CN114182025B - SNP molecular marker related to pig feed conversion rate and application thereof - Google Patents
SNP molecular marker related to pig feed conversion rate and application thereof Download PDFInfo
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- 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
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- 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
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Abstract
The application discloses SNP molecular markers related to the pig feed conversion rate and application thereof. The SNP molecular marker is located at the position of 7990990bp of international pig reference genome Ensembl Scrofa11.1 version pig No. 7 chromosome, and shows polymorphism of A/C. The application verifies the influence effect of the molecular marker on the feed conversion rate character, and establishes a high-efficiency and accurate molecular marker assisted breeding technology so as to be applied to the genetic improvement of the feed conversion rate character of the boar and help to reduce the feed conversion rate of the boar and improve the pork quality.
Description
Technical Field
The application relates to the technical field of molecular markers of pig feed conversion rate, in particular to SNP molecular markers related to the pig feed conversion rate and application thereof.
Background
The Feed Conversion Ratio (FCR) is the ratio of the weight of the feed consumed by animals to the weight gain, can be used as an important index for evaluating the feed efficiency, is widely used for estimating the feed efficiency of pig breeding, and is a research hotspot in the production of pigs at present. The pig strain with high feed conversion rate can not only make full use of feed nutrition, but also provide a high-quality basis for the meat quality. According to scientific data, the pig strain is selected and cultured, genetic improvement is carried out, the pig strain with low feed conversion rate can be obtained through breeding, not only can the help be provided for saving feed and reducing production cost, but also the economic benefit of the pig strain with high quality and meat quality can be brought into play. However, the prior art often analyzes and selects and matches according to the pig phenotype, not only the efficiency is low, but the breeding power is poor.
Modern molecular biology can utilize molecular markers associated with the pig feed conversion rate to breed pig lines with low feed conversion rate, however, the molecular markers possibly existing under the condition of the pig feed conversion rate associated with the multi-gene control are numerous and diverse, and the molecular markers which are fully researched and determined to be the pig feed conversion rate still have practical application significance.
Disclosure of Invention
In view of the above, the present application aims to provide at least one molecular marker associated with pig feed conversion rate, which is different from the prior art, so as to provide an aid for breeding pig lines with low feed conversion rate.
In a first aspect, the embodiment of the application discloses an SNP molecular marker related to the pig feed conversion rate, wherein the SNP molecular marker is positioned on the international pig reference genome Ensembl Scrofa11.1 version pig No. 7 chromosome 7990990bp and shows polymorphism A/C.
In the embodiment of the application, the upstream and downstream 100bp sequences of the SNP molecular marker mutation site are shown as SEQ ID NO. 1.
In a second aspect, the embodiments of the present application disclose a primer pair for detecting the SNP molecular marker of the first aspect, the nucleotide sequences of the primer pair are shown as SEQ ID No.2 and SEQ ID No. 3.
In a third aspect, the present application discloses a method for detecting the SNP molecular marker of the first aspect, which includes the following steps:
(1) Extracting the genome DNA of the pig to be detected;
(2) Carrying out PCR amplification on the genomic DNA of the pig to be detected so as to obtain a PCR amplification product;
(3) Sequencing the PCR amplification product so as to obtain a sequencing result;
(4) And judging the polymorphism of the 101 th nucleotide site in the sequence shown by the pig SEQ ID NO.1 based on the sequencing result.
In step (2) of the examples herein, the primer pair used for PCR amplification has the nucleotide sequence shown as SEQ ID NO.2 and SEQ ID NO. 3.
In a fourth aspect, the embodiments of the present application disclose a method for breeding a low-feed conversion rate pig line, the method comprising the following steps:
detecting the genotype of 7990990bp on the international pig reference genome Ensembll Sscrofa11.1 version pig No. 7 chromosome, and selecting an AA type individual with 7990990bp nucleotide sites as a pig.
In a fifth aspect, the embodiment of the application discloses application of SNP loci associated with the property of feed conversion rate of large white pigs in screening of pig lines with high meat-color redness value, wherein the SNP loci are nucleotide loci of 7990990bp of international pig reference genome Ensembl Scrofa11.1 version reference sequence pig No. 7 chromosome and have A/C polymorphism.
Compared with the prior art, the application has at least the following beneficial effects:
the application discloses a molecular marker associated with genes or genotypes related to the pig feed conversion rate and the pig feed conversion rate, the influence effect of the molecular marker on the feed conversion rate character is verified, and a high-efficiency and accurate molecular marker assisted breeding technology is established, so that the molecular marker assisted breeding technology is applied to genetic improvement of the pig feed conversion rate character, and the molecular marker assisted breeding technology can help to reduce the pig feed conversion rate and improve pork quality.
Drawings
FIG. 1 is a Manhattan chart relating to feed conversion ratio of white pigs provided in the examples of the present application.
Description of reference numerals: the black circles and arrows point to marked molecular markers for screening the feed conversion rate of the large white pigs, and the molecular markers are positioned on No. 7 chromosomes of the pigs.
FIG. 2 is a gel electrophoresis diagram of a large white pig genome DNA provided in the present application, wherein lane M represents 10000bp Marker molecular weight standard, and lanes 1-5 represent extracted genome DNA; wherein, the 10000bp Marker molecular weight strip is 10000bp, 7000bp, 4000bp, 2000bp, 1000bp, 500bp and 200bp from top to bottom in sequence.
FIG. 3 is a gel electrophoresis diagram of a DNA fragment with a selected molecular Marker provided in the present application, wherein lanes M represent a 2000bp Marker molecular weight standard, lanes 1-3 represent DNA products amplified at different annealing temperatures, and the annealing temperatures are sequentially as follows: 55 ℃, 58 ℃ and 60 ℃; wherein the 2000bp Marker molecular weight strip is 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom in sequence.
FIG. 4 is a diagram showing a sequence alignment of SNP mutation sites screened in an example of the present application, in which the mutation sites are indicated by arrows.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more apparent, the present application is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the present application.
1. Experimental sample Collection
The experimental herd was 1155 large white pigs from a certain pig farm. The swinery can feed and drink water freely, the whole feeding mode, feeding conditions and the like are always consistent, and the method is a conventional method. All the test pig ear tissue samples were collected and stored in 75% ethanol for later use in order to extract pig genomic DNA (the specific method refers to the instruction provided by the genomic DNA kit produced by Beijing Tiangen Biochemical technology Co., ltd.).
2. Determination of feed conversion ratio
All pigs in the experiment are raised in captivity, 10-12 pigs are raised in each pen in the growing and fattening stage, and only drinking water is taken, and the standard of uniform raising is met. Each circle is provided with a pig production performance measuring system which can detect the daily weight, daily feeding times and daily feeding amount of each pig; and data automatically generated by the pig production performance measuring system are processed, abnormal values are removed, and meanwhile, the data with missing or abnormal feed conversion rate are supplemented by using a method of using 4 balance mean values before and after R software.
3. Extraction and detection of pig genome DNA
The experiment adopts a genome DNA Kit (TIANAmp Genomic DNA Kit) produced by Beijing Tiangen Biochemical technology Limited company to extract the pig genome DNA from the pig ear tissue, and the specific operation steps are as follows:
1) The big white pig ear sample was cut into paste with an ophthalmic surgical scissors (wiped clean with alcohol cotton), 200. Mu.L of buffer GA was added, and the suspension was shaken to be thoroughly suspended.
2) Adding 20 μ L proteinase K solution, mixing, and digesting overnight in 56 deg.C water bath.
3) Add 200. Mu.L buffer GB, mix well by inversion, stand at 70 ℃ for 10 minutes, clear the solution, centrifuge briefly to remove beads on the inner wall of the tube cap.
4) Add 200. Mu.L of absolute ethanol, mix well with shaking for 15 seconds, at which time a flocculent precipitate may appear, and centrifuge briefly to remove water droplets on the inner wall of the tube cover.
5) Adding the solution and flocculent precipitate obtained in the previous step into an adsorption column CB3 (placing the adsorption column into a collecting pipe), centrifuging at 12000rpm for 30s, pouring off waste liquid, and placing the adsorption column CB3 back into the collecting pipe.
6) Add 500. Mu.L of buffer GD to adsorption column CB3, centrifuge at 12000rpm for 30s, pour off the waste, place adsorption column CB3 in the collection tube.
7) Adding 600 μ L of rinsing solution PW into adsorption column CB3, centrifuging at 12000rpm for 30s, pouring off waste liquid, and placing adsorption column CB3 into a collecting tube.
8) Operation 7 is repeated.
9) The adsorption column CB3 was returned to the collection tube, centrifuged at 12,000rpm for 2min, and the waste liquid was decanted. And (5) placing the adsorption column CB3 at room temperature for a plurality of minutes, and completely airing the residual rinsing 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 membrane, standing for 2-5 minutes at room temperature, centrifuging for 2 minutes at 12,000rpm, and collecting the solution into the centrifuge tube.
11 2 mul of the solution DNA solution obtained in the previous step and 1 mul of the loading buffer solution are mixed evenly, loaded on 1.2 percent agarose gel, electrophoresed for about 20 minutes at 120V, and the electrophoresis result is observed and photographed under an ultraviolet lamp to judge the integrity of the DNA. The quality of the extracted DNA was checked with a NanoDrop 2000 nucleic acid protein analyzer (Thermo Fisher Scientific, USA), and the ratio of A260/A280 was 1.7-2.1, and A260/A230 was 1.8-2.2, and was judged to be acceptable. 50-100 ng of extracted DNA was subjected to 2% agarose gel electrophoresis, and the result of observation under an imager showed that the bands were concentrated and bright, which is considered as the good extraction effect, as shown in FIG. 2.
4. SNP chip genotype judgment and genotype data quality control
Genomic DNA samples extracted from 1155 pig ear samples were hybridized on a PorcineSNP60 BeadChip whole genome chip developed by Illumina. The chip comprises 61565 SNP sites. Quality control test is carried out on the original genotype data of all individuals by adopting PLINK software, and indexes such as SNP genotype detection rate (SNP call rate) >90%, minimum Allele Frequency (MAF) >0.01, P value <10-6 of Hardy-Weinberg Equilibrium (HWE) test, sample call rate (sample call rate) >90% and the like are taken as standards.
5. Data collation and analysis
1) Phenotypic data analysis
And performing descriptive statistical analysis on the feed conversion rate measured value by using R statistical analysis software, wherein the descriptive statistical analysis comprises calculating the average value, standard deviation, maximum value and minimum value of the character.
2) Whole genome association analysis
And carrying out GWAS analysis by adopting PLINK software. Applicants analyzed the data using the following hybrid model. The model is as follows: yij = μ + Gi + epsilon ij; wherein YIj is the treated property value; mu is the mean value of the characters; gi is the genotype effect; ε ij is a random effect.
3) And (4) testing the significance of the SNP and the character association.
When a certain SNP meets P<10 -4 Under the conditions, the SNP is considered to reach the genome level of the whole genome remarkably.
4) SNP annotation
According to chip SNP information, a Variant Effect Predictor tool is applied to annotate the SNP in a Sus scrofa Buid 11.1 database of an Ensembl website (www.ensembl.org), namely, a chromosome where the SNP locus is located and the physical position of the SNP locus on the chromosome are determined, and therefore whether the significant SNPs are in the interior or the flanking region of a known gene in the Ensembl database is determined. Then, the genes in the target region are functionally annotated by bioinformatics based on information such as gene structures, gene types, gene functions, and pathways provided by websites such as Ensembl, NCBI (www.ncbi.nlm.nih.gov), and DAVID (DAVID. And finally, searching whether the locus falls into the reported QTLs related to the meat quality trait through a QTLdb (cn. Animal genome. Org/cgi-bin/QTLdb/index) website, and further determining the SNPs associated with the meat quality trait of the pig.
6. Analysis of results
As shown in figure 1, the Manhattan diagram shows that the SNP is located on chromosome 7, and provides a new genetic basis for researching factors influencing the pig feed conversion rate, further application of the factors and improving meat quality.
TABLE 1
Table 1 shows the effect of the pig chromosome 7 7990990 mutation site C/A on feed conversion rate in the Suhuai pig population; wherein, indicates that the difference is significant, P <0.05; * Indicates very significant difference P <0.01; the trait values in the table are mean ± standard error.
As can be seen from Table 1, the feed conversion efficiency was close for individuals with genotypes CC and AA; for individuals with genotype AA, the feed conversion efficiency is obviously low; the individual feed conversion rate of the genotype CA is obviously higher than that of the AA genotype and CC genotype individuals (p is less than 0.05). Therefore, in the pure white pig group, the AA type individuals at the 7990990 locus are subcultured and bred, the feed conversion rate of the white pig group can be gradually increased and reduced, and the purposes of saving the feed and improving the meat quality of the white pigs are achieved.
7. Detection of SNP molecular markers
Therefore, the embodiment of the application also discloses a method for detecting the SNP molecular marker associated with the feed conversion rate of the large white pigs, which comprises the following steps of extracting the genomic DNA of the pigs to be detected, carrying out PCR amplification on a DNA sample, sequencing the PCR amplification product, and judging the polymorphism of the 101 th nucleotide site in the sequence shown in the pig SEQ ID NO.1 according to the sequencing result.
Wherein, the PCR amplification process specifically comprises the following steps:
using the extracted DNA as a template, and carrying out PCR amplification according to the designed primer: taking 1 mu L of DNA template, 0.5 mu L of each primer shown in SEQ ID NO.2 and SEQ ID NO.3, and 10 mu L of PCR Mix reagent (2 XM 5 Taq HiFi PCR Mix, mei5 bio); setting a PCR amplification system: 94 ℃ for 3min;28cycles (94 ℃,25s, 55-60 ℃,25s, 72 ℃,10 s); 72 ℃ for 5min; at 4 ℃ and an infinite value.
As shown in FIG. 3, the PCR product was detected by electrophoresis in 2% agarose gel, and the amplified target fragment was about 544bp in size. As shown in FIG. 4, the remaining amplification products were sequenced, and the sequencing results were compared and analyzed with related gene fragments of swine in GenBank using Snapgene software to determine the genotype of 59747430 locus. Therefore, the SNP molecular marker and the primer sequence can detect the CC, CA or AA genotypes.
The above description is only for the preferred embodiment of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present application should be covered within the scope of the present application.
Sequence listing
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Claims (1)
1. The SNP molecular marker related to the pig feed conversion rate is applied to breeding of a pig strain with low feed conversion rate, the SNP locus is an international pig reference genome Ensembl Scrofa11.1 version reference sequence pig No. 7 chromosome 7990990bp nucleotide locus, and has A/C polymorphism, and the upstream and downstream 100bp sequences of the SNP molecular marker mutation locus are shown as SEQ ID NO. 1; the application method comprises the following steps:
detecting the genotype of 7990990bp on the international pig reference genome Ensembl Sscrofa11.1 version pig chromosome 7, and selecting an AA type individual with a 7990990bp nucleotide locus as a boar.
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| CN110317880A (en) * | 2019-07-12 | 2019-10-11 | 天津诺禾致源生物信息科技有限公司 | Molecular labeling relevant to pannage conversion ratio, identification and its application |
| CN113699246A (en) * | 2021-07-26 | 2021-11-26 | 华南农业大学 | SNP molecular marker influencing pig feed conversion efficiency traits and application thereof |
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| CN110317880A (en) * | 2019-07-12 | 2019-10-11 | 天津诺禾致源生物信息科技有限公司 | Molecular labeling relevant to pannage conversion ratio, identification and its application |
| CN113699246A (en) * | 2021-07-26 | 2021-11-26 | 华南农业大学 | SNP molecular marker influencing pig feed conversion efficiency traits and application thereof |
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