CN116287293A - Porcine SNP molecular marker and application thereof in swine litter size trait screening and/or swine breeding - Google Patents

Porcine SNP molecular marker and application thereof in swine litter size trait screening and/or swine breeding Download PDF

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CN116287293A
CN116287293A CN202310104779.0A CN202310104779A CN116287293A CN 116287293 A CN116287293 A CN 116287293A CN 202310104779 A CN202310104779 A CN 202310104779A CN 116287293 A CN116287293 A CN 116287293A
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litter size
pig
swine
molecular marker
breeding
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冯越
张宇
梅书棋
彭先文
吴俊静
乔木
周佳伟
李梓芃
徐忠
孙华
李良华
刘贵生
董斌科
宋忠旭
赵海忠
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Institute of Animal Science and Veterinary of Hubei Academy of Agricultural Sciences
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Institute of Animal Science and Veterinary of Hubei Academy of Agricultural Sciences
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
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    • Y02P60/87—Re-use of by-products of food processing for fodder production

Abstract

The invention discloses a pig SNP molecular marker and application thereof in pig litter size trait screening and/or pig breeding, and relates to the technical field of pig molecular markers. The sow litter size trait is total litter size, litter size and healthy litter size; the molecular marker is positioned in a third intron sequence of the pig SRRM3 gene, the length of the sequence is 447bp, the nucleotide sequence of the molecular marker is shown as SEQ ID NO.1 of a sequence table, and a G217-T217 base mutation exists at the 217bp position of the sequence. The invention also provides a primer pair for amplifying the molecular marker and a method for breeding by using the molecular marker. The invention discovers that one SNP molecular marker in the pig SRRM3 gene is related to the characteristics of total litter size, litter size and litter size of the sow for the first time, so that the invention can be used for pig marker assisted selection breeding, namely provides a new application of one SNP molecular marker in the pig SRRM3 gene in marker assisted breeding of litter size characteristics of the sow, and realizes early screening of litter size characteristics of the sow, and the screening method is simple and rapid.

Description

Porcine SNP molecular marker and application thereof in swine litter size trait screening and/or swine breeding
Technical Field
The invention relates to the technical field of pig molecular markers, in particular to a pig SNP molecular marker and application thereof in pig litter size trait screening and/or pig breeding.
Background
The reproductive performance of the sow is an important component for measuring the economic benefit of a pig farm, and the litter size is the most important index for evaluating the reproductive performance of the sow. The litter size trait belongs to low genetic power quantitative traits, and the selection effect is not obvious by the traditional selection method. Therefore, the key molecular genetic markers for controlling the litter size traits of pigs are searched, and the key molecular genetic markers are used for molecular marker assisted breeding, so that the method has important significance for improving the litter size traits of sows and increasing the economic benefits of pig farms.
A single nucleotide polymorphism (single nucleotide polymorphism, SNP), referred to as "third generation DNA molecular marker", refers to a DNA sequence polymorphism including four cases of base inversion, conversion, single base deletion or insertion caused by single nucleotide variation in a genome sequence, and is a DNA molecular marker directly marked by variation of the sequence. SNPs generally occur in three different regions, namely promoters, introns and exons, which have been shown to affect gene expression to varying degrees.
Serine/arginine-related nuclear matrix protein 3 (SRRM 3) gene is an alternative splicing factor required for motor coordination, possibly related to myocardial contractile movement. The gene protein domain includes a nuclear localization signal (nuclear localization signals, NLS) domain, and two domains similar to Ser-Arg-rich segments and SRRM 4. The influence of the gene on the litter size of pigs has not been reported yet.
Disclosure of Invention
The invention aims to overcome the technical defects and provide a pig SNP molecular marker and application thereof in pig litter size trait screening and pig breeding, and the invention discovers that one SNP in a third intron of an SRRM3 gene is associated with pig litter size trait and can be used as a molecular marker for pig litter size trait screening and pig breeding.
In a first aspect, the invention provides a pig SNP molecular marker applied to pig litter size trait screening and/or pig breeding, wherein the litter size trait is total litter size, live litter size and healthy litter size trait; the molecular marker is positioned in a third intron sequence of the pig SRRM3 gene, the length of the sequence is 447bp, the nucleotide sequence of the molecular marker is shown as SEQ ID NO.1 of a sequence table, and a G217-T217 base mutation exists at the 217bp position of the sequence.
Further, the G or T base polymorphism site at 217bp in the sequence SEQ ID NO.1 is represented as three genotypes GG, GT or TT, wherein the G allele is a dominant allele.
In a second aspect, the invention provides the use of a pig SNP molecular marker as set forth in the first aspect in the selection of litter size traits in pigs and/or in pig breeding.
In a third aspect, the present invention provides a primer pair for amplifying a molecular marker as described in the first aspect, the primer pair comprising: the nucleotide sequence of the upstream primer is shown as SEQ ID NO.2, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 3.
In a fourth aspect, the invention provides the use of a primer pair as described in the third aspect in swine litter size trait screening and/or swine breeding.
In a fifth aspect, the invention provides a kit for rapid breeding by using the swine SNP molecular markers for swine litter size trait screening and/or swine breeding as described in the first aspect, comprising the primer pair as described in the third aspect.
In a sixth aspect, the invention provides a method for screening swine litter size traits and/or breeding pigs, comprising the steps of:
s1, extracting genome DNA of a pig;
s2, carrying out PCR amplification by using the genomic DNA obtained in the step S1 as a template and adopting the primer pair as defined in claim 4 to obtain the molecular marker as defined in claim 1 and purifying;
s3, carrying out sequencing analysis on the molecular marker purified in the step S2, reserving individuals carrying G alleles at the 217bp position of the sequence, and eliminating the individuals carrying T alleles.
Further, in step S1, genomic DNA is extracted from the ear border tissue of the pig to be tested.
Further, in step S2, the PCR reaction system is 50. Mu.L, and the components in the system are as follows: 100ng of genomic DNA, 25. Mu.L of PCR mix, 1. Mu.L of each of the upstream primer and the downstream primer, and ddH 2 O was made up to a total volume of 50. Mu.L; the PCR was run as follows: pre-denaturation at 94℃for 3min; denaturation at 94℃for 30s, annealing at 58℃for 30s, elongation at 72℃for 30s,35 cycles; extending at 72 ℃ for 10min; preserving at 4 ℃. It is a second object of the present invention to provide a method for spreadingA primer pair for amplifying the molecular marker, comprising: the nucleotide sequence of the upstream primer is shown as SEQ ID NO.2, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 3.
Compared with the prior art, the invention has the beneficial effects that: the invention firstly discovers that one SNP molecular marker in the pig SRRM3 gene is related to the litter size trait of the pig, in particular to the total litter size, the number of live pigs and the litter size trait of the pig, so that the molecular marker can be used for screening the litter size trait of the pig and breeding, namely, the invention provides a new application of one SNP molecular marker in the pig SRRM3 gene in the marker assisted breeding of the litter size trait of the pig, and realizes the early screening of the litter size trait of the pig, and the screening method is simple and rapid.
Drawings
FIG. 1 is a diagram showing agarose gel electrophoresis detection of a PCR product in example 1 of the present invention, wherein lane M is DL5000Marker, lanes 1-7 are amplified fragments in a large white pig, and the fragment size is 447bp;
FIG. 2 is a sequencing map of g.217G > T sites in example 1 of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Example 1 acquisition of porcine SRRM3 Gene SNP detection fragment and establishment of method for detecting polymorphic site
1. Extraction of pig genomic DNA
Extraction of porcine genomic DNA an animal tissue genomic DNA extraction kit (PureLink) manufactured by Invitrogen company was used TM Pro 96Genomic DNA Purification Kit,K182104A), genomic DNA was extracted from the ear border tissue of the test pig according to the kit instructions. And (3) detecting the concentration and quality of the extracted DNA, and storing at-20 ℃ for standby.
2. Acquisition of pig SRRM3 gene SNP genetic marker detection fragment
(1) PCR amplification
A pair of primers is designed according to SNP genetic marker detection sequences (shown as SEQ ID NO. 1) in genome sequences of the pig SRRM3 genes, and fragments of polymorphic sites are amplified. The primers were as follows:
an upstream primer: 5'CGGGATCATTTATTCTTTCAGC 3' as shown in SEQ ID NO. 2;
a downstream primer: 5'TGCCCTCAGTCTTCTTGGAC 3' is shown in SEQ ID NO. 3.
By using the primers and taking the genome DNA of the large white pig as a template, carrying out PCR amplification, wherein the PCR reaction system comprises 50 mu L, and the components in the system are as follows: 100ng of genomic DNA, 25. Mu.L of PCR mix, 1. Mu.L of each of the above upstream and downstream primers, and ddH 2 O was made up to a total volume of 50. Mu.L.
The PCR was run as follows: pre-denaturation at 94℃for 3min; denaturation at 94℃for 30s, annealing at 58℃for 30s, elongation at 72℃for 30s,35 cycles; extending at 72 ℃ for 10min; preserving at 4 ℃. The PCR products were detected by 1.5% agarose gel electrophoresis, the detection result is shown in FIG. 1, wherein lane M is DL5000Marker, lanes 1-7 are amplified fragments in big white pigs, and the amplified fragments have 447bp.
(2) PCR product purification
The PCR amplified product was purified using a Gel Extraction Kit kit from Shanghai Biotechnology Co., ltd, and specific steps are shown in the kit specification.
3. Detection of molecular markers by direct sequencing of PCR products
The PCR purified product obtained above is directly sent to Beijing Oreg company for sequencing, and the genotype of the site in the detection population is judged according to the sequencing result. Analysis using DNAStar software revealed that there was a G217-T217 allele mutation at the 217bp in the sequence shown in SEQ ID NO.1, i.e., g.217G > T, which caused the polymorphism of the SRRM3 gene, as shown in FIG. 2.
EXAMPLE 2 detection of polymorphism distribution of molecular markers in white pigs
In this example, the polymorphism distribution rule of the g.217G > T locus of the SRRM3 gene of pigs was detected in 465 large white pigs, and the detection results are shown in Table 1.
TABLE 1SRRM3 Gene g.217G > T site polymorphism distribution rules
Figure BDA0004074382190000041
From the results in Table 1, it can be seen that: in large white pigs the SRRM3 gene g.217g > T locus appears as three genotypes GG, GT and TT, with more individuals of GT genotype and a G allele frequency of 65.6%.
Example 3 correlation analysis of molecular markers and litter size traits in pigs
In order to determine whether the g.217GT locus of the SRRM3 gene is related to the litter size trait differences of pigs, polymorphism detection was performed by the method set forth in example 1, and the correlation of the different genotypes of this polymorphic locus with the overall litter size, litter size and litter size traits of pigs was analyzed. The variance analysis of the different SNP genotype combinations was performed using SAS statistical software (SAS Institute Inc, version 9.1) GLM program and the significance test was performed using the following models:
Yij＝μ+Gi+Fj+eij；
yij is a trait phenotype value, mu is an average value, gi is a genotype effect (including a gene additive effect and a dominant effect; additive effect applications 1,0 and-1 represent GG, GT and TT genotypes respectively, dominant effect applications 1, -1 and 1 represent GG, GT and TT genotypes respectively); fj is the pig farm comprehensive effect; eij is the residual effect.
Correlation analysis between different genotypes and litter size traits was performed in large white pigs, and the statistical analysis results are shown in table 2:
TABLE 2 correlation analysis of SRRM3 Gene g.217G > T locus and litter size traits in pigs
Figure BDA0004074382190000051
Note that: the composition of the table neutral mean is mean ± standard deviation, a and B represent significant differences (P < 0.05), represent very significant differences (P < 0.01)
As can be seen from table 2, in large white pigs, the total litter size, the number of live-born and the number of healthy-born individuals of GG genotype at g.217g > T locus were significantly higher than those of GT and TT genotype individuals (P < 0.05), and the additive effects of total litter size and number of live-born reached a significant level (P < 0.05), so that the G allele was the dominant allele.
Example 4 application of SRRM3 Gene SNP molecular marker in porcine litter size trait screening and/or porcine breeding
The SNP molecular marker g.217G > T site on the SRRM3 gene intron is obviously related to the litter size trait of pigs, and the additive effect of G alleles reaches extremely obvious level. Therefore, in the process of breeding large white pigs, individuals with G alleles and good litter size traits can be selected in an auxiliary manner through the SNP molecular markers, and the individuals with the dominant alleles should be reserved in breeding, so that litter size traits of groups can be improved, genetic progress of sow litter size performance can be accelerated, and production benefits of high-fertility sows can be improved effectively.
The above-described embodiments of the present invention do not limit the scope of the present invention. Any other corresponding changes and modifications made in accordance with the technical idea of the present invention shall be included in the scope of the claims of the present invention.

Claims (9)

1. The pig SNP molecular marker applied to pig litter size trait screening and/or pig breeding is characterized in that the litter size trait is total litter size, live litter size and healthy litter size trait; the molecular marker is positioned in a third intron sequence of the pig SRRM3 gene, the length of the sequence is 447bp, the nucleotide sequence of the molecular marker is shown as SEQ ID NO.1 of a sequence table, and a G217-T217 base mutation exists at the 217bp position of the sequence.
2. The molecular marker of swine SNP for use in swine litter size trait screening and/or swine breeding according to claim 1, wherein the G or T base polymorphism site at 217bp in sequence SEQ ID No.1 is represented by three genotypes GG, GT or TT, wherein the G allele is a dominant allele.
3. Use of a pig SNP molecular marker according to any one of claims 1 or 2 in pig litter size trait screening and/or pig breeding.
4. A primer pair for amplifying the molecular marker of claim 1, wherein the primer pair comprises: the nucleotide sequence of the upstream primer is shown as SEQ ID NO.2, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 3.
5. The use of a primer pair according to claim 4 in swine litter size trait screening and/or swine breeding.
6. Kit for rapid breeding using the swine SNP molecular markers for swine litter size trait selection and/or swine breeding according to claim 1, characterized by comprising the primer pair according to claim 4.
7. A method for screening swine litter size traits and/or breeding pigs, comprising the steps of:
s1, extracting genome DNA of a pig;
s2, carrying out PCR amplification by using the genomic DNA obtained in the step S1 as a template and adopting the primer pair as defined in claim 4 to obtain the molecular marker as defined in claim 1 and purifying;
s3, carrying out sequencing analysis on the molecular marker purified in the step S2, reserving individuals carrying G alleles at the 217bp position of the sequence, and eliminating the individuals carrying T alleles.
8. The method for litter size trait selection and/or swine breeding according to claim 7, wherein in step S1, genomic DNA is extracted from the ear-border tissue of the swine to be tested.
9. The method for screening and/or breeding swine litter size trait according to claim 7, wherein in step S2, the PCR reaction system is 50 μAnd L, each component in the system is as follows: 100ng of genomic DNA, 25. Mu.L of PCR mix, 1. Mu.L of each of the upstream primer and the downstream primer, and ddH 2 O was made up to a total volume of 50. Mu.L; the PCR was run as follows: pre-denaturation at 94℃for 3min; denaturation at 94℃for 30s, annealing at 58℃for 30s, elongation at 72℃for 30s,35 cycles; extending at 72 ℃ for 10min; preserving at 4 ℃.
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