CN107988388B - One kind SNP marker relevant to pig dewclaw undue growth character and application thereof - Google Patents
One kind SNP marker relevant to pig dewclaw undue growth character and application thereof Download PDFInfo
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- CN107988388B CN107988388B CN201711310397.4A CN201711310397A CN107988388B CN 107988388 B CN107988388 B CN 107988388B CN 201711310397 A CN201711310397 A CN 201711310397A CN 107988388 B CN107988388 B CN 107988388B
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Abstract
The present invention provides a kind of SNP marker relevant to pig dewclaw undue growth character, the site of the SNP marker is international pig with reference to the 133470903rd nucleotide site on No. 13 chromosomes of 11.1 version of genome, and the base in the site is T or G.The present invention passes through the advantage allele of the preferably SNP, can reduce the generation of great Bai dewclaw undue growth, accelerates genetic progress, effectively improves the economic benefit of kind of pig breeding.
Description
Technical field
The present invention relates to a kind of SNP markers relevant to pig dewclaw undue growth character and application thereof.
Background technique
Pig is typical Artiodactyla mammal, and the 1st toe is degenerated completely；3rd, 4 toes are especially flourishing, and length is equal, claim
Be main hoof；2nd, 5 toes are undeveloped, hang stand in the sky under normal circumstances, referred to as dewclaw (see Fig. 1).The main function of dewclaw
It is the weight for assisting main hoof to bear pig and maintains its balance, (is greater than 7cm) when sow dewclaw is too long, easily occurs
Mechanical damage, in turn resulting in can not stand and diet, secondary to cause farrowing sow miscarriage, milking sow malnutrition etc. (see figure
2), so that the various aspects to sow have an impact, sow is directly eliminated when serious, brings huge economy to breeding enterprise
Loss.
Due to pig dewclaw final lengths after adult just it is observed that, and the seed selection of boar is before this, therefore early
The phase accuracy of selection is just particularly important.And separate and identify that pig dewclaw undue growth susceptibility loci is then that pig dewclaw is excessive
Grow the key of breeding for disease resistance.Currently, whole-genome association (Genome-wide association studies,
GWAS) it is widely used in finding with the sites of livestock and poultry complex character highlights correlations, the molecular labeling filtered out can be to being difficult to
The character (such as dewclaw undue growth) of measurement carries out molecular marker assisted selection (the marker assisted of early stage
Selection, MAS) breeding.Currently, this method has become the important means of large scale of pig farm industry molecular breeding.
Dewclaw undue growth is distributed widely in bacon hogs kind, is the determinant for causing the numerous sow of energy to be eliminated in advance
One of.Great Bai is one of bacon hogs kind famous in the world, because of its higher reproductive capacity, preferable growing and fattening and adaptation
The features such as property, often by as the synthetic female parent of many market pigs.Multiple studies have shown that there is apparent hang in large white sow group
Hoof undue growth phenomenon.Therefore, genetic improvement is carried out to the dewclaw growth traits of large white sow, will it is possible to prevente effectively from sow because
Limb hoof is abnormal and is eliminated in advance, improves the service life of sow to greatest extent, mentions to reduce sow because of toe hoof illness
Before be eliminated the economic loss of brought breeding enterprise.
Summary of the invention
The object of the present invention is to provide a kind of SNP marker relevant to pig dewclaw undue growth character and its use
On the way.
To achieve the above object, the technical solution used in the present invention: a kind of relevant to pig dewclaw undue growth character
SNP marker, the site of the SNP marker are international pig with reference to the 133470903rd on No. 13 chromosomes of 11.1 version of genome
Nucleotide site, the base in the site are T or G.
Preferably, the sequence of the SNP marker is as shown in SEQ ID NO:1, and sequence shown in the SEQ ID NO:1 is from 5 '
Having held the 332nd bit base is T or G.The SNP site of the molecular labeling corresponds to international pig and refers to 11.1 version of genome 13
T > G mutation at the 133470903rd on chromosome；The SNP site of the molecular labeling is SEQ ID NO1: shown, wherein sequence
M in column is T or G, leads to the difference of dewclaw growth traits.The present invention passes through the advantage allele of the preferably SNP, can subtract
The generation of few great Bai dewclaw undue growth, accelerates genetic progress, effectively improves the economic benefit of kind of pig breeding.
The present invention provides a kind of for detecting the primer pair of SNP marker described above, the nucleic acid sequence of the primer pair
Column are as shown in SEQ ID NO:2 and SEQ ID NO:3.
The present invention provides a kind of for detecting the kit of SNP marker described above, including primer described above
It is right.
The present invention provides a kind of methods for reducing pig dewclaw undue growth, the described method comprises the following steps:
The international pig of pig is detected with reference to the 133470903rd nucleotide site on No. 13 chromosomes of 11.1 version of genome
Genotype selects the GG type individual of the 133470903rd nucleotide site as boar.
Preferably, the international pig of the detection pig is with reference to the 133470903rd on No. 13 chromosomes of 11.1 version of genome
The method of the genotype of nucleotide site the following steps are included:
(1) genomic DNA of pig to be measured is extracted；
(2) primer pair as claimed in claim 3 is used, the genomic DNA of the pig to be measured is subjected to PCR amplification, so as to
Obtain pcr amplification product；
(3) pcr amplification product is sequenced, to obtain sequencing result；
(4) it is based on the sequencing result, determines the gene of the SNP marker as claimed in claim 1 or 2 of the pig to be measured
Type.
Preferably, the boar includes its synthesis system of Herba Phaii tankervilliaes.
The present invention provides SNP markers described above to reduce the purposes in pig dewclaw undue growth.
The present invention provides primer pairs described above or the kit to reduce the use in pig dewclaw undue growth
On the way.
The present invention provides SNP marker described above or the primer pairs or the kit in identification pig
Purposes in dewclaw undue growth character.
The beneficial effects of the present invention are:
Present invention research simultaneously determines the relevant molecular labeling of influence large white sow dewclaw undue growth, provides a kind of for reflecting
It is fixed the primer pair for ringing large white sow dewclaw undue growth trait molecular marker, finally establishes the molecular labeling auxiliary of efficiently and accurately
Breeding technique is applied in boar dewclaw growth traits genetic improvement, to reduce pig dewclaw undue growth, reduces sow
Mechanical damage occurs, it is secondary to cause farrowing sow miscarriage, milking sow malnutrition etc., thus to each of sow and offspring piglet
Aspect performance has an impact；Sow service life is improved, enterprise profit is improved, increases core competitiveness.
Detailed description of the invention
Fig. 1 is pig dewclaw schematic diagram；
Fig. 2 is that dewclaw is normal and dewclaw undue growth schematic diagram；
Fig. 3 is that large white sow is associated with (GWAS) analysis about the full-length genome of dewclaw growth traits on No. 13 chromosomes
Figure；Wherein: the chromosome numbers of abscissa expression pig；Ordinate expression-logP value.
Specific embodiment
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.
Foregoing invention purpose of the invention is specifically achieved in that
1, swinery is tested
Experiment swinery group used in the present invention is Guangdong Wen'S Foodstuffs Group Co., Ltd. boar branch company
475 purebred large white sows, are boar branch company core group, and group's pedigree record is detailed.Using identical feeding manner by special
Industry personnel raise and manage.Swinery is raised according to unified pig trophic level, is freely eaten and drinks water.It is strong to test pig
Health, well-grown, no clinical symptoms.
2, sample collection and phenotypic record
Above-mentioned pig ear tissue sample is collected, is soaked in 75% ethyl alcohol that be placed in -20 DEG C of refrigerators spare.
Record pig dewclaw growth traits: two back leg dewclaws measurement of all pigs is all made of moccasin ruler according to unification side
Method carries out precisive, and accuracy is millimeter.It is denoted as left and right directions with the direction that pig is eyed to the front, the dewclaw of hoof is surveyed behind left and right
The length of fixed outside dewclaw, each dewclaw measurement measure 3 times, its average value is taken to be recorded as the outstanding of the rear hoof dewclaw of pig or so respectively
Hoof length.
3, the extracting of tissue DNA and pig full-length genome 80K SNP sentence type
The ear tissue of above-mentioned large white sow is acquired, ear tissue is soaked in 75% ethyl alcohol to be placed in -20 DEG C of refrigerators standby in time
With.The complete genome DNA that large white sow is extracted referring to phenol chloroform method, with Nanodrop-ND1000 nucleic acid concentration instrument and agar
Sugared gel electrophoresis carries out concentration mensuration and quality testing to the DNA of large white sow.Specifically nucleic acid concentration instrument is measured
For A260/280 ratio in 1.8-2.0, A260/230 ratio is determined as purity qualification in 1.7-1.9, by concentration be higher than 300 nanograms/
Microlitre it is determined as concentration qualification；By the DNA sample of purity and concentration qualification be uniformly diluted to 50 nanograms/microlitre.6 μ l diluted again
DNA sample mixed with 2 μ lLoading Buffer, be loaded in 1% Ago-Gel, electrophoresis 25min under 150V voltage,
It observes and takes pictures under UV detector and gel imaging equipment, observe the integrity degree of DNA.By concentration, purity and complete
The all qualified DNA sample of degree is determined as up-to-standard sample.
GeneSeek Genomic Profiler Porcine 80K SNP parting platform, using Illumina
The operation instruction and normal process of Infinium carries out chip hybridization and result scans.It is read finally by GenomeStudio software
Take genotype data.Quality control is carried out with genotype data of the PLINK v1.07 to acquisition, specific standards are as follows:
(1) Hardy-Weinberg equilibrium (HWE, Hardy-Weinberg equilibrium) examines P value to be greater than 10-6
(2) SNP recall rate is greater than 90%
(3) individual SNP recall rate is greater than 90%
(4) SNP minimum gene frequency (MAF, Minor Allele Frequency) is greater than 5%
(5) SNP on unknown position and sex chromosome is deleted
4, full-length genome association (GWAS) analysis
Using case-control (Case-Control) whole-genome association based on SNP marker, by detecting full base
Because group within the scope of case individual and compare the difference of individual allelic and genotype frequency on each SNP marker site,
The site that linkage disequilibrium is in the gene locus of control objective trait is searched with this.This research selection comes from Michigan, United States
The GEMMA software that the Xiang Zhou of university and the Matthew Stephens of Chicago University are researched and developed jointly is outstanding to large white sow
Hoof undue growth group and normal dewclaw group carry out whole-genome association.GEMMA software is one and is carrying out full genome
The brute force software that group's layering and affiliation can be explained in group association analysis is suitble to use single variable linear mixed model
(Univariate Linear Mixed Model) is associated detection to single phenotype and label, explains that group is layered with this
With composition of sample effect, and the ratio for estimating genotype explanation phenotype.In view of affiliation and group's layering effect
A possibility that coping with false positive results caused by association analysis, this research are controlled using single argument mixed linear model, are led to
It crosses GEMMA software and carries out whole-genome association.Single argument mixed linear model:
Y=W α+x β+u+ ε
Y is phenotype vector；For age in days when W is indicated using field border effect and sampling as the oriental matrix of covariant, α is covariant
Corresponding related coefficient；The oriental matrix that x is SNP (for affiliation matrix after correction, is calculated by full-length genome marker genetype
And obtain), β is the effect of SNP；μ is the stochastic effects of prediction, and ε is residual error.Determine that full-length genome is associated with using Bonferroni method
Conspicuousness threshold value is analyzed to obtain, the remarkable threshold of genomic level is 0.05 divided by effective SNP site quantity, i.e. 0.05/50227=
9.9548E-07；Chromosome level remarkable threshold is 1 divided by effective SNP site quantity, i.e. 1/50227=1.9910E-05.
It is as shown in Figure 3 that GWAS analyzes result.As can be seen from Figure 3, outstanding in the presence of significantly affecting in No. 13 chromosomes in large white sow group
The site of hoof growth traits, most strongly connected SNP are g.332T > G (P=1.04E-06).
5, the association analysis of different genotype and dewclaw growth phenotype
According to table 1, the SNP site of molecular labeling g.332T > G and the extremely significant related (P=of dewclaw growth traits
1.04E-06), illustrate that this molecular labeling significantly affects the dewclaw growth traits of pig, the auxiliary of this SNP site to pig can be passed through
Selection is helped, to reduce group's dewclaw undue growth.In addition, normal growth group SNP g.332T > allele in the site G
The gene frequency of T is extremely significant to be lower than undue growth group (5.4% < 20.5%, P < 0.0001).Therefore, we are educating
It during kind, needs to eliminate the boar of TT and TG type, retains the boar of GG type, to improve the allele G in the site by generation
Frequency.
The different gene frequencies of table 1 and corresponding phenotypic difference
6, target DNA sequence amplification and sequencing
It (1) is 13 containing the amplification target fragment with the target fragment of the significant associated SNP positions of great Bai dewclaw undue growth
The nucleotide sequence of one section of 483bp in number chromosome, the upstream and downstream primer of sequence amplification are as follows:
Upstream primer 5 '-AGAAGAATCTATGGGAAACTAC-3 ' (SEQ ID NO:2)；
Downstream primer 5 '-GTAAGTGGGAGCGGGTAG-3 ' (SEQ ID NO:3).
(2) system and condition setting of PCR amplification
20ul system is configured, wherein DNA sample 3.5ul, upstream primer 0.6ul, downstream primer 0.6ul, PCR mix
10ul,ddH2O 5.3ul, PCR condition is 98 DEG C of initial denaturation 2min, and 98 DEG C of denaturation 30s, 50.8 DEG C of annealing 30s, 72 DEG C extend
30s, totally 35 circulations, finally extend to 72 DEG C of 10min.
(3) DNA sequence dna sequencing identification: sequence is carried out in Shenzhen Huada Genetic Technology Co., Ltd, and genetic fragment is surveyed
Positive and negative two reactions.By measured sequence and NCBI genomic sequence comparison, the mutation of corresponding SNP site is obtained.
Sequencing result is (SEQ ID NO:1) as follows:
Note: the M marked in sequence table is mutational site, (is mutating alkali yl in bracket, is equipotential with display is underlined
Gene mutation), primer sequence is shown as in the head and the tail overstriking of the sequence.
It should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than protects model to the present invention
The limitation enclosed, although the invention is described in detail with reference to the preferred embodiments, those skilled in the art should understand that,
It can be with modification or equivalent replacement of the technical solution of the present invention are made, without departing from the essence and model of technical solution of the present invention
It encloses.
Sequence table
<110>Agricultural University Of South China；Guangdong Wen'S Foodstuffs Group Co., Ltd.
<120>a kind of SNP marker relevant to pig dewclaw undue growth character and application thereof
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 483
<212> DNA
<213>pig
<220>
<221> misc_feature
<223>m=T or G
<400> 1
agaagaatct atgggaaact accaaaaccc cccacaaaac accaaggaga ggaaaaaaca 60
caagttgcgt ttgatttaaa aaatatttat aggacacggt ttttattttc atagaatgtc 120
aataaatatt ttaccaggat gatacggata tatcacaatg atgggaggaa gaatttggga 180
tcgctaaggg atggaaacat ttcgaccttt aatttaaacc tcgattcagc aggcttatcc 240
ttggcagtgg gggggtcttg aaatctggca gagaaagcgt ccttacgtgc acagaccaga 300
tttctaacgt gggcacgctg ccagatcagt amatggagga ccagtggcac cggctcttga 360
tgacctgaag caaaggcttc ctgaaagtga ggaagatgac agtgcaggcc accagcaggg 420
tgaggcagct gggaagaatg agcacgaggt acagcaggcc catgtctacc cgctcccact 480
tac 483
<210> 2
<211> 22
<212> DNA
<213>artificial sequence
<400> 2
agaagaatct atgggaaact ac 22
<210> 3
<211> 18
<212> DNA
<213>artificial sequence
<400> 3
gtaagtggga gcgggtag 18
Claims (6)
1. a kind of purposes of SNP marker relevant to pig dewclaw undue growth character in pig dewclaw growth genetic breeding, special
Sign is that the site of the SNP marker is international pig with reference to the 133470903rd core on No. 13 chromosomes of 11.1 version of genome
Thuja acid site, the base in the site are T or G.
2. SNP marker relevant to pig dewclaw undue growth character according to claim 1 is educated in the growth heredity of pig dewclaw
Purposes in kind, which is characterized in that the sequence of the SNP marker is as shown in SEQ ID NO:1, shown in the SEQ ID NO:1
Sequence the 332nd bit base from 5 ' ends is T or G.
3. SNP marker relevant to pig dewclaw undue growth character according to claim 1 or 2 grows heredity in pig dewclaw
Purposes in breeding, which is characterized in that a kind of for detecting the core of the primer pair of SNP marker as stated in claim 1 or 2
Acid sequence is as shown in SEQ ID NO:2 and SEQ ID NO:3.
4. a kind of method for reducing pig dewclaw undue growth, which is characterized in that the described method comprises the following steps:
Detect gene of the international pig with reference to the 133470903rd nucleotide site on No. 13 chromosomes of 11.1 version of genome of pig
Type selects the GG type individual of the 133470903rd nucleotide site as boar；
The method is treated for non-disease and diagnostic purpose.
5. according to the method described in claim 4, it is characterized in that, the international pig of the detection pig refers to 11.1 version of genome
On No. 13 chromosomes the genotype of the 133470903rd nucleotide site method the following steps are included:
(1) genomic DNA of pig to be measured is extracted；
(2) using primer pair described in claim 3, the genomic DNA of the pig to be measured is subjected to PCR amplification, to obtain
Obtain pcr amplification product；
(3) pcr amplification product is sequenced, to obtain sequencing result；
(4) it is based on the sequencing result, determines the gene of the SNP marker as stated in claim 1 or 2 of the pig to be measured
Type.
6. according to the method described in claim 4, it is characterized in that, the boar includes its synthesis system of Herba Phaii tankervilliaes.
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Families Citing this family (3)
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| CN108359735B (en) * | 2018-05-19 | 2020-12-25 | 华中农业大学 | SNP genetic marker of VTCN1 gene related to sow limb hoof bone density |
| CN108359736B (en) * | 2018-05-23 | 2020-12-29 | 华中农业大学 | SNP genetic marker for sow limb hoof bone density character |
| CN113637768B (en) * | 2021-08-06 | 2023-07-14 | 华南农业大学 | SNP molecular marker related to number of sow-produced malformed piglets on chromosome 13 of pig and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040241676A1 (en) * | 2001-04-24 | 2004-12-02 | Leif Andersson | Methods |
| CN103421769A (en) * | 2012-10-23 | 2013-12-04 | 华中农业大学 | SNP molecular marker related to pig carcass trait and application thereof |
-
2017
- 2017-12-11 CN CN201711310397.4A patent/CN107988388B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040241676A1 (en) * | 2001-04-24 | 2004-12-02 | Leif Andersson | Methods |
| CN103421769A (en) * | 2012-10-23 | 2013-12-04 | 华中农业大学 | SNP molecular marker related to pig carcass trait and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| dbSNP登记号：rs81210648;WU_ABGC;《NCBI》;20130315;参见GeneView、序列部分 * |
| 猪的全基因组水平SNP研究现状;陈慧勇 等;《生命科学》;20110430;第23卷(第4期);第390-393页 * |
| 蹄部异常对泌乳母猪行为的影响;温鹏;《今日养猪业》;20111231(第3期);第3、4、14页 * |
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Address after: 510642 No. five, 483 mountain road, Guangzhou, Guangdong, Tianhe District Co-patentee after: WENS FOODSTUFF GROUP Co.,Ltd. Patentee after: SOUTH CHINA AGRICULTURAL University Address before: 510642 No. five, 483 mountain road, Guangzhou, Guangdong, Tianhe District Co-patentee before: GUANGDONG WENS FOODSTUFF GROUP Co.,Ltd. Patentee before: SOUTH CHINA AGRICULTURAL University |