SNP (Single Nucleotide Polymorphism) molecular marker located on pig chromosome 1 and related to lean meat percentage and application of SNP molecular marker

Abstract

The invention discloses an SNP molecular marker located on a pig chromosome 1 and related to lean meat percentage and application of the SNP molecular marker, the site of the SNP molecular marker is located at the 49th site from the 5'end of a nucleotide sequence shown in SEQ ID NO: 1 and corresponds to the 161507722bp site on the chromosome 1 of the international pig reference genome version 11.1, and the site has Agt; t mutation. The SNP molecular marker provided by the invention is remarkably related to the lean meat percentage character of the pig, the lean meat percentage character can be identified by identifying the genotype of the SNP molecular marker, and the breeding process of the pig can be accelerated and the genetic improvement of the pig can be realized by breeding the pig with the genotype of the SNP molecular marker AA.

Description

Technical Field

[0001] The invention belongs to the field of molecular biotechnology and relates to a SNP molecular marker associated with lean meat rate and application thereof. The SNP molecular marker is located on chromosome 1 of pigs. Background Art

[0002] Lean Meat Percentage (LMP) refers to the percentage of lean meat mass in the entire carcass mass, and is the main indicator for evaluating the quality grade of pig carcasses. With the improvement of living standards, the quality of pork has received more and more attention from consumers. In most parts of my country, pork has traditionally been the main source of meat in the entire meat food structure. Therefore, high-quality lean pigs have great breeding demand and economic value. High fat content in pigs not only endangers the health of consumers, but also causes a lot of economic losses to the breeding industry. Therefore, how to improve the lean meat rate and increase lean meat yield in breeding production has become a focus of attention for breeding companies.

[0003] Lean meat rate is a complex quantitative trait with a relatively complex genetic structure. It is difficult to achieve the desired effect only through phenotypic breeding. It is necessary to improve this type of trait by exploring genetic markers related to growth traits. Summary of the invention

[0004] The purpose of the present invention is to provide a SNP molecular marker located on pig chromosome 1 and related to lean meat rate and its application.

[0005] According to one aspect of the present invention, a SNP molecular marker related to lean meat rate and located on chromosome 1 of pig is provided, wherein the site is located at the 49th position from the 5' end of the nucleotide sequence shown in SEQ ID NO:1, corresponding to the 161507722bp on chromosome 1 of the international pig reference genome version 11.1, and there is an A>T mutation (single base mutation, named: NC010443.5:g.161507722A>T) at the site, the nucleotide type is A or T, and the genotype of the SNP molecular marker is AA, TA or TT.

[0006] The SNP molecular marker provided by the present invention is significantly correlated with the lean meat rate trait of pigs, wherein the lean meat rate of pigs with genotype AA is significantly greater than the lean meat rate of pigs with genotype TA and genotype TT. By identifying the single nucleotide polymorphism of the SNP molecular marker and / or the genotype of the SNP molecular marker, the lean meat rate trait of pigs can be identified, and by breeding pigs with the genotype AA of the SNP molecular marker, the breeding process of pigs can be accelerated to achieve genetic improvement of pigs.

[0007] Therefore, the SNP molecular markers related to lean meat rate on chromosome 1 of pigs provided by the present invention can be applied to:

[0008] (1) Identify the lean meat percentage of pigs;

[0009] (2) preparing products for identifying lean meat percentage traits of pigs;

[0010] (3) Pig genetic improvement, based on the selection of pigs with SNP molecular marker genotype AA to achieve pig genetic improvement;

[0011] (4) Prepare a product for assisting pig genetic improvement, which assists pig genetic improvement based on identifying the genotype of pig SNP molecular markers.

[0012] In some embodiments, the pig is preferably American Duroc and its synthetic strains.

[0013] According to another aspect of the present invention, a primer pair is provided, which can specifically amplify an amplified fragment containing the 49th single nucleotide polymorphism from the 5' end of the nucleotide sequence shown in SEQ ID NO:1, the nucleotide sequence of the upstream primer is shown in SEQ ID NO:2, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:3.

[0014] The primer pair provided by the present invention can specifically amplify an amplified fragment containing the SNP molecular marker related to lean meat rate located on chromosome 1 of pig provided by the present invention, and can be used to identify whether the 49th single nucleotide from the 5' end in the nucleotide sequence shown in SEQ ID NO: 1 is A or T. Therefore, the primer pair provided by the present invention can be used to identify the genotype of the SNP molecular marker related to lean meat rate located on chromosome 1 of pig and / or prepare a product for identifying the genotype related to lean meat rate located on chromosome 1 of pig.

[0015] According to a third aspect of the present invention, a kit is provided, comprising a primer pair having nucleotide sequences as shown in SEQ ID NO:2 and SEQ ID NO:3.

[0016] Applications of the primer pairs and kits provided by the present invention include but are not limited to:

[0017] (1) To identify the genotype of the SNP molecular markers on chromosome 1 associated with lean meat percentage;

[0018] (2) preparing a product for identifying the genotype of a SNP molecular marker located on chromosome 1 of pigs and associated with lean meat percentage;

[0019] (3) Identifying the lean meat percentage trait of pigs, based on identifying the genotype of a SNP molecular marker associated with lean meat percentage located on chromosome 1 of pigs;

[0020] (4) preparing a product for identifying the lean meat percentage trait of pigs, wherein the product is based on identifying the genotype of a SNP molecular marker associated with lean meat percentage located on chromosome 1 of pigs to identify the lean meat percentage trait of pigs;

[0021] (5) Genetic improvement of pigs, based on breeding pigs with AA genotype of SNP molecular markers related to lean meat rate located on chromosome 1;

[0022] (6) Prepare a product for assisting pig genetic improvement, wherein the product assists pig genetic improvement based on identifying the genotype of a SNP molecular marker located on chromosome 1 of the pig and associated with lean meat rate.

[0023] In some embodiments, the kit provided by the present invention may also include: dNTPs, DNA polymerase, Mg 2+ The components of conventional PCR reaction systems such as PCR reaction buffer and PCR reaction buffer can be directly referenced or used for the relevant components of conventional commercially available PCR amplification kits.

[0024] According to a fourth aspect of the present invention, there is provided a method for genetic improvement of pigs, comprising the following steps:

[0025] (1) Determine the genotype of the SNP molecular marker associated with lean meat percentage located on chromosome 1 of pigs;

[0026] (2) Select individuals with the AA genotype of the SNP molecular marker and eliminate individuals with the TA and TT genotypes, thereby increasing the frequency of the allele A at the SNP molecular marker site generation by generation; thereby improving the lean meat rate of future generations of pigs and increasing the meat production of future generations of pigs.

[0027] In some embodiments, in step (1), the method for determining the genotype of a SNP molecular marker associated with lean meat rate located on chromosome 1 of a pig may include the following steps:

[0028] The whole genome DNA of the pig is extracted, PCR amplification is performed using the primer pair with nucleotide sequences such as SEQ ID NO: 2 and SEQ ID NO: 3, the amplified product is sequenced, and the genotype of the SNP molecular marker related to the lean meat rate located on chromosome 1 of the pig is determined based on the sequencing results.

[0029] In some embodiments, the pig is an American Duroc or its synthetic strains.

[0030] Compared with the prior art, the beneficial effects of the present invention include:

[0031] (1) The present invention provides a SNP molecular marker located on the nucleotide sequence of chromosome 1 of pigs and associated with the lean meat percentage of pigs, and verifies its effect on the lean meat percentage trait of pigs, which is helpful to establish a molecular marker-assisted selection breeding technology for rapid improvement of the lean meat percentage trait of pigs, improve the breeding process of Duroc and its synthetic lines, so as to meet the needs of the breeding pig market, increase the price of breeding pigs, and reduce breeding costs.

[0032] (2) The present invention provides a primer pair that can be used to identify a SNP molecular marker located on chromosome 1 of pigs and associated with lean meat rate. Through this primer pair, an efficient and accurate molecular marker-assisted breeding technology can be established to quickly and accurately select traits and accelerate the breeding process. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the Manhattan plot of genome-wide association analysis (GWAS) of lean meat rate on chromosome 1 of American Duroc. The horizontal axis represents the distribution of chromosome 1 of pigs. The vertical axis represents the statistical significance - log P value.

[0034] Figure 2 This is an analysis chart of the phenotypic proportion results of the lean meat percentage trait of pigs with different genotypes. DETAILED DESCRIPTION

[0035] The present invention is further described in detail below in conjunction with the embodiments. The examples are only for explanation and are not intended to limit the present invention in any way. Unless otherwise specified, the raw materials and reagents used in the examples are conventional products that can be obtained commercially; the experimental methods in the examples where specific conditions are not specified are usually carried out according to conventional conditions in the art or according to the conditions recommended by the manufacturer.

[0036] Example 1 Identification and verification of SNP sites associated with lean meat rate

[0037] (1) Experimental pig herd

[0038] The experimental pig population used in the present invention is 3760 purebred American Duroc pigs of the pig breeding experimental department of Wen's Food Group Co., Ltd., which is the core group of the pig breeding department, and the herd genealogy is recorded in detail. During the breeding process, the pigs are free to eat and drink water, and the entire feeding method, breeding conditions, etc. are always consistent, which is a conventional method.

[0039] (2) Lean meat percentage measurement method

[0040] The present invention estimates lean meat percentage (LMP) based on backfat thickness (BF) and eye muscle depth (LMD), and the specific method is as follows:

[0041] When the live weight of pigs reached 100 ± 5 kg, the back fat thickness and eye muscle depth between the 10th and 11th ribs were measured using an Aloka 500V SSD B-type ultrasonic instrument (Corometrics Medical Systems, USA), and the lean meat percentage was estimated according to the following formula:

[0042] LMP(%)=61.21920-0.77665*BF+0.15239*LMD

[0043] (3) Extraction of pig genomic DNA

[0044] The ear tissues or tail-docked tissues of the above 3760 American Duroc pigs were collected, immersed in a 75% by volume ethanol solution, and stored in a -20°C refrigerator for later use.

[0045] The whole genome DNA of pigs was extracted by the standard phenol-chloroform method, and the concentration and OD ratio (OD260 / 280, OD260 / 230) of each sample were accurately determined by Nanodrop2000 / 2000C nucleic acid protein detector. The qualified DNA samples were tested by NanoDrop2000 / 2000C nucleic acid protein detector, and the DNA was diluted to about 50ng / μL according to the tested concentration. Then 6μL of the extracted DNA sample to be tested was mixed with 2μL Loading Buffer, loaded into a 1% agarose gel with a mass volume ratio, electrophoresed at 150V for 25min, and observed and photographed under a UV spectrophotometer and gel imaging equipment to observe the integrity of the DNA.

[0046] (4) Pig whole genome 50K SNP genotype detection

[0047] The DNA samples were sent to Neogen Biotech (Shanghai) Co., Ltd., and the genotype was determined using the porcine whole genome 50K SNP chip (Illumina, USA) on the Illumina Beadstration platform according to the company's standard procedures.

[0048] PLINK v1.9 software was used to perform quality control on the 50K chip scanning typing data of all samples. Individuals with a detection rate lower than 90%, a genotype missing rate greater than 0.1, a family Mendelian error rate higher than 0.1, a minimum allele frequency lower than 0.05, and a Hardy-Weinberg equilibrium significance level lower than 10 were excluded. -6 SNPs.

[0049] Then, the SWIM database (https: / / swim.scau.pigselection.com / swim / ) was used to fill in the genotypes, and finally valid genotype data of 561,032 SNPs were obtained.

[0050] (5) Genome-wide association study (GWAS)

[0051] In order to eliminate the population stratification effect, the present invention uses the linear mixed model in the GEMMA software to perform GWAS analysis, adds kinship to the model to correct the population structure, and uses the similarity of the genome between individuals in the analysis model to correct the stratification effect. Because the Bonferroni correction method is too strict, and the present invention has a large number of hypotheses to be tested, the significance level after correction will be significantly reduced, which will cause many results to fail the significance test. Therefore, the present invention selects a higher significance level of 1e-5 (1×10 -5 ) as the threshold to avoid overly strict correction, making it easier to find significant results.

[0052] GWAS analysis results Figure 1 shown.

[0053] from Figure 1 It can be seen that there is a SNP locus in chromosome 1 of American Duroc that significantly affects lean meat rate. The strongest associated SNP is NC 010443.5:g.161507722A>T (P=5.69×10 -7 ), i.e., the 49th nucleotide from the 5' end in SEQ NO.1, corresponds to the A>T mutation at 161507722bp on chromosome 1 of the international pig reference genome version 11.1.

[0054] (6) Analyze the association between different genotypes and lean meat percentage phenotypes to verify the effect of SNP loci on lean meat percentage traits

[0055] The results are shown in Table 1. Figure 2 As shown in Table 1, Figure 2 It can be seen that:

[0056] (1) The molecular marker SNP site NC 010443.5:g.161507722A>T was extremely significantly correlated with the lean meat percentage trait (P<0.001), indicating that this molecular marker significantly affects the lean meat percentage trait of pigs. Through auxiliary selection of this SNP site in pigs, the breeding process of pigs with lean meat percentage trait can be accelerated.

[0057] (2) The lean meat percentage of pigs with AA SNP molecular marker genotype is significantly higher than that of TA and TT SNPs, and the lean meat percentage of TA is higher than that of TT SNPs, indicating that allele T is not conducive to the lean meat percentage of breeding pigs, so AA breeding pigs should be retained first. Lean meat percentage is an important indicator for measuring carcass quality grade, and improving the lean meat percentage of pigs is conducive to increasing economic benefits. Therefore, gradually retaining breeding pigs with AA genotype in breeding to increase the frequency of allele A at this locus generation by generation can significantly improve the lean meat percentage trait of breeding pigs and bring greater economic benefits to the enterprise.

[0058] Table 1 Correlation analysis between SNP locus NC 010443.5:g.161507722A>T and lean meat rate

[0059]

[0060] (7) Effect analysis

[0061] The present invention provides a SNP molecular marker significantly associated with the Duroc lean meat rate trait, and using the SNP molecular marker for marker-assisted selection can accelerate the lean meat rate breeding process of Duroc breeding pigs. Through molecular marker-assisted selection, if all TT-type individuals with molecular markers that affect the lean meat rate trait of pigs are selected into AA-type individuals, the lean meat rate trait of Duroc breeding pigs will be increased by 0.34%.

[0062] Example 2 Method for genetic improvement of pigs

[0063] The target fragment containing the SNP site significantly associated with the lean meat rate trait of American Duroc is a 128bp nucleotide sequence in chromosome 1, the specific sequence of which is shown in SEQ ID NO:1, and the primer pair for PCR amplification is shown in SEQ ID NO:2 and SEQ ID NO:3.

[0064] SEQ ID NO:1

[0065]

[0066] The W marked in the sequence is the mutation site, and A or T is the allele mutation; the bold at the beginning and end of the sequence is the primer sequence binding position.

[0067] Upstream primer primer-F: 5'-TCATCTGCCAGTAGACATCT-3' (SEQ ID NO: 2);

[0068] Downstream primer primer-R: 5′-ATGACTCAGCAATTCCACTT-3′ (SEQ ID NO: 3).

[0069] The method for genetic improvement of pigs includes the following steps:

[0070] S1. Determine the genotype of SNP molecular markers associated with pig lean meat rate

[0071] (1) Collect pig ear tissue or piglet tail tissue, extract the whole genome DNA of the pig according to the standard phenol-chloroform method, and then perform quality inspection and concentration measurement on the extracted DNA.

[0072] (2) PCR amplification

[0073] Prepare a 10 μL system, including: 1 μL DNA sample, 0.3 μL upstream primer, 0.3 μL downstream primer, 5 μL PCR mix, ddH 2 O 3.4μL; PCR mix can be commercially available 2×TaqPCR Star Mix (Dye) (manufacturer: GenStar, catalog number: A012-101), which includes dNTPs, DNA polymerase, Mg 2+ Components of conventional PCR reaction systems such as PCR reaction buffer.

[0074] PCR reaction program: 94°C pre-denaturation for 5 min, 94°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 45 s, a total of 35 cycles, and a final extension at 72°C for 5 min.

[0075] (3) DNA sequence identification

[0076] The PCR amplification products were sequenced, and the gene fragments were tested for both positive and negative reactions. The genotype of the pig SNP site NC 010443.5:g.161507722A>T was determined based on the sequencing results.

[0077] S2. Select pigs with AA genotype at the SNP site as parents for breeding, and increase the frequency of allele A at this site generation by generation.

[0078] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements can be made without departing from the creative concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A SNP molecular marker related to lean meat rate located on chromosome 1 of pigs, characterized in that: The site of the SNP molecular marker is located at 161507722bp on chromosome 1 of the international pig reference genome version 11.1, the nucleotide type of the site is A or T, and the genotype of the SNP molecular marker is AA, TA or TT; the single nucleotide polymorphism of the SNP molecular marker site affects the lean meat rate trait, wherein the lean meat rate of pigs with the SNP molecular marker genotype of AA is greater than the lean meat rate of pigs with the genotype of TA and the genotype of TT.

2. The SNP molecular marker according to claim 1, characterized in that: The pigs are American Duroc and its synthetic strain.

3. The use of SNP molecular markers according to claim 1 or 2, characterized in that: The applications include: (1) Identify the lean meat percentage of pigs; (2) preparing products for identifying lean meat percentage traits of pigs; (3) Genetic improvement of pigs, based on breeding pigs with AA genotype of SNP molecular markers related to lean meat rate on chromosome 1; (4) Prepare a product for assisting pig genetic improvement, wherein the product assists pig genetic improvement based on identifying the genotype of a SNP molecular marker located on chromosome 1 of the pig and associated with lean meat rate.

4. A primer pair, characterized in that The primer pair specifically amplifies an amplified fragment containing the 49th single nucleotide polymorphism from the 5' end of the nucleotide sequence shown in SEQ ID NO: 1; wherein, The 49th position from the 5' end of the nucleotide sequence shown in SEQ ID NO:1 is A or T, and the corresponding genotype of the SNP molecular marker related to lean meat rate located on chromosome 1 of pig is AA, TA or TT, and the lean meat rate of pigs with genotype AA is greater than the lean meat rate of pigs with genotype TA and genotype TT; In the primer pair, the nucleotide sequence of the upstream primer is shown as SEQ ID NO:2, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO:

3.

5. The use of the primer pair according to claim 4, characterized in that: The applications include: (1) To identify the genotype of the SNP molecular markers on chromosome 1 associated with lean meat percentage; (2) preparing a product for identifying the genotype of a SNP molecular marker located on chromosome 1 of pigs and associated with lean meat percentage; (3) Identifying the lean meat percentage trait of pigs, based on identifying the genotype of a SNP molecular marker associated with lean meat percentage located on chromosome 1 of pigs; (4) preparing a product for identifying the lean meat percentage trait of pigs, wherein the product is based on identifying the genotype of a SNP molecular marker associated with lean meat percentage located on chromosome 1 of pigs to achieve identification of the lean meat percentage trait of pigs; (5) Genetic improvement of pigs, based on breeding pigs with AA genotype of SNP molecular markers related to lean meat rate located on chromosome 1; (6) Prepare a product for assisting pig genetic improvement, wherein the product assists pig genetic improvement based on identifying the genotype of a SNP molecular marker located on chromosome 1 of the pig and associated with lean meat rate.

6. A kit, characterized in that Its composition includes the primer pair described in claim 4.

7. Use of the kit according to claim 6, characterized in that, The applications include: (1) To identify the genotype of the SNP molecular markers on chromosome 1 associated with lean meat percentage; (2) preparing a product for identifying the genotype of a SNP molecular marker located on chromosome 1 of pigs and associated with lean meat percentage; (3) Identifying the lean meat percentage trait of pigs, based on identifying the genotype of a SNP molecular marker associated with lean meat percentage located on chromosome 1 of pigs; (4) preparing a product for identifying the lean meat percentage trait of pigs, wherein the product is based on identifying the genotype of a SNP molecular marker associated with lean meat percentage located on chromosome 1 of pigs to achieve identification of the lean meat percentage trait of pigs; (5) Genetic improvement of pigs, based on breeding pigs with AA genotype of SNP molecular markers related to lean meat rate located on chromosome 1; (6) Prepare a product for assisting pig genetic improvement, wherein the product assists pig genetic improvement based on identifying the genotype of a SNP molecular marker located on chromosome 1 of the pig and associated with lean meat rate.

8. A method for genetic improvement of pigs, characterized in that: The steps include: (1) Determine the genotype of the SNP molecular marker associated with lean meat percentage located on chromosome 1 of pigs; (2) Select individuals with the AA genotype of the SNP molecular marker, eliminate individuals with the TA and TT genotypes, and increase the frequency of allele A at the SNP molecular marker site generation by generation; The site of the SNP molecular marker associated with lean meat rate on pig chromosome 1 is located at the 49th position from the 5' end of the nucleotide sequence shown in SEQ ID NO:1, corresponding to the 161507722bp position on chromosome 1 of the international pig reference genome version 11.

1. The nucleotide type of this site is A or T, and the genotype is AA, TA or TT.

9. The method for genetic improvement of pigs according to claim 8, characterized in that: In step (1), the method for determining the genotype of the SNP molecular marker associated with lean meat rate located on chromosome 1 of the pig comprises the following steps: The whole genome DNA of the pig is extracted, PCR amplification is performed using the primer pair with nucleotide sequences such as SEQ ID NO: 2 and SEQ ID NO: 3, the amplified product is sequenced, and the genotype of the SNP molecular marker related to the lean meat rate located on chromosome 1 of the pig is determined based on the sequencing results.

10. The method for genetic improvement of pigs according to claim 8 or 9, characterized in that: The pigs are American Duroc and its synthetic strain.

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