CN115820879B - Molecular marker related to intramuscular fat traits of pigs in pig AOPEP gene and application thereof - Google Patents
Molecular marker related to intramuscular fat traits of pigs in pig AOPEP gene and application thereof Download PDFInfo
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- 108020004414 DNA Proteins 0.000 claims abstract description 10
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- 235000015277 pork Nutrition 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 description 2
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- 108090000623 proteins and genes Proteins 0.000 description 2
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- 239000011669 selenium Substances 0.000 description 2
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- QSBGWDDCOJYQGY-KOQODJNWSA-N Angiotensin IV Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)NC(=O)[C@@H](N)C(C)C)C1=CC=C(O)C=C1 QSBGWDDCOJYQGY-KOQODJNWSA-N 0.000 description 1
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
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Abstract
The invention discloses a molecular marker related to intramuscular fat traits of pigs in a pig AOPEP gene and application thereof, wherein the molecular marker is a nucleotide corresponding to 194bp of a DNA molecule with a sequence shown as SEQ ID NO.1 in a second exon of the pig AOPEP gene, and is a C/A polymorphism. Experiments prove that the molecular marker provided by the invention is obviously related to the intramuscular fat trait of pigs, which indicates 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 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 molecular marker related to intramuscular fat traits of pigs in a second 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 second 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 molecular marker related to intramuscular fat traits of pigs in a pig AOPEP gene is a nucleotide corresponding to 194bp of a DNA molecule with a sequence shown as SEQ ID NO.1 in a second exon of the pig AOPEP gene, and is C > A polymorphism.
The invention also provides a substance for detecting the molecular marker.
The substances marked by the detection molecules 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 the use of the molecular markers or substances for detecting the molecular markers, as A1), A2) or A3) below:
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 method for detecting the genotype of the pig, wherein the genotype is AA genotype, AC genotype and CC genotype, and the method comprises the following steps: detecting 194bp nucleotides corresponding to the DNA molecule shown in SEQ ID NO.1 in the genome of the 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 of CC 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 an AC genotype;
g1 A) the 194bp position corresponding to the sequence shown in SEQ ID NO.1 is A;
g2 A nucleotide at 194bp corresponding to the sequence shown in SEQ ID NO.1 is C.
In the detection method, the nucleotide at 194bp corresponding to the sequence shown in SEQ ID NO.1 in the pig chromosome 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 AC genotype and the CC 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 3470100435) which is positioned in a second 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 rs3470100435 locus of the AOPEP gene of the pig in the invention; wherein, the A diagram is AA genotype, the B diagram is AC genotype, and the C diagram is CC 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'-GGCGTGAGGAAAGTCAGTAG-3' (SEQ ID NO. 2),
amplifying a primer downstream of the SEQ ID NO.1 sequence: 5'-CAGATTCAAGAAACAGCGTAG-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 62℃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 556bp, the sequence of which is shown as SEQ ID NO.1, and the agarose gel electrophoresis pattern of which is shown as figure 1.
EXAMPLE 2 detection of polymorphism distribution of molecular markers in selenium-Du-black pigs
This example detects the polymorphism at the rs3470100435 locus (194 bp of the sequence shown in SEQ ID NO. 1) of the second exon 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 rs3470100435 locus in selenium Du black pigs
The results in table 1 show that: in selenium-all black pigs, the locus rs3470100435 of the AOPEP gene is expressed as three genotypes of AA, AC or CC; wherein the C allele frequency is 0.54, 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 rs3470100435 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 rs3470100435 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 For genotype effects (including gene additive effects and dominant effects; additive effects apply 1,0 and-1 to AA, AC and CC genotypes, respectively, dominant effects apply 1, -1 and 1 to AA, AC and CC 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 rs3470100435 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 AA genotype at the rs3470100435 locus had significantly higher intramuscular fat than the AC genotype and CC genotype (P < 0.01). However, the C 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 3470100435) 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 second 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 the AC genotype and CC genotype in selenium-black pigs, so that the molecular marker can be used for screening or predicting the 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 (5)
1. The application of a molecular marker related to the intramuscular fat traits of pigs in a pig AOPEP gene or a primer for detecting the molecular marker is characterized in that the molecular marker is a nucleotide corresponding to 194 th bp of a DNA molecule with a sequence shown as SEQ ID NO.1 in a second exon of the pig AOPEP gene, and is a C/A polymorphism; the application is any one of the following:
the application in identifying or assisting in identifying the intramuscular fat traits of pigs;
application in pig breeding;
use in screening pigs with high intramuscular fat content.
2. The use according to claim 1, wherein the sequence of the primer is shown in SEQ ID No.2 and SEQ ID No. 3.
3. The method for detecting the genotype of the pig is characterized in that the genotypes are AA genotype, AC genotype and CC genotype, and the method for detecting the genotype of the pig comprises the following steps: detecting nucleotide corresponding to 194bp of DNA molecule shown in SEQ ID NO.1 in genome of a pig to be detected, wherein the two chromosomes of the pig to be detected are the chromosomes g 1) as follows, and 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 of CC 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 an AC genotype;
g1 A) the nucleotide at position 194bp corresponding to the sequence set forth in SEQ ID No.1 is a;
g2 A nucleotide corresponding to the 194 th bp position of the sequence shown in SEQ ID NO.1 is C.
4. A method for identifying or aiding in the identification of a trait of intramuscular fat in a pig, comprising the steps of: a method according to claim 3 for determining the genotype of a test pig, wherein the AA genotype test pig has a higher intramuscular fat content than or a candidate higher than the AC genotype and the CC genotype.
5. A method of breeding pigs, characterized in that the genotype of the pig to be tested is detected according to the method of claim 3, and the AA genotype pig is selected for breeding.
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| CN113549700A (en) * | 2021-07-16 | 2021-10-26 | 山东农业大学 | Molecular marker related to porcine intramuscular fat character and application of molecular marker in pig breeding |
| CN113913536A (en) * | 2021-11-30 | 2022-01-11 | 湖北省农业科学院畜牧兽医研究所 | Genetic marker for intramuscular fat content character by using variation of first exon of pig EEPD1 gene and application |
| CN114959066A (en) * | 2022-06-29 | 2022-08-30 | 湖北省农业科学院畜牧兽医研究所 | Molecular marker influencing intramuscular fat content traits of pigs and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113549700A (en) * | 2021-07-16 | 2021-10-26 | 山东农业大学 | Molecular marker related to porcine intramuscular fat character and application of molecular marker in pig breeding |
| CN113913536A (en) * | 2021-11-30 | 2022-01-11 | 湖北省农业科学院畜牧兽医研究所 | Genetic marker for intramuscular fat content character by using variation of first exon of pig EEPD1 gene and application |
| CN114959066A (en) * | 2022-06-29 | 2022-08-30 | 湖北省农业科学院畜牧兽医研究所 | Molecular marker influencing intramuscular fat content traits of pigs and application |
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| Line K M Lybech 等.Suicide Related Phenotypes in a Bipolar Sample: Genetic Underpinnings.Genes.2021,第12卷(第10期),摘要. * |
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