CN116042861A - SNP molecular marker related to identification of infant killing behavior of sow and application of SNP molecular marker in genetic breeding - Google Patents

SNP molecular marker related to identification of infant killing behavior of sow and application of SNP molecular marker in genetic breeding Download PDF

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CN116042861A
CN116042861A CN202310145148.3A CN202310145148A CN116042861A CN 116042861 A CN116042861 A CN 116042861A CN 202310145148 A CN202310145148 A CN 202310145148A CN 116042861 A CN116042861 A CN 116042861A
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刘欣
王立贤
颜华
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Institute of Animal Science of CAAS
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Abstract

The invention discloses SNP molecular markers related to identification of infant killing frequency of sows and application thereof in genetic breeding. The invention aims to solve the problem of identifying or assisting in identifying the frequency of infant killing behaviors of sows. The invention relates to the technical fields of molecular biology and genetic breeding, in particular to SNP molecular markers related to identification of infant killing behaviors of sows and application of SNP molecular markers in genetic breeding. The SNP is a locus on a pig chromosome 3, the nucleotide type of the SNP is T or C, and the SNP is 89 th nucleotide of a sequence 3 in a sequence table. The frequency of infant killing of individuals of the sow to be detected with the genotype of TT or CC of the SNP is lower than or the candidate is lower than that of the pig to be detected with the genotype of TC. The method can be used for assisting in identifying the frequency of the infant killing behavior of the sow, can be used for early screening of the pig variety, improves the survival rate of the piglet, and can assist in pig breeding by combining with molecular markers related to the infant killing behavior frequency of other sows.

Description

SNP molecular marker related to identification of infant killing behavior of sow and application of SNP molecular marker in genetic breeding
Technical Field
The invention relates to the technical fields of molecular biology and genetic breeding, in particular to SNP molecular markers related to identification of infant killing behaviors of sows and application of SNP molecular markers in genetic breeding.
Background
The quality of sow maternal behavior has important influence on the survival, growth and development and welfare of piglets. The infant killing behavior of a sow refers to the behavior of the sow in biting or even biting a newborn piglet during delivery or within 24 hours after birth of the piglet, also referred to as the biting behavior of the sow. This abnormal behaviour of sows is an extremely severe maternal behaviour. In the commercial sow group, the frequency of infant killing of sows reaches 7-12%, and the occurrence frequency of primiparous sows is higher, so that great loss is caused to production.
The infant killing behavior of sows is regulated by nerves and endocrine. The nervous system of the brain regulates hormone secretion and thus the occurrence of behavior. The YWHAG gene has been reported to regulate various cell signaling pathways by being highly expressed in the brain. And it was found that mutation of the gene resulted in the occurrence of developmental encephalopathy, epilepsy, and developmental retardation or degeneration. Therefore, the related research is carried out on the YWHAG gene of the sow and the maternal behavior of the sow, obvious related sites are found, and an effective means is provided for early identification of the infant killing behavior of the sow by utilizing a molecular biological means.
Disclosure of Invention
The invention aims to solve the problem of identifying or assisting in identifying the frequency of infant killing behaviors of sows.
In order to solve the technical problems, the invention firstly provides application of a substance for detecting polymorphism or genotype of SNP in pig genome.
The application provided by the invention is the application of a substance for detecting the polymorphism or genotype of SNP in pig genome,
(1) Detecting a single nucleotide polymorphism or genotype related to the frequency of infant killing activity of the sow;
(2) Identifying or aiding in identifying a product associated with a frequency of infant killing activity in a sow;
(3) A product for pig breeding;
(4) Preparing a product for identifying or assisting in identifying the infant killing frequency of the sow;
(5) Preparing a product for screening or breeding a pig variety with low infant killing frequency of a sow;
the SNP is a site on a pig chromosome 3, the nucleotide type of the SNP is T or C, and the SNP is 89 th nucleotide of a sequence 3 in a sequence table.
The pig genome sequence (version 10.2 (http:// asia. Ensembl. Org/sus_scrofa/Info/Index)) is used as a reference genome, and the SNP is 9907599bp (specifically, 89 rd position of the sequence 3 in the sequence table) on a pig chromosome 3.
The invention also provides a method for identifying or assisting in identifying the frequency of infant killing behaviors of the sow.
The method for identifying or assisting in identifying the frequency of the infant killing behavior of the sow comprises the steps of detecting the genotype of the SNP in the genome of the to-be-detected pig, and identifying or assisting in identifying the frequency of the infant killing behavior of the sow according to the genotype of the SNP of the to-be-detected pig: the SNP is a site on a pig chromosome 3, the nucleotide type of the SNP is T or C, and the SNP is 89 th nucleotide of a sequence 3 in a sequence table; genotype TT, TC or CC, wherein genotype TT is homozygous for the SNP T, genotype CC is homozygous for the SNP C, and genotype TC is heterozygous for the SNP T and C;
the method for identifying or assisting in identifying the infant killing frequency of the sow comprises the following steps of:
1) The pig to be detected with the genotype of TT or CC of SNP is or is candidate to be a pig variety with low infant killing frequency of the sow;
2) The pig to be detected with the genotype of TC of the SNP is or is candidate to be a pig variety with high infant killing behavior frequency of the sow;
3) The frequency of infant killing of individuals of the sow to be tested with the genotype of the SNP being TT or CC is lower than or the candidate is lower than that of the pig to be tested with the genotype of the SNP being TC.
As an embodiment, the method of identifying or aiding in identifying the frequency of infant killing activity of a sow may comprise the steps of:
(1) Taking genome DNA of a pig to be detected as a template, and sequencing by adopting a primer composition; the primer composition consists of a primer U and a primer D;
the primer U is a single-stranded DNA molecule with a nucleotide sequence of sequence 1 in a sequence table;
the primer D is a single-stranded DNA molecule with a nucleotide sequence of sequence 2 in a sequence table;
(2) After the step (1) is completed, determining the genotype of the SNP locus of the pig to be tested;
(3) And identifying the frequency of infant killing behaviors of the pigs to be tested according to genotype results: and the frequency of infant killing behaviors of individuals of the sow to be detected with the genotype of the SNP being TT or CC is lower than or the candidate is lower than that of the pig to be detected with the genotype of the SNP being TC.
In the above method, the PCR amplification system: 200ng of genome DNA, 5 mu L of 10 XPCR amplification buffer solution, 10mM of final concentration of dNTPs, 50ng of each of the upstream and downstream primers, 0.75U of Taq DNA polymerase, and Mg 2+ 2.5mmol/L with ddH 2 O complements the system to 50. Mu.L.
In the above method, the PCR amplification procedure: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 20s, annealing at 60.5℃for 30s, elongation at 72℃for 30s for 35 cycles; finally, the extension is carried out for 10min at 72 ℃.
The application of the method in pig breeding also belongs to the protection scope of the invention.
The invention also provides a pig breeding method.
The pig breeding method provided by the invention comprises the steps of detecting genotypes of SNP loci in a pig genome, selecting pigs with the genotypes of the SNPs being TT or CC as parents for breeding, wherein the TT genotypes are homozygous for the SNPs being T, and the CC genotypes are homozygous for the SNPs being C. And the frequency of infant killing behaviors of individuals of the sow to be detected with the genotype of the SNP being TT or CC is lower than or the candidate is lower than that of the pig to be detected with the genotype of the SNP being TC.
In the application or the method, the pig breeding is to cultivate a pig variety with low infant killing frequency of the sow.
As an implementation method, the method of pig breeding may comprise the steps of:
(1) Taking genome DNA of a pig to be detected as a template, and carrying out PCR amplification by adopting the primer composition;
(2) After the step (1) is completed, sequencing is carried out, and the genotype of the SNP of the pig to be tested is determined;
(3) Pig breeding is carried out by selecting pigs with genotype TT or CC of SNP.
The invention also provides a product for detecting the polymorphism or genotype of the aforementioned SNP in the pig genome.
The product for detecting the polymorphism or genotype of the SNP in the genome of the pig provided by the invention is a product containing the substance for detecting the polymorphism or genotype of the SNP in the genome of the pig and can be any one of the following G1) to G5):
g1 A product for detecting single nucleotide polymorphism or genotype related to the frequency of infant killing behavior of the sow;
g2 Identifying or aiding in identifying a product of a frequency of infant killing activity in a sow;
g3 A product for pig breeding;
c4 Screening or breeding a product of a pig variety with low infant killing frequency of the sow;
c5 Preparing a product for screening or breeding a pig variety with low infant killing frequency of a sow.
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 enzyme fragment length polymorphism, single-stranded conformational polymorphism, denaturing high performance liquid chromatography and SNP chips. The SNP chip comprises a chip based on nucleic acid hybridization reaction, a chip based on single base extension reaction, a chip based on allele specific primer extension reaction, a chip based on one-step method reaction, a chip based on primer connection reaction, a chip based on restriction enzyme reaction, a chip based on protein DNA binding reaction and a chip based on fluorescent molecule DNA binding reaction.
In the above application, method or product, the substance may be D1), D2) or D3) as follows:
d1 The substance is a primer composition for amplifying a pig genomic DNA fragment containing the SNP as described above;
d2 The substance is a PCR reagent containing the primer composition of D1);
d3 The substance is a kit containing the primer composition of D1) or the PCR reagent of D2).
Alternatively, the amplification may be PCR amplification.
Optionally, the primer composition consists of the primer U and the primer D.
Optionally, the primer U is a single-stranded DNA molecule with a nucleotide sequence of sequence 1 in a sequence table; the primer D is a single-stranded DNA molecule with a nucleotide sequence of sequence 2 in a sequence table.
The invention also provides a DNA molecule, and the nucleotide sequence is shown as a sequence 3 in a sequence table.
Alternatively, in the above application, the DNA molecule serves as a detection target.
In the above method or application, the pig is a white pig.
The invention also provides equipment for screening the frequency of infant killing behaviors of the sow, which comprises the following parts:
a sequencing device for sequencing a DNA fragment comprising the SNP of claim 1 in the genome of a swine to be tested to obtain a sequencing result;
the comparison device is connected with the sequencing device and is used for determining the genotype of the SNP based on the sequencing result;
and the analysis device is connected with the comparison device and is used for determining the frequency of the infant killing behavior of the sow based on the genotype.
According to the invention, the genotype of a pig individual is determined by detecting the 9907599 base of the 3 rd chromosome of a pig genome (Sscofa 10.2Primary Assembly), the characteristics of the sow in the infant killing behavior are further identified or assisted by the genotype, and the sow with low infant killing behavior occurrence frequency is selected, so that the sow with good sow quality is obtained, and the survival rate of piglets is improved.
The substance for detecting the SNP polymorphism or genotype in the pig genome provided by the invention is used for early detection of pigs, so that the problem of long selection time of excellent pigs in actual production is effectively solved, the breeding cost is reduced, sows with good maternal behaviors can be effectively selected, and the survival rate of piglets is improved. The method provided by the invention has the advantages of simple operation, low cost and high accuracy, can realize automatic direct detection, and plays a great role in the breeding work of sows. The substances for detecting the SNP polymorphism and the genotype can be combined with other substances (such as substances for detecting single nucleotide polymorphism or genotype of other molecular markers related to the frequency of the infant killing behavior of the sow) to prepare a product for identifying the frequency of the infant killing behavior of the sow.
Drawings
FIG. 1 is a diagram showing the sequencing result of the sequence around SNP site SSC3g.9907599.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The quantitative experiments in the following examples were performed in triplicate unless otherwise indicated.
The invention selects a large white pig (Sus scrofa) variety, and the pig is purchased from a Changping livestock and poultry experiment base of Beijing livestock and poultry veterinary research institute of China academy of agricultural sciences.
The PCR amplified sequences in the examples below were all referenced to the porcine genome (Sscofa 10.2Primary Assembly) sequence.
Example 1 development of sow infant killing behavior-related SNP markers
1. Determination of the polymorphic site of SSC3g.9907599T > C
Firstly, taking 20 big white pigs as experimental materials, respectively extracting genome DNA of the ear edge tissues, and determining that mutation exists at the site through mixing pools and individual sequencing.
(II) design and Synthesis of primers
Based on the YWHAG gene reference sequence (http:// asia. Ensembl. Org/sus_scrofa/Info/Index) in International pig genome version 10.2, primer 5 was used to design and synthesize the following primers:
u (upstream primer): 5'-GAAGCAGGAGTTGAGGCA-3' (SEQ ID No. 1);
d (downstream primer): 5'-AAGGAGGAAAGAAGACAGAGTT-3' (SEQ ID No. 2).
(III) PCR amplification
And (3) respectively carrying out PCR amplification by taking the genome DNA of the large white pig obtained in the step (one) as a template and taking U and D as primers to obtain a PCR amplification product.
PCR amplification system: 200ng of genome DNA, 5 mu L of 10 XPCR amplification buffer solution, 10mM of final concentration of dNTPs, 50ng of each of the upstream and downstream primers, 0.75U of Taq DNA polymerase, and Mg 2+ 2.5mmol/L with ddH 2 O complements the system to 50. Mu.L.
PCR amplification procedure: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 20s, annealing at 60.5℃for 30s, elongation at 72℃for 30s for 35 cycles; finally, the extension is carried out for 10min at 72 ℃.
(IV) sequencing and sequence analysis
Sequencing the PCR product to obtain the product with the sequence shown in SEQ ID No. 3. The SEQ ID No.3 has only one base difference, namely the 89 th position of the SEQ ID No.3 in the sequence table, and the nucleotide type is T or C (namely T/C polymorphism). Y in sequence 3 in the sequence table represents c or t. The SNP site is located at position 9907599 of the physical position 3 of the pig genome (Sscofa 10.2primary Assembly), as indicated by the arrow in FIG. 1, and is therefore designated SNP-SSC3g.9907599. The genotypes of the SNP loci are as follows: CC. TC or TT. The CC is homozygous for SNP-SSC3g.9907599 as C, the TT is homozygous for SNP-SSC3g.9907599 as T, and the TC is heterozygous for SNP-SSC3g.9907599 as T and C. That is, the individuals having T at the 9907599 th base of the Sscoffa 10.2Primary Assembly No.3 chromosome are individuals having homozygous TT genotypes, the individuals having C at the 9907599 th base of the Sscoffa 10.2Primary Assembly No.3 chromosome are individuals having homozygous CC genotypes, and the individuals having T and C at the 9907599 th bases of the Sscoffa 10.2Primary Assembly No.3 chromosome are individuals having heterozygous TC genotypes.
2. Correlation analysis of SNP locus SNP-SSC3g.9907599 and sow infant killing behavior
To determine whether SNP-ssc3g.9907599 is related to the infant killing behavior of sows, the following test was performed with 225 healthy white pigs as the experimental material:
genotyping based on SNP site SNP-SSC3g.9907599
Extracting genome DNA of the ear edge tissues of 225 pigs, carrying out PCR amplification by using the genome DNA as a template and using the primer pair consisting of U/D in the first step (II) to obtain each PCR amplification product, and sequencing the PCR products. Determining whether the SNP-SSC3g.9907599 genotype of each pig is homozygous TT, heterozygous TC or homozygous CC according to the method in the step one (four).
As a result, as shown in Table 1, 225 test animals obtained PCR amplification products with 209bp size by using both the U and D primer pairs, and the nucleotide sequences were sequence 3 of the sequence table.
Based on SNP locus SNP-SSC3g.9907599, 225 test animals are divided into three genotypes (shown in Table 3), CC genotype, TT genotype and TC genotype. The 89 th nucleotide of the sequence 3 in the sequence table in the TT genotype pig genome is homozygous for T, the 89 th nucleotide of the sequence 3 in the sequence table in the CC genotype pig genome is homozygous for C, and the 89 th nucleotide of the sequence 3 in the sequence table in the TC genotype pig genome is heterozygous for T and C. Of the 225 animals, 174 were TT genotypes, 46 were TC genotypes and 5 were CC genotypes.
(II) determination of the infant killing phenotype of 225 sows
Identification of the infant killing behaviour phenotype of the sow: and (3) installing a camera in the pig farm, recording the behavior of the sow from farrowing to piglet birth within 24 hours, counting whether the sow takes the piglet-biting behavior within the time range, recording as '1' if the piglet-biting behavior takes place, and recording as '0' if the piglet-biting behavior does not take place. The results are shown in Table 1.
Table 1, porcine SNP-SSC3g.9907599 genotype and phenotype information
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Figure BDA0004088922720000071
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Figure BDA0004088922720000081
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Figure BDA0004088922720000091
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Figure BDA0004088922720000101
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Figure BDA0004088922720000111
Note that: in the phenotype, 0 means that no infant killing of the sow occurs, and 1 means that infant killing of the sow occurs.
Correlation analysis of pig SNP-SSC3g.9907599 genotype and infant killing behavior of sow
Logit two-term regression analysis was performed on the correlation of SNP-SSC3g.9907599 genotype and the behavior of the sow against infant killing. The model used is as follows:
Y＝μ+G+e
wherein Y is an observed value of the infant killing behavior of the sow; mu is the mean value; g is genotype effect; e is a random error.
Table 2 correlation of porcine SNP-SSC3g.9907599 genotype with sow infant killing behavior
Figure BDA0004088922720000121
Note that: p <0.05 indicates that the different genotypes are significantly associated with phenotypes.
The results are shown in Table 2, with 174 TT genotypes, 46 TC genotypes and 5 CC genotypes in 225 animals. Wherein, the frequency of the infant killing of the individual sow with the genotype of the pig SNP-SSC3g.9907599 being TC genotype is 0.0870, the increase of the TC genotype can increase the infant killing probability of the sow, and the frequency of the infant killing of the individual sow with the genotype higher than or candidate higher than the TT genotype and the CC genotype is (P < 0.05). Combining the results of all pig genotypes and phenotypes, the P value of SNP-SSC3g.9907599 reached 0.0311 (P < 0.05) (Table 2)
In summary, in the actual sow breeding work, it is preferable to select a sow with the genotype of TT or CC as a parent for breeding, and eliminate individuals with the genotype of TC. By early genotype detection of the SSC3g.9907599 locus of the pig, the sow with good maternal behavior is effectively selected, and the survival rate of piglets is improved.
The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

Claims (10)

1. Use of a substance that detects a polymorphism or genotype of a SNP in the genome of a pig in any of the following:
(1) Detecting a single nucleotide polymorphism or genotype related to the frequency of infant killing activity of the sow;
(2) Identifying or aiding in identifying a product associated with a frequency of infant killing activity in a sow;
(3) A product for pig breeding;
(4) Preparing a product for identifying or assisting in identifying the infant killing frequency of the sow;
(5) Preparing a product for screening or breeding a pig variety with low infant killing frequency of a sow;
the SNP is a site on a pig chromosome 3, the nucleotide type of the SNP is T or C, and the SNP is 89 th nucleotide of a sequence 3 in a sequence table.
2. The method for identifying or assisting in identifying the infant killing frequency of the sow is characterized by comprising the following steps of: comprises the steps of detecting the genotype of the SNP in claim 1 in the genome of a pig to be detected, and identifying or assisting in identifying the frequency of infant killing of the sow according to the genotype of the SNP of the pig to be detected: the SNP is a site on a pig chromosome 3, the nucleotide type of the SNP is T or C, and the SNP is 89 th nucleotide of a sequence 3 in a sequence table; genotype TT, TC or CC, wherein genotype TT is homozygous for the SNP T, genotype CC is homozygous for the SNP C, and genotype TC is heterozygous for the SNP T and C;
the method for identifying or assisting in identifying the infant killing frequency of the sow comprises the following steps of:
1) The swine to be tested with genotype TT or CC of SNP as claimed in claim 1 is or is candidate to be a swine breed with low infant killing frequency of sow;
2) The swine to be tested with genotype TC of SNP as defined in claim 1 is or is candidate as a swine breed with high frequency of infant killing behavior by sow;
3) The test sow having a genotype of TT or CC of the SNP of claim 1 has a lower frequency of infant killing than or a candidate lower than a test sow having a genotype of TC of the SNP.
3. Use of the method of claim 2 in pig breeding.
4. The pig breeding method is characterized by comprising the following steps: the method comprises detecting genotypes of SNP loci in claim 1 in pig genome, selecting pigs with the genotypes of the SNP as TT or CC as parents for breeding, wherein the TT genotypes are homozygous for the SNP as T, and the CC genotypes are homozygous for the SNP as C.
5. Use according to claim 1 or 3, method according to claim 2 or 4, characterized in that: the pig breeding is to cultivate a pig variety with low infant killing frequency of the sow.
6. The product is characterized in that: the product is a product containing the substance for detecting polymorphism or genotype of pig genome SNP as set forth in claim 1, and may be any one of the following G1) to G5):
g1 A product for detecting single nucleotide polymorphism or genotype related to the frequency of infant killing behavior of the sow;
g2 Identifying or aiding in identifying a product of a frequency of infant killing activity in a sow;
g3 A product for pig breeding;
c4 Screening or breeding a product of a pig variety with low infant killing frequency of the sow;
c5 Preparing a product for screening or breeding a pig variety with low infant killing frequency of a sow.
7. The use according to claim 1 or the product according to claim 6, characterized in that: the substances are D1), D2) or D3) as follows:
d1 The substance is a primer composition for amplifying a porcine genomic DNA fragment comprising the SNP of claim 1;
d2 The substance is a PCR reagent containing the primer composition of D1);
d3 The substance is a kit containing the primer composition of D1) or the PCR reagent of D2).
8. The use or product according to claim 7, characterized in that: the primer composition consists of a primer U and a primer D; the primer U is a single-stranded DNA molecule with a nucleotide sequence of sequence 1 in a sequence table; the primer D is a single-stranded DNA molecule with a nucleotide sequence of sequence 2 in a sequence table.
A dna molecule characterized in that: the nucleotide sequence of the DNA molecule is sequence 3 in a sequence table.
10. An apparatus for frequency screening of infant killing activity in sows comprising:
a sequencing device for sequencing a DNA fragment comprising the SNP of claim 1 in the genome of a swine to be tested to obtain a sequencing result;
the comparison device is connected with the sequencing device and is used for determining the genotype of the SNP based on the sequencing result;
and the analysis device is connected with the comparison device and is used for determining the frequency of the infant killing behavior of the sow based on the genotype.
CN202310145148.3A 2023-02-21 2023-02-21 SNP molecular marker related to identification of infant killing behavior of sow and application of SNP molecular marker in genetic breeding Pending CN116042861A (en)

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