A SNP molecular marker related to piglet number trait and its application

Abstract

The present invention provides a SNP molecular marker related to the piglet number trait and its application, which relates to the field of biotechnology. The application includes applying a substance for detecting SNP sites to prepare a product for predicting the piglet number trait, and the SNP sites include: SNP site rs334299237 and SNP site rs325934988. These SNP sites are related to the piglet number trait, and can be used to predict the piglet number trait and for the screening and breeding of sows with high piglet number trait.

Description

Technical Field

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

[0002] The following statements merely provide background information related to the present disclosure and do not necessarily constitute prior art.

[0003] 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.

[0004] The trait of piglet number born alive is of great significance for the evaluation of sow reproductive efficiency and pig breeding. By studying the trait of piglet number born alive, we can screen out breeding pigs with high potential for piglet number born alive, 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.

[0005] Therefore, the identification of genes related to the number of piglets born alive 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.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The purpose of the present invention is to overcome the defects of the prior art and provide a SNP molecular marker related to the trait of piglet birth alive and its application.

[0008] In order to solve the above technical problems, the present invention particularly adopts the following technical solutions:

[0009] In a first aspect, a method for preparing a product for predicting the trait of piglet born alive is provided, wherein the SNP sites include: SNP site rs334299237 and SNP site rs325934988.

[0010] In a second aspect, a kit for predicting piglet number born alive is provided, the kit comprising at least one of the following primer pairs:

[0011] The primer pair used to amplify the SNP site rs334299237 has nucleotide sequences shown in SEQ ID NOs. 1 and 2, respectively; and, the primer pair used to amplify the SNP site rs325934988 has nucleotide sequences shown in SEQ ID NOs. 3 and 4, respectively.

[0012] In a third aspect, a method for predicting the piglet number trait is provided, comprising predicting a sow that meets (i) or (ii) as a sow with a high piglet number trait:

[0013] (i) The genotype of SNP site rs334299237 is TC,

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

[0015] In a fourth aspect, a method for screening sows with a high number of piglets born alive trait is provided, the screening method comprising screening sows with a high number of piglets born alive trait predicted by the method described in the third aspect.

[0016] In a fifth aspect, a method for breeding sows with a high live piglet number 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 rs325934988 is GA.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention uses low-depth resequencing and whole genome association analysis methods to find SNP molecular markers related to the piglet number trait, and finds that two SNP sites rs334299237 and rs325934988 are related. Sows with specific genotypes at the above two sites have a higher number of piglets born alive. This SNP molecular marker related to the piglet number trait can be used to predict the piglet number trait and screen sows with high piglet number trait, as well as a breeding method for obtaining sows with high piglet number trait. 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

[0021] 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.

[0022] Figure 1 This is a peak diagram of genotype detection obtained by capillary electrophoresis detection in an embodiment of the present invention. DETAILED DESCRIPTION

[0023] 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.

[0024] 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 the process of 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 number of piglets born alive is an important indicator for measuring the reproductive traits of sows. Therefore, the present invention explores the correlation between the single nucleotide polymorphism site of the CREBRF gene and the number of piglets born alive.

[0025] 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 piglet number trait. The present invention found that two SNP sites, rs334299237 and rs325934988, are associated with the piglet number trait.

[0026] 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 heterozygous of T and C, the sow has a high number of live piglets.

[0027] 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 number of live piglets.

[0028] In this article, "live piglets born" refers to the number of live piglets born by a sow after giving birth within a certain period of time. The live piglets born is one of the important parameters to measure the reproductive capacity of a sow. "High live piglets born" refers to the number of live piglets born that is higher than the average of the breed. It not only reflects the reproductive health of the sow itself, but is also closely related to feeding management, genetic factors, environmental conditions, etc. For farmers, increasing the number of live piglets means higher production efficiency, because more live piglets can be converted into more commercial pigs, thereby increasing economic benefits.

[0029] Based on the above findings, in a first aspect, a method for using a substance for detecting SNP sites in preparing a product for predicting the trait of piglet birth alive is provided, wherein the SNP sites include: SNP site rs334299237 and SNP site rs325934988.

[0030] In an optional embodiment, the pig live pig number trait includes the first-born live pig number and / or the multi-born live pig number.

[0031] In an optional embodiment, the product includes a substance for detecting the SNP site rs334299237, and the piglet number trait includes first birth or piglet number; and / or,

[0032] The product includes a substance for detecting the SNP site rs325934988, and the pig live piglet number trait includes the first-born live piglet number.

[0033] In an optional embodiment, the product includes substances for detecting: SNP site rs334299237 and SNP site rs325934988.

[0034] 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.

[0035] In an optional embodiment, the material for detecting SNP sites includes at least one 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; and a primer pair for amplifying SNP site rs325934988, the nucleotide sequences of which are shown in SEQ ID NOs. 3 and 4, respectively.

[0036] In the second aspect, a kit for predicting the number of piglets born alive is provided, which kit includes 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; and a primer pair for amplifying the SNP site rs325934988, the nucleotide sequences of which are shown in SEQ ID NOs. 3 and 4, respectively.

[0037] In a third aspect, a method for predicting the piglet number trait is provided, comprising predicting a sow that meets (i) or (ii) as a sow with a high piglet number trait:

[0038] (i) The genotype of SNP site rs334299237 is TC,

[0039] (ii) The genotype of SNP site rs325934988 is GA.

[0040] In an optional embodiment, the method comprises predicting the sows meeting the above (i) and / or (ii) as sows having a high number of live piglets born:

[0041] (i) The genotype of SNP site rs334299237 is TC,

[0042] (ii) The genotype of SNP site rs325934988 is GA.

[0043] The method for predicting the piglet number born alive provided in the third aspect includes using one or more of the SNP site rs334299237 and the SNP site rs325934988 as the only identification target; 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 piglet number born alive trait.

[0044] In a fourth aspect, a method for screening sows with a high number of piglets born alive trait is provided, comprising screening sows with a high number of piglets born alive trait predicted by the method described in the third aspect.

[0045] In a fifth aspect, a method for breeding sows with a high live piglet number trait is provided, the breeding method comprising enabling the sow to obtain at least one of the following genotypes:

[0046] (i) The genotype of SNP site rs334299237 is TC,

[0047] (ii) The genotype of SNP site rs325934988 is GA.

[0048] 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.

[0049] In an optional embodiment, the pig in any of the above embodiments is a Large White pig.

[0050] 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.

[0051] Example 1

[0052] 1. Experimental sample collection

[0053] The experimental population of this example 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.

[0054] 2. Extraction and library construction of porcine genomic DNA

[0055] (1) Cut an appropriate amount of pig ear tissue, chop it into pieces and place it into a 1.5 mL Axgen tube.

[0056] (2) Take a 50 mL BD centrifuge tube, mix the proteinase K with a final concentration of 0.4 mg / mL and the lysis buffer, and add 0.5 mL of lysis buffer to the 1.5 mL centrifuge tube containing the pig ear tissue for lysis.

[0057] (3) Place the centrifuge tubes evenly and parallely on the rocking plate of the constant temperature hybridization oven (close the tube caps 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 digested mixture is milky).

[0058] (4) After the sample is 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 ice bath for 15 minutes.

[0059] (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).

[0060] (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 precipitates appear in the solution (if no flocculent precipitates appear, the solution can be placed in a -20 °C refrigerator for 2 hours or in a 4 °C refrigerator overnight).

[0061] (7) Centrifuge at 12000 rpm at room temperature for 15 minutes and remove the supernatant.

[0062] (8) Add 0.5 mL of 70% ethanol to each centrifuge tube and gently invert to thoroughly rinse the precipitated DNA.

[0063] (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.

[0064] (10) Air-dry the DNA for 10 minutes.

[0065] (11) Use a pipette to add 0.1 mL of TE buffer to each tube, dissolve the DNA precipitate, and place the tube at 55°C for 2 hours, shaking it several times to ensure that the DNA is fully dissolved.

[0066] (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.

[0067] (13) Library construction: Prepare the sequencing library using the BGI library construction kit.

[0068] (14) On-line sequencing: After library preparation is completed and quality testing is performed, DNBSEQ-T7 is used for PE150 sequencing.

[0069] 3. SNP chip genotype determination and genotype data quality control

[0070] 590 DNA samples of Large White pigs were sent to Beijing Gezhi Boya Biotechnology Co., Ltd. for 20× deep sequencing and low-depth resequencing.

[0071] 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.

[0072] Table 1 Filter parameters

[0073]

[0074] 4. Data collation and analysis

[0075] (1) Phenotypic data analysis: The collected phenotypic data of the number of piglets born alive of 590 Large White pigs were sorted in Excel. Descriptive statistics were performed on the phenotypic data of the number of piglets born alive, and the sample size, mean, standard deviation, standard error, maximum value, minimum value and quartile of each variable were calculated.

[0076] (2) Statistical analysis: Use Excel 2021 to calculate various polymorphism indicators. The relevant polymorphism indicator calculation formula is as follows:

[0077] (2.1) Gene frequency (AF)

[0078] Formula (i);

[0079] Formula (ii).

[0080] (2.2) Genotype frequency (GF)

[0081] Formula (iii);

[0082] Formula (IV);

[0083] Formula (v).

[0084] (2.3) Genetic heterozygosity (He)

[0085] Formula (VI).

[0086] (2.4) Effective number of alleles (Ne)

[0087] Formula (ⅶ).

[0088] (2.5) Polymorphic Information Content (PIC)

[0089] Formula (ⅷ).

[0090] (2.6) Correlation analysis

[0091] The LSD method was used for "Anova" analysis. The phenotypes of the number of piglets born alive of different genotypes were expressed as mean ± standard deviation. The linear model of plink software was used for association analysis between genotype and the number of piglets born alive phenotype. The field effect was used as a covariate correction. P ≤ 0.05 was considered to be a significant difference, and P ≤ 0.01 was considered to be an extremely significant difference.

[0092] 5. Capillary electrophoresis detection of SNP

[0093] (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.

[0094] (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.

[0095] (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 of it into a PCR reaction tube. The primer sequences are shown in Table 3.

[0096] Table 2 Reaction system configuration

[0097]

[0098] Note: PCR products should be stored at 2-8°C and tested within 24 hours, or stored at -20°C±5°C and tested within 7 days. The number of freeze-thaw cycles should not exceed 3 times.

[0099] Table 3 Primer sequences

[0100]

[0101] (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.

[0102] (5) Place each reaction tube in the reaction tank of the PCR instrument and run the PCR program. The PCR amplification program is shown in Table 4.

[0103] Table 4 PCR program

[0104]

[0105] (6) Electrophoresis detection of PCR products: 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.

[0106] Capillary electrophoresis detection, the results are as follows Figure 1 As shown:

[0107] (1) Centrifuge the PCR amplification product at 3500 rpm for 2 min in a benchtop or plate centrifuge;

[0108] (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, and dispense 9 μL into a 96-well plate. Add 1 μL of PCR product supernatant (stock solution or dilution) to each well.

[0109] (3) Cover with a rubber pad and centrifuge briefly using a desktop or plate centrifuge;

[0110] (4) Denature at 95 °C for 5 min, then immediately place on ice for 3 min;

[0111] (5) Perform capillary electrophoresis using a genetic analyzer. Select the “Fragment” electrophoresis method. Use GeneMapper® analysis software (Applied Biosystems) for data analysis.

[0112] 6. Results Analysis

[0113] Table 5

[0114]

[0115] Table 6

[0116]

[0117] The genotype test results showed that:

[0118] 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 (rs325934988) site, the genotype of 263 pigs was GG genotype, and the genotype of 145 pigs was GA genotype.

[0119] The allele frequency and gene frequency results of the porcine CREBRF gene in the detected pig population are shown in Table 5:

[0120] For SNP (rs334299237), the genotype frequency of TT was 0.4155, the genotype frequency of TC was 0.3081, the genotype frequency of TT was higher than that of TC, and the TT allele was the dominant gene. For SNP (rs325934988), the genotype frequency of GG was 0.4630, the genotype frequency of GA was 0.2553, the genotype frequency of GG was higher than that of GA, and the GG allele was the dominant gene.

[0121] 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 number of first-born live pigs, and the TC type is greater than the TT 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 number of first-born live pigs, and the GA type is greater than the GG type.

[0122] The linear model of plink software was used to perform association analysis between genotype and live piglet number phenotype, and the field effect was used as a covariate correction. The results are shown in Table 6:

[0123] The SNP (rs334299237) locus has a significant effect on the number of first-born live pigs in pigs. The number of first-born live pigs of the TC genotype is significantly higher than that of the TT genotype. Therefore, in actual pig breeding, the TC genotype pigs have more first-born live pigs. The SNP (rs325934988) locus has a significant effect on the number of first-born live pigs in pigs. The number of first-born live pigs of the GA genotype is significantly higher than that of the GG genotype. Therefore, in actual pig breeding, the GA genotype pigs have more first-born live pigs.

[0124] 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 number of first-born live piglets: the number of first-born live piglets 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.

[0125] The nucleotides at the SNP (rs325934988) 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 live piglets: the number of first-born live piglets 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.

[0126] 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 reagent for detecting SNP sites in the preparation of a product for predicting the trait of piglet birth, wherein the SNP sites include: SNP site rs334299237 and SNP site rs325934988; Sows meeting (i) or (ii) are predicted to have the trait of high piglets born alive: (i) The genotype of SNP site rs334299237 is TC, (ii) The genotype of SNP site rs325934988 was GA; The reagents for detecting SNP sites are primers and / or probes.

2. The use according to claim 1, characterized in that: The pig live pig number trait includes the first-born live pig number and / or the perennial live pig number.

3. The use according to claim 2, characterized in that: The product includes a reagent for detecting the SNP site rs334299237, and the piglet number trait includes first birth or piglet number; and / or, The product includes a reagent for detecting the SNP site rs325934988, and the pig live piglet number trait includes the first-born live piglet number.

4. The use according to claim 1, characterized in that: The reagent 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 shown in SEQ ID NOs. 1 and 2, respectively; and, the primer pair used to amplify the SNP site rs325934988 has nucleotide sequences shown in SEQ ID NOs. 3 and 4, respectively.

5. A method for screening sows with high live piglet number trait, characterized in that: This includes screening sows that meet (i) or (ii): (i) The genotype of SNP site rs334299237 is TC, (ii) The genotype of SNP site rs325934988 is GA.

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