CN113373142B - Molecular marker-assisted selection method for pig backfat thickness and application thereof - Google Patents
Molecular marker-assisted selection method for pig backfat thickness and application thereof Download PDFInfo
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Abstract
The invention discloses a molecular marker-assisted selection method of pig backfat thickness and application thereof. The SNP marker related to the pig backfat thickness is positioned at 15,352,042 deoxynucleotides of chromosome No.2 of a reference sequence of version 10.2 of the international pig genome, the base type is C or T, the genotype at the position is an individual with CC, and the backfat thickness is obviously lower than that of individuals with TC and TT genotypes. The molecular marker-assisted selection method for the pig backfat thickness, provided by the invention, is simple to operate and high in accuracy, and can provide scientific basis for molecular marker-assisted selection of pig production traits.
Description
Technical Field
The invention belongs to the technical field of pig breeding, and particularly relates to a molecular marker-assisted selection method for pig backfat thickness and application thereof.
Background
The growth performance of the pigs is determined by indexes such as lean meat percentage, fat content, pork quality, feed utilization rate and the like, and the characters are commonly used for genetic improvement evaluation of the pigs. The backfat thickness is an important index for measuring the growth performance of the pigs, and is defined as the backfat thickness at the 3 rd to 4 th ribs of the pigs, which can not only influence the growth traits related to the lean meat percentage, but also be related to the reproductive traits such as litter size and the like. In the pig breeding work, the backfat thickness is an important reference index for measuring the quality of pig varieties and can be used for breeding excellent varieties.
The genetic factors are a main reason for influencing the thickness of the back fat of the pig, but Quantitative Trait Loci (QTL) related to the thickness of the back fat of the pig are more in number, more densely distributed and distributed on each chromosome, so that the functional genes really influencing the thickness of the back fat are difficult to identify through the QTL map. And SNP (single nucleotide polymorphism) as a third-generation molecular marker is easy to genotype, numerous in quantity, wide in distribution, strong in representativeness, good in genetic stability, high in detection flux, convenient for automatic analysis and the like. Therefore, SNP is widely applied to QTL positioning, livestock genetic map construction, marker-assisted selection, functional genome analysis and the like, and molecular marker-assisted breeding is greatly promoted.
Therefore, the improvement of the genetic rule of the backfat thickness has important significance for improving the meat quality traits of the pigs and improving the production efficiency, and the discovery of SNP (single nucleotide polymorphism) markers related to the backfat thickness of the pigs for molecular marker-assisted selection is urgently needed.
Disclosure of Invention
In order to overcome the problems, the inventor conducts intensive research to detect and obtain an SNP marker which is obviously related to the pig backfat thickness, is positioned on the sense strand of the chromosome 2 of a pig genome and has the base type of C or T, and is specifically positioned at the 15,352,042 deoxynucleotides of the chromosome 2 of the reference sequence version 10.2 of the international pig genome, the genotype of the individual at the position is CC, and the backfat thickness of the individual is obviously lower than that of the individuals with TC and TT genotypes. The method can assist in early selecting the individual pigs with low backfat thickness by selecting the pigs with the CC genotypes at the sites, shortens the breeding period, and improves the economic benefit and the social value of the pig breeding industry, thereby completing the invention.
Specifically, the present invention aims to provide the following:
the invention provides an SNP marker related to pig backfat thickness, wherein the SNP marker is positioned on a No.2 chromosome sense strand of a pig genome, and the base type is C or T.
Wherein the SNP marker is located at 15.35Mb of chromosome 2 of a pig genome and is located in a QTL region related to the traits of average backfat thickness, percentage of lean meat, daily feed intake, heart weight, tenth rib backfat meat and shoulder subcutaneous fat thickness.
Wherein the SNP marker related to the pig backfat thickness is located at 15,352,042 deoxynucleotides of the chromosome 2 of the reference sequence version 10.2 of the international pig genome.
Wherein, the CC genotype individual marked by the SNP at 15,352,042 deoxynucleotides of the chromosome 2 of the reference sequence of the international pig genome version 10.2 has the backfat thickness which is obviously lower than that of the TC and TT genotype individual.
The invention also provides a primer pair for detecting the SNP marker, wherein the primer pair comprises nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO. 2.
The invention also provides a kit for detecting the SNP marker, wherein the kit comprises a primer pair P1 and a primer pair P2, and the nucleotide sequence of the P1 comprises a nucleotide sequence shown in SEQ ID NO. 1;
the nucleotide sequence of the P2 comprises a nucleotide sequence shown in SEQ ID NO. 2.
Wherein, the kit also comprises a PCR amplification system, preferably comprises PCR buffer solution, dNTP and DNA polymerase.
The invention also provides a molecular marker-assisted selection method for the backfat thickness of pigs, wherein the method comprises the steps of determining the SNP marker of the 15,352,042 deoxynucleotides of the chromosome 2 of the international pig genome version 10.2 of the pigs in a core group of the pigs, and selecting according to the marker.
Wherein, the step of determining the SNP marker of the boar in the core group of the boar at 15,352,042 deoxynucleotides of the chromosome 2 of the international boar genome version 10.2 reference sequence comprises the substeps of amplifying and sequencing the genomic DNA of the boar.
The invention has the advantages that:
(1) The SNP marker related to the back fat thickness of the pig can be used for carrying out early selection on the back fat thickness, so that the breeding cost is reduced, the breeding period is shortened, and the breeding process of the pig is promoted;
(2) The SNP marker related to the pig backfat thickness can be used for screening a pig strain with lower backfat thickness by directly identifying the marker, so that the economic benefit and the social value of breeding enterprises and the whole live pig breeding industry are improved;
(3) The molecular marker-assisted selection method for the pig backfat thickness, provided by the invention, is simple to operate and high in accuracy, and can provide scientific basis for molecular marker-assisted selection of pig production traits.
Drawings
FIG. 1 is a box plot showing the genotype effect in example 1 of the present invention.
Detailed Description
The present invention is described in further detail below by way of preferred embodiments and examples. The features and advantages of the present invention will become more apparent from the description.
The word "exemplary" is used exclusively herein to mean "serving as an example, embodiment, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.
Whole genome association analysis is a major means of identifying genetic links between phenotype and genotype and can be used to discover SNPs that affect backfat thickness.
In one aspect, the invention provides an SNP marker related to pig backfat thickness, wherein the SNP marker is located on the sense strand of chromosome 2 of a pig genome, and the base type of the SNP marker is C or T.
The nucleotide sequence of the sense strand of the chromosome 2 of the pig genome refers to the reference sequence of the international version 10.2 (Sstrofa 10.2) of the pig genome, the SNP marker is positioned at 15.35Mb of the chromosome 2, and the SNP marker is in a QTL region related to properties such as average backfat thickness, lean meat percentage, daily feed intake, heart weight, tenth rib backfat and shoulder subcutaneous fat thickness compared with a QTL database.
Preferably, the SNP marker related to the pig backfat thickness is specifically positioned at 15,352,042 deoxynucleotides of the chromosome 2 of the international pig genome version 10.2 reference sequence.
Wherein the SNP marker is WU _10.2_2_15352042.
According to a preferred embodiment of the present invention, the SNP-labeled CC genotype individual located on chromosome 15,352,042 of reference sequence No.2 of International porcine genome version 10.2 has significantly lower backfat thickness than TC and TT genotype individuals.
In the present invention, the SNP markers correspond to three genotypes: CC. TC and TT, CC being a homozygote of base C, TT being a homozygote of base T, and TC being a heterozygote.
The research of the inventor finds that the backfat thickness of a CC genotype individual of the SNP marker of the pig is obviously lower than that of TC genotype and TT genotype individuals, so that the meat quality character of the pig can be genetically evaluated according to the genotype of the SNP site. Therefore, the inventor determines that the SNP marker located at 15,352,042 deoxynucleotides of chromosome No.2 of reference sequence of International pig genome version 10.2 is closely related to the pig backfat thickness property, can be effectively used for molecular marker-assisted breeding of pigs, and has the advantages of early screening, time saving, low cost and high accuracy.
Wherein, genotyping the SNP locus can eliminate the mistaken elutriation and the mistaken selection of excellent genes of pigs caused by factors such as feeding environment, feed, diseases and the like in phenotype selection to a certain extent, and enhance the accuracy of target character selection.
Furthermore, the invention preferably adopts a high-density SNP chip technology for genotyping, and compared with the traditional genotyping method (such as PCR, RFLP and the like), the method can be used for genotyping a large amount of SNP in a short time period, has high efficiency and can greatly reduce the cost.
Preferably, the invention employs the Neogen _ POR80K chip core genotyping of the Neogen corporation.
In still another aspect of the present invention, there is provided a primer pair for detecting the above SNP marker, which comprises the nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO. 2.
Preferably, the primer pair is P1 and P2, and the nucleotide sequences of the primer pair are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2.
In the invention, the primer is adopted to carry out PCR amplification on the nucleotide fragment containing the SNP marker related to the pig backfat thickness in the pig individual to be detected, so that the detection of the SNP locus is realized through sequencing, the genotype of the SNP locus is determined, and the pig backfat thickness of the pig individual to be detected is further determined.
In still another aspect of the present invention, there is provided a kit for detecting the SNP marker described above, which comprises the primer pair for detecting the SNP marker described above.
Preferably, the kit further comprises a PCR amplification system, preferably comprising PCR buffer, dntps and DNA polymerase.
In another aspect of the invention, the invention provides an application of the SNP marker, the primer pair or the kit in research, detection, identification, regulation, reduction of pig backfat thickness or pig molecular marker assisted breeding.
In another aspect of the present invention, there is provided a method for obtaining the SNP marker, including the steps of:
step 1, selecting a pig group, and carrying out backfat thickness measurement.
In the present invention, preferably, the live Backfat Thickness (BFT) is measured when the weight of the pig is in the range of 85-105 kg, and the Backfat Thickness between the 3 rd to 4 th intercostals of the pig in the population is measured by B-ultrasonic scanning, and is measured in millimeters. Then, the acquired data is subjected to phenotype data correction by adopting a genetic evaluation trait determination rule of a Hebei province local standard (DB 13/T2065-2014) file "technical specification for measuring production performance in a pig farm", and the in-vivo backfat thickness of 100kg of the pig is calculated according to the following correction formula:
corrected backfat thickness = measured backfat thickness × CF;
CF = a ÷ { a + [ B × (measured body weight-100) ] }.
Wherein, A and B are correction coefficients of backfat thicknesses of different pig species, and the unit of actually measured weight is kg.
And 2, extracting the genome DNA of the pig individual, and carrying out genotyping.
In the present invention, the genomic DNA of each individual in the pig population is extracted by a method or a kit commonly used in the prior art, and preferably, the extraction of the genomic DNA is performed by collecting the pig ear tissue.
After extracting the genomic DNA, preferably adopting a spectrophotometer and electrophoresis to carry out concentration determination and quality detection on the extracted porcine genomic DNA, wherein the ratio of A260/A280 of the extracted DNA is 1.8-2.0, and the ratio of A260/A230 is 1.7-1.9, and the purity is judged to be qualified; the concentration is judged to be qualified when the concentration is higher than 300 ng/. Mu.L.
Preferably, the DNA that is qualified for detection is genotyped using a high density SNP chip, such as the Neogen _ POR80K chip from New York corporation, preferably using the typing software GenCall version 7.0.0.
And 3, performing quality control on the backfat thickness data and the genotype typing data.
In the present invention, the quality control of the backfat thickness data is: individuals with a phenotype value deletion are eliminated, and individuals with a phenotype value greater than 3 times the standard deviation are eliminated.
The quality control of the genotyping data was: eliminating SNP sites with genotype detection rate less than 95%; eliminating individuals with a detection rate of less than 95 percent; (ii) individuals with clearance Minimum Allele Frequency (MAF) of less than 1%; clearing SNP sites with a Kazakh-Weinberg equilibrium (HWE) chi-square test P value less than 1.0E-4; eliminating SNP sites on sex chromosomes.
And 4, carrying out correlation analysis on the backfat thickness whole genome.
According to a preferred embodiment of the present invention, a whole genome association analysis of all typing SNP sites and corrected backfat thickness is preferably performed using the R language package GAPIT Version 3.
The statistical Model of the software package is a compressed Mixed linear Model, the design purpose of GAPIT is to accurately execute GWAS and genome prediction on a large data set, the Mixed Linear Model (MLM) comprises fixed and random effects, the Model takes the population structure as the fixed effect, and simultaneously brings individuals into the random effect to construct an individual genetic relationship matrix, and the statistical analysis Model is as follows:
Y＝Xβ+Zu+e
wherein Y is the value of the observed phenotype; β is an unknown value containing a fixed effect; u is an unknown value of random additive genetic effect from multiple background QTLs of an individual or line; x and Z are known design matrices; e is the residual vector that is not observed.
Through the correlation analysis, the SNP loci significantly related to the pig backfat thickness can be obtained, and the correlation results of different genotypes in the obtained SNP loci and the corrected backfat thickness also need to be further compared and analyzed.
In a further preferred embodiment, the genotype data of the obtained SNP sites and the corrected backfat thickness data are tested for differential significance using the Kruskal-Wallis method using Rstudio software to obtain a genotype type significantly correlated to the corrected backfat thickness of the pig.
In still another aspect of the present invention, there is provided a method for detecting the thickness of the back fat of a pig, which comprises the step of detecting the genotype of the SNP marker at chromosome 15,352,042 of the reference sequence No.2 of the international pig genome version 10.2 of the pig to be detected.
Preferably, the method comprises the steps of:
and step I, extracting the genome DNA of the pig to be detected.
Wherein, the DNA of the pig to be detected is extracted by adopting a method or a kit commonly used in the prior art, and the pig ear tissue is preferably collected.
And step II, performing PCR amplification by using the genome DNA as a template.
Wherein, the primer pair for detecting the SNP marker or the kit containing the primer pair is used for PCR amplification, and the obtained amplification product contains the SNP marker positioned at the 15,352,042 deoxynucleotide of the chromosome 2 of the reference sequence No. 10.2 version of the international swine genome.
And step III, determining the genotype of the SNP marker of the pig to be detected.
The detection method of the SNP marker is not particularly limited, and the direct sequencing method, the single-strand conformation polymorphism polymerase chain reaction (PCR-SSCP), the restriction fragment length polymorphism polymerase chain reaction (PCR-RFLP), the time-of-flight mass spectrometry and other technologies commonly used in the prior art can be adopted. The sequencing is a detection technology with high accuracy, strong flexibility, large flux and short detection period, and can directly detect the genotype of the SNP locus. Therefore, in the present invention, it is preferable to perform the detection by a direct sequencing method, i.e., directly sequence the PCR amplification product.
The sequencing method is not particularly limited, and only the sequence from which the PCR amplification product can be obtained, that is, the nucleotide sequence of the fragment in which the SNP marker is present. For example, HISEQ2000, SOLID, 454, single-molecule sequencing and the like can be adopted, so that sequencing results can be obtained quickly, efficiently and accurately in high throughput.
Based on the sequencing result, the genotype of the SNP marker of the pig to be detected can be effectively determined.
And IV, determining the backfat thickness of the pig to be detected according to the genotype.
Wherein, if the genotype of the SNP marker at the 15,352 and 042 deoxynucleotide of the chromosome 2 of the reference sequence of the 10.2 version of the international pig genome is CC, the pig has lower backfat thickness; if the genotype of the SNP marker at 15,352,042 deoxynucleotides of the chromosome 2 of the reference sequence of the international pig genome version 10.2 is TC or TT, the pig to be detected has higher backfat thickness.
The detection method provided by the invention can be used for rapidly, efficiently and accurately detecting the back fat thickness of the pig, and further can be effectively used for molecular marker-assisted breeding of the pig, so that the excellent variety of the pig can be bred in a short time, at low cost and with high accuracy at an early stage.
In still another aspect of the present invention, there is provided a molecular marker assisted selection method for pig backfat thickness, which comprises the steps of determining the SNP markers of pigs in the core group of pigs at 15,352,042 deoxynucleotides of chromosome 2 of reference sequence No. 10.2 of international pig genome, and making selections according to the markers.
Specifically, a primer pair capable of detecting the SNP marker related to the pig backfat thickness or a kit comprising the primer pair and the like are adopted to amplify and sequence the DNA of a boar genome so as to determine the genotype of the SNP marker, and then individuals with the genotype of the SNP marker at 15,352,042 chromosome of the No.2 chromosome of the reference sequence version 10.2 of the international boar genome are selected and bred successively, the genotype of the SNP locus is eliminated, and the genotype of the SNP locus is TC or TT, so that the frequency of the homozygous genotype CC at the locus is improved generation by generation, and the backfat thickness is reduced.
Examples
The present invention is further described below by way of specific examples, which are merely exemplary and do not limit the scope of the present invention in any way.
Example 1
1. Test pig population
The experimental swine herd used in this example was 1177 pigs from a north Hebei American Hopkins pig farm, and three breeds of big white pigs, long white pigs and Duroc pigs were included, wherein 597 pigs of big white pigs, 2 pigs of boars, 595 sows, 379 pigs of long white pigs, 15 pigs of boars, 364 pigs of sows, 201 Duroc pigs of boars, 23 pigs of boars and 178 pigs.
2. Backfat thickness measurement and correction
And (3) measuring the Back Fat Thickness (BFT) of the living body when the weight of the pig individual is in the range of 85-105 kg, and measuring the back fat Thickness between the 3 rd to 4 th intercostal of the pig individual in the group by adopting B-ultrasonic scanning, wherein the unit is millimeter. Then, the acquired data are subjected to phenotype data correction by adopting a genetic evaluation character determination rule of a local standard (DB 13/T2065-2014) file 'technical specification for measuring the production performance in the pig farm', and the back fat thickness of the living body of the pig reaching 100kg of body weight is calculated according to the following correction formula:
corrected backfat thickness = measured backfat thickness × CF;
CF = a ÷ { a + [ B × (measured body weight-100) ] }.
Wherein, A and B are correction coefficients of backfat thickness of different pig breeds, and the unit of actually measured body weight is kg.
The calibration table for the back fat thickness of the boars and sows of the three pig breeds is shown in the following table 1:
TABLE 1
3. Extraction of pig genomic DNA
The method comprises the following steps:
(1) Collecting pig ear tissue, cutting into pieces, placing into a 1.5ml centrifuge tube, adding 200 μ l tissue lysate TL, and blowing with large-caliber gun head to mix well;
(2) Adding 20 μ l proteinase K (20 mg/ml), shaking gently and mixing well by turning upside down;
(3) The lysed ear tissue was placed in a water bath at 55 ℃ for 3 hours or until the tissue digestion was complete, during which gentle shaking was several times to aid lysis;
(4) The lysate is blown and beaten for 2 to 3 times by a 1ml disposable transfusion device without a needle head;
(5) Adding 200 μ l of binding solution CB and 100 μ l of isopropanol, violently reversing, shaking gently, and mixing well;
(6) Centrifuging at 13000rpm for 5 min, adding the supernatant into an adsorption column AC (placing the adsorption column into a collection tube), centrifuging at 10000rpm for 30 s, and pouring off the waste liquid in the collection tube;
(7) Adding 500 μ l of inhibitor removing solution IR, centrifuging at 12000rpm for 30 s, and discarding the waste solution;
(8) Adding 700 mul of rinsing liquid WB (adding absolute ethyl alcohol in advance), centrifuging at 12000rpm for 30 seconds, and discarding waste liquid;
(9) Adding 500 mul of rinsing liquid WB, centrifuging at 12000rpm for 30 s, and discarding the waste liquid;
(10) Putting the adsorption column AC back into an empty collection pipe, centrifuging at 13000rpm for 2 minutes, and removing the rinsing liquid as much as possible so as to prevent residual ethanol in the rinsing liquid from inhibiting downstream reaction;
(11) Taking out the adsorption column AC, placing into a clean centrifuge tube, adding 100 μ l elution buffer EB (the elution buffer is preheated in water bath at 65-70 deg.C in advance) into the middle part of the adsorption membrane, standing at room temperature for 3-5 min, and centrifuging at 12000rpm for 1 min; adding the obtained solution into a centrifugal adsorption column again, standing at room temperature for 2 minutes, and centrifuging at 12000rpm for 1 minute; the larger the elution volume is, the higher the elution efficiency is, and if the DNA concentration is required to be higher, the elution volume can be reduced properly, but the minimum volume is not less than 50 mu l, and the too small volume reduces the DNA elution efficiency and the DNA yield;
(12) After the quality and the concentration are detected by a Nanodrop-100 spectrophotometer, the concentration is uniformly diluted to 50 ng/mu L, and the DNA can be temporarily stored at 2-8 ℃ and can be placed at-20 ℃ to prepare for DNA typing if the DNA is stored for a long time.
4. Genotyping
The DNA that was qualified was used to determine the genotype of each individual using a Neogen _ POR80K chip (catalog No.: 902148) from Neuger.
5. Quality control of data
In the backfat thickness phenotype value measurement process, eliminating individuals with phenotype values missing, and eliminating individuals with the phenotype values larger than 3 times of standard deviation;
in the genotyping process, SNP loci with genotype detection rate less than 95% are eliminated; eliminating individuals with a detection rate of less than 95 percent; individuals with a minimal allele frequency of less than 1% are eliminated; eliminating SNP sites with a P value less than 1.0E-4 by Hardy-Weinberg Pinchi square test; eliminating SNP sites on sex chromosomes.
6. The distribution of the genotype of the SNP sites in the test pig population is counted, and the results are shown in Table 2.
TABLE 2
As can be seen from Table 2, the TC genotype is the dominant genotype of the experimental population.
7. Adopting an R language package GAPIT Version 3 to carry out whole genome correlation analysis on all typing SNP loci and the corrected pig backfat thickness, wherein the adopted statistical analysis model is as follows:
Y＝Xβ+Zu+e
wherein Y is the value of the observed phenotype; β is an unknown value containing a fixed effect; u is an unknown value of random additive genetic effect from multiple background QTLs of an individual or line; x and Z are known design matrices; e is the residual vector that is not observed.
Statistical results show that the locus WU _10.2 \2 \15352042is significantly related to the pig backfat thickness trait.
8. The genotype data and the phenotype data were tested for differential significance using the Kruskal-Wallis method using the Rstudio software, wherein FDR _ Adjusted _ P-values, i.e., corrected P values, of 0.1-P-values < 0.05 indicated significant differences. The correlation analysis results are shown in table 3:
TABLE 3
As can be seen from Table 3, the backfat thickness of the CC genotype pigs was significantly lower than that of the TC and TT genotype individuals.
Further, box plots of the effects of the three genotypes were drawn using the R software, and the results are shown in fig. 1.
As can be seen from FIG. 1, the corrected backfat thickness of the individual with CC genotype was 0.69mm lower than that with TC genotype and 0.86mm lower than that with TT genotype.
Taken together, individuals homozygous for the CC at the WU _10.2_2 _15352042locus have advantages in backfat thickness. The WU _10.2_2 _15352042locus can be used as a genetic marker of backfat thickness, is applied to molecular marker-assisted selection of backfat thickness traits, and improves the accuracy of backfat thickness selection of pigs.
The invention has been described in detail with reference to specific embodiments and illustrative examples, but the description is not intended to be construed in a limiting sense. Those skilled in the art will appreciate that various equivalent substitutions, modifications or improvements may be made to the embodiments and implementations of the invention without departing from the spirit and scope of the invention, and are within the scope of the invention.
SEQUENCE LISTING
<110> Shenzhen agricultural genome institute of Chinese agricultural science institute
<120> molecular marker-assisted selection method for pig backfat thickness and application thereof
<130> 2020
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 18
<212> DNA
<213> primer P1 (Artificial sequence)
<400> 1
gagcaggaaa cacttgaa 18
<210> 2
<211> 18
<212> DNA
<213> primer P2 (Artificial sequence)
<400> 2
caaacgagaa tggaggtc 18
Claims (2)
1. The application of a molecular marker in individual selection of pigs with low backfat thickness is characterized by comprising the steps of determining SNP markers of 15,352,042 deoxynucleotides of pigs in a core group of pigs, wherein the pigs are positioned on the chromosome 2 of the reference sequence No. 10.2 version of the international pig genome, and selecting according to the markers;
the back fat thickness of the CC genotype individual marked by the SNP at 15,352,042 deoxynucleotides of the chromosome 2 of the reference sequence of the international pig genome version 10.2 is obviously lower than that of TC and TT genotype individuals;
the pig is selected from white pig, changbai pig and Duroc pig.
2. The use of claim 1, wherein the step of determining the SNP marker of a swine in the core population of swine located at chromosome 15,352,042 of chromosome 2 of reference sequence version 10.2 of the international swine genome comprises the sub-steps of amplifying and sequencing the swine genomic DNA.
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