SNP (Single Nucleotide Polymorphism) molecular marker located on pig chromosome 4 and related to hind leg weight and application of SNP molecular marker
By detecting the genotype of the SNP site g.161 T>C on pig chromosome 4, eliminating unfavorable genotypes and retaining favorable genotypes, and using molecular marker-assisted breeding technology, the problem of slow progress in genetic improvement in traditional breeding methods has been solved, resulting in a rapid increase in hind leg weight and improved economic benefits in pigs.
Patent Information
- Application Number
- CN202511194011.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-25
- Publication Date
- 2025-11-28
AI Technical Summary
Traditional methods make it difficult to quickly and accurately breed pigs with high hind leg weight, resulting in slow progress in genetic improvement and difficulty in meeting health consumption demands and improving economic efficiency.
Using SNP molecular markers located on pig chromosome 4 that are associated with hind leg weight, the TT and TC genotypes were eliminated and the CC genotype was retained by detecting the genotype of SNP site g.161 T>C. The frequency of allele C was increased generation by generation. Primer pairs were designed for amplification and sequencing to achieve molecular marker-assisted breeding.
It significantly increases the weight of pig hind legs, shortens the breeding cycle, improves economic benefits, and achieves efficient and accurate genetic improvement.
Smart Images
Figure CN121023031A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the fields of molecular biotechnology and molecular marker technology, and in particular relates to a SNP molecular marker located on chromosome 4 of pigs that is associated with hind leg weight and its application. Background Technology
[0002] With the continued growth of the consumer market and the increasing health awareness of consumers, the market for lean pork hind legs has maintained a steady growth trend. Pork hind leg meat is firm, low in fat, and high in lean meat, meeting people's demand for healthy, high-quality meat. Breeding pigs with high hind leg weight can directly increase the yield of high-value lean meat per unit carcass in offspring, aligning with the trend of healthy consumption and significantly improving the economic benefits of pig farming.
[0003] The weight of pig hind legs is a complex quantitative trait regulated by multiple genes. Traditional phenotype-based breeding methods are time-consuming, costly, and difficult to select early, resulting in slow genetic progress. With the continuous advancement of phenotyping technology and the development of high-throughput sequencing technology, using genome-wide association studies (GWAS) to discover molecular loci associated with these hard-to-collect and genetically complex traits and using them for marker-assisted selection can achieve early and precise selection of key genotypes, breaking through the bottleneck of traditional breeding and significantly accelerating the genetic improvement of pig hind leg weight.
[0004] Duroc × Landrace × Large White is a globally leading commercial pig hybridization system. By selecting and breeding pigs with hind leg weight traits in their parent and grandparent core groups, the genetic potential of high-quality breeding pigs can be transferred to the commercial pig population, which can significantly improve the production performance of commercial pigs and increase the economic benefits of pig farming enterprises. Summary of the Invention
[0005] The technical problem to be solved by the present invention is to provide a SNP molecular marker located on chromosome 4 of pigs that is associated with hind leg weight and its application.
[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:
[0007] The SNP molecular marker located on chromosome 4 of pigs and associated with hind leg weight corresponds to the T>C mutation at position 14657616 bp on chromosome 4 in the international pig reference genome Ensembl Sscrofa 11.1 version (hereinafter referred to as g.161 T>C molecular marker).
[0008] The nucleotide sequence of the SNP molecular marker is SEQ ID NO.1, where M in the sequence is T or C.
[0009] M is marked at position 161 in the SEQ ID NO:1 sequence (i.e., the SNP site is the T161-C161 nucleotide mutation marked at position 161 in the SEQ ID NO:1 sequence).
[0010] The primer pairs used to amplify the nucleotide sequences of the above-mentioned SNP molecular markers include primers P001-F and P002-R, whose base sequences are SEQ ID NO.2 and SEQ ID NO.3, respectively.
[0011] The application of the above-mentioned SNP molecular markers or primer pairs in pig assisted breeding.
[0012] Assisted breeding in pigs is a genetic improvement method to increase the weight of the hind legs in pigs.
[0013] The genetic improvement method for pigs involves detecting the aforementioned SNP molecular markers, eliminating individuals with the TC and TT genotypes at the SNP locus, retaining individuals with the CC genotype at the SNP locus as breeding pigs, and progressively increasing the frequency of the dominant allele C at this locus to improve the hind leg weight of offspring pigs.
[0014] The pigs are Duroc, Landrace, Large White, and Landrace crossbred commercial pigs.
[0015] To address the current challenge of genetic improvement of slaughter traits in pig assisted breeding, the inventors discovered a SNP molecular marker on pig chromosome 4 associated with hind leg weight. This SNP corresponds to a T>C mutation at position 14657616 bp on chromosome 4 in the international pig reference genome EnsemblSscrofa 11.1 (hereinafter referred to as the g.161 T>C molecular marker). The nucleotide sequence of this SNP molecular marker is SEQ ID NO.1, where M in the sequence represents either T or C. Studies have shown that polymorphism at this site leads to differences in hind leg weight in pigs. Preliminary association analysis between the genotype and hind leg weight trait was conducted by detecting the base mutation site, verifying its impact on hind leg weight. The inventors ultimately established an efficient and accurate molecular marker-assisted breeding technology, which can be applied to the genetic improvement of hind leg weight in pigs, thereby increasing the hind leg weight of offspring pigs and improving the economic profits of breeding enterprises. Based on this, the inventors established a corresponding genetic improvement method for pigs. By selecting the dominant allele of this SNP, the frequency of the dominant allele can be increased generation by generation, thereby increasing the weight of the pig's hind legs and accelerating the progress of pig genetic improvement, thus effectively improving the economic benefits of breeding pigs. In addition, the inventors also studied and designed primer pairs for amplifying the nucleotide sequence of the SNP molecular marker. Through these primer pairs, slaughter traits can be selected quickly and accurately, accelerating the breeding process. Attached Figure Description
[0016] Figure 1This is a Manhattan plot of genome-wide association analysis (GWAS) on hind leg weight in three-way crossbred commercial pigs on chromosome 4. In the plot: the horizontal axis represents the position of the SNP site on chromosome 4; the vertical axis represents the -logP value.
[0017] Figure 2 This is a graph showing the phenotypic ratios of hind leg weight in pigs of different genotypes.
[0018] Figure 3 This is a diagram of the sequencing results of the target DNA sequence. Detailed Implementation
[0019] Example 1: Screening of molecular markers and correlation analysis with hind leg weight
[0020] (1) Laboratory animals
[0021] The experimental pig herd used in this invention consisted of 382 three-way crossbred commercial pigs from a branch of Guangxi Yangxiang Group Co., Ltd. The pigs had free access to feed and water, and the feeding method and rearing conditions remained consistent throughout the process, following conventional methods.
[0022] (2) Sample collection
[0023] Collect the above-mentioned three-way crossbred pig ear tissue or muscle tissue, soak it in a 75% ethanol solution, and store it in a -20℃ refrigerator for later use.
[0024] (3) Phenotypic data collection
[0025] The phenotypic data of this invention came from the slaughterhouse of a branch of Guangxi Yangxiang Group Co., Ltd., and the slaughter traits of three-way crossbred commercial pigs, including hind leg weight, were determined using the AutoFom Ⅲ ultrasonic image analysis system.
[0026] (4) Pig genome 80K SNP genotyping
[0027] Ear or muscle tissue samples were collected from 382 three-way crossbred commercial pigs. Whole-genome DNA was extracted using the standard phenol-chloroform method. The DNA samples were then quality-assessed using a Nanodrop 2000 / 2000C nucleic acid and protein analyzer, and the DNA concentration and purity parameters (OD260 / 280 and OD260 / 230) of each sample were determined. DNA samples meeting the detection standards were uniformly diluted to a working concentration of 50 ng / μL with sterile deionized water according to the measured concentration. 6 μL of the DNA solution was mixed with 2 μL of loading buffer and loaded onto a 1% (w / v) agarose gel for electrophoresis analysis (150 V, 25 min). Finally, the DNA bands were observed and photographed using a UV spectrophotometer and gel imaging device to assess their clarity and integrity.
[0028] DNA samples were sent to Wuhan Yingzi Gene Technology Co., Ltd. for genotyping of porcine whole genome 80K functional sites using microarray analysis according to the company's standard procedures. Quality control was performed on the 80K microarray genotyping data of all samples using PLINK (V1.9), removing individuals with a detection rate below 90%, SNP detection rate below 90%, and minimum allele frequency less than 0.05, and Hardy's genotype. Weinberg equilibrium significance level is higher than The SNPs were analyzed. Ultimately, 124,265 valid genotype data for SNPs were obtained.
[0029] (5) Genome-wide association analysis (GWAS)
[0030] Genome-wide association analysis (GWA) of hind leg weight was performed using a linear mixture model in GCTA software. The Bonferrini method was used to determine the significance threshold for the association between SNPs and hind leg weight; the significance threshold was calculated as 1 divided by the number of valid SNP sites, i.e., a significance level threshold of 8.05 × 10⁻⁶. -6 This is 1 / 124265 (the number of valid SNPs). The analytical framework is based on the following model:
[0031]
[0032] In the formula: y is the phenotypic value vector; b is the covariate coefficient vector; α is the additive SNP effect vector; g is the random polygenic effect vector; e is the residual effect. X and Z are the corresponding matrices. , , I is the identity matrix, and G is the kinship matrix calculated from all autosomal SNPs.
[0033] GWAS analysis results are as follows Figure 1 As shown. From Figure 1 It was found that a site significantly affecting hind leg weight exists on chromosome 4 of three-way crossbred commercial pigs, and the significantly associated SNP is nucleotide g.161T>C at position 161 of SEQ NO. 1 (P=5.31×10⁻⁶). -06 ).
[0034] (6) Association analysis between different genotypes and hind leg weight phenotype
[0035] According to Table 1 and Figure 2 It was found that the SNP site g.161 T>C of the molecular marker was significantly correlated with the hind leg weight trait (P=5.31×10⁻⁶). -06The study found that pigs with the CC genotype of this molecular marker had higher hind leg weight than those with the TC and TT genotypes, indicating that C is a favorable allele and T is a unfavorable allele. Therefore, in the breeding process, it is necessary to gradually eliminate TT and TC genotype breeding pigs and retain CC genotype breeding pigs to increase the frequency of the C allele at this locus in each generation, thereby increasing the hind leg weight of pigs.
[0036]
[0037] Example 2: Target DNA Sequence Amplification and Sequencing
[0038] (1) Primer design
[0039] The DNA sequence of SEQ ID NO:1 on pig chromosome 4 was downloaded from the Ensembl website (http: / / asia.ensembl.org / index.html). Primers were designed using Primer Premier 6.0 software. The DNA sequences of the designed primers are shown below:
[0040] P001-F: 5'-TCGAAGCCCATTACCAGGAC-3',
[0041] P002-R: 5'-GAACGGCAGCTAAATGGTGG-3';
[0042] (2) PCR amplification
[0043] Prepare a 10μL system:
[0044] DNA sample 1.0 μL, upstream primer 0.3 μL, downstream primer 0.3 μL, PCR mix 5 μL, ddH2O 3.4 μL.
[0045] Configure amplification conditions:
[0046] First stage: Pre-denaturation at 95℃ for 5 minutes;
[0047] The second stage involves denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 45 seconds, for a total of 34 cycles.
[0048] The third stage involves final extension at 72°C for 5 minutes, followed by storage at 4°C.
[0049] (3) DNA sequencing
[0050] DNA sequence sequencing identification: Sequencing was performed at BGI Genomics Co., Ltd. in Shenzhen, with two sequencing reactions (positive and negative). The obtained sequences were compared with the NCBI genome sequence to identify mutations at corresponding SNP sites. The sequencing results are as follows: Figure 3As shown, M is the mutation site, marked with a red underline (the mutated base in parentheses represents the allele mutation). The positions of the designed primer sequences are indicated by bold underlines at the beginning and end of the sequence.
[0051] Example 3: Analysis of the SNP site g.161 T>C effect of molecular markers
[0052] This invention can significantly improve the SNP molecular markers of hind leg weight in pigs. Using these molecular markers for marker-assisted selection can accelerate the breeding process of hind leg weight traits in pigs.
[0053] The effect of the dominant allele CC at SNP site g.161 T>C significantly increased the phenotypic average weight by 0.37 kg and 0.49 kg compared to TC and TT alleles, respectively. By using marker-assisted selection to gradually eliminate individuals with the TC and TT alleles in the breeding pig population, the gene frequency of the dominant allele C can be continuously increased, thereby increasing the hind leg weight of the population and thus increasing the company's profits.
Claims
1. A SNP molecular marker located on chromosome 4 of pigs and associated with hind leg weight, characterized in that... Its SNP site corresponds to the T>C mutation at position 14657616bp on chromosome 4 of the international pig reference genome Ensembl Sscrofa 11.1 version.
2. The SNP molecular marker according to claim 1, characterized in that: The nucleotide sequence of the SNP molecular marker is SEQ ID NO.1, where M in the sequence is T or C.
3. The SNP molecular marker according to claim 1, characterized in that: The position of M in the SEQ ID NO:1 sequence is marked as position 161.
4. Primer pairs for amplifying the nucleotide sequence of the SNP molecular marker of claim 2, characterized in that... It includes primers P001-F and P002-R, whose base sequences are SEQ ID NO.2 and SEQ ID NO.3, respectively.
5. The application of the SNP molecular marker as described in claim 1 or 2 or the primer pair as described in claim 4 in pig-assisted breeding.
6. The application according to claim 5, characterized in that: The aforementioned pig assisted breeding is a genetic improvement method to increase the weight of the hind legs of pigs.
7. A method for genetic improvement of pigs, characterized in that: The SNP molecular markers described in claim 1 or 2 are detected, and individuals with SNP loci of TC genotype and TT genotype are eliminated, while individuals with SNP loci of CC genotype are retained as breeding pigs. The frequency of the dominant allele C at this locus is increased generation by generation, thereby increasing the hind leg weight of offspring pigs.
8. The method for genetic improvement of pigs according to claim 7, characterized in that: The pigs in question are Duroc, Landrace, Large White, and Landrace crossbred commercial pigs.