KR101686443B1

KR101686443B1 - SNP markers for discriminating Rhode Island Red breed of chicken and use thereof

Info

Publication number: KR101686443B1
Application number: KR1020150073270A
Authority: KR
Inventors: 이경태; 박미나; 허강녕
Original assignee: 대한민국
Priority date: 2015-05-26
Filing date: 2015-05-26
Publication date: 2016-12-14
Also published as: KR20160138856A

Abstract

The present invention relates to a SNP marker for judging the breed of a Rhode Island Red Chicken, and more particularly to a SNP marker for judging the breed of Rhode Island Red Chicken, a Rhode Island Red Chicken Kits, microarrays, and methods for determining the breed of Rhode Island Red Chickens. By using the SNP marker of the present invention, it is possible to quickly and accurately discriminate native chickens and to identify hybrids with native chickens.

Description

Rhode Island Red Chicken Varieties Judgmental SNP Markers and Their Uses {SNP markers for discriminating Rhode Island Red breed of chicken and use thereof}

The present invention relates to a SNP marker for judging the breed of a Rhode Island Red Chicken, and more particularly to a SNP marker for judging the breed of Rhode Island Red Chicken, a Rhode Island Red Chicken Kits, microarrays, and methods for determining the breed of Rhode Island Red Chickens.

Domestic chickens have been raised at the level of small farmers in Korea until 1960s, but most of them have disappeared after the introduction of highly improved practical systems, the scale of poultry facilities, and the full - scale industrialization. Recently, as the importance of genetic resources of domestic livestock has increased, interest in native chickens has increased in Korea. The National Livestock Research Institute of the Rural Development Administration carried out the restoration and system development project for 15 years based on native chickens collected from the whole country. Definitions of native chickens were defined at the council and public hearings in the "Research on Establishment of Domestic Chicken Breeding and Certification Standards" conducted by the National Livestock Academy of Rural Development in 2008 as follows. Domestic chickens have been cultivated in Korea since the past, and they have maintained pure lineage without incorporation of other varieties. Domestic chickens have been imported into Korea as a foreign cultivar, It is a varieties adapted to the climate and is classified into indigenous species with clear ascertainment, sub-breeding and generation-specific test records. In addition, we defined a group of native chickens that retained their genetic characteristics and maintained records and breeding characteristics maintained at least 7 generations by keeping the line at intervals of 1 generation every year. Based on the restored native chickens, the Rural Development Administration of the National Livestock Research Institute of the Rural Development Administration has developed a new breeding program for breeding, breeding, We succeeded in the industrialization by developing the 'Korean chicken'.

In order to evaluate the characteristics of molecular genetics varieties, identification and characterization methods using single nucleotide polymorphism (SNP) for each variety and microsatellite (MS) And genetic identification techniques using individual genotypes have been developed and utilized.

Prior art related to the present invention is disclosed in Korean Patent Laid-Open Publication No. 2013-0050832 entitled " Method for Analyzing Genetic Characteristics and Cultivar Identification of Domestic Chicken Poultry and Practical System (2013.05.16).

A number of patent documents are referenced and cited throughout the present invention. The disclosures of the cited patent documents are hereby incorporated by reference into the present invention in its entirety to better illustrate the state of the art to which the present invention pertains and the content of the present invention.

It is an object of the present invention to provide a Rhode Island Red Chicken breed-determining SNP marker that enables rapid and accurate breeding of Rhode Island Red chickens.

Another object of the present invention is to provide a composition for judging the breed of Rhode Island Red Chicken comprising an agent capable of detecting or amplifying the SNP marker.

It is another object of the present invention to provide a kit or a microarray for judging the breed of Rhode Island Red chicken comprising the above composition.

It is a further object of the present invention to provide a method for determining the breed of Rhode Island Red Chicken comprising the step of determining the polymorphic site of said SNP.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to an aspect of the present invention, there is provided a polynucleotide comprising: (a) a polynucleotide having a nucleotide sequence of C or T at position 202 and 5 to 401 consecutive nucleotides of SEQ ID NO: 1, A polynucleotide consisting of a base or a complementary polynucleotide thereof; (b) a polynucleotide consisting of 5 to 401 consecutive bases, wherein the 202st base is T or A in the polynucleotide shown in SEQ ID NO: 2, and the 202th base is the internal base sequence of SEQ ID NO: 2, or Complementary polynucleotides thereof; Or (c) a polynucleotide consisting of 5 to 401 consecutive bases, wherein the 202st base is A or G in the polynucleotide shown in SEQ ID NO: 3, and the 202th base is the internal base sequence of SEQ ID NO: 3 Or a complementary polynucleotide thereof, may be provided. &Lt; Desc / Clms Page number 7 >

According to another aspect of the present invention, there is provided a composition for judging the breed of Rhode Island Red Chicken comprising an agent capable of detecting or amplifying a SNP marker capable of judging the breed of chicken.

According to another aspect of the present invention, a kit for judging the breed of Rhode Island Red Chicken comprising the composition can be provided.

According to another aspect of the present invention, a microarray for judging the breed of Rhode Island Red Chicken comprising the polynucleotide may be provided.

According to another aspect of the present invention, there is provided a method for amplifying a polynucleotide comprising: amplifying at least one polynucleotide comprising the SNP marker from DNA of a sample separated from an individual; And determining the base of each polymorphic site amplified in the amplification step, a Rhode Island Red chicken breed determination method can be provided.

By utilizing the SNP marker according to an embodiment of the present invention, it is possible to quickly and accurately discriminate native chickens and to identify hybrids with native chickens.

According to one embodiment of the present invention, in the process of activating domestic native chicken market, it is possible to prevent the illegal fraud of the breeding transaction beforehand and establish the transparent distribution order.

According to one embodiment of the present invention, it is possible to develop an excellent trait-related breeding technique by analyzing the function of the genetic marker region.

According to one embodiment of the present invention, it is possible to develop a hybridization system using an excellent gene for native chicken line.

FIG. 1 shows the chromosomal location of the Rhodiola red variety-specific SNP according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail.

According to an aspect of the present invention, there is provided a polynucleotide comprising: (a) a polynucleotide having a nucleotide sequence of C or T at the 202nd nucleotide and 5 to 401 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 1, A polynucleotide consisting of a base or a complementary polynucleotide thereof; (b) a polynucleotide consisting of 5 to 401 consecutive bases, wherein the 202st base is T or A in the polynucleotide shown in SEQ ID NO: 2, and the 202th base is the internal base sequence of SEQ ID NO: 2, or Complementary polynucleotides thereof; Or (c) a polynucleotide consisting of 5 to 401 consecutive bases, wherein the 202st base is A or G in the polynucleotide shown in SEQ ID NO: 3, and the 202th base is the internal base sequence of SEQ ID NO: 3 Or a complementary polynucleotide thereof, may be provided. &Lt; Desc / Clms Page number 7 >

In the present invention, the length of the polynucleotide or its complementary polynucleotide is not limited, but may be 5 to 500 nucleotides.

In the present invention, SEQ ID NOS: 1 to 3 are located in the upstream region of the macrophage receptor with collagenous structure ( MARCO ) gene of chromosome 7. The nucleotide sequence of the MARCO gene can be obtained from a known database such as NCBI's GenBank and is represented by GeneBank Accession No. NC_006094 GPC_000000724.

The SNP markers of the present invention are the markers shown in Table 4. When the polymorphic site of the SNP marker of an individual is a base marked red, it can be judged to be Rhodiolae red.

In the present invention, the " agent " may further include a primer set capable of amplifying the gene, a reagent necessary for hybridization with the gene or the nucleic acid expression product expressed therefrom. In addition, the preparation may further comprise a buffer, a solvent, or the like serving as a medium for the reaction.

According to one embodiment of the present invention, the formulation comprises at least one primer set selected from the group consisting of primers set forth in SEQ ID NOS: 4 to 9 capable of amplifying the SNP markers, Lt; / RTI >

As used herein, the term "primer " refers to a single-stranded oligonucleotide sequence complementary to a nucleic acid strand to be copied, and may serve as a starting point for the synthesis of a primer extension product. The length and sequence of the primer should allow the synthesis of the extension product to begin. The primer preferably has a length of 15 to 30 base pairs.

The oligonucleotide used as the primer may also comprise a nucleotide analogue such as phosphorothioate, alkylphosphorothioate or peptide nucleic acid or an intercalating agent.

Primers for amplifying the DNA markers are shown in Table 1 below.

The primer set consists of one set of regular primer and inverse primer, the primer set for marker amplification of SEQ ID NO: 1 consists of the primer set of SEQ ID NOs: 4 and 5, the primer set for marker amplification of SEQ ID NO: 6, and 7, and the primer set for marker amplification of SEQ ID NO: 3 comprises the primer set of SEQ ID NOs: 8 and 9.

In the present invention, when applied to a PCR amplification process, a "kit" may optionally contain reagents necessary for PCR amplification, such as buffers, DNA polymerases (e.g., Thermus aquaticus (Taq), Thermus thermophilus , Thermisflavus, Thermococcus literalis or Pyrococcus furiosus (Pfu)), DNA polymerase joins and dNTPs. The kit may be made from a number of separate packaging or compartments containing the reagent components described above.

According to one embodiment of the present invention, the kit may be an RT-PCR kit or a DNA chip kit.

In the present invention, the RT-PCR kit may comprise a respective pair of primers specific for the marker gene, and may also contain other test tubes or other appropriate containers, reaction buffers (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.

In the present invention, the DNA chip kit is prepared by attaching nucleic acid species to a glass surface, which is generally not larger than a flat solid support plate, typically a slide for a microscope, in a gridded array. And is a tool for performing massive parallel analysis by performing hybridization reaction between the nucleic acid on the DNA chip and the complementary nucleic acid contained in the solution treated on the chip surface.

The term "microarray" in the present invention means a group of polynucleotides immobilized on a substrate at a high density, and the polynucleotide group means a microarray immobilized in a constant region. Such microarrays are well known in the art. The microarrays are described, for example, in U.S. Patent Nos. 5,445,934 and 5,744,305, the contents of which are incorporated herein by reference. Markers are as described above.

The term "substrate" refers to any substrate that has hybridization properties and to which the marker can be attached under conditions where the background level of hybridization is kept low. Typically, the substrate may be a microtiter plate, a film (e.g., nylon or nitrocellulose) or a microsphere (bead) or chip. Before application or fixation to the membrane, the nucleic acid probe may be modified to promote immobilization or improve hybridization efficiency. Such modifications may include homopolymer tailing, coupling with aliphatic groups, different reactive functional groups such as NH ₂ groups, SH groups and carboxyl groups, or coupling with biotin, haptens or proteins.

According to another aspect of the present invention, there is provided a method for amplifying DNA comprising the steps of: amplifying at least one polynucleotide comprising the SNP marker from DNA of a sample separated from an individual; And determining the base of each polymorphic site amplified in the amplification step, a Rhode Island Red chicken breed determination method can be provided.

In the present invention, the sample may be a genomic DNA of a chicken, and the genomic DNA may be obtained from various sources of a chicken, for example, from a muscle, epidermis, blood, bone, organs, Obtained from blood. In the method of the present invention, extraction of gDNA can be carried out according to conventional methods known in the art (see Rogers & Bendich (1994)).

Using the separated genomic DNA as a template, the target sequence can be amplified by performing amplification reaction using any one of the primer sets of SEQ ID NOS: 4 to 9. Methods for amplifying a target nucleic acid 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 primer set that specifically binds to a target nucleic acid using a polymerase. Such PCR methods are well known in the art, and commercially available kits may be used. The PCR can be carried out using a PCR reaction mixture containing various components known in the art necessary for the PCR reaction. The PCR reaction mixture includes a proper amount of DNA polymerase, dNTP, PCR buffer solution and water in addition to the genomic DNA extracted from ginseng to be analyzed and the primer set provided in the present invention. The PCR buffer comprises Tris -HCl (Tris-HCl), MgCl 2, KCl and the like. At this time, the MgCl ₂ concentration greatly affects the specificity and yield of the amplification, preferably 1.5-2.5 mM. Generally, if an excess of the Mg ² ⁺ increase the non-specific PCR amplification products, and reduce the yield of a PCR product if the Mg ² ⁺ insufficient. The PCR buffer solution may further contain an appropriate amount of Triton X-100 (Triton X-100).

According to one embodiment of the present invention, in the polynucleotide of SEQ ID NO: 1, when the allele of the 202nd base, which is a polymorphic site, is T; In the polynucleotide represented by SEQ ID NO: 2, when the allele of the 202 < th > base, which is a polymorphic site, is A; Or the polynucleotide of SEQ ID NO: 3, if the allele of the 202 < rd > base, which is a polymorphic site, is G, the chicken can be judged to be Rhode Island Red.

Hereinafter, the present invention will be described in more detail with reference to Examples. It should be understood, however, that these examples are for illustrative purposes only and are not to be construed as limiting the scope of the present invention.

Example 1: 5 varieties of Korean native chickens In 12 lines Securing Blood and DNA Extraction

Rhode Island Red: South Korea Rhode Island Red C, South Korea Rhode Island (South Korea) Rhode Island Red C, South Korea Rhode Island South Korea Rhode Island Red C, South Korea Rhode Island South Korea 5 female and 5 male of each strain were randomly selected from each line and 10 total of each line were selected from each line, from the red D; Cornish: Korean Brown Cornish S, Korean Black Cornish H; White Leghorn: Korean White Leghorn F, Korean White Leghorn K) Individuals were sampled and each 3 ml of blood was collected in EDTA-vacutainer. Genomic DNA was extracted from the collected blood using Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA). The concentration and purity of the extracted genomic DNA were confirmed by electrophoresis and spectrophotometer, and all genomic DNAs corresponded to Next Generation Sequencing (NGS).

Example 2: NGS Detection and mutation detection of whole nucleotide sequence by individual

A paired-end (PE) library with an average size of 300 bp was constructed according to the method provided by Illumina with high purity genomic DNA. Sequence decode of the constructed library was performed using a HiSeq 2000 instrument from Illumina (Illumina, San Diego, CA, USA). The generated nucleotide sequence (read) data was mapped to the chicken reference genome sequence Galgal4 using BWA (version 0.6.2) to form SAM files. Then, a read group was set in the SAM file mapped using PICARD (v1.90), sorted by position, duplicated read was removed, and non-paired mate read was corrected. In the following steps, GATK (v2.4-9) was used. We used Realigner TargetCreator and IndelRealigner to remove the incorrect mismatch and find the correct indel. We used BaseRecalibrator to correct sequencing context, platform, and position bias. For comparison of pooled genotyping data of a total of 120 individuals, UnifiedGenotyper was used and variant filtering was performed (MQ0 ≥ 500 && ((MQ0 / (1.0XDP)> 0.1), quality <100.0 ∥MQ <30.0 ∥ DP < 500 ∥DP> 10,000), diploid haplotype phasing was performed using Beagle (v3.3.2) with the Calling Variant Call format (VCF) file as an input file, and the non-phasing region was excluded (Table 2). Table 2 shows NGS data production for 12 species of native chickens.

Example 3: Varieties or Systematic Search for specific SNP

From the variant data provided, specific SNPs were determined on a systematic basis. The specific SNP was defined as the same genotype as the reference genotype sequence Galgal4 and the same genotype as the reference genotype sequence in other strains or cultivars. The number of individuals with heterozygous SNPs and the number of individuals with homozygous SNPs were indicated as the distance to the front and back, the read depth, the number of homozygous SNPs, and the number of individuals with heterozygous SNPs as the specific SNPs. As a result, GGA7_27994772C / T , GGA7_27995337T / A, and GGA7_28001545A / G were selected as Rhodiola redspecific SNPs (Tables 3 and 4). Table 3 below lists Rhode Island red variety-specific SNPs and genotypes. Table 4 below shows Rhodiola red varieties-specific SNP sequences (+/- 200 bp).

Example 4: Rhode Island Red Analysis of location characteristics for SNP varieties

GGA7_27994772C / T, GGA7_27995337T / A and GGA7_28001545A / G were located in the upstream region of the macrophage receptor with collagenous structure ( MARCO ) gene of chromosome 7 (FIG. 1). GGA7_27994772C / T was located at 550 bp from the exon start site of MARCO gene, 1,115 bp for GGA7_27995337T / A and 7,323 bp for GGA7_28001545A / G.

Example 5: Rhode Island Red Use of specific SNP

Because genotypes of Rhode Island red varieties-specific SNPs, GGA7_27994772C / T, GGA7_27995337T / A and GGA7_28001545A / G are found only in Rhode Island red varieties, the individuals in which these genotypes are found can be judged to be Rhodiola red varieties and hybrids, Can be used to preserve Rhode Island red cultivars.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> RURAL DEVELOPMENT ADMINISTRATION <120> SNP markers for discriminating breed of chicken and use thereof <130> NPF-27243 <160> 9 <170> Kopatentin 2.0 <210> 1 <211> 401 <212> DNA <213> Gallus gallus <400> 1 ggctgaacca gcctgaacca aacttctctt gctggtccag attcaggcat atgtcttccc 60 atctctcatg gctgtgttgt ggctgagcta ttctcaatgg ctcaggtgac acgggtgtta 120 aaatagggga acacattttg gcagttctaa tgtgccctga tccaatgacc agttctccac 180 atggctattt caggaaggtc tcctgtacac gccgctccta cattttgcca tctccaaaat 240 ccgtgtcagg gccagtttgt gttgtgtgga gggaagtgac tcacccaagc tggcaagtga 300 gagccatcag taggtaatgg gcagagccct ctgcacccac tgtgccaccg cctgctgtcc 360 cctctgaggt ttggatgagc cactgtcacc aggccctgtc c 401 <210> 2 <211> 401 <212> DNA <213> Gallus gallus <400> 2 taaaaggctt aaaccaaaaa taagcaggga ttttgaaatc cacttctgtg ttctcttttc 60 aagcactcag ctgtgacctg ggtcggcacc aagacagagg aagaagagat ctcccctatg 120 gtaacgttct gctacgtgct ccacgggaac tgggatgttt gcttcacggc aaagagctaa 180 gctaccccca tgagttaggg aggctgaacc agcctgaacc aaacttctct tgctggtcca 240 gattcaggca tatgtcttcc catctctcat ggctgtgttg tggctgagct attctcaatg 300 gctcaggtga cacgggtgtt aaaatagggg aacacatttt ggcagttcta atgtgccctg 360 atccaatgac cagttctcca catggctatt tcaggaaggt c 401 <210> 3 <211> 401 <212> DNA <213> Gallus gallus <400> 3 aaaggctggg agggatggtc cccctttagg gagagaaaaa catattagcc tgacttctgc 60 tttgaggtgc gtttaaaagt ctctgcttaa ggactatgga ttgatggaga gacctgaggt 120 gggaagaata ttaaaaaaga tgtcagcatg ccaataaact catgtgccag aaataaatgc 180 attatctatt agtccatcag ggaaagaaaa agaaaggcat tcctttatgg agagagacag 240 tccagtagct cacaccatta gggaaaaaaa aaagatcaaa gcagaaatta actaattccc 300 aagtgtcact tcaccttaga atcatagtaa catacaatca ttgaagttgg aaaaagaact 360 aagatcacct agcccgacca tccacctacc accaagatta c 401 <210> 4 <211> 20 <212> DNA <213> Gallus gallus <400> 4 gggaacacat tttggcagtt 20 <210> 5 <211> 20 <212> DNA <213> Gallus gallus <400> 5 tgacagtggc tcatccaaac 20 <210> 6 <211> 20 <212> DNA <213> Gallus gallus <400> 6 ggcaccaaga cagaggaaga 20 <210> 7 <211> 20 <212> DNA <213> Gallus gallus <400> 7 agccatgtgg agaactggtc 20 <210> 8 <211> 20 <212> DNA <213> Gallus gallus <400> 8 tcccccttta gggagagaaa 20 <210> 9 <211> 20 <212> DNA <213> Gallus gallus <400> 9 cttggtggta ggtggatggt 20

Claims

(a) a polynucleotide consisting of 5 to 401 consecutive bases, wherein the 202st base is C or T in the polynucleotide shown in SEQ ID NO: 1, and the 202th base is the internal base sequence of SEQ ID NO: 1; or Complementary polynucleotides thereof;
(b) a polynucleotide consisting of 5 to 401 consecutive bases, wherein the 202st base is T or A in the polynucleotide shown in SEQ ID NO: 2, and the 202th base is the internal base sequence of SEQ ID NO: 2, or Complementary polynucleotides thereof; And
(c) a polynucleotide consisting of 5 to 401 consecutive bases, wherein the 202nd base is A or G in the polynucleotide shown in SEQ ID NO: 3 and the 202th base is the internal base sequence of SEQ ID NO: 3; or A set of SNP markers capable of judging Rhode Island Red chicken varieties, including their complementary polynucleotides.

A composition for judging the breed of Rhode Island Red Chicken comprising an agent capable of detecting or amplifying a set of SNP markers capable of judging the breed of chicken of claim 1.

3. The method of claim 2,
The formulation
The primer set for marker amplification of SEQ ID NO: 1 comprises a primer set of SEQ ID NOs: 4 and 5;
The primer set for marker amplification of SEQ ID NO: 2 comprises a primer set of SEQ ID NOs: 6 and 7; And
A primer set for marker amplification of SEQ ID NO: 3 comprises a primer set of SEQ ID NOs: 8 and 9.

A Rhode Island Red Chicken Breeding kit comprising the composition of claim 2 or 3.

A microarray for judging the breed of Rhode Island Red Chicken comprising the polynucleotides of claim 1.

Amplifying polynucleotides comprising SNP markers of claim 1 from DNA of a sample isolated from an individual; And
And determining the base of each polymorphic site amplified in said amplification step.

The method according to claim 6,
In the polynucleotide represented by SEQ ID NO: 1, when the allele of the 202 < rd > base, which is a polymorphic site, is T;
In the polynucleotide represented by SEQ ID NO: 2, when the allele of the 202 < th > base, which is a polymorphic site, is A; And
In the polynucleotide shown in SEQ ID NO: 3, if the allele of the 202nd base, which is a polymorphism site, is G, the Rhode Island Red Chicken breed determination method judging that the chicken breed is Rhode Island Red.