SNP (Single Nucleotide Polymorphism) molecular marker located on pig chromosome 4 and related to waist meat thickness and application of SNP molecular marker

Abstract

The invention discloses an SNP molecular marker located on a pig chromosome 4 and related to the thickness of waist meat. The SNP site of the SNP molecular marker corresponds to Ggt at the position of 16572687bp of a chromosome 4 of a reference sequence of an international pig genome version 11.1; a mutation; the nucleotide sequence of the SNP molecular marker is SEQ ID NO.1, wherein M in the sequence is G or A. Researches show that the polymorphism of the basic group at the site leads to the difference of the thickness of the waist meat. Accordingly, the inventor establishes a corresponding pig genetic improvement method, and by optimizing the dominant alleles of the SNP, the frequency of the dominant alleles can be increased generation by generation, the thickness of pig waist meat can be increased, and the progress of pig genetic improvement can be accelerated, so that the economic benefit of breeding pig breeding can be effectively improved. Besides, the inventor also researches and designs a primer pair for amplifying the nucleotide sequence of the SNP molecular marker, and the carcass traits can be quickly and accurately bred through the primer pair, so that the breeding process is accelerated.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biotechnology and molecular marker technology, and in particular relates to a SNP molecular marker located on pig chromosome 4 and associated with loin thickness and its application. Background Art

[0002] According to public data released by the National Bureau of Statistics in 2024, my country's annual pig output exceeded 700 million head, and pork consumption continued to account for over 60% of total meat consumption. The pig industry holds a fundamental and strategic position in my country's national economy. It is not only a key pillar for ensuring national food security, but also a core industry for meeting basic livelihood needs and maintaining social stability. As people's living standards improve, consumers' demands for pork quality are increasing. In this context, meat quality traits, as key production indicators in the pig farming system, and the success of genetic improvement impacts the industry's economic profitability. The current core farming objectives are to meet consumer demand for both quantity and taste, effectively reducing carcass fat content and increasing lean meat percentage. Loin muscle thickness, a key metric, has attracted attention due to its significant positive correlation with lean meat percentage. This metric quantifies the thickness of the loin muscle; thicker loin muscles generally indicate higher lean meat yield potential.

[0003] With the iterative upgrades of high-throughput sequencing technology and the large-scale application of high-density single nucleotide polymorphism (SNP) chips, the cost of whole-genome testing in pigs continues to decrease, laying the foundation for large-scale genetic analysis of slaughter traits. Genome-wide association study (GWAS) technology can accurately locate functional loci and major genes that regulate loin thickness by systematically scanning the entire genome for statistical associations between SNPs and economically important phenotypes, thereby systematically analyzing the genetic mechanisms of loin thickness. Applying these key quantitative trait loci that can improve pig loin thickness to marker-assisted selection can significantly improve the efficiency of genetic improvement of pig loin thickness. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a SNP molecular marker located on pig chromosome 4 and associated with loin thickness and its application.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] The SNP molecular marker located on the pig chromosome 4 and related to loin eye muscle thickness, the SNP site corresponds to the G>A mutation at the position of 16572687bp on the chromosome 4 of the international pig genome reference sequence version 11.1.

[0007] The nucleotide sequence of the SNP molecular marker is SEQ ID NO. 1, wherein M in the sequence is G or A.

[0008] The SNP molecular marker according to claim 1, wherein M is at the position of 143 in the sequence of SEQ ID NO: 1 (i.e. the site of the SNP molecular marker is the nucleotide mutation of A143-G143 at the position of 143 in the sequence of SEQ ID NO: 1).

[0009] The primer pair for amplifying the nucleotide sequence of the above-mentioned SNP molecular marker, comprising primer P001-F and primer P002-R, and the base sequences thereof are SEQ ID NO. 2 and SEQ ID NO. 3.

[0010] The above-mentioned SNP molecular marker or primer pair is applied in pig assisted breeding.

[0011] The pig assisted breeding is a genetic improvement for increasing the loin eye muscle thickness of pigs.

[0012] The genetic improvement method of pigs, detecting the above-mentioned SNP molecular marker, eliminating the individuals with SNP site of GA genotype and AA genotype, and keeping the individuals with SNP site of GG genotype as breeding pigs, and increasing the frequency of the dominant allele G of the site generation by generation, so as to increase the loin eye muscle thickness of the offspring pigs.

[0013] The pig is Duroc × Landrace × Yorkshire commercial pig.

[0014] In view of the genetic improvement of the carcass traits in the current pig assisted breeding, the inventors find that the SNP molecular marker related to the loin thickness on the chromosome 4 of pig, the SNP site corresponds to the G>A mutation at the position of 16572687 bp of the chromosome 4 in the international pig genome 11.1 version reference sequence; the nucleotide sequence of the SNP molecular marker is SEQ ID NO. 1, wherein M in the sequence is G or A. Studies have shown that the polymorphism of the base at the site leads to the difference in the loin thickness. By detecting the base mutation site, the correlation between the genotype and the loin thickness trait of the pig is analyzed, and the effect of the SNP site on the loin thickness of the pig is verified. The inventors finally establish a high-efficiency and accurate molecular marker assisted breeding technology, which is applied to the genetic improvement of the loin thickness of the pig, so as to improve the loin thickness of the offspring pig, increase the lean meat rate, and improve the economic profit of the enterprise. Accordingly, the inventors establish a genetic improvement method of the pig, and by selecting the dominant allele of the SNP, the frequency of the dominant allele can be increased generation by generation, the loin thickness of the pig is improved, and the economic benefit of the pig breeding is effectively improved. In addition, the inventors also design a primer pair for amplifying the nucleotide sequence of the SNP molecular marker, and by the primer pair, the carcass traits can be quickly and accurately selected, and the breeding process is accelerated. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 is a Manhattan plot of the whole genome association analysis (GWAS) of the three commercial pigs on the chromosome 4 about the loin thickness, in which: the horizontal coordinate represents the position of the SNP site on the chromosome 4; and the vertical coordinate represents the -logP value.

[0016] Figure 2 is a phenotypic proportion result analysis diagram of the loin thickness of the pigs with different genotypes, in which: the horizontal coordinate represents the genotype category of the SNP site g.143 G>A; and the vertical coordinate represents the phenotypic value.

[0017] Figure 3 is a DNA sequence sequencing result diagram. DETAILED DESCRIPTION

[0018] Example 1: Screening of the molecular marker and correlation analysis with the loin thickness of the pig

[0019] (1) Experimental animals

[0020] The experimental pig population used in the present application is 506 three commercial pigs of the Pig Breeding Company of Guangxi Yangxiang Group Co., Ltd. The pigs are free to eat and drink, and the whole feeding method, feeding conditions and the like are always consistent with the conventional method.

[0021] (2) Sample collection

[0022] The muscle tissue and ear tissue of the three-way commercial pigs were collected, immersed in an ethanol solution with a volume fraction of 75%, and stored in a refrigerator at -20°C for standby use.

[0023] (3) Phenotype data collection

[0024] The phenotype data of the present application were collected from a slaughterhouse of a branch of Guangxi Yang Xiang Group Co., Ltd. The slaughter traits of the commercial pigs, including loin thickness, were determined by an AutoFom III ultrasonic image analysis system.

[0025] (4) 80K SNP genotyping of the whole genome of pigs

[0026] The ear tissue or muscle tissue samples of the above-mentioned 506 three-way commercial pigs were collected, and the whole genome DNA was extracted by a standard phenol-chloroform method. Then, the DNA samples were quality evaluated by a Nanodrop 2000 / 2000C nucleic acid protein detector to determine the DNA concentration and purity parameters (OD260 / 280 and OD260 / 230) of each sample. The DNA samples meeting the detection standard were uniformly diluted to a working concentration of 50 ng / μL with sterile deionized water according to the determined concentration. 6 μL of the DNA solution was mixed with 2 μL of Loading Buffer, and then loaded into an agarose gel with a mass / volume ratio of 1% for electrophoresis analysis (150 V, 25 min). Finally, the band clarity and integrity of the DNA were observed and photographed by an ultraviolet spectrophotometer and a gel imaging device.

[0027] The DNA samples were sent to Wuhan Shadow Gene Technology Co., Ltd. for genotype determination of the 80K functional site chip of the whole genome of pigs according to the standard process of the company. PLINK (V1.9) was used to perform quality control on the 80K chip scanning genotyping data of all samples, and SNPs with a detection rate of less than 90%, a minimum allele frequency of less than 0.05, and a Hardy-Weinberg equilibrium significance level higher than 1e-6 were excluded. Finally, 124265 effective genotype data of SNPs were obtained.

[0028] (5) Whole genome association (GWAS) analysis

[0029] The linear mixed model in the GCTA software was used to perform whole genome association analysis on the loin thickness. After data arrangement and invalid data exclusion, and based on the three standard deviation principle, abnormal values were excluded, and finally 384 effective individuals participated in the GWAS analysis. The Bonferrini method was used to determine the significance threshold of the association degree of SNPs and loin thickness, and the significant threshold was 1 divided by the number of effective SNP sites, i.e. the significant level threshold was 8.05x10 -6 ​​, which is 1 / 124,265 (the number of effective SNPs). The analysis framework is based on the following model:

[0030]

[0031] Where: y is the phenotypic value vector; β 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.

[0032] GWAS analysis results are as follows Figure 1 As shown. Figure 1 It can be seen that there is a site on chromosome 4 of Sanyuan commercial pigs that significantly affects loin thickness. The significantly associated SNP is the 143rd nucleotide g in SEQ NO.1. 143 G>A (P=1.55×10 -06 ).

[0033] (6) Correlation analysis between different genotypes and loin thickness phenotypes

[0034] According to Table 1, the molecular marker SNP site g.143 G>A was significantly correlated with loin thickness trait (P=1.55×10 -06 ), where GG pigs carrying this molecular marker had thicker loins than AG and AA pigs. This result indicates that G is a favorable allele for this molecular marker, while A is an unfavorable allele. Therefore, during breeding, it is necessary to gradually eliminate AA and AG genotypes and retain GG genotypes to increase the frequency of the G allele at this locus with each generation, thereby improving loin thickness.

[0035]

[0036] Example 2 Target DNA sequence amplification and sequencing

[0037] (1) Primer design

[0038] The DNA sequence of SEQ ID NO: 1 on porcine chromosome 4 was downloaded from the Ensembl website (http: / / asia.ensembl.org / index.html). Primers were designed using the primer design software Primer Premier 6.0. The DNA sequences of the designed primers are shown below:

[0039] P001-F: 5'-TCCCATGACAGTCCCTCTCC-3',

[0040] P002-R: 5'-TGGGAATGTTCGTGGTCCATT-3';

[0041] (2) PCR amplification

[0042] A 10μL PCR system was prepared containing 1.0μL DNA template, 0.3μL each of P001-F and P002-R, 5μL PCR mix, and 3.4μL ddH2O. The PCR reaction conditions were as follows: first stage: initial denaturation at 95°C for 5 min; second stage: denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 45 s, for 34 cycles; third stage: final extension at 72°C for 5 min, followed by storage at 4°C.

[0043] (3) DNA sequence determination

[0044] DNA sequencing identification: Sequencing was performed at Shenzhen BGI Genomics Co., Ltd., and the gene fragments were tested for both positive and negative reactions. The measured sequence was compared with the NCBI genome sequence to obtain the mutation of the corresponding SNP site. The sequencing results are as follows Figure 3 As shown, the marked M is the mutation site, which is underlined in red (the mutated base in brackets is the allele mutation), and the bold underlines at the beginning and end of the sequence are the positions of the designed primer sequences.

[0045] Example 3 Analysis of the molecular marker SNP site g.143 G>A effect

[0046] The present invention can significantly improve the SNP molecular marker of the thickness of the three-way commercial pig loin, and using the molecular marker for marker-assisted selection can accelerate the breeding process of the three-way commercial pig loin thickness trait.

[0047] The effect of the dominant allele GG at SNP locus g.143 G>A significantly increases the average phenotypic values ​​of AG and AA by 1.73 and 9.09, respectively. By gradually eliminating individuals with AG and AA phenotypes from the three-way commercial pig population through marker-assisted selection, the frequency of the dominant allele G can be continuously increased, thereby increasing loin thickness and thus increasing corporate profits.

Claims

1. A SNP molecular marker located on porcine chromosome 4 and associated with loin thickness, characterized in that Its SNP site corresponds to the G>A mutation at position 16572687bp on chromosome 4 of the International Porcine Genome Version 11.1 reference sequence.

2. The SNP molecular marker according to claim 1, wherein: The nucleotide sequence of the SNP molecular marker is SEQ ID NO.1, wherein M in the sequence is G or A.

3. The SNP molecular marker according to claim 1, wherein: The M is marked at position 143 in the SEQ ID NO: 1 sequence.

4. A primer pair for amplifying the nucleotide sequence of the SNP molecular marker according to claim 2, characterized in that The primer comprises primer P001-F and primer P002-R, and the base sequences thereof are SEQ ID NO.2 and SEQ ID NO.

3.

5. Use of the SNP molecular marker according to claim 1 or 2 or the primer pair according to claim 4 in pig assisted breeding.

6. The use according to claim 5, characterized in that: The pig assisted breeding is a genetic improvement to increase the thickness of pork loin.

7. A method for genetic improvement of pigs, characterized by: Detect the SNP molecular markers according to claim 1 or 2, eliminate individuals with GA genotype and AA genotype at the SNP site, retain individuals with GG genotype at the SNP site as breeding pigs, and increase the frequency of the dominant allele G at the site generation by generation, thereby increasing the thickness of the pork loin of the offspring.

8. The method for genetic improvement of pigs according to claim 7, characterized in that: The pig is a Duchangda three-way commercial pig.

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