KR101821541B1 - SNP marker regulating palmitoleic acid level in the pork and uses thereof - Google Patents
SNP marker regulating palmitoleic acid level in the pork and uses thereof Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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- A—HUMAN NECESSITIES
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- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The present invention relates to a composition for determining the content of palmitoleic acid in pork comprising an agent capable of detecting or amplifying SNP associated with the content of palmitoleic acid in pork, a composition for determining the content of palmitoleic acid in pork Kits, methods for determining the content of palmitoleic acid in pork, methods for producing pigs with increased palmitoleic acid content in pork, methods for increasing palmitoleic acid content in pork, methods for determining content of palmitoleic acid in pork Markers and microarrays. Since the SNP marker capable of determining the content of palmitoleic acid in the pork of the present invention is used as a means for objectively evaluating the content of palmitoleic acid in the pork which is not visually distinguishable from the naked eye, It can be utilized.
Description
The present invention relates to a SNP marker for controlling the content of palmitoleic acid in pork and a use thereof. More particularly, the present invention relates to a method for detecting the content of palmitoleic acid in pork, A composition for determining the content of palmitoleic acid, a kit for determining the content of palmitoleic acid in the pork containing the composition, a method for determining the content of palmitoleic acid in the pork, a method for preparing pigs having increased content of palmitoleic acid in pork A method for increasing the content of palmitoleic acid in pork, a marker for determining the content of palmitoleic acid in pork, and a microarray.
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. European varieties and Asian varieties are derived from Susscrofa on each continent. There are currently 200 varieties of varieties presently available. According to a recent FAO (Finance and Accounts Office) report, Asian cultivars account for 30% , And European varieties were reported to be around 33%. Differences in phenotype among these varieties are sought, size, and body shape.
Recently, in order to improve the quality of the pork meat, the pork is raised in a certain standard and is scientifically managed, and the pork meat obtained from the pork meat is branded, and various brands of pork meat are already commercially sold. However, since the branded pork meat and unbranded pork meat are difficult to identify with the naked eye, they can be used to sell the unbranded pork meat into branded pork meat, Disturbing events are occurring frequently. Indeed, since it is very difficult to distinguish between branded pigs and unbranded pork meat at the level of experts, studies are actively being conducted to establish objective criteria to judge the quality of genuine pork meat.
As a part of this research, a method for judging pig meat of a genuine brand was developed by analyzing the gene of pork meat. Randomly amplified polymorphic DNA (RAPD), single strand conformation polymorphisms, and other DNA analysis techniques to develop and develop a method for discriminating pork meat varieties. For example, Japanese Patent Application Laid-Open No. 2004-0039059 discloses a gene assay method capable of selecting pigs having excellent traits of pigs by using specific DNA markers related to the daily gain of body weight, backfat thickness, and meat quality traits of pigs. Patent Publication No. 2007-0113336 discloses a DNA marker for confirming the increase of porcine myocyte count using a mutation (SNP) caused by a single base sequence difference in the promoter region of the 5 'end of Myogenin gene known to be involved in the myocyte differentiation of pigs Japanese Patent Application Laid-Open No. 2011-0011443 discloses a technique for detecting an accurate Korean rice variety and other breeder pigs by detecting a Korean native pig-specific DNA marker for the KIT gene, -0050261 discloses a method of discriminating the varieties of black-bred pigs using haplotypes estimated from SNPs in the KIT gene region of black-bred pigs Patent Publication No. 2011-0139011 discloses a method for evaluating meat quality using a single-trait polymorphic biomarker for diagnosing fat content in pigs. In Patent Publication No. 2012-0046968, And a method of screening pigs of a trait of good quality using a gene of a pig is disclosed in Patent Publication No. 2012-0049624, (SNP) site of the PPARGC1A gene, which is involved in the meat quality of the pig, has been disclosed. In the patent publication No. 2012-0072871, there is disclosed a single gene for confirming the content of unsaturated fatty acids in pigs A method for identifying high quality pork meat using a base polymorphism (SNP) marker is disclosed.
On the other hand, palmitoleic acid is an unsaturated fatty acid of palmitic acid which shows various functionalities and is known to exhibit more excellent functionality than palmitic acid. The content of palmitoleic acid in pork is a criterion for judging the quality of pig However, a method for evaluating the content of palmitoleic acid in pork using a gene has not been developed yet.
Under these circumstances, the present inventors have made extensive efforts to develop a method for evaluating the content of palmitoleic acid in pork using a gene. As a result, it has been found that when SNPs present on chromosome 8 and chromosome 14 are used, And the present invention has been completed.
It is an object of the present invention to provide a composition for determining the content of palmitoleic acid in pork comprising a preparation capable of detecting or amplifying the SNP associated with the content of palmitoleic acid in pork.
Another object of the present invention is to provide a kit for determining the content of palmitoleic acid in pork comprising the above composition.
Another object of the present invention is to provide a method for determining the content of palmitoleic acid in pork using the above composition.
It is another object of the present invention to provide a method for producing pigs having increased palmitoleic acid content in pork.
It is another object of the present invention to provide a method for increasing the content of palmitoleic acid in pork.
Another object of the present invention is to provide a marker for determining the content of palmitoleic acid in pork.
It is still another object of the present invention to provide a microarray for determining the content of palmitoleic acid in pork containing the marker.
The present inventors searched for quantitative trait loci (QTL) related to saturated and unsaturated fatty acid contents of pigs in order to develop a method for reducing the saturated fatty acid content of pigs. As a result, SNPs located on chromosome 8 (MARC0025408) and SNP located on chromosome 14 (H3GA0042070) were found to be related to the content of palmitoleic acid in pork, and using this SNP as a gene marker to determine the content of palmitoleic acid in pork . Techniques using SNP (MARC0025408) located on chromosome 8 and SNP (H3GA0042070) located on chromosome 14 to determine the content of palmitoleic acid in pork have not been known until now and were developed for the first time by the present inventor.
In one embodiment, the present invention provides a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, wherein the base 501 is T or C, and the single nucleotide polymorphism (SNP) located at the base 501 is included A polynucleotide consisting of 5 to 1000 contiguous bases; A polynucleotide consisting of 5 to 1000 consecutive bases, wherein the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 is T or C at position 501, and the single nucleotide polymorphism (SNP) at position 501; Or an agent capable of detecting or amplifying complementary polynucleotides of the same or a complementary polynucleotide thereof.
The term "polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1" or "polynucleotide comprising nucleotide sequence of SEQ ID NO: 2" of the present invention refers to a polymorphic site of the gene involved in the content of palmitoleic acid in pork Polymorphic sequence " means a sequence comprising a polymorphic site comprising a SNP in a polynucleotide sequence. 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). Specific polymorphic markers have two or more alleles that exhibit an incidence 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.
The term "(palmitoleic acid) Lane palmitoleic acid (C16: 1)" of the present invention is, CH 3 having up to 16 carbon atoms, (CH 2) 5 CH = CH (CH 2) is represented by the formula of 7 COOH, 254.41Da By weight of unsaturated fatty acids.
In the present invention, the palmitoleic acid is used as a target for predicting the content of pork using the genotype of SNP (MARC0025408) located on chromosome 8 and SNP (H3GA0042070) located on chromosome 14, Pork containing an acid is not particularly limited, but can be pork derived from Jeju native pigs, land lace or their hybrid pigs.
The term "agent capable of detecting or amplifying a polynucleotide" of the present invention refers to an agent capable of specifically recognizing binding to a polymorphic site containing a SNP in a polynucleotide and amplifying the polymorphic site As a preparation, specifically, a probe capable of specifically binding to a polymorphic site containing a SNP, a polynucleotide comprising the SNP marker, or a primer capable of specifically amplifying a complementary polynucleotide of the polynucleotide may be used.
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 refers to a nucleotide sequence having a short free 3 'hydroxyl group, capable of forming a base pair with a complementary template, Means a short sequence functioning as a starting 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 one embodiment, the primer comprises a primer base pair consisting of SEQ ID NOS: 4 and 5 capable of amplifying a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1, and a nucleotide sequence of SEQ ID NO: 9 capable of amplifying a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: And 10, but is not limited thereto.
In the present invention, the content of palmitoleic acid in the pork is the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 or 2, wherein the pork derived from pigs having alleles of base No. 501 of T is all T, Can be judged to contain palmitoleic acid in a higher content than the pork derived from pigs having a C content
According to one embodiment of the present invention, in order to investigate the fatty acid content, the fattening of the fattened shrimp was sampled for the offspring of the Korean traditional pig and the land race reference group F2, The content of palmitoleic acid was the fifth highest in the total fatty acids, and the content was 3.15% (FIG. 1). In addition, it was confirmed that the SNP (MARC0025408) located on chromosome 8 and the SNP (H3GA0042070) located on chromosome 14 are related to the content of palmitoleic acid in pork (Fig. 2 and Table 1). As a result of analyzing the content of palmitoleic acid in pork according to its genotype, when the genotype of each SNP was TT, the content of palmitoleic acid was the highest , And when the respective genotypes were CC, the content of palmitoleic acid was the lowest and the content of palmitoleic acid was the intermediate value when the genotype was
Therefore, it can be seen that the SNPs of SEQ ID NO: 1 or 2 can be used as gene markers for determining the content of palmitoleic acid in pork.
In another aspect of the present invention, there is provided a kit for determining the content of palmitoleic acid in pork comprising the composition. Specifically, the kit may be 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 content of palmitoleic acid in pork by confirming the genotype of the SNP marker provided by the present invention using the above composition by amplification or by checking the expression level of mRNA. The kit provided in the present invention may be a kit containing essential elements necessary for performing RT-PCR.
For example, in addition to the respective primer pairs specific for the SNP for determining the content of palmitoleic acid in the pork, the RT-PCR kit can also be used in a test tube or other appropriate container, reaction buffer (pH and magnesium concentration Deoxyinucleotides (dNTPs), enzymes such as Taq polymerase and reverse transcriptase, DNase, RNAse inhibitors, DEPC-water, sterile water, and the like. It may also contain a primer pair specific for the gene used as a quantitative control.
As another example, the kit of the present invention may be a DNA chip kit for determining the content of palmitoleic acid in pork containing elements necessary for carrying out a DNA chip.
The term "DNA chip" of the present invention means one of DNA microarrays capable of confirming each base of hundreds of thousands of DNAs at a time.
The DNA chip kit is formed by attaching nucleic acid species to a glass surface, which is generally not larger than a flat solid support plate, typically a slide for a microscope, in a gridded array. The nucleic acid is uniformly arranged on the chip surface, It is a tool that enables multiple parallel hybridization reactions between the nucleic acid on the chip and the complementary nucleic acid contained in the treated solution on the chip surface.
In another embodiment for achieving the above object, the present invention relates to a method for producing a polynucleotide comprising the steps of: (a) selecting a polynucleotide consisting of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 and 2, Amplifying a polymorphic site of a complementary nucleotide; And (b) determining the base of the amplified polymorphic site. The present invention also provides a method for determining the content of palmitoleic acid in pork. At this time, DNA of the separated sample can be obtained from a sample isolated from an individual.
The term "individual" in the present invention means a pig to which the content of palmitoleic acid in pork is to be confirmed. By analyzing the genotype of the polymorphic site of the SNP using the sample obtained from the pig, The content of palmitoleic acid in the pork can be judged. Examples of the specimen include, but are not limited to, hair, urine, blood, various body fluids, separated tissues, separated cells or saliva, and the like.
The step of amplifying the polymorphic site of the SNP contained in the marker from the DNA of step (a) can be used in any method known to a person 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,
Determination of the base of the amplified polymorphic site in step (b) of the above method can be carried out by sequencing, hybridization by microarray, allele specific PCR, dynamic allelespecifichybridization, 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- (E.g., BioRad), CEQ and SNPstream system (Beckman), Molecular Inversion Probe array technology (e.g. Affymetrix GeneChip), and BeadArray Technologies (e.g. Illumina GoldenGate and Infinium assay) But is not particularly limited thereto. One or more alleles in a SNP marker comprising the mutation site can be identified by the methods described above or other methods available to those skilled in the art to which the invention pertains. Determination of the base at such a mutation site can be carried out specifically through a DNA chip.
The TaqMan method comprises the steps of (1) designing and preparing 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 polynucleotides of step (1) from the analysis results.
The sequencing analysis can be performed using a conventional method for nucleotide sequencing, and can be performed using an automated gene analyzer. In addition, allele-specific PCR means a PCR method in which a DNA fragment in which the mutation is located is amplified with a primer set including a primer designed with the base at the 3 'end at which the mutation site 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, When the base at the mutation position is A, the amplification reaction is normally performed and a band at a desired position is observed. When the base is substituted with G, the primer can be complementarily bound to the template DNA, And the amplification reaction is not performed properly due to the inability of complementary binding at the terminal. 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 single nucleotide polymorphism is located is amplified with a pair of primers, and all the nucleotides added to the reaction are deactivated by dephosphorylation, and a specific extension primer, dNTP And then performing a primer extension reaction by adding a mixture, a digoxinucleotide, a reaction buffer and a DNA polymerase. At this time, the extension primer has the 3 'end immediately adjacent to the 5' direction of the base where the mutation site is located, and the nucleic acid having the same base as the didyoxynucleotide is excluded in the dNTP mixture, and the didyoxynucleotide has a mutation ≪ / 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 kind of base showing the mutation by comparing the length of the extended primer.
At this time, as a detection method, when the extension primer or the didyxin nucleotide is fluorescently labeled, the mutation is detected by detecting fluorescence using a gene analyzer (for example, Model 3700 manufactured by ABI Co., Ltd.) used for general nucleotide sequence determination And when the unlabeled extension primer and the didyxin nucleotide are used, the genetic variation of the SNP can be detected by measuring the molecular weight using MALDITOF (matrix assisted laser desorption ionization-time of flight) technique.
Specifically, the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 or 2 among the nucleotide sequences determined in the step (b) above, all of the alleles of the 501 base are all T, It can be concluded that the content of palmitoleic acid increased from that of pork.
In another aspect of the present invention, the present invention provides a method of isolating SNP traits that are involved in the content of palmitoleic acid in pork, comprising the steps of: selecting from the group consisting of SEQ ID NOS: 1 and 2, And a step of immobilizing all the alleles of base No. 501 of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO:
In another aspect of the present invention, there is provided a method of isolating SNPs of genes involved in the content of palmitoleic acid in pork, comprising the steps of: , Wherein all the alleles of base number 501 of the polynucleotide consisting of the nucleotide sequence selected from SEQ ID NO: 1 and SEQ ID NO: 2 are all T-fixed.
Specifically, the step of immobilizing the SNP trait may be carried out by crossing an individual having the SNP trait and selecting an individual having the desired trait. More specifically, a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 or 2, involved in the content of palmitoleic acid in the pork, is crossed with an individual having a T / T of the 501st base, and in the progeny obtained therefrom, Or 2 is selected as the T / T-fixed pig of the polynucleotide base No. 501, pigs with increased content of palmitoleic acid in the pork can be obtained. The pig may be a hybrid of Jeju native pig, land lace or Jeju native pig and land lace.
In another aspect of the present invention, the present invention provides a polynucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 and 2, and combinations thereof, wherein the base 501 is T or C, And a marker for determining the content of palmitoleic acid in the pork,
In the present invention, using the marker, the pork pork in which the allele of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 or 2 is all T in the 501 base is more preferable than the pork pork in which the allele is all C It can be concluded that the content of palmitoleic acid is increased.
According to another aspect of the present invention, there is provided a microarray for determining the content of palmitoleic acid in pork comprising a marker for determining the content of palmitoleic acid in the pork.
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 producing 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 in which a polymorphic site exists in a nucleic acid fragment derived from two members of the same species and hybridizes to a DNA fragment derived from one member but does not hybridize to a fragment derived from another member . 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 determining the content of palmitoleic acid in pork by detecting an allele. The determination method includes detection methods based on hybridization of nucleic acids such as Southern blot, and may be provided in a form pre-bonded to a substrate of a DNA chip in a method using a DNA chip. The hybridization can usually be performed under stringent conditions, for example, a salt concentration of 1 M or less and a temperature of 25 ° C or higher. For example, conditions of 5x SSPE (750 mM NaCl, 50 mM Na Phosphate, 5 mM EDTA, pH 7.4) and 25-30 < 0 > C may be suitable for allele-specific probe hybridization.
The process of immobilizing the probe polynucleotide on the substrate in connection with the determination of the content of palmitoleic acid in the pork of the present invention can also be easily carried out 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.
Since the SNP marker capable of determining the content of palmitoleic acid in the pork of the present invention is used as a means for objectively evaluating the content of palmitoleic acid in the pork which is not visually distinguishable from the naked eye, It can be utilized.
1 is a schematic view showing the composition of fatty acids in pork;
FIG. 2 is a graph showing the result of analyzing a genome wide association study (GWAS) using a DNA chip as a chromosome containing a gene related to the content of palmitoleic acid in pork.
FIG. 3 is a schematic diagram showing various gene groups in the locus of the content of palmitoleic acid in the pork of chromosome 8.
FIG. 4 is a schematic diagram showing various gene groups in the locus of the content of palmitoleic acid in the pork of chromosome 14.
5 is a graph showing a result of identifying a genotype of the 501 (T / C) SNP contained in the polynucleotide of SEQ ID NO: 1 of chromosome 8 using pyrosequencing.
FIG. 6 is a graph showing the result of identifying the genotype of the 501 (T / C) SNP contained in the polynucleotide of SEQ ID NO: 2 of chromosome 14 using pyrosequencing.
Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.
Example One: In pork Fatty acid composition analysis
In order to analyze the composition of saturated and unsaturated fatty acids in pigs, the content of fatty acid composition was investigated in about 1,000 dogs in the second generation offspring of Jeju traditional pig and landrace mating reference group.
1 is a schematic view showing the composition of fatty acids in pork; As shown in FIG. 1, the content of palmitoleic acid was 3.15%, which is the fifth highest content of total fatty acids.
Example 2: In pork Palmitoleic acid Content association Gene locus Search
In order to analyze the gene region regulating the content of palmitoleic acid in pork, GWAS was analyzed with a large DNA chip (illumina 60k SNPchip) to search for the locus of saturated fatty acid content-related gene (Fig. 2).
FIG. 2 is a graph showing the result of analyzing a genome wide association study (GWAS) using a DNA chip as a chromosome containing a gene related to the content of palmitoleic acid in pork. As shown in FIG. 2, the locus associated with the content of palmitoleic acid in pork was found to have the highest significance on chromosome 8 and chromosome 14 of pigs. In addition, the effect of genotype on the chromosomes of chromosome 14 was relatively higher than that of chromosome 14 in relation to the content of palmitoleic acid in pork.
Example 3. In pork Palmitoleic acid Securing content-related SNP
It was confirmed that the content of palmitoleic acid in pork was determined by the loci on chromosomes 8 and 14 through the above Example 2. Based on this, SNPs having high correlation with palmitoleic acid content in pork were selected (Table 1).
8
8
8
14
14
14
14
ASGA0039203
ALGA0049249
ALGA0049254
H3GA0042070
ASGA0066315
ASGA0066314
ALGA0081396
90,557,819
20,972,820
120,996,107
121,721,589
127,025,126
127,001,828
129,795,414
21.797784
18.830326
18.830326
14.929593
14.775208
14.702239
14.532540
As shown in Table 1, the highest SNP associated with the content of palmitoleic acid in pork was found to be MARC0025408 on chromosome 8 and H3GA0042070 on chromosome 14 in chromosomes 8 and 14.
Example 4: In pork Palmitoleic acid Chromosome 8 and 14 Gene locus Identification of Genes in the Region
Based on the SNP list having a high correlation with the content of palmitoleic acid in the pork of Example 3, various gene groups of gene locus regions of chromosome 8 and 14 associated with the content of palmitoleic acid in pork were identified (FIGS. 3 and 4) 4).
Fig. 3 is a schematic diagram showing various gene groups in the locus of the content of palmitoleic acid in the pork of chromosome 8; Fig. As shown in FIG. 3, the genes related to palmitoleic acid content in the pork chromosome 8 contained PITX2C, ELOVL6, EGF, CASP6, LRIT3, PLA2G12A, SEC24B, RRH and CCDC109B.
Fig. 4 is a schematic diagram showing various gene groups in the locus of the content of palmitoleic acid in the pork of chromosome 8; Fig. As shown in FIG. 4, the gene group related to palmitoleic acid content in the pork chromosome 14 contained CNNM1, GOT1, NKX2-3, COX15, CPN1, SNORA12, HIF1AN, PAX2, SEMA4G and MRPL43 I could.
Example 5: Pyro-sequencing ( Phyro -sequencing In pork Palmitoleic acid Extraction and analysis of nucleotide sequences containing content-related SNPs
In Example 3, it was confirmed that MARC0025408 of chromosome 8 and H3GA0042070 of chromosome 14 were the most significant in relation to the content of palmitoleic acid in pork, and the respective SNPs present on the chromosome 8 and 14 1001 nucleotides (SEQ ID NO: 1 and SEQ ID NO: 2), respectively, were obtained and their nucleotide sequences were analyzed.
Example 5- 1: 8 times SNP analysis of chromosomes
In order to amplify the polynucleotide of SEQ ID NO: 1 of the chromosome 8, PCR was carried out using the DNA obtained from a number of pigs as a template and the following primers to obtain a polynucleotide fragment of SEQ ID NO: 1 (SEQ ID NO: 3, 249 bp).
Forward: 5'-GCT-TCC-TCC-ACC-TTC-AAA-GC-3 '(SEQ ID NO: 4)
Reverse: 5'-biotin-CCA-CTG-ACA-CCT-GAT-GTG-G-3 '(SEQ ID NO: 5)
The nucleotide sequence of the polynucleotide fragment of SEQ ID NO: 1 obtained using the above primer was determined and the genotype of the SNP contained therein was confirmed.
In order to confirm the 501 (T / C) SNP, the nucleotide sequence of the polynucleotide fragment was determined using the following primer (Mini-seq), and the primer of the input was used. / C) SNP genotype (Fig. 5).
ATCC-TAG-TAT-AA-3 ' (SEQ ID NO: 6)
Input: 5 '- [C / T] CTCTCCAGCTT-3' (SEQ ID NO: 7)
5 is a graph showing a result of identifying a genotype of the 501 (T / C) SNP contained in the polynucleotide of SEQ ID NO: 1 of chromosome 8 using pyrosequencing. As shown in FIG. 5, it was confirmed that polynucleotides of amplified SEQ ID NO: 1 contained TT, TC, or CC genotypes, respectively.
Example 5- 2: 14 SNP analysis of chromosomes
In order to amplify the polynucleotide of SEQ ID NO: 2 of the chromosome 14, PCR was carried out using the DNA obtained from a number of pigs as a template and the following primers to obtain a polynucleotide fragment of SEQ ID NO: 2 (SEQ ID NO: 8, 298 bp).
Forward: 5'-AGG-AGT-TTG-GGG-AGG-CTT-T-3 '(SEQ ID NO: 9)
Reverse: 5'-biotin-GCC-CAT-TAA-GGC-AAT-TGA-GA-3 '(SEQ ID NO: 10)
The nucleotide sequence of the polynucleotide fragment of SEQ ID NO: 2 obtained using the above primer was determined and the genotype of the SNP contained therein was confirmed.
In order to confirm the 501 (T / C) SNP, the nucleotide sequence of the polynucleotide fragment was determined using the following primer (Mini-seq), and the primer of the input was used. / C) SNP genotype (Fig. 6).
GGG-TGA-CCC-TGG-GA-3 '(SEQ ID NO: 11)
Input: 5 '- [C / T] TTGGGGTTGCT-3' (SEQ ID NO: 12)
FIG. 6 is a graph showing the result of identifying the genotype of the 501 (T / C) SNP contained in the polynucleotide of SEQ ID NO: 2 of chromosome 14 using pyrosequencing. As shown in FIG. 6, it was confirmed that polynucleotides of amplified SEQ ID NO: 1 contained TT, TC, or CC genotypes, respectively.
Example 6: 8 And the SNP genotype of chromosome 14 In pork Palmitoleic acid Analysis of association with content
The changes in the content of palmitoleic acid in pork according to the genotypes of the chromosome 8 SNPs of 8 and 14 identified in Example 5 were compared (Tables 2 and 3).
CT
CC
440
132
3.0601 + 0.4945
2.9214 + 0.4945
As shown in Table 2, the content of palmitoleic acid in pork was the highest when the genotype of chromosome 8 of TT was TT, and the content of palmitoleic acid in pork was the highest when the genotype was CC And the content of palmitoleic acid in the pork was found to be at an intermediate level when the genotype was TC.
CT
CC
384
23
3.0436 + - 0.5012
2.7291 + - 0.5012
As shown in Table 3, the content of palmitoleic acid in pork was the highest when the genotype of chromosome 14 was TT, and the content of palmitoleic acid in pork when the genotype was CC And the content of palmitoleic acid in the pork was found to be at an intermediate level when the genotype was TC.
Example 7: 8 The effect of combination of chromosome and chromosome 14 SNP
The change in the content of palmitoleic acid in the pork according to the genotype of the chromosome 8 of SEQ ID NO: 8 and 14 was confirmed through Example 6, and the change in the content of palmitoleic acid in the pork was confirmed according to the combination of the two SNPs ).
CT
CC
152
8
3.1766 ± 0.5007
2.6763 ± 0.5007
CT
CC
62
3
2.8190 ± 0.5007
2.1933 + - 0.5007
As shown in Table 4, when the genotypes of chromosome 8 and 14 were T, the content of palmitoleic acid was found to be higher than that of the genotype C.
Therefore, the content of palmitic acid in pork was determined by the SNP genotypes of chromosome 8 and 14.
<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> SNP marker regulating palmitoleic acid levels in the pork and uses the <130> KPA151107-KR <160> 12 <170> Kopatentin 2.0 <210> 1 <211> 1001 <212> DNA <213> Artificial Sequence <220> <223> SNP <400> 1 attttttttg accttggcat tctacacttt ttactctgca atatttcaac tataagaata 60 tttgaaacaa aagaaaattt aaaaatctaa cgtatttcat tacatctaaa gtgttttgac 120 gttgccactt ttttccttct tactagatgt atgcgtttcc tatagcaagt gtattaaatt 180 accacaaatt tagtgcctta aaattaacac agatgtattc ttttatagtt ctggaagtca 240 ctgggctctc aggagagaat ccacttcgtt gccattttca gtttcgagtg accacctgca ttctttcggc 360 ttatggcgct tcctccacct tcaaagcaca tcattctaac ctctgcttct atggtcactg 420 taccttctct gcaggctgac tcctccagtt tctaataagg atggttgtaa ttatatcagg 480 cccacccaga tctagtataa yctctccagc tttttaatcg cagctgcaaa ggtttcaggg 540 attaggaaag aatactgaga aggggacact atggggaagg gtacactatt cagcctacca 600 catcaggtgt cagtggcaga tttttcacaa ctataaggtg atttccacca attttgaaat 660 tccaccagtt ctaagatgca ccccaatttt agaggtgaaa atggtgaaaa aaaagtgtct 720 tagaaacaat gagtaactta attatcgcat ctaccaaaga gcataacaca gcatctcaca 780 aaaaaagcat caataaagaa accactgaaa aacagaaaaa ctcaggtaat acatagtctc 840 taagagaact gtaaattttt tcctccacta ctcataccca gatgagtaat atcaccatga 900 ggccagaact atacattttt aacttcaagt aagtatgtct gtataaaaga gggaaataga 960 ttattctaac acattatcta atcattatta aaatctgact t 1001 <210> 2 <211> 1001 <212> DNA <213> Artificial Sequence <220> <223> SNP <400> 2 aaagtgagac tgatttaaaa gtgttcttat gaggattaat gaactgatgg gtataacaca 60 taaaagcttt aaagccccct acaaatcagg gtcgttataa ccacttgccc caccctcagt 120 gtgaaaccaa attgtacctt gaagctcaca cttcacacag catctggatc ctgccctgat 180 tcccatccca gcaagaatat cgttaaggac atagaaagct tgatgaagag ctgttaaatg 240 aaaaattgaa gggatggaac acagtctaat ctctggtctc tgtgaagagg ccatactggt 300 ggagggtttg ggggagcaac gtagggaaga gagaaaactg gagtcagaac actggccagg 360 agtttgggga ggctttgggg gagtgggaaa aagtgggttc agggcatctg ggggtctctg 420 aagcagagtt tgtggtgcac agagggaaag tttgaggttg ggtgctgagg caagatgtgg 480 ggctgggt ggcaggtgga agatgggggt ccggccaacc caagtctctc tctgtccggc aggaggggac 600 tctggtgcag ggcgaggagg gagattagga ggcgctctca attgccttaa tgggctctaa 660 gaggcgggat cctattacag ctcggggtgc cccggggtgc cttgctggga agacaagcta 720 atctgccatg caaattaaat cctgcctcaa gacgcaaggc ctgcttaatg tgatccatta 780 cacagtttac ttgtttatat ctaatattgg aacaggcagc ccgggcgggc ggcagcggca 840 ggcggagcgg cccaaaagca ggagagtaaa ttaccagcaa aatgagcttt tccatcctta 900 gccgccgcca cccgcgcctt ggtggctctc tgtgtcctgg gcgcccccgc ccgcccgccc 960 tcccgcagcg tcccctcgct ctctgggctg tcccgctctc t 1001 <210> 3 <211> 249 <212> DNA <213> Artificial Sequence <220> <223> template <400> 3 gcttcctcca ccttcaaagc acatcattct aacctctgct tctatggtca ctgtaccttc 60 tctgcaggct gactcctcca gtttctaata aggatggttg taattatatc aggcccaccc 120 agatctagta taayctctcc agctttttaa tcgcagctgc aaaggtttca gggattagga 180 aagaatactg agaaggggac actatgggga agggtacact attcagccta ccacatcagg 240 tgtcagtgg 249 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 gcttcctcca ccttcaaagc 20 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 ccactgacac ctgatgtgg 19 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 cccacccaga tctagtataa 20 <210> 7 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 yctctccagc tt 12 <210> 8 <211> 298 <212> DNA <213> Artificial Sequence <220> <223> template <400> 8 aggagtttgg ggaggctttg ggggagtggg aaaaagtggg ttcagggcat ctgggggtct 60 ctgaagcaga gtttgtggtg cacagaggga aagtttgagg ttgggtgctg aggcaagatg 120 tggaaaaggg ggtgaccctg ggayttgggg ttgctgcttg aagggagagt tggggcctga 180 gctggcaggt ggaagatggg ggtccggcca acccaagtct ctctctgtcc ggcaggaggg 240 gactctggtg cagggcgagg agggagatta ggaggcgctc tcaattgcct taatgggc 298 <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 aggagtttgg ggaggcttt 19 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 gcccattaag gcaattgaga 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 aaaagggggt gaccctggga 20 <210> 12 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 yttggggttg ct 12
Claims (12)
The preparation comprises a primer pair consisting of SEQ ID NOS: 4 and 5 capable of amplifying a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1, SEQ ID NOS: 9 and 10 capable of amplifying a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2 Primer pairs, or combinations thereof.
Wherein said formulation is used to determine the content of palmitoleic acid in pork obtained from Jeju native pig, land lace or Jeju native pig and landrace hybrids.
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Non-Patent Citations (4)
| Title |
|---|
| Genetics Selection Evolution, Vol. 46, p. 28(1-10) (2014.04.23.)* |
| PLoS One, Vol. 8, p. e56664 (2013.06.07.)* |
| RefSNP Cluster Report No. rs80811639 (2009.05.01.)* |
| RefSNP Cluster Report No. rs81293549 (2008.11.26.)* |
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