CN115927658A - Pig SNP molecular marker and application thereof in pig meat quality character screening and breeding - Google Patents
Pig SNP molecular marker and application thereof in pig meat quality character screening and breeding Download PDFInfo
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
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Abstract
The invention discloses a pig SNP molecular marker and application thereof in pig meat quality character screening and breeding, wherein the nucleotide sequence of the molecular marker is shown as a sequence table SEQ ID NO.1, and an A/C polymorphic site exists at the 260bp position of the sequence, and a method for screening and breeding the pig meat quality character by using the molecular marker is further constructed. The invention discovers the SNP molecular markers related to the pork quality traits in the PVALB genes of the pigs for the first time, particularly related to the intramuscular fat content and marbling scoring traits of the pigs, can be used for marker-assisted selective breeding of the pigs, realizes early screening of the pork quality traits, has simple and rapid screening method and has potential important value for genetic improvement of the pork quality traits.
Description
Technical Field
The invention belongs to the technical field of molecular markers, and particularly relates to a pig SNP molecular marker and application thereof in pig meat quality character screening and pig breeding.
Background
The pork quality is an important economic shape of the pig, the improvement of the pork quality is one of the important targets of the current breeding work of the breeding pig, and simultaneously, the improvement of the pork quality is an important guarantee for meeting the requirements of consumers on the pork quality, the food health and the nutrition. The content of Intramuscular Fat (IMF) and marbling score are two important indexes for evaluating the pork quality, and are obviously related to the indexes of the pork such as flavor, tenderness and juiciness. Although the heritability of meat quality traits such as IMF is high, between 0.2 and 0.4, improvement by conventional breeding is difficult because in vivo measurement of meat quality traits is difficult. Therefore, the molecular Marker influencing the meat quality character is searched, and the molecular Marker Assisted breeding (MAS) has important significance for improving the pork quality.
MAS utilizes molecular markers associated with specific traits as an auxiliary means to perform selective breeding, has the advantages of rapidness, accuracy, no environmental influence and the like, can greatly reduce the manpower and material resource consumption of breeding, and can shorten the breeding time limit. Molecular markers for MAS include protein markers, microsatellite markers, single Nucleotide Polymorphism (SNP) markers, and the like.
Single nucleotide polymorphism refers to gene polymorphism caused by mutation of a single nucleotide in a genomic DNA sequence. Due to the universality and stability of SNP distribution, the method plays an important role in animal molecular marker-assisted selective breeding. The SNP related to the pork quality traits is excavated, and the process of genetic improvement of the pork quality traits can be effectively accelerated.
Disclosure of Invention
In view of the above, the present invention aims to provide a pig SNP molecular marker related to pork quality traits, and further construct an application method thereof in pork quality trait screening and pig breeding.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
the inventor discovers that the expression quantity and the chromatin opening region of PVALB (pvALB) in two populations are significantly different by RNA-seq and ATAC-seq integration analysis in the selenium-rich black pig population with extremely high/low IMF, and presumes that the PVALB gene has influence on meat quality traits such as pig IMF. The PVALB gene is a high-affinity calcium ion binding protein, is similar to calmodulin and troponin C in structure and function, and is involved in the muscle relaxation process in muscle tissues.
Based on the above, the invention identifies one SNP site related to the pork quality character in the PVALB gene through further research, and can be used as a molecular marker for pork quality character screening and pig breeding, wherein the nucleotide sequence of the molecular marker is shown as SEQ ID NO.1, and one A > C base mutation exists at the 260bp position of the sequence shown as SEQ ID NO. 1.
The sequence length of the molecular marker is 518bp, the molecular marker is specifically positioned in a partial sequence of a porcine PVALB gene, and the rs number of the SNP locus is rs45433252.
In the technical scheme, the A or C base polymorphism site at the 260bp position of the sequence described in SEQ ID NO.1 is specifically represented as three genotypes of AA, AC or CC, wherein an A allele is a dominant allele.
The invention further provides a primer pair for amplifying the molecular marker, which comprises an upstream primer with a nucleotide sequence shown as SEQ ID NO.2 and a downstream primer with a nucleotide sequence shown as SEQ ID NO. 3.
The invention also provides application of the molecular marker or the primer pair shown as SEQ ID NO. 2-3 in pork quality character screening and pig breeding, wherein the pork quality character is particularly preferably intramuscular fat content and marbling score.
Further, the method of the above application is specifically: taking the genome DNA of a pig to be detected as a template, obtaining a molecular marker shown as SEQ ID NO.1 through PCR amplification, purifying, carrying out sequencing analysis on the molecular marker, reserving an individual carrying an A allele at the 260bp position of the sequence, and eliminating the individual carrying a C allele.
In the above scheme, the genomic DNA of the test pig is preferably extracted from the ear margin tissue of the test pig.
In the above technical scheme, the upstream and downstream primers for PCR amplification are preferably primer pairs shown in SEQ ID NO. 2-3.
Based on the sequence characteristics of the pair of primers, the optimal system and procedure for PCR amplification are as follows:
the PCR amplification system is as follows: 50 μ L in total, 100ng of genomic DNA, 25 μ L of PCR mix, and 1 μ L, ddH for each of the upstream and downstream primers 2 The balance of O;
the procedure for PCR amplification was: pre-denaturation at 94 ℃ for 3min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 30s, and 35 cycles; extending for 10min at 72 ℃; storing at 4 ℃.
Compared with the prior art, the invention has the beneficial effects that: the invention discovers an SNP molecular marker in the PVALB gene of the pig related to the meat quality character of the pig for the first time, and the SNP molecular marker is used for screening the meat quality character of the pig, screening and breeding the meat quality character of the pig can realize early screening of the meat quality character of the pig, and the screening method is simple, rapid and high in accuracy.
Drawings
FIG. 1 is a diagram showing the results of agarose gel electrophoresis detection of the PCR amplification product of example 1;
FIG. 2 is a graph comparing the sequencing results of the rs45433252 polymorphic site in different genotype individuals.
Detailed Description
The technical solution of the present invention will be clearly and completely described below with reference to the following examples. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.
In the following examples, unless otherwise specified, all methods are conventional; the reagents and materials are commercially available unless otherwise specified.
Example 1
In this example, a method for detecting a polymorphic site was constructed, and the specific process was as follows:
1. and (3) extracting pig genome DNA.
The test pig variety is a selenium-Du black pig, and is a new nationally-examined live pig variety cultivated by Hubei academy of agricultural sciences.
Extraction of pig genomic DNA an animal tissue genomic DNA extraction kit (PureLink) manufactured by Invitrogen was used TM Pro 96 Genomic DNA Purification kit, K182104A), porcine Genomic DNA extraction was performed according to the kit instructions. And (4) detecting the concentration and quality of the extracted DNA, and storing at-20 ℃ for later use.
2. Obtaining a pig PVALB gene SNP genetic marker detection fragment.
(1) And (4) PCR amplification.
A pair of primers is designed according to the SNP genetic marker detection sequence (shown as SEQ ID NO. 1) in the genome sequence (GenBank ID: NC-010447) of the porcine PVALB gene, and the fragment of the polymorphic site is amplified. The primer sequences are as follows:
an upstream primer: 5'-CCTGAGCCTCGATTTTGGGT-3' (SEQ ID NO. 2),
a downstream primer: 5'-CGGCTCTAGCCATCTCGTTC-3' (SEQ ID NO. 3).
The primer pair is utilized, the genome DNA of the Se-bearing black pig is taken as a template to carry out PCR amplification, the PCR reaction system is 50 mu L, and each component in the system is: genomic DNA100ng, PCR mix 25. Mu.L, upstream and downstream primers 1. Mu.L each, plus ddH 2 O is added to the total volume of 50 mu L.
The running program of PCR was: pre-denaturation at 94 ℃ for 3min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 30s, and 35 cycles; extending for 10min at 72 ℃; storing at 4 ℃.
The PCR product was detected by 1.5% agarose gel electrophoresis, and the partial detection results are shown in FIG. 1, wherein lane M is DL2000Marker, lanes 1-3 are the amplified fragments of selenium-rich black pig, and the size of the amplified fragment is 518bp.
(2) And (5) purifying a PCR product.
The PCR amplification product is purified by using a Gel Extraction Kit of Shanghai biological engineering Co., ltd, and the specific steps are shown in the Kit instruction.
3. And detecting the molecular marker by using a PCR product direct sequencing method.
And directly sending the obtained PCR purified product to Beijing Okko company for sequencing, and judging the genotype of the site in the detection group according to a sequencing result.
The result of analysis by using SeqMan software is shown in FIG. 2, there is a mutation of A > C allele at 260bp in the sequence, namely rs45433252A > C, the mutation causes the polymorphism of PVALB gene,
example 2
In this embodiment, the polymorphism distribution rule of the porcine PVALB gene rs45433252A > C locus is detected in 300 selenium-rich black pigs, and the detection results are shown in Table 1.
TABLE 1 polymorphism distribution rule of rs45433252A >
From the results in Table 1, it can be seen that: in the black pig, PVALB gene rs45433252A > C loci are expressed into three genotypes of AA, AC and CC, wherein the AA genotypes have more individuals, and the AA allele frequency is 72.5%, so the A allele is a dominant allele.
Example 3
In order to verify whether the differences between the rs45433252A > C locus of the porcine PVALB gene and the pork quality traits are related or not, the method established in the embodiment 1 is adopted to carry out polymorphism detection, and the correlations between different genotypes of the polymorphic locus and the intramuscular fat content and marbling evaluation traits of the pigs are analyzed. And (3) carrying out association analysis on the mutation sites by adopting a GLM model of SAS 19.0 software. The model used is:
Y ijk ＝μ+G i +A j +S k +e ijk ，
wherein: y is ijk Representing a tabular value; μ represents the population mean; g i Indicating a genotype effect; a. The j Indicating a seasonal effect; s k Representing a paternal effect; e.g. of the type ijk Representing random residual effects. P<0.05 judges that the difference is significant; p<0.01 judges the difference to be extremely significant.
Correlation analysis between different genotypes and intramuscular fat content and marbling evaluation traits was performed in selenium-all-black pigs, and the statistical analysis results are shown in table 2:
TABLE 2 correlation analysis of the rs45433252A >
Note: the values of the property averages in the table are mean ± standard deviation, with a and B indicating very significant differences (P < 0.01).
As can be seen from Table 2, in the selenium-rich black pig, the intramuscular fat content and marbling score of the AA genotype individual at the rs45433252A > C site are significantly higher than those of the CC genotype individual (P < 0.01), so that the A allele is a dominant allele, and the individual carrying the dominant allele should be reserved in breeding, thereby being beneficial to improving the intramuscular fat content and marbling score traits of the population.
In conclusion, the molecular marker provided by the invention is closely related to the pork quality traits, and the individual with excellent pork quality traits can be accurately bred by detecting the genotype of the molecular marker.
While the invention has been described with reference to specific preferred embodiments, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the following claims.
Claims (10)
1. A pig SNP molecular marker is characterized in that the nucleotide sequence of the molecular marker is shown as SEQ ID No.1, and A/C polymorphic sites exist at the 260bp position of the sequence shown as SEQ ID No. 1.
2. The porcine SNP molecular marker of claim 1, wherein the a allele is a dominant allele.
3. A primer pair for amplifying the porcine SNP molecular marker of claim 1, wherein the sequence of the primer pair is shown as SEQ ID NO. 2-3.
4. The porcine SNP molecular marker according to claim 1 or the application of the primer pair according to claim 3 in meat quality trait screening of pigs.
5. The use of claim 4, wherein the pork quality trait is intramuscular fat content and marbling score.
6. The porcine SNP molecular marker according to claim 1, or the primer pair according to claim 3, for use in pig breeding.
7. The use according to claim 6, characterized in that it is in particular: the molecular marker of claim 1 is obtained by PCR amplification with the genome DNA of the pig to be tested as a template and purified, the molecular marker is subjected to sequencing analysis, the individual carrying the A allele at the 260bp position of the sequence is reserved, and the individual carrying the C allele is eliminated.
8. The use of claim 7, wherein the genomic DNA of the test pig is extracted from the ear margin tissue of the test pig.
9. The use according to claim 7, wherein the PCR amplification system is: 50. Mu.L total, genomic DNA100ng, PCR mix 25. Mu.L, upstream and downstream primers 1. Mu. L, ddH 2 And the balance of O.
10. The use of claim 7, wherein the PCR amplification procedure is: pre-denaturation at 94 ℃ for 3min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 30s, and 35 cycles; extending for 10min at 72 ℃; storing at 4 ℃.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108070662A (en) * | 2016-11-15 | 2018-05-25 | 大韩民国（农村振兴厅长） | Genetic marker for determining quality character of pork and application thereof |
| CN110734983A (en) * | 2019-10-08 | 2020-01-31 | 南京农业大学 | SNP markers related to intramuscular fat traits of Suhuai pigs, and detection method and application thereof |
| CN112176070A (en) * | 2020-08-03 | 2021-01-05 | 南京农业大学 | UCP3 gene related to pig intramuscular fat character, molecular marker and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108070662A (en) * | 2016-11-15 | 2018-05-25 | 大韩民国（农村振兴厅长） | Genetic marker for determining quality character of pork and application thereof |
| CN110734983A (en) * | 2019-10-08 | 2020-01-31 | 南京农业大学 | SNP markers related to intramuscular fat traits of Suhuai pigs, and detection method and application thereof |
| CN112176070A (en) * | 2020-08-03 | 2021-01-05 | 南京农业大学 | UCP3 gene related to pig intramuscular fat character, molecular marker and application thereof |
Non-Patent Citations (5)
| Title |
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| DBSNP: "rs332458243", ENSEMBL, 30 April 2020 (2020-04-30), pages 1 * |
| SOHYOUNG WON等: "Identification of genes related to intramuscular fat content of pigs using genome-wide association study", ASIAN-AUSTRALAS J ANIM SCI, vol. 31, no. 2, 28 February 2018 (2018-02-28), pages 157 - 162 * |
| YU SONG等: "DIA-based quantitative proteomic analysis on the meat quality of porcine Longissimus thoracis et lumborum cooked by different procedures", FOOD CHEM, vol. 371, 1 March 2022 (2022-03-01), pages 131206 * |
| 徐忠等: "猪PVALB基因变异筛选及其与肉质性状的关联分析", 中国畜牧杂志, vol. 59, no. 8, 31 August 2023 (2023-08-31), pages 202 - 206 * |
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