CN117965742A - SNP (Single nucleotide polymorphism) marker related to total litter size of large white pigs and application - Google Patents
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Abstract
The application discloses an SNP marker related to the total litter size of a white pig and application thereof. The SNP marker comprises: the nucleotide sequence formed by single nucleotide mutation of the 117307797 th base of the chromosome 7 of the international pig genome 11.1 version reference sequence, wherein the polymorphism of the single nucleotide mutation is represented as G/A polymorphism, and the total litter size of AG type individual pigs is obviously higher than that of AA type individuals and is obviously higher than that of GG type individuals. The SNP marker can be identified to screen the large white pig total litter size strain, and the obtained pig high total litter size strain has important edible value, economic benefit and social value.
Description
Technical Field
The application relates to the technical field of large white pig total litter size molecular markers, in particular to an SNP marker related to large white pig total litter size and application thereof.
Background
The high reproduction rate is an important selection standard of a live pig breeding scheme, and plays a vital role in live pig industry breeding. Reproductive performance is characterized by quantitative traits such as total birth number (TNB), birth Number (NBA), birth length, litter size, etc. Many Quantitative Trait Loci (QTLs) and candidate genes associated with reproductive performance have been identified by microsatellite markers and linkage mapping. The total litter size of pigs is an important index for measuring the reproductive capacity of pigs, and the conventional breeding method is used for breeding with great difficulty and slow progress.
With the development of molecular biology, the genome-wide SNPs molecular markers are gradually applied to selective breeding of pig litter size. Many genomic projects for identifying whole genome base pair sequences have rapidly progressed, and many genes can be identified in a short time by exhaustive analysis of hundreds of millions of Single Nucleotide Polymorphism (SNP) markers through selective hybridization on a solid surface based on genotypes due to development of DNA chip technology using microarrays. Researchers use whole genome association analysis technology to screen 160,124 SNP reaching significance level in Shengxian County flower pigs and Jinhua pig groups respectively, and also use whole genome SNPs chip to screen breast cancer antiestrogen resistant gene 3 (BCAR 3), N-ethylmaleimide sensitive factor (NSF) and other potential candidate genes of early day age in Duroc pigs.
Although some progress has been made, there are still problems and challenges in research. First, the total litter size of pigs is affected by a variety of factors, including factors such as genes, environment and management. Thus, in order to better predict the total litter size in pigs, multiple factors need to be considered in combination, not just the effect of a single molecular marker. Secondly, because of the large number of pig breeds, the genetic differences between different breeds are large, and therefore systematic studies are required in a plurality of different breeds to find more reliable and effective molecular markers.
Thus, the search for molecular markers related to the overall litter size of pigs has become one of the hot spots in current swine genomics research.
Disclosure of Invention
The application utilizes whole genome association analysis (GWAS) to detect genetic marker polymorphism of a plurality of individuals in a whole genome range to obtain genotypes, further carries out population level statistical analysis on the genotypes and phenotypes, screens SNP loci which are most likely to influence the traits according to significance detection, and finds out genes related to the total litter size of pigs. Therefore, the embodiment of the application at least discloses the following technical scheme:
A SNP marker associated with the total litter size of a white pig, the SNP marker comprising: the nucleotide sequence formed by single nucleotide mutation of the 117307797 th base of the chromosome 7 of the international pig genome 11.1 version reference sequence, wherein the polymorphism of the single nucleotide mutation is represented as G/A polymorphism, and the total litter size of AG type individual pigs is obviously higher than that of AA type individuals and is obviously higher than that of GG type individuals.
SNP marker primer related to total litter size of large white pigs, comprising DNA molecules shown as SEQ ID NO. 1-2.
An SNP marker related to the total litter size of a white pig is formed by amplification of the SNP marker primer.
A nucleic acid molecule having a nucleotide sequence formed by single nucleotide mutation of base 117307797 of chromosome 7 of version 11.1 of the international swine genome, said single nucleotide mutation polymorphism exhibiting a G/a polymorphism, wherein the total litter size of an individual pig of type AG is significantly higher than that of an individual of type AA, and significantly higher than that of an individual of type GG.
A kit for detecting the total litter size of a white pig comprises the SNP marker primer and other reagents required by PCR amplification.
A kit for screening a large white pig strain with total pig litter size comprises the SNP marker primer and other reagents required by PCR amplification.
The application of the molecular marker, or the nucleic acid molecule, or the SNP marker primer in screening of large white pig strains with high total pig yield.
A method for screening a large white pig strain of total litter size of a pig, comprising detecting the genotype of nucleotide locus of chromosome 117307797 of version 11.1 reference sequence pig chromosome 7 of the international pig genome of the large white pig, as represented by a G/a polymorphism.
Compared with the prior art, the application has at least one of the following beneficial effects:
The SNP marker provided by the application is related to the total litter size of the large white pigs, and the molecular marker and the primer developed based on the SNP can be used for detecting the SNP. Therefore, the SNP marker can be identified to screen the large white pig total litter size line, and the obtained pig high total litter size line has important edible value, economic benefit and social value.
The application perfects the breeding molecular marker related to the total litter size of the large white pig, utilizes a 60K gene chip to carry out SNP typing, screens and obtains the SNP related to the total litter size of the large white pig by using a whole genome correlation analysis technology (GWAS), and provides a new molecular marker resource and marker assisted selection application foundation for genetic breeding of pigs.
Drawings
Fig. 1 is a manhattan diagram provided by the present invention. Reference numerals illustrate: the study is that the pig total litter size trait, black circles point to molecular markers marked for screening of the invention, which are located on chromosome 7 of the large white pig.
FIG. 2 is a gel electrophoresis chart of a DNA fragment of amplified selected molecular markers provided in the embodiment of the present application, M represents a 500bp Marker molecular weight standard, 1-3 represents DNA products amplified at different annealing temperatures, and the annealing temperatures are sequentially: 55 ℃, 58 ℃ and 60 ℃. Wherein, the molecular weight bands of the 1000bp Marker are 500bp, 400bp, 300bp, 250bp, 200bp, 100bp and 50bp from top to bottom.
FIG. 3 is a sequence alignment pattern diagram for screening SNP mutation sites, wherein the arrow points represent mutation sites, provided by the embodiment of the application.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more apparent, the present application will be described in further detail with reference to the following examples. 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 application. The reagents not specifically and individually described in the present application are all conventional reagents and are commercially available; methods which are not specifically described in detail are all routine experimental methods and are known from the prior art.
It should be noted that, the terms "first," "second," and the like in the description and the claims of the present invention and the above drawings are used for distinguishing similar objects, and are not necessarily used for describing a particular sequence or order, nor do they substantially limit the technical features that follow. It is to be understood that the data so used may be interchanged where appropriate such that the embodiments of the invention described herein may be implemented in sequences other than those illustrated or otherwise described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
For a better understanding of the present application, and not to limit its scope, all numbers expressing quantities, percentages, and other values used in the present application are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. Each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
Screening and confirmation of SNP markers
1. Test sample collection
The experimental herd was 1182 pure white pigs (castrated, weighing about 90 kg) from a pig farm of some kind in Hubei province.
The pig flock can eat and drink water freely, and the whole feeding mode, feeding conditions and the like are kept consistent all the time, so that the method is a conventional method.
Before slaughtering, ear tissue samples of all test pigs are collected and put into 75% ethanol for preservation, so as to extract pig genome DNA for standby, and the specific method refers to a specification provided by genome DNA kit produced by Beijing Tiangen Biochemical technology Co.
2. Extraction and detection of pig genome DNA
The test adopts a genome DNA kit (TIANamp Genomic DNA Kit) produced by Beijing Tiangen Biochemical technology Co., ltd.) to extract pig genome DNA from pig ear tissues, and comprises the following specific operation steps:
1) The pig ear sample is sheared into paste by the ophthalmic surgical scissors after being sterilized by alcohol, 200 mu L of buffer GA (the kit is self-contained) is added, and the mixture is oscillated to thoroughly suspend.
2) Add 20. Mu.L of proteinase K solution (the kit is self-contained), mix well and place in 56℃water bath for digestion overnight.
3) 200. Mu.L of buffer GB (the kit is self-contained) was added, mixed well upside down, and left at 70℃for 10 minutes, and after the solution became clear, centrifuged briefly to remove the water droplets on the inner wall of the tube cap.
4) Adding 200 μl of absolute ethanol, shaking thoroughly, and mixing for 15s, wherein flocculent precipitate may appear, and centrifuging briefly to remove water droplets on the inner wall of the tube cover.
5) The solution obtained in the last step and flocculent precipitate are added into an adsorption column CB3 (the adsorption column is placed into a collecting pipe), the solution is centrifuged at 12000rpm for 30s, waste liquid is poured out, and the adsorption column CB3 is placed into the collecting pipe.
6) To the adsorption column CB3, 500. Mu.L of a buffer solution GD (the kit is self-contained), and the mixture is centrifuged at 12000rpm for 30s, and the waste liquid is poured off, and the adsorption column CB3 is placed in a collection tube.
7) 600. Mu.L of the rinse PW (the kit itself) was added to the column CB3, centrifuged at 12000rpm for 30s, the waste liquid was poured off, and the column CB3 was placed in a collection tube.
8) And repeating the operation step 7.
9) Placing the adsorption column CB3 back into the collecting pipe, centrifuging at 12000rpm for 2min, pouring out the waste liquid, placing the adsorption column CB3 at room temperature for several minutes, and thoroughly airing residual rinse liquid in the adsorption material.
10 Transferring the adsorption column CB3 into a clean centrifuge tube, suspending and dripping 50-200 mu L of elution buffer TE into the middle part of the adsorption film, standing for 2-5min at room temperature, centrifuging for 2min at 12000rpm, and collecting the solution into the centrifuge tube.
11 Taking 2 mu L of the DNA solution obtained in the last step and 1 mu L of the sample adding buffer solution, evenly mixing, loading the sample on 1.2% agarose gel, carrying out electrophoresis for about 20min at 120V voltage, observing the electrophoresis result under an ultraviolet lamp, and photographing to judge the integrity of the DNA. The quality of the extracted DNA was checked by a NanoDrop2000 nucleic acid protein analyzer (Thermo FISHER SCIENTIFIC, USA), the ratio of A260/A280 was 1.7-2.1, and A260/A230 was judged to be acceptable between 1.8-2.2. And (3) carrying out concentration measurement on qualified DNA, uniformly diluting the concentration to 200 ng/. Mu.L, and placing the DNA in a refrigerator at the temperature of minus 20 ℃. Failed DNA samples then need to be re-extracted. 3. SNP chip genotype determination and quality control of genotype data
Genomic DNA samples extracted from 1182 pig ear samples were hybridized on PorcineSNP60, 60 BeadChip 6 whole genome chips developed by Illumina corporation. The chip contains 61565 SNP loci. Quality control inspection is carried out on the original genotype data of all individuals by PLINK software, and indexes such as P value <10 -6, sample detection rate (SAMPLE CALL RATE) >90% and the like of SNP genotype detection rate (SNPCALL RATE) >90%, minimum allele frequency (minor allele frequency, MAF) >0.01, hardy-Winberg equilibrium (Hardy-Weinberg Equilibrium, HWE) inspection are used as standards.
4. Data arrangement and analysis
1) Phenotypic data analysis
Descriptive statistical analysis of the total litter size of pigs was performed using R statistical analysis software, including calculation of mean, standard deviation, maximum and minimum values for the trait.
2) Whole genome association analysis
GWAS analysis was performed using PLINK software.
The model is as follows: yij=μ+gi+εij;
Wherein YIj is the character value after treatment; mu is the mean value of each trait; gi is the genotypic effect; εij is a random effect.
3) Testing significance of association of SNPs with traits
When a certain SNP meets the P <10 -4 condition, we consider that this SNP reaches a significant genome level of the whole genome.
4) SNP annotation
Based on the chip SNP information, the SNP was annotated using VARIANT EFFECT Predictor tools in the Sus scrofa Buid 11.1.1 database of the Ensembl website (www.ensembl.org), i.e., the chromosome on which the SNP site was located and its physical location on the chromosome was determined, and thus whether these significant SNPs were within the interior or flanking regions of known genes in the Ensembl database. Then, the genes in the target region are functionally annotated based on information such as gene structure, gene type, gene function and pathway provided by Ensembl, NCBI (www.ncbi.nlm.nih.gov), DAVID (DAVID. Abcc. Ncifcrf. Gov), etc. by using a bioinformatics method. Finally, whether the site falls in the reported QTLs related to meat quality traits is searched through QTLdb (cn. Analogenome. Org/cgi-bin/QTLdb/index) websites, and SNPs related to the total litter size of the white pig are further determined.
6. Analysis of results
Table 1 shows SNP loci associated with total litter size of large white pigs. The genotype detection result of the pig gene polymorphic locus rs80823344 (ALGA 0045338, 117307797) shows that the AA genotypes are 440, the AG genotypes are 639 and the GG genotypes are 103 in 1182 individuals. For the characteristics of the total litter size of the large white pigs, the total litter size of individual pigs with the genotype AG is significantly higher than that of individuals with the genotype AA, the total litter size of individual pigs with the genotype AG is significantly higher than that of individuals with the genotype GG, and the total litter size of individual pigs with the genotype AA is not significantly different from that of individuals with the genotype GG.
TABLE 1 effect of polymorphism of ALGA0045338 and different genotypes on total litter size in pigs
| Number of individuals | Genotype of the type | Total litter size (head) |
| 440 | AA | 12.87±0.25 |
| 639 | AG | 13.69±0.23 |
| 103 | GG | 12.74±0.12 |
| P-value | ||
| AA-AG | 0.0324 | |
| AA-GG | 0.3522 | |
| AG-GG | 0.0281 |
Based on the results of table 1, the embodiment of the application discloses at least one SNP marker related to the total litter size of white pigs, which comprises: the nucleotide sequence formed by single nucleotide mutation of the 117307797 th base of the chromosome 7 of the international pig genome 11.1 version reference sequence, wherein the polymorphism of the single nucleotide mutation is represented as G/A polymorphism, and the total litter size of AG type individual pigs is obviously higher than that of AA type individuals and is obviously higher than that of GG type individuals.
SNP marker application
Based on the discovered SNP markers related to the total litter size of the white pig, the embodiment of the application also provides a SNP marker primer related to the total litter size of the white pig, which comprises DNA molecules shown as SEQ ID NO. 1-2.
Based on the SNP marker primer, the embodiment of the application also provides an SNP marker related to the total litter size of the white pig, which is formed by amplifying the SNP marker primer.
For example, the embodiment of the application also provides a nucleic acid molecule, which has a nucleotide sequence formed by single nucleotide mutation of the 117307797 th base of the chromosome 7 of the international pig genome 11.1 version reference sequence, wherein the polymorphism of the single nucleotide mutation is represented as a G/A polymorphism, and the total litter size of the pig of an AG type individual is significantly higher than that of an AA type individual and significantly higher than that of the pig of a GG type individual. In some embodiments, the nucleic acid molecule comprises a DNA molecule as set forth in SEQ ID No.3 and/or 4.
In addition, the embodiment of the application also provides a kit for detecting the total litter size of the large white pig, which comprises the SNP marker primer and other reagents required by PCR amplification.
The embodiment of the application also provides a kit for screening the large white pig strain with the total litter size of pigs, which comprises the SNP marker primer and other reagents required by PCR amplification.
In addition, the embodiment of the application also discloses application of the molecular marker or SNP marker primer in screening of large white pig strains with high total pig yield. In addition, the embodiment of the application also discloses a method for screening a large white pig strain with total pig litter size, which comprises detecting the genotype of nucleotide locus of chromosome 117307797 of chromosome 7 of the large white pig international pig genome 11.1 version reference sequence, wherein the genotype is expressed as G/A polymorphism.
In some specific embodiments, the analysis of the total litter size of large white pigs using these molecular markers, or SNP marker primers, comprises the steps of:
(1) Extraction of pig genomic DNA
Genomic DNA of the boar of the large white pig was extracted by referring to the above method.
(2) PCR amplification
According to the genotype of the 117307797 nucleotide locus of the chromosome 7 of the reference sequence pig version 11.1 of the international pig genome of the large white pig, which is expressed as G/A polymorphism, PCR primers are designed and synthesized near the single nucleotide polymorphism locus, and the obtained primer pair has a DNA molecule with a nucleotide sequence shown as SEQ ID NO. 1-2.
Taking the extracted large white piglet DNA as an amplification template, and carrying out PCR amplification according to the designed primer, wherein a PCR reaction system is as follows: 1. Mu.l of DNA template, 0.5. Mu.l of each of F and R primers, 10. Mu.l of PCR Mix reagent (2 XM 5 TAQ HIFI PCR Mix, mei5 bio); the PCR amplification procedure was: 94 ℃ for 3min;30cycles (94 ℃,25s; 55-60 ℃,25s;72 ℃,10 s); 72 ℃ for 5min;4 ℃ and infinity.
As shown in FIG. 3, the PCR products were electrophoretically detected in 2% agarose gel, and the amplified target fragment was about 185bp in size.
The 185bp target fragment is as follows:
aggaccttcaactgattgggtaaggcccaccatattctggctttcctcaaaatctattgagttaaatgctaagagctacacaacaccttcacaG/Acaacattgacaatggcgtttgactaaacaactgagccaccatagaccacccagggtgacacagaagtctatccataagagagtccaaggca, Wherein, the uppercase is the underlined SNP locus provided by the application, when the underlined is G, the underlined SNP locus is shown as SEQ ID NO.3, and when the underlined is A, the underlined SNP locus is shown as SEQ ID NO. 4.
(3) Genotyping
Sequencing the rest amplified products, comparing and analyzing the sequencing result with the relevant gene fragments of pigs in GenBank by Snapgene software, and judging the genotype of 117307797 locus. As shown in FIG. 3, the genotype of the large white pig is sequentially divided into AG type pig large white pig and boar with excellent litter size.
In conclusion, the SNP marker provided by the application is related to the litter size of the white pig, and the molecular marker and the primer developed based on the SNP can be used for detecting the SNP. Therefore, the SNP marker can be identified to screen the large white pig boar litter size line, and the obtained line of the large white pig boar litter size has important edible value, economic benefit and social value.
The present application is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present application are intended to be included in the scope of the present application.
Claims (9)
1. A SNP marker associated with the total litter size of a white pig, the SNP marker comprising: the nucleotide sequence formed by single nucleotide mutation of the 117307797 th base of the chromosome 7 of the international pig genome 11.1 version reference sequence, wherein the polymorphism of the single nucleotide mutation is represented as G/A polymorphism, and the total litter size of AG type individual pigs is obviously higher than that of AA type individuals and is obviously higher than that of GG type individuals.
2. SNP marker primer related to total litter size of large white pigs, comprising DNA molecules shown as SEQ ID NO. 1-2.
3. A SNP marker related to the total litter size of a large white pig, which is amplified from the SNP marker primer set forth in claim 2.
4. A nucleic acid molecule having a nucleotide sequence formed by single nucleotide mutation of base 117307797 of chromosome 7 of version 11.1 of the international swine genome, said single nucleotide mutation polymorphism exhibiting a G/a polymorphism, wherein the total litter size of an individual pig of type AG is significantly higher than that of an individual of type AA, and significantly higher than that of an individual of type GG.
5. The nucleic acid molecule according to claim 4, comprising a DNA molecule as shown in SEQ ID NO.3 and/or 4.
6. A kit for detecting the total litter size of a white pig, comprising the SNP marker primer of claim 2 and other reagents required for PCR amplification.
7. A kit for screening a white pig line for total litter size of pigs, comprising the SNP marker primer of claim 1, and other reagents required for PCR amplification.
8. Use of the molecular marker of claim 1 or 3, or the nucleic acid molecule of claim 4 or 5, or the SNP marker primer of claim 2, for screening large white pig lines of high total swine yield.
9. A method for screening a large white pig strain of total litter size of a pig, comprising detecting the genotype of nucleotide locus of chromosome 117307797 of version 11.1 reference sequence pig chromosome 7 of the international pig genome of the large white pig, as represented by a G/a polymorphism.
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| KR101855666B1 (en) * | 2016-11-10 | 2018-05-09 | 대한민국 | SNP markers for prediction of pigs total litter size and methods for prediction of pigs total litter size using the same |
| CN114045350A (en) * | 2021-12-14 | 2022-02-15 | 华中农业大学 | SNP molecular marker associated with porcine muscle drip loss character and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101855666B1 (en) * | 2016-11-10 | 2018-05-09 | 대한민국 | SNP markers for prediction of pigs total litter size and methods for prediction of pigs total litter size using the same |
| CN114045350A (en) * | 2021-12-14 | 2022-02-15 | 华中农业大学 | SNP molecular marker associated with porcine muscle drip loss character and application thereof |
Non-Patent Citations (1)
| Title |
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| 胡慧艳;贾青;赵思思;李晓敏;张伟峰;李书杨;赵坤峰;庞立欣;: "大白猪产仔数相关基因多态性及其遗传效应", 中国兽医学报, no. 11, 15 November 2017 (2017-11-15) * |
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