CN113718039B - SNP (Single nucleotide polymorphism) marker primer pair related to pig rib number character and application thereof - Google Patents
SNP (Single nucleotide polymorphism) marker primer pair related to pig rib number character and application thereof Download PDFInfo
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Abstract
The invention relates to a SNP (single nucleotide polymorphism) marker primer pair related to pig rib number traits and application thereof. The SNP marker is positioned on the nucleotide sequence of TRAM1 gene on the chromosome 4 of the pig, the locus of the SNP marker is the molecular marker of the locus of rs80949198 nucleotide of the chromosome 4 of the 11.1 version reference sequence of the international pig genome, and the locus has C/T polymorphism. A primer pair for detecting the SNP marker, wherein the upstream primer is: SEQ ID NO:2, the downstream primer is: SEQ ID NO:3. the SNP marker provided by the invention can be applied to marker-assisted selection of pig rib number traits, and a pig population or strain with multiple rib numbers can be screened by identifying the genotype of the SNP marker. The establishment of the group or the strain can improve the meat production performance of pigs and generate more social and economic benefits.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and relates to a SNP (single nucleotide polymorphism) marker primer pair related to pig rib number traits and application thereof.
Background
China is a major country of pork production and consumption, and in chinese dietary structure, pork is the major source of national animal protein. With the development of economy and the continuous improvement of life quality, the demand of people for pork is increasing, and how to improve the pork yield of pig carcasses to increase the demand of the market for pork is the key point of the current breeding work.
The pig spine can be divided into cervical vertebra, thoracic vertebra, lumbar vertebra, referral vertebra and coccyx, and the number of lumbar vertebra and thoracic vertebra of a general pig varies, while the number of cervical vertebra, referral vertebra and coccyx is constant. The ribs are connected to the thoracic vertebrae, the number of which determines the number of ribs. The number of ribs is obviously related to the carcass length of the pig, the carcass length of the pig is correspondingly increased along with the increase of the number of ribs, the carcass length can be increased by about 60mm each rib, and the meat yield of the pig is correspondingly increased. The pork chop is one of the carcass parts with highest selling price in the Chinese pork consumer market, and is popular with consumers due to high lean meat percentage and good meat quality. Therefore, the genetic molecular mechanism of the pig rib number is researched, and the related molecular markers are applied to production, so that the method has important economic benefit for breeding a population or a new line with multiple rib numbers and high meat yield.
Disclosure of Invention
The invention aims at providing a selective breeding molecular marker developed by SNP markers related to the number of pig ribs aiming at the problems of time and labor consumption and slow selective breeding effect of the traditional pig rib number selective breeding.
Another object of the present invention is to provide a primer set and a detection method for detecting the above SNP markers. It is another object of the present invention to provide the use of the SNP marker, the molecular marker and the primer.
The aim of the invention can be achieved by the following technical scheme:
a molecular marker related to pig rib number character, wherein the molecular marker sequence is shown in SEQ ID NO:1, wherein the gene contains a SNP marker locus related to pig rib number character, the locus is a nucleotide locus of a chromosome 4 rs80949198 of an international pig genome 11.1 version reference sequence, and the gene is shown in SEQ ID NO:1, wherein the SNP marker locus is positioned at the 501 st position and a C/T polymorphism exists.
A primer pair for detecting SNP markers related to swine rib number traits, the upstream primer being: SEQ ID NO:2, the downstream primer is: SEQ ID NO:3.
the molecular marker disclosed by the invention and the primer pair disclosed by claim 2 are applied to detection of pig rib number traits and/or pig breeding.
A method for detecting SNP markers related to pig rib number traits comprises the steps of amplifying a segment of sequence of a nucleotide locus of a pig No. 4 chromosome rs80949198 of a pig international pig genome 11.1 version reference sequence by PCR, sequencing an amplified product, and judging the C/T polymorphism of the locus.
Preferably, the pig is a Suhuai pig.
As a preferred mode of the invention, PCR amplification is carried out on the Suhuai pig genome DNA by using the primer pair.
As a further preferred aspect of the present invention, the method comprises the steps of:
(1) Taking a pig tissue sample to extract total DNA;
(2) Using the extracted pig genome DNA as a template, and carrying out PCR amplification by using the primer pair disclosed by the invention;
(3) Sequencing the amplified product, analyzing the sequencing result, and judging the sequence in SEQ ID NO: C/T polymorphism at position 501 of 1.
The molecular marker disclosed by the invention is applied to screening of multi-rib pig groups or new strains.
The primer pair provided by the invention is applied to screening of multi-rib pig groups or new strains.
A method for screening multi-rib pig groups comprises detecting genotypes of rs80949198 nucleotide loci of chromosome 4 of pig international pig genome 11.1 version reference sequence, and breeding CC type and CT type individuals of rs80949198 nucleotide loci to be preferentially used as reserve pigs for seed reserving.
As a preferred embodiment of the present invention, the swine used is Suhuai pig
As a preferred aspect of the invention, the method for detecting the genotype of the rs80949198 nucleotide site of the swine international swine genome version 11.1 reference sequence swine chromosome 4 is selected from PCR or genetic sequencing.
Advantageous effects
The invention develops SNP markers related to pig rib numbers, and provides primer pairs and methods for detecting the markers. Pig lines with multiple ribs were screened by identifying the genotype of the SNP marker. The establishment of the strain can improve the meat production performance of pigs and generate more social and economic benefits.
Drawings
FIG. 1 is a PCR amplification gel diagram of the rs80949198 locus of the TRAM1 gene of Suhuai pig No. 1.
Fig. 2 is an example of a rs80949198 locus typing map of the chromosome 1 TRAM1 gene of the su huai pig.
A is CC type, B is CT type, and C is TT type.
Detailed description of the preferred embodiments
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.
Example 1
1 test animal origin
Huaiyin new Huaihe pig farm in Huaishu, jiangsu province
2 extraction of pig genomic DNA
Collecting 457 parts of a tissue sample of the Su Huai pig ear for extracting individual DNA;
referring to the instruction book of the tissue DNA extraction kit of Tiangen biotechnology company, the extraction steps are as follows:
(1) 68mL of absolute ethyl alcohol and 200mL of absolute ethyl alcohol are respectively added into the buffer solution GD and the rinsing solution PW, and the mixture is fully and uniformly mixed.
(2) About 100mg of the collected ear tissue sample was placed in a 2mL EP tube, and after complete shearing, 200. Mu.L of buffer GA was added thereto, and the mixture was shaken until it was thoroughly suspended.
(3) Add 20. Mu.L proteinase K solution, mix well and digest overnight in 56℃metal bath until tissue sample dissolves and briefly centrifuge to remove water droplets from the inner wall of the tube cap.
(4) Adding 200 μl buffer GB, mixing, standing in 70 deg.C metal bath for 10min, clearing the solution strain, and centrifuging for a short time to remove water droplets on the inner wall of the tube cover.
(5) Adding 200 mu L of absolute ethyl alcohol, fully shaking and uniformly mixing for 15sec, wherein flocculent precipitation possibly occurs, and centrifuging briefly to remove water drops on the inner wall of the tube cover.
(6) The solution obtained in the previous step and the flocculent precipitate were both fed into an adsorption column CB3, the adsorption column was placed into a collection tube, and then centrifuged at 12,000rpm for 30sec, the waste liquid was discarded, and the adsorption column CB3 was returned into the collection tube.
(7) Adding 500 μl of buffer GD to the adsorption column CB3, centrifuging at 12,000rpm for 30sec, pouring out the waste liquid, and placing the adsorption column CB3 into a collection tube
(8) 600. Mu.L of the rinse PW was added to the adsorption column CB3, centrifuged at 12,000rpm for 30sec, and the waste liquid was poured off, and the adsorption column CB3 was placed in a collection tube.
(9) Repeating the operation step (8).
The adsorption column CB3 was returned to the collection tube and centrifuged at 12,000rpm for 2 minutes to discard the waste liquid. The adsorption column CB3 was left at room temperature for several minutes to thoroughly dry the residual rinse solution in the adsorption material.
The quality and concentration of DNA were measured using a Nanodrop-2000 spectrophotometer, and the DNA concentration was diluted to 50 ng/. Mu.L and stored at-20℃for further use.
3-purpose fragment PCR amplification and sequencing
PCR amplification is carried out by taking the Suhuai pig genome DNA as a template, and a reaction system comprises 1 mu L of DNA template and SEQ ID NO:2 and SEQ ID NO:3, 1. Mu.L of each of the primers shown in FIG. 3, and 22. Mu.L of each of the PCR mix; the amplification procedure was as follows:
the amplified product was subjected to agarose gel electrophoresis, the fragment size of the product was about 606bp, and the result of the electrophoresis was shown in FIG. 1. Sequencing the rest amplified products, comparing and verifying the accuracy of the sequence by DNAman software, and judging the rs80949198 locus by using Chromas software.
4 statistical analysis
The correlation analysis of genotype and phenotype was performed using a general linear model of SAS 9.4 software, the model being as follows: y is Y ijk ＝μ i +B j +G k +e jk
Wherein Yijk is the number of ribs of the individual; mu (mu) i Representing a population rib number average; b (B) j Representing the stationary effect of slaughter batches; g k Is the immobilization effect of SNP markers; e, e jk Is the residual.
5 results
Table 1 shows the effect of different genotypes at the rs80949198 locus on the rib count of Suhuai pigs. The results show that there is a very significant difference between the rib numbers of individuals of the three genotypes at the rs80949198 locus (P < 0.01). Wherein, the number of ribs of CC type individual is extremely more than those of CT type and TT type (P < 0.01), and the number of ribs of CT type individual is extremely more than those of TT type (P < 0.01). Therefore, CC type and CT type individuals at the rs80949198 locus are bred in the Suhuai pig offspring, which is beneficial to increasing the rib number of the Suhuai pig group and further improving the meat production performance of the Suhuai pig.
TABLE 1 analysis of the rs80949198 locus of the TRAM1 Gene of pig No. 4 chromosome and the rib count correlation of Suhuai pig
Note that: the different letters of the same row digital shoulder marks represent significant differences (P < 0.01)
Sequence listing
<110> Nanjing agricultural university
Huaian' an institute of Nanjing university of agriculture
<120> SNP marker primer pair related to pig rib number character and application thereof
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1001
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
tttttgcttt ttagggccac acccaaggca tatggaggtt cccaggctag gggttgaata 60
ggagctacag ctccctgtct acaccacagc cacagccaca gccacatgga atccgagctg 120
tgtccatgac cttcaccaca gctcaaggcc agatctttaa cccactgagc acagtcaggg 180
atggaacacg catcctcatg gatcctagtc gggttcatta actgctgagc cacaaaggga 240
actcctatct ctaatctttt aagtgtgaag gatcccacta ttctgccctt gaccttttag 300
acttctctca catcagttgt ctttcatatt ttcaggattc catgttttca ttccgtatct 360
cagaagcata aaacttaatt catcacctca tgcagctcta gactgctctg aaaatatcta 420
gatttcatat aggtgaatgg tacaagcttc tagccaatca gtggtgccag aaaatcttgg 480
atttcatttt ataatcctcc ctctctcacc cagttattct ttcagttcta acatctctcc 540
ccttgttgct gaaccattgc ataagctcag gccttcattc ttgttgctga accattgcat 600
aagcctcttc ccagcattcc ctgacttgaa tctcactgat tctgcctcct accctgtcca 660
tactccctcg ttccaaaatg aacattctaa cataaaacat ctgcaaaaaa aaacttcaat 720
gattttccca ttgctcctag tttaacatcg aaattctctt gcctttagac ctaaacattc 780
aaggaccttc aagatgttat ctcagcattt gagggccatt ggcattggct tacgggctca 840
tgacatactc taagcaatac tcagccatcc gtggtccctc agcttctccg tgtctttgtg 900
cacattgttc tctaatggaa atatgattcc tccacctgtt ttctgaatat gatctgctgg 960
ctgccagacc tgggaagcct tcatgatctt ctccagccag a 1001
<210> 2
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
gatcccacta ttctgccctt ga 22
<210> 3
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
gaccacggat ggctgagtat 20
Claims (3)
1. The application of the primer pair for SNP markers related to the rib number traits of the Suhuai pigs in detecting the rib number traits of the Suhuai pigs and/or breeding of the Suhuai pigs; the SNP marker related to the rib number character of the Suhuai pig is positioned at the rs80949198 nucleotide site of the chromosome 4 of the reference sequence pig version 11.1 of the international pig genome, and is shown in SEQ ID NO: 1. the SNP marker locus is positioned at the 501 th position, and a C/T polymorphism exists; the primer pair consists of SEQ ID NO:2 and the sequence of SEQ ID NO:3, and a downstream primer.
2. A method for screening multi-rib Suhuai pig groups is characterized by comprising the steps of detecting genotypes of rs80949198 nucleotide loci of a No. 4 chromosome of a pig international pig genome 11.1 version reference sequence, and breeding individuals with the rs80949198 nucleotide loci of CC and CT as reserve pigs.
3. The method according to claim 2, characterized in that the method for detecting the genotype of the rs80949198 nucleotide site of the swine international swine genome version 11.1 reference sequence swine chromosome 4 is selected from PCR or genetic sequencing.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107828891A (en) * | 2016-09-14 | 2018-03-23 | 华中农业大学 | The related molecular marker screening of pig rib number character and application |
| CN113046443A (en) * | 2020-11-18 | 2021-06-29 | 中国农业科学院北京畜牧兽医研究所 | SNP molecular marker influencing pig rib number and application thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107828891A (en) * | 2016-09-14 | 2018-03-23 | 华中农业大学 | The related molecular marker screening of pig rib number character and application |
| CN113046443A (en) * | 2020-11-18 | 2021-06-29 | 中国农业科学院北京畜牧兽医研究所 | SNP molecular marker influencing pig rib number and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| rs80949198.Ensemble.2021,1-2页. * |
| 枣庄黑盖猪群体中脊椎数候选基因的多态性与遗传分析;沈瑞玲 等;养猪(第04期);73-75页 * |
| 苏淮猪VRTN基因克隆、组织表达特征与多态性分析;吴一尘 等;中国农业科学(第18期);3639-3648页 * |
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