A pig SNP molecular marker and its application in pork quality trait screening and breeding
Through the screening of SNP molecular markers found in the PVALB gene of pigs, the problem of difficulty in improving pork qualitative traits was solved, and early and efficient screening of pork qualitative traits was achieved.
Patent Information
- Application Number
- CN202211147346.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-09-19
- Publication Date
- 2025-05-09
- Estimated Expiration
- 2042-09-19
AI Technical Summary
The prior art is difficult to improve the meat properties such as intramuscular fat content and marble score of pork through conventional breeding, and it is difficult to determine live life.
A SNP molecular marker related to pork plasmometry in the pig PVALB gene was discovered, and individuals carrying dominant alleles were screened through PCR amplification and sequencing analysis.
Early screening of pork qualitative traits is achieved, the method is simple, fast and has high accuracy, which can effectively improve the genetic improvement efficiency of pork qualitative traits.
Smart Images
Figure CN115927658B_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of molecular markers, and in particular relates to a pig SNP molecular marker and its application in the screening of pork quality traits and pig breeding. Background Art
[0002] Pork quality is an important economic shape of pigs. Improving pork quality is one of the important goals of current pig breeding work, and it is also an important guarantee to meet consumers' needs for pork quality, food health and nutrition. Among them, intramuscular fat (IMF) content and marbling score are two important indicators for evaluating pork quality, which are significantly correlated with pork flavor, tenderness and juiciness. Although meat quality traits such as IMF have a high heritability of 0.2-0.4, it is difficult to improve meat quality traits through conventional breeding because it is difficult to measure meat quality traits in vivo. Therefore, it is of great significance to find molecular markers that affect meat quality traits and improve pork quality through molecular marker-assisted selection (MAS).
[0003] MAS uses molecular markers associated with specific traits as an auxiliary means for selective breeding, which has the advantages of being fast, accurate, and not affected by the environment. It can not only greatly reduce the human and material consumption of breeding, but also shorten the breeding time. Molecular markers used in MAS include protein markers, microsatellite markers, single nucleotide polymorphism (SNP) markers, etc.
[0004] Single nucleotide polymorphism refers to the genetic polymorphism caused by the mutation of a single nucleotide in the genomic DNA sequence. Due to the wide distribution and stability of SNP, it plays an important role in animal molecular marker-assisted selection breeding. Mining SNPs related to pork quality traits can effectively accelerate the process of genetic improvement of pork quality traits. Summary of the invention
[0005] In view of this, the purpose of the present invention is to provide a pig SNP molecular marker related to pork quality traits, and further construct a method for using the marker in pork quality trait screening and pig breeding.
[0006] In order to achieve the above object, the technical solution of the present invention is as follows:
[0007] The inventors used RNA-seq and ATAC-seq integrated analysis to find that in the Selenium Black Pig population with extremely high / low IMF, PVALB gene (parvalbumin, PVALB ) had significant differences in expression and chromatin open regions between the two groups.PVALB Genes have an impact on pig meat quality traits such as IMF. PVALB The gene is a high-affinity calcium ion binding protein that is similar in structure and function to calmodulin and troponin C and is involved in the muscle relaxation process in muscle tissue.
[0008] Based on this, the present invention identified through further research PVALB There is a SNP site in the gene that is related to pork quality traits and can be used as a molecular marker for pork quality trait screening and pig breeding. The nucleotide sequence of the molecular marker is shown in SEQ ID NO.1, and there is an A>C base mutation at the 260th bp of the sequence shown in SEQ ID NO.1.
[0009] The sequence length of this molecular marker is 518 bp and is located in the PVALB Partial sequence of the gene.
[0010] In the above technical solution, the A or C base polymorphic site at the 260th bp of the sequence described in SEQ ID NO.1 is specifically expressed as three genotypes of AA, AC or CC, among which the A allele is the dominant allele.
[0011] The present invention further provides a primer pair for amplifying the above molecular marker, comprising an upstream primer having a nucleotide sequence as shown in SEQ ID NO.2, and a downstream primer having a nucleotide sequence as shown in SEQ ID NO.3.
[0012] The present invention also provides the use of the above molecular markers or primer pairs as shown in SEQ ID NO. 2-3 in pork quality trait screening and pig breeding, wherein the pork quality traits are preferably intramuscular fat content and marbling score.
[0013] Furthermore, the above-mentioned application method is specifically as follows: using the genomic DNA of the pig to be tested as a template, obtaining the molecular marker shown in SEQ ID NO.1 by PCR amplification and purifying it, sequencing and analyzing the molecular marker, retaining the individuals carrying the A allele at the 260bp of the sequence, and eliminating the individuals carrying the C allele.
[0014] In the above scheme, the genomic DNA of the pig to be tested is preferably extracted from the ear margin tissue of the pig to be tested.
[0015] In the above technical solution, the upstream and downstream primers used in PCR amplification are preferably the primer pair shown in SEQ ID NO.2~3.
[0016] According to the sequence characteristics of this pair of primers, the optimal system and procedure for PCR amplification are as follows:
[0017] The system for PCR amplification was: 50 μL in total, 100 ng of genomic DNA, 25 μL of PCR mix, 1 μL of each of the upstream and downstream primers, and the balance of ddH2O;
[0018] The PCR amplification program was as follows: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, 35 cycles; extension at 72°C for 10 min; and storage at 4°C.
[0019] Compared with the prior art, the invention has the following beneficial effects: the invention discovers for the first time the PVALB A SNP molecular marker in the gene that is related to the meat quality traits of pigs can be used to screen and breed pork quality traits, which can achieve early screening of pork quality traits. The screening method is simple, fast and highly accurate. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 The agarose gel electrophoresis detection result of the PCR amplification product in Example 1 is shown;
[0021] Figure 2 This is a comparison chart of sequencing results of polymorphic sites in individuals with different genotypes. DETAILED DESCRIPTION
[0022] The technical solution of the present invention will be described clearly and completely below in conjunction with the embodiments. It should be understood that the specific implementation methods described herein are only used to illustrate and explain the present invention and are not used to limit the present invention.
[0023] In the following examples, unless otherwise specified, all methods are conventional methods; the reagents and materials described, unless otherwise specified, can be obtained from commercial sources.
[0024] Example 1
[0025] This example constructs a method for detecting polymorphic sites, and the specific process is as follows:
[0026] 1. Extraction of pig genomic DNA.
[0027] The experimental pig variety of the present invention is the Xidu Black Pig, which is a new nationally approved pig variety bred under the lead of the Hubei Academy of Agricultural Sciences.
[0028] The porcine genomic DNA was extracted using the PureLink™ Pro 96 Genomic DNA Purification Kit, K182104A), and the porcine genomic DNA was extracted according to the instructions of the kit. The concentration and quality of the extracted DNA were tested and stored at -20°C for future use.
[0029] 2. Pig PVALB Obtaining gene SNP genetic marker detection fragments.
[0030] (1) PCR amplification.
[0031] According to the pig PVALB A pair of primers was designed based on the SNP genetic marker detection sequence (shown in SEQ ID NO.1) in the genomic sequence of the gene (GenBank ID: NC_010447) to amplify the fragment of the polymorphic site. The primer sequences are as follows:
[0032] Upstream primer: 5'-CCTGAGCCTCGATTTTGGGT-3' (SEQ ID NO. 2),
[0033] Downstream primer: 5'-CGGCTCTAGCCATCTCGTTC-3' (SEQ ID NO. 3).
[0034] PCR amplification was performed using the above primer pair and the genomic DNA of Xidu black pig as a template. The PCR reaction system was 50 μL, and the components in the system were: 100 ng genomic DNA, 25 μL PCR mix, 1 μL each of the above upstream and downstream primers, and ddH2O was added to make up the total volume to 50 μL.
[0035] The PCR operation program was as follows: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, 35 cycles; extension at 72°C for 10 min; and storage at 4°C.
[0036] The PCR products were detected by 1.5% agarose gel electrophoresis. Some of the test results are shown in Figure 1 As shown, lane M is DL2000 Marker, lanes 1-3 are amplified fragments from Xidu black pig, and the size of the amplified fragment is 518 bp.
[0037] (2) Purification of PCR products.
[0038] The PCR amplification product was purified using the Gel Extraction Kit from Shanghai Sangon Biotechnology Co., Ltd. For specific steps, see the kit instructions.
[0039] 3. Detect molecular markers using direct sequencing of PCR products.
[0040] The PCR purified products obtained above were directly sent to Beijing Aoke Company for sequencing, and the genotype of the site in the test population was determined based on the sequencing results.
[0041] The results were analyzed using SeqMan software. Figure 2 As shown, there is an A>C allele mutation at 260 bp in the sequence, which causes PVALB Gene polymorphism,
[0042] Example 2
[0043] This example detects pigs in 300 Selenium City black pigs. PVALB The polymorphism distribution pattern of gene mutation sites and the detection results are shown in Table 1.
[0044] Table 1 Distribution of polymorphisms at mutation sites
[0045]
[0046] From the results in Table 1, we can see that in black pigs PVALB The gene mutation site showed three genotypes: AA, AC and CC. Among them, the AA genotype individuals were more common and the AA allele frequency was 72.5%. Therefore, the A allele was the dominant allele.
[0047] Example 3
[0048] To verify the pig PVALB Whether the gene mutation site is related to the difference in pork quality traits, the polymorphism detection was performed using the method established in Example 1, and the correlation between different genotypes of this polymorphic site and the intramuscular fat content and marbling score traits of pigs was analyzed. The GLM model of SAS 19.0 software was used to perform association analysis on the mutation site. The model used was:
[0049] ,
[0050] Where: Y ijk represents the phenotypic value; μ represents the population mean; G i represents genotype effect; A j represents the year-season effect; S k represents the paternal effect; e ijk represents random residual effects. P <0.05 was considered significant difference; P <0.01 was considered to be extremely significant.
[0051] The association analysis between different genotypes and intramuscular fat content and marbling score traits was conducted in Sedu Black Pigs. The statistical analysis results are shown in Table 2:
[0052] Table 2 Association analysis between mutation sites and pork quality traits
[0053]
[0054] Note: The trait means in the table are mean ± standard deviation, A and B indicate extremely significant differences ( P <0.01).
[0055] As can be seen from Table 2, in Sedu Black Pig, the intramuscular fat content and marbling score of the AA genotype at the mutation site were significantly higher than those of the CC genotype ( P <0.01), so the A allele is the dominant allele, and individuals carrying this dominant allele should be retained in breeding, which is beneficial to improving the intramuscular fat content and marbling score traits of the population.
[0056] In summary, the molecular markers provided by the present invention are closely related to pork quality traits. Individuals with excellent pork quality traits can be accurately selected by detecting the genotype of the molecular markers. The present invention further provides primer pairs for specific amplification and constructs a detection method, laying a foundation for the application of the molecular markers.
[0057] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention.
Claims
1. Application of pig SNP molecular markers or primer pairs for amplifying the pig SNP molecular markers in screening for meat quality traits of Xidu black pigs, characterized in that: The nucleotide sequence of the molecular marker is shown in SEQ ID NO.1, and there is an A / C polymorphic site at the 260 bp of the sequence shown in SEQ ID NO.
1. The pork quality traits are intramuscular fat content and marbling score.
2. The use according to claim 1, characterized in that: The sequences of the primer pairs are shown in SEQ ID NOs. 2-3.
3. Application of pig SNP molecular markers or primer pairs for amplifying the pig SNP molecular markers in breeding of meat quality traits of Selenium City Black Pigs, characterized in that: The nucleotide sequence of the molecular marker is shown in SEQ ID NO.1, and there is an A / C polymorphic site at the 260 bp of the sequence shown in SEQ ID NO.
1. The pork quality traits are intramuscular fat content and marbling score.
4. The use according to claim 3, characterized in that: The sequences of the primer pairs are shown in SEQ ID NOs. 2-3.
5. The application according to claim 3, characterized in that: Specifically, the genomic DNA of the pig to be tested is used as a template, the molecular marker is obtained by PCR amplification and purified, the molecular marker is sequenced and analyzed, the individuals carrying the A allele at the 260bp of the sequence are retained, and the individuals carrying the C allele are eliminated.
6. The use according to claim 5, characterized in that: The genomic DNA of the pigs to be tested is extracted from the ear margin tissue of the pigs to be tested.
7. The use according to claim 5, characterized in that: The PCR amplification system is: a total of 50 μL, 100 ng of genomic DNA, 25 μL of PCR mix, 1 μL of upstream and downstream primers, and ddH2O as the balance.
8. The use according to claim 5, characterized in that: The PCR amplification procedure was as follows: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, 35 cycles; extension at 72°C for 10 min; and storage at 4°C.