CN109207611A - One kind SNP marker relevant to sheep heat character and its detection kit and application - Google Patents
One kind SNP marker relevant to sheep heat character and its detection kit and application Download PDFInfo
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Abstract
The present invention provides a kind of SNP marker relevant to sheep heat character and its detection kit and applications, belong to sheep SNP marker technical field, the SNP site is located at No. 10 chromosome 52551820bp (NC_019467.2 of sheep, based on ovine genome sequence information version number Oar_v4.0, in December, 2015), there are G/A base mutations in the SNP site, have significant correlation with sheep seasonal oestrus character.It can determine sheep heat character to be measured by carrying out parting to the SNP site.The method of SNP site genotype described in detection sheep provided by the invention, sensitivity, accuracy is higher, cost performance is higher, can to the SNP site realize automatic detection, during Sheep Breeding, can by with long-term heat character AA homozygous individual and GA heterozygous individual select and remain, to improve the rutting rate of sheep, sheep large-scale molecular breeding is had potential application.
Description
Technical field
The invention belongs to sheep SNP marker technical fields, more particularly to a kind of SNP relevant to sheep heat character
Molecular labeling and its detection kit and application.
Background technique
Single nucleotide polymorphism (SNP) is primarily referred to as in genome picodna (DNA) sequence by single deoxidation
The polymorphism of DNA fragmentation caused by the variation of nucleotide.The polymorphism of SNP relates only to the variation of single base, performance
Form has replacement, insertion and missing etc..The detection method of SNP, currently used to include, sanger sequencing, DNA chip, flight
The technologies such as time mass spectrum and newest high-throughput two generations sequencing.Genotype is detected by detection single nucleotide polymorphism
It is a kind of genotype detection method risen in recent years.SNP as genetic marker have been widely used for the assignment of genes gene mapping, gram
The research fields such as grand, genetic breeding and genetic diversity.Application of the molecular labeling in animal breeding for some time, phase
Than traditional breeding way, molecular marker breeding greatly accelerates Breeding Efficiency, saves breeding time, so that breeding scholar can be with
The simultaneously more excellent domestic animal kind of breeding is constantly explored on a molecular scale.
Seasonal breeding is that sheep adapts to the important evolution of existence as a result, but limiting production effect in actual production
Rate.Due to sheep only heat in the fall, the four seasons equilibrium supply of mutton is limited, this is that sheep production efficiency is low in global range
Under one of key constraints.The existing sheep number of bits in China is at the forefront in the world, but the rate of animals delivered to the slaughter-house is really lower than world average level,
It is extremely urgent to greatly develop sheep husbandry.Long-term heat is certain distinctive characters of feeding sheep kind, is educated by current routine
Kind technology is difficult to carry out genetic improvement to this character.Therefore, it from the key gene for genetically studying the long-term heat of sheep, utilizes
Modern biotechnology changes the seasonality of sheep heat, has a very important significance to the reproductive performance for improving sheep, applies
Prospect is very wide.But there is presently no the screenings that any special DNA molecular marker can be used for sheep heat character.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of SNP marker relevant to sheep heat character and its
Detection kit and application.
In order to achieve the above-mentioned object of the invention, the present invention provides a kind of SNP marker relevant to sheep heat character,
The SNP site is located at the site 52551820bp on No. 10 chromosome of sheep, and (NC_019467.2 is based on ovine genome
Sequence information version number Oar_v4.0, in December, 2015) there are G/A base mutations on the site, have with sheep heat character
There is significant correlation, when the site is A base, sheep has long-term heat character；When the site is G base,
The long-term heat character of sheep is unobvious.
The present invention provides utilize SequenomSNP technology detects the primer of the SNP site
Group, including upstream primer, downstream primer and extension primer；The nucleotide sequence of the upstream primer is as shown in SEQ IDNo.1；
The nucleotide sequence of the downstream primer is as shown in SEQ ID No.2；The nucleotide sequence of the extension primer such as SEQ ID
Shown in No.3.
The present invention provides utilize SequenomSNP technology detects the reagent of the SNP site
Box, including dNTPs, Taq archaeal dna polymerase, MgCl2, standard positive template DNA, PCR reaction buffer and SAP enzyme, feature
It is, further includes the primer sets.
Preferably, the use concentration of the primer sets middle and upper reaches primer and downstream primer stands alone as 0.45~0.55 μm of ol/
L；The concentration of extension primer in the primer sets is 0.6~1.3 μm of ol/L.
The present invention also provides utilize SequenomThe detection of SNP technology and the sheep heat
The method of the relevant SNP marker of shape, comprising the following steps:
1) genomic DNA of sheep to be measured is extracted；
2) using the genomic DNA of sheep to be measured as template, using in the primer sets upstream primer and downstream primer into
Row pcr amplification reaction obtains pcr amplification product；
3) digestion is carried out to the pcr amplification product obtained in step 2) with SAP enzyme and obtains postdigestive product；
4) using postdigestive product as template, extension is carried out using the extension primer in the primer sets and is extended
Product；
5) extension products are analyzed using Matrix-Assisted Laser Desorption Ionization Time of Flight technology, determined SNP described
The genotype of point.
Preferably, the reaction system that pcr amplification reaction described in step 2) uses includes: in terms of 5 μ L
The program of the pcr amplification reaction are as follows:
95 DEG C of 2min of initial denaturation；
It is denaturalized 95 DEG C of 30s；
Anneal 56 DEG C of 30s；
Extend 72 DEG C of 60s；
After the denaturation, annealing and extension step carry out 45 circulations；72 DEG C of holding 5min.
Preferably, system is digested described in step 3) includes: in terms of 2 μ L
SAPBuffer 0.17μL；
0.3 μ L of 1.7U/ μ L SAP enzyme；
1.53 μ L of deionized water；
The program of the digestion are as follows: 37 DEG C, 40min；85 DEG C, 5min.
Preferably, the system of extension described in step 4) includes: in terms of 2 μ L
The program of the extension are as follows:
94℃ 30s；
[94 DEG C of 5s, (52 DEG C of 5s, 80 DEG C of 5s)]；Wherein (52 DEG C of 5s, 80 DEG C of 5s) carry out 5 circulations, described
[94 DEG C of 5s, (52 DEG C of 5s, 80 DEG C of 5s)] carry out 40 circulations；
72℃ 3min。
The present invention also provides application of the SNP site in sheep assistant breeding, during Sheep Breeding, sieve
The sheep that the SNP site is A base is selected to carry out subsequent breeding.
The present invention also provides application of the kit in sheep assistant breeding, during Sheep Breeding, utilize
The kit screens the sheep that the SNP site is A base and carries out subsequent breeding.
Beneficial effects of the present invention: SNP marker relevant to sheep heat character provided by the invention, the SNP
Site is located at the site 52551820bp on No. 10 chromosome of sheep, and (NC_019467.2 is based on ovine genome sequence information
Version number Oar_v4.0, in December, 2015), there are G/A base mutations on the site, have with sheep heat character significant
Correlation, when the site is A base, sheep has the character of long-term heat；When the site is G base, sheep is normal
Year heat character is unobvious, shows as seasonal oestrus.The G/A base mutation of the SNP site and sheep heat character are close
Correlation can determine sheep heat character to be measured by carrying out parting to the SNP site.
It is provided by the invention to utilize SequenomSNP technology detects SNP site base described in sheep
Because of the method for type, sensitivity, accuracy is higher, and cost performance is higher, can be simultaneously to tens of to hundreds of in hundreds of to thousands of parts of samples
A SNP site is detected.
Heretofore described method can realize automatic detection to the SNP site, during Sheep Breeding, can incite somebody to action
AA homozygous individual and GA heterozygous individual with long-term heat character are selected and remain, so that the rutting rate of sheep is improved, it is right
Sheep large-scale molecular breeding has potential application.
Detailed description of the invention
Fig. 1 is that Sequenom is utilized in the embodiment of the present invention 1SNP technology is to FBXL3 gene
Three kinds of genotype, the i.e. testing result of GG, GA or AA.
Specific embodiment
The present invention provides a kind of SNP marker relevant to sheep heat character, the SNP site is located at sheep the
(NC_019467.2 is based on ovine genome sequence information version number Oar_ in the site 52551820bp on No. 10 chromosomes
V4.0, in December, 2015), there are G/A base mutations on the site, there is significant correlation with sheep heat character, when
When the site is A base, sheep has long-term heat character；When the site is G base, sheep is without long-term heat
Shape.Heretofore described SNP site is located in FBXL3 gene, in seasonal oestrus sheep mainly based on wild type G.
The present invention also provides utilize SequenomSNP technology detects drawing for the SNP site
Object group, including upstream primer, downstream primer and extension primer；The nucleotide sequence of the upstream primer such as SEQ ID No.1 institute
Show；The nucleotide sequence of the downstream primer is as shown in SEQ ID No.2；The nucleotide sequence of the extension primer such as SEQ ID
Shown in No.3.It is specific as follows: upstream primer 5 '-ACGTTGGATGACTCCAAATCCTTGTCCTCG-3 '；Downstream primer: 5 '-
ACGTTGGATGTCAGCAGCTTGAGTGTATCG-3'；Extension primer: 5 '-GCTCGCTGAAGATAGATGACA-3 '.
Sequenom of the present inventionSNP technology combination multiple PCR technique,
MassARRAYiPLEX Single base extension technology and matrix solid-dispersion flying time mass spectrum analysis mass-spectrometric technique
Parting detection is carried out to gene.Using the method automatic detection can be realized to the SNP site of FBXL3 gene.
The present invention also provides utilize SequenomSNP technology detects the examination of the SNP site
Agent box, including dNTPs, Taq archaeal dna polymerase, MgCl2, standard positive template DNA, PCR reaction buffer and SAP enzyme, also wrap
Include the primer sets.In the present invention, the use concentration of the primer sets middle and upper reaches primer and downstream primer is preferably stood alone as
0.45~0.55 μm of ol/L more preferably stands alone as 0.50 μm of ol/L；The concentration of extension primer in the primer sets is preferably
0.6~1.3 μm of ol/L.In the present invention, the use concentration of the dNTPs is preferably 20~30 μm of ol/L, more preferably 25 μ
mol/L；The use concentration of the Taq archaeal dna polymerase is preferably 4~6U/ μ L, more preferably 5U/ μ L；The MgCl2Use
Concentration is preferably 20~30mmol/L, more preferably 25mmol/L；The PCR reaction buffer is preferably that 10 × PCR reaction is slow
Fliud flushing；The enzyme activity of the SAP enzyme is preferably 1.7U/ μ L.Heretofore described kit preferably further includes SAP Buffer.?
In the present invention, the genotype of the standard positive template DNA is AA, and the standard positive template DNA increases as positive control
The accuracy of the SNP site detection.
The present invention also provides utilize SequenomThe detection of SNP technology and sheep heat character phase
The method in the SNP marker site closed, comprising the following steps: 1) extract the genomic DNA of sheep to be measured；2) with to be measured
The genomic DNA of sheep is template, utilizes the upstream primer and downstream primer progress pcr amplification reaction acquisition in the primer sets
Pcr amplification product；3) digestion is carried out to the pcr amplification product obtained in step 2) with SAP enzyme and obtains postdigestive product；4) with
Postdigestive product is template, carries out extension using the extension primer in the primer sets and obtains extension products；5) it utilizes
Matrix-Assisted Laser Desorption Ionization Time of Flight technology analyzes extension products, determines the genotype of the SNP site.
In the present invention, the genomic DNA of sheep to be measured is extracted first.The present invention does not have the type of the sheep to be measured
Particular/special requirement, any kind of sheep are in embodiments of the present invention sheep and Europe using breeds differentiation, Mongolia
Sheep；The present invention is not particularly limited the extracting method of the ovine genome to be measured, and the animal using this field routine is thin
Born of the same parents' Extraction Methods of Genome, in specific implementation process of the present invention, using erythrocyte cracked liquid cracking removal without DNA's
Red blood cell, nucleus lysate cracking packet cell release genomic DNA, and then albumen precipitation liquid selective precipitation removes egg
White, last pure genomic DNA is laid equal stress on by isopropanol precipitating is dissolved in DNA lysate.
The present invention is after obtaining the ovine genome DNA to be measured, using the genomic DNA of sheep to be measured as template, utilizes
Upstream primer and downstream primer in the primer sets carry out pcr amplification reaction and obtain pcr amplification product.It is heretofore described
The reaction system that pcr amplification reaction uses includes: in terms of 5 μ L
The program of the pcr amplification reaction are as follows:
95 DEG C of 2min of initial denaturation；
It is denaturalized 95 DEG C of 30s；
Anneal 56 DEG C of 30s；
Extend 72 DEG C of 60s；
After the denaturation, annealing and extension step carry out 45 circulations；72 DEG C of holding 5min.
Pcr amplification product is preferably stored in 4 DEG C after the completion of the PCR amplification by the present invention.PCR of the present invention
After amplified reaction, the DNA fragmentation where the site target SNPs is contained in the pcr amplification product.
The present invention carries out digestion acquisition with pcr amplification product of the SAP enzyme to acquisition after obtaining the pcr amplification product
Postdigestive product.The digestion system includes: in terms of 2 μ L in the present invention
SAP Buffer 0.17μL；
0.3 μ L of 1.7U/ μ L SAP enzyme；
1.53 μ L of deionized water；
The program of the digestion are as follows: 37 DEG C, 40min；85 DEG C, 5min.
Heretofore described postdigestive product is preferably stored in 25 DEG C.The effect of heretofore described digestion is digestion
Fall the primer sequence and remaining dNTPs in pcr amplification reaction system.
The present invention, using postdigestive product as template, utilizes prolonging in the primer sets after obtaining the digestion product
The object that extends carries out extension and obtains extension products.In the present invention, the system of the extension includes: in terms of 2 μ L
The program of the extension are as follows:
94℃ 30s；
[94 DEG C of 5s, (52 DEG C of 5s, 80 DEG C of 5s)]；Wherein (52 DEG C of 5s, 80 DEG C of 5s) carry out 5 circulations, described
[94 DEG C of 5s, (52 DEG C of 5s, 80 DEG C of 5s)] carry out 40 circulations；
72℃ 3min。
Iplex Buffer Plus, iplex Terminator, 0.6~1.3 μ in the present invention, in the extension system
Mol/Lprimer mix and iplex enzyme source inGold Reagent Set kit.Extension of the present invention
In reaction process, Single base extension is carried out to the SNP site to be checked in extension system, the extension primer of locus specificity will be prominent
Extend a base at displacement point and terminates.Extension primer will connect upper different ddNTPs according to the difference of mutation type,
Form molecular weight difference.The extension products are preferably passed through purifying resin after obtaining the extension products by son of the invention,
The present invention is not particularly limited the method for the purifying resin, using the purifying resin of this field routine.
The present invention utilizes Matrix-Assisted Laser Desorption Ionization Time of Flight technology point after obtaining the extension products
Extension products are analysed, determine the genotype of the SNP site.The extension products point sample to target on piece is used matter by the present invention
Spectrometer detects the molecular weight difference of different extension products, is analyzed by data, so that it may obtain the specific of each mutational site
Genotype.In the present invention, the mass spectrum point sample is related to be carried out using MassARRAY NanodispenserRS1000；It is described
Mass spectral analysis preferably uses MassARRAY Compact System to carry out；The present invention is after the mass spectral analysis, preferably
Using Typer4.0 software detection mass spectra peak, and according to each sample target site genotype of mass spectra peak map interpretation.
Heretofore described SequenomThe basic principle of SNP technology are as follows: use upstream first
Primer and the place downstream primer amplification target SNPs DNA fragmentation, are added SAP enzymic digestion in amplified production and fall in reaction system
Then primer sequence and remaining dNTPs carry out Single base extension to SNP site to be checked using extension primer simultaneously, site is special
Anisotropic extension primer will extend a base and be terminated at mutational site.Extension products by according to the difference of mutation type and
Different ddNTPs in connection forms molecular weight difference.Extension products are after purifying resin, by point sample to target on piece, and
It is detected using molecular weight difference of the mass spectrograph to different extension products, is analyzed by data, so that it may obtain each mutational site
Specific genotype.
The present invention also provides application of the SNP site in sheep assistant breeding, during Sheep Breeding, sieve
The sheep that the SNP site is A base is selected to carry out subsequent breeding.The screening of the SNP site preferably uses in the present invention
Above-mentioned SequenomSNP technology；Preferred screening SNP site is the silk floss of AA genotype and GA genotype
Sheep carries out subsequent breeding, more preferably screens the sheep that SNP site is AA genotype and carries out subsequent breeding.Application of the present invention can
To select and remain the frequency of genotypes AA homozygous individual with long-term heat character and genotype GA heterozygous individual, to improve sheep
Rutting rate, to sheep large-scale molecular breeding have very big application value.
Technical solution provided by the invention is described in detail below with reference to embodiment, but they cannot be understood
For limiting the scope of the present invention.
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
According to conventional laboratory conditions, such as Sambrook molecular cloning experiment handbook (Sambrook J&Russell DW,
Molecular cloning:a laboratory manual, 2001), or according to the condition of manufacturer's specification suggestion.
Embodiment 1
The identification of SNP marker relevant to sheep seasonal oestrus
Sequence, including three kinds of breeds differentiation are resurveyed by carrying out full-length genome to 10 sheep varieties, 99 sheep individuals
(mountain valley-type hiding sheep, grassland type hide sheep, OUra sheep), 6 Mongolia are sheep (Small-fat-tail sheep, black pearl Mu Qin, sheep known for its fine thick wool, sheep, Qira
Black sheep and Ba Yin Brooker) and 1 European sheep (Australia merino), obtain the ovine genome of maximum quantity so far
Genetic polymorphism site analyzes the genetic diversity and population genetic variations of these Sheep Populations, has screened and seasonal oestrus
Related gene.And obtain a large amount of effectively SNP site by the calculating of Fst value and screen and obtain some genes, including with
The SNP site of the relevant FBXL3 gene of sheep seasonal oestrus.It was found that FBXL3 gene is located at No. 10 chromosome
The site 52551820bp (NC_019467.2 is based on ovine genome sequence information version number Oar_v4.0, in December, 2015)
There are G/A mutation for base, have significant correlation with sheep seasonal oestrus.
Utilize SequenomThe SNP site of SNP technology detection sheep FBXL3 gene simultaneously verifies silk floss
The heat character of sheep
Experimental material
1 jumpbogroup of table verifies sheep variety used
| Kind | Number of elements | Remarks |
| Small-fat-tail sheep | 380 | Long-term heat |
| Du Bo | 30 | Long-term heat |
| Sheep known for its fine thick wool | 80 | Seasonal oestrus |
| Sunit ewes | 100 | Seasonal oestrus |
| Grassland type hides sheep | 131 | Seasonal oestrus |
| Suffolk | 39 | Seasonal oestrus |
2, reagent and instrument
Reagent: Complete Genotyping ReagentKit forCompact 384；
Gene magnification: ABI9700 384Dual；
Mass spectrum point sample: MassARRAY NanodispenserRS1000；
Mass spectral analysis: MassARRAY Compact System；
All reagents and instrument are purchased from Beijing Jun Nuode Bioisystech Co., Ltd (Beijing Genenode
Biotech Co.,Ltd)。
3, the extraction of genomic DNA
Sheep jugular vein blood collection 1ml, with EDTA anticoagulation.Erythrocyte cracked liquid cracking removal is without the red of DNA first
Cell, nucleus lysate cracking packet cell release genomic DNA, and then albumen precipitation liquid selective precipitation removes removing protein,
Last pure genomic DNA is laid equal stress on by isopropanol precipitating is dissolved in DNA lysate.
4、Sequenom SNP technology carries out Genotyping
For the 52551820th site on No. 10 chromosome of sheep (Oar4.0, Chr10, SNP g.52551820G > A)
Design primer combination.
The nucleotide sequence of PCR amplification primer is as follows:
Upstream primer F:5 '-ACGTTGGATGACTCCAAATCCTTGTCCTCG-3 '
Downstream primer R:5 '-ACGTTGGATGTCAGCAGCTTGAGTGTATCG-3 '
Extension primer sequence and extension products are as shown in table 1.
2 extension primer sequence of table and extension products
Above-mentioned primer is synthesized by Jun Nuo moral company.
Testing process is as follows:
1, the genomic DNA of sheep to be measured is extracted；
2, using the genomic DNA of sheep to be measured as template, it is anti-that PCR amplification is carried out using the upstream primer and downstream primer
It answers；
3, pcr amplification product is digested with SAP enzyme；
4, using postdigestive pcr amplification product as template, extension is carried out using the extension primer S1；
5, extension products are analyzed, to determine sheep FBXL3 genotype.
Wherein, the reaction system that pcr amplification reaction uses is calculated as with 5 μ L: 20-50ng/ μ L genomic DNA 1 μ L, 10 ×
0.5 μ L, 25mmol/L MgCl of PCR reaction buffer20.4 μ L, 25 μm of 0.1 μ L, PCR Primer mix of ol/L dNTPs, 1 μ
0.2 μ L of L, 5U/ μ L Taq archaeal dna polymerase, deionized water polishing to 5 μ L；
The amplification program of pcr amplification reaction are as follows: 95 DEG C of 2min；95 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 60s, 45 are followed
Ring；72℃ 5min.
Pcr amplification product is digested, mainly with the remaining primer and dNTP in SAP enzyme removal reaction product.Make
SAP enzymic digestion system is calculated as with 2 μ L: 0.17 μ L, SAP Enzyme of SAP Buffer, 0.3 μ L, deionized water polishing to 2
μL。
Reaction condition are as follows: 37 DEG C of 40min, 85 DEG C of 15min, 25 DEG C of preservations.
Extension system is calculated as with 2 μ L: 0.2 μ L, Terminator mix of iplex Buffer, 0.2 μ L,
0.94 μ L, iplex Enzyme of Extendprimermix, 0.041 μ L, deionized water polishing to 2 μ L；
Extension condition are as follows: 94 DEG C of 30s；[94 DEG C of 5s, (52 DEG C of 5s, 80 DEG C of 5s) 5 internal circulations], 40 outer
Portion's circulation；72℃ 3min.
Extension products after purifying resin are moved on 384 hole SpectroCHIP (Sequenom) chips, MALDI- is carried out
TOF-MS (Matrix-Assisted Laser Desorption Ionization Time of Flight) reaction, using Typer4.0 software detection mass spectra peak, and root
According to each sample target site genotype of mass spectra peak map interpretation.
Obtaining pcr amplification product size through mass spectral analysis is 133bp, and the Mass Spectrometer Method result of extension products is as shown in Figure 1.
Statistical result:
To genotype frequency and equipotential base of the site 52551820bp in sheep to be measured on No. 10 chromosome of sheep
Because frequency is counted respectively, and calculate polypeptide information content of this site in each group, heterozygosity and effectively etc.
Position gene number, and equilibrium state of the site in each group is detected with Chi-square Test, analysis statistical result is shown in Table 2.
The site 52551820bp different genes frequency and Population Genetics letter on table No. 10 chromosome of 2 sheep to be measured
Breath statistical analysis
Note: P > 0.05 indicates that site is in Hardy's Weinberg equilibrium state in the kind；P < 0.05 indicates site at this
Hardy's Weinberg equilibrium state is not in kind.
The site 52551820bp statisticallys analyze between different heat character Sheep Populations on No. 10 chromosome of table 3
Above-mentioned analysis can show that the site 52551820bp is in different heat phenotype sheep on No. 10 chromosome of sheep
Frequency distribution significant difference (P < 0.05).In long-term heat phenotype sheep based on saltant type A, and in seasonal oestrus sheep
In based on G.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Sequence table
<110>Institute of Animal Sciences, Chinese Academy of Agricultural Sciences
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acgttggatg actccaaatc cttgtcctcg 30
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acgttggatg tcagcagctt gagtgtatcg 30
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gctcgctgaa gatagatgac a 21
Claims (10)
1. a kind of SNP marker relevant to sheep heat character, which is characterized in that the SNP site is located at sheep the 10th
The site 52551820bp on number chromosome, NC_019467.2 are based on ovine genome sequence information version number Oar_v4.0,
In December, 2015, there are G/A base mutations on the site, have significant correlation with sheep heat character, when institute's rheme
When point is A base, sheep has long-term heat character；When the site is G base, sheep is without long-term heat character.
2. utilizing SequenomSNP technology detects the primer sets of SNP site described in claim 1,
It is characterised in that it includes upstream primer, downstream primer and extension primer；The nucleotide sequence of the upstream primer such as SEQ ID
Shown in No.1；The nucleotide sequence of the downstream primer is as shown in SEQ ID No.2；The nucleotide sequence of the extension primer is such as
Shown in SEQ ID No.3.
3. utilizing SequenomSNP technology detects the kit of SNP site described in claim 1,
Including dNTPs, Taq archaeal dna polymerase, MgCl2, standard positive template DNA, PCR reaction buffer and SAP enzyme, feature exist
In further including primer sets as claimed in claim 2.
4. kit according to claim 3, which is characterized in that the primer sets middle and upper reaches primer and downstream primer make
0.45~0.55 μm of ol/L is stood alone as with concentration；The concentration of extension primer in the primer sets is 0.6~1.3 μm of ol/L.
5. utilizing SequenomSNP technology detects relevant to sheep heat character in claim 1
The method of SNP marker, comprising the following steps:
1) genomic DNA of sheep to be measured is extracted；
2) using the genomic DNA of sheep to be measured as template, using in primer sets described in claim 2 upstream primer and downstream draw
Object carries out pcr amplification reaction and obtains pcr amplification product；
3) digestion is carried out to the pcr amplification product obtained in step 2) with SAP enzyme and obtains postdigestive product；
4) using postdigestive product as template, extension is carried out using the extension primer in primer sets described in claim 2 and is obtained
Obtain extension products；
5) extension products are analyzed using Matrix-Assisted Laser Desorption Ionization Time of Flight technology, determines the SNP site
Genotype.
6. according to the method described in claim 5, it is characterized in that, the reactant that pcr amplification reaction described in step 2) uses
It is to include: in terms of 5 μ L
The program of the pcr amplification reaction are as follows:
95 DEG C of 2min of initial denaturation；
It is denaturalized 95 DEG C of 30s；
Anneal 56 DEG C of 30s；
Extend 72 DEG C of 60s；
After the denaturation, annealing and extension step carry out 45 circulations；72 DEG C of holding 5min.
7. according to the method described in claim 5, it is characterized in that, digestion system described in step 3) includes: in terms of 2 μ L
SAPBuffer 0.17μL；
0.3 μ L of 1.7U/ μ L SAP enzyme；
1.53 μ L of deionized water；
The program of the digestion are as follows: 37 DEG C, 40min；85 DEG C, 5min.
8. according to the method described in claim 5, it is characterized in that, the system of extension described in step 4) is wrapped in terms of 2 μ L
It includes:
The program of the extension are as follows:
94℃30s；
[94 DEG C of 5s, (52 DEG C of 5s, 80 DEG C of 5s)]；Wherein described 5 circulations of (52 DEG C of 5s, 80 DEG C of 5s) progress, described [94 DEG C of 5s,
(52 DEG C of 5s, 80 DEG C of 5s)] carry out 40 circulations；
72℃3min。
9. application of the SNP site described in claim 1 in sheep assistant breeding, which is characterized in that in Sheep Breeding process
In, it screens the sheep that the SNP site is A base and carries out subsequent breeding.
10. application of the kit described in claim 3 in sheep assistant breeding, which is characterized in that during Sheep Breeding,
The sheep that the SNP site is A base, which is screened, using the kit carries out subsequent breeding.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114854870A (en) * | 2022-04-15 | 2022-08-05 | 中国农业科学院北京畜牧兽医研究所 | SNP (Single nucleotide polymorphism) marker related to goat heat-resistant character and detection method thereof |
| CN117894368A (en) * | 2023-11-20 | 2024-04-16 | 中国农业科学院北京畜牧兽医研究所 | SNP locus combination for molecular identification of 20 Tibetan sheep varieties based on XGBoost model and application |
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2018
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Non-Patent Citations (2)
| Title |
|---|
| NC_019467.1: "rs160521763", 《ENSEMBL GENOME BROWSER》 * |
| 博奥生物有限公司: "Sequenom SNP实验过程说明书", 《百度文库》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114854870A (en) * | 2022-04-15 | 2022-08-05 | 中国农业科学院北京畜牧兽医研究所 | SNP (Single nucleotide polymorphism) marker related to goat heat-resistant character and detection method thereof |
| CN117894368A (en) * | 2023-11-20 | 2024-04-16 | 中国农业科学院北京畜牧兽医研究所 | SNP locus combination for molecular identification of 20 Tibetan sheep varieties based on XGBoost model and application |
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