an object of the present invention to provide a composition for judging the level of muscle fiber type I in a muscle of a pig.

Another object of the present invention is to provide a kit for judging the level of muscle fiber type I in a pig, comprising a composition for judging the level of muscle fiber type I in the muscle of the pig.

It is yet another object of the present invention to provide a method for determining the level of muscle fiber type I in a muscle of a pig.

It is another object of the present invention to provide a method for producing pigs with increased levels of muscle fiber type I in the muscles of pigs.

It is another object of the present invention to provide a method for increasing the level of muscle fiber type I in the muscle of a pig.

Another object of the present invention is to provide a SNP marker for determining the level of muscle fiber type I in the muscle of a pig.

It is a further object of the present invention to provide a microarray for determining the level of muscle fiber type I in the pig, comprising a SNP marker for determining the level of muscle fiber type I in the muscle of the pig.

In one embodiment of the present invention, there is provided a polynucleotide comprising a polynucleotide having a nucleotide sequence of SEQ ID NO: 1, wherein the 501st nucleotide is G or A, and the polynucleotide comprising a nucleotide polymorphism (SNP) A composition capable of detecting or amplifying a polynucleotide consisting of 1 to 1000 consecutive bases or a complementary polynucleotide thereof, for determining the level of muscle fiber type I in a pig.

The "polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1" is a polymorphic sequence comprising a polymorphic site of a gene involved in the muscle fiber composition in the pig, wherein the polymorphic sequence is a polymorphic sequence comprising a SNP Quot; polymorphic < / RTI > site. The polynucleotide sequence may be DNA or RNA.

The term "polymorphism " of the present invention means a case where two or more alleles exist in one locus, and among polymorphic sites, only one single base is different from the single It is called single nucleotide polymorphism (SNP). Preferred polymorphic markers have two or more alleles with a frequency of occurrence of 1% or more, more preferably 5% or 10% or more in the selected population.

The term "allele " of the present invention refers to various types of a gene existing on the same locus of a homologous chromosome. Alleles are also used to represent polymorphisms, for example, SNPs have two kinds of bialles.

Specifically, the pig to be judged in the level of muscle fiber type I in the pig may be a hybrid of Jeju native pig, Duroc or Jeju native pig and duroc.

In one embodiment of the present invention, genes and SNP markers related to the types of muscle fibers were analyzed for the cross-reference groups of Korean traditional pigs and Duroc, and SNPs with the highest significance in relation to the ratio of muscle fiber type I were identified as ALGA0067099 , A nucleotide sequence was further extracted on the basis of the SNP using a next-generation nucleotide sequence analysis, and PCR was performed using the nucleotide sequence as a template. When the PCR product is treated with restriction enzyme Hha I, a single band of 499 bp, which is not cleaved, appears, resulting in expression of myofiber type I, and when the 501st base of the nucleotide sequence of SEQ ID NO: 1 is A, the level of muscle fiber type I is increased (Table 2).

The term "agents capable of detecting or amplifying SNP markers" in the present invention refers to agents capable of specifically recognizing binding to a polymorphic site containing a SNP in a gene and capable of amplifying the polymorphic site Specifically, a probe capable of specifically binding to a polymorphic site containing the SNP, a polynucleotide comprising the SNP marker, or a primer capable of specifically amplifying a complementary polynucleotide thereof.

More specifically, it may include, but is not limited to, a pair of primers consisting of SEQ ID NO: 2 and SEQ ID NO: 3 capable of amplifying the polynucleotide of SEQ ID NO: 1.

In the present invention, a probe used to recognize and bind to a SNP marker includes a sequence complementary to a polynucleotide sequence including a SNP, and may be a DNA, RNA, or DNA-RNA hybrid form . Further, fluorescent markers, radiation markers, and the like can be additionally attached to the 5 'or 3' ends of the probe so as to be visually recognizable.

The term " primer " of the present invention means a base sequence having a short free 3 'hydroxyl group and can form base pairs with a complementary template, It means a short sequence functioning as a point. The primers used in the present invention for the amplification of SNP markers can be amplified by PCR using appropriate conditions in suitable buffers (for example, 4 different nucleoside triphosphates and polymerase such as DNA, RNA polymerase or reverse transcriptase) Stranded oligonucleotide that can serve as a starting point for the directed DNA synthesis. The appropriate length of the primer may vary depending on the purpose of use, but it may be generally used in a size of 15 to 30 nucleotides. The primer sequence is not necessarily completely complementary to the polynucleotide comprising the SNP marker or its complementary polynucleotide, and can be used if it is sufficiently complementary to hybridize.

The primers can also be modified, for example, by methylation, capping, substitution of nucleotides or modifications between nucleotides, such as uncharged linkers (e.g., methylphosphonate, phosphotriester, phosphoramidate, Carbamates, etc.) or charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.).

In the present invention, the term " muscle fiber " refers to a fibrous unit structure constituting muscle or muscle tissue of an animal, and generally shows a polynuclear and elongated shape. Each muscle fiber is classified into oxidative metabolism, anaerobic metabolism, rate of contraction, fiber size, glycogen content, capillary ratio, and is closely related to the occurrence of meat quality, weight, post - metabolism, abnormal muscle.

In addition, the muscle fibers are classified into three types of muscle fiber types I, IIa and IIb, and the muscle fiber type I is also referred to as red muscle fiber. In addition, the muscle fiber type IIa exhibits an intermediate shape characteristic, and the muscle fiber type IIb exhibits white color and is also called white muscle fiber. Muscle fiber types I and IIa have a lot of mitochondria and myoglobin. Muscles containing a lot of muscle fibers show a lot of red, and muscles containing muscle fiber type IIb are relatively white.

With respect to meat quality, there is a difference in the meat quality level according to the composition of muscle fiber in muscle. As the ratio of muscle fiber type Ⅰ is lower and the ratio of muscle fiber type Ⅱb is higher, the meat quality level is lowered and the possibility of abnormal meat is increased.

In one embodiment of the present invention, genes and SNPs involved in muscle fiber type in the pigs were confirmed (FIG. 7), and it was confirmed that the level of muscle fiber type I could be increased by fixing the base at the SNP position with A . Further, it is obvious to those skilled in the art that the present invention can be applied to compositions and methods for improving the meat quality level of pigs by increasing the level of muscle fiber type I.

Another embodiment of the present invention provides a kit for determining the level of muscle fiber type I in a pig of the composition. Specifically, the kit may be, but not limited to, a PCR (Polymerase Chain Reaction) kit or a DNA analysis kit (for example, a DNA chip).

The kit of the present invention can determine the level of muscle fiber type I in the pigs by amplifying a gene involved in the muscle fiber composition of the pigs and then confirming the appearance of the band after cleavage with a restriction enzyme. The PCR kit may comprise a test tube or other appropriate container, a reaction buffer (varying in pH and magnesium concentration), deoxynucleotides (dNTPs), and the like, in addition to the respective primer pairs specific for polynucleotides comprising SNP markers or their complementary polynucleotides. ), Enzyme such as Taq polymerase, DNase, RNAse inhibitor, DEPC-water, sterilized water and the like. Specifically, the PCR kit may be a kit for performing PCR-RFLP.

In the present invention, the "PCR-RFLP" method is a technique for analyzing the restriction fragment length polymorphism (PCR) of gene fragments among various DNA analysis techniques for improving the genetic characteristics and capability of livestock. the presence of restriction enzyme recognition sites in the point mutation region allows the use of the fact that the lengths of the fragments generated by restriction enzymes vary according to the genotype difference of each individual, . In one embodiment of the present invention, a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 is amplified by PCR reaction, and the resulting PCR product is digested with restriction enzymes, followed by electrophoresis to observe the band. , It was judged that the base at the SNP position was A. When the allelic genotype was A / A, it was confirmed that the ratio of the muscle fiber type I in the pig was increased. Thus, when the base at the SNP position was A, Ⅰ (Fig. 8 and Table 2).

Specifically, the kit of the present invention may be a kit for determining the level of muscle fiber type I in a pig, which contains essential elements necessary for carrying out a DNA chip. DNA chip kits are those in which nucleic acid species are attached in a gridded array on a generally flat solid support plate, typically a glass surface not larger than a slide for a microscope, and nucleic acids are uniformly arranged on the chip surface, Hybridization reaction occurs between the nucleic acid on the surface of the chip and the complementary nucleic acid contained in the treated solution on the surface of the chip to enable massive parallel analysis and can be used to determine the level of muscle fiber type I in the pig have.

(A) amplifying a polymorphic site of a polynucleotide consisting of the nucleotide sequence of SEQ. ID. NO. 1 or its complementary polynucleotide from the DNA of the sample isolated from the individual; And (b) determining the base of each amplified polymorphic site of step (a). ≪ IMAGE > At this time, DNA of the separated sample can be obtained from a sample isolated from an individual.

The term " individual " of the present invention means a pig to which the level of muscle fiber type I in the muscle is to be inspected. By analyzing the SNP using the sample obtained from the pig, the composition of the muscle fiber in the muscle of the pig and the muscle fiber type I Can be determined. As the specimen, DNA can be obtained from hair, urine, blood, various body fluids, separated tissues, isolated cells, or saliva, but is not limited thereto. In particular, in order to determine the types of muscle fibers in the conventional pigs, in order to obtain the sirloin region of the pigs, measurement in the living body was impossible and slaughtering had to be performed. However, by using the method of the present invention, The type of muscle fiber can be judged.

The step of amplifying the polymorphic site of the SNP marker from the DNA of step (a) or hybridizing with the probe may be performed by any method known to those skilled in the art. For example, the target nucleic acid can be obtained by PCR amplification and purification thereof. Other ligase chain reaction (LCR) (Wu and Wallace, Genomics 4, 560 (1989), Landegren et al., Science 241, 1077 (1988)), transcription amplification (Kwoh et al., Proc. Natl. Acad. Sequence amplification based on nucleic acids (NASBA) can be used as well as self-sustaining sequence replication (Guatelli et al., Proc. Natl. Acad. Sci. USA 87, 1874 (1990)).

Determination of bases in the polymorphic site of step (b) of the above method can be carried out by sequencing, hybridization by microarray, allele specific PCR, dynamic allele-specifichybridization, DASH), PCR extension analysis, PCR-SSCP, PCR-RFLP analysis or TaqMan technique, SNPlex platform (Applied Biosystems), mass spectrometry (e.g. MassenRAY system of Sequenom), mini- But are not limited to, the BioRad system, the CEQ and SNPstream system (Beckman), the Molecular Inversion Probe array technology (e.g. Affymetrix GeneChip), and BeadArray Technologies (e.g. Illumina GoldenGate and Infinium analysis) Do not. One or more alleles in a polymorphic marker, including microsatellite, SNP or other types of polymorphic markers, can be identified by such methods or by other methods available to those skilled in the art to which this invention pertains. The determination of the base of such a polymorphic site can be carried out specifically through the SNP chip.

The term " SNP chip " means one of DNA microarrays capable of confirming each base of several hundred thousand SNPs at a time.

The TaqMan method comprises the steps of: (1) designing and constructing a primer and a TaqMan probe to amplify a desired DNA fragment; (2) labeling probes of different alleles with FAM dyes and VIC dyes (Applied Biosystems); (3) performing PCR using the DNA as a template and using the primer and the probe; (4) after completion of the PCR reaction, analyzing and confirming the TaqMan assay plate with a nucleic acid analyzer; And (5) determining the genotype of the polynucleotide of step (1) from the analysis result.

Sequencing analysis can be performed using conventional methods for sequencing and can be performed using an automated gene analyzer. The allele-specific PCR means a PCR method in which a DNA fragment in which the SNP is located is amplified with a primer set including a primer designed with the base at the 3 'end at which the SNP is located. The principle of the above method is that, for example, when a specific base is substituted by A to G, an opposite primer capable of amplifying a primer containing the A as a 3 'terminal base and a DNA fragment of an appropriate size is designed, In the case of performing the reaction, when the base at the SNP position is A, the amplification reaction is normally performed so that a band at a desired position is observed. When the base is substituted with G, the primer can complementarily bind to the template DNA , And 3 'ends do not perform complementary binding, so amplification reaction is not performed properly. DASH can be carried out by a conventional method, specifically, by the method by Prince et al.

On the other hand, in the PCR extension analysis, first, a DNA fragment containing a base in which a SNP is located is amplified as a primer pair, and then all nucleotides added to the reaction are deactivated by dephosphorylation, and SNP specific extension primer, dNTP mixture , Digoxinucleotide, reaction buffer, and DNA polymerase to perform primer extension reaction. At this time, the extension primer has a base immediately adjacent to the 5 'direction of the base in which the SNP is located at the 3' terminus, the nucleic acid having the same base as the didyxin nucleotide is excluded in the dNTP mixture, and the didyoxynucleotide indicates the SNP Base type. ≪ / RTI > For example, when dGTP, dCTP and TTP mixture and ddATP are added to the reaction in the presence of substitution from A to G, the primer is extended by the DNA polymerase in the base in which the substitution has occurred, The primer extension reaction is terminated by ddATP at the position where the base first appears. If the substitution has not occurred, the extension reaction is terminated at the position, so that it is possible to discriminate the type of the base representing the SNP by comparing the lengths of the extended primers.

As the detection method, when the extension primer or the dioxynucleotide is fluorescently labeled, the SNP is detected by detecting fluorescence using a gene analyzer (for example, Model 3700 of ABI Co., Ltd.) used for general nucleotide sequence determination And when the unlabeled extension primer and the didyxin nucleotide are used, the SNP can be detected by measuring the molecular weight using MALDI-TOF (matrix assisted laser desorption ionization-time of flight) technique.

Specifically, in the polynucleotide consisting of SEQ ID NO: 1 or its complementary polynucleotide, it can be judged that the level of muscle fiber type I in the muscle of the pig is increased when the allele of the 501st base, which is a polymorphic site, is all A. The pig may be a hybrid of Jeju native pig, Duroc or Jeju native pig and duroc.

In another embodiment of the present invention, there is provided a method for fixing an SNP trait of a gene involved in a muscle fiber type I level in a muscle of a pig, comprising the steps of: (a) isolating an allele of a polynucleotide comprising a nucleotide sequence of SEQ ID NO: Wherein all of the pigs are fixed at the level of A in the pigs.

In another embodiment of the present invention, there is provided a method for fixing an SNP trait of a gene involved in a muscle fiber type I level in a muscle of a pig, comprising the steps of: (a) isolating an allele of a polynucleotide comprising a nucleotide sequence of SEQ ID NO: All of which are immobilized to the muscle of the pig.

In particular, the step of immobilizing the SNP trait may be carried out by crossing an individual having all of the SNP traits, and selecting an individual having the desired trait. More specifically, an individual representing A / A, which is a genotype involved in increasing the level of muscle fiber type I, is crossed and among the offspring obtained therefrom, a polynucleotide of SEQ ID NO: To obtain pigs with increased levels of muscle fiber type I in the pig's muscles. The pig may be a hybrid of Jeju native pig, Duroc or Jeju native pig and duroc.

Another embodiment of the present invention provides a SNP marker for determining the level of muscle fiber type I in a pig, which is located at the 501st base, wherein the 501st base is G or A in the polynucleotide of SEQ ID NO: 1 . Specifically, the pig may be a hybrid of Jeju native pig, Duroc or Jeju native pig and duroc. Regarding the SNP marker of the present invention, when the base is A, the level of muscle fiber type I in the pig of the pig is increased, and thus it can be judged that the pig has a higher level of meat quality than the general pig.

Another embodiment of the present invention provides a microarray for determining the level of muscle fiber type I in muscle of a pig, which comprises the SNP marker.

The microarray may comprise DNA or RNA polynucleotides. The microarray comprises a conventional microarray except that the polynucleotide of the present invention is contained in the probe polynucleotide.

Methods for preparing microarrays by immobilizing probe polynucleotides on a substrate are well known in the art. The probe polynucleotide means a polynucleotide capable of hybridizing, and means an oligonucleotide capable of binding to the complementary strand of the nucleic acid in a sequence-specific manner. The probe of the present invention is an allele-specific probe, wherein a polymorphic site is present in a nucleic acid fragment derived from two members of the same species and hybridizes to a DNA fragment derived from one member but not to a fragment derived from another member Do not. In this case, the hybridization conditions show a significant difference in the intensity of hybridization between alleles, and should be sufficiently stringent to hybridize to only one of the alleles. This can lead to good hybridization differences between different allelic forms. The probe of the present invention can be used for detecting the allele gene to determine the composition of the muscle fiber in the muscle of the pig or the level of muscle fiber type I. The determination method includes detection methods based on hybridization of nucleic acids such as Southern blots, and may be provided in a form pre-bonded to a substrate of a DNA chip in a method using a DNA chip.

The process of immobilizing the probe polynucleotide on the substrate in connection with the determination of the muscle fiber muscle composition of the pig or the level of muscle fiber type I of the present invention can also be easily manufactured using this conventional technique. In addition, hybridization of nucleic acids on a microarray and detection of hybridization results are well known in the art. The detection can be accomplished, for example, by labeling the nucleic acid sample with a labeling substance capable of generating a detectable signal comprising a fluorescent material, such as Cy3 and Cy5, and then hybridizing on the microarray and generating The hybridization result can be detected.

The SNP markers of the present invention are specific markers for determining the types of muscle fibers in the pigs, respectively. These markers can be used as a means to objectively evaluate the level of muscle fiber type I that affects the meat quality of pigs Therefore, pigs with improved meat quality can be produced, and it can contribute to establishing the circulation order of pork meat.

FIG. 1 shows a process of sampling a sirloin of a pig.
Figure 2 shows muscle fiber types in the muscle by staining the sirloin tissue of pigs.
FIG. 3 shows the result of GWAS (genome-wide association study) analysis of the average cross-sectional area of muscle fiber type I in Jeju traditional pig and durok cross reference axis group.
FIG. 4 shows the result of a genome-wide association study (GWAS) analysis of the ratio of the area occupied by the muscle fiber type I in the reference group of the Jeju native pig and the duroc crossing.
FIG. 5 shows the result of GWAS (genome-wide association study) analysis of the area ratio of muscle fiber type IIb in Jeju traditional pig and durok cross reference axis group.
Figure 6 shows the gene families found in the QTL region related to the mean area and area ratio of muscle fiber type I in SSC12.
FIG. 7 shows additional nucleotide sequences extracted from the next generation nucleotide sequence analysis based on ALGA0067099, a SNP associated with muscle fiber type I.
FIG. 8 shows the result of reading the genotype of the myofiber type by digesting the PCR product using the gene having the nucleotide sequence shown in FIG. 7 as a template with restriction enzyme HhaI.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

Example  1. Characterization of muscle fiber

1-1. Sampling of pork loin tissue

The muscle of the pigs' sirloin tissue was cut and sampled using a knife machine. The procedure for sampling the sirloin of the above method is shown in Fig.

1-2. Types of Muscle Fibers through Dyeing Type ) division

(Red muscle fiber), Ⅱa (middle type muscle fiber) and Ⅱb (white muscle fiber) were classified into Microtome CRYOSTAT Microtome (microm HM525, Thermo, (OLYMPUS, BX43F, Japan) using an image pro (MediaCybernetics, USA) program at a magnification of 100x with the Acid Pre-incubation mATPase method.

Specifically, a section of 10 μm thickness was prepared using a freezing section and pre-incubation was performed in an acidic (pH 4.7) pre-incubation buffer (100 mM potassium chloride in 100 mM sodium acetate, adjusted to pH 4.7 with acetic acid) Respectively. The cells were washed with distilled water for 2 to 3 seconds and then washed with a washing buffer (20 mM glycine buffer containing 20 mM CaCl ₂ , pH 9.4) for 30 seconds for more smooth culture.

After incubation for 20 to 25 minutes in an incubation buffer (<40 mM glycine buffer containing 20 mM CaCl ₂ , pH 9.4> + 2.5 mM ATP disodium salt> adjust pH 9.4), the cells were treated with 1% calcium chloride aqueous solution (CaCl ₂ ) for 30 seconds After treatment with 2% cobalt chloride aqueous solution for 3 minutes, foreign matter was removed by washing with distilled water.

Then, the sample was treated with 1% aqueous ammonium sulfate solution for 10 seconds, washed with distilled water, and photographed. Statistical analysis was performed after analysis of muscle fiber characteristics using an image analysis program.

As shown in FIG. 2, the type I muscle fibers showed a dark color, the type IIa light color, and the type IIb muscle fibers had an intermediate color, and they were classified into the above three kinds of muscle fiber types.

In addition, type IIb muscle fibers were most distributed in distribution, followed by type IIa muscle fibers and type I muscle fibers in the order of type I muscle fibers.

Example  2. Large Capacity SNP chip For Type of Muscle Using GWAS ( genome - wise  association study ) analysis

A genome-wide association study (GWAS) analysis was performed on muscle fiber types I, IIa and IIb, which constitute the muscle tissue of pigs, using large SNP chips in Jeju traditional pig and duroc cross reference strains.

Specifically, a 60k SNP Beadchip commercially available from illumina was used to read the genotypes of the pigs, and genotypes were read from all chromosomes of the pigs, and the genotypes were used for phenotype and association analysis. At this time, we used the plink program to derive the results by analyzing the association between SNPs and phenotypes assigned to each chromosome.

From the GWAS analysis, the average cross-sectional area of the muscle fiber type I (FIG. 3), the area ratio of the muscle fiber type I (FIG. 4) and the area ratio of the muscle fiber type IIb (FIG. 5) Was found on chromosome 12. In the case of muscle fiber type Ⅱa, no significant locus was found regarding the number of myofibers and the average area and area ratio of myofibers. In case of myofiber type Ⅱb, the number of myofibers and the area occupied by the myofibers were not identified, The locus for the area was found to be significant in chromosome 12, which is the same as myofiber type I (Figs. 3 to 5).

In addition, as shown in FIG. 6, when the gene cluster found in the QTL region related to the average area and area ratio of the muscle fiber type I in SSC12 was found, it was found that 57,950,908 of chromosome 12 was located.

Example  3. Jeju traditional pig  base Black Pig  Analysis of muscle fiber composition in group

The composition of muscle fibers in the Korean black pork group based on Jeju native pig was analyzed using the same method as in Example 1-2.

As shown in Table 1, since the variation of the average area, area ratio and ratio of the number of myofibers in the muscle fiber type I and the muscle fiber type IIb is large, it is possible to control the expression at the gene level and the meat quality improvement effect I could have expected. SNPs that can control muscle fiber type and genes containing them were selected and analyzed.

Example  4. Next Generation Sequence Analysis ( Next Generation Sequencing , NGS ) Related to muscle fiber type Ⅰ SNP Sequence Extraction and Analysis

The SNPs with the highest significance in relation to the ratio of muscle fiber type Ⅰ in Jeju pork and durok mating reference group were identified as ALGA0067099 and the SNPs identified in the whole genome sequence obtained from Jeju pork A base sequence of 500 bp was further extracted on both sides, and further extracted base sequence (SEQ ID NO: 1) was as shown in FIG.

A gene amplification primer was prepared in order to determine the type of muscle fiber using the nucleotide sequence extracted above as a template.

Forward primer: 5'-CAT AGC TGA CTC CCC ACA CTG-3 '(SEQ ID NO: 2)

Reverse primer: 5'-GCC CTT TCT TCT CAA GTA GCT G-3 '(SEQ ID NO: 3)

In the sequence shown in FIG. 7, a red underlined portion indicates a primer position, and a red portion indicates a base mutation portion ([G / A]) for determining the type of muscle fiber. G or A depending on whether the composition of muscle fiber is different.

In particular, when the PCR product (size: 499 bp) generated after performing the PCR reaction using the primer prepared above as the recognition site of the restriction enzyme Hha I [GC / GC] is treated with a restriction enzyme, Two genomic fragments of 229 bp and 270 bp were cleaved and the genotypes were easily distinguishable.

1ul of DNA, 2ul of 10x buffer, 1.5ul of dNTP and 1ul of forward and reverse primers, respectively, were added as the composition of the above PCR reaction, and the remaining was added with distilled water to make a total of 20ul. The PCR reaction conditions were denaturation at 94 ° C for 5 minutes, followed by 1 minute at 94 ° C, 45 seconds at 60 ° C, 45 seconds at 72 ° C, followed by 35 cycles of reaction at 4 ° C.

5 μl of the generated PCR product, 0.3 μl of Hha1 (restriction enzyme), 1 μl of 10 × buffer, 1 μl of BSA and 2.7 μl of distilled water were reacted for at least 4 hours and then electrophoresed.

When the product of the PCR-RFLP method is treated with the restriction enzyme Hha I, a single band of 499 bp which is not cleaved when the 501st base of the nucleotide sequence of SEQ ID NO: 1 is A is shown, and non-invasively, It was confirmed that the type of muscle fiber can be judged (Fig. 8).

Example  5. Analysis of muscle fiber type distribution according to genotype

The present inventors confirmed whether the 501st base of the nucleotide sequence of SEQ. ID. NO. 1 is G or A through the above Example 4, so that the type of muscle fibers of pigs can be determined noninvasively. A / A, G / G, and G / G were used as the allele genotypes involved in the myofiber type to determine whether they could be involved in the regulation of the myofiber composition by fixing the base at the above position with a specific base of G or A. After fixation, the distribution of muscle fiber type was confirmed.

Specifically, after each genotype was fixed, the area, area ratio, and the ratio of the number of myofibers to the type of myofibers were measured, and the results are shown in Table 2 below.

The number of pigs with alleles of A / A was only 8.9% in the total of 223 pigs, but when fixed to A / A type, the average area of muscle fiber type Ⅰ was higher than that of pigs fixed to G / G type And the area ratio of muscle fiber type I increased by about 6%. Especially, it was found that the area ratio of muscle fiber type IIb decreased when fixed to A / A type.

From the above results, it can be seen that when fixed to A / A type, the proportion of muscle fiber type I increases and the ratio of muscle fiber type IIb decreases. By fixing alleles to A / A, It is possible to increase the level of Type I.

The proportion of muscle fibers in the pig affects the meat quality. The higher the ratio of muscle fiber type Ⅰ, the better the meat quality. The higher the ratio of muscle fiber type Ⅱb, , The above results suggest that high quality meat can be produced by fixing the allelic genotype to A / A.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Novel SNP marker for discriminating level of muscle fiber type I          within porcine muscle and use thereof <130> KPA140942-KR <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 1001 <212> DNA <213> porcine <220> <221> gene &Lt; 222 > (1) .. (1001) R = g or a <400> 1 aaagtccatt ttccttctcg ccaccacagg ggttggattg ttttccctga ggtgtccccc 60 ctccccccag cccccaactc actttttcct ctgttatgtg gagccattta gctccgcctt 120 ggacatgcat acttactcat ctggttactc aacaaaagtc ctcaagtgag gaagctcact 180 ttcatcctta gaaagtttgg gggccactat tttcaggcac atattccttt tgggggggcc 240 tcatctttat cccaccgcta tgccccatag tccatagctg actccccaca ctgccgtcct 300 gtcccttcca catctgagag gaagggtgtc cacgagggtt ttctctagtc ttgctgtaga 360 atcattttgt caactctaat gcaaagagaa ctttagtagt agtatccaaa gaagtcaagg 420 gaagtggtct tttcgagaga gaagatagaa taaaaaagct ccgtggagaa gctggagaaa 480 aggagggcct tgggaagggc rctgggttga tcagagaggt gatcctgaca ttgatgtcag 540 caggatggtg ggggtggaag ctttgaaggc aaggattaag gagcgagcag gtgtcacaga 600 ggaggaagga gctttcctga tttccctggg agcaaatctc tccaccaatc actgtctaag 660 acgcattcag gggaaatgca gagctggatg gagaaagcag tcgcagagcc aaataggcct 720 gggaaacgag cggaatcaag gtcattcctc agctacttga gaagaaaggg caattaattg 780 tgctgatagc aggatccttc cttacccctc acaaccgtca aagagcttat gtaacagatg 840 aaatttcaga aagcagcaag gatttcccca gccaggatca gaaatttctg ggccaaagtc 900 acaggacagg tacattaatg aatgatgaca aatttatatt taatcacaat taaaaattga 960 gtgagaatat tagattctag aactagagga ggccttgaat g 1001 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 2 catagctgac tccccacact g 21 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 3 gccctttctt ctcaagtagc tg 22

Claims (13)

A polynucleotide consisting of 5 to 1,000 consecutive bases including a single nucleotide polymorphism (SNP) located at the 501st base, or a polynucleotide consisting of the polynucleotide consisting of the polynucleotide having the nucleotide sequence of SEQ ID NO: A composition for determining the level of muscle fiber type I in a pig, comprising an agent capable of detecting or amplifying a polynucleotide of the present invention.
The method according to claim 1,
Wherein said preparation comprises a primer pair consisting of SEQ ID NO: 2 and SEQ ID NO: 3 capable of amplifying a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1.
The method according to claim 1,
Wherein the pig is a hybrid of Jeju native pig, Duroc or Jeju native pig and duroc.
A kit for determining the level of muscle fiber type I in a pig, comprising the composition of any one of claims 1 to 3.
(a) amplifying a polymorphic site of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 or its complementary polynucleotide from the DNA of the sample isolated from the individual; And
(b) determining the base of the polymorphic site amplified in step (a).
6. The method of claim 5,
Wherein the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 is judged to have increased levels of muscle fiber type I in the pig when allele of the polymorphic site 501 allele is A;
6. The method of claim 5, wherein the pig is a hybrid of Jeju native pig, Duroc or Jeju native pig and duroc.
Immobilizing all the alleles of the 501st base of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1, all of the alleles of the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, A method for producing pigs with increased levels of muscle fiber type I in the pigs' muscles.
9. The method of claim 8,
Wherein the step of immobilizing the SNP trait is carried out by crossing a subject having all of the SNP traits and selecting an individual having the trait of interest.
Immobilizing all the alleles of the 501st base of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1, all of the alleles of the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, A method for increasing the level of muscle fiber type I in a muscle of a pig.
11. The method of claim 10,
Wherein the step of immobilizing the SNP trait is carried out by crossing a subject having all of the SNP traits and selecting an individual having the trait of interest.
A polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, wherein the 501st base is G or A, and the SNP marker for determining the level of muscle fiber type I in the pig located in the 501st base.
A microarray for determining the level of muscle fiber type I in muscle of a pig, comprising the SNP marker of claim 12.

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