CN113736889B - SNP molecular marker related to pig stillbirth number and live litter rate on chromosome 7 and application thereof - Google Patents
SNP molecular marker related to pig stillbirth number and live litter rate on chromosome 7 and application thereof Download PDFInfo
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Abstract
The invention provides an SNP molecular marker related to the number of dead fetuses and the number of live piglets of a pig No. 7 chromosome and application thereof, wherein the SNP molecular marker corresponds to the 9581427 th C > T mutation on the No. 7 chromosome of an 11.1 version reference sequence of an international pig genome. According to the invention, the dominant allele of the SNP is optimized, so that the dominant allele frequency can be increased generation by generation, the dead number of pigs is reduced, the live-born rate is improved, the excellent germplasm of the two characters is cooperatively selected, and the progress of genetic improvement of the pigs is accelerated, so that the economic benefit of breeding of the breeding pigs is effectively improved.
Description
Technical Field
The invention relates to an SNP molecular marker related to the number of dead fetuses and the number of alive piglets on a chromosome 7 of a pig and application thereof.
Background
In pig breeding, improving the reproductive performance of sows is one of important ways for improving economic benefits, and is mainly reflected in indexes such as oestrus and breeding rate, nonpregnant rate, litter size, healthy litter size, weaning weight of piglets and the like of sows. By long-term breeding measures, the reproductive traits of sows have been greatly improved over the last 40 years. With the perfection of breeding targets, breeders do not need to breed more than one sow reproductive performance breeding, but carry out genetic improvement from key characters such as reducing dead fetus number, improving live offspring rate and the like so as to maximize economic benefit.
Dead fetus refers to a dead fetus produced by a fetus in utero at a stage of development that has been completed, that has approached the gestational period or the expiration of pregnancy, and that is similar in size to a litter size, with no sagging eyes. Dead fetus may die from antenatal stage, and sow delivery may be too long, and piglets cannot breathe in the delivery passage for too long, so that hypoxia is caused to die. The live piglet rate refers to the proportion of the number of weaned piglets to the total number of the piglets born after delivery, and is a key index for measuring the reproductive performance of sows. Meanwhile, the number of dead fetuses and the healthy young rate are also one of important factors affecting the economic benefit and the service life of the sow.
In the early stage, a certain genetic progress is obtained on the reproductive traits of the sow through a breeding mode based on data such as pedigree, phenotype and the like. However, the reproductive traits of sows belong to low genetic traits, and even though offspring data has been utilized, the breeding reliability level is still relatively low, which is one of the limiting factors that reduces the effectiveness of breeding programs. The reproductive traits of current commercial breeding pigs are hardly significantly improved compared with their bred progenitors, and as the genetic traits continue to progress, further improvements become increasingly slow and difficult. Therefore, the genotyping data is added on the basis of the original data, so that the dead number and the live number of the sow can be improved, and the breeding efficiency is improved, so that the breeding standard is objective and accurate.
Compared with the conventional breeding method, the new generation molecular breeding technology can carry out early breeding on the dead number and the alive number of the sows, quicken the breeding process of important economic characters and reduce the breeding cost. The most effective method for improving genetic progress is to incorporate molecular markers affecting the number of dead fetuses and the number of alive fetuses of sows into a breeding plan for breeding. Genome-wide association analysis (Genome-wide association study, GWAS) is one of the best strategies for screening important molecular markers. GWAS, by virtue of the numerous advantages of covering tens of thousands of molecular markers (e.g., single nucleotide polymorphisms Single nucleotide polymorphisms, SNPs) throughout the genome, eliminating the need for prior construction of families and easier realization of larger effective population sizes, gives rise to incomparable advantages in breeding improvement of breeding pigs. GWAS is currently an important means of molecular breeding in modern large-scale pig industry, and QTL and major genes affecting the economic traits of pigs can be identified, and mutation sites can be further mined. Therefore, in the genetic improvement of the characteristics of the dead number and the live number of the sow, the GWAS strategy can be fully utilized to identify the molecular marker, the molecular marker is brought into a breeding plan, molecular auxiliary marker selection and genome selection are carried out, a molecular foundation is laid for screening the sow with excellent reproductive performance, and a basis is provided for further analyzing the genetic mechanism of the characteristics of the dead number and the live number of the sow. In the method, a Single Nucleotide Polymorphism (SNP) site is identified in a Duroc pig breeding pig group by utilizing a GWAS strategy, and the result shows that the SNP site has a certain correlation with the number of dead fetuses and the number of live piglets of sows.
Disclosure of Invention
The invention aims to overcome the defects and the shortcomings of the prior art and provide an SNP molecular marker related to the number of dead pigs and the number of live pigs on chromosome 7 and application thereof, and also provides a primer pair and a kit for detecting the SNP molecular marker, and further provides a method for screening pig breeds with low number of dead pigs and high number of live pigs, and a method for genetic improvement of pigs.
In order to achieve the above purpose, the following technical scheme is adopted: a SNP molecular marker on a pig chromosome 7, wherein the SNP molecular marker is related to the number of dead fetus and the number of live piglets of the pig, and corresponds to a 9581427 th C > T mutation on a reference sequence chromosome 7 of the 11.1 version of the international pig genome. The polymorphism of the base at the locus leads to the difference of the number of dead pigs and the number of alive pigs.
Preferably, the nucleotide sequence of the SNP molecular marker is shown as SEQ ID NO. 1, wherein M in the sequence is T or C. The locus of the SNP molecular marker is the nucleotide mutation of T158-C158 at 158 th site marked by SEQ ID NO:1 sequence.
Preferably, the pig is a merozoite pig and its synthetic line.
The invention provides a primer pair for detecting the SNP molecular marker, which comprises a primer-F and a primer-R, wherein the nucleotide sequence of the primer-F and the primer-R is as follows:
P001-F:5’-AGCAGCGCTACCGATAGATTCA-3’
P001-R:5’-CCACAGTGTAGACCTTGTGCTAGAC-3’。
the invention provides a kit for detecting the SNP molecular marker, which comprises the primer pair.
The invention provides a method for screening pig breeds with low dead fetus number and high live litter size, which comprises the following steps:
detecting the SNP molecular marker related to the number of dead fetus and the number of alive offspring on the chromosome 7, eliminating the individual of which the 158 th single-core glutamic acid at the 5 'end of the SNP molecular marker is C, and reserving the individual of which the 158 th single-core glutamic acid at the 5' end of the SNP molecular marker is T.
The invention provides a method for genetic improvement of pigs, which comprises the following steps:
(1) Extracting genome DNA of a pig to be detected;
(2) Carrying out PCR amplification on the genome DNA of the pig to be detected by adopting the primer pair or the kit so as to obtain a PCR amplification product;
(3) Sequencing the PCR amplification product so as to obtain a sequencing result;
(4) Determining the genotype of the SNP molecular marker of the pig to be detected based on the sequencing result;
(5) And according to the genotype of the SNP marker, eliminating the individuals with the CC genotype of the pig to be detected, and reserving individuals with TT and TC genotypes so as to increase the frequency of the allele T of the locus generation by generation, thereby reducing the dead fetus number of the offspring pig and increasing the live offspring rate of the offspring pig.
Preferably, the pig is a merozoite pig and its synthetic line.
The invention also provides application of the SNP molecular marker, the primer pair or the kit in identifying the dead number and the litter size of pigs, screening pig varieties with low dead number and high litter size or reducing the dead number and improving the litter size of pigs.
The invention also provides application of the SNP molecular marker, the primer pair or the kit in pig genetic breeding.
The beneficial effects are that:
(1) The invention researches and determines that the molecular marker related to the dead number and the live number of the pig is positioned on a nucleotide sequence on chromosome 7 of the pig, verifies the influence effect of the molecular marker on the characteristics of the dead number and the live number, and finally establishes a high-efficiency and accurate molecular marker assisted breeding technology, and the molecular marker assisted breeding technology is applied to the genetic improvement of the breeding pig for reducing the dead number and increasing the live number, thereby improving the reproductive performance of offspring pigs, improving the economic benefit of enterprises and increasing the core competitiveness. By optimizing the dominant allele of the SNP, the dominant allele frequency can be increased generation by generation, the dead number of pigs is reduced, the live-birth rate is improved, the excellent germplasm of the two characters is selected in a synergistic way, and the progress of pig genetic improvement is accelerated, so that the economic benefit of breeding of the breeding pigs is effectively improved.
(2) The invention provides a primer pair for detecting SNP molecular markers related to dead fetus number and litter size on a No. 7 chromosome of pigs, and an efficient and accurate molecular marker assisted breeding technology can be established through the primer pair, so that breeding performance can be quickly and accurately bred, and the breeding process is accelerated.
Drawings
FIG. 1 is a full genome association analysis (GWAS) Manhattan diagram of Mesona Duroc on chromosome 7 for stillbirth number, litter size traits; wherein: the abscissa indicates chromosome number of pigs; the ordinate represents-logP value; a: number of dead tires, B: the young plant rate.
FIG. 2 is a graph showing analysis of phenotypic ratio results of the number of dead fetuses and the number of live births of pigs of different genotypes; wherein A: number of dead tires, B: the young plant rate.
Detailed Description
For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the following specific examples.
Example 1
(1) Experimental animal
The experimental pig group used in the invention is 604 heads of pure-breed Duroc sows of pig raising experimental part of Wen food group stock limited company, and is a core group of pig raising industry part.
The experiment selects the Duroc sows of the Mei-series in the resource group, the pig group freely feeds and drinks water, and the whole feeding mode, feeding conditions and the like are always kept, so that the method is a conventional method.
(2) Sample collection
Collecting the pigtail and ear tissues, soaking in 75% ethanol solution, and storing in a refrigerator at-20deg.C.
(3) Pig whole genome 50K SNP judgement
The tail tissue or ear tissue collected from each individual of the 604 Duroc species pigs is selected from the resource group, the whole genome DNA is extracted by a standard phenol-chloroform method, and the concentration and the OD ratio (OD 260/280, OD 260/230) of the DNA of each sample are accurately measured by a Nanodrop2000/2000C nucleic acid protein detector. And detecting a qualified DNA sample by a Nanodrop2000/2000C nucleic acid protein detector, and diluting the DNA to about 50 ng/. Mu.L according to the detected concentration. And mixing 6 mu L of the extracted DNA sample to be detected with 2 mu L of Loading Buffer, loading the mixture into agarose gel with the mass-volume ratio of 1%, carrying out electrophoresis for 25min at 150V, and observing and photographing under an ultraviolet spectrophotometer and gel imaging equipment to observe the integrity of the DNA.
DNA samples were sent to the New Biotechnology (Shanghai) Inc., and genotype determination was performed on a pig whole genome 50K SNP chip (Illumina, USA) on a Illumina Beadstration platform according to the company standard procedure. 50K cores for all samples using PLINK v1.9Quality control is carried out on slice scanning parting data, the reject and detection individual rate is lower than 90%, the family Mendelian error rate is higher than 0.1, the minimum allele frequency is lower than 0.05, and the Hady-Wenberg equilibrium significance level is higher than 10 -6 And finally obtaining the effective genotype data of 42141 SNPs.
(4) Whole genome association (GWAS) analysis
In order to eliminate the layering effect of the population, the linear hybrid model single-point regression analysis is adopted and is combined with GEMMA software to carry out GWAS analysis, and the layering effect is corrected by utilizing the genomic similarity among individuals in an analysis model. The Bonferini method is adopted to determine the significance threshold of the association degree of the SNP with the number of dead fetus and the sex of the live birth, the chromosome level significance threshold is 1 divided by the number of effective SNP loci, and the chromosome level threshold is 2.27e-7, namely 1/42141 (the number of effective SNP).
GWAS analysis results are shown. As can be seen from FIG. 1, duroc pigs have sites in chromosome 7 that significantly affect the number of dead fetus and the rate of live offspring, and the strongest associated SNP is g.158C > T (P values are 5.95e-6 and 1.05e-5, respectively).
(5) Correlation analysis of different genotypes of remarkably correlated sites and dead fetus number and litter size phenotype
As can be seen from Table 1, the molecular marker SNP locus g.158C > T (nucleotide at position 1 in SEQ NO:1, and C > T mutation at position 9581427 on chromosome 7 corresponding to version 11.1 reference base sequence of International pig) is extremely significantly correlated with the stillbirth number trait (P < 0.001), which indicates that the molecular marker significantly affects the stillbirth number trait of pigs, and the stillbirth number of the population can be reduced by auxiliary selection of the SNP locus of pigs, thereby accelerating the breeding process.
According to Table 2, it can be seen that the molecular marker SNP locus g.158C > T (nucleotide at position 1 in SEQ NO:1, and C > T mutation at position 9581427 on chromosome 7 corresponding to version 11.1 reference base sequence of International pig) is extremely significantly correlated with the litter size (P < 0.001), which indicates that the molecular marker significantly affects the litter size of pigs, and the litter size of the population can be increased by auxiliary selection of the SNP locus of pigs, thereby accelerating the breeding process.
As is clear from tables 1 to 2 and FIG. 2, the TT type and TC type of the SNP locus g.158C > T of the molecular marker are less in number of dead fetus and high in number of live fetus than the CC type. For the stillbirth trait, the average TT-type individual phenotype is 0.64 less than the average CC-type individual phenotype; for the liveness profile, the average TT-type individual phenotype was 8.18 greater than the average CC-type individual phenotype. Therefore, the TT and TC genotypes of the boars are gradually reserved in breeding so as to gradually increase the frequency of the allele T of the locus, thereby remarkably reducing the number of dead fetus and improving the survival rate, and bringing greater economic benefit to enterprises.
TABLE 1 correlation analysis of molecular marker loci g.158C > T and number of stillbirth
TABLE 2 correlation analysis of molecular marker loci g.158C > T and litter size
Example 2 amplification and sequencing of the DNA sequence of interest
(1) Primer design
The DNA sequence of SEQ ID NO. 1 on chromosome 7 of pigs was downloaded via the Ensembl website (http:// asia. Ensembl. Org/index. Html). And primers were designed using primer premier 6.0. The DNA sequence of the designed primers is as follows:
P001-F：5’-AGCAGCGCTACCGATAGATTCA-3’
P002-R：5’-CCACAGTGTAGACCTTGTGCTAGAC-3’
(2) PCR amplification
1. Mu.L of DNA template, 3.4. Mu.L of double distilled water, 2X Tag PCR StanMix with Loading Dye. Mu.L, and 0.3. Mu.L of each of the primers P001-F and P002-R were added to 10. Mu.L of the reaction system. The PCR reaction conditions were: after pre-denaturation at 94℃for 5min, denaturation at 94℃for 30s, annealing at 55℃for 30s, elongation at 72℃for 45s,35 cycles, and elongation at 72℃for 5min.
(3) DNA sequencing
Sequencing and identifying DNA sequences: the gene fragment was tested for both positive and negative responses in Shenzhen Dacron Gene technology Co. The sequence thus determined was aligned with the NCBI genomic sequence to give a mutation at the corresponding SNP site. The sequencing results are shown below:
note that: m marked in the sequence listing is a mutation site, shown by underline (mutation base in brackets, allelic mutation), and shown by thickening at the beginning and end of the sequence as a designed primer sequence position.
EXAMPLE 3 SNP site g.158C > T Effect analysis of molecular markers
The invention provides an SNP marker capable of remarkably reducing the dead fetus number of Duroc pigs and improving the live birth rate, and the SNP marker is used for marker-assisted selection, so that the breeding process of the reproduction-related characters of the Duroc pigs can be greatly influenced. Through molecular marker assisted selection, if all the molecular marker CC type individuals affecting the characteristics of the dead number and the litter size of pigs are selected and bred into TT type individuals, the dead number of each litter will be reduced by 0.64, and if the annual yield of sows is 2.5, the dead number of each sow will be reduced by 1.6 each year; according to the number of the piglets of the sow of 20, each sow will produce more 1.6 live piglets each year. Assuming that 2000 pigs can be bred in the pig farm, 6400 piglets can be produced annually by selecting the molecular marker.
The invention provides a new molecular marker for the molecular auxiliary selection of pigs by detecting 158 th base mutation sites in SEQ ID NO. 1 sequence and preliminarily carrying out the correlation analysis between the genotype and the number of dead pigs and the number of live pigs.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.
Sequence listing
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Claims (7)
1. A method for screening a low stillbirth number, high live litter size swine variety comprising the steps of: detecting the genotype of the C > T mutation at 9581427 on chromosome 7 of the international pig genome 11.1 version reference sequence of pigs, eliminating individuals with the C-type 5 'end of the C > T mutation at 9581427 and the C-type 158 single-core glutamic acid, and keeping the individuals with the T-type 5' end of the single-core glutamic acid at 158 as the genotype as breeding pigs, wherein the pigs are pure-bred American Duroc pigs.
2. A method for genetic modification of pigs comprising the steps of:
(1) Extracting genome DNA of a pig to be detected;
(2) Performing PCR amplification on the genome DNA of the pig to be detected in the step (1) by adopting a primer pair with nucleotide sequences shown as P001-F5'-AGCAGCGCTACCGATAGATTCA-3' and P001-R5'-CCACAGTGTAGACCTTGTGCTAGAC-3' to obtain a PCR amplification product;
(3) Sequencing the PCR amplification product to obtain a sequencing result;
(4) Determining the genotype of the 9581427C > T mutation of the pig to be tested based on the sequencing result;
(5) And according to the genotype of the 9581427C > T mutation, eliminating CC genotype individuals of the pig to be detected, and reserving TT and TC genotype individuals so as to increase the frequency of allele T of the locus generation by generation, thereby reducing the dead fetus number of the offspring pig and increasing the live offspring pig rate of the offspring pig.
3. The use of a primer pair for detecting a SNP molecular marker on chromosome 7 of a pig, which is related to the number of dead pigs and the number of live pigs, in breeding for identifying the number of dead pigs; the pig is a pure-bred Duroc pig, the locus of the SNP molecular marker is the 9581427 th C > T mutation on the chromosome 7 of the 11.1 version reference sequence of the international pig genome, and the molecular marker is TT type or TC type, and has less dead fetus number and high survival rate compared with CC type.
4. The use of a primer pair for detecting a SNP molecular marker on chromosome 7 of a pig, which is related to the number of dead pigs and the litter size of the pig, in the breeding of litter size; the pig is a pure-bred Duroc pig, the locus of the SNP molecular marker is the 9581427 th C > T mutation on the chromosome 7 of the 11.1 version reference sequence of the international pig genome, and the molecular marker is TT type or TC type, and has less dead fetus number and high survival rate compared with CC type.
5. The use of a primer pair for detecting a SNP molecular marker on chromosome 7 of a pig, which is related to the number of dead pigs and the number of live pigs, in the selection of breeding with low number of dead pigs; the pig is a pure-bred Duroc pig, the locus of the SNP molecular marker is the 9581427 th C > T mutation on the chromosome 7 of the 11.1 version reference sequence of the international pig genome, and the molecular marker is TT type or TC type, and has less dead fetus number and high survival rate compared with CC type.
6. The primer pair for detecting SNP molecular markers related to the number of dead pigs and the number of live pigs on chromosome 7 is used for breeding high-live pig breeds or reducing the number of dead pigs; the pig is a pure-bred Duroc pig, the locus of the SNP molecular marker is the 9581427 th C > T mutation on the chromosome 7 of the 11.1 version reference sequence of the international pig genome, and the molecular marker is TT type or TC type, and has less dead fetus number and high survival rate compared with CC type.
7. The application of the primer pair for detecting the SNP molecular marker related to the number of dead pigs and the litter size of the pigs on chromosome 7 in breeding for improving the litter size of the pigs; the pig is a pure-bred Duroc pig, the locus of the SNP molecular marker is the 9581427 th C > T mutation on the chromosome 7 of the 11.1 version reference sequence of the international pig genome, and the molecular marker is TT type or TC type, and has less dead fetus number and high survival rate compared with CC type.
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