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

Abstract

The invention provides an SNP (Single Nucleotide Polymorphism) molecular marker related to the total litter size character of pigs 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 total litter size character of pigs, and the SNP sites comprise one or more of an SNP site rs334299237, an SNP site rs318687930 and an SNP site rs325934988. The SNP sites are related to the pig total litter size character and can be used for predicting the pig total litter size character and screening and breeding sows with the high total litter size character.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a SNP molecular marker associated with the total litter size trait of pigs 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 total piglet number trait is of great significance for the evaluation of sow reproductive efficiency and pig breeding. By studying the total piglet number trait, we can screen out breeding pigs with high piglet number potential, accelerate the transmission and fixation of excellent genes, improve the reproductive capacity of the pig herd, and provide a scientific basis for breeding pigs and genetic improvement.

[0004] Therefore, the identification of genes related to total litter size in pigs 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 reproductive traits in pigs.

[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 total litter size trait of pigs 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 and using the method for preparing a product for predicting the total litter size trait of pigs is provided, wherein the SNP sites include one or more of SNP sites rs334299237, SNP sites rs318687930 and SNP sites rs325934988.

[0009] In a second aspect, a kit for predicting the total litter size trait of pigs is provided, the kit comprising at least one of the following primer pairs:

[0010] The primer pair used to amplify the SNP site rs334299237 has nucleotide sequences as shown in SEQ ID NOs. 1 and 2, respectively; the primer pair used to amplify the SNP site rs318687930 has nucleotide sequences as shown in SEQ ID NOs. 3 and 4, respectively; and, the primer pair used to amplify the SNP site rs325934988 has nucleotide sequences as shown in SEQ ID NOs. 5 and 6, respectively.

[0011] In a third aspect, a method for predicting the total litter size trait of pigs is provided, the method comprising predicting a sow that meets at least one of (i) to (iii) as a sow with a high total litter size trait:

[0012] (i) The genotype of SNP site rs334299237 is TC,

[0013] (ii) The genotype of SNP site rs318687930 was GA;

[0014] (iii) The genotype of SNP site rs325934988 is GA.

[0015] In a fourth aspect, a method for screening sows with a high total litter size trait is provided, the screening method comprising screening sows with a high total litter size trait predicted by the method described in the third aspect.

[0016] In a fifth aspect, a method for breeding sows with a high total litter size trait is provided, the breeding method comprising enabling the sow to obtain at least one of the following genotypes:

[0017] (i) The genotype of SNP site rs334299237 is TC,

[0018] (ii) The genotype of SNP site rs318687930 was GA;

[0019] (iii) The genotype of SNP site rs325934988 is GA.

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

[0021] The present invention uses low-depth resequencing and whole genome association analysis methods to find SNP molecular markers related to the total litter size trait of pigs, and finds that three SNP sites rs334299237, rs318687930 and rs325934988 are related to the total litter size trait of pigs. Sows with specific genotypes at the above three sites have a higher total litter size. This SNP molecular marker related to the total litter size trait of pigs can be used to predict the total litter size trait of pigs and screen sows with high total litter size traits, as well as a breeding method for obtaining sows with high total litter size traits. 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

[0022] 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.

[0023] Figure 1 This is a peak diagram of genotype detection obtained by capillary electrophoresis detection in an embodiment of the present invention. DETAILED DESCRIPTION

[0024] 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.

[0025] 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 the hormone level regulates the reproductive efficiency of sows. The total litter size is an important indicator for measuring the reproductive traits of sows, so the present invention explores the correlation between the CREBRF gene single nucleotide polymorphism site and the total litter size.

[0026] 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 in the pig genome that are significantly associated with the total litter size trait of pigs. The present invention found that three SNP sites, rs334299237, rs318687930 and rs325934988, are associated with the total litter size trait of pigs.

[0027] The SNP site rs334299237 of the porcine CREBRF gene is the nucleotide at position 51160002 on chromosome 16 of the pig (the reference genome is Sscrofa11.1). When the genotype of this SNP site is TC, that is, when the nucleotide at position 51160002 on chromosome 16 of the pig is a heterozygote of T and C, the sow has a high total number of piglets born at first birth.

[0028] 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 GA, that is, the nucleotide at position 51174074 on chromosome 16 of the pig is a heterozygous of G and A, the sow has the trait of high total litter size per parity.

[0029] The SNP site rs325934988 of the porcine CREBRF gene is the nucleotide at position 51225561 on chromosome 16 of the pig (the reference genome is Sscrofa11.1). When the genotype of this SNP site is GA, that is, the nucleotide at position 51225561 of the pig is a heterozygote of G and A, the sow has a high total litter size at first birth.

[0030] Herein, "total piglet number of first-born" means the total number of all piglets born by a sow during the first birth, including live piglets and dead piglets. "High total piglet number of first-born" means that the total piglet number of first-born is higher than the average total piglet number of first-born of the breed. In an optional embodiment, when the pig breed is Large White pig, "high total piglet number of first-born" means that the total piglet number of first-born is at least 12.

[0031] Herein, "total piglet number per parity" refers to the average of the total number of all piglets born by a sow from the second to the nth parity, where n is a positive integer ≥ 2. The total number of piglets born in each parity includes both live and dead pigs. "High total piglet number per parity" means that the total piglet number per parity is higher than the average total piglet number per parity of the breed. In an optional embodiment, when the pig breed is Large White pig, "high total piglet number per parity" means that the total piglet number per parity is at least 14.

[0032] Herein, "total litter size" includes at least one of the above-defined "total litter size for first-time births" and "total litter size for all-time births"; "high total litter size" includes at least one of the above-defined "high total litter size for first-time births" and "high total litter size for all-time births".

[0033] Based on the above findings, in the first aspect, a method for detecting SNP sites and using the method for preparing a product for predicting the total litter size trait of pigs is provided, wherein the SNP sites include one or more of SNP sites rs334299237, SNP sites rs318687930 and SNP sites rs325934988.

[0034] In an optional embodiment, the total piglet number trait includes the total piglet number per first parity and / or the total piglet number per multiparity.

[0035] In an optional embodiment, the product includes a substance for detecting the SNP site rs334299237, and the total piglet size trait includes the total piglet size of first birth.

[0036] In an optional embodiment, the product includes a substance for detecting the SNP site rs318687930, and the total piglet number trait includes the total piglet number of first parity and the total piglet number of multiparity.

[0037] In an optional embodiment, the product includes a substance for detecting the SNP site rs325934988, and the total piglet number trait includes the total piglet number of first births.

[0038] In an optional embodiment, the product includes substances for detecting: SNP site rs334299237, SNP site rs318687930 and SNP site rs325934988.

[0039] 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.

[0040] 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 rs334299237, the nucleotide sequences of which are shown in SEQ ID NOs. 1 and 2, respectively; a primer pair for amplifying SNP site rs318687930, the nucleotide sequences of which are shown in SEQ ID NOs. 3 and 4, respectively; and a primer pair for amplifying SNP site rs325934988, the nucleotide sequences of which are shown in SEQ ID NOs. 5 and 6, respectively.

[0041] In the second aspect, a kit for predicting the total litter size trait of pigs is provided, the kit comprising at least one of the following primer pairs: a primer pair for amplifying the SNP site rs334299237, the nucleotide sequences of which are shown in SEQ ID NOs. 1 and 2, respectively; a primer pair for amplifying the SNP site rs318687930, the nucleotide sequences of which are shown in SEQ ID NOs. 3 and 4, respectively; and a primer pair for amplifying the SNP site rs325934988, the nucleotide sequences of which are shown in SEQ ID NOs. 5 and 6, respectively.

[0042] In a third aspect, a method for predicting the total litter size trait of pigs is provided, the method comprising predicting a sow that meets at least one of (i) to (iii) as a sow with a high total litter size trait:

[0043] (i) The genotype of SNP site rs334299237 is TC,

[0044] (ii) The genotype of SNP site rs318687930 was GA;

[0045] (iii) The genotype of SNP site rs325934988 is GA.

[0046] In an optional embodiment, the method comprises predicting a sow meeting the above (i) to (iii) as a sow having a trait of high total litter size.

[0047] In an optional embodiment, the method for predicting the total piglet number trait comprises predicting a sow that meets at least one of (i) and / or (iii) as a sow with a high primiparous total piglet number trait:

[0048] (i) The genotype of SNP site rs334299237 is TC,

[0049] (iii) The genotype of SNP site rs325934988 is GA.

[0050] In an optional embodiment, the method for predicting the total piglet number trait comprises predicting the sows meeting (ii) as sows having a high total piglet number for first parity and a high total piglet number for multiparity:

[0051] (ii) The genotype of SNP site rs318687930 is GA.

[0052] The method for predicting the total pig litter size trait provided in the third aspect includes using one or more of the SNP site rs334299237, the SNP site rs318687930 and the SNP site rs325934988 as identification targets; it can also be combined with other targets, 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 pig litter size trait.

[0053] In a fourth aspect, a method for screening sows with a high total litter size trait is provided, comprising screening sows with a high total litter size trait predicted by the method described in the third aspect.

[0054] In a fifth aspect, a method for breeding sows with a high total litter size trait is provided, the breeding method comprising enabling the sow to obtain at least one of the following genotypes:

[0055] (i) The genotype of SNP site rs334299237 is TC,

[0056] (ii) The genotype of SNP site rs318687930 was GA;

[0057] (iii) The genotype of SNP site rs325934988 is GA.

[0058] In an optional embodiment, the breeding method includes allowing the sow to obtain a genotype of TC at the SNP site rs334299237 to increase the total number of piglets born in the first parity of the sow.

[0059] In an optional embodiment, the breeding method includes allowing the sow to obtain a genotype of GA at the SNP site rs318687930, so as to increase the total number of piglets born in the first parity and the total number of piglets born in each parity.

[0060] In an optional embodiment, the breeding method includes allowing the sow to obtain a genotype of GA at the SNP site rs325934988 to increase the total number of piglets born in the first parity of the sow.

[0061] 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.

[0062] In an optional embodiment, the pig in any of the above embodiments is a Large White pig.

[0063] 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.

[0064] Example 1

[0065] 1. Experimental sample collection

[0066] 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.

[0067] 2. Extraction and library construction of porcine genomic DNA

[0068] (1) Take an appropriate amount of pig ear tissue, chop it into small pieces and place it into a 1.5 mL Axgen tube.

[0069] (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.

[0070] (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).

[0071] (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.

[0072] (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).

[0073] (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).

[0074] (7) Centrifuge at 12000 rpm for 15 minutes at room temperature and remove the supernatant.

[0075] (8) Add 0.5 mL of 70% ethanol to each centrifuge tube and gently invert to fully rinse the precipitated DNA.

[0076] (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.

[0077] (10) Air-dry the DNA for 10 minutes.

[0078] (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.

[0079] (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.

[0080] (13) Library construction: The sequencing library was prepared using the BGI library construction kit.

[0081] (14) On-line sequencing: After library preparation was completed and quality was tested, DNBSEQ-T7 was used for PE150 sequencing.

[0082] 3. SNP chip genotype determination and genotype data quality control

[0083] 590 DNA samples of Large White pigs were sent to Beijing Gezhi Boya Biotechnology Co., Ltd. for 20× deep sequencing and low-depth resequencing.

[0084] 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.

[0085] Table 1 Filter parameters

[0086] 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

[0087] 4. Data collation and analysis

[0088] (1) Phenotypic data analysis: The collected phenotypic data of total litter size of 590 Large White pigs were collated in Excel. Descriptive statistics were performed on the total litter size phenotypic data, and the sample size, mean, standard deviation, standard error, maximum value, minimum value and quartile of each variable were calculated.

[0089] (2) Statistical analysis: Excel 2021 was used to calculate various polymorphism indicators. The relevant polymorphism indicator calculation formula is as follows:

[0090] (2.1) Gene frequency (AF)

[0091]

[0092] (2.2) Genotype frequency (GF)

[0093]

[0094] (2.3) Genetic heterozygosity (He)

[0095]

[0096] (2.4) Effective number of alleles (Ne)

[0097]

[0098] (2.5) Polymorphic Information Content (PIC)

[0099]

[0100] (2.6) Correlation analysis

[0101] 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.

[0102] 5. Capillary electrophoresis detection of SNP

[0103] (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.

[0104] (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.

[0105] (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.

[0106] Table 2 Reaction system configuration

[0107] 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

[0108] 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.

[0109] Table 3 Primer sequences

[0110]

[0111] (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.

[0112] (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.

[0113] Table 4 PCR program

[0114]

[0115] (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.

[0116] Capillary electrophoresis detection, the results are as follows Figure 1 As shown:

[0117] (1) The PCR amplification product was centrifuged at 3500 rpm for 2 min in a benchtop or plate centrifuge;

[0118] (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;

[0119] (3) Cover with a rubber pad and centrifuge briefly using a benchtop or plate centrifuge;

[0120] (4) 95°C, denature for 5 min, then immediately place on ice for 3 min;

[0121] (5) Perform capillary electrophoresis using a gene analyzer. Select the "Fragment" electrophoresis method. Data analysis was performed using ELISA software (Applied Biosystems).

[0122] 6. Results Analysis

[0123] Table 5

[0124]

[0125] Table 6

[0126]

[0127] The genotype test results showed that:

[0128] For the SNP (rs334299237) site, the genotype of 236 pigs was TT genotype, and the genotype of 175 pigs was TC genotype; 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 (rs325934988) site, the genotype of 263 pigs was GG genotype, and the genotype of 145 pigs was GA genotype.

[0129] The allele frequency and gene frequency results of the porcine CREBRF gene in the detected pig population are shown in Table 5: For the SNP (rs334299237) site, the TT genotype frequency is 0.4155, the TC genotype frequency is 0.3081, the genotype frequency of TT is higher than that of TC, and the TT allele is a dominant gene. For the SNP (rs318687930) site, the GG genotype frequency is 0.4331, the GA genotype frequency is 0.3275, the genotype frequency of GG is higher than that of GA, and the GG allele is a dominant gene. For the SNP (rs325934988) site, the GG genotype frequency is 0.4630, the GA genotype frequency is 0.2553, the genotype frequency of GG is higher than that of GA, and the GG allele is a dominant gene.

[0130] As can be seen from Table 6: Among 590 large white pigs, there are 236 TT genotypes and 175 TC genotypes at the SNP (rs334299237) site. There is a very significant difference between CREBRF and the total number of piglets born in the first parity, and the TC type is greater than the TT type. Among 590 large white pigs, there are 246 GG genotypes and 186 GA genotypes at the SNP (rs318687930) site. There is a very significant difference between CREBRF and the total number of piglets born in the first parity and the total number of piglets born in the multiparity, and the GA type is greater than the GG type. Among 590 large white pigs, there are 263 GG genotypes and 145 GA genotypes at the SNP (rs325934988) site. There is a very significant difference between CREBRF and the total number of piglets born in the first parity, and the GA type is greater than the GG type.

[0131] 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 Table 6: SNP (rs334299237) locus has a significant effect on the total litter size of pigs in the first birth, and the total litter size of TC genotype in the first birth is significantly higher than that of TT genotype, so in actual pig breeding, TC genotype pigs have more total litter size in the first birth. SNP (rs318687930) locus has a significant effect on the total litter size of pigs, and the total litter size of GA genotype in the first birth and the total litter size of multiparity are significantly higher than those of GG genotype, so in actual pig breeding, GA genotype pigs have more total litter size. SNP (rs325934988) locus has a significant effect on the total litter size of pigs in the first birth, and the total litter size of GA genotype in the first birth is significantly higher than that of GG genotype, so in actual pig breeding, GA genotype pigs have more total litter size in the first birth.

[0132] In summary, the nucleotides at the SNP (rs334299237) site of the pig CREBRF gene can be used to determine whether the pig individual has the TC genotype or the TT genotype, thereby assisting in identifying the total number of piglets born at the first birth: the total number of piglets born at the first birth with the TC genotype is significantly higher than that with the TT genotype. The TC genotype is a heterozygous T and C nucleotide at position 51160002 on chromosome 16 of the pig; the TT genotype is a homozygous T nucleotide at position 51160002 on chromosome 16 of the pig.

[0133] The nucleotides at the SNP (rs318687930) 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 the identification of the total number of first-born pigs and the total number of pigs born per parity: the total number of first-born pigs and the total number of pigs born per parity of the GA genotype are significantly higher than those of the GG 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.

[0134] The nucleotides at the SNP (rs325934988) site of the pig CREBRF gene can be used to determine whether the pig individual is GA genotype or GG genotype, thereby assisting in identifying the total number of first-born pigs: the total number of first-born pigs with the GA genotype is significantly higher than that with the GG genotype. The GA genotype is a heterozygous G and A nucleotide at position 51225561 on chromosome 16 of the pig; the GG genotype is a homozygous G nucleotide at position 51225561 on chromosome 16 of the pig.

[0135] 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 total litter size trait of pigs, wherein the SNP sites include: One or more of SNP site rs334299237, SNP site rs318687930 and SNP site rs325934988.

2. The use according to claim 1, characterized in that: The total piglet number trait includes the total piglet number of first parity and / or the total piglet number of multiparity.

3. The use according to claim 2, characterized in that: The product includes a substance for detecting the SNP site rs334299237, and the total piglet number trait includes the total piglet number of first birth; and / or, The product includes a substance for detecting the SNP site rs318687930, and the total piglet number trait includes the total piglet number of first parity and the total piglet number of multiparity; and / or, The product includes a substance for detecting the SNP site rs325934988, and the total piglet number trait includes the total piglet number of first birth.

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 rs334299237 has nucleotide sequences as shown in SEQ ID NOs. 1 and 2, respectively; the primer pair used to amplify the SNP site rs318687930 has nucleotide sequences as shown in SEQ ID NOs. 3 and 4, respectively; and, the primer pair used to amplify the SNP site rs325934988 has nucleotide sequences as shown in SEQ ID NOs. 5 and 6, respectively.

6. A kit for predicting the total litter size of pigs, characterized in that: Includes at least one of the following primer pairs: The primer pair used to amplify the SNP site rs334299237 has nucleotide sequences as shown in SEQ ID NOs. 1 and 2, respectively; the primer pair used to amplify the SNP site rs318687930 has nucleotide sequences as shown in SEQ ID NOs. 3 and 4, respectively; and, the primer pair used to amplify the SNP site rs325934988 has nucleotide sequences as shown in SEQ ID NOs. 5 and 6, respectively.

7. A method for predicting the total litter size of pigs, characterized in that: The method comprises predicting a sow that meets at least one of (i) to (iii) as a sow having a trait of high total litter size: (i) The genotype of SNP site rs334299237 is TC, (ii) The genotype of SNP site rs318687930 was GA; (iii) The genotype of SNP site rs325934988 is GA.

8. The method for predicting the total litter size of pigs according to claim 7, characterized in that: The method comprises predicting a sow that meets at least one of (i) and / or (iii) as a sow having a trait of high total litter size at first parity: (i) The genotype of SNP site rs334299237 is TC, (iii) the genotype of SNP site rs325934988 was GA; and / or, including predicting the sows meeting (ii) as sows having a high total litter size in first parity and a high total litter size in multiparity: (ii) The genotype of SNP site rs318687930 is GA.

9. A method for screening sows with a high total litter size trait, characterized in that: The method comprises screening sows having a trait of high total litter size predicted by the method of claim 7 or 8.

10. A method for breeding sows with a high total litter size trait, characterized in that: The method comprises providing the sow with at least one of the following genotypes: (i) The genotype of SNP site rs334299237 is TC, (ii) The genotype of SNP site rs318687930 was GA; (iii) The genotype of SNP site rs325934988 is GA.

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