CN102181582B - Method for detecting classical swine fever resistance character of swine and special kit thereof - Google Patents
Method for detecting classical swine fever resistance character of swine and special kit thereof Download PDFInfo
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Abstract
The invention discloses a method for detecting a classical swine fever resistance character of a swine and a special kit thereof. A specific primer pair which is provided by the invention for assisting detection of the classical swine fever resistance character of the swine is shown as (a) or (b): (a) the primer pair consisting of a DNA (Deoxyribonucleic Acid) fragment shown as a sequence 5 of a sequence table and a DNA fragment shown as the sequence 6 of the sequence table; and (b) the primer pair consisting of the DNA fragment shown as the sequence 3 of the sequence table and the DNA fragment shown as the sequence 4 of the sequence table. In the method, the primers are designed at a swine NEAT1 homologous sequence conservation region to amplify genome DNA fragments of different types of swine; the obtained fragment comprises a C/G mutation site; and the classical swine fever virus antibody level of the swine is detected by using polymorphism of a BfaI enzyme cutting site caused by the mutation site. Experiments prove that the mutation site is obviously related to the classical swine fever virus antibody level, so that the method for evaluating the classical swine fever virus antibody level by using the mutation site is accurate and feasible.
Description
Technical field
The present invention relates to detect method and the dedicated kit thereof of the anti-hog cholera proterties of pig.
Background technology
Pork is the topmost animal protein food source of China resident, and pig industry is produced in the chain in herding and occupied very important status.Recent decades, GENERALIZATION OF MODERN BREEDING TECHNIQUE is significantly improved the production performance of pig, but mainly for be the production traits (growth traits and reproductive trait).With respect to the production traits, but comparatively weak for the research of pig health and resistance against diseases.The breeding objective take high bacon hogs as guiding causes allelic lose or thereby frequency reduces the decline of physique and the resistibility cause the modern commerce kind, and along with popularizing of intensive farm mode, the transmissible disease that swine disease occurs is more repeatedly given to raise pigs to produce and has been brought heavy losses.Swine fever (Classical swine fever, CSF) is a kind of hyperinfection that is caused by Pestivirus suis and the epidemic disease of lethality rate, brings huge financial loss for whole world pig industry.It is not strict to be subject to vaccine quality, immune programme for children, and takes the factors such as viruliferous wild boar colony propagation, and swine fever is threatening the safety of pig industry always.
At present pig disease resistant breeding research mainly is to seek control disease resistance major gene or QTL.By the methods such as candidate gene, icp gene group and genome scanning identified comprise K88 and/or F18 acceptor, FUT, Interferon, rabbit and acceptor thereof, MHC, Mxl gene affect pig diarrhea and a plurality of functional genes and the QTL such as edema disease, anti-foot and mouth disease, anti-swine fever and resisiting influenza virus.Wherein: the cell-surface antigens that (1) K88 or F18 acceptor defect type can be blocked intestinal bacteria K88 or F18 bacterial strain is attached to the small intestinal epithelial cells surface receptor, thereby due to corresponding intestinal bacteria, suffer from diarrhoea or edema disease generation resistance (Bertschinger, H.and Pohlenz, J. (1983) Bacterial colonization andmorphology of the intestine in porcine Escherichia coli enterotoxemia (edema disease) .Veterinary pathology.20,99.).
Deng (
P., Meijerink, E., Fries, R., Neuenschwander, S., Vorl nder, N., Stranzinger, G.and Bertschinger, H. (1997) Amolecular test for the detection of E.coli F18 receptors:a breakthrough in the struggleagainst edema disease and post-weaning diarrhea in swine.Schweizer Archiv f ü rTierheilkunde.139,479.) also can be used as 1 candidate gene that control F18 adheres to by the linkage analysis discovering and location in salt algae glycosyltransferase gene FUT1 and the FUT2 of 6q11 section.U.S. ARS utilizes this to be marked on and announce to breed this sick resistance pig in July, 1999 in Yorkshire.(2) Interferon, rabbit of pig is proved to be breeding difficulty and respiratory syndrome virus, foot and mouth disease virus, the multiple serious infectious diseases virus such as Pestivirus suis all has wide spectrum defence and restraining effect (Buddaert, W., Van Reeth, K.and Pensaert, M. (1998) In vivo and in vitro interferon (IFN) studies with the porcine reproductive and respiratory syndrome virus (PRRSV) .Coronaviruses and arteriviruses.440,461.), thereby Interferon, rabbit and acceptor gene thereof are the important candidate genes of breed breeding.(3) MHC has been proved to be and the phagocytosis of the replying of plurality of antigens, bacterium, relevant to proterties such as Trichinella spiralis, toxoplasmatic infection and melanoma and tumour generations as the breeding for disease resistance molecule marker, is the important selection site of non-specific disease resistance.(4) studies have shown that, the transgenic pig that turns mouse MX1 gene has strengthened the resistibility (Muller of infected by influenza, M., Brenig, B., Winnacker, E.L.and Brem, G. (1992) Transgenic pigscarrying cDNA copies encoding the murine Mx1 protein which confers resistance toinfluenza virus infection.Gene.121,263-270.).(the Nakajima such as Nakajima, E., Morozumi, T., Tsukamoto, K., Watanabe, T., Plastow, G.and Mitsuhashi, T. (2007) A naturally occurringvariant of porcine Mx1 associated with increased susceptibility to influenza virus in vitro.Biochemical Genetics.45,11-24.) results of in vitro studies finds that a natural variation of pig MX1 gene may increase the susceptibility of suffering from influenza.(5) SLC11A1 (claiming again NRAMP1) studies a more disease-resistant gene recently.That reports the earliest SLC11A1 and tuberculosis, sarcoidosis has a relation (Bellamy, R., Ruwende, C., Corrah, T., McAdam, K.P.W.J., Whittle, H.C.and Hill, A.V.S. (1998) Variaions in theNRAMP1 gene and susceptibility to tuberculosis in West Africans.New England Journal ofMedicine.338,640-644.).This gene (Moisan that in the regulation and control of the signal transduction that Toll-like acceptor 7 aglucons are induced, plays a role, J., Thuraisingam, T., Henault, J., Sanctis, J.D.and Radzioch, D. (2006) Role ofSLC11A1 (formerly NRAMP1) in regulation of signal transduction induced by Toll-likereceptor 7 ligands.FEMS Immunology ﹠amp; Medical Microbiology.47,138-147.), thereby may relate to widely nonspecific immune response.Existing research is found, the SLC11A1 gene is the important molecular markers (Wu relevant with pig immune trait, production performance etc., H., Cheng, D.and Wang, L. (2008) Association ofpolymorphisms of Nramp1 gene with immune function and production performance of largewhite pig.Journal of Genetics and Genomics.35,91-95.).The Candidate Gene Study of further carrying out disease-resistant proterties in a deep going way will be the important content of from now on pig disease resistant breeding research.
Full genome analysis is found to have transcribed a lot of non-coding RNAs in the mammalian genes group, increasing evidence shows that these non-coding RNAs are not only " noise " of transcribing, but the regulation and control (Ponting that the precursor participation that can be used as structure RNA or little RNA is transcribed, C.P., Oliver, P.L.and Reik, W. (2009) Evolution and functions oflong noncoding RNAs.Cell.136,629-641.).NEAT1 is the (Peyman such as Peyman, J.A. the main a kind of potential non-coding RNA transcript of in the trophocyte, expressing of (1999) Repression of major histocompatibility complex genes by a human trophoblast ribonucleicacid.Biology of reproduction.60,23.) finding.Thereby people NEAT1 gene can suppress the expression that II class trans-activating factor promotor suppresses major histocompatibility complex, and the allogeneic reaction (Geirsson that significantly suppresses the B cell, A., Bothwell, A.L.M.and Hammond, G.L. (2004) Inhibition of alloresponse by a human trophoblastnon-coding RNA suppressing class II transactivator promoter III and majorhistocompatibility class II expression in murine B-lymphocytes.The Journal of heart andlung transplantation.23,1077-1081.).NEAT1 can with paraspeckle protein (P54nrb) in conjunction with (Murthy, U.and Rangarajan, P.N.Identification of protein interaction regions ofVINC/NEAT1/Men epsilon RNA.FEBS letters.584,1531.), formation and the delay of mRNA nuclear to paraspeckle play a crucial role, and its expression inhibiting is the major cause that does not exist mRNA nuclear to be detained in the embryonic stem cell of people source.The HeLa cell can cause losing of paraspeckle after striking and subtracting NEAT1, and carry that output strengthens (Chen between the karyon kytoplasm of mRNA of reverse Alu sequence, L.L.and Carmichael, G.G. (2009) Altered nuclearretention of mRNAs containing inverted repeats in human embryonic stem cells:functionalrole of a nuclear noncoding RNA.Molecular cell.35,467-478.).
Summary of the invention
An object of the present invention is to provide the Auele Specific Primer pair of the anti-hog cholera proterties of auxiliary detection pig.
The Auele Specific Primer of the anti-hog cholera proterties of auxiliary detection pig provided by the present invention pair, for following a) or b) institute not:
A) primer that is formed by the dna fragmentation shown in the sequence 6 of the dna fragmentation shown in the sequence 5 of sequence table and sequence table pair;
B) primer that is formed by the dna fragmentation shown in the sequence 4 of the dna fragmentation shown in the sequence 3 of sequence table and sequence table pair.
Described Auele Specific Primer also belongs to protection scope of the present invention to the application in the anti-hog cholera proterties of auxiliary detection pig.
Described Auele Specific Primer also belongs to protection scope of the present invention to the application in the test kit of the anti-hog cholera proterties for preparing the auxiliary detection pig.
Another object of the present invention provides the test kit of the anti-hog cholera proterties of a kind of auxiliary detection pig.
The test kit of the anti-hog cholera proterties of auxiliary detection pig provided by the present invention, contain following a) or b) shown in Auele Specific Primer pair:
A) primer that is formed by the dna fragmentation shown in the sequence 6 of the dna fragmentation shown in the sequence 5 of sequence table and sequence table pair;
B) primer that is formed by the dna fragmentation shown in the sequence 4 of the dna fragmentation shown in the sequence 3 of sequence table and sequence table pair.
Another purpose of the present invention provides the method for the anti-hog cholera proterties of auxiliary detection pig.
The method of the anti-hog cholera proterties of auxiliary detection pig provided by the present invention is following 1) or 2) shown in:
1) genotype of detection pig to be measured is CC genotype, GG genotype or CG genotype; Described CC genotype be in the sequence table in the genomic dna shown in the sequence 2 in the dna fragmentation from 5 ' end the 112nd Nucleotide be the homozygote of C; Described GG genotype be in the sequence table in the genomic dna shown in the sequence 2 in the dna fragmentation from 5 ' end the 112nd Nucleotide be the homozygote of G; Described CG genotype be in the sequence table in the genomic dna shown in the sequence 2 in the dna fragmentation from 5 ' end the 112nd Nucleotide be the heterozygote of C and G; Described CC genotype is compared with the genotypic pig of GG with the genotypic pig of CG, has stronger anti-hog cholera ability;
2) genotype of detection pig to be measured is CC genotype, GG genotype or CG genotype; Described CC genotype be in the sequence table in the genomic dna shown in the sequence 1 in the dna fragmentation from 5 ' end the 451st Nucleotide be the homozygote of C; Described GG genotype be in the sequence table in the genomic dna shown in the sequence 1 in the dna fragmentation from 5 ' end the 451st Nucleotide be the homozygote of G; Described CG genotype be in the sequence table in the genomic dna shown in the sequence 1 in the dna fragmentation from 5 ' end the 451st Nucleotide be the heterozygote of C and G; Described CC genotype is compared with the genotypic pig of GG with the genotypic pig of CG, has stronger anti-hog cholera ability.
The genotype of described detection pig to be measured is that CC genotype, GG genotype or the genotypic method of CG comprise the steps:
1) with the genomic dna of pig to be measured as template, to carrying out pcr amplification, obtain pcr amplification product with Auele Specific Primer; Described Auele Specific Primer to be following a) or b) shown in:
A) primer that is formed by the dna fragmentation shown in the sequence 6 of the dna fragmentation shown in the sequence 5 of sequence table and sequence table pair;
B) primer that is formed by the dna fragmentation shown in the sequence 4 of the dna fragmentation shown in the sequence 3 of sequence table and sequence table pair;
2) described pcr amplification product is detected, determine that according to detected result the genotype of described pig to be measured is CC genotype, GG genotype or CG genotype;
The described method that described pcr amplification product is detected is following A) or B) shown in:
A) described pcr amplification product is checked order, determine that according to sequencing result the genotype of described pig to be measured is CC genotype, GG genotype or CG genotype;
B) with the BfaI restriction enzyme described pcr amplification product is carried out enzyme and cut, detect enzyme and cut product; If enzyme is cut two fragments that product is 112bp and 108bp, determine that then the genotype of described pig to be measured is the CC genotype; If enzyme is cut the fragment that product is 220bp, determine that then the genotype of described pig to be measured is the GG genotype; If enzyme is cut three fragments that product is 112bp, 108bp and 220bp, determine that then the genotype of described pig to be measured is the CG genotype.
Described step 2) it is to detect by agarose gel electrophoresis that enzyme described in is cut product; If described enzyme is cut product and is shown as two bands of 100bp-200bp and 100bp-200bp at gel, determine that then the genotype of described pig to be measured is the CC genotype; If enzyme is cut product for be shown as the band of 200bp-300bp at gel, determine that then the genotype of described pig to be measured is the GG genotype; If enzyme is cut product for be shown as three bands of 100bp-200bp, 100bp-200bp and 200bp-300bp at gel, determine that then the genotype of described pig to be measured is the CG genotype.
Described hog cholera is caused by Pestivirus suis;
Described CC genotype is compared with the genotypic pig of GG with the genotypic pig of CG, has stronger anti-hog cholera ability to be: the antibody titer of the Pestivirus suis in the blood of described CC genotype and the genotypic pig of CG is higher than the genotypic pig of GG.
Described Auele Specific Primer also belongs to protection scope of the present invention to, described examination box and/or the application of described method in the breeding of pig.
Another purpose of the present invention provides the breeding method of a boar.
The breeding method of pig provided by the present invention is that the described method of selection detects the CC genotype or the genotypic pig of CG carries out breeding.
Method of the present invention is at pig NEAT1 homologous sequence conserved regions design primer amplification different varieties pig genomic DNA fragment, the fragment that obtains contains a C/G of place sudden change, and utilizes polymorphic detection pig immune trait---the antibody against swine fever virus level of the BfaI restriction enzyme site that this mutational site causes.Experimental results show that there are significant correlation in this mutational site and antibody against swine fever virus level, show that the method that the present invention utilizes this mutational site to assess the antibody against swine fever virus level is accurate feasible method.Method of the present invention can be used for assessing antibody against swine fever virus tires, thereby for the molecular breeding of pig provides the method for new its inherited character of detection, and will in the breeding of pig, play a significant role.
Description of drawings
Fig. 1 is the electrophoresis result of three kinds of genotypic samples.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
The method of the anti-hog cholera proterties of embodiment 1, pig NEAT1 Gene RFLP polymorphic detection pig
One, the identification of pig NEAT1 Gene RFLP pleomorphism site
According to pig NEAT1 gene conserved regions primers, primer sequence is as follows:
Neat1a:5 '-GGAGGTGGACAAAATTGACC-3 ' (sequence 3 in the sequence table),
Neat1b:5 '-GACACGGGACATTGGAGAAC-3 ' (sequence 4 in the sequence table).
Respectively take Tongcheng pig, Large White, landrace, Wuzhi Mountain pig, Laiwu Pigs, each genomic dna of 3 of the fragrant pig of bar horse as template, carry out pcr amplification take Neat1a and Neat1b as primer, PCR reaction cumulative volume is 20 μ L, and wherein template DNA is 50ng, contain 1 * Buffer, the MgCl of 1.5mmol/L
2, the dNTP final concentration is 200 μ mol/L, the primer final concentration is 0.2 μ mol/L, 1.0U Taq archaeal dna polymerase (TaKaRa).The pcr amplification program is: 95 ℃ of 5min denaturations then are 95 ℃ of 30s, 60 ℃ of 30s, and 72 ℃ of 30s circulate 35 times, and last 72 ℃ are extended 5min.
This primer amplification sheet segment length 559bp, i.e. sequence 1 in the sequence table.The purified recovery of amplified production and cloning and sequencing are compared to sequence and are analyzed possible polymorphic site with the SeqMan module in the DNAstar7.0 software.The analysis sequencing result is found, sequence 1 the 451st sudden change that has C or G from 5 ' end in the sequence table, and when the 451st Nucleotide was C, (C ↓ TAG) can cause in the sequence table sequence 1 interior BfaI endonuclease bamhi polymorphic 1 BfaI restriction enzyme site.
Two, the foundation of pig NEAT1 Gene RFLP pleiomorphism detecting method
1, design of primers
At this polymorphic site both sides design primer, primer sequence is as follows:
BfaIa:5 '-GTACCCGCTGAAAGCTACGC-3 ' (sequence 5 in the sequence table),
BfaIb:5 '-GGCCTAGACACGGGACATTG-3 ' (sequence 6 in the sequence table).
2, experiment material
Be used for the DNA sample of gene pleiomorphism detection from 7 swinerys (table 1).Adopt the phenol chloroform method to extract the pig blood genomic dna, save backup in-20 ℃.
Colony and the sample number of table 1RFLP polymorphic detection
(3) pcr amplification condition
PCR reaction cumulative volume 20 μ L, wherein the about 50ng of pig genomic dna contains 1 * Buffer, 1.5mmol/LMgCl
2, the dNTP final concentration is 200 μ mol/L, the primer final concentration is 0.2 μ mol/L, 1U Taq archaeal dna polymerase.The pcr amplification program is: 95 ℃ of 5min denaturations, follow 95 ℃ of 30s, and 60 ℃ of 30s, 72 ℃ of 30s circulate 35 times altogether, and last 72 ℃ are extended 5min.The PCR reaction product detects with the agarose gel electrophoresis of 2% concentration and takes pictures, and reclaims order-checking.Sequencing result shows that the nucleotide sequence of this primer amplification fragment is shown in the sequence 2 in the sequence table, and length is 226bp.The sequencing results shows sequence 2 the 112nd sudden change that has C or G from 5 ' end in the sequence table.When the Nucleotide in this site is the homozygote of C, there is 1 BfaI restriction enzyme site (C ↓ TAG).In addition, sequence 2 the 220th the-the 223rd another BfaI enzyme recognition site of existence from 5 ' end can by the small segment of 6bp under 3 ' end-grain cutting of BfaI enzyme sequence 2 in sequence table, can be ignored in RFLP detects in the sequence table.
(4) BfaI-RFLP detects
The PCR product is cut with the BfaI enzyme, and the endonuclease reaction system is 20 μ L, 10 * Buffer, 2 μ L wherein, and PCR product 8 μ L, BfaI restriction enzyme 1 μ L (available from NEB company) uses ddH
2O supplies 20 μ L.With centrifugal behind the sample blending, 37 ℃ of temperature are bathed 2.5h.Enzyme is cut product and is cut the result by the agarose gel electrophoresis detection enzyme of 2% concentration, and gel imaging system is taken pictures and recorded genotype, and the result is shown in Fig. 1 and table 2.
When sequence in the sequence table 2 Nucleotide of the 112nd from 5 ' end was the homozygote of C, this gene was by C allelotrope control (the BfaI enzyme is cut product 112bp and two fragments of 108bp); When sequence in the sequence table 2 Nucleotide of the 112nd from 5 ' end is the homozygote of G, this gene is by G allelotrope control (the BfaI enzyme is cut product and only had 220 1 fragments), when sequence in the sequence table 2 Nucleotide of the 112nd from 5 ' end is the heterozygote of C and G, this gene is by C allelotrope and G allelotrope control (the BfaI enzyme is cut product 112bp, 108bp and three fragments of 220bp), and these two allelotrope can form three kinds of genotype CC, GG and CG.
The BfaI enzyme of these three kinds of genotype samples is cut the result shown in Fig. 1 (M:DNA molecular weight standard (100bp-1500bpladder)), and sample shows 3 kinds of banding patterns altogether).The BfaI enzyme of sample is cut the result for obtaining two fragments of 112bp and 108bp, with its called after CC genotype; The BfaI enzyme of sample is cut the result for obtaining fragment of 220bp, with its called after genotype GG; The BfaI enzyme of sample is cut the result for obtaining three fragments of 112bp, 108bp and 220bp, with its called after genotype CG.
BfaI-RFLP detected result in the different swinery bodies of table 2
Show that according to the genotype of table 2 and the result of gene frequency in these several kinds that detect, except two kinds of allelotrope of landrace are more or less the same (C vs.G=0.412 vs.0.588), other several kinds all are that C allelotrope is preponderated.
Three, the association analysis of pig NEAT1 gene different genotype and anti-swine fever proterties
The experiment swinery that is used for association analysis is the resource family that is made of 376 purebred landraces (available from Guangdong Wen Shi group), and wherein the parent is 17 boars and 36 sows, and filial generation is 323.All filial generations are raised but be distributed in 2 production lines all from same pig farm.
Genotype detection: detect according to method described in the experiment two, respectively the PCR product is checked order and enzyme is cut, it is consistent that the genotype result that result order-checking draws and enzyme are cut the genotype result who draws.As shown in table 3.
Anti-hog cholera proterties detects: testing index comprises the normal blood indexes such as the tiring of hog cholera antibody in the blood (CSFV-AB), red corpuscle, white corpuscle and thrombocyte etc.
CSFV-AB tires by calculating after hog cholera antibody enzyme-linked immunosorbent assay kit (CSFV-AB ELISA Kit is available from Indexx) the detection hog cholera antibody blocking-up rate; Hog cholera antibody blocking-up rate (PI)=(OD
N-OD
T)/(OD
N-OD
P), OD wherein
N, OD
TAnd OD
PIt is respectively the OD value (negative control and positive control are that test kit carries) that negative control, specimen and positive control are measured; The method of calculation of CSFV antibody titer (Y) are: Log
2Y=-7.56+56.41PI-83.85PI
2+ 44.66PI
3
The mensuration of normal blood index (red corpuscle, white corpuscle and thrombocyte) is finished by blood analyser (available from sysmex, model is kx-21N).These indexs are subjected to the impact of sex, feeding environment and parity, belong to fixed effect; Also be subjected to simultaneously the impact of family and colony's inner structure, such as dam effect in male animal or the male animal, belong to stochastic effect.So according to group structure and the properties and characteristics of collecting sample, select mixture model analyze candidate gene the genotype effect and with the relation of proterties, modeling is as follows:
Y＝Xβ+Zb+ε
Wherein,
Y is the proterties observation vector;
X is the fixed effect incidence matrix;
β is the parameter vector of fixed effect, comprises genotype effect, sex-effects, parity effect and the environmental effect (Genotypes, Sex, Litter, and Environment) of candidate gene;
The incidence matrix of Z stochastic effect;
The parameter vector of b stochastic effect comprises dam (Sire, Dam (Sire) effect in male animal effect and the male animal;
ε is the random error effect vector, and is separate between ε.
Experiment swinery genotype and proterties association analysis are used SAS V8 GLM program and are finished, and the difference of proterties between different genotype represents with the least square variance.Eliminated kind, butcher batch and sex between difference after, the simple mean of proterties and standard error analytical results are summarized in table 3 between genotype.
The association analysis of table 3 pig NEAT1 genotype and colony's blood and immune indexes
Annotate: represent significant difference (P＜0.05) with different shoulder marks (a, b) in the delegation, asterisk represents the conspicuous level (P＜0.05 or P＜0.01) of genetic effect.
Found that the genotype effect all reaches conspicuous level (p＜0.05) to mensuration proterties; CC and CG genotype individuality 0 day and 17 age in days hog cholera antibodies are tired, and to be significantly higher than the GG genotype individual, and CC genotype individuality also shows higher erythrocyte parameter and lower platelet parameter (p＜0.05) than other genotype individualities.Judge that according to the association analysis result C allelotrope is the advantage allelotrope of swine fever virus resistant, the CC genotype is the beneficial gene type of swine fever virus resistant.The CC genotype is compared with the genotypic pig of GG with the genotypic pig of CG, has stronger anti-hog cholera ability.
Claims (2)
1. following Auele Specific Primer is to the application in the test kit of the anti-hog cholera proterties for preparing the auxiliary detection pig;
Described Auele Specific Primer pair, for following a) or b) shown in:
A) primer that is formed by the dna fragmentation shown in the sequence 6 of the dna fragmentation shown in the sequence 5 of sequence table and sequence table pair;
B) primer that is formed by the dna fragmentation shown in the sequence 4 of the dna fragmentation shown in the sequence 3 of sequence table and sequence table pair.
2. following Auele Specific Primer is to the application in the breeding of pig;
Described Auele Specific Primer pair, for following a) or b) shown in:
A) primer that is formed by the dna fragmentation shown in the sequence 6 of the dna fragmentation shown in the sequence 5 of sequence table and sequence table pair;
B) primer that is formed by the dna fragmentation shown in the sequence 4 of the dna fragmentation shown in the sequence 3 of sequence table and sequence table pair.
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