CN102816837A - Method for identifying Chinese and foreign pig breeds - Google Patents
Method for identifying Chinese and foreign pig breeds Download PDFInfo
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Abstract
The invention provides a method for identifying Chinese and foreign pig breeds. The Chinese pig breeds and the foreign pig breeds are identified according to differences of SNP loci in a sequence represented by SEQ ID NO.2 and of CYB5 gene promoters of pigs to be detected, wherein the SNP loci comprise one or more positioned in -660bp, -380bp or -69bp at the upstream of CYB5 gene initiators. Bases at the -660bp, the -380bp and the -69bp in the CYB5 gene sequences of the Chinese pig breeds are single base deletion, C and T respectively, and the bases at the -660bp, the -380bp and the -69bp in the CYB5 gene sequences of the foreign pig breeds are G, T and C respectively. The invention also provides a genetic marker and a PCR primer for identifying the Chinese and foreign pig breeds, and a kit containing the primer. The method provided by the invention enables the pig breeds of different genetic backgrounds, especially the Chinese and foreign pig breeds to be rapidly and accurately identified, has the advantages of easy operation in a laboratory, less pollution, low detection cost, and convenient basic popularization and use, and has a wide market application prospect.
Description
Technical field
The present invention relates to genetically engineered and biology field, specifically, relate to a kind of difference of the special haplotype of promoter region of utilizing and identify the method for pig kind at home and abroad.
Background technology
China is as world pork production and consumption big country, and abundant pig kind resource has been carried in vast region; With respect to external pig kind, characteristics such as it is slower that the Chinese Pigs kind shows growth, and lean ratio is lower, but advantages such as its prolificacy, good meat and crude feed tolerance also let external pig kind too far behind to catch up.The anabolism difference of lipid and steroid has much relations in the difference performance of China and foreign countries' pig kind aspect meat and its body.Existing research report is participated in the electron transfer protein Cyt b5 of organism lipid and the important anabolic reaction of steroid, in China and foreign countries' pig kind, shows genetic polymorphism.Therefore, the molecular genetic The Characteristics to electron transfer protein Cyt b5 encoding sox CYB5 pig kind in China and foreign countries will help to explain the metabolism difference of China and foreign countries' pig kind profoundly and for the pig kind improves foundation will be provided.
In recent years; Relevant report shows some human disease because the CYB5 gene is undergone mutation and produced, as discovers in the patient of congenital haemoglobinaemia, and 3 ' of the introne 1 of CYB5 gene is held the A → G that undergos mutation; Make the shearing site of cDNA change; Cause the cDNA disappearance of 16bp, thereby caused the generation (Giordano etc., 1994) of congenital haemoglobinaemia; CYB5 gene W27X sudden change can cause terminator codon in advance, causes losing of Cytb5 E48 and E49 amino-acid residue, thereby make 17 that 20-lyase inactivation causes sexual abnormality disease (Kok etc., 2005).In the research of pig CYB5 gene, present international research mainly concentrates on the pass of CYB5 and steroid hormone metabolism and fastens, and few at the research report of others.Discover sudden change through the PCR-SSCP CYB5 gene 5 ' non-translational region upper reaches G → T in the 8th base place; This sudden change can significantly reduce the proteic expression of CYB5; And then the level of reduction boar fat Androstanediol, but decline (Lin etc., 2005 that can not cause boar reproductivity; Peacock etc., 2008).If more than research and pertinent literature data owner are about the Genetic Variation Analysis of CYB5 gene coding region; Do not report the heritable variation situation of CYB5 upstream region of gene control region between different pig kinds, and do not see up to now yet and utilize the special haplotype combination of CYB5 gene promoter area China and foreign countries' pig kind to be carried out the research report of Genetic identification.
Summary of the invention
The objective of the invention is provides a kind of method of identifying China and foreign countries' pig kind based on to pig CYB5 gene order and diagnostic snp analysis.
Another object of the present invention provides genetic marker and the application thereof that is used to identify China and foreign countries' pig kind.
A purpose more of the present invention provides the test kit that is used to identify the PCR primer of China and foreign countries' pig kind and contains this primer.
In order to realize the object of the invention; The present invention at first provides a kind of genetic marker that is used to identify China and foreign countries' pig kind; It is positioned on pig CYB5 gene 5 ' promoter region; Its nucleotide sequence is identified Chinese Pigs kind and external pig kind according to the difference in SNP site in this sequence shown in SEQID NO.2, said SNP site be arranged in the CYB5 gene start codon upper reaches-660bp ,-380bp or-69bp one or more.Wherein, in the Chinese Pigs kind CYB5 gene promoter sequence-660bp ,-380bp ,-69bp place base is respectively single base deletion, C, T, in the external pig kind CYB5 gene promoter sequence-660bp ,-380bp ,-69bp place base is respectively G, T, C.
The present invention also provides a kind of method of identifying China and foreign countries' pig kind; Identify Chinese Pigs kind and external pig kind according to the difference in SNP site in the sequence shown in the SEQ ID No.2 of pig CYB5 gene promoter to be measured, said SNP site be arranged in the CYB5 gene start codon upper reaches-660bp ,-380bp or-69bp one or more.Wherein, in the Chinese Pigs kind CYB5 gene promoter sequence-660bp ,-380bp ,-69bp place base is respectively single base deletion, C, T, in the external pig kind CYB5 gene promoter sequence-660bp ,-380bp ,-69bp place base is respectively G, T, C.
Preceding method may further comprise the steps: the genomic dna that 1) extracts pig to be measured; 2) genomic dna with pig to be measured is a masterplate, utilizes Auele Specific Primer amplification to be comprised the fragment in different SNP site in the pig CYB5 gene promoter sequence to be measured; Wherein, said SNP site for be arranged in the CYB5 gene start codon upper reaches-660bp ,-380bp or-69bp is one or more; 3) detect pcr amplification product.
In the preceding method, step 2) primer is to being described in:
I) upstream primer 5 '-AAGGAGGAGGTAAGCAATG-3 ' and downstream primer 5 '-GAGATGAGCGGAACAGAGT-3 '; Or
Ii) upstream primer 5 '-TGGGTCAACAGCGAGATA-3 ' and downstream primer 5 '-CCTCCGTAGAACGAGTGTA-3 '; Or
Iii) upstream primer 5 '-CGCCTGCCACAATGAACC-3 ' and downstream primer 5 '-GCGCAGAAGGGGATGATG-3 '; Or
Iv) upstream primer 5 '-TGAGCGGAACAGAGTGGG-3 ' and downstream primer 5 '-AATCATCGGAGGTCGTGC-3 '.
In the preceding method, the method for the said detection pcr amplification product of step 3) is: the primer that amplification is used step 2) is to being i) or ii), the method that detects amplified production is order-checking; Step 2) primer that amplification is used in is to for iii), and the method that detects amplified production is that tetra-sodium checks order; Or step 2) primer that amplification is used in is to for iv), and the method that detects amplified production is the RFLP detection.
In the preceding method, the method for the said detection pcr amplification product of step 3) is: the primer that amplification is used step 2) is to being i) or ii), the method that detects amplified production is order-checking; Step 2) primer that amplification is used in is to for iii), and the method that detects amplified production is that tetra-sodium checks order; Or step 2) primer that amplification is used in is to for iv), and the method that detects amplified production is the RFLP detection.
When step 2) in the primer that uses of amplification to for iii) the time, the sequencing primer of use was 5 '-TGGAGTGGCTGGCGA-3 ' when amplified production was carried out the tetra-sodium order-checking.
When step 2) in the primer that uses of amplification to for iv) the time, cut amplified production with restriction enzyme FspBI enzyme, and enzyme cut product carry out agarose gel electrophoresis and detect, what electrophoresis result presented 2 bands is the Chinese Pigs kind, what be merely 1 band is external pig kind.
The present invention also is provided for identifying the PCR primer of China and foreign countries' pig kind, and it comprises one or more pairs of in the following primer:
I) upstream primer 5 '-AAGGAGGAGGTAAGCAATG-3 ' and downstream primer 5 '-GAGATGAGCGGAACAGAGT-3 ';
Ii) upstream primer 5 '-TGGGTCAACAGCGAGATA-3 ' and downstream primer 5 '-CCTCCGTAGAACGAGTGTA-3 ';
Iii) upstream primer 5 '-CGCCTGCCACAATGAACC-3 ' and downstream primer 5 '-GCGCAGAAGGGGATGATG-3 '; Or
Iv) upstream primer 5 '-TGAGCGGAACAGAGTGGG-3 ' and downstream primer 5 '-AATCATCGGAGGTCGTGC-3 '.
The present invention also provides the test kit that is used to identify China and foreign countries' pig kind that contains above-mentioned primer.Preferably, said test kit also comprises dNTPs, Taq archaeal dna polymerase, Mg
2+, in the PCR reaction buffer one or more.More preferably, said test kit also comprises standard positive template.
The present invention further provides the said application of the genetic marker of China and foreign countries' pig kind at the pig molecule mark assistant breeding that be used for identifying.
Among the present invention, mainly through pig CYB5 gene order and diagnostic snp analysis are explained the genetic diversity of pig kind CYB5 gene at home and abroad.Research object comprises 11 Chinese native pig breeds in Chinese native pig breed 6 major types (blue pool pig, Wuzhi Mountain pig, people pig, the black pig of lining trouble, Han River pig, go up high pig, Jinhua pig, the black pig in Jiaxing, the black pig of Qian Bei, inland river pig, hide pig), 3 kinds of wild boars (northeastern wild boar, Zhejiang wild boar, Jiangxi wild boar) and 3 external pig kinds (Da Bai, in vain long and Du Luoke) totally 532 individuals (affinity-less relation).
Adopt the imitative extraction process of phenol from the pig ear tissue, to extract genomic dna, detect concentration and the purity of its DNA with NanoDrop ND-1000 spectrophotometer.Plan is utilized the control region at the GenBank information design specific primers amplification CYB5 gene 5 ' upper reaches, whether has the SNP site with the special distribution of pig kind to seek.
The pig CYB5 gene order of announcing according to NCBI (accession number: NW_001885867), its 5 ' flank head of district 4521bp.This section is carried out promotor and Transcription Factor Binding Sites Prediction with websites such as Softberry and MatInspector, take all factors into consideration the back find upstream from start codon-957bp～-exist the possibility of promotor core area bigger in the section of 41bp.According to the primer of the designs specificity that predicts the outcome of each promotor prediction website, amplification upstream from start codon 917bp fragment.Adopt 20 μ l systems to carry out pcr amplification; The PCR product carries out cloning and sequencing after detecting through 1% agarose electrophoresis; Find that through order-checking in Chinese Pigs kind and external pig kind, have the remarkable different SNP site, 3 place that distributes, they are respectively: single base deletion in the PolyG of-660bp place sequence; T → the C of-380bp place, the C → T of-69bp place.Going up the sequence of announcing with NCBI is wild-type, and then the Chinese Pigs kind is mutant, and external pig kind is wild-type.
Through detection, affirmation, correction and the statistics to three SNP sites, discovery allelotrope distributes in colony and meets hardy weinberg equilibrium (χ
2＞χ
2 2,0.05) and the genotype in three sites all identical in same individuality, have tangible chain situation.Therefore in China and foreign countries' pig kind, only find two kinds of haplotypes: A type (G-T-C) and Type B (
).Analysis revealed: in the external local pig breed, the advantage haplotype all is the A type, and in the Chinese Pigs kind, the black pig kind of trouble, the advantage haplotype is Type B in the North China type with external pig kind gene infiltration.
The invention has the advantages that the pig variety that can fast, accurately differentiate different genetic backgrounds, especially differentiate Chinese Pigs kind and external pig variety, this method is easy to laboratory operation, pollutes few; Testing cost is low, is convenient to basic unit's popularization and use, has wide market application prospect, and in addition, the detection kit based on the inventive method exploitation can produce considerable economic and good social benefit.In addition, molecular genetic marker provided by the invention does not receive restrictions such as the age, sex of pig, can be used for the early stage seed selection of pig, even when pig just is born, can screen exactly, has accelerated the breeding process of pig.
Description of drawings
Fig. 1 is for carrying out the sampling Detection result behind 1% agarose gel electrophoresis to the pig genomic dna; Wherein M is DNA Marker, and 1-10 is respectively high pig, inland river pig, hides pig, people pig, Jinhua pig, Wuzhi Mountain pig, northeastern wild boar, duroc, landrace, Large White.
Fig. 2 utilizes DNAMAN software that the dna fragmentation (917bp) that amplification obtains 8 pig kinds is carried out the result that sequence alignment is analyzed.
Fig. 3 is the order-checking peak figure of 3 SNP of place homozygous genotypes on the pig CYB5 gene, and wherein A is-660bp place corresponding wild-type and absence type, and B is-corresponding TT type and the CC type in 380bp place that C is-corresponding CC type and the TT type in 69bp place.
Fig. 4 is the PCR product agarose gel electrophoresis figure that little satellite detects; Wherein M is DNAMarker, and 1-12 is respectively high pig, the black pig of Qian Bei, hides pig, people pig, Jinhua pig, blue pool pig, northeastern wild boar, Zhejiang wild boar, Jiangxi wild boar, duroc, landrace, Large White.
Fig. 5 is three kinds of genotypic little satellite detected peaks figure.
Fig. 6 is the PCR product agarose gel electrophoresis figure of tetra-sodium order-checking; Wherein M is DNAMarker, and 1-11 is respectively high pig, the black pig of Qian Bei, Tibetan pig, people pig, the black pig of lining trouble, Jinhua pig, Wuzhi Mountain pig, northeastern wild boar, duroc, landrace, Large White.
Fig. 7 is three kinds of genotype tetra-sodium order-checking peak figure.
Fig. 8 is PCR product agarose gel electrophoresis result in the RFLP detection; Wherein M is DNAMarker, and 1-12 is respectively high pig, inland river pig, hides pig, people pig, Jinhua pig, Wuzhi Mountain pig, northeastern wild boar, Zhejiang wild boar, Jiangxi wild boar, duroc, landrace, Large White.
Fig. 9 is part PCR product RFLP polyacrylamide gel electrophoresis figure, and wherein M is 50bpDNA Ladder, is followed successively by 500bp, 400bp, 350bp, 300bp, 250bp, 200bp, 150bp, 100bp, 50bp from top to bottom.
Embodiment
Following examples are used to explain the present invention, but are not used for limiting scope of the present invention.If do not specialize the conventional means that used technique means is well known to those skilled in the art among the embodiment, the raw materials used commercial goods that is.
Adopt chloroform extraction method to extract pig ear tissue genomic dna in the present embodiment,,, get the part dna solution again it is diluted to 100ng/ μ l according to the concentration of measuring with concentration and the purity of its DNA of NanoDrop ND-1000 spectrophotometer detection.Stoste is stored to-20 ℃ of refrigerators, get 2 μ l diluents and carry out the detection of 1% agarose gel electrophoresis, detected result is seen Fig. 1.
The promotor of embodiment 2 prediction pig CYB5 genes
The pig CYB5 gene order of announcing according to NCBI (accession number: NW_001885867), its 5 ' flank head of district 4521bp.This section is carried out promotor and Transcription Factor Binding Sites Prediction with websites such as Softberry and MatInspector, and employed promotor prediction website comprises:
Matinspector(http://www.genomatix.de/online_help/help_matinspector/matinspector_help.html)
Berrysoft(http://linuxl.softberry.com/berry.phtml)
TF?Search(http://molsunl.cbrc.aist.go.jp/research/db/TFSEARCH.html)
Promoter?Scan(http://www-bimas.cit.nih.gov/cgi-bin/molbio/proscan)
BDGP(http://fruitfly.org：9005/seq_tools/promoter.html
Through biological software such as genomatix, TF search predictions, find that upstream of coding region 1000bp exists the possibility of promotor core area maximum, therefore the fragment of wherein 917bp has been carried out cloning and sequencing.According to the research to 5 ' flanking region of people's CYB5 gene such as Li (1995), people's CYB5 gene promoter sub-element also is positioned at-450～-1500bp and-300～-the 150bp section, transcription initiation site is positioned at-100～-110bp.The 917bp fragment of therefore cloning among the present invention also comprises the promotor core area probably.
The detection of embodiment 2 pig CYB5 gene 5 ' upstream promoter district SNP
Utilize Primer5.0 design primer, design earlier detects the primer of 5 ' upstream promoter district SNP, and primer information is as shown in table 1.Amplification system is as shown in table 2, and amplification program is: 94 ℃ 3 minutes; 94 ℃ 30 seconds, annealed 30 seconds, 72 ℃ 2 minutes, totally 33 circulations; 72 ℃ 5 minutes.
The amplimer of table 1CYB5 predictive genes promotor
Table 2PCR amplification system
(pig is hidden on peaceful pig, Xiao Mei mountain to random choose Chinese Pigs kind; Northeastern wild boar, each 1 of Zhejiang wild boar) and external pig kind (landrace, Large White; Each 1 of Du Luoke) DNA sample carries out pcr amplification, and (the segmental base sequence of 917bp of Chinese Pigs kind and external pig kind is respectively shown in Seq ID No.1 and Seq ID No.2, wherein to have obtained the 917bp fragment of CYB5 predictive genes promoter region; Last base C is positioned at the pig CYB5 gene start codon upper reaches-41bp place among the SEQ ID No.2), but owing to have the PolyG structure in this sequence, when having heterozygote; The PCR product directly checks order to be difficult to survey and leads to, so need carry out cloning and sequencing.The 917bp fragment purification is cut glue reclaim, be connected into the PMD18-T carrier, transform, identify.Find that through order-checking in Chinese Pigs kind and external pig kind, have the remarkable different SNP site, 3 place that distributes, they are respectively and are arranged in the CYB5 gene start codon upper reaches: the single base deletion of the PolyG of-660bp place sequence, the T → C of-380bp place, the C → T of-69bp place.Going up the sequence of announcing with NCBI is wild-type, and then the Chinese Pigs kind is mutant, and external pig kind is wild-type (Fig. 2 and Fig. 3; Wherein, among Fig. 2 290-300bp corresponding to pig CYB5 gene promoter area upstream from start codon-667～-657bp, 570～580bp corresponding to-387～-377bp, 880～890bp corresponding to-77～-67bp).
The colony in 3 SNP sites of embodiment 3 pig CYB5 genes is detected
Through behind the cloning and sequencing, preliminary screening is to having allochoric 3 SNP sites of significance difference in China and foreign countries' pig kind, for it still is the general character in the colony for the chance of random choose, in the present embodiment and then carried out colony's checking.Choose 11 Chinese native pig breeds (blue pool pig, Wuzhi Mountain pig, people pig, lining trouble are deceived pig, Han River pig, upward high pig, Jinhua pig, the black pig in Jiaxing, Qian Bei deceive pig, inland river pig, hide pig), 3 Chinese wild boar kind (northeastern wild boars; Zhejiang wild boar, Jiangxi wild boar) and 3 foreign pig kinds (Da Bai, long white and Du Luoke) totally 532 individuals carry out colony and detect.The group structure of laboratory animal is as shown in table 3.
Table 3 laboratory animal group structure
1 ,-the little satellite of 660bp detects
Because the G base deletion at CYB5 gene-660bp place is in the tandem repetitive sequence, so can adopt the method for little satellite to detect.Can the increase primer of the short segments that comprises this site of design, the specifying information of primer is seen table 4.Amplification system is as shown in table 5, and response procedures is: 95 ℃ 5 minutes; 95 ℃ 30 seconds, annealed 30 seconds, 72 ℃ 30 seconds, totally 35 circulations; 72 ℃ 7 minutes.
The amplimer of the little satellite of table 4-660bp
Annotate: * representes that upstream primer 5 ' end adds biotin labeling
The pcr amplification system that the little satellite of table 5 detects
162bp PCR product to each sample carries out agarose electrophoresis; Electrophoresis result is as shown in Figure 4; Single by the visible amplification of Fig. 4 back band, be suitable for carrying out little satellite and detect, then get 7 μ l amplified productions and deliver to Beijing and hold up section's bio tech ltd; Accomplish little satellite by it and detect, detected peaks figure is as shown in Figure 5.Be single base deletion in the PolyG sequence in this site Chinese Pigs kind (trouble is deceived the pig kind in the North China type with external pig kind gene infiltration), external pig kind does not then have base deletion.
2 ,-order-checking of 380bp tetra-sodium
The primer that the tetra-sodium order-checking is used is as shown in table 6.Because of institute's amplified fragments GC content higher (68.4%),, increase amplification efficiency so adopt trimethyl-glycine (Betaine Solution) to reduce its Tm value.The pcr amplification system of tetra-sodium order-checking is as shown in table 7.Response procedures is: 95 ℃ 2 minutes; 95 ℃ 30 seconds, annealed 30 seconds, 72 ℃ 20 seconds, totally 34 circulations; 72 ℃ 5 minutes.The fragment of each PCR of tetra-sodium order-checking is carried out agarose detect, Fig. 6 is the electrophorogram of an intercepting wherein.Visible by Fig. 6, PCR product band is single and efficient is higher, can carry out the tetra-sodium order-checking.Qualified PCR product is carried out the tetra-sodium order-checking, and peak figure is as shown in Figure 7 in order-checking.Because sequencing primer is reverse, so the result is A/A (a TT type) in showing, A/G (CT type), G/G (CC type).Be C in this site Chinese Pigs kind (trouble is deceived the pig kind in the North China type with external pig kind gene infiltration), external pig kind is T.
Table 6 tetra-sodium sequencing primer
Annotate: * representes that upstream primer 5 ' end adds biotin labeling
The pcr amplification system of table 7 tetra-sodium order-checking
3 ,-69bp restriction fragment length polymorphism (RFLP)
Use the primer5.0 software analysis, the base at discovery-69bp place has increased the restriction enzyme site of a FspBI restriction endonuclease after the sudden change of C → T takes place, so can adopt the method for RFLP to detect this site mutation.The recognition site of restriction enzyme FspBI is:
5’-C^TAG-3’
3’-G?AT^C-5’
Design primer on the restriction enzyme site both sides with Primer5.0 software, primer information is as shown in table 8, and amplification system is as shown in table 2.Response procedures is: 95 ℃ 5 minutes; 95 ℃ 30 seconds, annealed 30 seconds, 72 ℃ 30 seconds, totally 34 circulations; 72 ℃ 7 minutes.
The amplimer of table 8-69bp
Institute's amplification PCR products is carried out agarose gel electrophoresis detect, the good PCR product 37 ℃ of enzymes that spend the night in constant incubator of amplification are cut.Because after enzyme was cut, the PCR product of mutant was cut to two bar segment of 25bp and 100bp, and, therefore select for use the method for polyacrylamide gel electrophoresis to detect with the difficult difference in length that detects 25bp of agarose electrophoresis.PCR product to 125bp carries out the sepharose detection, and the result is as shown in Figure 8.Glue figure result is good, proves that the PCR product can be used for carrying out RFLP and detect.The good PCR product of expanding effect is carried out doing after enzyme is cut polyacrylamide gel electrophoresis and with after the EB dyeing, effect is as shown in Figure 9.Be T in this site Chinese Pigs kind (trouble is deceived the pig in the North China type with external pig kind gene infiltration), external pig kind is C.
Through detection, confirm that proofread and correct and statistics, discovery allelotrope distributes and meets hardy weinberg equilibrium (χ in colony to three SNP sites
2＞χ
2 2,0.05), and the genotype in three sites is all identical in same individuality, has tangible chain situation.Therefore in China and foreign countries' pig kind, only find two kinds of haplotypes: A type (G-T-C) and Type B (
).From table 9, can find out: in the foreign local pig breed, the advantage haplotype all is the A type, and in the Chinese Pigs kind, the black pig of trouble, the advantage haplotype all is a Type B in the North China type with external pig kind gene infiltration.
Table 9 China and foreign countries pig kind haplotype distributes
Selected pig kind has comprised local variety and 3 dissimilar Chinese wild boar kinds of 6 traditional types in the present embodiment, and individuality is a random choose, and external pig kind is representative, so the inventive method is applicable to the evaluation of all Chinese Pigs kinds and external pig kind.
Though, the present invention has been done detailed description in the preceding text with general explanation and specific embodiments, on basis of the present invention, can to some modifications of do or improvement, this will be apparent to those skilled in the art.Therefore, these modifications or the improvement on the basis of not departing from spirit of the present invention, made all belong to the scope that requirement of the present invention is protected.
Claims (10)
1. be used to identify the genetic marker of China and foreign countries' pig kind; It is characterized in that; It is positioned on the pig CYB5 gene 5' promoter region; Its nucleotide sequence is identified Chinese Pigs kind and external pig kind according to the difference in SNP site in this sequence shown in SEQ ID NO.2, said SNP site be arranged in the CYB5 gene start codon upper reaches-660bp ,-380bp or-69bp one or more;
Wherein, in the Chinese Pigs kind CYB5 gene promoter sequence-660bp ,-380bp ,-69bp place base is respectively single base deletion, C, T, in the external pig kind CYB5 gene promoter sequence-660bp ,-380bp ,-69bp place base is respectively G, T, C.
2. identify the method for pig kind at home and abroad for one kind; It is characterized in that; Identify Chinese Pigs kind and external pig kind according to the difference in SNP site in the sequence shown in the SEQ ID No.2 of pig CYB5 gene promoter to be measured, said SNP site be arranged in the CYB5 gene start codon upper reaches-660bp ,-380bp or-69bp one or more;
Wherein, in the Chinese Pigs kind CYB5 gene promoter sequence-660bp ,-380bp ,-69bp place base is respectively single base deletion, C, T, in the external pig kind CYB5 gene promoter sequence-660bp ,-380bp ,-69bp place base is respectively G, T, C.
3. method according to claim 2 is characterized in that, may further comprise the steps:
1) genomic dna of extraction pig to be measured;
2) genomic dna with pig to be measured is a masterplate, utilizes Auele Specific Primer amplification to be comprised the fragment in different SNP site in the pig CYB5 gene promoter sequence to be measured;
Wherein, said SNP site for be arranged in the CYB5 gene start codon upper reaches-660bp ,-380bp or-69bp is one or more;
3) detect pcr amplification product.
4. method according to claim 3 is characterized in that step 2) described in primer to being:
I) upstream primer 5 '-AAGGAGGAGGTAAGCAATG-3 ' and downstream primer 5 '-GAGATGAGCGGAACAGAGT-3 '; Or
Ii) upstream primer 5 '-TGGGTCAACAGCGAGATA-3 ' and downstream primer 5 '-CCTCCGTAGAACGAGTGTA-3 '; Or
Iii) upstream primer 5 '-CGCCTGCCACAATGAACC-3 ' and downstream primer 5 '-GCGCAGAAGGGGATGATG-3 '; Or
Iv) upstream primer 5 '-TGAGCGGAACAGAGTGGG-3 ' and downstream primer 5 '-AATCATCGGAGGTCGTGC-3 '.
5. method according to claim 3 is characterized in that, the method for the said detection pcr amplification product of step 3) is:
Step 2) primer that amplification is used in is to being i) or ii), the method that detects amplified production is order-checking;
Step 2) primer that amplification is used in is to for iii), and the method that detects amplified production is that tetra-sodium checks order; Or
Step 2) primer that amplification is used in is to for iv), and the method that detects amplified production is the RFLP detection.
6. method according to claim 5 is characterized in that, when step 2) in the primer that uses of amplification to for iii) the time, the sequencing primer of use was 5 '-TGGAGTGGCTGGCGA-3 ' when amplified production was carried out the tetra-sodium order-checking.
7. method according to claim 5; It is characterized in that; When step 2) in the primer that uses of amplification to for iv) the time, cut amplified production with restriction enzyme FspBI enzyme, and enzyme cut product carry out the agarose gel electrophoresis detection; What electrophoresis result presented 2 bands is the Chinese Pigs kind, is merely the external pig kind that is of 1 band.
8. be used to identify the PCR primer of China and foreign countries' pig kind, it comprises one or more pairs of in the following primer:
I) upstream primer 5 '-AAGGAGGAGGTAAGCAATG-3 ' and downstream primer 5 '-GAGATGAGCGGAACAGAGT-3 ';
Ii) upstream primer 5 '-TGGGTCAACAGCGAGATA-3 ' and downstream primer 5 '-CCTCCGTAGAACGAGTGTA-3 ';
Iii) upstream primer 5 '-CGCCTGCCACAATGAACC-3 ' and downstream primer 5 '-GCGCAGAAGGGGATGATG-3 '; Or
Iv) upstream primer 5 '-TGAGCGGAACAGAGTGGG-3 ' and downstream primer 5 '-AATCATCGGAGGTCGTGC-3 '.
9. the test kit that is used to identify China and foreign countries' pig kind that contains the said primer of claim 8.
10. the said application that is used for identifying the genetic marker of China and foreign countries' pig kind at the pig molecule mark assistant breeding of claim 1.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103966349A (en) * | 2014-05-29 | 2014-08-06 | 江苏省农业科学院 | Screening method and application of multiple-allele PCR (Polymerase Chain Reaction) amplified fragment |
| CN105255878A (en) * | 2015-11-16 | 2016-01-20 | 上海市农业科学院 | SNP molecular markers used in chromosome 6 of pig for traceability and application thereof |
| CN105255877A (en) * | 2015-11-16 | 2016-01-20 | 上海市农业科学院 | SNP molecular markers used in chromosome 6 of pig for traceability and application thereof |
| CN108130375A (en) * | 2017-12-04 | 2018-06-08 | 漳州傲农现代农业开发有限公司 | A kind of method for differentiating Jinhua Pigs, Landrace based on SNP site |
| CN113207808A (en) * | 2021-06-03 | 2021-08-06 | 贵州大学 | Breeding method of Kele pigs |
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| CN1900311A (en) * | 2006-07-20 | 2007-01-24 | 上海交通大学 | Separating and identifying method for pig follicle-stimulating hormone beta sub-gene monomer |
| CN1900310A (en) * | 2006-07-20 | 2007-01-24 | 上海交通大学 | Separating and identifying method for pig female hormone receptor gene monomer |
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| CN1337469A (en) * | 2000-08-08 | 2002-02-27 | 华中农业大学 | Typing method of pig monosperm microsatellite DNA mark |
| CN1900311A (en) * | 2006-07-20 | 2007-01-24 | 上海交通大学 | Separating and identifying method for pig follicle-stimulating hormone beta sub-gene monomer |
| CN1900310A (en) * | 2006-07-20 | 2007-01-24 | 上海交通大学 | Separating and identifying method for pig female hormone receptor gene monomer |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103966349A (en) * | 2014-05-29 | 2014-08-06 | 江苏省农业科学院 | Screening method and application of multiple-allele PCR (Polymerase Chain Reaction) amplified fragment |
| CN105255878A (en) * | 2015-11-16 | 2016-01-20 | 上海市农业科学院 | SNP molecular markers used in chromosome 6 of pig for traceability and application thereof |
| CN105255877A (en) * | 2015-11-16 | 2016-01-20 | 上海市农业科学院 | SNP molecular markers used in chromosome 6 of pig for traceability and application thereof |
| CN105255878B (en) * | 2015-11-16 | 2018-01-26 | 上海市农业科学院 | It is used for SNP marker and its application traced to the source on No. 6 chromosomes of pig |
| CN105255877B (en) * | 2015-11-16 | 2018-02-13 | 上海市农业科学院 | It is used for SNP marker and its application traced to the source on No. 6 chromosomes of pig |
| CN108130375A (en) * | 2017-12-04 | 2018-06-08 | 漳州傲农现代农业开发有限公司 | A kind of method for differentiating Jinhua Pigs, Landrace based on SNP site |
| CN113207808A (en) * | 2021-06-03 | 2021-08-06 | 贵州大学 | Breeding method of Kele pigs |
| CN113207808B (en) * | 2021-06-03 | 2023-09-29 | 贵州大学 | Breeding method of cola pigs |
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