CN116083598A - SNP molecular marker related to intramuscular fat traits of pigs and application thereof - Google Patents
SNP molecular marker related to intramuscular fat traits of pigs and application thereof Download PDFInfo
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Abstract
The invention discloses a SNP molecular marker related to pig intramuscular fat characters and application thereof, wherein the SNP molecular marker is a nucleotide corresponding to 373bp of a DNA molecule with a sequence shown as SEQ ID NO.1 in a fourth exon of a pig AOPEP gene, and is a G/A polymorphism. Experiments prove that the SNP molecular marker provided by the invention is obviously related to the intramuscular fat trait of pigs, which proves that the molecular marker and the detection substance thereof can identify or detect the intramuscular fat trait of pigs, and have important significance for screening or predicting the pig varieties with excellent intramuscular fat traits.
Description
Technical Field
The invention belongs to the technical field of molecular markers, and particularly relates to a SNP molecular marker related to intramuscular fat traits of pigs in a fourth exon of an AOPEP gene of the pigs and application thereof.
Background
Intramuscular fat content is an important assessment index of pork quality, and the intramuscular fat content directly influences the taste and flavor of the pork. Intramuscular fat traits belong to moderate genetic traits, with genetic transmission between 0.2 and 0.4, but since the in vivo measurement of meat quality traits is difficult, improvement by conventional breeding is difficult. Therefore, the molecular markers affecting the meat quality traits are sought, and the genetic improvement of the intramuscular fat traits of pigs can be accelerated by a method combining conventional breeding with marker-assisted selection (MAS).
MAS utilizes molecular markers associated with specific traits as auxiliary means for selective breeding, has the advantages of rapidness, accuracy, no environmental influence and the like, can reduce the workload of field measurement personnel, and can shorten the breeding time limit. Single Nucleotide Polymorphisms (SNPs) refer to genetic polymorphisms caused by mutation of a single nucleotide in a genomic DNA sequence, which are widely present in the genome and often used in breeding practice by the method of MAS, have a key role in genetic improvement of important traits.
The AOPEP gene is a member of the family of encoding M1 amino zinc oxide peptidases. The encoded protein is a zinc-dependent metallopeptidase, is involved in peptide hormone metabolism, protein metabolism and other related pathways, and can play a role in the generation of angiotensin IV and dystonia.
Disclosure of Invention
In view of the above, the invention provides a SNP molecular marker which is positioned in a fourth exon of a pig AOPEP gene and is related to the intramuscular fat trait of pigs, and the molecular marker can provide a reference basis for the auxiliary selection of pork quality trait markers.
The technical scheme of the invention is as follows:
a SNP molecular marker related to the intramuscular fat trait of pigs is a nucleotide corresponding to 373bp of a DNA molecule with a sequence shown as SEQ ID NO.1 in a fourth exon of an AOPEP gene of pigs, and is a G > A polymorphism.
The invention also provides a substance for detecting the SNP molecular marker.
The substances for detecting SNP molecular markers comprise primers shown in SEQ ID NO.2 and SEQ ID NO. 3.
The substance can be a kit containing the primer, and the kit can also comprise conventional reagents for PCR amplification and conventional reagents for sequencing.
The invention also provides application of the SNP molecular marker or a substance for detecting the SNP molecular marker, which is A1), A2) or A3) as follows:
a1 Identification or assisted identification of the intramuscular fat trait of pigs;
a2 Pig breeding;
a3 Screening pig breeds with high intramuscular fat content.
The invention also provides a detection method of the pig genotype, wherein the genotypes are AA genotype, AG genotype and GG genotype, and the detection method comprises the following steps: detecting the nucleotide at 373bp of a DNA molecule shown in SEQ ID NO.1 in the genome of a pig to be detected, wherein if two chromosomes of the pig to be detected are the chromosomes g 1), the pig to be detected is AA genotype; the two chromosomes of the pig to be tested are the chromosomes of g 2), and the pig to be tested is GG genotype; one of the two chromosomes of the pig to be tested is the chromosome of g 1), and the other chromosome of g 2), and the pig to be tested is AG genotype;
g1 A) the nucleotide at 373bp corresponding to the sequence shown in SEQ ID NO.1 is A;
g2 A nucleotide at 373bp corresponding to the sequence shown in SEQ ID NO.1 is G.
In the detection method, the nucleotide at 373bp corresponding to the sequence shown in SEQ ID NO.1 in the chromosome of the pig to be detected is detected by adopting a primer pair shown in SEQ ID NO.2 and SEQ ID NO. 3.
The invention also provides a method for identifying or assisting in identifying intramuscular fat traits of pigs, which comprises the following steps: and detecting the genotype of the pig to be detected, wherein the intramuscular fat content of the pig to be detected with the AA genotype is higher than or the intramuscular fat content of the pig to be detected with the AA genotype is higher than the AG genotype and the GG genotype.
The invention also provides a pig breeding method, which specifically comprises the following steps: detecting the genotype of the pig to be detected, and selecting the AA genotype pig for breeding.
The invention has the beneficial effects that: the invention discovers a molecular marker (rs 340610840) which is positioned in a fourth exon of the pig AOPEP gene and is related to the intramuscular fat trait of the pig, and the Single Nucleotide Polymorphism (SNP) can be used as the molecular marker of the intramuscular fat trait of the pig, so that a novel molecular breeding marker is provided for the marker-assisted breeding of the intramuscular fat trait of the pig.
Drawings
FIG. 1 is an agarose gel electrophoresis pattern of a sequence fragment of the porcine AOPEP gene as set forth in SEQ ID NO. 1;
FIG. 2 is a diagram showing the result of SANGER sequencing and reading at the rs340610840 locus of the AOPEP gene of the pig in the invention; wherein, A is AA genotype, B is AG genotype, C is GG genotype.
Detailed Description
In order to make the technical problems, technical schemes and beneficial effects to be solved more clear, the invention is further described in detail below with reference to the accompanying drawings and embodiments. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention.
In the following examples, unless otherwise specified, the methods are conventional; the reagents and materials described, unless otherwise specified, are commercially available.
Example 1 acquisition of pig AOPEP Gene fragment and establishment of polymorphism detection method
1. Extraction of pig genome DNA.
The test pig variety of the invention is a new variety of selenium-rich black pigs which are jointly cultivated by the national academy of agricultural sciences of Hubei province, livestock and veterinary institute and Living pig breeding Limited company in Hubei days.
Pig genomic DNA was extracted from porcine ear margin tissue using an animal tissue genomic DNA kit (operated according to the kit instructions) produced by Beijing Optimu Biotechnology Co., ltd.
2. Obtaining the pig AOPEP gene fragment.
(1) And (3) PCR amplification:
the following primer pairs were designed based on the genomic sequence of the porcine AOPEP gene (GenBank accession number: NC-010452.4):
amplification of the SEQ ID NO.1 sequence upstream primer: 5'-ACTTCACGGAGCATTTCAT-3' (SEQ ID NO. 2),
amplifying a primer downstream of the SEQ ID NO.1 sequence: 5'-TCAAGCACCACCCTTTATT-3' (SEQ ID NO. 3).
PCR amplification was performed in selenium-both black pig genomic DNA using the above primers, 50. Mu.L of a PCR reaction system, and the concentrations of the components in the system were 100ng of template DNA, 10 Xbuffer (containing Mg 2+ ) 4. Mu.L of each of the above-mentioned upstream and downstream primers was 0.5. Mu.M, 2.5. Mu.M dNTPs, 1U of Taq DNA polymerase.
The PCR was run as follows: pre-denaturation at 94℃for 3min; denaturation at 94℃for 30s, annealing at 61℃for 30s, elongation at 72℃for 45s,35 cycles; extending at 72 ℃ for 10min; preserving at 4 ℃.
(2) And (3) purifying a PCR product: the purification is carried out by adopting a Gel Extraction Kit kit of Shanghai biological engineering Co., ltd, and specific steps are shown in a kit instruction book.
3. And (3) delivering the purified and recovered PCR product to Beijing ao Dingsheng biotechnology Co., ltd for SANGER sequencing to detect molecular markers.
The PCR product has a length of 653bp, the sequence of which is shown as SEQ ID NO.1, and the agarose gel electrophoresis pattern of which is shown as FIG. 1.
EXAMPLE 2 detection of polymorphism distribution of molecular markers in selenium-Du-black pigs
This example detects the polymorphism at the fourth exon rs340610840 site (i.e., 373bp of the sequence shown in SEQ ID NO. 1) of the porcine AOPEP gene in selenium-both black pigs according to the method established in example 1, and the detection results are shown in Table 1 and FIG. 2.
TABLE 1 genotype frequencies and allele frequencies of AOPEP Gene rs340610840 locus in selenium Du black pigs
The results in table 1 show that: in selenium-all black pigs, the locus rs340610840 of the AOPEP gene is represented by three genotypes AA, AG or GG; wherein the G allele frequency is 0.58, is the dominant allele.
Example 3 correlation analysis of molecular markers and intramuscular fat traits of pigs and application
In order to determine whether the rs340610840 locus of the porcine AOPEP gene is related to the intramuscular fat difference of the pig, polymorphism detection is carried out by adopting the method established in the example 1, and the correlation between different genotypes of the rs340610840 locus of the porcine AOPEP gene and the intramuscular fat trait of the pig is analyzed.
The variance analysis of the different SNP genotype combinations was performed using SAS statistical software (SAS Institute Inc, version 9.1) GLM program and the significance test was performed using the following models:
Y ij ＝μ+G i +F j +e ij ；
wherein Y is ij Is the character phenotype value, mu is the average value, G i Is genotype effect (including gene additive effect and dominant effectEffects; additive effects 1,0 and-1 represent the AA, AG and GG genotypes, respectively, dominant effects 1, -1 and 1 represent the AA, AG and GG genotypes, respectively; f (F) j Is a pig farm comprehensive effect; e, e ij Is a residual effect.
Correlation analysis between different genotypes and intramuscular fat traits was performed in selenium-all black pigs, and the statistical analysis results are shown in table 2:
TABLE 2 correlation analysis of the rs340610840 locus of the AOPEP Gene and the intramuscular fat Properties of pigs
Note that: a and B represent extremely significant differences (P < 0.01), representing significant differences (P < 0.01), and representing significant differences (P < 0.05).
As can be seen from table 2: in selenium-all black pigs, the intramuscular fat of the AA genotype at the rs340610840 locus was significantly higher than the AG genotype and the GG genotype (P < 0.01). However, the G allele is the dominant allele in this population and, therefore, should be used in breeding to preserve individuals carrying the a allele, thereby facilitating an increase in intramuscular fat content of the population.
In conclusion, the molecular marker (rs 340610840) related to the intramuscular fat traits of the pigs, which is discovered by the invention, is positioned in a partial DNA sequence (SEQ ID NO. 1) of the fourth exon of the AOPEP gene of the pigs, and the intramuscular fat of the AA genotype of the molecular marker is extremely higher than that of AG genotype and GG genotype in selenium-black pigs, so that the molecular marker can be used for screening or predicting pig breeds with excellent intramuscular fat traits.
The present invention is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present invention are intended to be included in the scope of the present invention.
Claims (10)
1. A SNP molecular marker related to the intramuscular fat trait of pigs is characterized in that the SNP molecular marker is a nucleotide corresponding to 373bp of a DNA molecule with a sequence shown as SEQ ID NO.1 in a fourth exon of an AOPEP gene of pigs, and is a G/A polymorphism.
2. A substance for detecting the SNP molecular marker according to claim 1.
3. A substance according to claim 3, characterized in that it comprises primers of the sequences shown in SEQ ID No.2 and SEQ ID No. 3.
4. Use of the SNP molecular marker of claim 1 or the substance of claim 2 for the identification or assisted identification of intramuscular fat traits in pigs.
5. Use of the SNP molecular marker of claim 1 or the substance of claim 2 in pig breeding.
6. Use of the SNP molecular marker of claim 1 or the substance of claim 2 for screening pigs with high intramuscular fat content.
7. A method for detecting a pig genotype, wherein the genotypes are an AA genotype, an AG genotype and a GG genotype, and the method comprises the following steps: detecting the nucleotide at 373bp of a DNA molecule shown in SEQ ID NO.1 in the genome of a pig to be detected, wherein if two chromosomes of the pig to be detected are the chromosomes g 1), the pig to be detected is AA genotype; the two chromosomes of the pig to be tested are the chromosomes of g 2), and the pig to be tested is GG genotype; one of the two chromosomes of the pig to be tested is the chromosome of g 1), and the other chromosome of g 2), and the pig to be tested is AG genotype;
g1 A) the nucleotide at 373bp corresponding to the sequence shown in SEQ ID NO.1 is A;
g2 A nucleotide at 373bp corresponding to the sequence shown in SEQ ID NO.1 is G.
8. The method according to claim 7, wherein the detection of the nucleotide at 373bp corresponding to the sequence shown in SEQ ID NO.1 in the chromosome of a pig to be tested is carried out using the substance according to claim 2 or 3.
9. A method for identifying or aiding in the identification of a trait of intramuscular fat in a pig, comprising the steps of: the method according to claim 7 or 8, wherein the genotype of the pig to be tested is determined and the intramuscular fat content of the pig to be tested is higher than or alternatively higher than the AG genotype and GG genotype.
10. A method for breeding pigs, characterized in that the genotype of the pig to be tested is detected according to the method of claim 7 or 8, and the AA genotype pig is selected for breeding.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117363741A (en) * | 2023-10-13 | 2024-01-09 | 湖北省农业科学院畜牧兽医研究所 | Molecular marker related to pig carcass and meat quality traits in pig GPX3 gene 5' UTR and application thereof |
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