CN110564867B

CN110564867B - SNP molecular marker of Qinchuan cattle CFL1 gene and detection method thereof

Info

Publication number: CN110564867B
Application number: CN201910957607.1A
Authority: CN
Inventors: 孙雨佳; 徐崇; 杨章平
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 2019-10-10
Filing date: 2019-10-10
Publication date: 2022-06-24
Anticipated expiration: 2039-10-10
Also published as: CN110564867A

Abstract

The invention belongs to the field of molecular genetics, and provides an SNP molecular marker of a Qinchuan cattle CFL1 gene and a detection method thereof, wherein a to-be-detected Qinchuan cattle whole genome DNA containing a CFL1 gene is used as a template, a primer pair P is used as a primer, and the Qinchuan cattle CFL1 gene is amplified by PCR; digesting the PCR amplification product by using restriction enzyme HinfI, and then carrying out agarose gel electrophoresis on the amplified fragment subjected to enzyme digestion; and identifying the base polymorphism of the 2052 th site of the Qinchuan cattle CFL1 gene according to the electrophoresis result. Because the mutation site is closely related to the Qinchuan cattle meat traits (body length, chest width, chest depth and weight), and the method is a method for screening and detecting molecular genetic markers closely related to the Qinchuan cattle growth traits on the DNA level, the method can be used for auxiliary selection and molecular breeding of Qinchuan cattle to accelerate the fine breed breeding speed of Qinchuan cattle.

Description

SNP molecular marker of Qinchuan cattle CFL1 gene and detection method thereof

Technical Field

The invention belongs to the field of molecular genetics, and relates to a SNP molecular marker taking Single Nucleotide Polymorphism (SNP) of a functional gene of Qinchuan cattle as a molecular genetic marker, in particular to an SNP molecular marker of a CFL1 gene of Qinchuan cattle and a detection method thereof.

Background

In beef cattle breeding, people expect to achieve the purposes of early seed selection and improvement of accuracy of breeding values through selection of DNA markers which are closely related to growth traits and closely linked with quantitative traits, thereby obtaining greater genetic progress in livestock breeding.

Molecular Marker Assisted Selection (MAS) technology is used for selecting genetic resources or breeding materials by means of DNA Molecular markers and improving the comprehensive characters of livestock and poultry, and is a method combining modern Molecular biology and traditional genetic breeding to breed new species.

Genetic polymorphism refers to differences in genomic sequence between different species or between individuals within the same species, which are caused by nucleotide changes in DNA alleles in chromosomes, mainly including base substitutions, insertions, deletions, and changes in copy number of repeated sequences.

Single Nucleotide Polymorphism (SNP) is a genetic marker system proposed by the scholars Lander (1996) of the human genome research center of the American college of science and technology, Massachusetts, and refers to a Polymorphism in a genomic DNA sequence caused by the substitution of a Single Nucleotide (A/T/C/G). SNP has been widely used as a new genetic marker for gene mapping, cloning, genetic breeding and diversity studies. SNPs are a very abundant variant form present in the genome, accounting for over 90% of the genetic polymorphisms in the human genome. SNPs are distinct from rare variations, and usually such variations with a frequency of 1% or less in a population are called mutations, while only those with a frequency of more than 1% are called single nucleotide polymorphisms. Its variants are: transversions, transitions, insertions, deletions, and the like, are caused mainly by transitions or transversions of a single base. SNPs having base variations of the transition type account for about 2/3.

Based on the location of the single nucleotide polymorphism in the genome, the following 3 classes can be assigned: coding-region single nucleotide polymorphisms (cSNPs), gene-peripheral single nucleotide polymorphisms (pSNPs), and Intergenic single nucleotide polymorphisms (iSNPs).

Research has shown that the cSNP located in the coding region is relatively small, and the research of the cSNP in the coding region is more concerned because it has important significance in the research of genetic diseases. The cSNP within the coding region of a gene can be divided into 2 types: one is synonymous cSNP (synonymus cSNP) in the coding region, i.e., a change in the coding sequence caused by a SNP does not affect the amino acid sequence of the protein translated by the SNP; another is nonsynonymous cSNP (Non-synnyms cSNP) in the coding region, i.e.a change in the base sequence will result in a change in the encoded amino acid and thus in the amino acid sequence in the protein, possibly ultimately affecting the function of the protein.

Since SNPs are bi-allelic molecular markers, in a diploid organism population, theoretically, SNPs may be composed of 2, 3 or 4 alleles, but in practice, SNPs of 3 or 4 alleles are rare, and thus SNPs are generally referred to simply as bi-allelic molecular markers. Currently, several different routes are mainly used to discover SNPs: namely, a DNA sequencing method, a Polymerase Chain Reaction-Single Strand Conformation Polymorphism (PCR-SSCP) and DNA sequencing combination method, an Allele Specific PCR (AS-PCR) method, a primer extension method, an oligonucleotide ligation Reaction, and the like. Among these SNP detection techniques, DNA sequencing is the most accurate SNP detection method, but it is extremely expensive, requires a large-scale instrument such as a DNA sequencer, and requires highly skilled technicians and experience in the sequencing process, and therefore, is not an ideal SNP detection method for practical use; certainly, the detection cost can be properly reduced by using the PCR-SSCP and DNA sequencing combination method to detect the SNP, but the PCR-SSCP has longer experimental process and more complicated operation, and the experimental process has the problem of false positive, so the method is not an ideal SNP detection means; as a novel SNP detection method, the AS-PCR method has a very wide prospect in the future application field, but the method needs to design a special primer and only can aim at a specific gene locus, and meanwhile, the probability of false detection also exists in the detection process, so that the AS-PCR method does not have the characteristic of universal application at present; the primer extension method and the oligonucleotide ligation reaction technology for detecting SNP sites need detection platforms such as a plate reading instrument, a gene chip, a microsphere array technology and a mass spectrometer, and are not strong in implementability for general molecular laboratories.

The Restriction Fragment Length Polymorphism-Polymerase Chain Reaction (RFLP-PCR) method used for detecting the SNPs is an effective technology for detecting the SNPs, and after the SNP sites are found, an upstream primer and a downstream primer are designed to be cut by Restriction endonuclease, and then agarose and polypropylene gel electrophoresis analysis is carried out, so that the SNP sites can be accurately identified. The RFLP-PCR method not only has the accuracy of the DNA sequencing method, but also overcomes the defects of high cost, complicated operation and false positive, and the detected sequence sites have no special requirements.

Cofilin is a low molecular weight actin-binding protein, with a molecular weight of 20kDa, that regulates actin assembly in vivo, and is a family of proteins widely distributed in various cells in organisms, including muscle cells and non-muscle cells. The CFL1(Cofilin 1) gene is a new member of the Cofilin family of actin-binding proteins and plays an important role in cell migration, proliferation, phagocytosis and the occurrence and development of various cancers. It has been shown that cofilin expressionThe form of the gene is changed correspondingly with the growth and development of muscle cells of healthy mice, namely, the skeletal muscle can detect the expression of the CFL1 gene and the CFL2 gene in the embryonic development period, but as the muscle growth and development progress, when the myogenic cells are completely differentiated to the terminal stage, the CFL2 gene gradually replaces the expression of the CFL1 gene until the CFL1 gene expression disappears and the CFL1 gene gradually replaces the CFL1 gene expression^-/-The knockout embryo is shown as death of the mouse embryo after 9.5 days of development, and the embryo cannot normally survive to complete the development of the embryo. Therefore, the genetic variation or SNP locus of the CFL1 gene plays an important role in various growth and development related traits in animal production practice.

The research on the genetic variation of the CFL1 gene of the animal is mostly found in animals such as human, mice and the like at home and abroad, and the genetic variation or SNP research of the CFL1 gene of large-sized livestock is not reported. Because the research in the genetic variation field of the CFL1 gene of Qinchuan cattle in China is deficient, the functional research of the gene locus and the research of the correlation between the genetic variation of the gene and economic traits (such as meat production, growth and development and the like) become blanks.

Disclosure of Invention

The invention aims to provide a method for detecting single nucleotide polymorphism of a cattle CFL1 gene and application thereof, and detects the process of mRNA stability and positioning change, activity change of cis-form regulatory elements in genes and gene transcription strength change possibly caused by synonymous mutation on a gene locus by utilizing a PCR-RFLP method, so that individuals generating the synonymous mutation are eliminated in advance, and the establishment of cattle populations with high-quality economic characters is accelerated.

The invention is realized by the following technical scheme:

an SNP molecular marker of a Qinchuan cattle CFL1 gene, the nucleotide sequence of the molecular marker is shown as SEQ ID NO.1, the 2052 th site of the sequence from the 5' end is an SNP locus, and the basic group is T or C.

Furthermore, the growth speed of CC genotype individuals marked by the SNP molecular marker is obviously higher than that of TT genotype individuals and TC genotype individuals.

The invention also provides a primer pair, which comprises an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is shown as SEQ ID NO.2, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 3.

The present invention also provides a kit for the above SNP marker, which comprises the primer set according to claim 3.

The invention also provides application of the SNP molecular marker of the Qinchuan cattle CFL1 gene in animal breeding.

The invention also provides application of the SNP molecular marker of the Qinchuan cattle CFL1 gene in identifying animal growth traits.

A detection method of single nucleotide polymorphism of Qinchuan cattle CFL1 gene comprises the following steps:

PCR amplification is carried out on the Qinchuan cattle CFL1 gene by taking the CFL1 gene-containing to-be-detected Qinchuan cattle whole genome DNA as a template and a primer pair P as a primer; digesting the PCR amplification product by using restriction enzyme HinfI, and then carrying out agarose gel electrophoresis on the amplified fragment subjected to enzyme digestion; identifying the single nucleotide polymorphism of 2052 th site of the Qinchuan cattle CFL1 gene according to an electrophoresis result;

the primer pair P is as follows:

an upstream primer: 5'-CTTTTTCTTTGGCTGCTATTC-3'

A downstream primer: 5'-TCTCCTTGCCCTCCTCCAGGATGAG-3'

Further, the PCR amplification reaction program is as follows:

pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 68 ℃ for 30s, and extension at 72 ℃ for 25s for 18 cycles at-1 ℃ per cycle; denaturation at 94 ℃ for 30s, annealing at 50 ℃ for 30s, and extension at 72 ℃ for 25s, for 20 cycles; extension at 72 ℃ for 10 min.

Further, the mass concentration of the agarose gel is 3%.

Furthermore, the 2052 base polymorphism of the CFL1 gene is as follows: the TT type is expressed as follows: 367bp and 28 bp; TC type expression: 395bp, 367bp and 28 bp; type CC shows: 395 bp.

Has the beneficial effects that:

the invention utilizes an RFLP-PCR method to detect the mononucleotide polymorphism which can possibly generate the change of coding protein conformation when the mutation on the 2052 site of the cattle CFL1 gene is mutated from T to C, and asparagine at the 138 site of a peptide chain is subjected to synonymous mutation in the transcription process to change the stability and the positioning of mRNA (messenger ribonucleic acid), influence the activity of cis-form regulatory elements in the gene and change the transcription intensity of the gene. Due to the high conservation of cofilin, the mutation of the CFL1 gene site directly causes the corresponding change of the spatial configuration of the protein coded by the CFL1 gene with important physiological functions, thereby causing the change of the biological functions related to the muscle growth and development.

The invention discloses a nucleotide polymorphism of a functional gene CFL1 related to growth traits of Qinchuan cattle, which can be used as a molecular genetic marker, and utilizes marker locus information and phenotypic information of quantitative traits to more accurately estimate breeding values of individual animals, improve selection efficiency and accelerate breeding progress.

Aiming at the SNP polymorphism of the CFL1 gene, the invention also discloses a detection method thereof, and the single nucleotide polymorphism can be simply, quickly, cheaply and accurately detected by the RFLP-PCR method by designing a specific PCR primer amplification fragment.

The SNP of the CFL1 gene is subjected to genotyping and gene frequency analysis, and is subjected to association analysis with the growth traits of Qinchuan cattle; the result shows that the nucleotide polymorphic site of the CFL1 gene can be a marker for molecular genetic assisted breeding.

The detection method provided by the invention lays a foundation for establishing the relation between the SNP of the CFL1 gene and the growth traits, so that the detection method is conveniently used for marker-assisted selection of the growth traits of the Qinchuan cattle in China and can be used for quickly establishing the Qinchuan cattle population with excellent genetic resources.

Drawings

FIG. 1 is a map of the DNA electrophoresis detection of a Qinchuan cattle blood sample genome;

FIG. 2 is the electrophoresis diagram of 395bp fragment PCR amplified from Qinchuan cattle CFL1 gene;

FIG. 3 is a diagram showing the result of electrophoresis in which CFL1 gene polymorphism is detected by electrophoresis after the 395bp PCR product of exon 2 of the Qinchuan cattle CFL1 gene is cut with the restriction enzyme HinfI; 28bp is smaller, so the gene cannot be seen in agarose gel electrophoresis analysis;

FIG. 4 is a diagram of sequencing peaks of different genotypes of the 2052 SNP of the Qinchuan cattle CFL1 gene.

Detailed Description

In the invention, CFL1 gene conserved sequence design primer pair P is used for amplifying CFL1 gene exon 2 395bp fragment, Qinchuan cattle genome DNA is used as a template for PCR amplification, and the amplified product is sequenced to search the mononucleotide polymorphic site of the amplified fragment; the growth character relevance analysis is carried out aiming at the discovered mononucleotide polymorphic sites, and a detection method is provided, so that the nucleotide polymorphism of the CFL1 gene becomes a molecular genetic marker which can be quickly and conveniently detected, and a basis is provided for accelerating the establishment of Qinchuan cattle population with high-quality economic characters.

a. Detection of Qinchuan cattle CFL1 gene polymorphism

1. Collection and treatment of Qinchuan cattle blood sample

Taking 10mL of Qinchuan cattle blood sample, adding 500 mu L of EDTA (ethylene diamine tetraacetic acid) with the concentration of 0.5mol/L for anticoagulation, slowly reversing for 3 times, and placing into an ice box for preservation at-80 ℃ for later use.

The invention adopts Qinchuan cattle breed, which is shown in the table 1.

TABLE 1 Qinchuan cattle sample source table

2. Extraction of genomic DNA from blood samples

(1) Thawing frozen blood sample at room temperature, transferring 500. mu.L to 1.5mL Eppendorf tube, adding equal volume of PBS buffer solution, mixing well, centrifuging at 12000r/min for 10min (4 ℃), discarding supernatant, repeating the above steps until supernatant is transparent and 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. Preparation of DNA extraction buffer: 0.6057g of Tris, 18.612g of EDTA and 2.5g of SDS were added to 500mL of ultrapure water, sterilized, adjusted to pH 8.0, and stored at 4 ℃ for further use.

(3) Adding protease K3 μ L (20mg/mL) and mixing, standing overnight at 55 deg.C until the mixture is clear, adding protease K1 μ L, mixing, and digesting.

(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; centrifugation was carried out at 12000r/min for 10min at 4 ℃ and the supernatant was transferred to another 1.5mL centrifuge tube and repeated 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 0.1 volume time NaAc buffer solution and 2 volume times ice-cold absolute ethyl alcohol, mixing and rotating the centrifuge tube until white flocculent precipitate is separated out, and preserving at-20 ℃ for 30-60 min.

(7) Centrifugation was carried out at 12000r/min at 4 ℃ for 10min, the supernatant was discarded, and the DNA precipitate was rinsed 2 times with 70% ice-cold ethanol.

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

(9) And dissolving the dried DNA in 80-100 mu L of TE-buffer solution or ultrapure water, preserving at 4 ℃ until the DNA is completely dissolved, detecting the mass of the DNA by 0.8% agarose gel electrophoresis, and preserving at-80 ℃.

(10) To 500. mu.L of the DNA solution, 10% SDS was added to give a final concentration of 0.1%, and proteinase K was added to give a final concentration of 50. mu.g/mL.

(11) Keeping the temperature at 5 ℃ for about 10 h.

(12) Phenol, chloroform, isoamyl alcohol (25: 24: 1) and chloroform were extracted once respectively in equal volumes.

(13) Centrifuging at 12000r/min for 5min, and sucking the upper water phase into another centrifuge tube.

(14) 1/10 volumes of 3mol/L sodium acetate and 2 volumes of ice-cold absolute ethanol were added to precipitate the DNA.

(15) Pouring out the liquid, washing with 70% ethanol, air drying, adding 60 μ L sterilized ultrapure water for dissolving, and detecting at 4 deg.C.

3. Construction of DNA pools

(1) Detection by 1% agarose gel electrophoresis

And (3) selecting a part of DNA samples to carry out agarose gel electrophoresis detection, and selecting the samples with uniform DNA sample bands, no tailing and no degradation to construct a DNA pool.

(2) Determination of OD value

The OD values of the DNA samples at 260nm and 280nm were measured by an ultraviolet photometer. Calculation of DNA content and OD₂₆₀/OD₂₈₀The ratio of (a) to (b). Such as OD₂₆₀/OD₂₈₀The ratio is less than 1.6, which indicates that the sample contains more protein or phenol, and purification is required; if the ratio is greater than 1.8, then RNA purification removal should be considered.

DNA mass (ng) ═ 50 XOD₂₆₀Value x dilution factor

(3) Construction of variety DNA pools

After DNA detection is finished, taking out a certain amount of DNA to be diluted to 50 ng/mu L, and then taking 10 mu L of DNA samples of Qinchuan cattle with the concentration of 50 ng/mu L to be mixed to construct a variety DNA pool;

the detection result of the Qinchuan cattle blood sample genome DNA is shown in figure 1, and the quality of the Qinchuan cattle genome DNA can be seen to be very high.

4. PCR amplification

Taking a Qinchuan cattle DNA pool as a template, and carrying out PCR amplification on P by using a designed primer, wherein the total PCR reaction system is 25 mu L, which is shown in a table 2; the PCR general reaction program is shown in Table 3.

TABLE 2 PCR reaction System

Components of the System	Volume (μ L)
		2*Reaction Mix	12.5
Upstream primer (10pmol/L)	1.0
		Downstream primer (10pmol/L)	1.0
Taq DNA polymerase (0.5U/. mu.L)	0.3
		DNA template (50 ng/. mu.L)	1.0
Sterilized ultrapure water (H)₂O)	9.2
		Total volume	25.0

TABLE 3 PCR reaction procedure

5. PCR product purification and sequencing

After the PCR amplification is finished, agarose gel electrophoresis is carried out, and the electrophoresis result is shown in figure 2, and a 395bp band can be clearly seen; then, gel cutting recovery and purification of PCR products are carried out: cutting off the gel containing the target fragment from the agarose gel under an ultraviolet lamp, putting the gel into a 1.5mL centrifuge tube, then purifying the PCR product by using a PCR product recovery and purification kit (Beijing Tiangen Biotech), and operating according to the kit specification, wherein the specific steps are as follows:

(1) first, 500. mu.L of the equilibration solution BL was added to the adsorption column, centrifuged at 12000rpm for 1 minute, the waste solution in the collection tube was discarded, and the adsorption column was replaced in the collection tube.

(2) A single band of the target DNA was cut from the agarose gel and placed in a clean centrifuge tube and weighed.

(3) Adding the equal volume of the solution PC into the gel block, placing the gel block in a water bath at 60 ℃ for about 10 minutes, and continuously and gently turning the centrifugal tube up and down to ensure that the gel block is fully dissolved.

(4) Adding the solution obtained in the previous step into an adsorption column, centrifuging at 12000rpm for 1 min, pouring the waste liquid in the collecting tube, and putting the adsorption column into the collecting tube again.

(5) Add 700. mu.L of the rinse to the column, centrifuge at 12000rpm for 1 minute, discard the waste, replace the column in the collection tube.

(6) Adding 500 μ L of rinsing solution into adsorption column, centrifuging at 12000rpm for 1 min, pouring off waste liquid, placing the adsorption column into collection tube, centrifuging at 12000rpm for 2 min, and removing rinsing solution as much as possible. The adsorption column was placed in an incubator at room temperature or 50 ℃ for several minutes and completely dried.

(7) And (3) putting the adsorption column into a clean centrifugal tube, suspending and dropwise adding a proper amount of elution buffer solution into the middle position of the adsorption film, and standing at room temperature for 2 minutes. The DNA solution was collected by centrifugation at 12000rpm for 1 minute.

(8) In order to increase the recovery amount of DNA, the solution obtained by centrifugation may be added back to the centrifugal adsorption column and step 7 may be repeated.

And (3) sending the PCR purified product taking the Qinchuan cattle DNA pool as a template to Shanghai bioengineering company Limited for bidirectional sequencing. The sequencing result of 395bp of the Qinchuan cattle CFL1 gene target fragment is shown in figure 4.

Analyzing the sequencing peak map, wherein two different peaks at the same site are subjected to single nucleotide mutation; t, C two detection results appear at the 2052 th site of the Qinchuan cattle CFL1 gene, namely the screened SNP polymorphism of the Qinchuan cattle CFL1 gene, and the site is the base polymorphism of T or C.

b. RFLP-PCR detection of Qinchuan cattle CFL1 gene T > C mutation polymorphism

The base polymorphism which is screened is an unnatural enzyme cutting site, so that the PCR-RFLP can not be carried out by the commonly used incision enzyme for identification. Therefore, the restriction enzyme HinfI cleavage site is introduced by changing the mutation site to the next base A into C. When the 2052 th site of the Qinchuan cattle CFL1 gene does not generate T > C mutation, namely T before mutation, enzyme digestion is introduced, a CFL1 gene sequence gaatc amplified by a primer pair P is used as a recognition site of restriction enzyme HinfI, and the amplified 395bp target fragment can be directly subjected to enzyme digestion by HinfI for genotyping.

1. RFLP-PCR primer design

Aiming at the T > C mutation at the 2052 th site contained in a sequencing peak diagram, designing an enzyme digestion primer pair P by utilizing primer design software Primer5.0, and designing primers in upstream and downstream sections of a mutation site through primer enzyme digestion, wherein the specific primer design is as follows:

an upstream primer: 5'-CTTTTTCTTTGGCTGCTATTC-3'

A downstream primer: 5'-TCTCCTTGCCCTCCTCCAGGATGAG-3'

The primer can amplify 395bp segment of exon 2 of the Qinchuan cattle CFL1 gene.

2. RFLP-PCR reaction conditions

The PCR product amplification system and the reaction conditions are respectively shown in Table 2 and Table 3, the 1.5% agarose gel electrophoresis pattern of the PCR amplification product is shown in FIG. 2, and it can be seen that the designed primer pair P can amplify a fragment of 395 bp.

3. HinfI digestion of PCR amplification product

(1)20 μ L HinfI digestion reaction: 10 μ L of PCR product, 10 XBuffer (buffer)

2.0. mu.L of HinfI (10U/. mu.L) was 0.6. mu.L, and 7.4. mu.L of sterilized purified water (H)₂O)。

(2) Digestion conditions of enzyme digestion: digesting for 12-16 h in a constant temperature incubator at 37 ℃.

(3) PCR products were digested with HinfI and analyzed by agarose gel electrophoresis.

Electrophoresis is carried out for 1 hour by using 3.0 percent agarose Gel at the voltage of 120V, the restriction enzyme digestion result is detected by dyeing with nucleic acid dye, the analysis is carried out by using a BIO-RAD Gel Doc 2000 Gel imaging analysis system for photographing, and the genotype is judged and recorded;

because 395bp fragments amplified by PCR-RFLP do not contain other HinfI restriction enzyme cutting recognition sites, when the 2052 position of CFL1 gene does not generate T > C mutation, after a CFL1 gene product amplified by PCR is recognized by restriction enzyme HinfI, the amplified fragments are cut by enzyme cutting at g ^ aatc, and the amplified fragments are cut into 2 segments; when the 2052 th site of the CFL1 gene is mutated, a new restriction enzyme HinfI restriction enzyme cutting recognition site cannot be formed, and an amplified fragment cannot be cut by enzyme;

as Qinchuan cattle are 2-ploid animals, when T > C mutation occurs, 3 different genotypes can be formed, namely TT, TC and CC, and the gel result graph of PCR-RFLP detection is shown in figure 3:

the TT genotype is wild type, SNP sites of two DNA chains of the TT genotype can be cut by HinfI enzyme, and the TT genotype is represented by 367bp and 28bp bands; SNP sites of two strands of the mutated genotype CC cannot be cut by enzyme, and are represented as 395bp bands; one SNP site in two strands of the heterozygote TC can be identified, while the other SNP site can not be identified, and the SNP sites are represented as 395bp, 367bp and 28bp bands; 28bp is smaller and therefore cannot be seen in agarose gel electrophoresis analysis, but fragments of 395bp and 367bp can identify TT type and TC type, and according to the number of bands and the size of the bands, the gel electrophoresis detection result shown in figure 3 can clearly judge whether point mutation occurs or not, so that three genotypes can be distinguished, and the SNP polymorphism can be detected.

(4) Sequencing verification of PCR products of individuals with different genotypes

Respectively carrying out positive and negative bidirectional sequencing on individual PCR products of different genotypes by using ABI 377 and ABI 3730 sequencers; meanwhile, SNP position analysis is carried out, and the result shows that the sequence diagram of 2052 bit of a heterozygote TC genotype individual containing 395bp, 367bp and 28bp bands is really represented as T or C, and as shown in figure 4C, the 4 th peak from left to right is a double peak; the TT genotype and the CC genotype are T, C respectively, as shown in FIG. 4a and FIG. 4b respectively.

c. Application of SNP (single nucleotide polymorphism) at 2052 position of Qinchuan cattle CFL1 gene as molecular marker in different Qinchuan cattle group polymorphisms

1. Detection of single nucleotide polymorphisms in populations

The SNP polymorphism detection method is utilized to identify the SNP polymorphism of 488 DNA samples of Qinchuan cattle; and (5) counting the frequency distribution of the SNP sites.

2. Statistical analysis of frequency of SNP loci

Genotype frequency refers to the ratio of the number of individuals with a certain genotype for a trait to the total number of individuals in a population. P is_AA＝N_AAN, wherein P_AARepresenting the AA genotype frequency of a certain locus; n is a radical of hydrogen_AARepresenting the number of individuals in the population having the AA genotype; and N is the total number of detection groups.

Gene frequency refers to the relative ratio of a certain number of genes in a population to the total number of its alleles. The formula for the calculation can be written as: p_A＝(2N_AA+N_Aa1+N_Aa2+……+N_Aan) and/2N. In the formula, P_AIndicates allele A frequency, N_AARepresenting the number of individuals with AA genotype in the population, N_AaiRepresenting the number of individuals having the Aai genotype in the population, a1-an being n different multiple alleles of allele A; the statistical results are shown in Table 4.

TABLE 4 Qinchuan cattle CFL1 gene 2213 th SNP gene frequency distribution table

As can be seen from Table 4, the frequencies of the C alleles were much higher than those of the T alleles in Qinchuan cattle, respectively, indicating that the C allele may be associated with a growth trait.

3. Association analysis of gene effects

Genotype data: genotype recognized by HinfI (TT, TC and CC)

Growth trait data: body size data (height of body, height of cross, length of body, bust, chest width, chest depth,

Long, wide seated position, wide waist and heavy)

And (3) correlation analysis model:

the SPSS (16.0) software was used to analyze the correlation of gene loci, sires, field effects, age and breed effects with growth traits. Firstly, performing description analysis on data to determine whether an outlier exists, and then correcting the data by using least square analysis; and analyzing the genotype effect by using a multivariate linear model according to the data characteristics.

The model is as follows:

y_ijklmn＝μ+Genotype_i+S_j+B_k+F_l+Age_m+Xn+e_ijklmn

wherein: y is_ijklm(ii) recording the phenotype of the individual; f_lField effect; s. the_jIs a male breeding effect; b is_k: variety effect; age_mIs an age effect; xn is various second-level and above-level interaction effects, such as: age × Genottype, S_jXGenotype, etc.; e.g. of a cylinder_ijklmnIs a random error; the data were analyzed using SPSS (16.0) software and the differences significance was examined for each genotype intermediate scale index by least squares fitting to a linear model.

The results show (see table 5): for the SNP locus of 2052 which can be identified by HinfI, the CC genotype is the dominant genotype; for body length, chest width, chest depth and weight, the numerical value of the CC genotype individual is obviously higher than that of TT and TC genotype individuals, and research shows that the weight trait and the meat production trait are in positive correlation, which shows that the CC genotype can become a molecular genetic marker for improving the breeding speed of the meat production trait of Qinchuan cattle.

TABLE 5 analysis of variance between HinfI polymorphic sites and the size of the Qinchuan cattle

Note: capital letters differ to indicate a very significant difference (P <0.01) and lower case letters differ to indicate a significant difference (P < 0.05).

Sequence listing

<110> Yangzhou university

<120> SNP molecular marker of Qinchuan cattle CFL1 gene and detection method thereof

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catctgggcg cgcgagaggg aaagggggcg caacgtcggc accctcttcc ccagctcctg 180

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cacagagcgg accgcgaagg agtcctcggc tgtcctccca ggctggcgag cccctgaagg 720

cgagccgggt tcgcggagta cacccggcgg agactgggac ggcgctcccg gccggcgcgt 780

gcgcacacgg tcaggcctgg ccgctgggcc agaagccgtg gactaacggg gccttcggct 840

ctcccggatg gggcctgagg gtggagtctc gtttagggaa gtgacccagg cgggcaccgc 900

ccggaaacca ccccgtcccc tgtgttcggc ccgagggctg cgagagccga aggttgtgta 960

acttgcccag gcctgcagcg cagggggccg ataagctcgt cggcgcaggc gattccattg 1020

ggttgaggcc ttccgagatg cagctgccgc tgggtatgag tttttttttt ttttcgggga 1080

gggggctgat tctagccgat ctgctttcac cgcctactcc ggaagcggct caatcacgtg 1140

gccggcttcc tctgcagttc cgggagcggg ggcagatttc accgtggacc gtcactccct 1200

ccctggctag ggtgtcccgc aacacgacct acaagtttaa agagctctca gagactgtgg 1260

gcacttgact ttggaccgct gactcctgtc agcgacttgg gggcctgcat ttttcccttg 1320

tgaggcctta attaggggtt tgttgttgct ttggtttggg gagaccccgg cagagaagga 1380

aatgtgtaat aactgctttg aaccgtgctg atgggcttgc tggaactgtt gagtactttt 1440

ggtgggtcct ggttctatgg agatgactag gatctggatc cctttctgct ctacgtgaag 1500

ttctgagtca gcactttatg caaaagctga gactgcgtag aaaacggggt cagtagtctg 1560

tcggttcttt taatttggac tcacatcttg gccgaggctc tgcttctcac aggtcttgga 1620

tctctagcct gcaggatgtc ggggtgggga ggaatgctgt tgaattatgc gatcaaaggt 1680

ctctttttct ttggctgcta ttctgggatt gtgtcacatc tgatcctttg acggtgacct 1740

gggtgggaaa atagtgtcac tagtcctagg aaggggatga gtagtgacct cttggggctg 1800

aggactgtac tttgttggcc tctagacaga tctcctaaca aagcaaaaga gggacttcag 1860

ggcatgtccc agaaactgtc tcctcacgag gtgcgtgtgg tgtggctcat cctctaggcc 1920

tccggtgtgg ctgtctctga tggggtcatc aaagtgttca acgacatgaa agtgcgtaag 1980

tcgtcgacac cagaggaagt gaagaagcgc aagaaggcgg tgctcttctg cctgagtgag 2040

gacaagaaga atatcatcct ggaggagggc aaggagatcc tggtgggtga cgtgggccag 2100

acggtagacg acccctatgc cacctttgtc aagatgctgc cagacaagga ctgccgctac 2160

gccctctatg atgcaaccta cgagaccaag gagagcaaga aggaggacct ggtgttcatc 2220

ttctggtgag ctcctcctgc aggcactttc ccctttgacc tgctctagct ctgccccacc 2280

cccctttccc aggacaggaa ggtctgtggc tccggcttaa tccagacgcc tgcgtgtcag 2340

aggagggtgt tgtaataaaa cacttgtcaa tgaggaactt gatgaaagcg tgaatgctgg 2400

ggcctagctc aggggtttct ggaagttatt tatcgcttga gggcacttgc tggatttgga 2460

ggcaagttct cttgattctt ttctcttcca gggcccctga gtgtgcaccc cttaagagca 2520

aaatgatcta tgccagctcc aaggacgcca tcaagaagaa gctgacgggt aagggccagc 2580

attggtggcg ccatatgccc ttgtgcttgg gccttggctg ctgggtgggg tgaatggcat 2640

ggcccaggag atccctgccc gctggaaggc agcctggtcc cctccatctg ttcagactgg 2700

ctccttccca gctgcagcac cccccacccc cacccttcct gctcacagat gctccctctt 2760

cttccctaca gggatcaagc atgaattaca agcaaactgc tacgaggagg tcaaggaccg 2820

ctgcaccctt gcagagaagc tggggggcag cgctgtcatc tccctggagg gcaagccttt 2880

gtgagccccc tccagccccc tgcctggagc atctggcagc cccagacctg cccacggggg 2940

ttgcaggctg cccccttcct gccagaccgg aggggctggg gggaatccca gcagggggag 3000

ggcagtccct tcaccccagt tgccaaacag cccccccgac cccctggacc ttcctcctcc 3060

ctccacccct gacggttctg gccttcccaa accgcttttg atcttgattc ctcttgggtt 3120

gaaacagacc aagttccccc cccaggaacc cctgtttggg gggggcctgt atttttttta 3180

acgacacccc agttcc 3196

<210> 2

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

ctttttcttt ggctgctatt c 21

<210> 3

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

tctccttgcc ctcctccagg atgag 25

Claims

1. Qinchuan cattleCFL1Detection primer of SNP molecular marker of gene in Qinchuan cattle breeding or Qinchuan cattle identificationThe application of the SNP molecular marker in the growth traits of the cattle is characterized in that the base of the 2052 th site of the sequence shown as SEQ ID No.1 from the 5' end is T or C, the nucleotide sequence of the upstream primer of the detection primer is shown as SEQ ID No.2, and the nucleotide sequence of the downstream primer is shown as SEQ ID No. 3; the body length, chest width, chest depth and body weight of the Qinchuan cattle with the SNP molecular marker of the CC genotype are all higher than those of the Qinchuan cattle with the SNP molecular marker of TT and TC genotypes, and the Qinchuan cattle with the SNP molecular marker of the CC genotype is bred.

2. Use according to claim 1, characterized in that it comprises the following steps:

to compriseCFL1The Qinchuan cattle whole genome DNA to be detected of the gene is taken as a template, and the detection primer is used for PCR amplification of the Qinchuan cattleCFL1A gene; using restriction endonucleasesHinfI, digesting a PCR amplification product, and then carrying out agarose gel electrophoresis on the amplified fragment subjected to enzyme digestion; identifying Qinchuan cattle according to electrophoresis resultCFL1Single nucleotide polymorphism at position 2052 of gene.

3. The use according to claim 2, wherein the agarose gel is present at a mass concentration of 3%.

4. Use according to claim 2, wherein the electrophoresis results as follows: the electrophoresis bands of the Qinchuan cattle with the TT genotype are represented by 367bp and 28 bp; the electrophoresis bands of the Qinchuan cattle with the TC genotype are represented as 395bp, 367bp and 28 bp; the electrophoresis band of Qinchuan cattle with CC genotype is represented as 395 bp.