KR102304998B1 - Snp makers of identification of whole black hair in woori black porcine and method for identifying whole black hair using the same - Google Patents

Abstract

The present invention is a SNP marker for the identification of whole-body black hair of guinea pigs comprising any one of the polynucleotides represented by SEQ ID NOs: 1 to 10 or polynucleotides complementary thereto.

Description

SNP marker for whole-body black hair identification of Korean black pigs and a whole-body black hair identification method using the same

The present invention relates to a SNP marker for whole-body black hair identification of Korean black pigs and a whole-body black hair identification method using the same.

Recently, in order to improve the quality of pig meat, pigs are raised and scientifically managed under a certain standard, and the pork meat obtained therefrom is branded, and various brands of pork are already commercially sold. In this way, since it is very difficult even for an expert to distinguish branded and unbranded pork, research to establish an objective standard for judging genuine branded pork is being actively conducted.

As part of this research, a method for judging genuine brand pork by analyzing the gene of pig meat was developed. As such, the method of analyzing the gene is RAPD (random amplified polymorphic DNA) using PCR technology, single strand conformation (SSCP) Research using various DNA analysis techniques such as polymorphisms) is being actively conducted.

However, a method for accurately determining the black hair color of pigs has not yet been developed.

Under this background, the present inventors confirmed that the quality of pig meat can be determined by determining the SNP contained in the gene involved in the hair color of pigs, and completed the present invention.

An object of the present invention is to provide a SNP marker for whole-body black-haired identification of Korean black pigs capable of identifying with high accuracy a breed of Korean black pig having a whole-body black hair color, and a whole-body black hair color identification method using the same.

In addition, an object of the present invention is to provide a SNP marker for whole-body black-hair color identification of Korean black pigs capable of fixing the hair color by crossbreeding with Korean black pigs through discrimination and classification, and a whole-body black hair color identification method using the same.

Furthermore, as described above, the purpose of improving the brand differentiation is by fixing the color of the black pig as described above, and by using it for the selection of the gray scale of the Korean black pig and the self-reproduction of the supplying farms.

In order to achieve the above object, the present invention is a SNP marker for the identification of the whole body black hair of guinea pigs comprising any one of the polynucleotides represented by SEQ ID NOs: 1 to 10 or polynucleotides complementary thereto.
The polynucleotides represented by SEQ ID NOs: 1 to 10 are: polynucleotides represented by SEQ ID NO: 1 consisting of 10 to 100 consecutive nucleic acids comprising a single nucleotide polymorphism (SNP) site in which the 51st base is A or G nucleotides; a polynucleotide consisting of 10 to 100 consecutive nucleic acids including a single nucleotide polymorphism (SNP) site in which the 51st base is A or G in the polynucleotide represented by SEQ ID NO: 2; a polynucleotide consisting of 10 to 100 consecutive nucleic acids including a single nucleotide polymorphism (SNP) site in which the 51st base is A or G in the polynucleotide represented by SEQ ID NO: 3; a polynucleotide consisting of 10 to 100 consecutive nucleic acids including a single nucleotide polymorphism (SNP) site in which the 51st base is A or G in the polynucleotide represented by SEQ ID NO: 4; a polynucleotide consisting of 10 to 100 consecutive nucleic acids including a single nucleotide polymorphism (SNP) site in which the 51st base is A or G in the polynucleotide represented by SEQ ID NO: 5; a polynucleotide consisting of 10 to 100 consecutive nucleic acids including a single nucleotide polymorphism (SNP) site in which the 51st base is A or G in the polynucleotide represented by SEQ ID NO: 6; a polynucleotide consisting of 10 to 100 consecutive nucleic acids including a single nucleotide polymorphism (SNP) site in which the 51st base is A or G in the polynucleotide represented by SEQ ID NO: 7; a polynucleotide consisting of 10 to 100 consecutive nucleic acids including a single nucleotide polymorphism (SNP) site in which the 51st base is A or C in the polynucleotide represented by SEQ ID NO: 8; a polynucleotide consisting of 10 to 100 consecutive nucleic acids including a single nucleotide polymorphism (SNP) site in which the 51st base is A or C in the polynucleotide represented by SEQ ID NO: 9; and a polynucleotide consisting of 10 to 100 consecutive nucleic acids including a single nucleotide polymorphism (SNP) site in which the 51st base is A or C in the polynucleotide represented by SEQ ID NO: 10.

In addition, another embodiment of the present invention is a composition for the identification of whole-body black hair of guinea pigs comprising an agent capable of detecting or amplifying the SNP marker for identification of the whole body black hair of guinea pigs.

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In addition, another embodiment of the present invention is a kit for identifying the whole body black hair of guinea pigs comprising the composition for identifying the black shading of the whole body of guinea pigs.

In another embodiment of the present invention, the kit is an RT-PCR kit or a DNA chip kit.

In addition, another embodiment of the present invention is a microarray for whole-body black hair identification of guinea pigs including the SNP marker for whole-body black hair color identification of guinea pigs.

Furthermore, another embodiment of the present invention, a sample extraction step of extracting a sample from our heukdon; a polymorphism amplification step of amplifying the polymorphic site of the SNP marker from the DNA of the sample; and a base determination step of determining the base of the amplified polymorphic site.

In another embodiment of the present invention, the polymorphic site is present on chromosome 6 of the black pig.

In another embodiment of the present invention, the step of determining the base is a step of discriminating the allele of the base of the polymorphic site in the SNP marker for identification of the whole body black hair of the guinea pig.

In another embodiment of the present invention, in the polynucleotide represented by SEQ ID NO: 1, when the allele of the 51st base, which is the polymorphic site, is A/A, it is identified that the guinea pig has a whole black hair color.

In another embodiment of the present invention, in the polynucleotide represented by SEQ ID NO: 2, when the allele of the 51st base, which is a polymorphic site, is G/G, the black pig is identified as having a whole black hair color.

In another embodiment of the present invention, in the polynucleotide represented by SEQ ID NO: 3, when the allele of the 51st base, which is a polymorphic site, is G/G, it is identified that the guinea pig has a whole black hair color.

In another embodiment of the present invention, in the polynucleotide represented by SEQ ID NO: 4, when the allele of the 51st base, which is a polymorphic site, is G/G, it is identified that the guinea pig has a whole black hair color.

In another embodiment of the present invention, in the polynucleotide represented by SEQ ID NO: 5, when the allele of the 51st base, which is a polymorphic site, is G/G, the black pig is identified as having a whole black hair color.

In another embodiment of the present invention, in the polynucleotide represented by SEQ ID NO: 6, when the allele of the 51st base, which is a polymorphic site, is G/G, it is identified that the guinea pig has a whole black hair color.

In another embodiment of the present invention, in the polynucleotide represented by SEQ ID NO: 7, when the allele of the 51st base, which is a polymorphic site, is G/G, it is identified that the guinea pig has a whole black hair color.

In another embodiment of the present invention, in the polynucleotide represented by SEQ ID NO: 8, when the allele of the 51st base, which is the polymorphic site, is G/G, it is identified that the guinea pig has a whole black hair color.

In another embodiment of the present invention, in the polynucleotide represented by SEQ ID NO: 9, when the allele of the 51st base, which is a polymorphic site, is G/G, it is identified that the guinea pig has a whole black hair color.

In another embodiment of the present invention, in the polynucleotide represented by SEQ ID NO: 10, when the allele of the 51st base, which is the polymorphic site, is G/G, it is identified that the guinea pig has a whole black hair color.

The details of other embodiments are included in the detailed description and drawings.

By utilizing the SNP marker for whole-body black-haired identification of Korean black pigs according to an embodiment of the present invention and the whole-body black-haired identification method using the same, there is an effect that can identify with high accuracy the breed of Korean black pigs with whole-body black hair.

In addition, there is an effect that can fix the hair color by crossbreeding the black pigs through the discrimination and classification of Korean black pigs.

Furthermore, as described above, by fixing the hair color of Korean black pigs, it is possible to improve the brand differentiation by using the gray scale selection and propagation farms of Korean black pigs for their own breeding.

1 is a graph showing a dielectric dispersion ratio for each SNP window according to an embodiment of the present invention.

Since the present invention can apply various transformations and can have various embodiments, specific embodiments are illustrated in the drawings and described in detail in the detailed description. However, this is not intended to limit the present invention to specific embodiments, and it should be understood to include all modifications, equivalents and substitutes included in the spirit and scope of the present invention.

Advantages and features of the present invention and methods of achieving them will become apparent with reference to the embodiments described below in detail in conjunction with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in various different forms, and only these embodiments allow the disclosure of the present invention to be complete, and common knowledge in the art to which the present invention pertains It is provided to fully inform those who have the scope of the invention, and the present invention is only defined by the scope of the claims.

The terms used in the present application are only used to describe specific embodiments, and are not intended to limit the present invention. The singular expression includes the plural expression unless the context clearly dictates otherwise. In the present application, terms such as “comprise” or “have” are intended to designate that a feature, number, step, operation, component, part, or combination thereof described in the specification exists, but one or more other features It should be understood that this does not preclude the existence or addition of numbers, steps, operations, components, parts, or combinations thereof.

The SNP marker for whole-body black-haired identification of guinea pigs according to an embodiment of the present invention is an invention for identifying the whole-body black-haired characteristics of pigs, and specifically targets woori heuk swine.

As used herein, the term “Korean heukdon” refers to synthetic pigs produced by crossing Duroc and Korean native pigs. Specifically, a hybrid first-generation female obtained by crossing a Duroc female with a male native pig, and a second-generation hybrid female obtained by outcrossing a Duroc male, and the obtained second-generation female and the first hybrid male are outcrossed. Synthetic money obtained Woori Heuk Pork is a synthetic pig that has only the advantages of Duroc (excellent growth characteristics) and the advantages of traditional Korean pigs (excellent meat quality). Our heukdon may have the A (Asn) allele as the Asp298Asn single nucleotide mutation in the MC4R gene, and may have G as the 1846th base based on the ATG start codon in the PRKAG3 gene, and as the 586th nucleotide in the KIT gene. It is possible to have T. A more detailed definition and production method of Korean black pig is as described in Korean Patent Application No. 10-2015-0058980. Woori Heuk Pork is a synthetic pig that possesses both the excellent growth characteristics of Duroc and the excellent meat quality of Korean native pigs. has been acknowledged

As used herein, the term "Durok" means Duroc or Duroc species of pigs, and is a concept including Durocjersey species or Korean Chukjin Duroc species. The durok jersey is a species of pig belonging to a large species, having a country of origin in the United States and a color ranging from pink to dark red. The head is relatively small and the face is slightly concave but close to a straight line. The above-mentioned Chukjin Duroc is a brand name of the Duroc item developed by the National Institute of Livestock Science and has the meaning of promoting the livestock industry. The Chukjin Duroc was developed in response to consumer demand and the demand for development of seed pork for differentiation from imported pork. The above-mentioned Chukjin Duroc was completed in 1997, and the system was established in 1998 and trademark registration was completed in 2008. Compared to other species, general Duroc species have superior growth rate and growth rate, and Chukjin Duroc species have superior meat quality compared to general Duroc species.

In addition, as used herein, the term "Korean native pig" is a breed of pigs inhabiting the Korean Peninsula, and is a mammal of the Somok family. Generally, it is defined as an individual with black hair, black body parts, long and sharp head, with mountain-shaped wrinkles on the forehead, long and straight nose, straight chin, and upright ears. have. The Korean traditional pig may include general conventional pigs, black pigs, Jeju native black pigs, and the like.

In the present invention, by analyzing the genotype of the SNP marker using the sample obtained from the guinea pig, the whole body black hair color characteristic of the guinea pig can be identified. The sample may be a sample such as hair, urine, blood, various body fluids, isolated tissue, isolated cells, or saliva, but is not particularly limited thereto.

According to one embodiment of the present invention, there is provided a SNP marker for the identification of the black hair color of the entire body of guinea pigs comprising any one of the polynucleotides represented by SEQ ID NOs: 1 to 10 or polynucleotides complementary thereto.

a polynucleotide consisting of 10 to 100 consecutive nucleic acids including a single nucleotide polymorphism (SNP) site in which the 51st base is A or G in the polynucleotide represented by SEQ ID NO: 1;

a polynucleotide consisting of 10 to 100 consecutive nucleic acids including a single nucleotide polymorphism (SNP) site in which the 51st base is A or G in the polynucleotide represented by SEQ ID NO: 2;

a polynucleotide consisting of 10 to 100 consecutive nucleic acids including a single nucleotide polymorphism (SNP) site in which the 51st base is A or G in the polynucleotide represented by SEQ ID NO: 3;

a polynucleotide consisting of 10 to 100 consecutive nucleic acids including a single nucleotide polymorphism (SNP) site in which the 51st base is A or G in the polynucleotide represented by SEQ ID NO: 4;

a polynucleotide consisting of 10 to 100 consecutive nucleic acids including a single nucleotide polymorphism (SNP) site in which the 51st base is A or G in the polynucleotide represented by SEQ ID NO: 5;

a polynucleotide consisting of 10 to 100 consecutive nucleic acids including a single nucleotide polymorphism (SNP) site in which the 51st base is A or G in the polynucleotide represented by SEQ ID NO: 6;

a polynucleotide consisting of 10 to 100 consecutive nucleic acids including a single nucleotide polymorphism (SNP) site in which the 51st base is A or G in the polynucleotide represented by SEQ ID NO: 7;

a polynucleotide consisting of 10 to 100 consecutive nucleic acids including a single nucleotide polymorphism (SNP) site in which the 51st base is A or C in the polynucleotide represented by SEQ ID NO: 8;

a polynucleotide consisting of 10 to 100 consecutive nucleic acids including a single nucleotide polymorphism (SNP) site in which the 51st base is A or C in the polynucleotide represented by SEQ ID NO: 9; and

A polynucleotide consisting of 10 to 100 consecutive nucleic acids comprising a single nucleotide polymorphism (SNP) site in which the 51st base is A or C in the polynucleotide represented by SEQ ID NO: 10.

As used herein, the term "polymorphism" refers to a case in which two or more alleles exist at one locus, and among polymorphic sites, only a single base differs from one person to another. It is called a single nucleotide polymorphism (SNP). Preferred polymorphic markers have two or more alleles that exhibit an incidence of at least 1%, more preferably at least 5% or 10% in a selected population.

As used herein, the term “allele” refers to several types of one gene present at the same locus on homologous chromosomes. Alleles are also used to indicate polymorphisms, for example, SNPs have two types of alleles.

The "SEQ ID NOs. 1 to 10" is a polymorphic sequence including a polymorphic site, and the polymorphic sequence is represented by y in SEQ ID NOs: 1 to 10. The polymorphic sequence refers to a sequence including a polymorphic site including a SNP in a polynucleotide sequence. The polynucleotide sequence may be DNA or RNA.

In addition, in another embodiment of the present invention, there is provided a composition for the identification of the whole body black hair of guinea pigs comprising an agent capable of detecting or amplifying the SNP marker for identification of the whole body black hair of guinea pigs.

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As used herein, the term "agent capable of detecting or amplifying a SNP marker" refers to a composition capable of determining the whole-body black hair characteristic of Korean heuk swine by confirming the polymorphic region of the gene as described above through amplification, preferably means a primer capable of specifically amplifying the polynucleotide of the SNP marker.

As used herein, the term "primer" refers to a base sequence having a short free 3' hydroxyl group, which can form a base pair with a complementary template and is a start for template strand copying. A short sequence that functions as a branch. Primers are capable of initiating DNA synthesis in the presence of reagents for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in appropriate buffers and temperatures. In this case, PCR conditions, the length of the sense and antisense primers can be modified based on those known in the art.

Further, in another embodiment of the present invention, there is provided a kit for identifying the whole body black hair of guinea pigs comprising the composition for discrimination of the whole body black hair of guinea pigs. The kit may be provided as a rotational polymerase chain reaction (RT-PCR) kit or a DNA chip kit.

The kit of the present invention can determine the whole-body black hair characteristic of guinea pigs by amplifying the SNP marker, which is a marker for judging the whole-body black hair characteristic of guinea pigs, or by checking the mRNA expression level by checking the expression level of the SNP marker. .

As a specific example, in the present invention, the kit for measuring the mRNA expression level of the SNP marker for determining the whole-body black hair color characteristic of guinea pigs may be a kit including essential elements necessary for performing RT-PCR. The RT-PCR kit contains, in addition to each primer pair specific for the gene of the SNP marker, a test tube or other suitable container, reaction buffer (pH and magnesium concentration vary), deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNAse inhibitors, DEPC-water, sterile water, and the like. It may also include a pair of primers specific for a gene used as a quantitative control.

Also preferably, the kit of the present invention may be a kit for judging the black hair color characteristics of the whole body of a guinea pig including essential elements necessary for performing a DNA chip. As used herein, the term "DNA chip" refers to one of the DNA microarrays capable of confirming each base of hundreds of thousands of DNA at once. A DNA chip kit is a method in which nucleic acid species are attached in a gridd array to a generally flat solid support plate, typically a glass surface no larger than a microscope slide, and the nucleic acids are uniformly arranged on the chip surface, so that the DNA chip It is a tool that allows multiple hybridization reactions between the nucleic acids on the chip surface and the complementary nucleic acids contained in the solution treated on the chip surface to enable massively parallel analysis.

In addition, in another embodiment of the present invention, a microarray for the identification of the whole body black hair of guinea pigs including the SNP marker for the identification of the whole body black hair of guinea pigs may be provided.

The microarray may include a DNA or RNA polynucleotide, and may consist of a conventional microarray. Methods for preparing microarrays by immobilizing probe polynucleotides on a substrate are well known in the art. The probe polynucleotide refers to a hybridizable polynucleotide, and refers to an oligonucleotide capable of sequence-specific binding to a complementary strand of a nucleic acid.

Furthermore, in another embodiment of the present invention, a sample extraction step of extracting a sample from our heukdon; a polymorphic amplification step of amplifying the polymorphic site of the SNP marker of claim 2 from the DNA of the sample; And there is provided a whole-body black hair color identification method of guinea pigs comprising a base determination step of determining the base of the amplified polymorphic site.

The polymorphic site is present on chromosome 6 of the black pig.

The step of determining the base is a step of discriminating the allele of the base of the polymorphic site in the SNP marker for identification of the black hair color of the whole body of the guinea pig.
In the base determination step, when the allele of the 51st base (451,496th base of chromosome 6), which is the polymorphic site in the SNP marker for whole-body black hair color identification of guinea pigs represented by SEQ ID NO: 1, is A/A, the cage Black pigs can be identified as having a full-body black coat.
In addition, in the SNP marker for whole-body black hair identification of guinea pigs represented by SEQ ID NO: 2, when the allele of the 51st base (base 489,077 of chromosome 6), which is a polymorphic site, is G/G, it is shown as SEQ ID NO: 3 When the allele of the 51st base (the 592,593th base of chromosome 6), which is a polymorphic site, is G/G in the SNP marker for the identification of whole-body black shading of guinea pigs, the whole-body black shading of guinea pigs represented by SEQ ID NO: 4 In the SNP marker for identification, if the allele of the 51st base (base 2,770,269 of chromosome 6), which is the polymorphic site, is G/G, in the SNP marker for identification of the whole body black shaggy color of guinea pigs represented by SEQ ID NO: 5, the polymorphism When the allele of the 51st base (base 2,882,725 of chromosome 6), which is the site, is G/G, in the SNP marker for whole-body black hair identification of guinea pigs represented by SEQ ID NO: 6, the polymorphic site at the 51st base (6 If the allele of the 3,055,343 base of chromosome No. 3) is G/G, the 51st base (base 3,119,282 of chromosome 6), which is the polymorphic site, in the SNP marker for whole-body black hair identification of guinea pigs represented by SEQ ID NO: 7 If the allele of is G/G, the allele of the 51st base (base 3,165,397 of chromosome 6), which is the polymorphic site, is G/G in the SNP marker for whole-body black hair identification of guinea pigs represented by SEQ ID NO: 8 If the allele of the 51st base (base 4,017,367 of chromosome 6), which is a polymorphic site, in the SNP marker for whole-body black hair color identification of guinea pigs represented by SEQ ID NO: 9, is G/G, as SEQ ID NO: 10 In the displayed SNP marker for whole-body black hair color identification, if the allele of the 51st base (base 4,034,234 of chromosome 6), which is a polymorphic site, is G/G, the black pig is identified as having a whole body black hair color can do.

At this time, the dominant genotype of the SNP marker represented by SEQ ID NO: 1 is AA, the dominant genotype of the SNP marker represented by SEQ ID NO: 2 is AA, the dominant genotype of the SNP marker represented by SEQ ID NO: 3 is AA, The dominant genotype of the SNP marker represented by SEQ ID NO: 4 is GG, the dominant genotype of the SNP marker represented by SEQ ID NO: 5 is AA, the dominant genotype of the SNP marker represented by SEQ ID NO: 6 is CC, SEQ ID NO: 7 The dominant genotype of the SNP marker shown in The dominant genotype is CC.

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In the sample extraction step, a sample obtained from guinea pig is used as a sample. The sample may include, but is not limited to, pig hair, urine, blood, various body fluids, isolated tissues, isolated cells, or saliva.

In the polymorphism amplification step, any method known to those skilled in the art for amplifying the polymorphic site of the SNP marker from DNA may be used. For example, it can be obtained by amplifying a target nucleic acid through PCR and purifying it.

In the nucleotide determination step, methods for determining the base of the polymorphic site include sequence analysis, microarray hybridization, allele specific PCR (allele specific PCR), dynamic allelespecific hybridization (DASH), and PCR Extension analysis, PCR-SSCP, PCR-RFLP analysis or TaqMan technique, SNPlex platform (Applied Biosystems), mass spectrometry (eg Sequenom's MassARRAY system), mini-sequencing method, Bio-Plex system ( BioRad), CEQ and SNPstream systems (Beckman), Molecular Inversion Probe array technology (eg, Affymetrix GeneChip), and BeadArray Technologies (eg, Illumina GoldenGate and Infinium assays). Not limited.

One or more alleles in the SNP marker comprising the polymorphic site may be identified by the above methods or other methods available to those skilled in the art to which the present invention pertains. Determination of the base of such a polymorphic site can be preferably performed through a DNA chip.

Using the above-described SNP marker for whole-body black-haired identification and the whole-body black-haired identification method of the present invention, as the hair color is fixed by crossbreeding among Korean black pigs having a whole-body black-haired color, the selection and distribution of the grayscale composition of the Korean black pig itself It has the advantage that it can be used for breeding.

The present invention may be better understood by the following examples, which are for illustrative purposes of the present invention and are not intended to limit the scope of protection defined by the appended claims.

Example: Full-length genome analysis test

1. Test purpose

Duroc-derived brown stripes were expressed in less than 20% of Korean black pigs developed by crossbreeding Duroc and native pigs.

Therefore, in the present invention, a whole-genome sequencing (WGS) test was performed for verification and selection of genes for fixing black pigs in whole-body black hair.

Whole genome analysis comprehensively studies the genetic factors for disease and drug responsiveness, determines the individual SNP genotype, and calculates the probability of coexistence with a disease or specific phenotype among the determined genotypes, and the most significant genotype -This is a test to discover SNPs of the phenotype.

2. Test method

(1) Whole-body black hair expression ratio analysis

kind phenotype Number of genomes (SNP chip) analysis head number Whole body dark hair proportions Duroc 7,768 0% 878 traditional pig 741 100% 195 our black money 2,836 83% 697 Sum 11,345 1,770

Table 1 shows the basic statistics of the expression rate of whole-body black hair for Duroc, conventional pigs, and Korean black pigs, respectively. Referring to Table 1, Korean black pig was developed through hybridization of Duroc and conventional pigs, and the expression of whole-body black hair color is shown in Table 1. The ratio was found to be about 83%.

(2) Estimation of heritability

The season (test year - 2 months) was set as a random effect for estimating heritability, and the BLUPF90 Family program was used to analyze categorical data using a single step gibbs sampling method.

The genetic parameters (genetic properties of the population) were estimated with the final 1000 samples by sampling the 10th unit out of 10,000 samples.

hereditary parameters average Additive dielectric dispersion 68.76 residual variance 0.74 phenotypic variance 69.50 Heritability (standard deviation) 0.99
(0.002)

Table 2 shows the results of estimating the genetic parameters for the expression of black hair color throughout the body. The genetic parameters are the genetic properties of the population that are estimated through statistics, the numerical values obtained from actual measurements in the sample when interpreting the genetic quantity trait. say

Referring to Table 2, it was estimated that the heritability was about 99% as a result of estimating the genetic parameters.

(3) Whole-genome analysis for whole-body black hair expression

The genome map information was Sus crofa 11.1, and sex chromosome SNPs were excluded from the analysis. Sample and SNP QC were set under the conditions of minimum allele frequencies (MAF) <0.9, genotype call rate <0.9, animal missing rate >0.9, Hardy-Weinberg equilibrium (HWE) 0.15. Finally, 33,892 SNPs and 1,730 heads were used for whole genome analysis.

In order to utilize the single step genome-wide association study (single step GWAS) method, the SNP window was set in units of 0.4Mbp using the BLUPF90 Family program, and the genetic variance by window (phenotype in one population) Among the variances, the portion caused by the genetic variation of the individual) was detected.

Genetic variance (Vg) was calculated as the sum of variance due to additive effect (Va), variance due to dominant effect (Vd), and variance due to upper gene effect (Vi). (Vg=Va+Vd+Vi)

The top three windows (40 SNP markers) were screened for the ratio of genetic variance.

3. Test results

(1) Analysis of the genetic variance ratio by SNP window

1 is a graph showing a dielectric dispersion ratio for each SNP window according to an embodiment of the present invention.

In the graph, the X-axis represents chromosomes and the Y-axis represents the genetic variance ratio, and it can be seen that the higher the genetic variance ratio, the greater the heritability.

As shown in FIG. 1 , in the case of chromosome 6, it was found that the genetic variance value was significantly larger than that of other chromosomes. That is, it was confirmed that the polymorphic site involved in the whole-body dark hair trait exists in chromosome 6.

(2) SNP marker candidate selection

Table 3 below shows 40 SNP marker candidates selected according to the genetic variance ratio for each SNP window.

turn chromosome SNP position (bp) window
(dispersion ratio) One 6 ALGA0119273 451,496 TOP3
(0.7%) 2 6 H3GA0055463 489,077 3 6 H3GA0053623 592,593 4 6 H3GA0053053 2,770,269 TOP1
(2.0%) 5 6 MARC0031342 2,848,500 6 6 ASGA0093316 2,853,231 7 6 M1GA0024473 2,873,515 8 6 ASGA0100675 2,879,708 9 6 ASGA0105361 2,882,725 10 6 ALGA0122740 2,893,272 11 6 MARC0076503 2,937,815 12 6 MARC0077625 2,978,592 13 6 H3GA0056764 3,055,343 14 6 ALGA0107773 3,087,452 15 6 H3GA0053448 3,104,691 16 6 ASGA0083249 3,119,282 17 6 ASGA0094031 3,144,723 18 6 M1GA0027268 3,165,397 19 6 MARC0039494 3,931,665 TOP2
(1.8%) 20 6 ALGA0121836 3,945,296 21 6 ASGA0104016 3,973,212 22 6 ASGA0103013 3,976,460 23 6 ASGA0090800 3,982,654 24 6 ALGA0116736 3,985,810 25 6 MARC0010736 4,009,114 26 6 ALGA0117051 4,017,367 27 6 ALGA0110953 4,034,234 28 6 MARC0032184 4,038,902 29 6 H3GA0055717 4,087,033 30 6 ALGA0102593 4,104,584 31 6 MARC0018463 4,106,208 32 6 ALGA0118793 4,106,407 33 6 ASGA0090309 4,117,942 34 6 MARC0012731 4,119,340 35 6 H3GA0056185 4,157,449 36 6 ASGA0082474 4,218,083 37 6 H3GA0054369 4,255,006 38 6 ASGA0096526 4,255,502 39 6 ASGA0083801 4,301,625 40 6 H3GA0053829 4,316,622

(3) Final SNP marker selection

Through full-length genome analysis of 1,770 heads, 40 candidate SNP markers related to systemic black hair in chromosome 6 were screened, and the top 3 SNP windows with high genetic variance were selected.

Of the 40 SNP markers, 10 SNP markers in which the dominant homogenotype expresses 95% or more of the total black hair color ratio were finally selected.

turn SNP chromosome position (bp) window Woo Sung Homo
genotype Black hair percentage (%) Woo Sung Homo hetero recessive homo One ALGA0119273 6 451,496 TOP3 AA 95% 83% 16% 2 H3GA0055463 6 489,077 TOP3 AA 95% 83% 13% 3 H3GA0053623 6 592,593 TOP3 AA 96% 83% 16% 4 H3GA0053053 6 2,770,269 TOP1 GG 96% 70% 6% 5 ASGA0105361 6 2,882,725 TOP1 AA 96% 77% 17% 6 H3GA0056764 6 3,055,343 TOP1 CC 100% 96% 30% 7 ASGA0083249 6 3,119,282 TOP1 AA 100% 97% 13% 8 M1GA0027268 6 3,165,397 TOP1 GG 99% 95% 14% 9 ALGA0117051 6 4,017,367 TOP2 AA 96% 68% 23% 10 ALGA0110953 6 4,034,234 TOP2 CC 97% 83% 28%

Table 4 shows the SNP markers finally selected according to the whole-body black hair color ratio among the SNP marker candidates. Referring to Table 4, the dominant genotypes of the 10 SNPs are markers that are differentiated from the hair color of swine pigs, especially dominant homo. In the case of the genotype, it was confirmed that about 95% or more of the whole body black hair was expressed. Therefore, the pig individual having the above-mentioned fixed genotype combination has the effect of discriminating with high accuracy that it is a breed of black pig with a whole body black hair color.

The whole-body black SNP marker of the present invention as described above is a genome selection method (single step genomic BLUP, ssGBLUP) with higher accuracy than the existing genome-wide association study (GWAS) using only genome analysis individuals. It was discovered using

In addition, the whole body black hair color SNP marker of the present invention utilized not only genome analysis individuals (1,770 heads) but also color information (11,345 heads) of ancestors, descendants, and contemporaneous groups. The genetic effect from the root cultivar was also taken into consideration by the integrated analysis of the durok (brown color).

The above description of the present invention is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

<110> REPUBLIC OF KOREA, REPUBLIC OF KOREA <120> SNP MAKERS OF IDENTIFICATION OF WHOLE BLACK HAIR IN WOORI BLACK PORCINE AND METHOD FOR IDENTIFYING WHOLE BLACK HAIR USING THE SAME <130> FP-1909103-KR <160> 10 <170> KoPatentIn 3.0 <210> 1 <211> 51 <212> DNA <213> Unknown <220> <223> WOORI BLACK PORCINE <400> 1 cccctagtgc ccttcccaag gaagcccagg gacctcagac ttaactctac y 51 <210> 2 <211> 51 <212> DNA <213> Unknown <220> <223> WOORI BLACK PORCINE <400> 2 tttctcagga gtttccattg tggctcaggt gctttaaaaa ccccacatag y 51 <210> 3 <211> 51 <212> DNA <213> Unknown <220> <223> WOORI BLACK PORCINE <400> 3 aagacgtgtc tgagttacag cgaattctgg ccgcagagac ggggactccc y 51 <210> 4 <211> 51 <212> DNA <213> Unknown <220> <223> WOORI BLACK PORCINE <400> 4 agaaatacac atgcgtgaat gagcatactg ctgggccttc tggaaggttc y 51 <210> 5 <211> 51 <212> DNA <213> Unknown <220> <223> WOORI BLACK PORCINE <400> 5 cccctagtgc ccttcccaag gaagcccagg gacctcagac ttaactctac y 51 <210> 6 <211> 51 <212> DNA <213> Unknown <220> <223> WOORI BLACK PORCINE <400> 6 cttggaacac gggaaacaaa aggttctcta ctgaaccaga aaatccccaa y 51 <210> 7 <211> 51 <212> DNA <213> Unknown <220> <223> WOORI BLACK PORCINE <400> 7 ggctctgaaa tcggatgaga cgttgtcact gctgatctgg tccccacctc y 51 <210> 8 <211> 51 <212> DNA <213> Unknown <220> <223> WOORI BLACK PORCINE <400> 8 ttttatggtc accacacaca tcaaagacat tcccggcact ggcccggccc y 51 <210> 9 <211> 51 <212> DNA <213> Unknown <220> <223> WOORI BLACK PORCINE <400> 9 gcgattgacg ttcgccaaga ctctagcagg cggtgcgact ggaacattgt y 51 <210> 10 <211> 51 <212> DNA <213> Unknown <220> <223> WOORI BLACK PORCINE <400> 10 gcccacaaat taattccaaa ggacacggaa tgctaccctt cgaagacctt y 51

Claims (18)

a polynucleotide consisting of 10 to 100 consecutive nucleic acids including a single nucleotide polymorphism (SNP) site in which the 51st base is A or G in the polynucleotide represented by SEQ ID NO: 1;
a polynucleotide consisting of 10 to 100 consecutive nucleic acids including a single nucleotide polymorphism (SNP) site in which the 51st base is A or G in the polynucleotide represented by SEQ ID NO: 2;
a polynucleotide consisting of 10 to 100 consecutive nucleic acids including a single nucleotide polymorphism (SNP) site in which the 51st base is A or G in the polynucleotide represented by SEQ ID NO: 3;
a polynucleotide consisting of 10 to 100 consecutive nucleic acids including a single nucleotide polymorphism (SNP) site in which the 51st base is A or G in the polynucleotide represented by SEQ ID NO: 4;
a polynucleotide consisting of 10 to 100 consecutive nucleic acids including a single nucleotide polymorphism (SNP) site in which the 51st base is A or G in the polynucleotide represented by SEQ ID NO: 5;
a polynucleotide consisting of 10 to 100 consecutive nucleic acids including a single nucleotide polymorphism (SNP) site in which the 51st base is A or G in the polynucleotide represented by SEQ ID NO: 6;
a polynucleotide consisting of 10 to 100 consecutive nucleic acids including a single nucleotide polymorphism (SNP) site in which the 51st base is A or G in the polynucleotide represented by SEQ ID NO: 7;
a polynucleotide consisting of 10 to 100 consecutive nucleic acids including a single nucleotide polymorphism (SNP) site in which the 51st base is A or C in the polynucleotide represented by SEQ ID NO: 8;
a polynucleotide consisting of 10 to 100 consecutive nucleic acids including a single nucleotide polymorphism (SNP) site in which the 51st base is A or C in the polynucleotide represented by SEQ ID NO: 9; and
In the polynucleotide represented by SEQ ID NO: 10, the 51st base is A or C for single nucleotide polymorphism (SNP) region comprising a polynucleotide consisting of 10 to 100 continuous nucleic acids for identification of black guinea pigs SNP marker composition. [Claim 1] A composition for the identification of whole-body black hair of black pigs, comprising an agent capable of detecting or amplifying the SNP marker composition for identification of the black hair of black pigs of claim 1. A kit for the identification of a whole body black porcine of a guinea pig comprising the composition for identification of the whole body tan color of the guinea pig of claim 2 . 4. The method of claim 3,
The kit is an RT-PCR kit or a DNA chip kit, a kit for the identification of the black fur of Korean black pigs. A microarray for whole-body black-haired identification of Korean black pigs comprising the SNP marker composition for whole-body black-haired identification of Korean black pigs of claim 1. A sample extraction step of extracting a sample from Woori Heukdon;
A polymorphic amplification step of amplifying the polymorphic site of the SNP marker composition for the identification of the whole body black hair of claim 1 from the DNA of the sample; and
A whole-body black hair identification method of guinea pigs comprising a base determination step of determining the base of the amplified polymorphic site. 7. The method of claim 6,
The polymorphic site is a method for identifying the black hair color of the whole body of the black pig, which is present on the chromosome 6 of the black pig. 8. The method of claim 7,
The step of determining the base is a step of discriminating the allele of the base of the polymorphic site in the SNP marker for whole-body black hair identification of the black pig, the whole body black hair identification method of the black pig. 9. The method of claim 8,
When the allele of the 51st base, which is a polymorphic site, is A/A in the SNP marker for whole-body black hair identification of Woori heukdon represented by SEQ ID NO: 1, the whole body of Woori heukdon identified as having a whole body black hair color. How to identify black hair. 9. The method of claim 8,
When the allele of the 51st base, which is a polymorphic site, is G/G in the SNP marker for whole-body black hair identification of guinea pigs represented by SEQ ID NO: 2, the whole body of guinea pigs identified as having a whole body black shading. How to identify black hair. 9. The method of claim 8,
When the allele of the 51st base, which is a polymorphic site, is G/G in the SNP marker for whole-body black shading of guinea pigs represented by SEQ ID NO: 3, the whole body of guinea pigs identified as having whole-body black shading. How to identify black hair. 9. The method of claim 8,
In the SNP marker for the identification of whole-body black shading of guinea pigs represented by SEQ ID NO: 4, when the allele of the 51st base, which is a polymorphic site, is G/G, the whole body of guinea pigs identified as having a whole-body black shading. How to identify black hair. 9. The method of claim 8,
When the allele of the 51st base, which is a polymorphic site, is G/G in the SNP marker for whole-body black hair identification of guinea pigs represented by SEQ ID NO. How to identify black hair. 9. The method of claim 8,
In the SNP marker for the identification of whole-body black shading of guinea pigs represented by SEQ ID NO: 6, when the allele of the 51st base, which is a polymorphic site, is G/G, the whole body of guinea pigs identified as having a whole-body black shading. How to identify black hair. 9. The method of claim 8,
In the SNP marker for whole-body black hair identification of guinea pigs represented by SEQ ID NO: 7, when the allele of the 51st base, which is a polymorphic site, is G/G, the whole body of guinea pigs identified as having a whole body black shading. How to identify black hair. 9. The method of claim 8,
When the allele of the 51st base, which is a polymorphic site, is G/G in the SNP marker for whole-body black hair identification of guinea pigs represented by SEQ ID NO: 8, the whole body of guinea pigs identified as having a whole body black shading. How to identify black hair. 9. The method of claim 8,
When the allele of the 51st base, which is a polymorphic site, is G/G in the SNP marker for whole-body black hair identification of guinea pigs represented by SEQ ID NO: 9, the whole body of guinea pigs identified as having a whole body black shading. How to identify black hair. 9. The method of claim 8,
When the allele of the 4,034,234 nucleotide, which is a polymorphic site, is G/G in the SNP marker for whole-body black shading of guinea pigs represented by SEQ ID NO: 10, the whole body of guinea pigs identified as having whole-body black shading. How to identify black hair.

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