CN105349674B - A kind of detection method and application growing relevant CNV label to Qinchuan Cattle - Google Patents
A kind of detection method and application growing relevant CNV label to Qinchuan Cattle Download PDFInfo
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Abstract
The invention discloses a kind of detection methods and application that relevant CNV label is grown to Qinchuan Cattle: the CNV label refers to the copy number variation of ox SHH gene candidate region Chr 4:118264951-118267170, the detection method is using the genomic DNA of Qinchuan Cattle as template, using primer pair P1 and P2 as primer, the region gene C NV Qinchuan Cattle SHH and reference gene BTF3 are expanded respectively by real-time fluorescence quantitative PCR, according to log22‑ΔΔCtResult is divided into insert type, deletion form and normal type, to identify the copy number variation of Qinchuan Cattle SHH gene.The present invention can detect on DNA level to be marked with the closely related CNV of the Qinchuan Cattle production traits, can be used as the important candidate molecular marker of the marker assisted selection of Qinchuan Cattle growth traits.
Description
Technical field
The present invention relates to domestic animal molecular Biological Detection fields, and in particular to a kind of detection Qinchuan Cattle growth traits is relevant
The detection method and application of SHH gene C NV label.
Background technique
Copy number variation (Copy Number Variations, CNVs) refers to the missing of larger segment in genomic DNA
Or polyisomenism.CNVs is a kind of structure variation of Asia microscopic level on genome, and the clip size being related to is in 1kb to multiple Mb
Between, including copy number increases (Copy number gain) and copy number reduces (Copy number loss).Some copies
Number variation belongs to polymorphism scope, does not impact to the phenotype of animals and plants, and some copy number variations then pass through upset base
Gene expression is influenced because of sequence and change gene content, to cause phenotypic difference and phenotypic adaptation.
The technology for being applied to copy number variation detection at present mainly has: (1) comparative genome hybridization (CGH): CGH can be complete
On portion's chromosome or chromosome subzone level, the copy number of DNA sequence dna between different genes group is detected, so that discovery is copied
Shellfish number variation.However the technology resolution ratio, in Mb level, the copy number segment of more small fragment is then not easy to detect.Technology behaviour simultaneously
Make cumbersome, flux is low, time-consuming and expensive, needs relatively large number of template DNA, is unfavorable for promoting on a large scale.(2) more
Reconnecting probe amplification technology (MLPA): MLPA is a kind of copy number detection method to grow up for 2002.The technology has
Accurate relative quantification function, but this method probe prepares complex while complex for operation step, time-consuming.And
Using Capillary Electrophoresis as analysis means, flux is lower, higher cost and belongs to open-sky technique, easily causes PCR product
Pollution.(3) high-resolution fusion curve analysis (HRM): HRM in 2003 invent, by accurate Cooling rate control with
And the instruction of DNA saturable dye, it realizes and PCR product is identified by the melting temperature for studying PCR product sequence.It should
Technology has many advantages, such as quick, cheap, high throughput, has the following deficiencies simultaneously: the premise that this method is realized is that mutational site must
It must be heterozygosis, to increase the cost of experiment and the difficulty of design, and reduce the flux of detection.Also, single core
Influence of the difference of thuja acid for melting curve be smaller or even some differences have little influence on the offset of melting curve, causes to examine
It is lower to survey sensitivity.(4) real-time fluorescence quantitative PCR (qPCR): the difference of fluorescence chemical method according to used in qRT-PCR,
It is broadly divided into two class of fluorescent dye embedding inlay technique and fluorogenic hybridization probe method.Excessive SYBR Green is added in PCR reaction system
Dye molecule can specifically penetrate into DNA double chain and emit fluorescence signal, and free dye molecule then only has very low fluorescence
Background hardly shines, so that it is guaranteed that the increase of signal is synchronous with the increase of PCR product, can pass through the intensity of detection fluorescence signal
To reflect the quantity of genomic DNA.By to target gene (there is copy number variation) and with reference to gene (no copy number variation)
Relative quantification is carried out, according to 2-ΔΔCtThe copy number of method statistic detection sample candidate gene.The advantages of dye method is experimental cost
It is low, without designing synthesising probing needle, easy to use, can detecte the absolute copy number of target fragment, but be not suitable for large sample
High throughput detection.
Molecular breeding, i.e. molecular marker assisted selection (molecular mark-assist selection, MAS), the skill
Art is to select by DNA molecular marker genetic resources or breeding material, carries out breed improvement to the Comprehensive Traits of livestock and poultry.
In Animal Breeding, by closely related to growth traits, and the selection with the DNA marker of quantitative character tight association, it reaches
The purpose that breeding value accuracy is chosen seeds and improved to early stage, to obtain bigger genetic progress in Animal Breeding.
SHH gene is the important regulatory factor of embryonic development and Various Tissues orga- nogenesis, can promote mescenchymal stem cell
To at flesh or osteoblast differentiation, and inhibit Adipose Differentiation, and regulate and control the development of four limbs, nerve and digestive system, to influence
The growth and development of economic animal.High-flux sequence finds to contain in SHH gene there are the copy number variation (CNVs) of 2220bp
The Gene Partial exon, introne and 3 ' UTR critical function regions may change the function of the gene and its related target gene
It plays, and then influences the growth and development of body.Currently, there is not yet related SHH gene CNVs is to Qinchuan Cattle growth traits shadow
Loud document report.Therefore, it studies the gene copy number variation and extremely closes itself and the important growth traits association analysis of Qinchuan Cattle
It is important, theoretical foundation can be provided for China's Qinchuan Cattle molecular breeding, assist choosing convenient for the label of Chinese Qinchuan Cattle growth traits
It selects, quickly establishes the excellent Qinchuan Cattle population of genetic resources.
Summary of the invention
The purpose of the present invention is to provide a kind of detection methods and application that relevant CNV label is grown to Qinchuan Cattle.
To achieve the goals above, the invention adopts the following technical scheme:
A kind of detection method growing relevant CNV label to Qinchuan Cattle, comprising the following steps: with Qinchuan Cattle genome
DNA is template, using primer pair P1 and primer pair P2 as primer, expands Qinchuan Cattle SHH gene respectively by real-time fluorescence quantitative PCR
The region CNV and reference gene BTF3 identify the copy number variation of Qinchuan Cattle SHH gene according to quantitative result.
The CNV label refers to that the copy number of ox SHH gene candidate region Chr 4:118264951-118267170 becomes
It is different.
The copy number variation is according to log22-ΔΔCtQuantitative result is divided into three classes: insert type, Log22-ΔΔCt>
0.5；Deletion form, Log22-ΔΔCt<-0.5；Normal type, Log22-ΔΔCt≤|±0.5|。
The primer pair P1 are as follows:
Upstream primer F1:5 '-CGCACGCAATGAGACTTTA-3 ',
Downstream primer R1:5 '-AATAGCCAGGAGAGGTGAA-3 '；
The primer pair P2 are as follows:
Upstream primer F2:5 '-AACCAGGAGAAACTCGCCAA-3 ',
Downstream primer R2:5 '-TTCGGTGAAATGCCCTCTCG-3 '.
Amplification system used in the real-time fluorescence quantitative PCR is calculated as with 20 μ L: 1 μ L of 50ng/ μ L template DNA,
Each 1 μ L, 2 × SYBR Green qPCR Mix10 of upstream and downstream primer corresponding to the primer pair P1 or primer pair P2 of 10pmol/L
7 μ L of μ L and deionized water.
Response procedures used in the real-time fluorescence quantitative PCR are as follows: (1) 95 DEG C of 30s of initial denaturation；(2) amplified reaction: 95
DEG C denaturation 10s, 60 DEG C of annealing 30s, 39 recycle.
It is above-mentioned to grow application of the detection method of relevant CNV label in Qinchuan Cattle molecular breeding to Qinchuan Cattle.
The CNV label applies the Qin Chuan cattle breeds outstanding in early molecule marker assisted selection growth traits.
The region SHH gene C NV difference copy number is significant related to the growth and development of Qinchuan Cattle, wherein insert type individual
Growth traits be significantly higher than deletion form individual.
The present invention is according to the copy number variation of ox SHH gene candidate region Chr 4:118264951-118267170, specifically
Ground, the CNV label are located at the 7776bp- of ox SHH gene (GenBank Accession No.AC_000161) sequence
In the region 9995bp, using the genomic DNA of ox to be measured as template, genome C NVs is detected using real-time fluorescence quantitative PCR, according to
log22-ΔΔCtResult is divided into three classes: Log22-ΔΔCt> 0.5 is insert type；Log22-ΔΔCt< -0.5 is deletion form, Log22-ΔΔCt≤ | ± 0.5 | it is then normal type.It is to be measured if copy number is insert type according to the testing result of cow genome group CNVs
Ox phenotype is more excellent；If copy number is deletion form, test individual phenotype is poor.According to an embodiment of the invention, with ox
SHH gene candidate region Chr 4:118264951-118267170 is candidate locus, is examined by Real-Time Fluorescent Quantitative PCR Technique
Copy number variation situation of the site in Qinchuan Cattle group, group is surveyed, and is closed with important economical traits such as weight and busts
Connection analysis；If the copy number variation type for detecting SHH gene candidate site is insert type, ox phenotype to be measured is more excellent；Such as
Fruit copy number variation type is deletion form, then test individual phenotype is poor.
Compared with prior art, the present invention has the following advantages:
(1) Qinchuan Cattle SHH gene copy number variation detection method provided by the invention, is not limited by the age, be can be used for
The early stage breeding of cow, or even just can be chosen in rigid birth；
(2) method for detecting ox SHH gene copy number variation is accurate and reliable, easy to operate；
(3) detection in ox SHH gene copy number variation site, the molecular marker assisted selection for ox growth and development provide section
Learn foundation.
Detailed description of the invention
Fig. 1 is the amplification curve that qPCR drafting is carried out in the present invention；
Fig. 2 is the solubility curve that qPCR drafting is carried out in the present invention.
Specific embodiment
It elaborates with reference to the accompanying drawings and examples to the present invention.
The present invention is detected using copy number variation of the real-time fluorescence quantitative PCR to Qinchuan Cattle SHH gene and is used for point
Sub- breeding, generally includes following steps:
(1) the copy number variation situation using real-time fluorescence quantitative PCR (qPCR) technology detection candidate locus in group,
Screen CNV label relevant to Qinchuan Cattle growth traits；
(2) copy number variation type and ox growth traits etc. are associated analysis using 19.0 software of SPSS；
(3) the excellent Qinchuan Cattle breeding of growth traits is carried out according to copy number variation type.
1, Qinchuan Cattle sample collection
The present invention is specifically using place of china Qin Chuan cattle breeds as test object, the blood sample sample collection of 208 Qinchuan Cattles
From Shaanxi Province Qinchuan Cattle stock breeding center.
2, separation, extraction, the purifying of genomic DNA
Bibliography Sambrock et al (2002) method.
3, the amplification of target sequence and reference sequences
The ox SHH gene order (GenBank announced with ncbi database (http://www.ncbi.nlm.nih.gov/)
Accession No.AC_000161) it is reference sequences, real-time fluorescence quantitative PCR primer pair, which is designed, using Primer 5.0 detects
SHH gene copy number variation, primer pair sequence information are as shown in table 1.By drawing amplification curve (Fig. 1) and dissolution peak come really
Determine whether primer is suitable for qPCR analysis.According to the solubility curve of drafting, each sample curve coincide together, and curve tendency is flat
It slides, peak height and point, miscellaneous peak (Fig. 2) caused by primer free dimer or non-specific amplification.
Internalcontrol sequence is the known sequence that copy number variation is not present, the i.e. sequence of one section of 166bp in BTF3 gene.
The primer information of 1 real-time fluorescence quantitative PCR of table
Wherein, carry out amplification system used in real-time fluorescence quantitative PCR to be calculated as with 20 μ L: 50ng/ μ L template DNA (extracts
From the genomic DNA of blood sample sample) each 1 μ L, 2 × SYBR Green qPCR Mix of upstream and downstream primer of 1 μ L, 10pmol/L
10 μ L, 7 μ L of deionized water.
The response procedures of PCR amplification are as follows: (1) initial denaturation: 95 DEG C of 30s；(2) amplified reaction: 95 DEG C of denaturation 10s, 60 DEG C are moved back
Fiery 30s, 39 circulations；(3) drafting solubility curve: 95 DEG C of 5s, -0.01 DEG C/s, 65 DEG C of 1min.
4, the deduction of number variation is copied
Each sample is expanded with the primer of target sequence and reference sequences respectively, and 3 repetitions of each pair of primer.Root
According to 2-ΔΔCtThe analysis of method progress copy number.Wherein Δ Δ Ct=(CT target gene-CT reference gene)Experimental group-(CT target gene-CT reference gene)Control group。
Experimental group is to be detected whether there is or not the sample of CNVs, and control group is the known sample without copy number variation.2-ΔΔCtIndicate be
The opposite multiple with control group of the copy number of experimental group target sequence.Then the gene expression abundance of gene is carried out logarithmic transformed (with 2
For bottom 2-ΔΔCtLogarithm) be allowed to meet normal distribution, after carrying out homogeneity test of variance, the difference between statistical check each group.
When target sequence is normal type sequence, according to Log22-ΔΔCtCalculate 0 or so (Log of normalized value2
2-ΔΔCt≤|±0.5|).When target sequence is deletion form sequence, Log22-ΔΔCtCalculate normalized value Log22-ΔΔCt<-0.5.When target sequence is insert type sequence, Log22-ΔΔCtCalculate normalized value Log22-ΔΔCt>0.5。
5, the association analysis in the site Qinchuan Cattle SHH gene C NV and growth traits
The association analysis of table 2.1 SHH gene C NV and Qin Chuan cattle breeds growth traits
The association analysis of table 2.1 SHH gene C NV and Qin Chuan cattle breeds growth traits
Creation data: body length, body height, weight, bust, chest breadth, chest depth and hip cross are high.
Relation analysis model: analysis first is described to data, it is determined whether there are outliers, recycle least square point
Analysis is to Data correction；According to data characteristics, the production traits effect between each genotype is analyzed using 19 software of SPSS.To base
Fixed model is used when being analyzed because of type effect:
Yijk=μ+Ai+CNVj+eijk
Wherein: YijkFor character observation value, μ is population mean, AiFor i-th individual age, CNVjFor j-th of copy number
The fixed effect of variation type, eijkFor random error.Otherness between each group of data is tested using LSD Multiple range test, is tried
Test result is indicated in the form of Mean ± SE.
Association analysis is the result shows that (being shown in Table 2): the site Qinchuan Cattle SHH gene C NV can significantly affect body height, the body at 24 monthly ages
Length, weight, hip cross be high and body length, bust, weight, chest breadth and the chest depth at 30 monthly ages.Also, it is excellent in two age levels
Gesture copy number variation type is insert type, illustrates that the SHH gene site CNV can be used as a raising Qinchuan Cattle growth traits
Candidate molecules genetic marker.
6, above-mentioned CNV marks the application in Qinchuan Cattle breeding
The CNV of acquisition can be used as candidate molecules genetic marker, find influence ox growth associated therewith or close linkage
The quantitative trait locus of shape, to carry out molecular marker assisted selection to Qinchuan Cattle, to accelerate the choosing of Qinchuan Cattle breed improvement
Educate process.
Claims (6)
1. a kind of detection method for growing relevant CNV label to Qinchuan Cattle, it is characterised in that: the following steps are included: with Qin Chuan
Cow genome group DNA is template, expands the region gene C NV Qinchuan Cattle SHH and reference gene respectively by real-time fluorescence quantitative PCR
BTF3 identifies the copy number variation of Qinchuan Cattle SHH gene according to quantitative result, and wherein the growth traits of insert type individual is significantly high
In deletion form individual；
The CNV label refers to the copy number variation of ox SHH gene candidate region Chr4:118264951-118267170；
The amplimer pair in the region the Qinchuan Cattle SHH gene C NV are as follows:
Upstream primer F1:5 '-CGCACGCAATGAGACTTTA-3 ',
Downstream primer R1:5 '-AATAGCCAGGAGAGGTGAA-3 '.
2. a kind of detection method for growing relevant CNV label to Qinchuan Cattle as described in claim 1, it is characterised in that: described
Copy number variation be according to log2 2-ΔΔCtQuantitative result is divided into three classes: insert type, Log2 2-ΔΔCt>0.5；Deletion form,
Log2 2-ΔΔCt<-0.5；Normal type, Log2 2-ΔΔCt≤|±0.5|。
3. a kind of detection method for growing relevant CNV label to Qinchuan Cattle as described in claim 1, it is characterised in that: described
The amplimer pair of reference gene BTF3 are as follows:
Upstream primer F2:5 '-AACCAGGAGAAACTCGCCAA-3 ',
Downstream primer R2:5 '-TTCGGTGAAATGCCCTCTCG-3 '.
4. a kind of detection method for growing relevant CNV label to Qinchuan Cattle as described in claim 1, it is characterised in that: described
Real-time fluorescence quantitative PCR used in amplification system be calculated as with 20 μ L: the amplification Qin of 50ng/ μ L template DNA 1 μ L, 10pmol/L
Upstream and downstream primer corresponding to the primer pair of the river region gene C NV ox SHH and reference gene BTF3 each 1 μ L, 2 × SYBR
7 μ L of 10 μ L of Green qPCR Mix and deionized water.
5. a kind of detection method for growing relevant CNV label to Qinchuan Cattle as described in claim 1, it is characterised in that: described
Real-time fluorescence quantitative PCR used in response procedures are as follows: (1) 95 DEG C of 30s of initial denaturation；(2) amplified reaction: 95 DEG C of denaturation 10s, 60
DEG C annealing 30s, 39 circulation.
6. application of the method as described in any one of claim 1-5 claim in Qinchuan Cattle molecular breeding, special
Sign is: the CNV label applies the Qin Chuan cattle breeds outstanding in early molecule marker assisted selection growth traits.
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| CN106755444A (en) * | 2016-12-31 | 2017-05-31 | 东北农业大学 | A kind of soybean gene copy number analysis of variance method |
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| CN107119117B (en) * | 2017-04-25 | 2019-10-29 | 西北农林科技大学 | A kind of method and its application of detection Qinchuan Cattle GBP2 gene C NV label |
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