KR101312480B1

KR101312480B1 - Novel snp marker for discriminating number of rib of pig and use thereof

Info

Publication number: KR101312480B1
Application number: KR1020120158428A
Authority: KR
Inventors: 조인철; 임현태; 한상현; 이준헌; 고문석; 이성수
Original assignee: 대한민국(농촌진흥청장); 충남대학교산학협력단; 경상대학교산학협력단
Priority date: 2012-12-31
Filing date: 2012-12-31
Publication date: 2013-10-01

Abstract

PURPOSE: A single nucleotide polymorphism (SNP) marker is provided to objectively evaluate the number of ribs in a pig, which is hard to distinguish with naked eyes, thereby establishing distribution of pork. CONSTITUTION: An SNP marker for determining the number of ribs in a pig contains a polynucleotide with 5-100 continuous bases including 374th base which is A or C in exon 32 of LTBP2 gene or a complementary polynucleotide thereof. A composition for determining the number of ribs in a pig contains an agent for determining or amplifying the SNP marker. A kit for determining the number of ribs in a pig contains the composition. [Reference numerals] (AA) C/C genotype; (BB) A/C genotype; (CC) A/A genotype

Description

SNP marker for determining the number of ribs in pigs and its use {Novel SNP marker for discriminating number of rib of Pig and use approximately}

The present invention relates to a SNP marker for determining the number of ribs in pigs and its use, and more particularly, the present invention includes an SNP marker capable of determining the number of ribs in pigs, and an agent capable of detecting or amplifying the SNP markers. The present invention relates to a method for determining the number of ribs of a pig, comprising determining a polymorphic region of the pork rib number determining kit or microarray and the SNP marker.

Since breeding in the eastern part of India about 9,000 years ago, pigs have been raised as the most basic animal for the worldwide consumption of protein that people need, according to their age, situation and people's preferences. In general, European and Asian varieties are derived from wild boar (Susscrofa) of each continent, and there are about 200 varieties that exist so far. According to the latest FAO (Finance and Accounts Office) report, 30% of Asian varieties are Asian. In Europe, 33% of European varieties have been reported. Differences in phenotype among these varieties are sought, size, and body shape.

Recently, in order to improve the quality of the pork meat, the pork is raised in a certain standard and is scientifically managed, and the pork meat obtained from the pork meat is branded, and various brands of pork meat are already commercially sold. However, since the branded pork and the unbranded pork are difficult to be identified by the general public, the distribution order of pork meat is misused by selling the unbranded pork as branded pork. Disruptive events are occurring frequently. Indeed, since it is very difficult to distinguish between branded pigs and unbranded pork meat at the level of experts, studies are actively being conducted to establish objective criteria to judge the quality of genuine pork meat.

As a part of this research, a method for judging pig meat of a genuine brand was developed by analyzing the gene of pork meat. Randomly amplified polymorphic DNA (RAPD), single strand conformation polymorphisms, and other DNA analysis techniques to develop and develop a method for discriminating pork meat varieties. For example, Japanese Patent Application Laid-Open No. 2004-0039059 discloses a gene testing method capable of selecting pigs having excellent traits of pigs by using specific DNA markers related to the daily gain of the pigs, the backfat thickness, Patent Publication No. 2007-0113336 discloses a DNA marker for confirming the increase of porcine myocyte count using a mutation (SNP) caused by a single base sequence difference in the 5'promoter region of Myogenin gene known to be involved in the myocyte differentiation of pigs , Patent Publication No. 2011-0011443 discloses a technique for detecting the Korean native pig-specific DNA marker for the KIT gene to discriminate an accurate variety of native Korean pig and other improved pig from the genome, and discloses a technique disclosed in Patent Publication No. 2011-0050261 Discloses a method for identifying a variety of black-bred pigs using haplotypes estimated from SNPs of the KIT gene region of black-bred pigs, Patent Publication No. 2011-0139011 discloses a method for evaluating meat quality by using a single-trait polymorphic biomarker for diagnosing fat content in pigs. Patent Document No. 2012-0046968 discloses a method for evaluating meat quality using a pig gene, Discloses a method for screening pigs having excellent color quality using a gene of a pig, and Patent Publication No. 2012-0049624 discloses a method for screening pigs having an excellent color of meat. In Patent Publication No. 2012-0052796, (SNP) site of the PPARGC1A gene involved in the characteristics of the pig, and Patent Publication No. 2012-0072871 discloses a DNA marker for confirming the increase in the meat quality of a pig, and a single base polymorphism (SNP) markers are used to identify high quality pork meat. However, a method for accurately determining the number of ribs of pigs has not been developed.

Under these backgrounds, the present inventors have made intensive studies to develop a method for judging the quality of pork based on the number of ribs of pigs. As a result, when using the SNP included in the LTBP2 gene involved in the number of ribs of pigs, By determining the genetically determined number of pork ribs, it was confirmed that the quality of the pig can be determined and completed the present invention.

One object of the present invention is to provide a SNP marker that can determine the number of ribs of pigs.

It is another object of the present invention to provide a composition for determining the number of ribs of a pig comprising an agent capable of detecting or amplifying the SNP marker.

Still another object of the present invention is to provide a kit or microarray for determining the number of ribs of pork comprising the composition.

Still another object of the present invention is to provide a method for determining the number of ribs of a pig comprising determining a polymorphic site of the SNP marker.

As one aspect for achieving the above object, the present invention provides a single nucleotide polymorphism (SNP) marker that is a single nucleotide polymorphism capable of determining the number of ribs of pigs.

The marker may preferably be all or part of a polynucleotide comprising the SNP region of the LTBP2 gene, more preferably all or part of a polynucleotide of the polynucleotide consisting of SEQ ID NO: 1, and most preferably the LTBP2 gene 374th base of the polynucleotide consisting of SEQ ID NO: 1 corresponding to the 4481th base of the SNP position of the (NPON exon 32 of the LTBP2 gene) is A or C, consisting of 5 to 100 consecutive bases containing the SNP position It may be a SNP marker capable of determining the number of ribs of a pig, including a polynucleotide or a complementary polynucleotide thereof.

The term "LTBP2 gene" of the present invention refers to a gene encoding Latent-transforming growth factor beta-binding protein 2 (LTBP2), which is a type of extracellular matrix protein, wherein the LTBP2 gene is composed of 37 exons, and It has a length of 5,463bp based on the nucleotide sequence, and has a length of 1,821aa based on the amino acid sequence. The base sequence of the LTBP2 gene can be obtained from a known database such as GenBank of NCBI. For example, GenBank Accession No. It may be a gene represented by XM_003987875.1, preferably may include a polynucleotide of SEQ ID NO: 1. The LTBP2 has a high homology with fibrillin and is known to act as a component of microfibrils or to play a role in cell adhesion.

The term "polymorphism" of the present invention refers to the case where two or more alleles exist in one locus, and among the polymorphic sites, only a single base differs according to a person. It is called single nucleotide polymorphism (SNP). Preferred polymorphic markers have two or more alleles with a frequency of occurrence of 1% or more, more preferably 5% or 10% or more in the selected population.

The term "allele" in the present invention means several types of genes that exist at the same locus of homologous chromosomes. Alleles are also used to represent polymorphisms, for example, SNPs have two kinds of bialles.

The "SEQ ID NO. 1" is a polymorphic sequence comprising a polymorphic site. A polymorphic sequence means a sequence comprising a polymorphic site comprising a SNP in a polynucleotide sequence. The polynucleotide sequence may be DNA or RNA.

In the SNP marker of the present invention, when the polymorphic region of the SNP marker is C / C genotype, it can be determined that the pig including the same has more ribs than the normal pig.

According to one embodiment of the present invention, as a result of analyzing the distribution of ribs in the carcasses of 1,041 pigs representing the shipping age of about 200 days, the pigs have a total of 14-17 ribs and 15 It was confirmed that about 57% of the rib-numbered conductors, about 16% of the ribs, about 14%, about 14% of the ribs and about 1% As a result of analyzing the relationship between the number of ribs and the length of the carcass, it can be seen that the pigs having 16 and 17 ribs occupy the majority (Fig. 1), and the result of analyzing the relationship between the length of the carcass and the weight of the carcass. It was found that the length of the carcass and the carcass weight were proportional to each other, and the pigs having the carcass length of 96 to 110 cm and the carcass weight of 74 to 86 kg occupy the majority (Table 2). On pork ribs Genes that are involved in a number of QTL results pork ribs you perform the analysis, or GWAS analysis confirmed the presence in the No. 7 chromosome (Figures 2a and 2b). In addition, through QTL mapping on the number of pork ribs, the genes involved in the number of pork ribs were identified as VRTN, VSX2, NPC2, ENSSSCG00000002359, ENSSSCG00000002362, ISCA2, SYNDIG1L, or LTBP2 (Fig. 3). The number of ribs was changed by the SNP (c.4481A> C) mutation of 32 (FIGS. 4 and 5). As a result of genotyping using a Pyro-sequencer, the SNP (c.4481A> C) of the LTBP2 gene was determined. ) The C / C genotype, the A / C genotype, and the A / A genotype were shown to have different results, respectively (FIG. 6). The SNP of the LTBP2 gene was used to determine whether the number of pork ribs can be determined. As a result, the number of ribs significantly increased as the number of C copies in the SBP of the LTBP2 gene increased, and the number of ribs per C copy increased by about 0.5 (Table 3).

Therefore, by detecting the SNP (c.4481A> C) mutation of the axon 32 of the LTBP2 gene of the present invention or using a primer capable of detecting the mutation, it is expected that the number of ribs of the pig can be accurately determined. The SNP (c.4481A> C) mutation was first identified by the inventors.

In another aspect, the present invention provides a composition for determining the number of ribs of pigs comprising an agent capable of detecting or amplifying the SNP marker.

The term "agent capable of detecting or amplifying an SNP marker" as used herein means a composition capable of determining the level of the sperm cross section of a pig by confirming the polymorphic site of the gene by amplification, Means a primer capable of specifically amplifying the polynucleotide of the SNP marker. The primers used for the SNP marker amplification can be amplified using appropriate conditions in suitable buffers (for example, four different nucleoside triphosphates and polymerase such as DNA, RNA polymerase or reverse transcriptase) and template-directed DNA Stranded oligonucleotide which can serve as a starting point of synthesis. The appropriate length of the primer may vary depending on the intended use, but is usually 15 to 30 nucleotides. Short primer molecules generally require a lower temperature to form a stable hybrid with the template. The primer sequence need not be completely complementary to the SNP marker but should be sufficiently complementary to hybridise with the SNP marker, preferably including the polynucleotide sequence of SEQ ID NOS: 2 to 4, It does not.

As used herein, the term "primer" refers to a base sequence having a short free 3 'hydroxyl group, capable of forming complementary templates and base pairs and starting for template strand copying. By a short sequence that functions as a point. The primer can initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i.e., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures. At this time, the length of PCR conditions, sense and antisense primers can be modified based on what is known in the art.

The primers of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences may also be modified using many means known in the art. Non-limiting examples of such modifications include, but are not limited to, methylation, "capping ", replacement of natural nucleotides with one or more homologues, and modifications between nucleotides, such as uncharged linkers, such as methylphosphonate, Phosphoamidates, carbamates, etc.) or charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.).

In another aspect, the present invention provides a kit for determining the number of ribs of pork comprising the composition. The kit may be an RT-PCR kit or a kit for DNA analysis (eg, DNA chip).

The kit of the present invention can determine the number of ribs in pigs by checking the SNP markers, which are markers for determining the number of ribs in pigs, or confirming the expression levels of mRNAs. As a specific example, the kit for measuring the mRNA expression level of the SNP markers for determining the rib count of pigs in the present invention may be a kit containing the necessary elements necessary to perform RT-PCR. RT-PCR kits, in addition to each primer pair specific for the gene of the SNP marker, RT-PCR kits can be used in test tubes or other suitable containers, reaction buffers (variable pH and magnesium concentrations), deoxynucleotides (dNTPs), Enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNAse inhibitors, DEPC-water, sterile water, and the like. It may also contain a primer pair specific for the gene used as a quantitative control. Also preferably, the kit of the present invention may be a kit for determining the number of pork ribs including essential elements necessary for performing a DNA chip. DNA chip kits are those in which nucleic acid species are attached in a gridded array on a generally flat solid support plate, typically a glass surface not larger than a slide for a microscope, and nucleic acids are uniformly arranged on the chip surface, Hybridization reaction occurs between the nucleic acid on the surface and the complementary nucleic acid contained in the solution treated on the surface of the chip to enable a mass parallel analysis.

In still another aspect, the present invention provides a microarray for determining the pork rib number including the polynucleotide of the SNP marker for determining the pork rib number.

The microarray may comprise DNA or RNA polynucleotides. The microarray comprises a conventional microarray except that the polynucleotide of the present invention is contained in the probe polynucleotide.

Methods for producing microarrays by immobilizing probe polynucleotides on a substrate are well known in the art. The probe polynucleotide means a polynucleotide capable of hybridizing, and means an oligonucleotide capable of binding to the complementary strand of the nucleic acid in a sequence-specific manner. The probe of the present invention is an allele-specific probe in which a polymorphic site exists in a nucleic acid fragment derived from two members of the same species and hybridizes to a DNA fragment derived from one member but does not hybridize to a fragment derived from another member . In this case, the hybridization conditions show a significant difference in the intensity of hybridization between alleles, and should be sufficiently stringent to hybridize to only one of the alleles. This can lead to good hybridization differences between different allelic forms. The probe of the present invention can be used in the method of determining the number of ribs of pigs by detecting alleles. The determination method includes detection methods based on hybridization of nucleic acids such as Southern blot and the like, and may be provided in a form that is pre-bound to the substrate of the DNA chip in a method using a DNA chip. The hybridization can usually be performed under stringent conditions, for example, a salt concentration of 1 M or less and a temperature of 25 ° C or higher. For example, conditions of 5 × SSPE (750 mM NaCl, 50 mM Na Phosphate, 5 mM EDTA, pH 7.4) and 25-30 ° C. may be suitable for allele specific probe hybridization.

The process of immobilizing the probe polynucleotides associated with the determination of the number of ribs of the pigs of the present invention on a substrate can also be easily performed using this conventional technique. In addition, hybridization of nucleic acids on a microarray and detection of hybridization results are well known in the art. The detection can be accomplished, for example, by labeling the nucleic acid sample with a labeling substance capable of generating a detectable signal comprising a fluorescent material, such as Cy3 and Cy5, and then hybridizing on the microarray and generating The hybridization result can be detected.

In another aspect, the present invention provides a method for amplifying a polynucleotide comprising: (a) amplifying a polymorphic site of the SNP marker from DNA of a sample isolated from an individual; And (b) determining the base of the amplified polymorphic site of step (a).

The term "individual" of the present invention refers to a pig which is the target to check the number of ribs, by using a sample obtained from the pig, the number of ribs of the pig can be determined by analyzing the genotype of the SNP marker. The sample may be hair, urine, blood, various body fluids, separated tissues, samples such as isolated cells or saliva, but is not particularly limited thereto.

Amplifying the SNP region of the SNP marker from the DNA of step (a) can be used by 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) (Wu and Wallace, Genomics 4, 560 (1989), Landegren et al., Science 241, 1077 (1988)), transcription amplification (Kwoh et al., Proc. Natl. Acad. Sequence amplification based on nucleic acids (NASBA) can be used as well as self-sustaining sequence replication (Guatelli et al., Proc. Natl. Acad. Sci. USA 87, 1874 (1990)).

Determining the base of the SNP site in step (b) of the method is sequence analysis, hybridization by microarray, allele specific PCR, dynamic allele-specific hybridization, DASH), PCR prolongation analysis, PCR-SSCP, PCR-RFLP analysis or TaqMan technique, SNPlex platform (Applied Biosystems), mass spectrometry (e.g. Sequenom's MassARRAY system), mini-sequencing method, Bio -Plex system (BioRad), CEQ and SNPstream system (Beckman), Molecular Inversion Probe array technology (e.g. Affymetrix GeneChip), and BeadArray Technologies (e.g. Illumina GoldenGate and Infinium assays) However, it is not particularly limited thereto. One or more alleles in the SNP marker comprising the SNP site can be identified by the above methods or other methods available to those skilled in the art to which the present invention pertains. Determining the base of such an SNP site may be preferably performed through a DNA chip.

The term "DNA chip" of the present invention means one of the DNA microarray which can identify each base of hundreds of thousands of DNA at a time.

The TaqMan method comprises the steps of: (1) designing and constructing a primer and a TaqMan probe to amplify a desired DNA fragment; (2) labeling probes of different alleles with FAM dyes and VIC dyes (Applied Biosystems); (3) performing PCR using the DNA as a template and using the primer and the probe; (4) after completion of the PCR reaction, analyzing and confirming the TaqMan assay plate with a nucleic acid analyzer; And (5) determining the genotype of the polynucleotides of step (1) from the analysis results.

The sequencing assay can use conventional methods for sequencing and can be performed using an automated genetic analyzer. In addition, allele-specific PCR refers to a PCR method of amplifying a DNA fragment in which the SNP is located with a primer set including a primer designed with the base at which the SNP site is located as the 3 'end. The principle of the above method is that, for example, when a specific base is substituted by A to G, an opposite primer capable of amplifying a primer containing the A as a 3 'terminal base and a DNA fragment of an appropriate size is designed, In the case where the base at the SNP position is A, the amplification reaction is normally performed and a band at a desired position is observed. When the base is substituted with G, the primer can be complementarily bound to the template DNA, And the amplification reaction is not performed properly due to the inability of complementary binding at the terminal. DASH can be performed by a conventional method, preferably by a method such as Prince et al.

On the other hand, in the PCR extension analysis, first, a DNA fragment containing a base in which a single nucleotide polymorphism is located is amplified with a pair of primers, and all the nucleotides added to the reaction are deactivated by dephosphorylation, and a specific extension primer, dNTP And then performing a primer extension reaction by adding a mixture, a digoxinucleotide, a reaction buffer and a DNA polymerase. At this time, the extension primer is a 3 'end of the base immediately adjacent to the 5' direction of the base where the SNP site is located, the dNTP mixture excludes a nucleic acid having the same base as the didioxynucleotide, the didioxynucleotide is a SNP It is selected from one of the base types shown. For example, if there is a substitution from A to G, when a mixture of dGTP, dCTP and TTP and ddATP is added to the reaction, the primer is extended by DNA polymerase at the base where the substitution has occurred, and after several bases A The primer extension is terminated by ddATP at the position where the base first appears. If the substitution has not occurred, the extension reaction is terminated at the position, so that it is possible to discriminate the type of the base representing the SNP by comparing the lengths of the extended primers.

In this case, as the detection method, when fluorescently labeling an extension primer or didioxynucleotide, the SNP can be detected by detecting fluorescence using a gene analyzer (for example, Model 3700, manufactured by ABI, etc.) which is used for general sequencing. In the case of using unlabeled extension primers and didioxynucleotides, the SNP may be detected by measuring molecular weight using a matrix assisted laser desorption ionization-time of flight (MALDI-TOF) technique.

Preferably, in exon 32 of the LTBP2 gene described in SEQ ID NO: 1 in the base sequence determined in step (b), when the 374th base, which is the SNP region, is C, the pig has a greater number of ribs than the normal pig. You can judge.

The SNP marker of the present invention is a specific SNP marker that determines the number of ribs of pigs, and is used as a means for objectively evaluating the number of ribs of pigs not visually distinguished, and thus can contribute to establishing a distribution order of pork meat. will be.

1 is a graph showing the relationship between the number of ribs of pigs and the length of the conductor.
Figure 2a is a graph showing the results of performing a QTL analysis on the number of ribs of the progeny using an MS marker.
Figure 2b is a graph showing the results of performing GWAS analysis on the number of ribs of the offspring using a large capacity SNP chip.
Figure 3 is a schematic diagram showing the QTL mapping results for the number of pork ribs.
Figure 4 is a schematic diagram showing the generation of the SNP of the LTBP2 gene in pig carcase samples having different ribs.
FIG. 5 is a schematic diagram showing that the SNP causes p.1494H> P mutation at the protein level by SNP (c.4481A> C) mutation of axon 32 of LTBP2 gene.
6 is a graph showing the results of detecting the SNP (c.4481A> C) mutation by performing genotyping of the LTBP2 gene using a Pyro-sequencer.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1: the number of pork ribs Conductor Length and Carcass Association analysis

Example 1-1: Distribution of Pork Ribs

The distribution of the number of ribs was analyzed for the carcases of 1,041 pigs representing the age of shipment before and after 200 days (Table 1).

Number of ribs Ribs can Conductor 14
15
16
17 115
594
318
14

As shown in Table 1, the pig has a total number of ribs 14-17, about 57% of conductors having 15 ribs, about 31% of conductors having 16 ribs, and about 14 ribs It was confirmed that the conductor had about 11% and the number of 17 ribs had about 1%.

Example 1-2: the number of pork ribs Conductor Correlation Analysis of Length

Using the pig carcasses representing the days of shipment before and after 200 days, the relationship between the number of ribs and the length of the carcass was analyzed (FIG. 1). 1 is a graph showing the relationship between the number of ribs of pigs and the length of the conductor. As shown in Figure 1, it can be seen that pigs with 16 and 17 ribs occupy a large number.

Example 1-3: Conductor Length and Carcass Association analysis

The relationship between the length of the carcass and the weight of the carcass was analyzed using the pig carcass showing the shipping age of 200 days before and after (Table 2).

Relationship between conductor length and conductor weight Conductor length (cm) frequency Conductor weight (kg) <90
91-95
96-100
101-105
106-110
111-115
116-120
120-125 12
90
263
288
198
64
13
2 55.33 ± 11.14
67.21 ± 11.58
74.71 ± 8.67
80.75 ± 9.64
86.19 ± 10.21
95.55 ± 10.52
95.62 ± 9.34
114.0 ± 8.49

As shown in Table 2, it was confirmed that the conductor length and the carcass weight were in a proportional relationship, and the pigs having the carcass length of 96 to 110 cm and the carcass weight of 74 to 86 kg were mostly occupied.

Example 2: number of pork ribs QTL Analysis

QTL or GWAS analysis was performed on the number of ribs of pigs using MS (microsatellite) markers or high-capacity SNP chips (illumina, USA) for the offspring obtained by crossing Jeju native black pigs and Landrace pigs (FIGS. 2A and FIG. 2). 2b). Figure 2a is a graph showing the results of performing a QTL analysis of the number of ribs of the offspring using the MS marker, Figure 2b is a graph showing the result of performing a GWAS analysis of the number of ribs of the offspring using a large capacity SNP chip. . As shown in Figures 2a and 2b, it was confirmed that the gene involved in the number of ribs of pigs is present on chromosome 7.

On the other hand, through the QTL mapping for the number of pork ribs, the rib number regulatory locus was compressed in the gene region from VRTN to LTBP2 gene (Fig. 3). Figure 3 is a schematic diagram showing the QTL mapping results for the number of pork ribs. As shown in Figure 3, the gene involved in the number of pork ribs was found to be VRTN, VSX2, NPC2, ENSSSCG00000002359, ENSSSCG00000002362, ISCA2, SYNDIG1L or LTBP2.

In addition, the number of ribs was changed by the SNP of the LTBP2 gene in the gene (Figs. 4 and 5). Figure 4 is a schematic diagram showing the generation of SNP of the LTBP2 gene in pig carcass samples having different rib number, Figure 5 is a protein level by the SNP (c.4481A> C) mutation of the axon 32 of the LTBP2 gene Is a schematic showing that the p.1494H> P mutation occurs. As shown in Figures 4 and 5, it can be seen that the number of ribs in pigs is changed by SNP (c.4481A> C) mutation of the axon 32 of the LTBP2 gene.

Example 3: Pyro - sequencer Through analysis LTBP2 Genotyping of Genes

Genotyping with a pyro-sequencer was performed to prepare primers capable of detecting the SNP (c.4481A> C) mutation as follows:

pLTe32F: 5'-GGAAACTGAGGCAAGGAGAA-3 '(SEQ ID NO: 2)

B-pLTe32R: 5'-TGTCCTCACCCTCACACTTG-3 '(SEQ ID NO: 3)

s-pLTe32: 5'-CGAGTGTGTGATGTTCGGGC-3 '(SEQ ID NO: 4)

Among the primers, B-pLTe32R was prepared in the form of binding of biotin to its 5'-end.

PCR was performed using the primers and genomic DNA obtained from pigs as a template to obtain mutated gene samples (PCR amplification conditions: 96 ° C. → 30 s, 60 ° C. → 30 s, 72 ° C. → 30 s 30 cycles). The obtained mutated gene samples were subjected to genotyping using a Pyro-sequencer (FIG. 6). 6 is a graph showing the results of detecting the SNP (c.4481A> C) mutation by performing genotyping of the LTBP2 gene using a Pyro-sequencer. As shown in Figure 6, the SNP (c.4481A> C) mutations of the LTBP2 gene was confirmed that the C / C genotype, A / C genotype and A / A genotype each shows different results.

From the above results, it was found that genotyping using Pyro-sequencer using the primers can easily detect the SNP of the LTBP2 gene.

Example 4: LTBP2 Gene Association between SNP Genotype and Pork Rib Count

Genotyping was carried out using the primers on 1,066 second-generation offspring obtained by crossing Jeju native black pigs and Landrace pigs, and the results of the carcass weight, back fat thickness, fillet area, number of ribs and body length By comparison, it was confirmed whether the primers could be used for the determination of the number of pork ribs (Table 3).

Analysis of association between SNP (c.4481A> C) mutations in the LTBP2 gene and the number of pork ribs LTBP2 genotype Distribution Conductor weight (kg) Backfill thickness
(mm) Sirloin cross section
(Cm / cm 2) Number of Ribs (Number) Length (cm) A / A
A / C
C / C 260
540
266 77.2 ± 12.1
80.1 ± 13.1
80.2 ± 11.0 22.3 ± 7.2
23.1 ± 6.8
23.4 ± 6.5 21.2 ± 3.8
21.5 ± 4.4
21.3 ± 3.8 14.7 ± 0.5
15.2 ± 0.5
15.8 ± 0.6 101.4 ± 5.6
103.0 ± 6.0
103.5 ± 5.3

As shown in Table 3, it was confirmed that the number of ribs per C copy was increased by about 0.5 depending on the LTBP2 genotype without affecting the backfat thickness and the fillet cross-sectional area of the pig carcass.

Attach an electronic file to a sequence list

Claims

In exon 32 of the LTBP2 gene represented by SEQ ID NO: 1, the 374th base is A or C and comprises a polynucleotide consisting of 5 to 100 consecutive bases comprising the 374th base or a complementary polynucleotide thereof Single nucleotide polymorphism (SNP) marker for determining the number of ribs in pigs.

A composition for determining the number of ribs in pigs, comprising an agent capable of detecting or amplifying the SNP marker for determining the number of ribs in pigs of claim 1.

3. The method of claim 2,
Wherein the agent is a primer represented by a polynucleotide selected from the group consisting of SEQ ID NOS: 2-4.

Kit for determining the number of ribs of pigs comprising the composition of claim 2 or 3.

5. The method of claim 4,
The kit is a kit for determining the number of ribs of pork that is RT-PCR kit or DAN chip kit.

Microarray for determining the number of pork ribs comprising the polynucleotide of claim 1.

(a) amplifying the polymorphic site of the SNP marker of claim 1 from DNA of a sample isolated from the individual; And
(b) determining the base of the amplified polymorphic site of step (a), the method for determining the number of pork ribs.

The method of claim 7, wherein
Wherein, in the polynucleotide of SEQ ID NO: 1, if the allele of the 374th base, which is the SNP position, is C / C, the number of ribs of the pig is determined to be higher than that of the normal pig.