SNP (Single Nucleotide Polymorphism) molecular marker related to number of live piglets on pig chromosome 1 and application of SNP molecular marker

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) molecular marker related to the number of live piglets on a No.1 chromosome of a pig. SNP loci of the SNP molecular marker correspond to 13th, 051th and 624th Ggt on a No.1 chromosome of a reference sequence of an international pig genome version 11.1; a mutation is carried out, and the polymorphism of the base at the site leads to the difference of live piglets born by pigs. By verifying the influence effect of the molecular marker on the number of live piglets of sows, an efficient and accurate molecular marker-assisted breeding technology is finally established, and the molecular marker is applied to genetic improvement of increasing the number of live piglets of boars, so that the reproductive performance of offspring pigs is improved, and the economic profit of enterprises is increased. Accordingly, a corresponding genetic improvement method is established, and by optimizing the dominant alleles of the SNP, the frequency of the dominant alleles can be increased generation by generation, the number of live piglets born by sows can be increased, and the progress of genetic improvement of pigs can be accelerated, so that the economic benefit of breeding of boars can be effectively improved. In addition, the inventor also researches and designs a primer pair for amplifying the nucleotide sequence of the SNP molecular marker, and through the primer pair, breeding traits can be rapidly and accurately bred, and the breeding process is accelerated.

Description

Technical Field

[0001] The present invention belongs to the fields of molecular biotechnology and molecular marker technology, and particularly relates to an SNP molecular marker related to the number of live piglets on porcine chromosome 1 and its application. Background Art

[0002] According to the data of the National Bureau of Statistics in 2024, the annual slaughter volume of live pigs in China exceeds 700 million, and the consumption of pork continues to account for more than 60% of the total meat consumption, fully demonstrating the important strategic position of the live pig industry as a basic industry of the national economy. The live pig industry is not only an important pillar of the national food security system, but also a key industrial support for maintaining the basic needs of people's livelihood and social stability. As a key production index in the live pig breeding system, the genetic improvement effect of reproductive traits directly determines the economic benefits of the industry. Among them, the number of live piglets born is the core parameter to measure the reproductive efficiency of sows. A higher number of live piglets can directly increase the annual slaughter volume of sows, spread the fixed breeding costs, and increase the income of breeding enterprises. The genetic improvement level of the number of live piglets has a significant impact on the unit production capacity and operating efficiency of breeding enterprises.

[0003] The genetic progress of the number of live piglets born has been in a low-speed state for a long time. On the one hand, the number of live piglets born is a low-heritability (h 2 ≈0.10) trait controlled by minor polygenes, which is easily affected by various factors such as environment and parity, greatly restricting the breeding efficiency; on the other hand, the high-fertility breeding pig resources have long relied on imports. Under this background, establishing a molecular breeding system based on genomic selection and breaking through the technical barriers of genetic improvement of reproductive traits have become the only way to realize the revitalization of the live pig seed industry and ensure national germplasm security.

[0004] With the iterative upgrade of high-throughput sequencing technology and the large-scale application of high-density single nucleotide polymorphism (SNP) chips, the cost of whole-genome detection of live pigs has been continuously reduced, laying a foundation for large-scale genetic analysis of reproductive traits. The breakthrough development of genome-wide association study (GWAS) technology has provided a new path for revealing the molecular genetic mechanism of reproductive traits. By integrating high-throughput SNP chips and phenotypic data, GWAS can accurately locate the functional loci and major genes related to the number of live piglets born, and then systematically analyze the genetic mechanism of the number of live piglets born trait and accurately locate the key quantitative trait loci. Applying the loci that can increase the number of live piglets born by sows to marker-assisted selection can significantly improve the annual genetic progress of the number of live piglets born. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide an SNP molecular marker related to the number of live piglets on porcine chromosome 1 and its application.

[0006] To solve the above technical problems, the present invention adopts the following technical solutions:

[0007] An SNP molecular marker related to the number of live piglets on Sus scrofa chromosome 1, where the SNP locus corresponds to a G>A mutation at position 13,051,624 on chromosome 1 of the Sus scrofa genome reference sequence version 11.1.

[0008] The nucleotide sequence of the SNP molecular marker is SEQ ID NO.1, where M in the sequence is G or A (i.e., the nucleotide mutation of G153-A153 at position 153, and the polymorphism of the base at this site leads to differences in the number of live piglets produced by pigs).

[0009] A primer pair for amplifying the nucleotide sequence of the SNP molecular marker, including primer P001-F and primer P002-R, whose base sequences are SEQ ID NO.2 and SEQ ID NO.3.

[0010] The application of the SNP molecular marker or primer pair related to the number of live piglets on Sus scrofa chromosome 1 in pig assisted breeding.

[0011] Pig assisted breeding is a genetic improvement to increase the number of live piglets born to breeding pigs.

[0012] A genetic improvement method for pigs, detecting the above SNP molecular marker, eliminating individuals with the AG genotype and AA genotype at the SNP locus, retaining individuals with the GG genotype at the SNP locus as breeding pigs, and gradually increasing the frequency of the dominant allele G at this locus, thereby increasing the number of live piglets born to the offspring pigs.

[0013] The pig is a Large White pig.

[0014] In view of the problems existing in the current genetic improvement technology for the number of live piglets born to sows, the inventors discovered an SNP molecular marker related to the number of live piglets on Sus scrofa chromosome 1. The SNP locus corresponds to a G>A mutation at position 13,051,624 on chromosome 1 of the reference sequence of the international pig genome version 11.1. The nucleotide sequence of the SNP molecular marker is SEQ ID NO.1, where M in the sequence is G or A (i.e., the nucleotide mutation of G153-A153 at position 153). The polymorphism of the base at this locus results in differences in the number of live piglets born to pigs. By verifying its effect on the number of live piglets born to sows, the inventors finally established an efficient and accurate molecular marker-assisted breeding technology and applied it to the genetic improvement of breeding pigs to increase the number of live piglets, thereby improving the reproductive performance of offspring pigs and increasing the economic profit of enterprises. Accordingly, the inventors established a corresponding genetic improvement method for pigs. By selecting the advantageous allele of this SNP, the frequency of the advantageous allele can be increased generation by generation, the number of live piglets born to sows can be increased, and the progress of pig genetic improvement can be accelerated, thereby effectively improving the economic benefits of breeding pig breeding. In addition, the inventors also designed a primer pair for amplifying the nucleotide sequence of the SNP molecular marker. Through this primer pair, the reproductive traits can be selected quickly and accurately, and the breeding process can be accelerated. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is a Manhattan plot of genome-wide association study (GWAS) related to the number of live piglets born on chromosome 1 of Large White pigs. In the figure: the abscissa represents the position of the SNP locus on chromosome 1, and the ordinate represents the -logP value.

[0016] Figure 2 It is a sequencing result diagram of the target DNA sequence. DETAILED DESCRIPTION OF THE INVENTION

[0017] Example 1 Screening of Molecular Markers and Association Analysis with the Number of Live Piglets

[0018] (1) Experimental Animals

[0019] The experimental pig population used in the present invention is 839 purebred Large White pigs from the breeding pig branch of Guangxi Yangxiang Group Co., Ltd. The pigs were allowed to eat and drink freely, and the entire feeding method and breeding conditions were conventional methods and remained consistent throughout.

[0020] (2) Sample Collection

[0021] The ear tissues of the above-mentioned Large White pigs were collected and soaked in an ethanol solution with a volume fraction of 75% and stored in a -20°C refrigerator for later use.

[0022] (3) Collection of Phenotypic Data

[0023] The phenotypic trait data of the present invention comes from the breeding data of all pigs recorded by the company, including the number of live-born piglets (the number of live piglets in the same litter within 24 hours after birth), etc.

[0024] (4) Genotyping of 80K SNPs in the whole genome of pigs

[0025] Ear tissue samples of the above 839 Large White sows were collected. After extracting genomic DNA using the standard phenol-chloroform method, the quality of the DNA samples was evaluated using a Nanodrop2000 / 2000C nucleic acid and protein detector, and the DNA concentration and purity parameters (OD260 / 280 and OD260 / 230) of each sample were measured. The DNA samples that met 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% agarose gel by mass / volume for electrophoresis analysis (150 V, 25 min). Finally, it was observed and photographed under an ultraviolet spectrophotometer and a gel imaging device to observe the band clarity and integrity of the DNA.

[0026] The DNA samples were sent to Wuhan Shadow Gene Technology Co., Ltd. for genotyping of 80K functional site chips in the whole genome of pigs according to the company's standard procedures. PLINK (V1.9) was used to perform quality control on the 80K chip scanning genotyping data of all samples, and SNPs with an individual detection rate lower than 90%, an SNP detection rate lower than 90%, a minor allele frequency less than 0.05, and a Hardy-Weinberg equilibrium significance level higher than 10 -6 were excluded. The Beagle (V5.2) software was used to impute the 80K SNPs chip data of the above 839 individuals and then perform the same quality control conditions as above. Finally, effective genotype data of 102,094 SNPs were obtained.

[0027] (5) Genome-wide association (GWAS) analysis

[0028] Since the phenotypic data of the number of live-born piglets contains multiple litter records of each sow individual at different times and different parities. To explain this structure, the GMAT software was used to perform genome-wide association analysis of longitudinal traits. The Bonferrini method was used to determine the significance threshold for the association degree between SNPs and the number of live-born piglets. The significant threshold was 1 divided by the number of effective SNP loci, that is, the significant level threshold was 9.79×10 -6 , that is, 1 / 102,094 (the number of effective SNPs). This analysis framework is based on the following model:

[0029] Y = Xb + Qa + Zp + e

[0030] Where: y is the vector of phenotypic values; b is the vector of fixed effects, including the sow's farrowing year and farrowing season; a and p are the vectors of random regression polynomial coefficients for additive genetic effects and individual-specific permanent environmental effects; e is the vector of random residuals. X, Q, and Z are the corresponding matrices. Among them K is the marker-based relationship matrix; I is the identity matrix; is the Kronecker product; ∑ a is the (co)variance matrix of the random regression coefficients of additive polygenic effects; ∑ p is the variance-covariance matrix of the random regression coefficients of permanent environmental effects; R is a diagonal matrix with different values at different time periods.

[0031] The results of GWAS analysis are as Figure 1 shown. From Figure 1 it can be seen that there is a locus on chromosome 1 of Large White pigs that significantly affects the number of live born piglets. The significantly associated SNP is the 153rd nucleotide g.153G>A in SEQ NO.1 (P = 9.79×10 -06 ).

[0032] (6) Association analysis between different genotypes and the phenotype of the number of live born piglets

[0033] According to Table 1, the SNP locus g.153G>A of the molecular marker is extremely significantly correlated with the trait of the number of live born piglets (P = 9.79×10 -06 ). Among them, the GG genotype pigs of this molecular marker have a higher number of live born piglets than AG and AA genotype pigs. The results show that the G of this molecular marker is the favorable allele and A is the unfavorable allele. Therefore, during the breeding process, it is necessary to gradually eliminate AA and AG genotype breeding pigs and retain GG genotype breeding pigs to gradually increase the frequency of allele G at this locus and improve the number of live born piglets of pigs.

[0034] Table 1 Correlation between the SNP locus g.153G>A of the molecular marker and the number of live born piglets

[0035]

[0036] Example 2 Amplification and sequencing of the target DNA sequence

[0037] (1) Primer design

[0038] Download the DNA sequence of SEQ ID NO:1 on chromosome 1 of pigs through the Ensembl website (http: / / asia.ensembl.org / index.html). And use the primer design software primer premier 6.0 to design primers. The DNA sequences of the designed primers are as follows:

[0039] P001-F: 5’-ACTCTGCTACCTGTTTGCGG-3’,

[0040] P002-R: 5’-GTGCTGACCTCTGGAAACCT-3;

[0041] (2) PCR amplification

[0042] Prepare a 10 μL system: 1.0 μL of DNA sample, 0.3 μL of upstream primer, 0.3 μL of downstream primer, 5 μL of PCR mix, and 3.4 μL of ddH2O. Configure the amplification conditions: The first stage: pre-denaturation at 95 °C for 5 min; the second stage: denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s, extension at 72 °C for 45 s, for 34 cycles; the third stage: final extension at 72 °C for 5 min, and store at 4 °C.

[0043] (3) DNA sequence determination

[0044] DNA sequence sequencing identification: The sequence sequencing was carried out at BGI-Shenzhen Co., Ltd. The gene fragment was sequenced in both forward and reverse reactions. The obtained sequence was compared with the NCBI genomic sequence to obtain the mutations at the corresponding SNP sites. The sequencing results are as Figure 2 shown. The marked M is the mutation site (the mutated base is in the parentheses, which is an allelic gene mutation). The designed primer sequence positions are underlined and bolded at the beginning and end of this sequence.

[0045] Example 3 Analysis of the effect of the SNP site g.153G>A of the molecular marker

[0046] Using this molecular marker for marker-assisted selection can accelerate the breeding process of the number of live piglets born per large white sow.

[0047] The effects of the dominant allele genotype GG of the SNP site g.153G>A were significantly increased by 0.6 and 1.45 respectively compared with the average phenotypes of the AG and AA types. By marker-assisted selection, gradually eliminating the individuals of the AG type and AA type in the large white pig population can continuously increase the gene frequency of the dominant allele G, thereby increasing the number of live piglets born in the population and increasing the enterprise's income.

Claims

1. A SNP molecular marker on chromosome 1 of pigs related to live piglet number, characterized in that Its SNP site corresponds to the G>A mutation at position 13,051,624 on chromosome 1 of the international porcine genome version 11.1 reference sequence.

2. The SNP molecular marker on pig chromosome 1 associated with live piglet number according to claim 1, characterized in that: The nucleotide sequence of the SNP molecular marker is SEQ ID NO.1, wherein M in the sequence is G or A.

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

3.

4. Use of the SNP molecular marker associated with live piglet number on pig chromosome 1 as described in claim 1 or 2 or the primer pair as described in claim 3 in pig assisted breeding.

5. The use according to claim 4, characterized in that: The pig assisted breeding is a genetic improvement for increasing the number of piglets born alive.

6. A method for genetic improvement of pigs, characterized in that: Detect the SNP molecular markers described in claim 1 or 2, eliminate individuals with AG 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 number of live piglets born in subsequent generations.

7. The method for genetic improvement of pigs according to claim 6, characterized in that: The pig is a Large White pig.

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