CN105543362B - Detection method and molecular breeding method for single nucleotide polymorphism of cattle PPAR β gene - Google Patents

Abstract

The invention relates to a method for detecting single nucleotide polymorphism of a cattle PPAR β gene, which takes the whole genome DNA of the cattle to be detected containing a PPAR β gene as a template, takes a primer pair P as a primer, amplifies the cattle PPAR β gene by PCR, then uses restriction endonuclease PvuII to digest the PCR amplification product, then carries out non-denaturing polyacrylamide gel electrophoresis on the enzyme-cut fragment, and finally identifies the single nucleotide polymorphism of 71616 th site of the cattle PPAR β gene according to the electrophoresis result.

Description

Detection method and molecular breeding method for single nucleotide polymorphism of cattle PPAR β gene

Technical Field

The invention relates to a method for detecting single nucleotide polymorphism of a gene and a molecular breeding method, in particular to a method for detecting single nucleotide polymorphism of a cattle PPAR β gene and a molecular breeding method.

Background

Single Nucleotide Polymorphism (SNP) mainly refers to a Polymorphism of a DNA sequence caused by variation of a Single Nucleotide at a specific site on the genome level. The polymorphism exhibited by SNPs is caused by a single base transition or transversion, and there is a possibility that any base may be mutated in genomic DNA. Thus, SNPs may be present in both gene sequences and non-coding sequences. SNPs occur most frequently in CG-rich sequences and are mostly C-cytosine to T-thymine, since C in CG deaminates spontaneously to thymine upon methylation.

The SNP as a third generation molecular genetic marker has the following characteristics: (1) the density is high. SNPs are the most ubiquitous and frequent genetic markers in genomic DNA. The research shows that the occurrence frequency of SNP is 1/1000-10/1000, the total SNPs of more than 300 ten thousand in 30 hundred million bases of human beings have higher density than the microsatellite DNA marker. (2) And automatic analysis is easy to realize. The DNA consists of 4 bases, but the SNP generally consists of only two bases, so that the SNP marker is a biallelic marker which is not the same when in detection, the length of the fragment is not analyzed when in genome screening, and +/-analysis is often only needed, thereby being beneficial to realizing the SNPs screened and detected by an automatic technology. (3) Has wide application. SNPs serve as one of molecular genetic markers and have a wide range of functions in animal genetic breeding. The method is mainly used for researches such as genetic mapping, disease detection, site identification, evolution analysis and the like.

Currently, methods for detecting SNPs can be divided into two broad categories: one is a traditional method based on gel electrophoresis, which mainly comprises a PCR-SSCP and DNA sequencing combined method, a PCR-RFLP method, an AS-PCR method, denaturing gradient gel electrophoresis and the like. Among them, the PCR-SSCP experiment process is relatively long, the operation is relatively complicated, and only suitable for small DNA fragments with length less than 500bp, if the DNA fragments are too long, the DNA single strand is difficult to form a stable hairpin structure, and the experiment process has false negative problem, so the method is not an ideal SNP detection means. The AS-PCR method needs to design special primers and can only aim at specific gene loci, but is not suitable for the detection of high-throughput SNP due to poor stability of the specific primers, strict requirements on amplification conditions and complicated operation. Denaturing gradient gel electrophoresis also allows only a relatively coarse detection of mutations and does not allow the determination of mutation location and type. PCR-RFLP has high site specificity, simple operation, low cost and other advantages, and is widely used in plant genotyping, gene mapping, molecular identification and other research. The other is to use a high-throughput, highly automated detection method. High throughput detection methods include direct sequencing, DNA chips, denaturing high performance liquid chromatography, and the like. Wherein, direct sequencing is the most direct and reliable method for detecting SNP, and the DNA chip can screen SNP in large scale, but the detection cost of both is extremely expensive; the denaturing high performance liquid chromatography method has high requirements on reagents and environment, and can not detect homozygous mutation, so the method is not the first choice for detecting SNP. As described above, the PCR-RFLP method is probably the most ideal genetic marker method for detecting SNP at present.

Cattle are one of main livestock breeding resources in China, but the cattle face the pressure of insufficient provenance and poor population genetic quality of excellent cattle varieties. Traditional breeding methods are based on determining the phenotype of an animal and using the genetic information of its parents, progenitors and other relatives in animal models to perform genetic evaluation, typically assuming that the trait is affected by many genetic differences of the genes, and therefore considering that each gene has a relatively small contribution to the trait, the result is an inevitable bias in the estimation of the trait. Molecular genetic markers are one of the fastest-developing fields of modern genetics in recent years, and with the continuous emergence of new genomics methods and new technologies, new livestock and poultry varieties can be reconstructed by integrating various technologies and designing on the gene level, namely the molecular level. Based on the generation and application of DNA genetic markers, a new field of molecular breeding is created, a new technical support is provided for animal breeding and improvement, and a good prospect is brought to animal genetic breeding.

The PPAR family can be divided into three members, namely PPAR α, β and gamma, PPAR β widely exists in animals and is highly expressed in tissues with active lipid metabolism, such as heart, fat cell, skeletal muscle and the like, at present, a large amount of research proves that PPAR β plays an important role in the differentiation of fat cells and the metabolism of fatty acid, the research of PPAR β gene is mostly carried out on model animals such as human and mice, but few reports are carried out on cattle, the research of genetic variation of cattle in China has no literature reports, therefore, the development of a simple, quick, low-cost and accurate detection method of PPAR β gene mononucleotide polymorphism of cattle PPAR 35 β gene is urgently needed, the research of PPAR β gene genetic variation and the genetic variation of cattle in China has no literature reports, and the research of genetic variation of PPAR gene β gene and the genetic variation of cattle in China has a wide correlation with the theoretical genetic weight gain of cattle, and the theoretical growth of cattle, and the establishment of cattle growth and the early-stage genetic variation of cattle, and the research of cattle with high yield and high yield of cattle.

Disclosure of Invention

The invention aims to solve the technical problem of providing a simple, quick, low-cost and accurate detection method and a molecular breeding method for single nucleotide polymorphism of PPAR β gene of cattle, searching SNP (single nucleotide polymorphism) associated with economic traits as a molecular marker, and accelerating the establishment of cattle population with high-quality economic traits.

The technical scheme adopted for solving the technical problems is that a method for detecting single nucleotide polymorphism of PPAR β gene of cattle is characterized by taking whole genome DNA of cattle to be detected containing PPAR β gene as a template and a primer pair P (containing P1 and P2) as primers, carrying out PCR amplification on the PPAR β gene of cattle, then digesting the PCR amplification product by using restriction enzyme Pvu II, carrying out non-denaturing polyacrylamide gel electrophoresis on the amplified fragment after enzyme digestion, and finally identifying the single nucleotide polymorphism of 71616 th site of the PPAR β gene of cattle according to the result of the polyacrylamide gel electrophoresis, wherein the primer pair P is as follows:

upstream primer P1: 5' -TCCTTCCAGCAGCTACACAGCT-3'22bp, SEQ ID NO: 1;

the downstream primer P2: 5'-GGGAGACAACTCGCCCAAGA-3'20bp, see SEQ ID NO: 2.

Note: the "_" in the primer indicates a mismatched base introduced to form a cleavage site.

Further, the PCR amplification reaction program is as follows: pre-denaturation at 95 ℃ for 10 min; denaturation at 94 ℃ for 30-50 s, annealing at 63 ℃ for 30s, extension at 72 ℃ for 30-50 s, and 30-40 cycles; extending for 10min at 72 ℃; storing at 4 ℃.

Further, the polyacrylamide gel electrophoresis is polyacrylamide gel with the mass concentration of 12%.

Furthermore, the single nucleotide polymorphism of 71616 bit of the cattle PPAR β gene is that the genotype of AG (heterozygous type) shows four bands of 167bp, 147bp, 28bp and 20bp, the genotype of GG (mutant type) shows three bands of 147bp, 28bp and 20bp, and the genotype of AA (normal type) is absent.

The invention further solves the technical problem by adopting the technical scheme that the molecular breeding method based on the single nucleotide polymorphism of the cattle PPAR β gene comprises the following steps:

(1) the method for detecting the single nucleotide polymorphism of the cattle PPAR β gene according to claims 1 to 4, wherein the genotype of the 71616 th single nucleotide polymorphism of the cattle PPAR β gene is determined;

(2) through correlation analysis of the genotype of the 71616 site single nucleotide polymorphism of the cattle PPAR β gene and the growth traits of cattle, the 71616 site single nucleotide polymorphism of the cattle PPAR β gene is determined to be a molecular marker for improving the early growth traits of cattle and is used for molecular breeding of cattle;

(3) the GG genotype in the 71616 single nucleotide polymorphism of the cattle PPAR β gene can be used as a candidate molecular genetic marker for improving the early growth traits of cattle.

The invention designs primers according to the sequence of the published cattle PPAR β gene (NCBI: AC _000180.1), respectively takes the genome DNA of 5 cattle varieties as templates to carry out PCR amplification, sequences the PCR products, compares the partial sequence of the cattle PPAR β gene obtained after the sequencing with the sequence published by the NCBI,PCR amplification of PPAR β gene product, the mutation has no natural enzyme cutting site, one mismatched base needs to be introduced into the primer to form enzyme cutting site CAGCTG of PvuII at the mutation site, PCR amplified fragment size is 195bp, and other enzyme cutting site of PvuII exists besides SNP site, therefore, even normal genotype can be cut into two bands of 167bp and 28 bp. in the cutting process, when A occurs at 71616 site, SNP polymorphism of PPAR β gene is detected by enzyme cutting identification with specific restriction enzyme after PCR amplification>When G is mutated, the 71611 bp-71616 bp of PCR amplified PPAR β gene is CAGCTGWhen the protein is used, the protein is identified by PvuII enzyme; when position 71616 does not occur>G mutation, when the 71611 bp-71616 bp of the PCR amplified PPAR β gene is CAGCTA, the PCR amplified PPAR β gene is not recognized by PvuII enzyme, so that the SNP polymorphism of the site can be detected.

The invention determines the genotype of 71616 th single nucleotide polymorphism of the PPAR β gene of cattle by a PPAR β gene SNP detection method, further performs correlation analysis on partial growth traits (height, weight, oblique length and the like) of cattle, determines that the site can be used as a molecular marker for improving the early growth trait of cattle, is convenient for Marker Assisted Selection (MAS) of the growth trait for Chinese cattle meat, and quickly establishes a cattle population with excellent genetic resources.

Drawings

FIG. 1 is an electrophoresis test of 195bp PCR products of cattle PPAR β gene containing 71616 th polymorphic site, wherein lanes 1-5 are agarose electrophoresis test of 195bp fragments of PPAR β gene containing 71616 th polymorphic site, and lane M is Marker DL1000(1000bp,700bp,500bp,400bp,300bp,200bp,100 bp).

FIG. 2(a) is a PvuII PCR-RFLP method for detecting 71616 th SNP enzyme cutting electrophoresis of cattle PPAR β gene, wherein, Lane 1 is DL500(500bp, 400bp,300bp,200bp, 150bp, 100bp, 50bp), Lane 2-4 is AG genotype individual (167bp, 147bp, 28bp, 20bp), Lane 5 is GG genotype individual (147bp, 28bp, 20bp), and AA-free genotype individual (b) is a reverse sequencing diagram of cattle PPAR β gene 71616 site AG and GG genotype individual.

FIG. 3 is a diagram showing the sequence analysis of the SNP (AC _000180.1_ g.71616A > G) of the second intron of the PPAR β gene of cattle detected by PvuII PCR-RFLP method (wherein the boxes respectively represent the upstream and downstream primer sequences, and the site with single letter plus blue background represents the SNP site).

FIG. 4 shows a 195bp fragment of the PPAR β gene from 71594bp to 71788 bp.

Detailed Description

The invention is further illustrated by the following examples and figures.

1. Biochemical and biological reagents: golden DNA polymerase (available from beijing tiangen biochemistry technologies, ltd); 2 × Reaction Mix contains Mg2+, dNTPs, etc. (available from Mix, Biotechnology, Inc., Beijing Tian, N.C.); restriction enzyme PvuII (available from TAKARA); proteinase K (available from huamei bioengineering); marker (DL500, DL1000) from Dalibao bioengineering, Inc.

2. General reagents: purchased from huamei bioengineering, inc, as an imported split-filled product: citric acid, sodium citrate, glucose, Tris, acrylamide, methylene bisacrylamide, TEMED, ammonium persulfate, EDTA, NaCl, NaOH, KCl, Na₂HPO₄、KH₂PO₄Tris-saturated phenol, chloroform, isoamyl alcohol, absolute ethyl alcohol, sodium acetate, Sodium Dodecyl Sulfate (SDS), Ethidium Bromide (EB), bromophenol blue, acetic acid, sucrose, boric acid, agarose, and the like.

3. Solution and buffer: all solutions and buffers were prepared with deionized ultrapure water under autoclaving conditions of 15bf/in (1.034X 10)⁵Pa), 25 min; the reagents were prepared according to the molecular cloning protocol described in Sambrook et al.

(1) Solutions for sample collection

Anticoagulant ACD: 2.4g of citric acid; trisodium citrate 6.6 g; glucose 7.35g, constant volume to 50mL ddH₂And O, autoclaving. 0.2mL of ACD solution was added to each 10mL of fresh blood. This anticoagulant is superior to heparin and is used during blood storageCan better preserve the DNA of the macromolecule. The anticoagulated blood can be preserved at 0 deg.C for several days or at-80 deg.C for a long period.

(2) Solution for separating genomic DNA of blood sample

① PBS buffer solution of NaCl 8g, KCl 0.2g, Na₂HPO₄1.44g，KH₂PO₄0.24g, adding ultrapure water to 1000mL, adjusting pH to 7.4, and autoclaving.

② 10% SDS 10g SDS was dissolved in 90mL ultrapure water, dissolved in a 68 ℃ water bath, adjusted to pH 7.2 with HCl, and made to volume of 100 mL.

③ 0.5.5 mol/L EDTA, 186.1g EDTA, dissolved in 800mL ultrapure water, adjusted to pH 8.0 with NaOH, made to volume of 1000mL, autoclaved, stored at 4 ℃.

④ 1mol/L Tris & Cl 121.14g Tris dissolved in 800mL ultrapure water, HCl adjusted pH to 8.0, constant volume to 1000mL, autoclaving, and storing at 4 ℃.

⑤ 5mol/L NaCl 292.2g was dissolved in 1000mL of ultrapure water.

⑥ DNA extraction buffer solution, 0.5mmol/L EDTA 40mL, 1mmol/L Tris. Cl 10 mL.

⑦ 5mmol/L NaCl 4mL, 10% SDS 10mL to 100mL, actual concentration 200mmol/L EDTA, pH 8.0: 100mmol/L Tris & HCl, pH 8.0, 200mmol/L NaCl, 2% SDS. RNase 20. mu.g/mL.

⑧ NaAc buffer solution NaAc 3H₂20.4g of O; 40mL of ultrapure water; adjusting the pH value of the diluted HAc to 7.4; the volume is up to 50 mL.

⑨ TE buffer Tris-Cl buffer (pH 8.0)10mmol/L, EDTA buffer (pH 8.0)0.1mmol/L, autoclaving, and storing at 4 deg.C.

⑩ protease K, 20mg/mL with ultrapure water, and storing at-20 deg.C.

(3) Solutions for polyacrylamide gel electrophoresis analysis

① 1 XTBE buffer 10 XTBE 100mL was taken and the volume was adjusted to 1000 mL.

② Loading buffer 0.25% bromophenol blue, 0.25% xylene blue FF, 40.0% (w/v) sucrose in water.

Example 1 method for detecting Single nucleotide polymorphism of cattle PPAR β Gene

Design of cattle PPAR β gene PCR primer

A195 bp gene fragment of 71594 bp-71788 bp of a PPAR β gene is shown in FIG. 4 and SEQ ID NO:5 by taking a bovine PPAR β (AC _000180.1) sequence published by NCBI as a reference, and a PCR Primer capable of amplifying a 71616 region of a bovine PPAR β gene is designed by using Primer 5.0, wherein the Primer sequence is as follows:

p3 (upstream primer): 5'-TCCTGTCTTCCCTTTCGTCC-3'20bp as shown in SEQ ID No. 3;

p4 (downstream primer): 5'-GGAGACAACTCGCCCAAGAT-3'20bp as shown in SEQ ID No. 4;

PCR amplification is carried out on the cattle genome by adopting primers P3 and P4, a 427bp gene fragment containing 71616 th site of cattle PPAR β gene (AC _000180.1 sequence) can be amplified, and after sequencing and identification are carried out on the amplified fragment, the 71616 th site of the PPAR β gene is analyzed to find that SNP polymorphism exists.

Because the mutation position has no natural enzyme cutting site, the enzyme cutting primer needs to be redesigned, and a mismatched base is introduced on the primer, so that the enzyme cutting site of the restriction enzyme PvuII can be formed at the mutation position. The primer pair P sequences redesigned are as follows:

p1 (upstream primer): 5' -TCCTTCCAGCAGCTACACAGCT-3'22bp

P2 (downstream primer): 5'-GGGAGACAACTCGCCCAAGA-3'20bp

Note: the "_" in the primer indicates a mismatched base introduced to form a cleavage site.

When PPAR β gene product is amplified by PCR, the mutation has no natural restriction site, one mismatched base needs to be introduced into the primer to form the restriction site CAGCTG of PvuII at the mutation site, PCR amplified fragment has 195bp, and other restriction site of PvuII exists besides SNP site, therefore, even normal genotype can be cut into two bands of 167bp and 28 bp. in the cutting process, when A occurs at 71616 site, A occurs>When G is mutated, the 71611 bp-71616 bp of PCR amplified PPAR β gene is CAGCTGThen, it is recognized by PvuII enzyme. When position 71616 does not occur>G mutation, PCR amplification of 71611 bp-71616 bp of PPAR β geneWhen CAGCTA is adopted, the SNP polymorphism at the site can be detected without being recognized by PvuII enzyme.

Secondly, carrying out PCR amplification on the to-be-detected cattle PPAR β gene fragment by using the primer pair P

1. And (3) collection of cattle samples: 454 individuals in total of 5 cattle breeds are used as detection objects, and specific collected samples are shown in table 1: shaanxi Qin Sichuan cattle (QC, 30), Henan Pingting mountain Jiaxian county red cattle (JX, 141), Henan south Yang cattle (NY, 139), Shandong province Shandong Wen cattle (LX, 114), and Shandong province Bohai Black cattle (BH, 30).

TABLE 1 cattle sample Collection

2. Separation, extraction and purification of blood sample genome DNA

(1) Thawing frozen blood sample (mainly blood cells) at room temperature, transferring 500 mu L to a 1.5mL Eppendorf centrifuge tube, adding equal volume of PBS (phosphate buffer solution) to the tube, fully mixing the solution, centrifuging the solution at 12000r/min for 10min (4 ℃), discarding supernatant, and repeating the steps until the supernatant is transparent and the precipitate is light yellow;

(2) adding 500 mu L of DNA extraction buffer solution into a centrifuge tube, shaking to separate the blood cell sediment from the tube wall of the centrifuge tube, and carrying out water bath at 37 ℃ for 1 h;

(3) adding protease K to 3 μ L (20mg/mL), mixing, standing overnight at 55 deg.C until the mixture is clear, adding 1 μ L protease K, mixing, and digesting until the mixture is clear;

(4) cooling the reaction solution to room temperature, adding 500 mu L of Tris-saturated phenol, and gently shaking the centrifuge tube for 20min to fully mix the Tris-saturated phenol and the centrifuge tube; centrifuging at 4 ℃ at 12000r/min for 10min, transferring the supernatant into another 1.5mL centrifuge tube, and repeating once;

(5) adding 500 μ L chloroform, mixing well for 20min, centrifuging at 4 deg.C and 12000r/min for 10min, and transferring the supernatant into another 1.5mL centrifuge tube;

(6) adding 500 μ L of chloroform and isoamylol mixture (24:1), mixing well for 20min, centrifuging at 4 deg.C and 12000r/min for 10min, transferring the supernatant into another 1.5mL centrifuge tube;

(7) adding 0.1 time volume of NaAc buffer solution and 2 times volume of ice-cold absolute ethyl alcohol, mixing, rotating the centrifuge tube until white flocculent precipitate is separated out, and preserving at-20 ℃ for 30-60 min;

(8) centrifuging at 4 deg.C and 12000r/min for 10min, discarding supernatant, rinsing DNA precipitate with 70% ice cold ethanol for 2 times;

(9) centrifuging at 4 deg.C and 12000r/min for 10min, discarding supernatant, and volatilizing ethanol at room temperature;

(10) dissolving the dried DNA in 80-100 mu L of TE solution, storing at 4 ℃ until the DNA is completely dissolved, detecting the quality of the DNA by 0.8% polyacrylamide gel electrophoresis, and storing at-80 ℃.

(11) Adding 10% SDS into 500. mu.L DNA solution to make its final concentration be 0.1%, adding proteinase K to make its final concentration be 50. mu.g/mL;

(12) keeping the temperature at 5 ℃ for about 10 h;

(13) respectively extracting with equal volume of phenol, chloroform, isoamyl alcohol (25:24:1) and chloroform;

(14) centrifuging at 12000r/min for 5min, and absorbing the upper water phase into another centrifuge tube;

(15) adding 1/10 volumes of 3mol/L sodium acetate and 2 times volumes of ice-cooled absolute ethyl alcohol to precipitate DNA;

(16) pouring out the liquid, washing with 70% ethanol, air drying, adding 60 μ L sterilized ultrapure water for dissolving, measuring the concentration by ultraviolet spectrophotometry, and diluting into 50ng/μ L working solution.

3. PCR amplification

The PCR reaction system adopts a mixed sample adding method, namely the total amount of various reaction components is calculated according to the quantity of various components required by each reaction system and the quantity of PCR reactions required by 1 reaction, the reaction components are added into a 1.5mL centrifuge tube, the mixture is instantly centrifuged after being evenly mixed, the mixture is subpackaged into each 0.2mL Eppendorf PCR tube, then template DNA is added, and PCR amplification is carried out after the instant centrifugation. The PCR reaction system is shown in Table 2.

TABLE 2 PCR reaction System

Component Reaction Components	Volume of usage
		2×Reaction Mix	7.5
Sense Primer(10μM)	0.1
		Anti-sense Primer(10μM)	0.1
Golden DNA polymerase	0.1
		DNA template	0.5
ddH2O	6.7
		Total	15

The 15. mu.L Reaction system included Golden DNA polymerase 0.1. mu.L (Beijing Tiangen science and technology Co., Ltd.), 2 × Reaction Mix 7.5. mu.L (including Mg)²⁺dNTPs, etc.) (Mix, Beijing Tiangen science and technology Co., Ltd.), 50 ng/. mu.L of cattle genomic DNA containing PPAR β gene 0.5. mu.L, 10 pmol/. mu.L of each of the upstream and downstream primers 0.1. mu.L.

PCR amplification procedure: pre-denaturation at 95 ℃ for 10 min; denaturation at 94 ℃ for 30-50 s, annealing at 63 ℃ for 30s, extension at 72 ℃ for 30-50 s, and 30-40 cycles; final extension at 72 deg.C for 10 min; storing at 4 ℃.

The genomic DNA of 454 samples of 5 cattle breeds is amplified by PCR, and 195bp DNA fragments containing the SNP sites in the cattle PPAR β genes of 454 individuals are obtained.

Third, PvuII enzyme digestion PCR amplified PPAR β gene fragment

1. PvuII digestion system (20. mu.L): mu.L of PCR product, 2.0. mu.L of 10 XM buffer, 0.3. mu.L of PvuII (10U/. mu.L), sterilized ultrapure water (H)₂O)9.7μL。

2. Digestion conditions of enzyme digestion: digesting for 10-12 h in a constant temperature incubator at 37 ℃.

Gel electrophoresis analysis of tetra (polyacrylamide)

1. Preparing 12.0% polyacrylamide gel, performing 250V pre-electrophoresis for 10min, performing electrophoresis at 200V for 2h after sample application, and dyeing with silver nitrate after electrophoresis is finished;

2. analysis of SNP polymorphism based on Polyacrylamide gel electrophoresis results

The polymorphism of SNP was judged according to the silver staining results, as shown in FIG. 3. When the gene product is amplified by PCR, when the 71616 th site has A > G mutation, the A > G mutation is recognized by PvuII enzyme for cutting; when the site is not mutated to A > G, it cannot be recognized by the restriction enzyme PvuII, and the SNP polymorphism at the site can be detected.

A PvuII PCR-RFLP method is used for detecting a 71616 th SNP enzyme cutting electrophoresis picture of a cattle PPAR β gene, as shown in a picture 2(a), the polyacrylamide gel electrophoresis result of the 71616 th SNP polymorphism of the cattle PPAR β gene of a cattle genome is that an AG (heterozygous type) genotype shows four bands of 167bp, 147bp, 28bp and 20bp, a GG (mutant type) genotype shows three bands of 147bp, 28bp and 20bp, and no AA (normal type) genotype exists, as can be known from the picture 2 (a).

3. Sequencing verification of PCR products of individuals with different genotypes

Because the introduced mismatched bases are positioned in the forward primer, the PCR products of individuals with different genotypes are subjected to reverse sequencing; meanwhile, SNP position analysis is performed. The results showed that the sequencing pattern at position 71616 of the individual heterozygote AG genotype containing the 167bp, 147bp, 28bp and 20bp bands is indeed represented as A or G, as shown in FIG. 2(b), while the sequencing pattern is G, and the sequencing pattern is a single peak.

Statistical analysis of frequency of SNP sites of cattle PPAR β genes

1. Gene and genotype frequency

Genotype frequency in population genetics means in onePopulationThe percentage of a certain genotype in the gene.

P_AA＝N_AA/N

In the formula, P_AAFrequency of AA genotype at one SNP site; n is a radical of_AARepresenting the number of individuals possessing the AA genotype; n is the total number of the whole population.

The frequency of a gene is that a certain gene is in a certain positionPopulationThe ratio of the number of gene individuals to the total number of genes is shown in (1).

P_A＝(2N_AA+N_Aa1+N_Aa2+N_Aa3+……N_Aai+……+N_Aan)/2N

In the formula, P_AIndicates the frequency of allele A, N_AAIndicates the number of AA genotypes possessed in the population, N_AaiRepresenting the number of individuals possessing the Aai genotype; a is₁-a_nRepresents n different multiple alleles of allele a.

The allele involved in the invention is G and A, so the specific gene frequency calculation formula is as follows:

P_G＝(2N_GG+N_AG)/2N

P_A＝(2N_AA+N_AG)/2N

in the formula, P_G，P_AIndicates the frequency of the alleles G and A, respectively, N_GG、N_AGAnd N_AAThe numbers of individuals for the GG, AG and AA genotypes, respectively, are indicated, and N is the total population number.

The gene frequency distribution is shown in Table 3, and it can be seen from Table 3 that the variation range of the G allele frequency in the SNP of the PPAR β gene of different cattle varieties is 65-84.2%, and the variation range of the A allele frequency is 15.8-35%, which is in accordance with the definition of the SNP with the allele frequency of more than 1.0%, so that the locus is a single nucleotide polymorphic locus.

TABLE 3 frequency distribution Table of PvuII PCR-RFLP at 71616 th SNP site of cattle PPAR β gene

Example 2 molecular Breeding method based on Single nucleotide polymorphism of cattle PPAR β Gene

Genotype data: PvuII-recognized genotype (AG and GG)

Production data: the birth weight of red cattle in Nanyang and Jiaxian county, and the body weight, height, body slant length, chest circumference, width of ischium, and daily gain of 6-month-old, 12-month-old, 18-month-old and 24-month-old.

And (3) correlation analysis model: firstly, performing description analysis on data to determine whether an outlier exists, and then correcting the data by using least square analysis; from the data characteristics, SPSS (17.0) software was used to analyze the effect of production traits between genotypes. A fixed model was used in the analysis of genotype effects:

Y_ijkl＝μ+BF_i+Month_j+G_k+e_ijkl

wherein: y is_ijklFor trait observations, μ is the overall mean, BF_iFor the fixed effect of the ith breed and farm, Month_jFixed effect for month j observation, G_kFor the fixed effect of the kth single SNP marker genotype, e_ijklIs a random error.

The results of the analysis of variance between the 71616 polymorphic site of the PPAR β gene and production indices of Nanyang cattle and Jiaxian cattle at different ages in month are shown in Table 4. from Table 4, it is clear that the individual body weight of the GG genotype is significantly higher than that of the AG genotype (P <0.01) at 0 day age, 6 month age and 24 months age, the individual body height of the GG genotype is significantly higher than that of the AG genotype (P <0.05) at 6 month age and 24 months age, and the individual body height of the GG genotype is significantly higher than that of the AG genotype (P <0.01) at 18 months age, and the hip end width of the GG genotype is significantly higher than that of the AG genotype (P <0.01) at 0 day age, 6 months age, 12 months age, 18 months age and 24 months age.

The correlation analysis result shows that the GG genotype can be used as a candidate molecular genetic marker for improving the early weight, the body height and the width of the ischium of the cattle.

TABLE 4 analysis of variance between the 71616 polymorphic site of PPAR β gene and the production index of Nanyang cattle, Jiaxian cattle at different ages of the month

Note: those with numeric shoulders with different letters (e.g., a and B, a, B, etc.) indicate that the difference is either very significant (P <0.05), or very significant (P < 0.01).

Claims (2)

1. A method for detecting single nucleotide polymorphism of cattle PPAR β gene is characterized in that whole genome DNA of cattle to be detected containing PPAR β gene is used as a template, a primer pair P is used as a primer, the cattle PPAR β gene is amplified by PCR, then a PCR amplification product is digested by restriction endonuclease PvuII, then non-denaturing polyacrylamide gel electrophoresis is carried out on the digested fragment, and finally single nucleotide polymorphism at 71616 th site of cattle PPAR β gene is identified according to the result of the polyacrylamide gel electrophoresis, wherein the primer pair P is:

upstream primer P1: 5' -TCCTTCCAGCAGCTACACAGCT-3'22bp；

The downstream primer P2: 5'-GGGAGACAACTCGCCCAAGA-3'20 bp;

the PCR amplification reaction program is pre-denaturation at 95 ℃ for 10 min; denaturation at 94 ℃ for 30-50 s, annealing at 63 ℃ for 30s, extension at 72 ℃ for 30-50 s, and 30-40 cycles; extending for 10min at 72 ℃; storing at 4 deg.C;

the polyacrylamide gel electrophoresis selects polyacrylamide gel with the mass concentration of 12%;

the single nucleotide polymorphism of 71616 th site of the cattle PPAR β gene is identified according to the result of polyacrylamide gel electrophoresis, wherein the genotype of AG is represented by four bands of 167bp, 147bp, 28bp and 20bp, the genotype of GG is represented by three bands of 147bp, 28bp and 20bp, and the genotype of GG is free of AA.

2. A molecular breeding method based on single nucleotide polymorphism of cattle PPAR β gene is characterized by comprising the following steps:

(1) the method for detecting the single nucleotide polymorphism of the cattle PPAR β gene according to claim 1, wherein the genotype of the 71616 th single nucleotide polymorphism of the cattle PPAR β gene is determined;

(2) through correlation analysis of the genotype of the 71616 site single nucleotide polymorphism of the cattle PPAR β gene and the growth traits of cattle, the 71616 site single nucleotide polymorphism of the cattle PPAR β gene is determined to be a molecular marker for improving the early growth traits of cattle and is used for molecular breeding of cattle;

(3) the GG genotype in the 71616 single nucleotide polymorphism of the cattle PPAR β gene can be used as a candidate molecular genetic marker for improving the early growth traits of cattle.

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