CN105543362A - Detection method for single nucleotide polymorphism of cattle PPARbeta gene and molecular breeding method - Google Patents

Detection method for single nucleotide polymorphism of cattle PPARbeta gene and molecular breeding method Download PDF

Info

Publication number
CN105543362A
CN105543362A CN201610007765.7A CN201610007765A CN105543362A CN 105543362 A CN105543362 A CN 105543362A CN 201610007765 A CN201610007765 A CN 201610007765A CN 105543362 A CN105543362 A CN 105543362A
Authority
CN
China
Prior art keywords
gene
ppar
cattle
single nucleotide
polymorphism
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201610007765.7A
Other languages
Chinese (zh)
Other versions
CN105543362B (en
Inventor
马云
张琼琼
李芬
郝瑞杰
李俊雅
付玮玮
刘锦妮
马福军
石奎林
李何
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Xinyang Normal University
Original Assignee
Xinyang Normal University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Xinyang Normal University filed Critical Xinyang Normal University
Priority to CN201610007765.7A priority Critical patent/CN105543362B/en
Publication of CN105543362A publication Critical patent/CN105543362A/en
Application granted granted Critical
Publication of CN105543362B publication Critical patent/CN105543362B/en
Expired - Fee Related legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention discloses a detection method for single nucleotide polymorphism of a cattle PPARbeta gene. The detection method comprises the steps that PCR amplification is performed on the cattle PPARbeta gene by taking to-be-detected cattle whole genome DNA containing the PPARbeta gene as a template and taking a primer pair (P) as primers; a PCR amplification product is digested with a restriction enzyme (Pvu II), and unmodified polyacrylamide gel electrophoresis is performed on segments obtained through enzyme digestion; the polymorphism of the 71616<th> single nucleotide of the cattle PPARbeta gene is identified according to an electrophoresis result. The invention further relates to a method for constructing a cattle molecular breeding index system by means of the single nucleotide polymorphism of the PPARbeta gene. Due to the fact that the PPARbeta gene has the important biological function, the gene single nucleotide polymorphism has the significant influence on the weight, the withers height and the ischium end width of a cattle in the early stage and can be used for cattle beef and molecular marker-assisted selection of growth characteristics, and then a cattle population which has excellent genetic resources can be quickly constructed.

Description

A kind of detection method of ox PPAR β gene mononucleotide polymorphism and molecular breeding method
Technical field
The present invention relates to a kind of detection method and molecular breeding method of gene mononucleotide polymorphism, be specifically related to a kind of detection method and molecular breeding method of ox PPAR β gene mononucleotide polymorphism.
Background technology
Single nucleotide polymorphism (SingleNucleotidePolymorphism, SNP) mainly refers to the polymorphism of the DNA sequence dna of the specific site in genomic level caused by the variation of single core thuja acid.The polymorphism that SNP shows to be changed by single base or caused by transversion, in genomic dna, any base all has the possibility morphed.Therefore, SNP had both likely been present in gene order, was also likely positioned on non-coding sequence.The frequency that SNP occurs in the sequence being rich in CG is the highest, and is mostly that cytosine(Cyt) C is converted to thymus pyrimidine T, and reason is that the C in CG spontaneously can become thymus pyrimidine by deamination after methylating.
SNP, as third generation molecular genetic marker, has following characteristics: (1) density is large.SNP is ubiquity and the highest genetic marker of frequency in genomic dna.There are some researches show, the frequency that SNP occurs is between 1/1000 ~ 10/1000, and have the SNPs of more than 3,000,000 in the mankind's 3,000,000,000 bases, its density is higher than microsatellite DNA mark.(2) easily automated analysis is realized.The base of composition DNA has 4 kinds, but SNP generally only has two kinds of based compositions, therefore, SNP marker is two equipotential marks, show as either-or when detecting, need not analyze the length of fragment when genome screens, often only need the analysis of +/-, this is just beneficial to and realizes automatic technology screening and detect SNPs.(3) of many uses.SNP, as the one of molecular genetic marker, has function widely in Animal Genetics.Be mainly used in the researchs such as genetic mapping, disease detection, site mark, evolutionary analysis.
At present, the method detecting SNP can be divided into two large classes: a class is the traditional method based on gel electrophoresis, mainly comprises PCR-SSCP and DNA sequencing combined techniques, PCR-RFLP method, AS-PCR method, denaturing gradient gel electrophoresis etc.Wherein, the experimentation of PCR-SSCP is long, operates more loaded down with trivial details, be only suitable for the little DNA fragmentation that length is less than 500bp, if DNA fragmentation is oversize, DNA single chain is difficult to be formed stablizes hairpin structure, and there is Problem of False Negative in experimentation, therefore also and nonideal SNP detection means.AS-PCR method needs to design special primer, and can only for specific gene locus, but due to the poor stability of Auele Specific Primer, requires relatively stricter, operate more loaded down with trivial details, be therefore unsuitable for the detection of high-throughput SNP amplification condition.Denaturing gradient gel electrophoresis also can only detect sudden change comparatively roughly, can not determine mutated site and type.PCR-RFLP because locus specificity is high, the comparatively simple and cost of operation is lower etc., therefore, is widely used the research in aspects such as plant gene somatotype, the assignment of genes gene mapping, Molecular Identification.Another kind of is the detection method utilizing high-throughput, level of automation higher.High-throughout detection method comprises direct Sequencing, DNA chip, denaturing high-performance chromatography etc.Wherein, direct Sequencing detects SNP the most reliable method, and DNA chip can carry out large-scale examination to SNP, but the testing cost of the two is all extremely expensive; And denaturing high-performance chromatography method for reagent and environmental requirement higher, homozygous mutation can not be detected, therefore neither detect the prefered method of SNP.In sum, PCR-RFLP method is utilized may to be the optimal genetic marking method of current detection SNP.
Ox is one of main poultry kind resource of China, but is faced with now the pressure of the not enough and population genetic inferior quality of outstanding yellow cattle breed provenance.Traditional breeding method based on measure animal phenotype and utilize its parental generation, for generations and the genetic information of other relatives in animal model realize genetic evaluation, generally, imagination proterties is subject to the impact of much gene genetic difference, therefore think that each gene pairs proterties has relatively little contribution, result causes the estimation of proterties some deviation inevitable.Molecular genetic marker is one of field that modern genetics is with fastest developing speed in recent years, along with continuing to bring out of genomics novel method and new technology, by integrating every technology, can carry out design to reconstruct livestock and poultry new variety from gene and molecular level.Based on generation and the application of DNA genetic marker, start the frontier of molecular breeding, for improvement of breed and improvement provide new technical support, bring fine prospect to Animal Genetics.
Peroxisome proliferation-activated receptors (PeroxisomeProliferatorActivatedReceptor, PPAR) steroid/Tiroidina/retinoid receptor superfamily is belonged to, having important regulative for the sugar balance in insulin sensitivity, body and Adipose Differentiation and generation aspect, is the focus of research at present.This family can be divided into three members, PPAR α, β and γ, and PPAR β extensively exists in animal body, and tissue such as heart, adipocyte, the skeletal muscle etc. active at lipid metabolism organize camber to express.At present, PPAR β plays a significant role in the differentiation of adipocyte and the metabolism of lipid acid to have large quantifier elimination to prove, and the research overwhelming majority of PPAR β gene carries out on the animal pattern such as people and mouse, and ox but rarely has report.The research of native Chinese cattle PPAR β gene genetic variation there is no bibliographical information at present.Therefore, need simple, quick, low cost, the accurate detection method of a kind of ox PPAR β gene mononucleotide polymorphism of exploitation badly, again associating of the SNP of PPAR β gene and heritable variation thereof and ox growth traits index (as: body weight, day weight gain, height, point of the buttocks wide, chest measurement etc.) is studied, for use in the marker assisted selection of the meat growth traits of Chinese Cattle, reach the object of early stage seed selection and raising breeding value accuracy, thus the yellow cattle breed setting up genetic resources fast excellent is provided fundamental basis.
Summary of the invention
Technical problem to be solved by this invention is, a kind of simple, quick, low cost, accurate detection method and molecular breeding method of ox PPAR β gene mononucleotide polymorphism are provided, the SNP that searching associates with economic characters, as molecule marker, accelerates the foundation with Quality and economy proterties ox population.
The technical scheme that the present invention solves the employing of its technical problem is, a kind of method detecting ox PPAR β gene mononucleotide polymorphism, to comprise the ox complete genome DNA to be measured of PPAR β gene for template, with primer pair P (comprising P1 and P2) for primer, pcr amplification ox PPAR β gene; Then digest pcr amplification product with restriction enzyme Pvu II, the amplified fragments after then cutting enzyme carries out native polyacrylamide gel electrophoresis; Finally according to the single nucleotide polymorphism of polyacrylamide gel electrophoresis result qualification ox PPAR β gene the 71616th; Wherein, described primer pair P is:
Upstream primer P1:5'-TCCTTCCAGCAGCTACACAG ct-3'22bp, is shown in SEQIDNO:1;
Downstream primer P2:5'-GGGAGACAACTCGCCCAAGA-3'20bp, is shown in SEQIDNO:2.
Note: " _ " in primer represents the base mismatch forming restriction enzyme site and introduce.
Further, described pcr amplification reaction program is: 95 DEG C of denaturation 10min; 94 DEG C of sex change 30 ~ 50s, 63 DEG C of annealing 30s, 72 DEG C extend 30 ~ 50s, 30 ~ 40 circulations; 72 DEG C extend 10min; 4 DEG C of preservations.
Further, described polyacrylamide gel electrophoresis is mass concentration is 12% polyacrylamide gel.
Further, the single nucleotide polymorphism of described ox PPAR β gene the 71616th is: AG (heterozygous) genotypic expression is 167bp, 147bp, 28bp and 20bp tetra-band; GG (saltant type) genotypic expression is 147bp, 28bp and 20bp tri-band; Without AA (normal type) genotype.
The technical scheme that the present invention solves the employing of its technical problem is further that a kind of molecular breeding method based on ox PPAR β gene mononucleotide polymorphism, comprises the following steps:
(1) detection method of ox PPAR β gene mononucleotide polymorphism according to claims 1 to 4, determines the genotype of ox PPAR β gene the 71616th single nucleotide polymorphism;
(2) association analysis is carried out by the genotype of ox PPAR β gene the 71616th single nucleotide polymorphism and ox growth traits, determine that the single nucleotide polymorphism of ox PPAR β gene the 71616th can as the molecule marker improving ox early growth traits, for the molecular breeding of ox;
(3) in the single nucleotide polymorphism of ox PPAR β gene the 71616th, GG genotype can be used as the candidate molecules genetic marker improving ox early growth traits.
The present invention is according to the primers announcing ox PPAR β gene (NCBI:AC_000180.1), respectively with the genomic dna of 5 yellow cattle breed for template, carry out pcr amplification, and PCR primer is checked order, the sequence that the partial sequence of ox PPAR β gene obtained after order-checking and NCBI are announced is contrasted, finds that the 71616th at PPAR β gene exists SNP polymorphism.For PPAR β gene SNP polymorphism, after the present invention carries out pcr amplification, carry out enzyme with specific restriction enzyme and cut qualification, can simply, fast, low cost, accurately detect the polymorphism of its mononucleotide.During pcr amplification PPAR β gene product, this sudden change does not have nature restriction enzyme site, need introduce a base mismatch and make it form the restriction enzyme site CAGCTG of Pvu II at mutational site place on primer.Pcr amplified fragment size is 195bp, also there is the restriction enzyme site of another Pvu II except SNP site.Therefore, even normal genotype also can be cut into two band 167bp and 28bp in cutting process.When there is A>G sudden change for the 71616th, the 71611bp ~ 71616bp of pcr amplification PPAR β gene is CAGCT gtime, then can by the identification of Pvu II enzyme; When the 71616th A>G sudden change does not occur, when the 71611bp ~ 71616bp of pcr amplification PPAR β gene is CAGCTA, then by the identification of Pvu II enzyme, so just can not detect this site SNP polymorphism.
The present invention, by the detection method of PPAR β gene SNP, determines the genotype of ox PPAR β gene the 71616th single nucleotide polymorphism; And then carry out association analysis with ox some growth proterties (height, body weight and Body steep length etc.), determine that this site can as the molecule marker improving ox early growth traits, for use in the marker assisted selection (MAS) of the meat growth traits of Chinese Cattle, set up the ox population that genetic resources is excellent fast.
Accompanying drawing explanation
Fig. 1 is the 195bpPCR product electrophoresis detection figure that ox PPAR β gene comprises the polymorphic site of the 71616th, and wherein, swimming lane 1-5 is the agarose electrophoresis detection figure that PPAR β gene comprises the 195bp fragment of the polymorphic site of the 71616th; Swimming lane M is MarkerDL1000 (1000bp, 700bp, 500bp, 400bp, 300bp, 200bp, 100bp).
Fig. 2 (a) is Pvu II PCR-RFLP method detection ox PPAR β gene the 71616th SNP restriction enzyme digestion and electrophoresis figure, wherein, and swimming lane 1:M road: DL500 (500bp, 400bp, 300bp, 200bp, 150bp, 100bp, 50bp); Swimming lane 2-4:AG genotype individuals (167bp, 147bp, 28bp, 20bp); Swimming lane 5:GG genotype individuals (147bp, 28bp, 20bp); Without AA genotype individuals.B backward sequencing figure that () is ox PPAR β gene the 71616th site AG and GG genotype individuals.
Fig. 3 is that Pvu II PCR-RFLP method detects ox PPAR β gene intron 2 SNP (AC_000180.1_g.71616A>G) sequential analysis figure (wherein, band frame represents upstream and downstream primer sequence respectively, and the site that single letter adds blue background represents SNP site).
Fig. 4 is the gene fragment figure of the 195bp of PPAR β gene 71594bp ~ 71788bp.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is illustrated further.
1, biochemical reagents and biological reagent: GoldenDNA polysaccharase (purchased from Beijing Tian Gen biochemical technology company limited); 2 × ReactionMix includes Mg2+, dNTPs etc. (purchased from Beijing Tian Gen biochemical technology company limited Mix); Restriction enzyme Pvu II (purchased from TAKARA company); Proteinase K (purchased from Huamei Bio-Engrg Co.); Marker (DL500, DL1000), purchased from the precious biotechnology company limited in Dalian.
2, general reagent: buying from Huamei Bio-Engrg Co., is import packing product: citric acid, Trisodium Citrate, glucose, Tris, acrylamide, methylene diacrylamide, TEMED, ammonium persulphate, EDTA, NaCl, NaOH, KCl, Na 2hPO 4, KH 2pO 4, the saturated phenol of Tris, chloroform, primary isoamyl alcohol, dehydrated alcohol, sodium-acetate, sodium laurylsulfonate (SDS), ethidium bromide (EB), tetrabromophenol sulfonphthalein, acetic acid, sucrose, boric acid, agarose etc.
3, solution and damping fluid: all solution and damping fluid all adopt deionization ultrapure water to prepare, autoclave conditions is 15bf/in (1.034 × 10 5pa), 25min; Preparation of reagents method is all with reference to " Molecular Cloning: A Laboratory guide " that Sambrook etc. writes.
(1) sample collecting solution used
Antithrombotics ACD: citric acid 2.4g; Trisodium citrate 6.6g; Glucose 7.35g, is settled to 50mLddH 2in O, autoclaving.The ACD liquid of 0.2mL is added in every 10mL fresh blood.This antithrombotics is better than heparin, can preserve high molecular DNA better in blood storage process.Blood through its anti-freezing can preserve a couple of days or-80 DEG C of preservations for a long time at 0 DEG C.
(2) blood sample genomic dna is separated solution used
1. PBS damping fluid: NaCl8g, KCl0.2g, Na 2hPO 41.44g, KH 2pO 40.24g, adds ultrapure water to 1000mL, adjusts pH to 7.4, autoclaving.
2. 10%SDS:10gSDS is dissolved in the ultrapure water of 90mL, and 68 DEG C of water-baths are dissolved, and adjust pH to 7.2, be settled to 100mL with HCl.
3. 0.5mol/LEDTA:EDTA186.1g, is dissolved in the ultrapure water of 800mL, adjusts pH to 8.0, be settled to 1000mL, autoclaving, 4 DEG C of preservations with NaOH.
4. 1mol/LTrisCl:121.14gTris, be dissolved in 800mL ultrapure water, HCl regulates pH to 8.0, is settled to 1000mL.Autoclaving, 4 DEG C of preservations.
5. 5mol/LNaCl:NaCl292.2g is dissolved in 1000mL ultrapure water.
6. DNA extraction buffer: get 0.5mmol/LEDTA40mL, 1mmol/LTrisCl10mL.
7. 5mmol/LNaCl4mL, 10%SDS10mL are settled to 100mL.Actual concentrations is 200mmol/LEDTA, pH8.0:100mmol/LTrisHCl, pH8.0,200mmol/LNaCl, 2%SDS.RNase20μg/mL。
8. NaAc damping fluid: NaAc3H 2o20.4g; Ultrapure water 40mL; Rare HAc adjusts pH to 7.4; Be settled to 50mL.
9. TE damping fluid: TrisCl damping fluid (pH8.0) 10mmol/L, edta buffer liquid (pH8.0) 0.1mmol/L, autoclaving, 4 DEG C of preservations.
10. Proteinase K: be made into 20mg/mL with ultrapure water ,-20 DEG C of preservations.
(3) Polyacrylamide Gel Electrophoresis solution used
1. 1 × tbe buffer liquid: get 10 × TBE100mL, be settled to 1000mL.
2. sample-loading buffer: 0.25% tetrabromophenol sulfonphthalein, the blue or green FF of 0.25% dimethylbenzene, 40.0% (w/v) aqueous sucrose solution.
Embodiment 1: the detection method of ox PPAR β gene mononucleotide polymorphism
One, the design of ox PPAR β gene PCR primer
Ox PPAR β (AC_000180.1) sequence announced with NCBI is reference, and the gene fragment of the 195bp of PPAR β gene 71594bp ~ 71788bp is shown in Fig. 4 and SEQIDNO:5.Utilize Primer5.0 design to increase and comprise the PCR primer in ox PPAR β gene the 71616th region, its primer sequence is as follows:
P3 (upstream primer): 5'-TCCTGTCTTCCCTTTCGTCC-3'20bp, see SEQIDNO:3;
P4 (downstream primer): 5'-GGAGACAACTCGCCCAAGAT-3'20bp, see SEQIDNO:4;
P3 and P4 primer pair ox genome is adopted to carry out pcr amplification; can be increased the gene fragment of the 427bp comprising ox PPAR β gene (AC_000180.1 sequence) the 71616th; after order-checking qualification is carried out to the fragment of amplification; by analysis, find that PPAR β gene the 71616th exists SNP polymorphism.
Due at this place's mutated site without natural restriction enzyme site, therefore need redesign enzyme cut primer, on primer introduce a base mismatch, make it can form the restriction enzyme site of restriction enzyme Pvu II at mutated site.The primer pair P sequence redesigned is as follows:
P1 (upstream primer): 5'-TCCTTCCAGCAGCTACACAG ct-3'22bp
P2 (downstream primer): 5'-GGGAGACAACTCGCCCAAGA-3'20bp
Note: " _ " in primer represents the base mismatch forming restriction enzyme site and introduce.
During pcr amplification PPAR β gene product, this sudden change does not have nature restriction enzyme site, need introduce a base mismatch and make it form the restriction enzyme site CAGCTG of Pvu II at mutational site place on primer.Pcr amplified fragment size is 195bp, also there is the restriction enzyme site of another Pvu II except SNP site.Therefore, even normal genotype also can be cut into two band 167bp and 28bp in cutting process.When there is A>G sudden change for the 71616th, the 71611bp ~ 71616bp of pcr amplification PPAR β gene is CAGCT gtime, then can by the identification of Pvu II enzyme.When the 71616th A>G sudden change does not occur, when the 71611bp ~ 71616bp of pcr amplification PPAR β gene is CAGCTA, then by the identification of Pvu II enzyme, so just can not detect this site SNP polymorphism.
Two, pcr amplification ox PPAR to be measured beta gene fragment is carried out with primer pair P
1, the collection of ox sample: amount to 454 individualities as detected object using 5 cattle breeds, concrete collecting sample is in table 1: Shaanxi Qinchuan Cattle (QC, 30), control in Pingdingshan City, Henan Province growth traits in Jiaxian red cattle (JX, 141), Nanyang, henan ox (NY, 139), Shandong Province western Shandong ox (LX, 114), Shandong Province's Bohai Black Cattle (BH, 30).
The collection of table 1-ox sample
2, the separation of blood sample genomic dna, extraction, purifying
(1) freezing blood sample (being mainly hemocyte) thaw at RT, transferase 45 00 μ L to 1.5mLEppendorf centrifuge tube, add equal-volume PBS liquid, abundant mixing, the centrifugal 10min of 12000r/min (4 DEG C), abandoning supernatant, repetition above-mentioned steps is transparent to supernatant liquor, precipitation is faint yellow;
(2) in centrifuge tube, add DNA extraction buffer 500 μ L, shake, hemocyte is precipitated and departs from centrifuge tube tube wall, 37 DEG C of water-bath 1h;
(3) add Proteinase K to 3 μ L (20mg/mL) and mix, 55 DEG C are spent the night to clarification, not yet defecator, can add 1 μ L Proteinase K mixing and continue digestion until clarification;
(4) reaction solution is cooled to room temperature, adds Tris-saturated phenol 500 μ L, gentle shake centrifuge tube 20min, makes it fully mix; 4 DEG C, the centrifugal 10min of 12000r/min, proceeds to supernatant liquor in another 1.5mL centrifuge tube, repeats once;
(5) add chloroform 500 μ L, fully mix 20min, 4 DEG C, the centrifugal 10min of 12000r/min, supernatant liquor is proceeded in another 1.5mL centrifuge tube;
(6) add chloroform, primary isoamyl alcohol mixed solution (24:1) 500 μ L, fully mix 20min, 4 DEG C, the centrifugal 10min of 12000r/min, supernatant liquor is proceeded in another 1.5mL centrifuge tube;
(7) add the NaAc damping fluid of 0.1 times of volume and the ice-cold dehydrated alcohol of 2 times of volumes, centrifuge tube is rotated in mixing, until the flocks of white is separated out, preserves 30 ~ 60min for-20 DEG C;
(8) 4 DEG C, the centrifugal 10min of 12000r/min, abandoning supernatant, precipitates 2 times with 70% ice cold ethanol rinsing DNA;
(9) 4 DEG C, the centrifugal 10min of 12000r/min, abandoning supernatant, makes ethanol volatilize clean under room temperature;
(10) dried DNA is dissolved in the TE liquid of 80 ~ 100 μ L, preserves until DNA dissolves completely for 4 DEG C, 0.8% its quality of polyacrylamide gel electrophoresis electrophoresis detection ,-80 DEG C of preservations.
Adding 10%SDS in the DNA solution of (11) 500 μ L makes its final concentration be 0.1%, adds Proteinase K to final concentration and reaches 50 μ g/mL;
(12) 5 DEG C of insulation about 10h;
(13) extracting of equal-volume phenol, chloroform, primary isoamyl alcohol (25:24:1) and chloroform difference once;
(14) the centrifugal 5min phase-splitting of 12000r/min, draws upper strata aqueous phase in another centrifuge tube;
(15) 1/10 volume 3mol/L sodium-acetate and 2 times of volumes ice cold dehydrated alcohol precipitation DNA are added;
(16) outwell liquid, dry after 70% washing with alcohol, add 60 μ L sterilizing ultrapure waters and dissolve, its concentration surveyed by ultraviolet spectrophotometer, is then diluted to the working fluid of 50ng/ μ L.
3, pcr amplification
PCR reaction system adopts mixing application of sample method, the number that the quantity of the various components namely needed for each reaction system and the PCR needed for 1 secondary response react, calculate the total amount of various reactive component, join in 1.5mL centrifuge tube, brief centrifugation after mixing, be dispensed in each 0.2mLEppendorfPCR pipe, then add template DNA, the more laggard performing PCR amplification of brief centrifugation.PCR reaction system is in table 2.
Table 2-PCR reaction system
Composition Reaction components Usage quantity Volume
2×Reaction Mix 7.5
Sense Primer(10μM) 0.1
Anti-sense Primer(10μM) 0.1
Golden archaeal dna polymerase 0.1
DNA profiling 0.5
ddH 2O 6.7
Total 15
15 μ L reaction systems comprise GoldenDNA polysaccharase 0.1 μ L (Beijing Tian Gen Science and Technology Ltd.), and 2 × ReactionMix7.5 μ L (includes Mg 2+, dNTPs etc.) (Beijing Tian Gen Science and Technology Ltd. Mix), 50ng/ μ L is containing the ox genomic dna 0.5 μ L of PPAR β gene, each 0.1 μ L of 10pmol/ μ L upstream and downstream primer.
Pcr amplification program: 95 DEG C of denaturation 10min; 94 DEG C of sex change 30 ~ 50s, 63 DEG C of annealing 30s, 72 DEG C extend 30 ~ 50s, 30 ~ 40 circulations; 72 DEG C of ends extend 10min; 4 DEG C of preservations.
The genomic dna 5 yellow cattle breed being amounted to 454 samples carries out pcr amplification, obtains the DNA fragmentation comprising the 195bp of this SNP site in 454 individual ox PPAR β genes.
Three, the PPAR beta gene fragment of Pvu II digestions pcr amplification
1, Pvu II endonuclease reaction digestion system (20 μ L): 8 μ LPCR products, 10 × M damping fluid 2.0 μ L, Pvu II (10U/ μ L) 0.3 μ L, sterilizing ultrapure water (H 2o) 9.7 μ L.
2, digestions condition: digest 10 ~ 12h in 37 DEG C of constant incubators.
Four, Polyacrylamide Gel Electrophoresis
1, make the polyacrylamide gel of 12.0%, 250V prerunning 10min, 200V electrophoresis 2h after point sample, electrophoresis terminates rear cma staining;
2, according to polyacrylamide gel electrophoresis interpretation of result SNP polymorphism
Judge the polymorphism of SNP according to silver dye result, see Fig. 3.During pcr amplification gene product, when there is A>G sudden change for the 71616th, then can be cut by the identification of Pvu II enzyme; When A>G sudden change does not occur in this site above-mentioned, now can not identify by being limited property restriction endonuclease Pvu II, so just can detect this site SNP polymorphism.
Pvu II PCR-RFLP method detects ox PPAR β gene the 71616th SNP restriction enzyme digestion and electrophoresis figure, sees Fig. 2 (a).From Fig. 2 (a), the polyacrylamide gel electrophoresis result of the SNP polymorphism of ox genomic PPAR β gene the 71616th is: AG (heterozygous) genotypic expression is 167bp, 147bp, 28bp and 20bp tetra-band; GG (saltant type) genotypic expression is 147bp, 28bp and 20bp tri-band; Without AA (normal type) genotype.
3, the sequence verification of the individual PCR primer of different genotype
Because the base mismatch introduced is positioned at forward primer, therefore, backward sequencing is carried out to the PCR primer of different genotype individuality; Meanwhile, SNP position analysis is carried out.Result shows that its 71616 the sequencer map of heterozygote AG genotype individuals comprising 167bp, 147bp, 28bp and 20bp band is expressed as A or G really, and as shown in Fig. 2 (b), and GG genotype is G, and order-checking peak figure is simple spike.
Five, the frequency statistics analysis of ox PPAR β gene SNP site
1, gene and genotype frequency
Genotype frequency, in population genetics, refers at one populationin per-cent shared by certain genotype.
P AA＝N AA/N
In formula, P aAwhat represent a SNP site is the genotypic frequency of AA; N aArepresent and have the genotypic number of individuals of AA; N is total number of whole colony.
Gene frequency is that certain gene is at certain populationthe ratio of middle appearance is the ratio that certain genetic entities number accounts for full gene number.
P A＝(2N AA+N Aa1+N Aa2+N Aa3+……N Aai+……+N Aan)/2N
In formula, P arepresent the frequency of allelotrope A, N aArepresent in colony and have the genotypic number of AA, N aairepresent and have the genotypic individual amount of Aai; a 1-a nrepresent the individual different multiple allelomorphos of the n of allelotrope A.
Allelotrope involved in the present invention is G and A, and therefore concrete gene frequency calculation formula is:
P G＝(2N GG+N AG)/2N
P A＝(2N AA+N AG)/2N
In formula, P g, P arepresent the allelic frequency of allelotrope G and A respectively, N gG,n aGand N aArepresent the genotypic individual amount of GG, AG and AA respectively, N represents total group number.
Gene frequency distribution is shown in Table 3, as shown in Table 3, the G gene frequency rangeability in different yellow cattle breed PPAR β gene SNP in 65% ~ 84.2%, A gene frequency rangeability between 15.8% ~ 35%.Meet the definition that gene frequency is greater than the SNP of 1.0%, therefore this site is mononucleotide polymorphic site.
The frequency distribution table of Pvu II PCR-RFLP of table 3-ox PPAR β gene the 71616th SNP site
Embodiment 2: based on the molecular breeding method of ox PPAR β gene mononucleotide polymorphism
The genotype (AG and GG) that genotype data: Pvu II identifies
Production data: Nanyang and growth traits in Jiaxian red cattle ox birth weight, and 6 monthly ages, 12 monthly ages, the body weight at 18 monthly ages and 24 monthly ages, height, Body steep length, chest measurement, point of the buttocks are wide, day weight gain.
Relation analysis model: be first described analysis to data, determines whether there is outlier, and recycling Least square analysis is to Data correction; According to data characteristics, the production traits effect between each genotype of application SPSS (17.0) software analysis.Fixed model is have employed when analyzing genotype effects:
Y ijkl＝μ+BF i+Month j+G k+e ijkl
Wherein: Y ijklfor character observation value, μ is population mean, BF ibe the fixing effect on i-th kind and farm, Month jfor the fixed effect of a jth month observation, G kfor a kth genotypic fixed effect of single SNP marker, e ijklfor random error.
The results of analysis of variance between PPAR β gene the 71616th polymorphic site and Nanyang cattle, Jiaxian County ox Different Month productive capacity is in table 4.As shown in Table 4, in 0 age in days, 6 monthly ages and 24 monthly ages, the genotypic whose body weight pole of GG is significantly higher than AG genotype (P<0.01); In 6 monthly ages and 24 monthly ages, the genotypic individual height of GG is significantly higher than AG genotype (P<0.05); AG genotype (P<0.01) is significantly higher than in the 18 genotypic individual height poles of monthly age GG.In 0 age in days, 6 monthly ages, 12 monthly ages, 18 monthly ages and 24 monthly ages, the wide all pole of the point of the buttocks of GG genotype individuals is significantly higher than AG genotype individuals (P<0.01).
Association analysis result illustrates that GG genotype can improve the wide candidate molecules genetic marker of the early stage body weight of ox, height and point of the buttocks as one.
Table 4PPAR β gene the 71616th polymorphic site and Nanyang cattle, variance analysis between the ox Different Month productive capacity of Jiaxian County
Note: digital shoulder has different alphabetical person (as a and b, A, B etc.) and represents that difference is different significantly (P<0.05), or difference extremely significantly (P<0.01).

Claims (5)

1. a detection method for ox PPAR β gene mononucleotide polymorphism, is characterized in that, to comprise the ox complete genome DNA to be measured of PPAR β gene for template, with primer pair P for primer, and pcr amplification ox PPAR β gene; Then digest pcr amplification product with restriction enzyme Pvu II, the fragment after then cutting enzyme carries out native polyacrylamide gel electrophoresis; Finally according to the single nucleotide polymorphism of polyacrylamide gel electrophoresis result qualification ox PPAR β gene the 71616th; Wherein, described primer pair P is:
Upstream primer P1:5'-TCCTTCCAGCAGCTACACAG ct-3'22bp;
Downstream primer P2:5'-GGGAGACAACTCGCCCAAGA-3'20bp.
2. the detection method of ox PPAR β gene mononucleotide polymorphism according to claim 1, is characterized in that, described pcr amplification reaction program is 95 DEG C of denaturation 10min; 94 DEG C of sex change 30 ~ 50s, 63 DEG C of annealing 30s, 72 DEG C extend 30 ~ 50s, 30 ~ 40 circulations; 72 DEG C extend 10min; 4 DEG C of preservations.
3. the detection method of ox PPAR β gene mononucleotide polymorphism according to claim 1, is characterized in that, described polyacrylamide gel electrophoresis select mass concentration be 12% polyacrylamide gel.
4. the detection method of ox PPAR β gene mononucleotide polymorphism according to claim 1, it is characterized in that, describedly identify that the single nucleotide polymorphism of ox PPAR β gene the 71616th is according to polyacrylamide gel electrophoresis result: AG genotypic expression is 167bp, 147bp, 28bp and 20bp tetra-band; GG genotypic expression is 147bp, 28bp and 20bp tri-band; Without AA genotype.
5., based on a molecular breeding method for ox PPAR β gene mononucleotide polymorphism, it is characterized in that, comprise the following steps:
(1) detection method of ox PPAR β gene mononucleotide polymorphism according to claims 1 to 4, determines the genotype of ox PPAR β gene the 71616th single nucleotide polymorphism;
(2) association analysis is carried out by the genotype of ox PPAR β gene the 71616th single nucleotide polymorphism and ox growth traits, determine that the single nucleotide polymorphism of ox PPAR β gene the 71616th can as the molecule marker improving ox early growth traits, for the molecular breeding of ox;
(3) in the single nucleotide polymorphism of ox PPAR β gene the 71616th, GG genotype can be used as the candidate molecules genetic marker improving ox early growth traits.
CN201610007765.7A 2016-01-07 2016-01-07 Detection method and molecular breeding method for single nucleotide polymorphism of cattle PPAR β gene Expired - Fee Related CN105543362B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201610007765.7A CN105543362B (en) 2016-01-07 2016-01-07 Detection method and molecular breeding method for single nucleotide polymorphism of cattle PPAR β gene

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201610007765.7A CN105543362B (en) 2016-01-07 2016-01-07 Detection method and molecular breeding method for single nucleotide polymorphism of cattle PPAR β gene

Publications (2)

Publication Number Publication Date
CN105543362A true CN105543362A (en) 2016-05-04
CN105543362B CN105543362B (en) 2020-04-03

Family

ID=55822919

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201610007765.7A Expired - Fee Related CN105543362B (en) 2016-01-07 2016-01-07 Detection method and molecular breeding method for single nucleotide polymorphism of cattle PPAR β gene

Country Status (1)

Country Link
CN (1) CN105543362B (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106636429A (en) * 2017-01-23 2017-05-10 西北农林科技大学 Tetra-primer amplification refractory mutation system-PCR (polymerase chain reaction) method for detecting cattle ADNCR gene single nucleotide polymorphism and application of tetra-primer amplification refractory mutation system-PCR method
CN106947826A (en) * 2017-04-27 2017-07-14 西北农林科技大学 A kind of method for detecting ox SERPINA3 gene mononucleotide polymorphisms and its application

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104278083A (en) * 2014-07-23 2015-01-14 信阳师范学院 Method for detecting single nucleotide polymorphisms of cattle 17HSDB8 gene

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104278083A (en) * 2014-07-23 2015-01-14 信阳师范学院 Method for detecting single nucleotide polymorphisms of cattle 17HSDB8 gene

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ANTHONY G DORAN等: "Whole genome association study identifies regions of the bovine genome and biological pathways involved in carcass trait performance in Holstein-Friesian cattle", 《BMC GENOMICS》 *
樊月圆: "肉牛PPARs家族和PLIN基因遗传变异及其与秦川牛胴体、肉质性状相关分析", 《中国博士学位论文全文数据库农业科技辑》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106636429A (en) * 2017-01-23 2017-05-10 西北农林科技大学 Tetra-primer amplification refractory mutation system-PCR (polymerase chain reaction) method for detecting cattle ADNCR gene single nucleotide polymorphism and application of tetra-primer amplification refractory mutation system-PCR method
CN106636429B (en) * 2017-01-23 2019-12-13 西北农林科技大学 Four-primer amplification hindered mutation system PCR detection method for single nucleotide polymorphism of cattle ADNCR gene and application thereof
CN106947826A (en) * 2017-04-27 2017-07-14 西北农林科技大学 A kind of method for detecting ox SERPINA3 gene mononucleotide polymorphisms and its application

Also Published As

Publication number Publication date
CN105543362B (en) 2020-04-03

Similar Documents

Publication Publication Date Title
CN105624314A (en) Method for detecting goat TMEM95 gene subtle copy number variation through PCR technology and application thereof
CN101921856B (en) Method for detecting cattle ANGPTL4 gene single nucleotide polymorphism
CN103290123B (en) Detecting method and kit of cattle IGF2 (Insulin-like Growth Factor 2) gene mononucleotide polymorphism
CN103233001B (en) The detection method of Qinchuan Cattle FoxO1 gene mononucleotide polymorphism molecular marker and application
CN101921852B (en) Method for detecting single nucleotide polymorphism of cattle AdPLA gene
CN101671726B (en) Method for detecting single nucleotide polymorphism (SNP) of ox PRDM16 gene
CN101921848B (en) Method for detecting single nucleotide polymorphism (SNP) of cattle MGAT2 gene
CN105543362A (en) Detection method for single nucleotide polymorphism of cattle PPARbeta gene and molecular breeding method
CN102296110A (en) Method for detecting mononucleotide polymorphism of FGF 21 gene of yellow cattle
CN101921857A (en) PCR-RFLP test method for mononucleotide polymorphism of Chinese local cattle Pax7 gene
CN103320429A (en) Method for detecting Qinchuan cattle Wnt7a gene single nucleotide polymorphism, and application thereof
CN105671189A (en) Molecular breeding method based on single nucleotide polymorphism of cattle Angpt18 genes
CN103290113B (en) A kind of RFLP method of fast detecting ox SIRT1 gene SNP
CN103695416B (en) A kind of method and its application for the SNP for detecting Qinchuan Cattle CFL2 genes
CN104278083B (en) A kind of method detecting ox 17HSDB8 gene mononucleotide polymorphism
CN104498610A (en) RFLP method and kit for detecting SNP locus of MEF2C gene of cattle
CN103789407A (en) Method for quickly detecting polymorphism of cattle APOA2 gene and application thereof
CN102649962B (en) The mononucleotide polymorphism site of cattle WNT10B gene and detection method thereof
CN101875977B (en) Method for detecting mononucleotide polymorphism of scalper SREBP1c gene
CN106011273A (en) Method for detecting Zaosheng cattle GH gene mononucleotide polymorphism
CN106755422A (en) A kind of detection method of MEG3 gene SNP related to ox growth traits and its application
CN102747150B (en) Method for detecting body sizes of Qinchuan cattle through using ANAPC13 gene
CN102094081A (en) Method for detecting single nucleotide polymorphism of cattle SH2B1 gene
CN107130037B (en) Method and special kit for auxiliary detection of cattle growth and carcass traits through TNNI1 gene
CN105463104A (en) Method for detecting single nucleotide polymorphism of PPAR alpha genes of cattle

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant
CF01 Termination of patent right due to non-payment of annual fee

Granted publication date: 20200403

CF01 Termination of patent right due to non-payment of annual fee