SNP molecular marker related to identifying sow infanticide behavior and application thereof in genetic breeding

Abstract

The application discloses a SNP molecular marker related to identifying the frequency of sow killing infant behavior and application thereof in genetic breeding. The application aims to identify or assist in identifying the frequency of sow killing infant behavior. The application relates to the technical field of molecular biology and genetic breeding, and particularly relates to a SNP molecular marker related to identifying the frequency of sow killing infant behavior and application thereof in genetic breeding. The SNP is a site on a pig chromosome 3, and the nucleotide type is T or C, which is the 89th nucleotide in sequence 3 in the sequence table. The frequency of the sow individual to be detected with the genotype of TT or CC killing infant behavior is lower than or lower than the candidate pig to be detected with the genotype of TC. The application can be used for assisting in identifying the frequency of sow killing infant behavior, early screening of pig breeds can be performed, the survival rate of piglets can be improved, and the application can be combined with other molecular markers related to the frequency of sow killing infant behavior to assist in pig breeding.

Description

Technical Field

[0001] This invention relates to the fields of molecular biology and genetic breeding technology, and in particular to SNP molecular markers associated with identifying sow infanticide behavior and their application in genetic breeding. Background Technology

[0002] The quality of a sow's maternal behavior has a significant impact on the survival, growth, development, and welfare of piglets. Sow infanticide refers to the behavior of a sow biting or even killing her newborn piglets during farrowing or within 24 hours of birth; it is also known as sow biting behavior. This abnormal behavior is an extremely egregious form of maternal behavior. Studies have reported that the frequency of infanticide in commercial sow populations reaches 7-12%, with an even higher frequency in primiparous sows, causing significant losses in production.

[0003] Sow infanticide behavior is regulated by both neurological and endocrine factors. The nervous system in the brain regulates hormone secretion, thereby controlling the occurrence of this behavior. The YWHAG gene has been reported to be highly expressed in the brain, regulating multiple cellular signaling pathways. Mutations in this gene have been found to lead to developmental encephalopathy, epilepsy, and developmental delays or regressions. Therefore, we conducted an association study between the sow YWHAG gene and sow maternal behavior, identifying significant association sites. By utilizing molecular biology techniques, we aim to provide an effective means for the early identification of sow infanticide behavior. Summary of the Invention

[0004] The problem this invention aims to solve is how to identify or assist in identifying the frequency of sow infanticide behavior.

[0005] To address the above technical problems, this invention first provides an application of a substance for detecting SNP polymorphisms or genotypes in the pig genome.

[0006] The application provided by this invention is the use of substances for detecting SNP polymorphisms or genotypes in the porcine genome in any of the following applications.

[0007] (1) Products that detect single nucleotide polymorphisms or genotypes associated with the frequency of sow infanticide behavior;

[0008] (2) Identify or assist in the identification of products related to the frequency of sow infanticide behavior;

[0009] (3) Products used in pig breeding;

[0010] (4) Prepare products for identifying or assisting in identifying the frequency of sow infanticide behavior;

[0011] (5) Prepare products for screening or breeding pig breeds with low frequency of infanticide behavior in sows;

[0012] The SNP is a site on pig chromosome 3, and its nucleotide type is T or C, which is the 89th nucleotide of sequence 3 in the sequence listing.

[0013] Using the pig genome sequence (version 10.2 (http: / / asia.ensembl.org / Sus_scrofa / Info / Index)) as the reference genome, the SNP is located at 9907599 bp on pig chromosome 3 (specifically, position 89 of sequence 3 in the sequence listing).

[0014] This invention also provides a method for identifying or assisting in the identification of the frequency of infanticide behavior in sows.

[0015] The method for identifying or assisting in identifying the frequency of sow infanticide behavior provided by this invention includes detecting the genotype of the aforementioned SNP in the genome of the pig to be tested, and identifying or assisting in identifying the frequency of sow infanticide behavior based on the genotype of the SNP of the pig to be tested: the SNP is a locus on pig chromosome 3, its nucleotide type is T or C, and it is the 89th nucleotide of sequence 3 in the sequence listing; the genotype is TT, TC or CC, wherein the genotype TT is homozygous for the SNP being T, the genotype CC is homozygous for the SNP being C, and the genotype TC is heterozygous for the SNP being both T and C;

[0016] The method for identifying or assisting in identifying the frequency of sow infanticide behavior is as follows:

[0017] 1) Pigs with the genotype TT or CC of the SNP mentioned above are or candidates for pig breeds with low frequency of sow infanticide behavior.

[0018] 2) Pigs with the genotype TC of the SNP mentioned above are or candidate pig breeds with a high frequency of sow infanticide behavior;

[0019] 3) The frequency of infanticide behavior in the tested sows with the SNP genotype TT or CC mentioned above is lower or lower than that in the tested sows with the SNP genotype TC.

[0020] As one implementation method, the method for identifying or assisting in identifying the frequency of sow infanticide behavior may include the following steps:

[0021] (1) Sequencing was performed using the genomic DNA of the pig to be tested as a template and a primer composition was used; the primer composition consisted of primer U and primer D;

[0022] The primer U is a single-stranded DNA molecule whose nucleotide sequence is sequence 1 in the sequence listing;

[0023] The primer D is a single-stranded DNA molecule whose nucleotide sequence is sequence 2 in the sequence listing;

[0024] (2) After completing step (1), determine the genotype of the SNP locus of the pig to be tested;

[0025] (3) Identify the frequency of infanticide behavior in the test pigs based on the genotype results: the frequency of infanticide behavior in the test sows with the SNP genotype TT or CC is lower or lower than that in the test pigs with the SNP genotype TC.

[0026] In the above method, the PCR amplification system consists of: 200 ng genomic DNA, 5 μL 10× PCR amplification buffer, dNTPs final concentration of 10 mM, 50 ng each of forward and reverse primers, 0.75 U Taq DNA polymerase, and Mg... 2+ 2.5 mmol / L, add ddH2O to make up the volume to 50 μL.

[0027] In the above method, the PCR amplification program is as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 20 s, 60.5℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 35 cycles; and finally 72℃ extension for 10 min.

[0028] The application of the above-described methods in pig breeding also falls within the scope of protection of this invention.

[0029] This invention also provides a method for pig breeding.

[0030] The pig breeding method provided by this invention includes detecting the genotype of the SNP locus mentioned above in the pig genome, selecting pigs with the genotype TT or CC of the SNP as parents for breeding, wherein the TT genotype is homozygous for the SNP T, and the CC genotype is homozygous for the SNP C. The breeding objective of this method includes selecting pigs with a low frequency of infanticide behavior in sows. The frequency of infanticide behavior in the tested sows with the genotype TT or CC of the SNP is lower than or lower than that in the tested pigs with the genotype TC of the SNP.

[0031] In the above applications or methods, the pig breeding refers to the development of pig breeds with a low frequency of infanticide behavior in sows.

[0032] As an implementation method, pig breeding methods may include the following steps:

[0033] (1) Using the genomic DNA of the pig to be tested as a template, PCR amplification was performed using the above primer combination;

[0034] (2) After completing step (1), sequencing is performed to determine the genotype of the SNP in the pig to be tested;

[0035] (3) Select pigs with SNP genotypes of TT or CC for pig breeding.

[0036] This invention also provides products for detecting polymorphisms or genotypes of the aforementioned SNPs in the pig genome.

[0037] The product provided by this invention for detecting the polymorphism or genotype of the aforementioned SNPs in the pig genome is a product containing the aforementioned substance for detecting the polymorphism or genotype of SNPs in the pig genome, and may be any one of the following G1)-G5):

[0038] G1) Products that detect single nucleotide polymorphisms or genotypes associated with the frequency of sow infanticide behavior;

[0039] G2) Products used to identify or assist in identifying the frequency of sow infanticide behavior;

[0040] G3) Products used in pig breeding;

[0041] C4) Products made from pig breeds with low frequency of infanticide behavior in sows;

[0042] C5) Prepare products for screening or breeding pig breeds with low frequency of infanticide behavior in sows.

[0043] In the above applications, methods, and products, the substance may be a reagent and / or instrument required to determine the polymorphism or genotype of the SNP site by at least one of the following methods: DNA sequencing, restriction fragment length polymorphism, single-strand conformation polymorphism, denaturing high-performance liquid chromatography, and SNP chips. The SNP chips include chips based on nucleic acid hybridization reactions, chips based on single-base extension reactions, chips based on allele-specific primer extension reactions, chips based on one-step reactions, chips based on primer ligation reactions, chips based on restriction endonuclease reactions, chips based on protein-DNA binding reactions, and chips based on fluorescent molecule-DNA binding reactions.

[0044] In the above applications, methods, or products, the substance may be D1), D2), or D3):

[0045] D1) The substance described is a primer composition for amplifying porcine genomic DNA fragments containing the SNPs described above;

[0046] D2) The substance described is a PCR reagent containing the primer composition described in D1);

[0047] D3) The substance is a kit containing the primer composition described in D1) or the PCR reagent described in D2).

[0048] Optionally, the amplification may be PCR amplification.

[0049] Optionally, the primer composition consists of primer U and primer D.

[0050] Optionally, primer U is a single-stranded DNA molecule whose nucleotide sequence is sequence 1 in the sequence listing; primer D is a single-stranded DNA molecule whose nucleotide sequence is sequence 2 in the sequence listing.

[0051] The present invention also provides a DNA molecule, the nucleotide sequence of which is shown in Sequence 3 of the sequence listing.

[0052] Optionally, in the above applications, the DNA molecule serves as a detection target.

[0053] In the above methods or applications, the pig is a Large White pig.

[0054] The present invention also provides a device for screening the frequency of infanticide behavior in sows, comprising the following parts:

[0055] A sequencing device, wherein the sequencing device is used to sequence DNA fragments in the genome of a pig to be tested that include the SNP of claim 1, and to obtain sequencing results;

[0056] A comparison device, connected to the sequencing device, is used to determine the genotype of the SNP based on the sequencing results;

[0057] An analysis device, connected to the comparison device, is used to determine the frequency of sow infanticide behavior based on the genotype.

[0058] This invention determines the genotype of a pig by detecting the base at position 9907599 of chromosome 3 in the pig genome (Sscrofa10.2 Primary Assembly). The genotype is then used to further identify or assist in the identification of sows exhibiting infanticide behavior. Sows with a lower frequency of infanticide behavior are selected, thereby obtaining sows with good maternal instincts and improving piglet survival rates.

[0059] The substances provided by this invention for detecting SNP polymorphisms or genotypes in the pig genome are used for early detection in pigs, effectively alleviating the problem of long selection time for superior breeding pigs in actual production, reducing breeding costs, and enabling the effective selection of sows with good maternal behavior, thereby improving piglet survival rates. The method provided by this invention is simple to operate, low in cost, highly accurate, and can achieve automated direct detection, playing a significant role in sow breeding. The substances for detecting the aforementioned SNP polymorphisms and genotypes can be combined with other substances (such as substances for detecting single nucleotide polymorphisms or genotypes of other molecular markers related to the frequency of sow infanticide behavior) to prepare products for identifying the frequency of sow infanticide behavior. Attached Figure Description

[0060] Figure 1 The sequencing results are shown for the sequence near the SNP site SSC3g.9907599. Detailed Implementation

[0061] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0062] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0063] Unless otherwise specified, all quantitative experiments in the following examples are performed in triplicate.

[0064] This invention uses the Large White pig (Sus scrofa) breed, which was purchased from the Changping Livestock and Poultry Experimental Base of the Beijing Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences.

[0065] The PCR amplification sequences in the following examples all refer to the pig genome (Sscrofa10.2 Primary Assembly) sequence.

[0066] Example 1: Discovery of SNP markers associated with sow infanticide behavior

[0067] I. Determination of the SSC3g.9907599T>C polymorphism site

[0068] (a) Using 20 Large White pigs as experimental materials, genomic DNA was extracted from their ear margin tissues, and the presence of mutations at the site was determined by pooling and individual sequencing.

[0069] (II) Primer Design and Synthesis

[0070] Using the YWHAG gene as a reference sequence in the International Swine Genome Project, version 10.2 (http: / / asia.ensembl.org / Sus_scrofa / Info / Index), the following primers were designed and synthesized using Primer 5:

[0071] U (upstream primer): 5'-GAAGCAGGAGTTGAGGCA-3' (SEQ ID No. 1);

[0072] D (downstream primer): 5'-AAGGAGGAAAGAAGACAGAGTT-3' (SEQ ID No. 2).

[0073] (III) PCR Amplification

[0074] Using the genomic DNA of the Large White pig obtained in step (I) as a template, PCR amplification was performed with U and D as primers to obtain PCR amplification products.

[0075] PCR amplification system: 200 ng genomic DNA, 5 μL 10× PCR amplification buffer, dNTPs final concentration of 10 mM, 50 ng each of forward and reverse primers, 0.75 U Taq DNA polymerase, Mg 2+ 2.5 mmol / L, add ddH2O to make up the volume to 50 μL.

[0076] PCR amplification program: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 20 s, 60.5℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 35 cycles; final extension at 72℃ for 10 min.

[0077] (iv) Sequencing and Sequence Analysis

[0078] The PCR product was sequenced, and the sequence is shown in SEQ ID No. 3. SEQ ID No. 3 differs by only one base, at position 89 of SEQ ID No. 3 in the sequence listing, and its nucleotide type is either T or C (i.e., T / C polymorphism). In sequence 3 of the sequence listing, y represents c or t. This SNP site is located at physical position 9907599 on chromosome 3 of the pig genome (Sscrofa10.2 Primary Assembly). Figure 1 As indicated by the middle arrow, this SNP locus is therefore named SNP-SSC3g.9907599. This SNP locus has three possible genotypes: CC, TC, or TT. CC is the homozygous genotype of SNP-SSC3g.9907599 as C, TT is the homozygous genotype of SNP-SSC3g.9907599 as T, and TC is the heterozygous genotype of SNP-SSC3g.9907599 as both T and C. Individuals with all T bases at position 9907599 of chromosome 3 in the pig genome (Sscrofa10.2 Primary Assembly) are homozygous TT individuals; individuals with all C bases at position 9907599 of chromosome 3 in the pig genome (Sscrofa10.2 Primary Assembly) are homozygous CC individuals; and individuals with both T and C bases at position 9907599 of chromosome 3 in the pig genome (Sscrofa10.2 Primary Assembly) are heterozygous TC individuals.

[0079] II. Association Analysis of SNP Locus SNP-SSC3g.9907599 with Sow Infanticide Behavior

[0080] To determine whether SNP-SSC3g.9907599 is associated with the sow's infanticide behavior trait, the following experiment was conducted using 225 healthy Large White pigs:

[0081] (I) Genotyping based on the SNP locus SNP-SSC3g.9907599

[0082] Genomic DNA was extracted from the ear margin tissue of 225 pigs. Using the genomic DNA as a template, PCR amplification was performed using the U / D primer pair from step one (ii). The PCR products were then sequenced. The SNP-SSC3g.9907599 genotype of each pig was determined to be homozygous TT, heterozygous TC, or homozygous CC, according to the method in step one (iv).

[0083] The results are shown in Table 1. PCR amplification products of 209 bp were obtained from 225 experimental animals using both U and D primer pairs, and their nucleotide sequences were all sequence 3 in the sequence listing.

[0084] Based on the SNP locus SNP-SSC3g.9907599, 225 experimental animals were divided into three genotypes (as shown in Table 3): CC, TT, and TC. In the TT genotype, nucleotide 89 of sequence 3 in the pig genome was homozygous for T; in the CC genotype, nucleotide 89 of sequence 3 in the pig genome was homozygous for C; and in the TC genotype, nucleotide 89 of sequence 3 in the pig genome was heterozygous for both T and C. Of the 225 animals, 174 were TT genotype, 46 were TC genotype, and 5 were CC genotype.

[0085] (II) Phenotypic determination of infanticide behavior in 225 sows

[0086] Phenotypic identification of infanticide behavior in sows: Cameras were installed in the pig farm to record the behavior of sows from farrowing to the birth of piglets within 24 hours. Whether the sow bit piglets during this time period was recorded as "1" if it occurred, and "0" if it did not. The results are shown in Table 1.

[0087] Table 1. Genotypic and phenotypic information of porcine SNP-SSC3g.9907599

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094] Note: In the phenotype, 0 indicates that no sow infanticide occurred, and 1 indicates that sow infanticide occurred.

[0095] (III) Association analysis between porcine SNP-SSC3g.9907599 genotype and sow infanticide behavior.

[0096] Logit binomial regression analysis was performed to investigate the correlation between the SNP-SSC3g.9907599 genotype and infanticide behavior in sows. The model used is as follows:

[0097] Y = μ + G + e

[0098] Where Y is the measured value of the sow's infanticide behavior trait; μ is the mean; G is the genotype effect; and e is the random error.

[0099] Table 2. Association between porcine SNP-SSC3g.9907599 genotype and sow infanticide behavior.

[0100]

[0101] Note: P<0.05 indicates that different genotypes are significantly associated with different phenotypes.

[0102] The results are shown in Table 2. Of the 225 animals, 174 were of the TT genotype, 46 were of the TC genotype, and 5 were of the CC genotype. Among them, the frequency of infanticide behavior in sows with the TC genotype (SNP-SSC3g.9907599) was 0.0870. The increase in the TC genotype significantly increased the probability of infanticide behavior in sows, exceeding or potentially exceeding the frequency of infanticide behavior in sows with the TT and CC genotypes (P<0.05). Combining the results of all pig genotypes and phenotypes, the P-value for SNP-SSC3g.9907599 reached 0.0311 (P<0.05) (Table 2).

[0103] In conclusion, in practical sow breeding, it is best to select sows with the genotype TT or CC for the SNP as parents, and cull individuals with the genotype TC for the SNP. Early genotyping at the SSC3g.9907599 locus in pigs can effectively select sows with good maternal behavior and improve piglet survival rates.

[0104] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. The application of substances for detecting SNP polymorphisms or genotypes in the pig genome in any of the following: (1) To identify or assist in identifying the frequency of sow infanticide behavior; (2) Prepare products for identifying or assisting in identifying the frequency of sow infanticide behavior; (3) Prepare products from pigs that have been screened or bred with a low frequency of infanticide behavior in sows; Using the pig genome Sscrofa10.2 Primary Assembly sequence as a reference genome, the SNP is located at position 9907599 bp on pig chromosome 3, and its nucleotide type is T or C. The genotype of this SNP site is one of the following three types: CC, TC or TT. in, Sows with the TC genotype had a higher or candidate higher frequency of infanticide than sows with the TT and CC genotypes. The substance is either D1), D2), or D3). D1) The substance is a primer composition for amplifying porcine genomic DNA fragments containing the SNP; D2) The substance is a PCR reagent containing the primer composition described in D1); D3) The substance is a kit containing the primer composition described in D1) or the PCR reagent described in D2).

2. The application according to claim 1, characterized in that: The primer composition consists of primer U and primer D; primer U is a single-stranded DNA molecule with a nucleotide sequence as shown in SEQ ID NO.1; Primer D is a single-stranded DNA molecule with the nucleotide sequence shown in SEQ ID NO.

2.

3. A method for identifying or assisting in identifying the frequency of infanticide behavior in sows, characterized in that: This includes detecting the genotype of SNPs in the genome of the pig to be tested, and identifying or assisting in identifying the frequency of sow infanticide behavior based on the genotype of the SNPs in the pig to be tested. Using the pig genome Sscrofa10.2 Primary Assembly sequence as a reference genome, the SNP is located at position 9907599 bp on pig chromosome 3, and its nucleotide type is T or C. The genotype of this SNP site is one of the following three types: CC, TC or TT. The genotype TT is homozygous for the SNP being T, the genotype CC is homozygous for the SNP being C, and the genotype TC is heterozygous for the SNP being both T and C. The genotyping or auxiliary identification of the frequency of sow infanticide behavior based on the SNP of the pig to be tested is any one of the following: 1) Pigs with the SNP genotype TT or CC are or candidates for pigs with a low frequency of sow infanticide behavior; 2) The test pigs with the SNP genotype TC are or candidates for pigs with a high frequency of sow infanticide behavior; 3) The frequency of infanticide behavior in the tested sows with the SNP genotype TT or CC is lower or lower than that in the tested sows with the SNP genotype TC.

4. The application of the method according to claim 3 in pig breeding, characterized in that, The pig breeding mentioned refers to the selection and breeding of pigs with a low frequency of infanticide behavior in sows.

5. A method for pig breeding, characterized by: The method includes detecting the genotype of the SNP locus as described in claim 1 in the pig genome, selecting pigs with the genotype of TT or CC of the SNP as parents for breeding, wherein the TT genotype is homozygous for the SNP being T, and the CC genotype is homozygous for the SNP being C, and the breeding objective of the method is to select pigs with a low frequency of sow infanticide behavior.

6. Equipment for screening the frequency of sow infanticide behavior, including: A sequencing device for sequencing DNA fragments in the genome of a pig to be tested that include the SNPs as described in claim 1, and obtaining sequencing results; A comparison device is connected to the sequencing device and is used to determine the genotype of the SNP based on the sequencing results; An analysis device, connected to the comparison device, is used to determine the frequency of sow infanticide behavior based on the genotype; wherein, the frequency of infanticide behavior in test sows with the SNP genotype TT or CC is lower or lower than that in test sows with the SNP genotype TC.

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