CN112760387A - SNP molecular marker related to total number of nipples of pig and application - Google Patents
SNP molecular marker related to total number of nipples of pig and application Download PDFInfo
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Abstract
The invention relates to an SNP molecular marker related to total pig papilla number and application thereof. The SNP molecular marker with the SNP locus corresponding to the G > T mutation at the 123564644 th site of the chromosome 2 of the reference sequence of the 11.1 version of the international genome is obtained by screening, and the dominant allele of the SNP is screened generation by generation, so that the frequency of the dominant allele can be improved, the total number of nipples of pigs is further improved, the reproductive performance of the pigs is improved, the survival rate of piglets is improved, the genetic improvement progress of the pigs is accelerated, and the economic benefit of pig breeding is effectively improved.
Description
Technical Field
The invention relates to the technical field of pig Marker Assisted Selection (MAS), in particular to an SNP molecular marker related to the total number of pig papillae and application thereof.
Background
The nipples of sows are important organs for piglets to obtain nutrition and immunity in the lactation period. In recent years, the breeding for increasing the number of litter pigs increases the burden of more piglets fed by sows. Therefore, the fact that the sows have a sufficient number of nipples is always one of the important criteria for breeding the breeding pigs. Total number of nipples: (total teat numberTTN) is a very important reproductive trait of pigs, more piglets can be fed by sows with more nipples, so that the increase of the total number of nipples of the sows is beneficial to improving the survival rate of the piglets and promoting the weight gain of the piglets, and the method is a very important technical means. Therefore, the breeding of the total number of the nipples is very necessary for improving the production performance of the pigs.
The breeding performance of the sows has very important influence on the production benefit of a pig farm, and the total number of nipples of the pigs is one of important indexes for evaluating the breeding performance of the pigs. Enough papillary number is possessed to better guarantee the survival rate of piglets. The number of nipples is a medium heritability character, however, in actual production work, breeding workers generally select the number of nipples directly from the phenotype, but simple elimination and seed reservation selection cannot meet daily production needs, and the production potential of pigs is not fully developed. Therefore, the character improvement of the total papilla number of the core swinery by adopting a genetic means can accelerate the breeding progress of the target character. The method is beneficial to fully exploiting the breeding performance of the pigs and improving the survival rate and the growth performance of the piglets, thereby improving the economic benefit and enhancing the core competitiveness of the commercial pig production.
Papillary number is a typical quantitative trait controlled by multiple genes. Utilization of researchers at home and abroadQTLPositioning and candidate basesGenetic pairs of quantitative trait loci affecting papillary number: (Quantitative trait loci，QTL) The study was conducted, but the results were not ideal.
Disclosure of Invention
The invention aims to provide an SNP molecular marker related to the total nipple number of a pig and application thereof in aspects of genotype identification, genetic breeding and the like, and aims to solve the technical problem of insufficient total nipples of sows.
Porcine whole genome sequence-based publishing and high-density SNPs (II)single nucleotide polymorphismDevelopment of single nucleotide polymorphism) chip to allow genome-wide association analysis: (Genome-wide association studyGWAS) has a powerful detection tool in identifying molecular markers and candidate genes affecting complex traits; the research of the invention identifies some molecular markers related to the total number of the pig nipples based on a GWAS analysis strategy, for example, SNP with large effect is applied to molecular marker auxiliary selection and genome selection, thereby accelerating the genetic improvement progress of the total number of the pig nipples and improving the economic benefit. The specific technical scheme is as follows:
screening to obtain a SNP molecular marker which is positioned on a swine No. 2 chromosome and is related to the total number of swine papillae, wherein the SNP locus of the SNP molecular marker corresponds to the 123564644G > T mutation of the chromosome No. 2 of the reference sequence version 11.1 of the international genome.
The nucleotide sequence of the SNP site of the molecular marker is shown as SEQ ID NO.1, wherein M in the sequence is G or T, which causes the difference of the total number of the pig nipples.
The molecular marker influencing the effective pig papilla number character can be applied to the identification of the total pig papilla number character and the genetic breeding of pigs.
The primer pair for identifying the SNP molecular marker related to the number of the nipples on the pig chromosome 2 comprises a primer P001-F and a primer P002-R, and the nucleotide sequences of the primer P001-F and the primer P002-R are shown as follows:
primers P001-F: 5'-TTTTCCCTTTGCTGATTTGG-3'
Primers P002-R: 5'-TGTCTCTTGGAGTTCCCATTG-3' are provided.
A method for identifying the SNP sites associated with total pig papilla number comprising the steps of:
(1) taking a tissue sample of a pig and extracting the genome DNA of the tissue sample;
(2) performing PCR amplification by using the primers by using the genomic DNA as a template;
(3) sequencing the amplified product, checking 123564644 th nucleotide site on the No. 2 chromosome based on the reference sequence of the international pig genome version 11.1, and judging the G > T polymorphism of the site.
The breeding method of the pig strain with the increased nipple number comprises the following steps:
determining the positions of the SNP molecular markers, which are related to the number of the nipples, on the chromosome 2 of the pig in the core group of the pig, and making corresponding selection according to the molecular markers:
selecting a boar individual with TG and TT genotypes at 123564644 th site on chromosome No. 2 of version 11.1 of the international pig reference genome, and eliminating a boar individual with GG genotype at 123564644 th site in the boar core group to improve the frequency of allele T at the site generation by generation so as to improve the total number of nipples of offspring pigs.
The boar is a white pig and a synthetic line thereof.
Compared with the prior art, the invention has the main beneficial technical effects that:
1. the invention researches and determines that the molecular marker related to the total number of the pig nipples is positioned on the nucleotide sequence of the chromosome 2 of the pig, verifies the influence effect of the molecular marker on the total number of the nipples, establishes an efficient and accurate molecular marker assisted breeding method, is applied to the genetic improvement of the number of the pig nipples, can quickly and accurately carry out breeding on the pig, quickens the breeding process of the pig, improves the reproductive performance of offspring pigs, and further achieves the purposes of improving enterprise profits and increasing the core competitiveness. The method specifically comprises the following steps: by screening dominant alleles of the SNP generation by generation, the frequency of the dominant alleles is improved, the total number of the nipples of the pigs is further improved, the reproductive performance of the pigs is improved, the progress of genetic improvement of the pigs is accelerated, and therefore the economic benefit is effectively improved.
2. The invention provides a primer pair for identifying the SNP molecular marker related to the total papilla number on the pig chromosome 2, and a high-efficiency and accurate molecular marker-assisted breeding technical system can be established through the primer pair, so that the pig is rapidly and accurately bred, and the breeding process is accelerated.
Drawings
FIG. 1 is a Global genome Association analysis (GWAS) Manhattan plot for Total papillary number trait on chromosome 2 of a white pig in an example of the present invention; wherein the abscissa represents the pig chromosome number and the ordinate represents the-logP value.
FIG. 2 shows the results of gene detection in the examples of the present invention; the agarose gel concentration is 1%; lanes in the figure: m is DL2000 marker; lane 1 is genotype TT, 233 bp; lane 2 is genotype GG, 233 bp; lane 3 is genotype TG, 233 bp.
FIG. 3 is a peak diagram of sequencing results of different genotypes of effective total papilla number trait major mutation sites G, 114G > T of large white pigs in the embodiment of the invention; wherein (a) is a sequencing result peak diagram of the TT genotype; (b) is a sequencing result peak diagram of GG genotype; (c) peak images of sequencing results of TG genotypes.
FIG. 4 is a diagram showing the sequencing results of the PCR amplification products using the primers P001-F and the primers P002-R in the present example; in the figure, M is a mutation site, underlined (wherein the parentheses in the mutant base, for allelic mutation), in the sequence of the first bold, indicated as designed primer sequence position.
Detailed Description
The following examples are given to illustrate specific embodiments of the present invention, but are not intended to limit the scope of the present invention in any way.
The instruments and devices referred to in the following examples are conventional instruments and devices unless otherwise specified; the related reagents and raw materials are all conventional products sold in the market if not specified; the test methods involved are conventional methods unless otherwise specified.
Example (b): cloning of SNP markers and genotype verification
1. Source of experimental animals
The experimental population used in the present invention was 1118 pure white pig populations from Henan Xinxin animal husbandry GmbH.
The large white pigs in the resource group selected in the experiment are fed and drunk freely, and the whole feeding mode, feeding conditions and the like are always kept consistent, so that the method is a conventional method.
2. Sample collection and phenotype recording
Collecting ear tissue of the above pig, storing in 75% alcohol at-20 deg.C, and recording the number of left papilla, the number of right papilla and total number of papilla.
3. Pig whole genome 50K SNP (single nucleotide polymorphism) genotyping
From the ear tissues of the large white pig group, the whole genome was extracted using an animal tissue DNA extraction kit (purchased from shanghai agilent bioengineering, ltd., code GK 0122). The concentration and OD value of each sample are measured by an ultraviolet spectrophotometer, 100ng of DNA is extracted and mixed with 1 mu of Loading Buffer, the mixture is loaded into 1% agarose gel, electrophoresis is carried out for 30min at 120V, the gel imaging equipment observes and photographs, and the integrity of the DNA is detected, as shown in figure 2.
DNA was diluted to about 20 ng/. mu.L and sampled to Nyquist Biotechnology (Shanghai) LtdIllumina BeadstrationCarrying out pig whole genome 50K SNP chip on a platform according to the standard process of a companyIlluminaUnited states, usa) genotype determination. Quality control is carried out on 50K chip data by using plink 1.9, individuals and sites with the rejection rate lower than 90%, the minimum allele frequency is less than 0.05, and the significance level of the Harden-Weinberg balance is higher than 10-6Excluding the sites that do not correspond to the site on the pig 11.1 reference genome and the sites on the X and Y chromosomes, finally obtaining effective genotype data of 1118 effective individuals and 46033 SNPs.
4. Whole genome association analysis
Performing principal component analysis by using GCTA v1.24 software; GWAS analysis was performed using GEMMA v0.94 software.
And (3) carrying out correlation analysis on the chip data after quality control and a nipple number phenotype by using a univariate mixed linear model, wherein the model is as follows:
y is a phenotypic value of the corresponding reproductive traits, n samples correspond to n traits, and w is a fixed effect matrix comprising the first two PCAs and seasons; α is the c-vector of the corresponding coefficient including the intercept; x is an n vector of marker genotypes; β is the magnitude of the effect of the label; u is the n vector of the random effect; is an n vector of errors; t is t-1Is the residual variance; λ is the ratio of the two variance components; k is a known n × n correlation matrix, lnIs an nxn identity matrix; MVNnRepresenting an n-dimensional multivariate normal distribution.
The GWAS analysis results are shown in fig. 1 and fig. 2. As can be seen from fig. 1 and 2, in the large white pig population, there are sites in chromosome 2 that significantly affect the total papilla number, and the most strongly associated SNP is g.114G > T (P ═ 1.31748 e-05).
5. Correlation analysis of different genotypes with Total papillary number phenotype
According to table 1, the SNP site g.114G > T (nucleotide in SEQ No.1, corresponding to 123564644G > T mutation on chromosome 2 of international pig genome version 11.1 reference sequence) of the molecular marker is significantly related to the total number of papillae and right number of papillae (P <0.01), which indicates that the molecular marker significantly affects the total number of papillae of pigs, and the total number of papillae of the population can be increased by auxiliary selection of the SNP site of the pig, thereby accelerating the breeding process.
In addition, as is clear from table 1, the average total papilla numbers of TT type and GT type are higher than those of GG type, TT type is more prominent than those of GG type and GT type, and GG type and GT type are also prominent, indicating that allele T is unfavorable for the total papilla number. The total number of nipples is an important index for measuring the reproductive performance of the pigs, and the higher total number of nipples indicates that the reproductive performance of the pigs is better. Therefore, during breeding, GG type pigs need to be eliminated, TT type and GT type breeding pigs are reserved, the frequency of the allele T of the locus is improved generation by generation, and further more economic benefits are brought.
TABLE 1 analysis of SNP sites g.114G > T-Effect of molecular markers
6. Amplification and sequencing of DNA sequences of interest
The designed primer DNA sequences are shown below:
primers P001-F: 5'-TTTTCCCTTTGCTGATTTGG-3'
Primers P002-R: 5'-TGTCTCTTGGAGTTCCCATTG-3', respectively;
and (3) PCR amplification: mu.L of DNA template, 8.75. mu.L of double distilled water, 12.5. mu.L of 2 XTag PCR StanMix with Loading Dye, and 1.25. mu.L of each of primers P001-F and P002-R were added to 25. mu.L of the reaction system.
The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 65 ℃ for 30s, and extension at 72 ℃ for 40s, wherein the annealing temperature is reduced by 1 ℃ per cycle, and the annealing temperature is reduced from 65 ℃ to 54 ℃ for 12 cycles; denaturation at 94 ℃ for 40 s; annealing at 54 ℃ for 30s, and 72 extending for 40 s; 30 cycles, 72 extension 10 min.
DNA sequencing: the two reactions of gene fragment were measured in Biotechnology engineering (Shanghai) GmbH. Combining the measured sequences withEnsemblAnd comparing the sequences of the upper genome to obtain the mutation of the corresponding SNP site. The sequencing results are shown in FIG. 4.
SNP site g.114G > T effect analysis of molecular marker:
the invention detects the 114 th base mutation site in the SEQ ID NO.1 sequence, preliminarily analyzes the association between the total number of the pig nipples and the genotype, and provides a new molecular marker for the auxiliary selection of the pig molecular marker. As can be seen from Table 1, the total number of papillae of TT type pigs is 0.223 more than that of GG type pigs on average, and the difference is very significant. Through the molecular marker-assisted selection, the breeding process of the white breeding pigs can be promoted, the reproductive performance of the pigs is increased, the pork production is promoted, and the economic benefit of the pig raising industry is increased. The concrete expression is as follows:
the pigs with GG gene types in the group are eliminated through molecular marker-assisted selection, the total number of nipples of the group is obviously improved, wherein the effective total number of nipples of each pig is improved between 0.104 and 0.223, 1040 to 2230 piglets can be suckled by 10000 sows per fetus, the piglets are calculated by directly fattening the piglets to 100kg and appearing on the market, and according to the estimation of 80 percent of slaughter rate, 83.2 to 178.4 tons of pork can be provided by ten thousand sows per fetus, so that huge economic income and effect can be brought to enterprises, and the core competitiveness of the sows is improved.
Although the present invention has been described with reference to specific preferred embodiments, it will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and various other changes, modifications, substitutions, combinations and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof and are intended to be included in the scope of the present invention.
SEQUENCE LISTING
<110> agriculture and animal husbandry Co., Ltd in yellow Pan area, Henan province; university of Henan agriculture
<120> SNP molecular marker related to total number of pig nipples and application
<130> 1
<160> 3
<170> PatentIn version 3 .3
<210> 1
<211> 233
<212> DNA
<213> Artificial
<220>
<223> /
<400> 1
ttttcccttt gctgatttgg ttttgtatcc tttggcttcc acaagcatta 50
gtcgtgaaca caactatatg cttagtcctt tgagcctttc tagggagtca 100
ttgaagctgg gtggggtcat ggggatcccc tttacaggaa taaataggaa 150
aaatgacctt ttcatatatc atagaatttc tataccagta ataaaaccaa 200
gataaaaatt attgtctctt ggagttccca ttg 233
<210> 2
<211> 21
<212> DNA
<213> Artificial
<220>
<223> primer P001-F
<400> 2
ttttcccttt gctgatttgg 20
<210> 3
<211> 21
<212> DNA
<213> Artificial
<220>
<223> primer P002-R
<400> 3
tgtctcttgg agttcccatt g 21
Claims (7)
1. A SNP molecular marker related to the total number of papillae of pigs, which is characterized in that a single nucleotide polymorphism variant of G > T transition type exists at the 123564644 th base on the chromosome corresponding to the reference sequence No. 2 of the 11.1 version of the international pig genome.
2. A primer for identifying the SNP molecular marker of claim 1, comprising:
the upstream primer P001-F: 5'-TTTTCCCTTTGCTGATTTGG-3'
The downstream primer P002-R: 5'-TGTCTCTTGGAGTTCCCATTG-3' are provided.
3. A kit for detecting the SNP marker according to claim 1, which comprises the primer according to claim 2.
4. Use of the primer according to claim 2 or the kit according to claim 3 in pig molecular marker assisted breeding.
5. A method for identifying SNP sites associated with total pig papilla number as claimed in claim 1, comprising the steps of:
(1) taking a tissue sample of a pig and extracting the genome DNA of the tissue sample;
(2) performing PCR amplification using the genomic DNA as a template and the primer according to claim 2;
(3) sequencing the amplified product, checking 123564644 th nucleotide site on the No. 2 chromosome based on the reference sequence of the international pig genome version 11.1, and judging the G > T polymorphism of the site.
6. A breeding method of a pig strain capable of increasing the number of nipples is characterized by comprising the following steps:
(1) detecting and determining the genotype of the 123564644 th site on the chromosome of the international pig reference genome 11.1 version 2 of a boar in the core group of the boar;
(2) selecting 123564644 th boar individuals with TG and TT genotypes from the boar core group, eliminating boar individuals with GG genotypes to increase the frequency of allele T of the loci generation by generation, thereby increasing the number of nipples of offspring pigs.
7. The method for breeding a pig line with increased papillary count according to claim 6, wherein the pig is a white pig or a synthetic line thereof.
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| CN113736889A (en) * | 2021-07-30 | 2021-12-03 | 华南农业大学 | SNP molecular marker related to pig stillbirth number and survival rate on pig chromosome 7 and application thereof |
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| KR101929391B1 (en) * | 2017-07-24 | 2018-12-17 | 대한민국 | Novel SNP marker for discriminating increasedthe number of nipples of pigs and use thereof |
| CN110144408A (en) * | 2019-05-15 | 2019-08-20 | 华南农业大学 | SNP marker relevant to total number of nipples and application on No. 7 chromosomes of pig |
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|---|---|---|---|---|
| KR101929391B1 (en) * | 2017-07-24 | 2018-12-17 | 대한민국 | Novel SNP marker for discriminating increasedthe number of nipples of pigs and use thereof |
| CN110144408A (en) * | 2019-05-15 | 2019-08-20 | 华南农业大学 | SNP marker relevant to total number of nipples and application on No. 7 chromosomes of pig |
Non-Patent Citations (3)
| Title |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113736889A (en) * | 2021-07-30 | 2021-12-03 | 华南农业大学 | SNP molecular marker related to pig stillbirth number and survival rate on pig chromosome 7 and application thereof |
| CN113736889B (en) * | 2021-07-30 | 2023-07-11 | 华南农业大学 | SNP molecular marker related to pig stillbirth number and live litter rate on chromosome 7 and application thereof |
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