KR101701105B1 - Genetic marker for discriminating plumage color of Korean native duck and uses thereof - Google Patents

Abstract

The present invention relates to a genetic marker for distinguishing coat colors of Korean native duck, and to a use thereof. By using the molecular marker developed to distinguish coat colors of Korean native duck, it is possible to provide genetic information associated with coat color traits of the duck, thereby enabling an examination of first filial generation, variety protection, and variety discrimination of duck in an efficient way. Furthermore, a variety which ensures enhanced user preference can be developed by changing the coat colors, and varieties can be improved through manipulation of coat color traits of Korean native duck as well. To this end, the genetic marker includes: a polynucleotide or a complementary polynucleotide thereof, made up of at least 8 consecutive nucleotides including a single nucleotide polymorphism (SNP) at the 726^th base in base sequences of a dopachrome tautomerase (DCT) gene consisting of base sequences represented by sequence number 1; and a polynucleotide or a complementary polynucleotide thereof, made up of at least 8 consecutive nucleotides including an insertion-deletion (InDel) mutation at the 82^nd to 85^th base in intron 4 in the DCT gene consisting of base sequences represented by sequence number 2.

Description

Genetic markers for discrimination of native ducks of Korean native ducks and their uses (Discrimination of genetic markers for Korean native ducks and uses thereof)

The present invention relates to a genetic marker and a use thereof for color classification of Korean native ducks.

Uncertainty in breeding and production of livestock can be considered as one of the major economic traits. In general, wild animals generally have various colors of color in order to survive in the ecosystem. Individuals with white picolor tend to be found by predators and tend to be disadvantaged in terms of hunting. However, in the improvement of livestock, white picolour has a considerable advantage in the processing of meat, and there is no trace of pigmentation on the appearance of slaughtered meat. This is an economically important trait because it is possible. Therefore, it is very important to search the candidate genes related to the coloring in the breeding of livestock as well as to the poultry, and to confirm the phenotype of this gene.

One of the factors that directly affect this coloration is melanin, which has a major influence on pigmentation. Because the complex synthesis pathway and the regulating action of melanin affect the phenotype of various colors, the function of melanin Affected gene studies have been studied extensively in a variety of varieties. It is known that melanin produces various colors due to complex interactions between pheomelanin, which is involved in yellow deposition, and eumelanin, which is involved in deposition of black and brown. Thus, identifying genetic variants of various genes that may affect melanogenesis and revealing genetic variation and phenotypic relationships can have a significant impact on livestock improvement and can be used to increase economic value. have.

Among the genes involved in the melanin production, genes that are reported to be related include ASIP (Agouti signaling protein), MC1R (Melanocortin 1 receptor), MITF (Microphthalmia-associated transcription factor) and DCT (Dopachrome tautomerase) genes. The ASIP gene encodes secretory signaling molecules for the synthesis of peromelanin instead of eumelanin. In addition, the ASIP gene plays a role in regulating melanogenesis in the central nervous system by participating in the endogenous confluence and peripheral systems of the melanocortin system. The MC1R gene is well known as a gene that affects the pigmentation of various vertebrates. The MITF gene is a Tyr (Tyrosinase) family gene that plays an important role in the staining process and is known to play an important role in pigmentation. The MITF gene has also been reported to produce patterns and albumin as a result of loss and down-regulating of pigmentation in dogs and cows. Expression of this gene can activate transcription of Tyr gene by regulation of Scf-Kit signal. Chang et al. (2006, BMC Genomics, 7-19) have identified that the causative mutation of the recessive white allele of chicken is the result of insertion of the intron 4 of the Tyr gene as a retroviral sequence. The Tyr family includes TYR (tyrosinase), TYRP1 (tyrosinase-related protein-1) and DCT (also referred to as TYRP2) genes. DCT enzyme functions to convert melanin biosynthesis process from melanocyte, Dopachrome to DHICA (5,6, dihydroxyindole-2-carboxylic acid).

On the other hand, recent advances in molecular genetic techniques have led to the development of genetic markers that are associated with genetic diseases at the DNA level or with certain traits of livestock. DNA polymorphisms enable the development of markers by major genes and quantitative trait loci (QTL) that determine important economic traits and include linkage analysis, genetic maps, parentage testing has been used as a powerful tool to analyze distinction of breeds, pedigree analysis, genetic diversity and relationship. Especially in the field of genetic and breeding of livestock, it is used for marker assisted selection (MAS) through genetic markers based on DNA polymorphism.

Most economic traits in livestock are known to be quantitative traits due to the influence of various genes, and studies are being actively conducted to search for genetic loci and genetic loci involved in these traits. Especially, in order to discover the gene related to the trait, a single nucleotide polymorphism (SNP) and trait related researches have been actively carried out on functional genes. Various trait related SNPs have been discovered to date. However, there have been no studies to confirm the genetic information of native ducks until now, and research on the development of markers that can be used to identify individuals and identify varieties has been limited. Therefore, it is required to shorten the period for breeding of native ducks and to develop genetic markers to accurately distinguish cultivars.

Korean Patent No. 1538052 discloses a method for distinguishing breeds of chickens using a single nucleotide polymorphism marker in the TYRP1 gene and Korean Patent No. 1450792 discloses a SNP marker for judging the black staining of pigs and its use However, as in the present invention, the genetic markers for distinguishing the native ducks of Korean native ducks and their uses have not been disclosed.

The present invention has been made in view of the above needs, and it is an object of the present invention to confirm the DCT gene mutation involved in melanin biosynthesis between Korean native ducks of colored (Mallard or black) Among them, the present invention has been completed by identifying two kinds of gene mutation markers capable of discriminating the coloring traits of Korean native ducks.

In order to solve the above-described problems, the present invention provides a polynucleotide comprising a nucleotide sequence of DCT (Dopachrome tautomerase) comprising the nucleotide sequence of SEQ ID NO: 1 and consisting of 8 or more consecutive nucleotides comprising a single nucleotide polymorphism (SNP) Polynucleotides or complementary polynucleotides thereof; Or a polynucleotide consisting of 8 or more consecutive nucleotides comprising an InDel mutation located in the 82nd to 85th bases in intron 4 of the DCT (Dopachrome tautomerase) gene consisting of the nucleotide sequence of SEQ ID NO: 2 or a complementary poly A nucleotide sequence of SEQ ID NO: 1;

The present invention also provides a genetic marker composition for discriminating the color of ducks from ducks, comprising a preparation capable of detecting or amplifying a gene marker for distinguishing white ducks from ducks.

The present invention also provides a primer set for discriminating the color of a duck for amplifying a genetic marker for discriminating the color of the duck.

Further, the present invention provides a primer set comprising: the primer set; And a reagent for carrying out an amplification reaction.

In addition, the present invention provides a method for isolating genomic DNA comprising the steps of: isolating genomic DNA from a duck sample;

Amplifying the target sequence by using the separated genomic DNA as a template and carrying out an amplification reaction using the primer set of the present invention; And

A single nucleotide polymorphism (SNP) located at the 726th base in the base sequence of the DCT (Dopachrome tautomerase) gene comprising the nucleotide sequence of SEQ ID NO: 1 in the above amplification product; Or confirming the genotype of the InDel mutation site located in the 82nd to 85th bases in the intron 4 of the DCT (Dopachrome tautomerase) gene consisting of the nucleotide sequence of SEQ ID NO: 2 ≪ / RTI >

The DCT gene mutation marker according to the present invention provides genetic information related to the color of the duck, thereby enabling efficient identification of the duck, protection of the variety, and testing of one hybrid, and improvement of the consumer's preference by changing the color of the duck It is expected that it will be possible to develop varieties that can be cultivated in Korea,

FIG. 1 shows PCR-RFLP genotyping results of the mutation regions in the three-coloring-related genes ( DCT , MITF , and MC1R ) in duck samples having different genotypes. The right side of the M (marker) at the top of the electrophoresis image shows the genotype of the mutation region in the gene.

In order to accomplish the object of the present invention, the present invention provides a DNA sequence encoding a DNA sequence comprising a nucleotide sequence of DCT (Dopachrome tautomerase) consisting of 8 or more consecutive nucleotides (SNPs) ≪ / RTI > or a complementary polynucleotide thereof; Or a polynucleotide consisting of 8 or more consecutive nucleotides comprising an InDel mutation located in the 82nd to 85th bases in intron 4 of the DCT (Dopachrome tautomerase) gene consisting of the nucleotide sequence of SEQ ID NO: 2 or a complementary poly A nucleotide sequence of SEQ ID NO: 1;

In the present invention, the term " marker " refers to a base sequence used as a reference point when identifying genetically unrelated loci.

In the present invention, the term " mutation region " means a place where a change of a gene or a chromosome occurs, and in the present invention, it may be mixed with a Dense mutation block (DMB). That is, it may mean a region where there is a difference in gene between cultivars. In the present invention, it may mean a region where SNVs are dense. More specifically, the present invention defines mutation regions when the number of single base mutations (SNVs) that differ from standard dielectric genome information is four or more per 10 kb.

In the present invention, the term "single nucleotide polymorphism " (SNP) means a polymorphism in a single base. That is, in some groups, some bases in the entire genome are different from each other in chromosomes. Normally, SNP exists in about 300 to 1000 bases, but is not limited thereto.

As used herein, the term "homozygotic genotype " at the SNP position means a case where allelic bases on the homologous chromosome corresponding to the SNP mutation position are the same base, The term " heterozygotic genotype "at the SNP mutation position means that the homologous base on the homologous chromosome corresponding to the SNP mutation position is a different base.

The SNP marker of the present invention can be used for discriminating the color of ducks based on the fact that the 726th base at the SNP mutation position of the nucleotide sequence of the gene represented by SEQ ID NO: 1, which is the DCT gene of duck, is different from C or T. For example, the SNP corresponding to the 726th nucleotide of SEQ ID NO: 1 indicates a base polymorphism with C or T. In the case of a SNP having the C / T dicotyledon junction type, it is a Korean native duck having a colored pigment, When a homozygous genotype is present, it can be judged as a duck having a white color.

The term " InDel " in the present invention refers to a mutation in which some bases are inserted or deleted in the base sequence of DNA. The InDel marker searches for insertion or deletion regions of the standard genome by comparing and analyzing the genomic information of the genotypes used in the experiment and the genome of the experiment, and generates a primer based on the information do. Therefore, the amplification result can show two types of insertion and deletion in comparison with the standard genome.

The indel marker of the present invention can be used for discriminating the color of ducks based on the insertion or deletion of the AATC base located at the 82nd to 85th bases in intron 4 of the DCT gene consisting of the nucleotide sequence of SEQ ID NO: For reference, the nucleotide sequence of SEQ ID NO: 2 is a sequence corresponding to intron 4 of the DCT gene, not the entire DCT gene. For example, when the 82nd to 85th bases in the intron 4 of the DCT gene consisting of the nucleotide sequence of SEQ ID NO: 2 are genotypes deleted from the nucleotide sequence, an AATC base located at 82nd to 85th bases Can be judged to be a white unpigmented duck when the genotype is an inserted genotype.

The present invention also provides a genetic marker composition for discriminating the color of ducks from ducks, comprising a preparation capable of detecting or amplifying a gene marker for distinguishing white ducks from ducks. The agent capable of detecting or amplifying the gene marker for distinguishing white dyed color of the duck may be, but is not limited to, a primer or a probe.

The present invention also provides a primer set for discriminating the color of a duck for amplifying a genetic marker for discriminating the color of the duck.

In one embodiment of the present invention, the primer set for discriminating the color of the ducks for amplifying the genetic markers for discriminating the color of the ducks may be an oligonucleotide set consisting of the nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 4, But is not limited thereto. Wherein the oligonucleotide primer has 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more in the sequence of SEQ ID NO: 3 or SEQ ID NO: 4 according to the sequence length of each oligonucleotide primer. More than 23, and more than 24 consecutive nucleotides of the oligonucleotide. In addition, the oligonucleotide primer may also include an addition, deletion or substitution sequence of the nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 4.

In the present invention, a "primer" refers to a single strand oligonucleotide sequence complementary to a nucleic acid strand to be copied, and may serve as a starting point for synthesis of a primer extension product. The length and sequence of the primer should allow the synthesis of the extension product to begin. The specific length and sequence of the primer will depend on the complexity of the desired DNA or RNA target as well as the primer usage conditions such as temperature and ionic strength.

In the present invention, the oligonucleotide used as a primer may also include a nucleotide analogue such as phosphorothioate, alkylphosphorothioate, or peptide nucleic acid, or And may include an intercalating agent.

Further, the present invention provides a primer set comprising: the primer set; And a reagent for carrying out an amplification reaction.

In the kit of the present invention, the reagent for carrying out the amplification reaction may include DNA polymerase, dNTPs, buffer and the like. In addition, the kit of the present invention may further include a user guide describing optimal reaction performing conditions. The manual is a printed document that explains how to use the kit, for example, how to prepare PCR buffer, the reaction conditions presented, and so on. The manual includes instructions on the surface of the package including a brochure or leaflet in the form of a brochure, a label attached to the kit, and a kit. In addition, the brochure includes information that is disclosed or provided through an electronic medium such as the Internet.

In addition,

Separating the genomic DNA from the duck sample;

Amplifying the target sequence by performing amplification reaction using the separated genomic DNA as a template and using the primer set of the present invention; And

A single nucleotide polymorphism (SNP) located at the 726th base in the base sequence of the DCT (Dopachrome tautomerase) gene comprising the nucleotide sequence of SEQ ID NO: 1 in the above amplification product; Or confirming the genotype of the InDel mutation site located in the 82nd to 85th bases in the intron 4 of the DCT (Dopachrome tautomerase) gene consisting of the nucleotide sequence of SEQ ID NO: 2 ≪ / RTI >

The method of the present invention comprises isolating genomic DNA from a duck sample. As a method for isolating the genomic DNA from the sample, a method known in the art may be used. For example, the CTAB method may be used, or the PrimePrep (TM) genomic DNA isolation kit (GenetiBio, Korea) may be used. Using the separated genomic DNA as a template, an oligonucleotide primer set according to an embodiment of the present invention may be used as a primer to perform an amplification reaction to amplify the target sequence. Methods for amplifying the target sequence include polymerase chain reaction (PCR), ligase chain reaction, nucleic acid sequence-based amplification, transcription-based amplification system, Strand displacement amplification or amplification with Q [beta] replicase, or any other suitable method for amplifying nucleic acid molecules known in the art. Among them, PCR is a method of amplifying a target nucleic acid from a pair of primers that specifically bind to a target nucleic acid using a polymerase. Such PCR methods are well known in the art, and commercially available kits may be used.

In the method of the present invention, the amplified target sequence may be labeled with a detectable labeling substance. In one embodiment, the labeling material can be, but is not limited to, a fluorescent, phosphorescent or radioactive substance. Preferably, the labeling substance is Cy-5 or Cy-3. When the target sequence is amplified, PCR is carried out by labeling the 5'-end of the primer with Cy-5 or Cy-3, and the target sequence may be labeled with a detectable fluorescent labeling substance. When the radioactive isotope such as ³² P or ³⁵ S is added to the PCR reaction solution, the amplification product may be synthesized and the radioactive substance may be incorporated into the amplification product and the amplification product may be labeled as radioactive. The set of oligonucleotide primers used to amplify the target sequence are as described above.

The method of the present invention comprises identifying said genotype. The genotyping can be performed by gene sequencing, hybridization analysis using an allele-specific microarray, allele-specific PCR, capillary electrophoresis, single-strand conformation polymorphism (SSCP), denaturation gradient gel electrophoresis ), Denaturing HLPC, mass spectrometry, restriction fragment length polymorphism (RFLP), or TaqMan SNP genetyping assay.

In the method according to one embodiment of the present invention, when the amplification product using the oligonucleotide set consisting of the nucleotide sequences of SEQ ID NOS: 3 and 4 is the nucleotide sequence of the DCT gene consisting of the nucleotide sequence of SEQ ID NO: 1, the nucleotide at position 726 is TT Homozygotic genotype; Or in the case of a genotype in which the 82nd to 85th nucleotides are inserted in the intron 4 of the DCT gene consisting of the nucleotide sequence of SEQ ID NO: 2, it can be judged as a white unpigmented duck.

In the method according to one embodiment of the present invention, the amplification product using the oligonucleotide set consisting of the nucleotide sequences of SEQ ID NOS: 3 and 4 is the nucleotide sequence of the nucleotide sequence of the nucleotide sequence of DCT (Dopachrome tautomerase) Heterozygotic genotype with base CT; Or a deletion of the 82nd to 85th bases in the intron 4 of the DCT (Dopachrome tautomerase) gene consisting of the nucleotide sequence of SEQ ID NO: 2.

In the method according to one embodiment of the present invention, the ducks may preferably be Korean native ducks or utility ducks, but are not limited thereto.

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

Materials and Methods

The practical duck group used in the present invention is a Peking species duck of Cherryvalley Co., which is a group having a white pigmented phenotype, and the native duck group is a colored (black duck / black) pigmented phenotype maintained and preserved at National Livestock Academy . The color of these two populations represents a distinct phenotypic difference between black (dark color) and white.

For the polymorphism test, samples of 20 domestic ducks (Mallard color, black) and 20 practical ducks (white, Cherryvally) were supplied from the National Livestock Academy and genomic DNA was obtained from PrimePrep ™ Genomic DNA Isolation Kit (GenetiBio, Korea). In order to identify the location of mutation, sampled samples were subjected to direct - sequencing using three samples each to confirm the presence or absence of mutation in the gene. Primers for PCR amplification for sequencing were prepared using Primer3 program (ver 0.4.0) with reference sequence of Anas Plathyrhychos (Domestic duck, Mallard Duck) reference sequence in NCBI. 100 ng of genomic DNA was used for the PCR reaction. For smooth reaction, the total reaction liquid volume was adjusted to 25 μl by combining 10X buffer, 10 nM dNTP mixture, 10 pmol primer set, and HS Prime Taq (GenetBio, Korea) Amplification reaction. PCR amplification was carried out using an My-Genie 96 thermal block (Bioneer, Korea) at 95 ° C for 10 minutes of initial denaturation followed by denaturation at 95 ° C for 30 seconds, annealing at 65 ° C for 30 seconds, , Followed by 35 cycles of extension at 72 ° C for 30 seconds and final extension at 72 ° C for 10 minutes. The size of the amplified products was confirmed by ultraviolet light in the EtBr stained 2% (w / v) agarose gel. The amplified product was purified and directly sequenced to obtain the nucleotide sequence.

The sequence of the obtained nucleotide sequence was confirmed by alignment using ClustalW program and the restriction enzyme which can cleave each site was searched for the genotyping of all the samples at the obtained mutation position. The RFLP method of the PCR amplification product was performed to confirm the mutation position of the obtained candidate gene, and this information was provided in detail in Table 1 and FIG. The restriction enzyme reaction was carried out in a total volume of 20 μl by mixing 5 μl of the restriction enzyme reaction mixture with 15 μl of the PCR amplification product. The sectioned fragment was electrophoresed on EtBr stained 3% (w / v) agarose gel to determine the size of the section. The genotypes and phenotypes of the 7 SNPs obtained and phenotypes were compared using the SAS (ver. 9.3) and Chi-square test and Fisher's test. The significance level was P <0.05 Respectively.

Example  One. Uncolored  Identification of Genetic Polymorphisms in Related Genes

Information on the total of 65 non-coding region mutations and 11 mutations in total of the chromatin- related genes ( ASIP , DCT , MITF , MC1R ) are shown in Tables 2 and 3, respectively.

A total of 6,290 bp long ASIP (NCBI Gene ID: 101795392) gene was confirmed to contain three exons, an intron and a flanking region. As a result, six intron regions Mutation and 2 3'-UTR region mutations (Table 2).

A total of 17,316 bp long DCT (NCBI Gene ID: 101792699) gene confirmed nucleotide sequences capable of analyzing 8 exons and some introns and adjacent sequences, resulting in 19 mutations in intron and adjacent regions Table 2). Among them, the InDel region of g.10585-88 region was also confirmed. A total of 11 mutation regions were identified in the exon region (Table 3).

A total of 45,505 bp MITF (NCBI Gene ID: 101795047) genes were identified as 8 exons and introns and 8 nucleotides in the 5 'UTR region, 5 nucleotides in the exon (coding) region, (22) and one indel (InDel) in the region (Table 2 and Table 3).

A total of 1,229 bp long MC1R (NCBI Gene ID: 101796989) genes were also identified to identify one exon region and adjacent region. As a result, mutations in two exon regions and seven 3'UTR mutations were confirmed, Nine mutation regions were identified (Table 2 and Table 3).

Example 2. Uncolored  Related genes DCT  My genetic polymorphism Uncolored  Check the association with traits

As shown in Table 3 above, the mutations of a total of 18 exon regions are identified as mutations that can affect amino acid changes and protein structure due to changes in the base sequence, so that the genotypes of the samples with different phenotypes are identified The restriction enzymes that can cleave the mutation region were identified and as a result, restriction enzymes were identified to confirm the genotypes of the seven regions (Table 1). The obtained restriction enzymes were subjected to genotyping analysis by a restriction fragment length polymorphism (RFLP) method to identify genotypes of samples having different phenotypes (FIG. 1).

As a result of analysis of the seven mutations that can identify the genotypes, there was a significant difference between the native duck (colored) group and the utility duck (white) group in all exon regions except the DCT gene c.726C> T (Table 4). The 4 bp indel (InDel) of the non-coding region g.10585-88 (AATC) of the other DCT genes was also identified as a mutation showing a significant correlation with the unpigmented trait (Table 4). Taken together, these two results indicate that a significant correlation is found between the c.726 region, which is the coding region of the DCT gene, and the non-coding region, g.10585, the c.726 mutation of the coding region, And the mutation of g.10585 indel (InDel) acts as a genetic marker for the causative gene DCT gene.

<110> The Industry & Academic Cooperation in Chungnam National University (IAC) <120> Genetic marker for discriminating plumage color of native native          duck and uses thereof <130> PN15190 <160> 12 <170> Kopatentin 2.0 <210> 1 <211> 2146 <212> DNA <213> Anas platyrhynchos <400> 1 acgcgggacc cgacaccagc atcctcagta gtgagagcga ttatcccagg ggagctgctg 60 ccaggccagg ttgtcccaca gaggagagct ctgtgctccc ctcactcggg tccaggctgc 120 acccagcagc aatgggtgcc cggaggtggc ttttttgggc tgtgctgagc tgcctgagct 180 gctgttgtcc ccgcggggca gaggcacagt tcccccgtgt ctgcatgacg gtggaggcca 240 ttcggtcgaa gcgctgctgc ccggccttgg ggccggagcc aggcaatgtg tgtgggtccc 300 tgcagggcag aggacgctgc cagggggtgc aggtggacac tcagccctgg agtggcccct 360 acactcttcg aaatgtggat gaccgggaat ggtggccact caagttcttc aaccagagct 420 gttggtgcac aggaaacttt gctggctaca actgtggtga ctgcaaattt ggctggacag 480 gccccgactg caatgtcagg aagccgccag ttgtcaggaa gaacatccat tccttgacag 540 cagaggagag ggagcagttc ttcgatgctc tagaccgtgc aaagactacc attcacccgg 600 actatgtaat tacaactcag caccggataa gtctgcttgg tcccactgga gaggaaccgc 660 aaattgccaa ttgtagcatt tataactact ttgtctggct tcattactac tcagtcagag 720 acacactact agggccaggc cgtcccttca cagctattga tttctcccat caaggacctg 780 cctttgtgac ttggcatagg taccatttgc tgttgctgga gagagacctg cagcgactgc 840 tgggcaatga ctcctttgca ctgccctact gggactttgc cacaggtaga aacgtgtgtt 900 ttgtgtgcac agaccagctc tttggagcat cgcggccaga tgacctgggg ctaatcagcc 960 tgaattccag attctcccgg tggcaaatag tttgtaacag cttggatgat tacaaccgcc 1020 tggtgacact gtgcaatggg agtgatgagg ggccactgcg gcggagacca cagagggact 1080 cgggagagca gttgcccact gtggaagatg tcaggaggtg cctttccctg caggagttcg 1140 acagcccccc atttttccgc aattccagtt tcagcttcag gaatgcactg gaaggcttca 1200 ataaaccaga gggagccctg aactcgccaa tgctaagcct ccataatttg gctcactctt 1260 tcctgaatgg gaccagtgtt ctccctcatg cagctgccaa tgatcccatc tttgtggttc 1320 ttcactcctt cactgatgcc atctttgacg agtggaagaa gcggtttaac ccccctgaca 1380 atgcctggcc tgaggagctg gcccctatcg gtcacaacag actgtacaat atggtccctt 1440 ttttccctcc tgtgaccaat gaccaactct tccaaactgc ggagcagctt ggctacacct 1500 atgccattga cctgccaggt tcacttgaag aaacccaagc atgggctgtc aagattggat 1560 ctacgattgg aggagcattt atagctttgg ccatactcat tctgctgctt gtactttttc 1620 agcaccgaag gcagagaaaa ggttttgaac cactggtgga tgtgagcttc agccccaaaa 1680 agtacatggg agaggcctag tgcccttgct tcgtttcctt gcttattctt gtaggaaatg 1740 accttgattg atttattcag atcttagctg agtttagcta actttatcac acacaccttt 1800 ggcagctgac tacattgttc tcatttagtt ttccagttat cttgtgaatt gtgatataca 1860 aagtctctgg gtgcatatgt gcaaggccca agacagtcca gggttttctc attaccagac 1920 attatttgga gtgtgacatt cagaagtcat cctctataga tttagatcaa gacagctaag 1980 aatcaaaata gatgaaaaac aacttgaata agcaagatat ttttttcatc tcagtgtgat 2040 tttgatttca tttagaagcc ctgggatgcg ctgaaaatta taaactctac atatatcttt 2100 gaataacata ttattttaca gttgtcaata aatactctga ataatg 2146 <210> 2 <211> 2339 <212> DNA <213> Anas platyrhynchos <400> 2 gtactggaat acctagggca agcctgtgta ctgaccaaat cttgttttgg cattatagaa 60 agtaagaggt gggctgagaa aaatctgttt ccttgtatag ggtgagatcc taactgatat 120 atttagagtg gctttgggac tatggaaact atagcactcc tggaaatttt gggactcctc 180 aaaactttgc taacacacat ggaaaacaga gatgattagt gacagccacc atggcttcac 240 taaaggcaaa ttgtgcctga caaattgttc accctctgtg atggaattac agcagaaggg 300 aagagcaact agcatcacct acctggactt gtgaaaagca tttggcactg tcctgtctaa 360 aatggagaga catggatttg atgtatgaac cactcagtag gtaaggaatt gtttgaatgg 420 ttgcattcaa agagttgcag tcaatggatc gatgtccagg tggagaccag tgatgagtgg 480 tgtccctaag ggggccgtac tgggaccggt actatttaac atctttttcg gcaatatggt 540 ctatattggg gaccacctga actggggtaa tcccaaatgt gactgaaggc tggatgacaa 600 gtggatagag agcagccctg cagagaagga ctttgggatg ttggttaatg aaaaactgaa 660 tatgagccag caatgtgcat ttgcagcaca gaaagccagc cttatcctgg gctgtatcaa 720 aagaagcatg gccagcaggt caagggaggt gattctgctc ctctactctg ttcctgtgag 780 accccacctg gagtgctaca ttcaactctg aagtccccca gcacaagaag aacatggaag 840 tgtgagaatg agtacagaga agggcgatga ggatgctcag agggctggag cacctctcct 900 atgaagacag gctgagggag ttggggttgt tcatcctgga gaagagaagg ctcctgggag 960 gctttatggt ggtcttctaa taacaaaagt aggcctacag gaaagctggg gagggactct 1020 ttgtcttctt ctttactatg agagtgaggc attggtgcag gttgcccaca gaatctgtgt 1080 tgccccatcc ctggaggtgc tcaaggccag gctggatggg gctttgggca gcccagtgtg 1140 gtgggaagtg tccctgctca tagaagaggg gttggaatga ggtgattttt aaggtctctt 1200 ctaacccaag ccattctgtg attttgattc agtgactgac cttgaaatgg ccaagacttc 1260 acacaggaaa gaacataaag ataacaacat tcttgctgcc agctattagt aggcatttat 1320 tgatttgttt aagtgcaggg ctttgatgat gtgttcatgt ccaggaccag ggcagtatga 1380 cataggacgc tgccagttct aatgctcata tcagctccgg tcagatctgg ggcagcgtgg 1440 agccagccag atgaaggtac tgagagctca aggctttaca ggaaagtctc tttatctggg 1500 tatctaaagt gctacttgaa tttctagctt taaaggcacc agttggcttt aaccccagtc 1560 cctacttttt ctctaagatt tttttttttt cttggaaact ggaatgagta aatttttgcc 1620 ttgctaaagt taaagagaga aggaatttat gaaaattcca ttttgtgtta aataacacag 1680 attttttttt tttcctgagt gttacatttt tctcctcctc tgtccttctt tttgtccctg 1740 atactagaga gtttcccctc actaactagt aggcagcccc tgcaacagca gactatcact 1800 gaggtcctga aaaagaaggt gcaatgcatt gcagttgctt ttcttctaaa agagatgaag 1860 gatggaaact gaaaatcagt tttgtgtcat atattgcttt tctctaccac agcccacagt 1920 gggactttcc tgcttttttg ctttttattg agttagaaaa attgtccttg ttcaggactg 1980 gcagttcagc aataacttcc acttccagtc catgggatgt gaattgggcc tgcccatggg 2040 gtgtctgtcc ctaagctctg ctctctcaca cctgcagctg tggccccaga atcacagcat 2100 cacagcatgg ttgaggttgg caggaaccta tggagctcat ctgatccacc ccctgctaca 2160 gcagggccat ccagagcagg ttgcccagga ccatgtcctg gtggcttttg aagatctcca 2220 aggatacctt acaacctgac tggtcaacct gtttgtcttt tatcctatat gaaaaaaagc 2280 ctacaagcct ggcttcacca cctttcccta acttgtgcct ctttctgttt cacgctcag 2339 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 gcagaataca ggatgcccag c 21 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 gctatagttt ccatagtccc aaagc 25 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 ctcagctgct gtcccttcc 19 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 cctctccttt acctgcaaag c 21 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 caccaccttt ccctaacttg tgc 23 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 ttgaagccac agccactgac 20 <210> 9 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 gcatttggaa acattcggtc cc 22 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 gcaaatcaag ccacagtgca gc 22 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 gctcttcatg ctgctgatgg 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 ggcaggtgac gatgaggatg 20

Claims (9)

A polynucleotide consisting of 8 or more consecutive nucleotides or a complementary polynucleotide thereof comprising a single nucleotide polymorphism (SNP) located at the 726th base in the nucleotide sequence of a Dopachrome tautomerase (DCT) gene consisting of the nucleotide sequence of SEQ ID NO: 1; Or a polynucleotide consisting of 8 or more consecutive nucleotides comprising an InDel mutation located in the 82nd to 85th bases in intron 4 of the DCT (Dopachrome tautomerase) gene consisting of the nucleotide sequence of SEQ ID NO: 2 or a complementary poly A gene marker for discriminating the color of ducks, including nucleotides. A gene marker composition for discriminating the color of ducks from ducks, which comprises the agent according to claim 1 capable of detecting or amplifying a genetic marker for discriminating the color of duck. A primer set for discriminating the color of a duck for amplifying a genetic marker for discriminating the color of the duck according to claim 1. 4. The primer set for discriminating the color of ducks according to claim 3, wherein the primer set is an oligonucleotide set consisting of the nucleotide sequences of SEQ ID NOS: 3 and 4. A primer set of claim 3 or 4; And a reagent for carrying out an amplification reaction. [Claim 6] The kit for discriminating ducks from poultry according to claim 5, wherein the reagent for carrying out the amplification reaction comprises DNA polymerase, dNTPs and a buffer. Separating the genomic DNA from the duck sample;
Amplifying the target sequence by performing amplification reaction using the separated genomic DNA as a template and using the primer set of the third or fourth aspect; And
A single nucleotide polymorphism (SNP) located at the 726th base in the base sequence of the DCT (Dopachrome tautomerase) gene comprising the nucleotide sequence of SEQ ID NO: 1 in the above amplification product; Or confirming the genotype of the InDel mutation site located in the 82nd to 85th bases in the intron 4 of the DCT (Dopachrome tautomerase) gene consisting of the nucleotide sequence of SEQ ID NO: 2 Way. The method according to claim 7, wherein the step of identifying the genotypes comprises the steps of: sequencing of genes, hybridization analysis using an allele-specific microarray, allele-specific PCR, capillary electrophoresis, single-strand conformation wherein the amplification is performed by at least one method selected from the group consisting of polymorphism, denaturation gradient gel electrophoresis (DGGE), denaturing HLPC, mass spectrometry, restriction fragment length polymorphism (RFLP) and TaqMan SNP genetyping assay How to determine the color of a duck. [Claim 7] The method according to claim 7, wherein the method comprises: homozygotic genotype in which the 726th base is T / T in the nucleotide sequence of DCT (Dopachrome tautomerase) comprising the nucleotide sequence of SEQ ID NO: 1; Or a nucleotide sequence in which the 82nd to 85th nucleotides are inserted in the intron 4 of the DCT (Dopachrome tautomerase) gene comprising the nucleotide sequence of SEQ ID NO: 2, the nucleotide sequence of SEQ ID NO: 1 A heterozygotic genotype in which the 726th base is C / T in the nucleotide sequence of the DCT (Dopachrome tautomerase) gene; Or a deletion of the 82nd to 85th bases in the intron 4 of the DCT (Dopachrome tautomerase) gene consisting of the nucleotide sequence of SEQ ID NO: 2. / RTI &gt;

Images