SNP (Single Nucleotide Polymorphism) molecular marker related to characters of mummy number and stillbirth number of pigs and application of SNP molecular marker

Abstract

The invention provides an SNP (Single Nucleotide Polymorphism) molecular marker related to swine mummy number and stillbirth number characters and application, and relates to the technical field of biology. According to the application, a substance for detecting the SNP sites is applied to preparation of a product for predicting the death number of a pig fetus, and the SNP sites comprise one or more of an SNP site rs318687930, an SNP site rs337505213, an SNP site rs321068825 and an SNP site rs340473552. The SNP sites are related to the character of the pig fetal death number, and can be used for predicting the pig fetal death number and screening and breeding sows with the character of the low-birth dead fetal number.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a SNP molecular marker associated with the number of pig mummies and the number of stillbirths and an application thereof. Background Art

[0002] Pig fertility traits are important indicators for measuring the reproductive capacity of sows, including litter size, piglet birth weight, number of teats, lactation capacity (21-day-old litter weight) and number of finished piglets at weaning, etc. These traits are extremely important for the pig farming industry. Good fertility performance of sows means that each sow can produce more healthy piglets within a certain period of time, which can reduce breeding costs and improve production efficiency.

[0003] The number of pig mummies and stillbirths is of great significance for the evaluation of sow reproductive efficiency and pig breeding. By studying the number of mummies and stillbirths, the transmission and fixation of excellent genes can be accelerated, the reproductive capacity of the pig herd can be improved, and a scientific basis can be provided for breeding and genetic improvement of breeding pigs.

[0004] Therefore, the identification of genes related to the number of pig mummies and stillbirths can provide important clues for explaining the genetic mechanisms of fetal growth and development in pigs and other mammals, and provide a theoretical basis for the genetic improvement of pig reproductive traits.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to overcome the defects of the prior art and provide a SNP molecular marker related to the number of pig fetal deaths and its application.

[0007] In order to solve the above technical problems, the present invention particularly adopts the following technical solutions:

[0008] In a first aspect, a method for detecting SNP sites is provided for preparing a product for predicting the number of pig fetal deaths, wherein the SNP sites include one or more of SNP sites rs318687930, SNP site rs337505213, SNP site rs321068825 and SNP site rs340473552.

[0009] In a second aspect, a kit for predicting the number of pig fetal deaths is provided, comprising at least one of the following primer pairs:

[0010] The primer pairs used to amplify the SNP site rs318687930 have nucleotide sequences as shown in SEQ ID NOs. 1 and 2, respectively; the primer pairs used to amplify the SNP site rs340473552 have nucleotide sequences as shown in SEQ ID NOs. 3 and 4, respectively; the primer pairs used to amplify the SNP site rs321068825 have nucleotide sequences as shown in SEQ ID NOs. 5 and 6, respectively; and the primer pairs used to amplify the SNP site rs337505213 have nucleotide sequences as shown in SEQ ID NOs. 7 and 8, respectively.

[0011] In a third aspect, a method for predicting the number of pig fetal deaths is provided, comprising predicting a sow that meets at least one of (i) to (iv) as a sow with low fetal mortality:

[0012] (i) The genotype of SNP site rs318687930 is GG,

[0013] (ii) the genotype of SNP site rs337505213 was CC;

[0014] (iii) the genotype of SNP site rs321068825 was GG;

[0015] (iv) The genotype of SNP site rs340473552 is CT.

[0016] In a fourth aspect, a method for screening sows with low-birth and fetal mortality is provided, the screening method comprising screening sows with low-birth and fetal mortality predicted by the method described in the third aspect.

[0017] In a fifth aspect, a breeding method for sows with low fetal death numbers is provided, the breeding method comprising enabling the sow to obtain at least one of the following genotypes:

[0018] (i) The genotype of SNP site rs318687930 is GG,

[0019] (ii) the genotype of SNP site rs337505213 was CC;

[0020] (iii) the genotype of SNP site rs321068825 was GG;

[0021] (iv) The genotype of SNP site rs340473552 is CT.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention uses low-depth resequencing and whole genome association analysis methods to find SNP molecular markers related to the number of pig fetal deaths, that is, the number of mummified fetuses and the number of stillbirths, and finds that four SNP sites rs318687930, rs337505213, rs321068825 and rs340473552 are related. Sows with specific genotypes at the above four sites have a lower number of dead fetuses. This SNP molecular marker related to the number of pig fetal deaths can be used to predict the number of pig fetal deaths and screen sows with low numbers of dead fetuses, as well as a breeding method for obtaining sows with low numbers of dead fetuses. The use of this SNP site helps to improve the reproductive performance of sows and bring higher economic benefits to the pig farming industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 This is a peak diagram of genotype detection obtained by capillary electrophoresis detection in an embodiment of the present invention. DETAILED DESCRIPTION

[0026] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] cAMP response element binding protein 3 (CREB3 regulatory factor, CREBRF), also known as Luman / CREB3 recruiting factor (Luman recruiting factor, LRF), is a regulatory factor of Luman / CREB3 protein (cyclic adenosinemonophosphate response element binding protein 3, Luman / CREB3). Studies have shown that the prolactin level of CREBRF knockout mice is significantly lower than that of normal mice, glucocorticoid signaling is affected, maternal behavior is severely defective, and the instinct to care for pups is lacking. The CREBRF gene is expressed at a high level in the ovaries and uterus during estrus and metestrus, suggesting that CREBRF may be involved in cell differentiation, embryonic development and implantation. Another study showed that CREBRF can participate in regulating the apoptosis of GCs by mediating the ERS pathway and plays a key role in mouse follicle selection. The expression of CREBRF can be inhibited by E2, but can be induced by P4, and CREBRF can regulate decidualization during pregnancy by regulating the proliferation of mouse uterine stromal cells. Therefore, the CREBRF gene plays an important regulatory role in animal growth, development and reproduction, especially in follicle growth and development, cell differentiation, embryonic development and implantation. Moreover, the polymorphism of the CREBRF gene SNP site is related to the estrogen level, and then the hormone level is used to regulate the reproductive efficiency of sows. The number of fetal deaths is an important indicator for measuring the reproductive traits of sows, so the present invention explores the correlation between the single nucleotide polymorphism site of the CREBRF gene and the number of fetal deaths.

[0028] The present invention uses low-depth resequencing and whole-genome association analysis methods to conduct association analysis on the polymorphism of the CREBRF gene and important reproductive traits, and finds SNP molecular markers significantly associated with the number of pig fetal deaths in the pig genome. The present invention found that four SNP sites, rs318687930, rs337505213, rs321068825 and rs340473552, are associated with the number of pig fetal deaths.

[0029] The SNP site rs318687930 of the porcine CREBRF gene is the nucleotide at position 51174074 on chromosome 16 of the pig (the reference genome is Sscrofa11.1). When the genotype of this SNP site is GG, the sow has a low fetal mortality rate.

[0030] The SNP site rs337505213 of the porcine CREBRF gene is the nucleotide at position 51208314 on chromosome 16 of the pig (the reference genome is Sscrofa11.1). When the genotype of this SNP site is CC, the sow has a low fetal mortality rate.

[0031] The SNP site rs321068825 of the porcine CREBRF gene is the nucleotide at position 51195295 on chromosome 16 of the pig (the reference genome is Sscrofa11.1). When the genotype of this SNP site is GG, the sow has a low fetal mortality rate.

[0032] The SNP site rs340473552 of the porcine CREBRF gene is the nucleotide at position 51177550 on chromosome 16 of the pig (the reference genome is Sscrofa11.1). When the genotype of this SNP site is CT, the sow has a low fetal mortality rate.

[0033] Herein, "fetal deaths" refers to the death of pig fetuses in utero. Depending on the time and specific circumstances of occurrence, it can be further subdivided into different types. For example, it can be, but not limited to, mummified fetuses and / or stillbirths. "Low-birth fetal deaths" refers to fetal deaths that are lower than the average fetal deaths of the breed.

[0034] Based on the above findings, in the first aspect, a method for preparing a product for predicting the number of pig fetal deaths by using a substance for detecting SNP sites is provided, wherein the SNP sites include one or more of SNP sites rs318687930, SNP sites rs337505213, SNP sites rs321068825 and SNP sites rs340473552.

[0035] In an optional embodiment, the pig fetal death includes the number of mummified fetuses and / or the number of stillbirths.

[0036] In an optional embodiment, the product includes a substance for detecting the SNP site rs318687930, and the number of pig fetal deaths includes the number of primiparous mummies.

[0037] In an optional embodiment, the product includes a substance for detecting the SNP site rs337505213, and the number of pig fetal deaths includes the number of primiparous mummies.

[0038] In an optional embodiment, the product includes a substance for detecting the SNP site rs321068825, and the number of pig fetal deaths includes the number of primiparous stillbirths.

[0039] In an optional embodiment, the product includes a substance for detecting the SNP site rs340473552, and the number of pig fetal deaths includes the number of stillbirths.

[0040] In an optional embodiment, the product includes substances for detecting: SNP site rs318687930, SNP site rs337505213, SNP site rs321068825 and SNP site rs340473552.

[0041] In an optional embodiment, the substance for detecting SNP sites includes, but is not limited to, one or more of reagents for nucleic acid amplification, reagents for detecting nucleic acid amplification products, reagents for constructing sequencing libraries, and reagents for sequencing. More specifically, the substance for detecting SNP sites includes, but is not limited to, primers, probes, enzymes for nucleic acid amplification reactions, fluorescent markers, buffer reagents, dNTPs, salts, etc. Depending on the specific detection means, those skilled in the art can select the above-mentioned reagents for nucleic acid amplification, reagents for detecting nucleic acid amplification products, reagents for constructing sequencing libraries, and reagents for sequencing according to general and more specific methods recorded in teaching materials, references, process manuals, product descriptions, and standard documents, and the present invention does not limit this.

[0042] In an optional embodiment, the material for detecting SNP sites includes at least one pair of the following primer pairs: a primer pair for amplifying SNP site rs318687930, the nucleotide sequences of which are shown in SEQ ID NOs. 1 and 2, respectively; a primer pair for amplifying SNP site rs340473552, the nucleotide sequences of which are shown in SEQ ID NOs. 3 and 4, respectively; a primer pair for amplifying SNP site rs321068825, the nucleotide sequences of which are shown in SEQ ID NOs. 5 and 6, respectively; and a primer pair for amplifying SNP site rs337505213, the nucleotide sequences of which are shown in SEQ ID NOs. 7 and 8, respectively.

[0043] In a second aspect, a kit for predicting the number of pig fetal deaths is provided, which kit includes at least one of the following primer pairs: a primer pair for amplifying SNP site rs318687930, the nucleotide sequences of which are shown in SEQ ID NOs. 1 and 2, respectively; a primer pair for amplifying SNP site rs340473552, the nucleotide sequences of which are shown in SEQ ID NOs. 3 and 4, respectively; a primer pair for amplifying SNP site rs321068825, the nucleotide sequences of which are shown in SEQ ID NOs. 5 and 6, respectively; and a primer pair for amplifying SNP site rs337505213, the nucleotide sequences of which are shown in SEQ ID NOs. 7 and 8, respectively.

[0044] In a third aspect, a method for predicting the number of pig fetal deaths is provided, the method comprising predicting a sow that meets at least one of (i) to (iv) as a sow with low fetal mortality:

[0045] (i) The genotype of SNP site rs318687930 is GG,

[0046] (ii) the genotype of SNP site rs337505213 was CC;

[0047] (iii) the genotype of SNP site rs321068825 was GG;

[0048] (iv) The genotype of SNP site rs340473552 is CT.

[0049] In an optional embodiment, the method comprises predicting the sows meeting (i) and / or (ii) as sows with low mummified litter numbers:

[0050] (i) The genotype of SNP site rs318687930 is GG,

[0051] (ii) The genotype of SNP site rs337505213 is CC.

[0052] In an optional embodiment, the method for predicting the number of low-birth fetal deaths in pigs comprises predicting sows that meet (iii) and / or (iv) as sows with low-birth fetal deaths:

[0053] (iii) the genotype of SNP site rs321068825 was GG;

[0054] (iv) The genotype of SNP site rs340473552 is CT.

[0055] The third aspect provides a method for predicting the number of fetal deaths in low-birth pigs, including using one or more of the SNP sites rs318687930, rs337505213, rs321068825, and rs340473552 as the only identification target; other targets may also be used, such as other polymorphic molecular markers (such as SSR molecular markers, STR molecular markers, or InDel molecular markers), or other screening methods known in the art to assist in the prediction of the total litter size trait of pigs.

[0056] In a fourth aspect, a method for screening sows with low-birth and fetal death numbers is provided, comprising screening sows with low-birth and fetal death numbers predicted by the method described in the third aspect.

[0057] In a fifth aspect, a breeding method for sows with low production and low fetal death is provided, the breeding method comprising enabling the sow to obtain at least one of the following genotypes:

[0058] (i) The genotype of SNP site rs318687930 is GG,

[0059] (ii) the genotype of SNP site rs337505213 was CC;

[0060] (iii) the genotype of SNP site rs321068825 was GG;

[0061] (iv) The genotype of SNP site rs340473552 is CT.

[0062] The specific means for obtaining a specific genotype in a sow can be achieved by a person skilled in the art using conventional methods known in the art, and the present invention is not limited thereto. In an optional embodiment, an offspring with a target genotype is obtained by obtaining a parent with a known genotype. In an optional embodiment, an offspring with a target genotype is obtained by a gene editing method.

[0063] In an optional embodiment, the pig in any of the above embodiments is a Large White pig.

[0064] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0065] Example 1

[0066] 1. Experimental sample collection

[0067] The experimental population of this embodiment is 226 British Large White pigs from Shandong Rizhao Breeding Farm, 165 Canadian Large White pigs from Xinjiang Tiankang Jiamei Breeding Branch, and 199 Danish Large White pigs from the great-grandparent breeding farm of Xinjiang Tiankang Animal Husbandry Technology Co., Ltd. A total of 590 Large White pigs were used for CREBRF gene SNP typing. Ear tissue samples were collected one by one, placed in 75% alcohol and stored at -20°C for DNA extraction.

[0068] 2. Extraction and library construction of porcine genomic DNA

[0069] (1) Take an appropriate amount of pig ear tissue, chop it into small pieces and place it into a 1.5 mL Axgen tube.

[0070] (2) Take a 50 mL BD centrifuge tube, mix proteinase K with a final concentration of 0.4 mg / mL and lysis buffer, and add 0.5 mL of lysis buffer to the 1.5 mL centrifuge tube containing pig ear tissue for lysis.

[0071] (3) Place the centrifuge tubes evenly and in parallel on the rocking plate of the constant temperature hybridization oven (close the tube lid tightly to prevent liquid leakage). Place at 55°C for more than 6 hours (it is very important to fully mix the samples during the digestion process. The basis for judgment is that there is no obvious pig ear tissue visible to the naked eye and the mixed solution after digestion is milky).

[0072] (4) After the samples are fully lysed, remove the centrifuge tubes and add 0.3 mL of saturated sodium chloride solution to each tube. Invert and mix thoroughly 6-8 times, then place on ice and place in an ice bath for 15 minutes.

[0073] (5) After the ice bath, centrifuge at 12,000 rpm for 15 minutes at room temperature. Carefully and slowly transfer the supernatant to a new 1.5 mL Axgen centrifuge tube (be careful to avoid pouring out the precipitate with the supernatant. Keep the pouring technique consistent and keep the amount of supernatant in each tube the same).

[0074] (6) Add 0.7 mL of isopropanol (the amount of isopropanol added varies with the amount of supernatant poured out, and the two are equal in volume) to each tube and invert until flocculent precipitation appears in the solution (if no flocculent precipitation occurs, the solution can be placed in a -20°C refrigerator for 2 hours or in a 4°C refrigerator overnight).

[0075] (7) Centrifuge at 12000 rpm for 15 minutes at room temperature and remove the supernatant.

[0076] (8) Add 0.5 mL of 70% ethanol to each centrifuge tube and gently invert to fully rinse the precipitated DNA.

[0077] (9) Centrifuge at 10,000 rpm for 30 seconds. Use a 200 μL micropipette to remove the ethanol in the centrifuge tube, leaving the precipitated DNA in the tube.

[0078] (10) Air-dry the DNA for 10 minutes.

[0079] (11) Use a pipette to add 0.1 mL of TE buffer to each tube, redissolve the DNA precipitate, and place it at 55°C for 2 hours, shaking it several times periodically to fully dissolve the DNA.

[0080] (12) After the DNA is fully dissolved, the concentration is measured by UV spectrophotometer, and the quality is tested by agarose gel electrophoresis. The quality of the DNA is further tested using Qbuit, and qualified samples are tested for library construction.

[0081] (13) Library construction: The sequencing library was prepared using the BGI library construction kit.

[0082] (14) On-line sequencing: After library preparation was completed and quality was tested, DNBSEQ-T7 was used for PE150 sequencing.

[0083] 3. SNP chip genotype determination and genotype data quality control

[0084] 590 DNA samples of Large White pigs were sent to Beijing Gezhi Boya Biotechnology Co., Ltd. for 20× deep sequencing and low-depth resequencing.

[0085] The default parameters of fastp v0.23.2 were used to remove low-quality sequencing data and obtain clean reads. The default parameters of bwa-mem v0.7.17 were used to align clean reads to the reference genome, and then the alignment results were converted into bam files and sorted using Samtools software. Picard MarkDuplicates was used to remove duplicate reads from the sorted bam files. GATK-HaplotypeCaller software was used to detect genomic variations and generate gvcf files for single samples. GATK CombineGVCFs and GenotypeGVCFs were then used to merge gvcf and perform genotyping. The filtering parameters for SNPs and Indels are shown in Table 1.

[0086] Table 1 Filter parameters

[0087] Filter parameters SNP Indel QD ＜2.0 ＜2.0 QUAL ＜30.0 ＜30.0 MQ ＜40.0 ＜40.0 SOR ＞3.0 FS ＞60.0 ＞200.0 MQRankSum ＜-12.5 ReadPosRankSum ＜-8.0 ＜-20.0

[0088] 4. Data collation and analysis

[0089] (1) Phenotypic data analysis: The collected phenotypic data of the number of mummies and stillbirths of 590 Large White pigs were sorted in Excel. Descriptive statistics were performed on the phenotypic data of the number of mummies and stillbirths, and the sample size, mean, standard deviation, standard error, maximum, minimum, and quartile of each variable were calculated.

[0090] (2) Statistical analysis: Excel 2021 was used to calculate various polymorphism indicators. The relevant polymorphism indicator calculation formula is as follows:

[0091] (2.1) Gene frequency (AF)

[0092]

[0093] (2.2) Genotype frequency (GF)

[0094]

[0095] (2.3) Genetic heterozygosity (He)

[0096]

[0097] (2.4) Effective number of alleles (Ne)

[0098]

[0099] (2.5) Polymorphic Information Content (PIC)

[0100]

[0101] (2.6) Correlation analysis

[0102] The LSD method was used for "Anova" analysis, and the reproductive phenotypes of different genotypes were expressed as mean ± standard deviation. The linear model of plink software was used for association analysis between genotype and reproductive phenotype, and the field effect was used as a covariate correction. P ≤ 0.05 was considered a significant difference, and P ≤ 0.01 was considered an extremely significant difference.

[0103] 5. Capillary electrophoresis detection of SNP

[0104] (1) Determine the number of reactions N. During the test, a no-template control (nuclease-free pure water) should be tested simultaneously: N = number of samples to be tested (n) + no-template control (1) + 1.

[0105] (2) Place the enzyme mixture on ice and thaw the remaining components at room temperature until they are completely melted. Mix using a vortex oscillator for 10 seconds and centrifuge using a microcentrifuge for 10 seconds to concentrate the liquid at the bottom of the tube.

[0106] (3) Prepare the reaction system except the template according to Table 2. Take a centrifuge tube, add each component, mix using a vortex oscillator for 10 seconds, centrifuge using a microcentrifuge for 10 seconds, and take 9 μL and dispense it into a PCR reaction tube. The primer sequences are shown in Table 3.

[0107] Table 2 Reaction system configuration

[0108] Serial number Component name Standard system addition amount (μL) Reaction number N Addition volume (μL) 1 2.5×PCR reaction solution 4 4N 2 Primer mix 1 1N 3 Nuclease-free water 4 4N total 9 9N

[0109] Note: PCR products should be stored at 2-8℃ and tested within 24h, or stored at -20℃±5℃ and tested within 7 days. The number of freeze-thaw cycles should not exceed 3 times.

[0110] Table 3 Primer sequences

[0111]

[0112] (4) Add 1 μL of the sample to be tested to each tube of the aliquoted reaction system. Record the number sequence, mix using a vortex oscillator for 10 seconds, and centrifuge using a microcentrifuge for 10 seconds.

[0113] (5) Place each reaction tube in the reaction tank of a PCR instrument and run the PCR program. The PCR amplification program is shown in Table 4.

[0114] Table 4 PCR program

[0115]

[0116] (6) PCR product electrophoresis detection: Take an appropriate amount of PCR product and load it on a 2% to 3% agarose gel for electrophoresis to detect whether the amplification is successful.

[0117] Capillary electrophoresis detection, the results are as follows Figure 1 As shown:

[0118] (1) The PCR amplification product was centrifuged at 3500 rpm for 2 min in a benchtop or plate centrifuge;

[0119] (2) Prepare a mixture of HiDi and Liz500, mix the mixture using a vortex oscillator for 10 seconds, centrifuge it using a microcentrifuge for 10 seconds, dispense 9 μL into a 96-well plate, and add 1 μL of PCR product supernatant (stock solution or dilution) to each well;

[0120] (3) Cover with a rubber pad and centrifuge briefly using a benchtop or plate centrifuge;

[0121] (4) 95°C, denature for 5 min, then immediately place on ice for 3 min;

[0122] (5) Perform capillary electrophoresis using a gene analyzer. Select the "Fragment" electrophoresis method. Data analysis was performed using ELISA software (Applied Biosystems).

[0123] 6. Results Analysis

[0124] Table 5

[0125]

[0126] Table 6

[0127]

[0128] Table 7

[0129]

[0130] The genotype test results showed that:

[0131] For the SNP (rs318687930) site, the genotype of 246 pigs was GG genotype, and the genotype of 186 pigs was GA genotype; for the SNP (rs340473552) site, the genotype of 261 pigs was CC genotype, and the genotype of 144 pigs was CT genotype; for the SNP (rs321068825) site, the genotype of 266 pigs was GG genotype, and the genotype of 140 pigs was GA genotype; for the SNP (rs337505213) site, the genotype of 232 pigs was CC genotype, and the genotype of 198 pigs was CT genotype.

[0132] The allele frequency and gene frequency results of the porcine CREBRF gene in the detected pig population are shown in Table 5:

[0133] For the SNP (rs318687930) site, the GG genotype frequency was 0.4331, the GA genotype frequency was 0.3275, the GG genotype frequency was higher than that of GA, and the GG allele was the dominant gene; for the SNP (rs340473552) site, the CC genotype frequency was 0.4595, the CT genotype frequency was 0.2535, the CC genotype frequency was higher than that of CT, and the CC allele was the dominant gene; for the SNP (rs321068825) site, the GG genotype frequency was 0.4683, the GA genotype frequency was 0.2465, the GG genotype frequency was higher than that of GA, and the GG allele was the dominant gene; for the SNP (rs337505213) site, the CC genotype frequency was 0.4085, the CT genotype frequency was 0.3486, the CC genotype frequency was higher than that of CT, and the CC allele was the dominant gene.

[0134] As can be seen from Table 6: Among 590 Large White pigs, there are 246 GG genotypes and 186 GA genotypes at the SNP (rs318687930) site. There is a significant difference between CREBRF and the number of first-born pig mummies, with the GA type being greater than the GG type. Among 590 Large White pigs, there are 232 CC genotypes and 198 CT genotypes at the SNP (rs337505213) site. There is a significant difference between CREBRF and the number of first-born pig mummies, with the CT type being greater than the CC type.

[0135] As can be seen from Table 7: Among 590 Large White pigs, there are 261 CC genotypes and 144 CT genotypes at the SNP (rs340473552) site. There is a very significant difference between CREBRF and the number of stillbirths during pregnancy, with CC genotype being greater than CT genotype. Among 590 Large White pigs, there are 266 GG genotypes and 140 GA genotypes at the SNP (rs321068825) site. There is a very significant difference between CREBRF and the number of primiparous stillbirths, with GA genotype being greater than GG genotype.

[0136] The linear model of plink software was used to perform association analysis between genotype and total litter size phenotype, and the field effect was used as a covariate correction. The results are shown in Tables 6 and 7:

[0137] The SNP (rs318687930) locus has a significant effect on the number of first-born mummies in pigs. The number of first-born mummies of the GA genotype is significantly higher than that of the GG genotype. Therefore, in actual pig breeding, the GG genotype pigs have fewer first-born mummies. The SNP (rs337505213) locus has a significant effect on the number of first-born mummies. The number of mummies and stillbirths of the CT genotype are significantly higher than those of the CC genotype. Therefore, in actual pig breeding, the CC genotype pigs have fewer first-born mummies. The SNP (rs340473552) locus has a significant effect on the number of stillbirths in pigs. The number of stillbirths of the CC genotype is significantly higher than that of the CT genotype. Therefore, in actual pig breeding, the CT genotype pigs have lower number of stillbirths. The SNP (rs321068825) locus has a significant effect on the number of stillbirths in pigs. The number of first-born stillbirths in GA genotype is significantly higher than that in GG genotype. Therefore, in actual pig breeding, GG genotype pigs have a lower number of first-born stillbirths.

[0138] In summary, the nucleotides at the SNP (rs318687930) site of the pig CREBRF gene can be used to determine whether the pig individual has the GA genotype or the GG genotype, thereby assisting in identifying the number of mummies: the number of first-born mummies with the GG genotype is significantly lower than that with the GA genotype. The GA genotype is a heterozygous G and A nucleotide at position 51174074 on chromosome 16 of the pig; the GG genotype is a homozygous G nucleotide at position 51174074 on chromosome 16 of the pig.

[0139] The nucleotides at the SNP (rs337505213) site of the pig CREBRF gene can be used to determine whether the pig individual has the CT genotype or the CC genotype, thereby assisting in identifying the number of mummies: the number of first-born mummies with the CC genotype is significantly lower than that with the CT genotype. The CT genotype is a heterozygous C and T nucleotide at position 51208314 on chromosome 16 of the pig; the CC genotype is a homozygous C nucleotide at position 51208314 on chromosome 16 of the pig.

[0140] The nucleotides at the SNP (rs340473552) site of the pig CREBRF gene can be used to determine whether the pig individual has the CC genotype or the CT genotype, thereby assisting in identifying the number of stillbirths per parity: the number of stillbirths per parity of the CT genotype is significantly lower than that of the CC genotype. The CT genotype is a heterozygous C and T nucleotide at position 51177550 on chromosome 16 of the pig; the CC genotype is a homozygous C nucleotide at position 51177550 on chromosome 16 of the pig.

[0141] The nucleotides at the SNP (rs321068825) site of the pig CREBRF gene can be used to determine whether the individual pig has the GA genotype or the GG genotype, thereby assisting in identifying the number of first-born stillbirths: the number of first-born stillbirths with the GG genotype is significantly lower than that with the GA genotype. The GA genotype is a heterozygous form of G and A at the 51195295th nucleotide on chromosome 16 of the pig; the GG genotype is a homozygous form of G at the 51195295th nucleotide on chromosome 16 of the pig.

[0142] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of a substance for detecting SNP sites in the preparation of a product for predicting the number of pig fetal deaths, wherein the SNP sites include: One or more of SNP site rs318687930, SNP site rs337505213, SNP site rs321068825 and SNP site rs340473552.

2. The use according to claim 1, characterized in that: The pig fetal deaths include the number of mummified fetuses and / or stillbirths.

3. The use according to claim 2, characterized in that: The product includes a substance for detecting the SNP site rs318687930, and the number of pig fetal deaths includes the number of primiparous mummified fetuses; and / or, The product includes a substance for detecting the SNP site rs337505213, and the number of pig fetal deaths includes the number of primiparous mummified fetuses; and / or, The product includes a substance for detecting the SNP site rs321068825, and the number of pig fetal deaths includes the number of primiparous stillbirths; and / or, The product includes a substance for detecting the SNP site rs340473552, and the number of pig fetal deaths includes the number of stillbirths.

4. The use according to any one of claims 1 to 3, characterized in that: The substance for detecting SNP sites includes one or more of a reagent for nucleic acid amplification, a reagent for detecting nucleic acid amplification products, a reagent for constructing a sequencing library, and a reagent for sequencing.

5. The use according to claim 4, characterized in that: The substance for detecting the SNP site includes at least one of the following primer pairs: The primer pair used to amplify the SNP site rs318687930, the nucleotide sequences are shown in SEQ ID NOs. 1 and 2 respectively; the primer pair used to amplify the SNP site rs340473552, the nucleotide sequences are shown in SEQ ID NOs. 3 and 4 respectively; The primer pair used to amplify the SNP site rs321068825, the nucleotide sequences are shown in SEQ ID NOs. 5 and 6 respectively; and the primer pair used to amplify the SNP site rs337505213, the nucleotide sequences are shown in SEQ ID NOs. 7 and 8 respectively.

6. A kit for predicting the number of pig fetal deaths, characterized in that: Includes at least one of the following primer pairs: The primer pair used to amplify the SNP site rs318687930, the nucleotide sequences are shown in SEQ ID NOs. 1 and 2 respectively; the primer pair used to amplify the SNP site rs340473552, the nucleotide sequences are shown in SEQ ID NOs. 3 and 4 respectively; The primer pair used to amplify the SNP site rs321068825, the nucleotide sequences are shown in SEQ ID NOs. 5 and 6 respectively; and the primer pair used to amplify the SNP site rs337505213, the nucleotide sequences are shown in SEQ ID NOs. 7 and 8 respectively.

7. A method for predicting the number of pig fetal deaths, characterized in that: The method comprises predicting that a sow meeting at least one of (i) to (iv) is a sow having a low-birth fetal mortality rate: (i) The genotype of SNP site rs318687930 is GG, (ii) the genotype of SNP site rs337505213 was CC; (iii) the genotype of SNP site rs321068825 was GG; (iv) The genotype of SNP site rs340473552 is CT.

8. The method for predicting the number of pig fetal deaths according to claim 7, characterized in that: Including predicting sows meeting (i) and / or (ii) as sows with low mummified litters: (i) The genotype of SNP site rs318687930 is GG, (ii) the genotype of SNP site rs337505213 was CC; and / or, including predicting sows meeting (iii) and / or (iv) as sows with a low number of stillbirths: (iii) the genotype of SNP site rs321068825 was GG; (iv) The genotype of SNP site rs340473552 is CT.

9. A method for screening sows with low production and low fetal mortality, characterized in that: The method comprises screening sows predicted to have a low-birth number of fetal deaths using the method of claim 7 or 8.

10. A method for breeding sows with low fetal mortality, characterized in that: The method comprises providing the sow with at least one of the following genotypes: (i) The genotype of SNP site rs318687930 is GG, (ii) the genotype of SNP site rs337505213 was CC; (iii) the genotype of SNP site rs321068825 was GG; (iv) The genotype of SNP site rs340473552 is CT.

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