KR20150050499A - A method for discriminating of shank color of korean native chicken - Google Patents

Abstract

The present invention relates to a development of a DNA marker engaging in a shank color of Korean native chicken, and aims to develop the DNA marker for predicting and identifying an inheritance ability for the shank color of Korean native chicken in early stage, thereby being used for a breeding improvement or economic trait improvement program of the Korean native chicken. In case of using the DNA marker as an early selecting tool of the Korean native chicken to check the shank color of Korean native chicken, the present invention enables to more effectively and economically propel the breeding improvement into a Korean native chicken having a darker color, that is, having a low brightness shank color, compared to an imported broiler having the shank color that a customer requires to the Korean native chicken, specifically, a yellow color close to a bright yellow color. Therefore, the present invention enables to contribute to an income increase and a management improvement of a native chicken farm.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for identification of Korean traditional chickens,

The present invention relates to a method for distinguishing the tongue of a Korean native chicken and a novel DNA marker for distinguishing the tongue from the chicken.

Until recently, various efforts to preserve animal genetic resources have continued. In particular, animal genetic resources around the world are being documented, centered on the Livestock Diversity Information System (DAD-IS) run by the United Nations Food and Agriculture Organization (FAO). In Korea, conservation of livestock genetic resources is being carried out mainly in the National Livestock Academy. In relation to chickens, starting with the process of collecting traditional chickens scattered all over the country since 1992, The breeding project was started and the selection of the traditional chicken was carried out based on the economic trait for 15 years. As a result, five Korean native chicken lines were constructed and listed in DAD-IS under the names of white native, black native, dark brown native, yellow native and red-brown native species.

The Korean chicken selected as the Korean traditional chicken line is produced and consumed in a practical system such as 'Woorimachi chicken'. When compared to other practical broilers, our chicken is superior in nutrition, unique flavor and texture, .

The Korean native chickens are classified into five lines mainly by feather color. When constructing these lines, individuals with dark colored tongue color such as gray black or dark green were selected for differentiating from broiler having yellow tongue different color. However, in spite of repeated generations of generations, individuals with various tongue alternatives appear on all lines.

Korea 's consumers prefer to have darker tongue of traditional chicken, so tongue is considered as one of the important economic traits. In particular, consumers expect that traditional chickens, unlike ordinary broilers, will have a dark tongue. Therefore, when the consumers distinguish between broiler and conventional chicken, the difference of the stool is considered to be an important index.

It is known that chickens are affected by genes such as TYR (Tyrosinase), SLC45A2, SOX10, PMEL (Premelanosome protein), MC1R (Melanocortin 1 receptor) and MLPH (Melanophilin).

Among them, the MC1R gene is known to be an important gene that regulates the search for mammals and the feather color of a chicken. The MC1R gene is known to play a role in regulating eumelanin (black / brown) and pheomelanin (red / yellow).

Skin color, such as chicken tongue, is associated with carotenoid content, such as xanthophyll in feed, and previous studies have used the BCDO2 gene as a causative gene for yellow skin color. The BCDO2 gene is known to encode a beta-carotene dioxygenase 2 enzyme and cleave a colorless carotenoid into a colorless apocarotenoid, resulting in yellow or white skin tone. This pigmentation provided information that the red jungle fowl is not the only origin of domesticated chickens.

Previous studies using Korean native chickens have involved studies on the MC1R gene in relation to the chicken mutation, but the causative genes related to the mutation in the chicken stomach have not been identified to date. Therefore, it is required to identify the genetic factors related to the Korean traditional chicken tongue dichotomy in order for the consumers to select the desired tongue-like individuals and to maintain the tongue-related genes of these individuals.

However, studies on genetic information such as genetic information, such as marker-assisted selection, have been conducted mainly in relation to cattle or pigs, and studies on chickens, especially Korean native chickens, to be. In particular, there has been little research on the relationship between MC1R specific genotypes or polymorphisms and chicken tongue dysplasia in relation to chicken tongue dysplasia.

KR 10-1258186 B

As described above, the present invention provides a method for selecting Korean traditional chickens having a different color of the stool, which is deemed to be darker than yellow, that is desired by the consumer, In order to improve profitability, it is aimed to provide a genetic marker capable of identifying the tongue color of Korean native chicken, specifically a DNA marker.

It is another object of the present invention to provide a method for identifying the tongue color of Korean native chicken using the DNA marker.

In order to achieve the above object, the present invention provides a genetic marker capable of selecting chickens from Korean native chickens, specifically DNA markers.

In addition, the present invention relates to a genetic marker capable of identifying a chicken from Korean native chicken, and a composition capable of detecting or amplifying a DNA marker, to provide.

In addition, the present invention provides a kit for distinguishing the Korean traditional chicken from the Korean traditional chicken, which comprises a composition for identifying the tongue of Korean native chicken.

In addition, the present invention provides a discrimination method for identifying the tongue differentiation of Korean native chicken, specifically, a genetic marker for confirming the tongue differentiation of Korean native chicken, and a method for discriminating the native tongue of Korean native chicken using DNA marker do.

The inventors of the present invention conducted research on improving the breeds of Korean native chickens or Korean native chickens and found that Korean traditional chickens And to investigate DNA markers related to the demand for dichromatite required for the diagnosis of the disease.

The inventors of the present invention have conducted intensive researches on this subject and found that the genetic analysis of five Korean native chickens constructed through conservation work of livestock genetic resources on chickens has shown that the DNA sequences of native chickens Variations were detected. In particular, there are four single nucleotide polymorphisms (SNPs) in the MC1R (Melanocortin 1 receptor) gene of Korean native chicken, and the only single nucleotide polymorphism, specifically the 427th nucleotide sequence of the MC1R gene (A> G) locus was closely related to the tongue dynamics of Korean native chicken. From this, it was confirmed that the single nucleotide polymorphism of MC1R gene of Korean native chicken Can be used as a DNA marker for confirming the tongue coloration of Korean native chicken. The present invention has been completed based on this finding.

In the present invention, polymorphism refers to a case where the same species of organism has various features or unique characteristics, or a case where two or more alleles exist in one locus.

Differences in the traits due to mutations of a single base are referred to as single nucleotide polymorphism (SNP) in the polymorphic sites, depending on the individual. Single nucleotide polymorphism by the single gene mutation is widely used to judge various animal breeds. Various studies using SNP DNA analysis using single nucleotide polymorphism for the study of genetic resources in Korea and conservation of genetic resources However, research on pigs has been increasing recently. However, studies on Korean native or native chickens have been limited.

The polymorphism marker for confirming the polymorphism may have two or more alleles showing a frequency of occurrence of 1% or more, preferably 5% or more, or 10% or more in the selected population.

In the present invention, an allele means various types of a gene existing on the same locus of a homologous chromosome. The alleles are also used to represent polymorphisms, for example, single base polymorphisms have two kinds of biallele.

In the present invention, a primer is a nucleotide sequence having a short free 3 'hydroxyl group, which is a starting point for template strand copy capable of forming base pairs with a complementary template Quot; short sequence " The primers are designed to be capable of undergoing a polymerization reaction (Polymerase Chain Reaction, PCR) by four nucleoside triphosphates different from enzymes such as DNA polymerase or reverse transcriptase at appropriate buffer solutions and temperatures it means. The PCR conditions, the lengths of the sense and antisense primers in the polymerase chain reaction, and the like can be modified based on those known in the art.

In the present invention, the term " an individual " refers to a chicken, which is a subject to be tested for tongue color of an individual, specifically, a Korean native chicken. By analyzing the genotype of the SNP marker using a sample obtained from the Korean native chicken, It is possible to judge whether or not the color of the shin is dark, specifically the color of the shin. 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.

In the present invention, a DNA chip means one of DNA microarrays capable of confirming each base of hundreds of thousands of DNAs at a time.

Hereinafter, the present invention will be described in detail.

In one aspect of the present invention, the present invention relates to a genetic marker or a DNA marker for identification of chicken stomach, particularly Korean native chicken or Korean native chicken.

Preferably, the genetic marker for confirming the dangling dangling of Korean native chicken is preferably selected from the group consisting of whether or not the dangling color of Korean native chicken is confirmed, that is, whether the dangling dangling is darker than other genotypes of Korean native chicken, more specifically, That is, it is possible to confirm or determine whether the lightness of the native chicken of Korean native chicken is lower than the lightness (L value) or the lightness (L value) of the tongue of the native Korean chicken having different genotype And provides a single nucleotide polymorphism (SNP) marker.

The marker may preferably be all or some of the polynucleotides comprising the SNP region of the MC1R gene, more preferably all or some polynucleotides of the polynucleotide consisting of SEQ ID NO: 1, 1 is a polynucleotide consisting of 5 to 500 consecutive bases or a complementary polynucleotide thereof containing the 427th base at the position of the single nucleotide polymorphism (SNP) of the MC1R gene represented by SEQ ID NO: It may be a DNA marker which can confirm the high and low brightness of the chicken stomach, especially the lightness of the chicken stomach of Korean native chicken.

In this regard, the DNA marker may be a polynucleotide consisting of 5 to 500 consecutive bases, wherein the 427th base is A or G in the nucleotide sequence of the MC1R gene represented by SEQ ID NO: 1, or It is a complementary polynucleotide of chicken, and it can be a DNA marker which can confirm the high and low brightness of chicken liver of Korean native chicken.

The DNA marker may be a single nucleotide polymorphism (SNP) marker for confirming whether or not the Korean native chicken is unique to the stomach, specifically, whether it is yellow or red.

In addition, the polynucleotide of the single nucleotide polymorphism (SNP) marker for Korean native chicken identification, which has a lower lightness than the other genotypes having a different lightness of the stool specimen, has a nucleotide sequence of nucleotide 427 of the MC1R gene Is A or G and is a polynucleotide consisting of 5 to 500 consecutive bases comprising the 427th base or a complementary polynucleotide thereof.

Preferably, the polynucleotide of the single nucleotide polymorphism (SNP) marker for Korean native chicken identification is lower in the nucleotide sequence of the MC1R gene than in the nucleotide sequence of the nucleotide sequence of SEQ ID NO: 1, 427 The second base is A or G, and the second base is a polynucleotide composed of 20 to 500 or 220 to 300 consecutive bases including the 427th base, or a complementary polynucleotide thereof.

In addition, the single nucleotide polymorphism (SNP) marker for identification of Korean native chicken having a low lightness of the stomach, i.e., a low specificity of the stool differs from the other genotypes having different lightness of the stool specimen, is represented by SEQ ID NO: 1 in the nucleotide sequence of the MC1R gene represented by SEQ ID NO: The genotype of the MC1R gene may be a DNA marker for confirming whether the 427th genotype of the MC1R gene is a heterozygote genotype of AG. The DNA marker may be a DNA marker capable of identifying the genotypes of Korean native chicken and confirming the dangling nature of Korean native chicken.

According to one embodiment of the present invention, a total of 88 families of fifty male and fifty-seven females of Korean native chicken were crossed and 596 male hatchlings were crossed with each other. Analysis of base polymorphism and tongue dichromatism showed that the 427th base of the MC1R gene is closely related to the darkness of the stool tongue, that is, the density of the stool tongue, and the 427th genotype is a heterozygote genotype of AG And it was confirmed that whether or not the tongue is dark or not depends on whether or not the tongue is dark.

Therefore, when the 427th genotype of the MC1R gene of the present invention is detected as a heterozygote genotype of AG or using a primer capable of confirming the genotype, it is possible to determine whether the tongue color of Korean native chicken is darker Can be accurately identified.

In another aspect, the present invention provides a composition for identifying dangers of Korean native chicken, comprising a preparation capable of detecting or amplifying the DNA marker.

The composition for screening can be a composition for confirming the composition of Korean traditional chicken for duck tongue identification, specifically, whether or not the duck color of Korean native chicken is dark, that is, the brightness of duck tongue is lower than that of other genotypes.

Wherein the DNA marker is a polynucleotide consisting of 5 to 500 consecutive bases, wherein the 427th base is A or G in the base sequence of the MC1R gene represented by SEQ ID NO: 1, the complementary poly Nucleotides. It is possible to confirm whether or not the Korean traditional chicken has a different color of the stomach, preferably the stomach, or more preferably the lightness of the stool is lower than that of the other genotypes Which is a DNA marking factor for confirming the dysplasia of Korean native chicken.

The agent capable of detecting or amplifying the DNA marker may be a composition capable of detecting the shin color of Korean traditional chicken, specifically the darkness of the shin color, by detecting or amplifying the polymorphic site of the gene. Specifically, it may be a primer capable of specifically amplifying the polynucleotide of the single nucleotide polymorphism marker, that is, the SNP marker.

The primer used for the SNP marker amplification may be a single strand oligonucleotide capable of serving as a starting point of template-directed DNA synthesis under appropriate conditions and a suitable temperature change, using an appropriate buffer.

The appropriate length of the primer may vary depending on the purpose of use. In addition, the primer sequence need not be completely complementary to the SNP marker, but should be sufficiently complementary to hybridize with the SNP marker.

Thus, the primers are Korean native chickens having an average or other genotype of Korean native chickens, for example, Korean traditional chickens having homozygous genotype, for example, L value) is low. The primer may be one that can confirm the single base polymorphism of the gene marker, specifically, the MC1R gene of Korean native chicken, preferably the MC1R gene of SEQ ID NO: 1.

The primers can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. The nucleic acid sequence of the primer can also be modified using many means known in the art. Non-limiting examples of such modifications include, but are not limited to, methylation, capping, substitution of one or more natural nucleotides with an analog, and modification between nucleotides, such as methylphosphonate, phosphotriester, phosphoramidate, carbamate, Non-conjugated linkages or transformations into charged linkages such as phosphorothioates, phosphorodithioates, and the like.

In another aspect, the present invention provides a Korean traditional chicken having an average or different genotype of Korean native chickens, specifically, Korean traditional chicken having a homozygous genotype of Korean native chicken, The present invention relates to a kit for distinguishing a Korean traditional chicken from a Korean traditional chicken, which comprises a composition for discriminating between the darkness and the lightness of the dorsal root of a Korean native chicken, will be.

The kits for distinguishing the Korean traditional chicken's tongue can identify the presence or absence of the DNA marker or gene marker or the expression level of the DNA marker or the expression level of the DNA marker, It is possible to judge whether or not the chicken stool can be identified.

In this respect, the kit for discriminating the stool of Korean native chicken may be a kit for DNA analysis or a PCT kit. The DNA analysis kit may be, for example, a DNA chip, and the PCT kit may be, for example, an RT-PCR kit.

The DNA analysis kit may be a DNA chip kit including essential elements necessary for performing a DNA chip. The DNA chip means one of DNA microarrays capable of confirming each base of hundreds of thousands of DNAs at a time. For example, the DNA chip kit may include a flat solid support plate, specifically a chip having a glass surface on a glass surface that is not larger than a microscope slide, with a nucleic acid species attached in a gridded array, Nucleic acid is uniformly arranged on the surface of the chip and hybridization reaction between the nucleic acid on the DNA chip and the complementary nucleic acid contained in the solution treated on the surface of the chip enables the mass parallel analysis related to the DNA in a short time .

The RT-PCR kit may be a kit for measuring the mRNA expression level of DNA markers for dysplasia identification of Korean native chicken, and may be a kit containing essential elements necessary for carrying out conventional RT-PCR. The RT-PCR kit may comprise a respective pair of primers specific for the gene of the DNA marker, and may further comprise a test tube or other appropriate container, a reaction buffer (pH and magnesium concentrations vary), deoxynucleotides (dNTPs), enzymes such as Taq polymerase and reverse transcriptase, DNase, RNAse inhibitor, DEPC-water, sterile water, and the like. It may also contain a primer pair specific for the gene used as a quantitative control.

In another embodiment, the present invention provides a method for identifying a Korean traditional chicken having a homozygous chromosome of Korean native chicken, specifically Korean native chicken having an average or other genotype of Korean native chicken, Korean native chicken having homozygous genotype, (L value) is low or not, preferably, a low lightness value (L value) is low.

Amplifying a specific region of the MC1R gene represented by SEQ ID NO: 1 including the 427th base of the DNA marker from the DNA of the sample separated from the individual; Analyzing a nucleotide sequence of a specific site of the amplified MC1R gene; And identifying the 427th base from the result of the analyzed nucleotide sequence.

The nucleotide sequence of the specific region of the MC1R gene represented by SEQ ID NO: 1 including the 427th base of the DNA marker of claim 1 was analyzed from the DNA of the sample isolated from the individual ; And identifying the 427th base of the MC1R gene of SEQ ID NO: 1 from the result of the analyzed nucleotide sequence.

The step of confirming the base from the result of the analyzed nucleotide sequence can be performed by confirming that the 427th genotype in the nucleotide sequence of the MC1R gene represented by SEQ ID NO: 1 is a homozygous genotype of AG, The method can be performed by confirming that the 427th genotype of the MC1R gene represented by SEQ ID NO: 1 is a heterozygote genotype of AG from the result of the analyzed nucleotide sequence.

The screening method includes a marker-assisted selection (MAS) using a marker or a primer set capable of genotyping a marker, or a marker using a marker or primer set related to the SNP site of the MC1R gene of Korean native chicken It can be a help selection method.

The DNA sample separated from the individual can be a genome DNA obtained from the chicken, preferably Korean native chicken to be searched. More specifically, the DNA sample separated from the genome can be a MC1R gene of a chicken isolated from the genomic DNA, It may be a site corresponding to the MC1R gene.

The DNA sample may be a DNA sample collected from a chicken, specifically a Korean native chicken. More specifically, it may be a genomic DNA obtained from the blood, semen, juice, or chicken meat of Korean native chicken.

The MC1R gene represented by SEQ ID NO: 1 may be a sequence comprising a polymorphic site containing a single nucleotide polymorphism (SNP) in a polynucleotide sequence, preferably a nucleotide polymorphism in a 427th nucleotide Polynucleotide < / RTI > The polynucleotide may be DNA or RNA.

The step of analyzing the nucleotide sequence of the specific region of the MC1R gene represented by SEQ ID NO: 1 may be carried out by a marker-assisted selection method using a marker or primer set capable of genotyping.

The marker-assisted selection method comprises performing a PCR reaction using a primer set capable of amplifying a sequence including the specific region using a DNA sample of chicken as a template, sequencing the PCR reaction product After the PCR reaction product is treated with a restriction enzyme and then subjected to electrophoresis using an agarose gel and a polyacrylamide gel, a DNA band resulting from the electrophoresis (DNA band).

The step of amplifying a specific region of the MC1R gene represented by SEQ ID NO: 1 can be used in 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), transcription amplification, self-sustaining sequence replication and nucleic acid based sequence amplification (NASBA) can be used.

The step of analyzing the nucleotide sequence of the specific region of the amplified MC1R gene or the nucleotide sequence of the specific region of the MC1R gene represented by SEQ ID NO: 1 may be performed by sequencing, hybridization by microarray, allele specific PCR, dynamic allele specific amplification (DASH), PCR extension analysis, PCR-SSCP, PCR-RFLP analysis or TaqMan technique, SNPlex platform (Applied Biosystems), mass spectrometry (for example, Sequenom's MassARRAY Systems, mini-sequencing methods, Bio-Plex systems (BioRad), CEQ and SNPstream systems (Beckman), Molecular Inversion probe array technologies (e.g. Affymetrix GeneChip), and BeadArray Technologies , Illumina GoldenGate, and Infinium assay), but the present invention is not particularly limited thereto.

One or more of the alleles in a DNA marker that comprises the nucleotide sequence of a specific region of the MC1R gene can be identified by the methods described above or other methods available to those skilled in the art to which the invention pertains. The nucleotide sequence of the specific region of the amplified MC1R gene or the nucleotide sequence of the specific region of the MC1R gene represented by SEQ ID NO: 1 can be determined through a DNA chip.

For example, the TaqMan method includes the steps of: designing and preparing a primer and a TaqMan probe to amplify a DNA fragment to be analyzed, for example, a DNA fragment containing a nucleotide sequence of a specific region of the MC1R gene represented by SEQ ID NO: 1; Labeling the probes of different alleles with FAM dyes and VIC dyes (Applied Biosystems); Performing PCR using the DNA as a template and using the primer and the probe; After completion of the above PCR reaction, the TaqMan assay plate is analyzed and confirmed with a nucleic acid analyzer; And determining the genotype of the polynucleotides of the DNA fragment to be analyzed from the analysis result.

The sequencing analysis can be performed using a conventional method for nucleotide sequencing, and can be performed using an automated gene analyzer. Also, the allele-specific PCR is a primer set containing a primer designed with the SNP or the 3 'end of a DNA fragment containing the nucleotide sequence of a specific region of the MC1R gene represented by SEQ ID NO: 1 as the SNP or the MC1R gene Means a PCR method of amplifying a DNA fragment in which a specific site is located.

The DNA marker of the present invention is a specific SNP marker which can identify Korean native chicken having a darker and darker color than conventional broiler which is desired by consumers among Korean native chickens. In case of using the DNA marker, , It is possible to quickly and easily identify whether or not the shade of the shank is difficult to distinguish. Therefore, it is possible to predict and identify the genetic ability of the Korean traditional chicken, Therefore, by using the DNA marker of the present invention in the economic trait improvement or breeding improvement program of Korean native chicken, breeding improvement of Korean native chicken can be promoted more efficiently and economically, thereby increasing the income of domestic chicken farmers It is estimated that it will contribute to the improvement of profitability and sales of farmers who raise Korean native chickens by helping the competitiveness and efficiency of breeding farms that require international competitiveness by trade treaties such as FTA.

FIG. 1 is a diagram illustrating a nucleotide sequence of the MC1R gene of SEQ ID NO: 1 according to an embodiment of the present invention. In the region indicated by the bold type, Means a result of detecting a mutation.
FIGS. 2 and 3 are photographs of the Korean traditional chicken provided with a DNA sample used as a sample (FIG. 2) by feather color.

Hereinafter, the present invention will be described in more detail with reference to production examples and examples. However, the following Preparation Examples and Examples are merely illustrative examples for the purpose of facilitating understanding of the present invention, and can be modified into various other forms, and the scope of the present invention is not limited to the following Examples.

Example

Example  One. DNA  Sampling

The National Livestock Research Institute of Korea (NIAS) has 597 Korean native chicken stocks of 5 lines, 88 specimens of 15 male and 73 female specimens, We used a sample of 597 Korean traditional chicken breeders and their children who mated them. This strain was maintained by standard breeding procedures at the National Livestock Academy.

These 596 F1 individuals had 110 grayish brown lines (G), 90 black lines (L), 135 reddish brown lines (R), 126 white lines (W), and 135 yellowish brown lines Y), and the tongue color of each individual was measured. All the animals were kept in the same conditions as the same conditions in order to minimize environmental variation.

Each tibial dichroism was measured at different positions with a minimum of 3 repetitions. Lightness, redness, and yellowness were measured using a spectrophotometer for color measurement.

A DNA sample of the Korean native chicken was obtained in the following manner.

First, a blood sample was obtained using a vacutainer tube containing EDTA from an umbilical vein located on chicken wings of Korean native chicken at 22 weeks of age, and DNA was extracted from the blood sample using a manual extraction method.

Specifically, manual extraction was performed as follows. First, Buffer A (0.32 M Sucrose, 1 mM TrisHCl, 5 mM MgCl ₂ , 1% Triton X-100, dH ₂ O) was diluted with 4-fold volume per 5 ml of the blood sample and then spun down The process of carefully removing the supernatant was repeated 3 times to remove the supernatant. 250 μl of Buffer B (400 mM NaCl, 2 mM EDTA, 10 mM TriHCl, dH ₂ O) and Buffer C (2 mg / ml of 5% SDS, 2.6 ml of Proteinase K) were added to the blood sample from which the supernatant was removed , Inverted and immersed in a 50 shaking incubator for 24 h. After the immersion, 675 μl of 6 M NaCl in a saturated state was added, and the mixture was vigorously shaken for 15 seconds, centrifuged at 3000 rpm for 15 minutes in a centrifuge, and the supernatant was transferred to a new tube. A volume of 99% ethanol (EtOH) aqueous solution was added to the tube to which the supernatant was transferred to precipitate the DNA, and the 99% aqueous ethanol solution was removed. The precipitate from which the 99% aqueous ethanol solution was removed was washed with a 70% aqueous ethanol solution, DNA was obtained by pipette-tip-hook method and transferred to a new microtube.

500 μl of TE buffer (TE buffer) was added to the obtained DNA pellet, and the DNA was dissolved to obtain a final DNA sample

Example  2. MC1R  Gene SNP  Confirm

The SNP genotype of Korean native chicken was genotyped by the BioMark HD system (Fluidigm® 192.24 SNPtype ^™ , USA).

The major amplification site (STA: SNP location) was selected by selecting four SNPs of the MC1R gene, and 14 amplifications were performed using 25 ng / μl of DNA. The STA used for amplification was diluted using an IFC controller and the mixture was subjected to an amplification cycle using a BioMark 192.24 array. Finally, mutation of the MC1R gene in the offspring generation group was confirmed by SNP genotyping analysis.

The measured results were tested using Minitab 14.0 (Minitab, USA) to determine whether they could be used for significance analysis. The variance analysis model was tested for significance between individuals or lines using a general linear model (GLM). The effect of the MC1R gene on SNP-induced body weight gain was calculated by applying to all 591 Korean native chicken 5 lines. The general linear analysis was analyzed using the following model.

[formula]

In the above formula 1, Y _ijklmno the phenotype of the individual, and the average of μ group, G _i is a fixed effect of the genotype, S _j is a fixed effect due to gender, B _k is a fixed effect of the group, L _l is caused by Systems fixing effect, F _{m (l)} is any effect of the paternal belonging to the first system, M _{n (lm)} is any effect of maternal line belongs to a mating group of the first grid and paternity, □ _ijklmno is of the parents group It was used as the residual of association analysis. Tukey's test was used to distinguish these genotypes.

As a result of the above analysis, among the six base polymorphisms identified in Example 1, there were four base polymorphisms associated with dorsal root dysplasia, and three significant polymorphic base polymorphisms among the dorsal root were identified as yellow, In particular, the nucleotide polymorphism, which is significantly related to the light intensity of the dark stool, was significantly different from that of the stool specimen (Table 1). Specifically, the base polymorphism was confirmed to be c.427 G> A SNP.

In Table 1, the P-value represents a significant value, the L value represents brightness, the a value represents redness, and the b value represents yellowness.

As shown in Table 1, c.212 C> T SNP, c.274 A> G SNP, and c.427 A> G SNP among the base polymorphisms were found to be related to dorsal dichroism, T SNP and c.274 A> G SNP were associated only with yellow color, and c.427 A> G was found to be associated only with the lightness of darkness of Korean traditional chicken, that is, darkness. In the 427th genotype of the gene, it was confirmed that the shingles color was the darkest color in the homozygous genotype of AG compared to the homozygous.

In case of Korean traditional chicken, it is recognized as superior to consumers in terms of superior nutrition and unique flavor and texture as compared with other commercial broilers. However, in case of chicken, which is characteristic of Korean traditional chicken, And it is not easy to distinguish Korean native chickens. Therefore, consumers perceive chickens with dark shank color as Korean native chickens, compared to imported broilers.

In this respect, SNP associated with lightness of shin color which was first identified in the present invention, that is, c. 427 A> G, may be useful for improving economic traits such as dark shin color in relation to Korean native chicken Respectively.

<110> Foundation of University-Industry Research Collaboration <120> A METHOD FOR DISCRIMINATION OF SHANK COLOR OF KOREAN NATIVE          CHICKEN <130> DP20140423 <150> 2013-0130321 <151> 2013-10-30 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 945 <212> DNA <213> The MC1R gene <400> 1 atgtcgatgc tggcccccct gcgcctgctg cgcgagccct ggaacgccag tgagggcaac 60 cagagcaayg ccacggccgg ggccggaggt gcctggtgcc aggggctgga catccccaat 120 gagctcttcc tgacgctggg gctggtgagc ctggtggaga acctgctggt ggtggccgcc 180 atcctcaaga acaggaatct gcactcgccc aygtactact tcatctgctg cctggccgtc 240 tccgacatgc tggtgagcgt cagcaacctg gccragacgc tcttcatgct gctgatggag 300 ccggcgtgc tggtgatccg cgccagcatc gtccgccaca tggacaatgt catcgacatg 360 ctcatctgca gctccrtcgt gtcctccctc tccttcctgg gggtcatcgc cgtggaccgc 420 tacatcrcca tcttctatgc gctgcgctac cacagcatca tgacgctgca gcgcgccgtg 480 gtcaccatgg ccagcgtctg gctggccagc accgtctcca gcaccgtctt aatcacctac 540 taccgcaaca acgccatcct gctctgcctc attggcttct tcctcttcat gctggtcctc 600 atgctggtgc tctacattca catgttcgcg ctggcaygcc accacgtgcg cagcatctcc 660 agccagcaga agcagcccac catctaccgc accagcagcc tgaagggagc cgtcacgctc 720 accatcctgc tgggagtctt cttcatctgc tgggggccct tcttcttcca cctcatcctc 780 atcgtcacct gccccaccaa ccccttctgc acctgcttct tcagctattt caacctcttc 840 ctcatcctca tcatctgcaa ttcagtggtc gatcccctga tctatgcctt ccggagccag 900 ggctccggc ggacgctgcg ggaggtggtg ctgtgctcct ggtag 945

Claims (9)

Wherein the 427th base in the base sequence of the MC1R gene represented by SEQ ID NO: 1 is A or G, the polynucleotide consisting of 5 to 500 consecutive bases comprising the 427th base or a complementary polynucleotide thereof A DNA marker for confirming the tongue differentiation of Korean native chicken, characterized by having at least one selected polynucleotide selected from the group consisting of Korean native chicken tongue. The method according to claim 1,
Wherein the polynucleotide is a polynucleotide consisting of 5 to 500 consecutive bases, wherein the 427th base is A or G in the nucleotide sequence of the MC1R gene represented by SEQ ID NO: 1, the polynucleotide comprising the 814th nucleotide or a complementary polynucleotide DNA Marker Factor for the Identification of Korean Stalled Chicken. The method according to claim 1,
The DNA markers for identification of the tongue in Korean native chickens are characterized by the fact that the lightness of the tongue of Korean native chicken is low. A composition for identifying dangers of Korean traditional chicken, comprising a substance capable of detecting or amplifying a DNA marker for confirming the dangling dangling of the chicken of claim 1. The kit for distinguishing the Korean traditional chicken from the Korean traditional chicken, which comprises the composition for identifying the tongue of the Korean traditional chicken, which can identify the tongue of the Korean native chicken according to the item 4. 6. The method of claim 5,
The kit for distinguishing the Korean traditional chicken from the stool is a DNA chip kit or a PCT kit. Amplifying a specific region of the MC1R gene represented by SEQ ID NO: 1, which comprises the 427th base of the DNA marker of claim 1, from the DNA of the sample separated from the individual;
Analyzing a nucleotide sequence of a specific site of the amplified MC1R gene; And
From the result of the analyzed nucleotide sequence, the step of confirming the 427th base
The Korean Society of Food Science and Nutrition. Analyzing the nucleotide sequence of a specific site of the MC1R gene represented by SEQ ID NO: 1, which comprises the 427th base of the DNA marker of claim 1, from the DNA of the sample separated from the individual; And
Identifying one or more bases selected from the group consisting of the 427th base of the MC1R gene represented by SEQ ID NO: 1 from the result of the analyzed base sequence
The Korean Society of Food Science and Nutrition. 9. The method of claim 8,
The step of confirming the base from the result of the analyzed nucleotide sequence is performed by a method of confirming that the 427th genotype in the nucleotide sequence of MC1R gene shown in SEQ ID NO: 1 is a homozygote genotype of AG Discrimination method of Korean traditional chicken.

Images