SNP (Single Nucleotide Polymorphism) molecular marker related to pig weaning litter number character and application of SNP molecular marker

Abstract

The invention belongs to the technical field of pig molecular markers, and particularly discloses an SNP (Single Nucleotide Polymorphism) molecular marker related to a pig weaning litter number character and application of the SNP molecular marker. Gene typing is carried out by utilizing a gene chip technology, whole genome association analysis is carried out on the basis of SNP molecular markers, and 11 SNP molecular marker sites related to the weaning litter number character of the binary hybrid sow are screened out through the analysis. The sites of the SNP molecular marker are located on a WDR25 gene segment and a BEGAIN gene segment of a pig chromosome 7, and the SNP molecular marker can be used for marker-assisted breeding of the pig weaning litter number character.

Description

Technical Field

[0001] The present invention belongs to the technical field of pig molecular markers, and in particular relates to SNP molecular markers related to pig weaning litter size traits and applications thereof. Background Art

[0002] In modern pig production, the number of piglets at weaning is a key trait for measuring the reproductive efficiency of sows and the survival rate of piglets. It refers to the number of piglets successfully weaned in each litter. This trait comprehensively reflects the sow's lactation ability, maternal behavior and the health status of the piglets. It is one of the goals with important economic value in the selection of breeding pigs. The higher the number of piglets at weaning, the more piglets the sow can successfully raise, and the higher the success rate, which helps to increase the annual number of piglets slaughtered per sow, thereby significantly improving the breeding efficiency. With the continuous advancement of genetic breeding technology, incorporating the number of piglets at weaning into the breeding evaluation system will help improve the reproductive performance of the group and provide strong support for the efficient development of the pig industry (Chi Lan 2021).

[0003] With the rapid development of molecular biology and multi-omics sequencing technologies, marker-assisted selection (MAS) has become an important tool in modern pig breeding (Zhang et al. 2011). Litter size at weaning (i.e., the number of piglets per litter that survive weaning) is a key trait for measuring sow reproductive performance and piglet survival, and is influenced by multiple factors, including sow lactation, maternal behavior, and piglet health. Increasing litter size not only helps increase the total number of piglets reared per sow annually but also significantly improves breeding efficiency. In recent years, domestic and international researchers have conducted in-depth studies on pig reproductive traits using genome-wide association studies (GWAS), identifying numerous single nucleotide polymorphisms (SNPs) and candidate genes associated with reproductive performance (Hu et al. 2022). For example, a GWAS analysis of reproductive performance in a Large White pig population identified 14 candidate genes (e.g., BHLHA15 and OCM2), providing potential genetic markers for molecular marker-assisted selection breeding in pigs (Wang et al. 2018). In addition, the study also found that there is a significant correlation between birth litter weight and indicators such as total piglet size, number of live piglets born, number of healthy piglets, and number of piglets at weaning, further emphasizing the importance of piglet size at weaning in evaluating sow reproductive performance (Dou Yaqing et al. 2021). In summary, as an important trait that comprehensively reflects the sow's lactation ability and piglet survival rate, its genetic improvement is of great significance for improving the overall reproductive performance and breeding efficiency of the pig herd. By using molecular breeding technologies such as GWAS, identifying genetic markers related to piglet size at weaning will help accelerate the genetic improvement process of this trait.

[0004] In modern pig production systems, two-way hybrid sows, as the terminal female parent used directly in production, shoulder the core task of improving fertility and piglet survival. Although these sows do not participate in further breeding, their reproductive performance directly determines production efficiency and economic benefits (Szulc et al. 2023). Traditional genetic breeding focuses primarily on purebred populations, emphasizing the accumulation of additive genetic effects. However, in hybrid sows, many important economic traits are often influenced by dominant effects. Therefore, identifying and utilizing dominant genetic loci associated with these traits in two-way hybrid sows is of great practical significance. Using molecular markers to select parental pig breeds carrying favorable dominant alleles allows for more precise breeding of sows, maximizing heterosis and thus improving per-sow production efficiency. This not only helps optimize breeding structure and improve sow utilization efficiency, but also provides a new technical path for the precise improvement of functional traits and efficient utilization of breeding pig resources.

[0005] The present invention screened out 11 SNP sites that were significantly associated with the pig litter size trait through genome-wide association analysis fitting dominant effects, providing a theoretical basis and application approach for molecular marker-assisted selection breeding of pigs. Summary of the Invention

[0006] The purpose of the present invention is to overcome the defects of the existing technology, use gene chip technology for genotyping, conduct genome-wide association analysis with the sow weaning litter size trait, screen out 11 SNPs that are significantly associated with the weaning litter size trait of binary hybrid sows, and provide a new SNP molecular marker resource for pig reproductive traits.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The applicant screened for SNP molecular markers significantly associated with litter size at weaning in binary hybrid sows through genome-wide association analysis, and obtained the nucleotide sequences 100bp upstream and downstream of each SNP based on the Ensmble database pig version 11.1 reference genome, as follows:

[0009] The nucleotide sequence containing the SNP1 marker is shown in SEQ ID NOs. 1 and 2. The SNP1 marker is located at position 101 of the sequence, corresponding to position 121318719 of chromosome 7 of the porcine genome. The corresponding gene is WDR25, with the accession number in the dbSNP database being rs3470320794. The polymorphic site is A or G, and the favorable allele is A.

[0010] The nucleotide sequence containing the SNP2 marker is shown in SEQ ID NOs. 3 and 4. The SNP2 marker is located at position 101 of the sequence, corresponding to position 121373289 of chromosome 7 of the porcine genome. The corresponding gene is WDR25, with the accession number in the dbSNP database being rs3476692836. The polymorphic site is A or G, and the favorable allele is A.

[0011] The nucleotide sequence containing the SNP3 marker is shown in SEQ ID NOs. 5 and 6. The SNP3 marker is located at position 101 of the sequence, corresponding to position 121407584 of chromosome 7 of the porcine genome. The corresponding gene is BEGAIN, and the accession number in the dbSNP database is rs336965142. The polymorphic site is G or C, and the favorable allele is G.

[0012] The nucleotide sequence containing the SNP4 marker is shown in SEQ ID NOs. 7 and 8. The SNP4 marker is located at position 101 of the sequence, corresponding to position 121407608 of chromosome 7 of the porcine genome. The corresponding gene is BEGAIN, and the accession number in the dbSNP database is rs328268058. The polymorphic site is T or A, and the favorable allele is T.

[0013] The nucleotide sequence containing the SNP5 marker is shown in SEQ ID NOs. 9 and 10. The SNP5 marker is located at position 101 of the sequence, corresponding to position 121407645 of chromosome 7 of the porcine genome. The corresponding gene is BEGAIN, and the accession number in the dbSNP database is rs340592418. The polymorphic site is T or C, and the favorable allele is T.

[0014] The nucleotide sequence containing the SNP6 marker is shown in SEQ ID NOs. 11 and 12. The SNP6 marker is located at position 101 of the sequence, corresponding to position 121407660 of chromosome 7 of the porcine genome. The corresponding gene is BEGAIN, and the accession number in the dbSNP database is rs329947679. The polymorphic site is T or C, and the favorable allele is T.

[0015] The nucleotide sequence containing the SNP7 marker is shown in SEQ ID NOs. 13 and 14. The SNP7 marker is located at position 101 of the sequence, corresponding to position 121439050 of chromosome 7 of the porcine genome. The accession number in the dbSNP database is rs330902464. The polymorphic site is G or A, and the favorable allele is G.

[0016] The nucleotide sequence containing the SNP8 marker is shown in SEQ ID NOs. 15 and 16. The SNP8 marker is located at position 101 of the sequence, corresponding to position 121439103 of chromosome 7 of the porcine genome. The accession number in the dbSNP database is rs790454899. The polymorphic site is C or G, and the favorable allele is C.

[0017] The nucleotide sequence containing the SNP9 marker is shown in SEQ ID NOs. 17 and 18. The SNP9 marker is located at position 101 of the sequence, corresponding to position 121439130 of chromosome 7 of the porcine genome, and its accession number in the dbSNP database is rs336167182. The polymorphic site is G or A, and the favorable allele is G.

[0018] The nucleotide sequence containing the SNP10 marker is shown in SEQ ID NOs. 19 and 20. The SNP10 marker is located at position 101 of the sequence, corresponding to position 121439147 of chromosome 7 of the porcine genome, and its accession number in the dbSNP database is rs81397284. The polymorphic site is A or G, and the favorable allele is A.

[0019] The nucleotide sequence containing the SNP11 marker is shown in SEQ ID NO. 21 and 22. The SNP11 marker is located at position 101 of the sequence, corresponding to position 121460440 of chromosome 7 of the porcine genome. The accession number in the dbSNP database is rs321743370. The polymorphic site is T or C, and the favorable allele of the SNP11 marker is T.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] Compared to traditional screening methods, this method offers significant advantages, such as simplicity and speed. Its application in selective breeding for litter size in sows can increase the total number of weaned piglets, effectively reducing economic losses for pig farming enterprises and increasing production revenue. From a national perspective, it can also increase the effective scale of the pig farming industry, improve domestic pig breeding standards, and accelerate the breeding process. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 : This is a Manhattan plot produced by the present invention, which visualizes the results of GWAS analysis.

[0023] Figure 2 : This is the QQ graph produced by the present invention, by comparing the actual -log 10 (P) value and expected-log 10 The quantile of the (P) value probability distribution is used to compare the two probability distributions and further judge the reliability of the GWAS results. DETAILED DESCRIPTION

[0024] Example 1 Genotyping Detection and Data Processing

[0025] (1) DNA extraction

[0026] ① Ear tissue was collected from 16,906 two-way hybrid sows. The collected ear tissue samples were minced and placed in a glass homogenizer. An equal volume of cell lysis buffer consisting of 100 mmol / L Tris-saturated phenol, 500 mmol / L disodium ethylenediaminetetraacetic acid (EDTA), 20 mmol / L sodium chloride (NaCL), 10% sodium dodecyl sulfate (SDS), and 20 μg / ml pancreatic RNase was added. 10 ng / mL proteinase K was then added. The mixture was mixed and placed in a 65°C constant temperature water bath for 30 minutes. The centrifuge tube was gently shaken for 15 minutes and centrifuged at 12,000 rpm for 5 minutes. The supernatant was then transferred to another centrifuge tube.

[0027] ② Add equal volumes of phenol, chloroform, and isoamyl alcohol (volume ratio of 25:24:1), shake and mix, centrifuge at 12,000 rpm for 5 minutes, and transfer the supernatant to another centrifuge tube;

[0028] ③ Add equal volumes of phenol, chloroform, and isoamyl alcohol (volume ratio of 25:24:1), shake and mix thoroughly, centrifuge at 12,000 rpm for 10 min, and transfer the supernatant to another centrifuge tube;

[0029] ④ Add 2 times the volume of pre-cooled anhydrous ethanol, let it stand until the ethanol evaporates, pick out the DNA precipitate and use ultrapure water to dissolve the DNA;

[0030] ⑤ Use a DNA concentration meter and agarose gel electrophoresis to detect DNA quality.

[0031] (2) Genotyping

[0032] Based on the DNA extracted above, genotyping was performed using the pig 80K functional site gene chip produced by Wuhan Yingzi Gene Technology Co., Ltd.

[0033] Example 2 Genome-wide association analysis of SNP molecular markers and litter size at weaning in binary hybrid sows

[0034] (1) Phenotype correction

[0035] A total of 39,876 litter sizes at weaning were collected from 16,906 two-way hybrid sows at a pig farm. Phenotypic data were processed using the mean ± 3 standard deviations. A single-trait repeatability model was fitted using the R package lme4, with farrowing site, year, season, and parity as fixed effects to adjust for environmental influences on phenotype. The sum of the breeding value and the residual was used as the adjusted phenotypic value.

[0036] (2) SNP molecular marker quality control

[0037] Based on the chip data of binary hybrid sows, PLINK v1.9 software was used to perform quality control on the obtained SNP molecular marker loci. SNPs with a site missing rate greater than 10%, a sample missing rate greater than 10%, and a minimum allele frequency less than 0.01 were eliminated. Finally, 178,746 autosomal SNPs and 16,906 samples were used for genome-wide association analysis.

[0038] (3) Genome-wide association analysis of the dominant effect of litter size in hybrid sows

[0039] The experimental pig population used for the genome-wide association analysis in this example was a 16,906-strong population of two-way hybrid sows. Based on the litter size data recorded for the experimental pig population, a linear mixed model approach was used using the remma analysis module in GMAT software. The first three principal components were used as covariates to perform a genome-wide association analysis fitting a dominant effect between SNP molecular markers and litter size in two-way hybrid sows. The specific model is shown below:

[0040] y=Wα+ZG a a a +ZG d a d +e

[0041] Where y is the phenotype vector; α is the fixed effect vector, W is the fixed effect design matrix; Z is the random effect correlation matrix, a a is the additive SNP effect, a d is a dominant SNP effect, G a is the additive effect matrix, G d is the dominant effect matrix, e is the random residual vector, which conforms to in is the residual variance, and I is the identity matrix.

[0042] According to the results of GWAS, the P value of each SNP site was calculated as -log 10 (P), calculated according to the significant threshold line formula -log 10 (1 / SNP number) calculation, the present invention calculates each SNP site -log 10When the (P) value is greater than or equal to 5.252236, it indicates that there is a significant association between the SNP and the litter size trait, and the Manhattan plot and QQ plot are drawn using the CMplot package of R language ( Figure 1 , Figure 2 ).

[0043] Table 1: Candidate SNPs for litter size in hybrid sows identified based on GWAS

[0044]

[0045] Table 2: Genotype frequencies and mean litter size of candidate SNPs in hybrid sows

[0046]

[0047]

[0048] Table 2 shows the genotype frequencies and mean number of weaned sows of the 11 candidate SNP loci identified based on genome-wide association analysis in the binary hybrid sow population. It can be seen from this that the favorable allele of the rs3470320794 marker is A, the favorable allele of the rs3476692836 marker is A, the favorable allele of the rs336965142 marker is G, the favorable allele of the rs328268058 marker is T, the favorable allele of the rs340592418 marker is T, the favorable allele of the rs329947679 marker is T, the favorable allele of the rs330902464 marker is G, the favorable allele of the rs790454899 marker is C, the favorable allele of the rs336167182 marker is G, the favorable allele of the rs81397284 marker is A, and the favorable allele of the rs321743370 marker is T. The above SNP markers can be used alone or in combination for selective breeding of the litter size trait of binary hybrid sows, thereby increasing the total number of weaned piglets.

[0049] Main references:

[0050] [1] Chi Lan. Investigation and improvement strategies for sow farrowing performance in large-scale pig farms. China Pig Industry, 2021, 16: 27-31.

[0051] [2] Deng Xiaoming, Wang Wenyue, Wang Xiaolong, Ge Yiqiang. Practical ideas for supporting the modernization of animal husbandry through scientific and technological innovation. China Animal Husbandry Journal, 2024, 60: 282-285.

[0052] [3] Dou Yaqing, Qi Kunlong, Liu Yingke, Song Chenglei, Li Chenlei, Li Xinjian, Qiao Ruimin, Wang Kejun, Li Xiuling, Han Xuelei. Effects of different factors on reproductive performance of American Landrace sows. Henan Agricultural Sciences, 2021, 50: 142-148.

[0053] [4] Zhang Zhe, Zhang Qin, Ding Xiangdong. Research progress on genomic selection in livestock and poultry. Science Bulletin, 2011, 56: 2212-2222.

[0054] [5]Hu Z, Park CA, Reecy JM. Bringing the animal qtldb and corrdb into the future: meeting new challenges and providing updated services. NucleicAcids Res, 2022,50:D956-D961.

[0055] [6]Wang Y, Ding X, Tan Z, Xing K, Yang T, Wang Y, Sun D, Wang C. Genome-wide association study for reproductive traits in a large white pigpopulation. Anim Genet, 2018, 49: 127-131.

[0056] [7] Szulc K, Skrzypczak E, Arszylo M. Analysis of reproduction performance traits in sows of the cg36 hybrid line, including the effect of selected factors and phenotypic correlations. Agriculture (Basel), 2023, 13.

Claims

1. Application of SNP markers in selection of pig litter size at weaning, characterized in that: Includes at least one of the following SNP markers: The nucleotide sequence containing the SNP1 marker is shown in SEQ ID NOs. 1 and 2. The SNP1 marker is located at position 101 of the sequence, and the polymorphic site is A or G; The nucleotide sequence containing the SNP2 marker is shown in SEQ ID NOs. 3 and 4. The SNP2 marker is located at position 101 of the sequence, and the polymorphic site is A or G; The nucleotide sequence containing the SNP3 marker is shown in SEQ ID NOs. 5 and 6. The SNP3 marker is located at position 101 of the sequence, and the polymorphic site is G or C; The nucleotide sequence containing the SNP4 marker is shown in SEQ ID NOs. 7 and 8. The SNP4 marker is located at position 101 of the sequence, and the polymorphic site is T or A; The nucleotide sequence containing the SNP5 marker is shown in SEQ ID NOs. 9 and 10. The SNP5 marker is located at position 101 of the sequence, and the polymorphic site is T or C; The nucleotide sequence containing the SNP6 marker is shown in SEQ ID NOs. 11 and 12. The SNP6 marker is located at position 101 of the sequence, and the polymorphic site is T or C; The nucleotide sequence containing the SNP7 marker is shown in SEQ ID NOs. 13 and 14. The SNP7 marker is located at position 101 of the sequence, and the polymorphic site is G or A; The nucleotide sequence containing the SNP8 marker is shown in SEQ ID NOs. 15 and 16. The SNP8 marker is located at position 101 of the sequence, and the polymorphic site is C or G; The nucleotide sequence containing the SNP9 marker is shown in SEQ ID NOs. 17 and 18. The SNP9 marker is located at position 101 of the sequence, and the polymorphic site is G or A; The nucleotide sequence containing the SNP10 marker is shown in SEQ ID NOs. 19 and 20. The SNP10 marker is located at position 101 of the sequence, and the polymorphic site is A or G; The nucleotide sequence containing the SNP11 marker is shown in SEQ ID NOs. 21 and 22. The SNP11 marker is located at position 101 of the sequence, and the polymorphic site is T or C.

2. The use according to claim 1, characterized in that: The favorable allele marked by SNP1 is A, the favorable allele marked by SNP2 is A, the favorable allele marked by SNP3 is G, the favorable allele marked by SNP4 is T, the favorable allele marked by SNP5 is T, the favorable allele marked by SNP6 is T, the favorable allele marked by SNP7 is G, the favorable allele marked by SNP8 is C, the favorable allele marked by SNP9 is G, the favorable allele marked by SNP10 is A, and the favorable allele marked by SNP11 is T.

3. The use according to claim 1 or 2, characterized in that The pig is a two-purpose hybrid sow.

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