CN103571952B - Primer, kit and method for detecting genetype on black skin locus of chick - Google Patents
Primer, kit and method for detecting genetype on black skin locus of chick Download PDFInfo
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Abstract
The invention discloses a primer, kit and method for detecting a genetype on a black skin locus of a chick, belonging to the technical field of biology. The method comprises the steps: designing a primer pair P for an encoding gene EDN3; extracting the genomic DNA (Deoxyribose Nucleic Acid) of the chick to be detected; carrying out fluorogenic quantitative PCR (Polymerase Chain Reaction); detecting a relative copy number of the gene EDN3 on the black skin locus of the chick to be detected; and furthermore, judging the genetype on the black skin locus of the chick to be detected. The method is simple and convenient, accurate in typing, low in cost, high in efficiency, free of sequencing and specific instruments and easy to popularize. The genetype on the black skin locus of the chick to be detected can be judged through detecting the relative copy number of the gene EDN3 on the black skin locus, so that chicken are selected and remained to improve the consistency of skin colors of descendants; and the method is widely applied to the variety breeding of a black skin chick.
Description
Technical field
The present invention is specifically related to a kind of primer for detection of chicken crow skin loci gene type, and the test kit that comprises this primer and detection method, belongs to biological technical field.
Background technology
Gallus Domesticus is medicinal poultry well-known at home and abroad, within 1874, is admitted in the world for standard variety, and it has special medicinal, nutrition and ornamental value.Research shows, the pharmaceutical use of black-bone chicken is mainly its melanic effect, and black skin proterties is that human consumer expresses the most directly and judges melanochrome.
Crow skin locus Fm is the locus that affects chicken crow skin proterties, and the dominant allele Fm on its seat is the reason that causes chicken fibrid pigment hyperplasia (fibromelanosis), presents black look thereby make to carry this genotypic individual whole skin.At present, confirm chicken crow skin gene locus coding ATP5E(ATP synthase epsilonsubunit), TUBB1(tubulin, beta1), SLMO2(slowmo homolog2) and EDN3(endothelin3) four albumen.Research shows: chicken crow skin site allelotrope is due to ATP5E(ATP synthase epsilonsubunit), TUBB1(tubulin, beta1), SLMO2(slowmo homolog2) and EDN3(endothelin3) etc. gene copy number increase cause.Wherein, Endothelin3(EDN3) albumen is to affect the principal element that pigment cell is grown.On this locus, the variation of EDN3 gene copy number causes the excessive generation of pigment cell, and excessive pigment cell produces a large amount of melanochrome.Dominant allele Fm is four genotype that the relative copy number of gene is 2 such as EDN3, and Recessive alleles fm is four genotype that the relative copy number of gene is 1 such as EDN3.For the gene fragment of copy number variation, after conventional pcr amplification, agarose gel electrophoresis cannot carry out somatotype to homozygote and heterozygote, must use the method for quantitative fluorescent PCR that copy number is made a variation and identified, and still lack at present a kind of effective authentication method.
Summary of the invention
The object of this invention is to provide a kind of primer for detection of chicken crow skin loci gene type.
Meanwhile, the present invention also provides a kind of test kit for detection of chicken crow skin loci gene type.
Finally, the present invention also provides a kind of method that detects chicken crow skin loci gene type.
In order to realize above object, the technical solution adopted in the present invention is:
For detection of a primer for chicken crow skin loci gene type, comprise primer pair P,
Described primer pair P is:
Upstream primer P-F:5 '-CTTGACTTTTGGGATTTACCT-3 ' (as shown in SEQ ID No.1);
Downstream primer P-R:5 '-ACTGGTGCAGTATTTTCTGTT-3 ' (as shown in SEQ ID No.2).
For detection of a test kit for chicken crow skin loci gene type, mainly comprise primer pair P:
Upstream primer P-F:5 '-CTTGACTTTTGGGATTTACCT-3 ';
Downstream primer P-R:5 '-ACTGGTGCAGTATTTTCTGTT-3 '.
Preferably, a kind of test kit for detection of chicken crow skin loci gene type, also comprises quantitative fluorescent PCR 2 × damping fluid, sterilizing ultrapure water and fluorescent quantitation standard substance.
Described quantitative fluorescent PCR 2 × damping fluid mainly comprises 5U/ μ L Taq DNA Polymerase, 10 × Buffer, 25mM MgCl
2, 2mM dNTPs, SYBR Green I.
Described fluorescent quantitation standard substance are white skin chicken Whole Blood Genomic DNA, and concentration is respectively 80,40,20,10,5,2.5ng/ μ L.
A method that detects chicken crow skin loci gene type, comprises the following steps: taking the chicken Whole Blood Genomic DNA to be measured that comprises EDN3 gene as template, carry out fluorescent quantitative PCR taking primer pair P as primer, the DNA sequence dna of amplified fragments is as shown in SEQ ID No.3.The total length of this sequence is 134bp, is the molecule marker being positioned on the EDN3 gene of chicken crow skin site, can detect chicken crow skin gene locus EDN3 gene and whether have copy number variation.
Described fluorescent quantitative PCR reaction system is: quantitative fluorescent PCR 2 × damping fluid 10 μ L, ddH
2o7 μ L, P-F0.5 μ L, P-R0.5 μ L, DNA profiling 2 μ L.
Described fluorescent quantitative PCR response procedures is: 95 DEG C of denaturation 2min; 40 circulations: 95 DEG C of sex change 30s, 57 DEG C of annealing 30s, 72 DEG C are extended 30s; 72 DEG C are extended 10min; 20 DEG C of preservations.
Concrete, a kind of method that detects chicken crow skin loci gene type, further comprising the steps of: to utilize typical curve to ask the relative copy number of chicken EDN3 gene to be measured, according to the genotype in its relative copy number qualification chicken crow skin site.
The making method of described typical curve is: using white skin chicken Whole Blood Genomic DNA as fluorescent quantitation standard substance, concentration is respectively 80,40, and 20,10,5,2.5ng/ μ L, carries out fluorescent quantitative PCR taking primer pair P as primer, taking the Ct value of above-mentioned six standard substance as ordinate zou, taking the logarithmic value of concentration as X-coordinate, Criterion curve, tries to achieve typical curve equation.
The described typical curve that utilizes asks the method for the relative copy number of chicken EDN3 gene to be measured to be: before fluorescent quantitative PCR, first the DNA of chicken to be measured is diluted to same concentration 10ng/ μ L, again by the Ct value substitution typical curve equation of chicken to be measured, obtain its corresponding logarithm concentration, be converted into again concentration, be the relative copy number of this chicken EDN3 gene to be measured.
Described identifies that according to relative copy number the genotypic method in chicken crow skin site is: in the time that the relative copy number of chicken EDN3 gene to be measured is 2, this individuality is black skin homozygote (FmFm); In the time that the relative copy number of chicken EDN3 gene to be measured is 1.5, this individuality is black skin heterozygote (Fmfm); In the time that the relative copy number of chicken EDN3 gene to be measured is 1, this individuality is non-black skin homozygote (fmfm).
Beneficial effect of the present invention:
The present invention is taking the DNA full length sequence of chicken crow skin site EDN3 gene as template, design primer pair P, carry out quantitative fluorescent PCR, calculate the relative copy number of chicken crow skin to be measured site EDN3 gene, and then judge the genotype in chicken to be measured crow skin site, thereby group selects and remain to chicken, improve the consistence of offspring's skin color, in the seed selection of black skin chicken breed, there is widespread use.
The present invention, by extracting the genomic dna of chicken to be identified, is adjusted to its concentration unanimously (10ng/ μ L); Use primer pair P, carry out quantitative fluorescent PCR taking the DNA adjusting after concentration as template, utilize calibration curve method to calculate; The relative copy number of the EDN3 gene of white skin individuality is made as to 1, and in the time that the relative copy number of the EDN3 of chicken to be measured gene is 1.5, individuality to be measured is black skin heterozygote (Fmfm), and phenotype is black skin; In the time that the relative copy number of the EDN3 of chicken to be measured gene is 2, individuality to be measured is black skin homozygote (FmFm), and phenotype is black skin.The method is easy, and somatotype is accurate, and cost is low, and efficiency is high, does not need order-checking, does not need special instrument, easily popularizes.
The present invention is by detecting the relative copy number of EDN3 gene in chicken different skin phenotype colony, and the relative copy number of finding purebred black skin chicken kind EDN3 gene is 2 times of relative copy number of purebred white skin chicken kind EDN3 gene; And purebred black skin chicken kind and the relative copy number of the EDN3 gene of the filial generation of white skin chicken kind are 1.5 times of relative copy number of purebred white skin chicken kind EDN3 gene.
The present invention is by carrying out fluorescence quantitative PCR detection and sentence type and verified its accuracy Different Chicken varietal population and different genotype colony.Research shows that the method can diagnose the genotype in chicken crow skin site, the molecular marker assisted selection that can judge for chicken crow skin loci gene type effectively.
Brief description of the drawings
Fig. 1 is the technological line schematic diagram of the embodiment of the present invention 1;
Fig. 2 is the canonical plotting in embodiment 1;
Fig. 3 is the canonical plotting in embodiment 2.
Embodiment
Following embodiment is only described in further detail the present invention, but does not form any limitation of the invention.
Embodiment 1
The method that detects four different varieties chicken crow skin loci gene types in the present embodiment, technological line schematic diagram as shown in Figure 1, specifically comprises the following steps:
(1) preparation of sample and preservation
5 gu-shi chickens of Breeding Chicken Station, Henan Agricultural Univ. (white skin), 5 silk plumage Gallus Domesticuss (black skin), 5 Lushi chickens (white skin) and 5 chicken wings & legs with brown sauce (white skin) wing venous blood collection, add 1/3 antithrombotics, with chloroform-phynol method extraction blood DNA, DNA sample retention is for subsequent use in 4 DEG C of Refrigerator stores.
(2) DNA sample concentration is adjusted
Utilize micro-spectrophotometer to carry out concentration determination the DNA of testing sample, according to surveyed concentration, each testing sample DNA concentration is added to TE and be diluted to 10ng/ μ L, after dilution, measure with micro-ultraviolet spectrophotometer, 4 DEG C of Refrigerator stores are for subsequent use.
(3) preparation of standard substance
Getting at random a gu-shi chicken DNA sample, measure after concentration, be diluted to 80ng/ μ L, as standard substance mother liquor, is 80,40,20,10,5,2.5ng/ μ L by mother liquor doubling dilution, as 6 standard substance of production standard curve.
(4) design of primers
Taking the DNA sequence dna of chicken EDN3 gene (GenBank accession number as: NC_006107.3) as template, design primer pair P:
Upstream primer P-F:5 '-CTTGACTTTTGGGATTTACCT-3 ';
Downstream primer P-R:5 '-ACTGGTGCAGTATTTTCTGTT-3 '.
(5) fluorescent quantitative PCR
Taking primer pair P as amplimer, set up 20 μ L systems: quantitative fluorescent PCR 2 × damping fluid (mainly comprises 5U/ μ LTaq DNA Polymerase, 10 × Buffer, 25mM MgCl
2, 2mM dNTPs, SYBR Green I) and 10 μ L, ddH
2o7 μ L, P-F0.5 μ L, P-R0.5 μ L, DNA profiling 2 μ L; Response procedures is: 95 DEG C of denaturation 2min; 95 DEG C of sex change 30s, 57 DEG C of annealing 30s, 72 DEG C are extended 30s, 40 circulations; 72 DEG C are extended 10min; 20 DEG C of preservations.
(6) judgement of fluorescent quantitative PCR data processing and genotype result
Taking 80,40,20,10,5, the Ct value of 6 standard substance of 2.5ng/ μ L is as ordinate zou, taking the logarithm of concentration as X-coordinate, Criterion curve, as shown in Figure 2.Obtain the equation y=-0.285x+7.665 of typical curve, R
2=0.9986 and the amplification efficiency 92.72% of this reaction.
Bring the Ct value of individuality to be measured into equation, obtain its corresponding logarithm concentration, then be converted into concentration, be the relative copy number of this chicken to be measured.Test-results is as shown in table 1～4.
The relative copy number of table 1 gu-shi chicken crow skin site EDN3 gene
Note: GS represents gu-shi chicken.
A table 2 relative copy number of plumage Gallus Domesticus crow skin site EDN3 gene
Note: WJ represents a plumage Gallus Domesticus.
The relative copy number of table 3 Lushi chicken crow skin site EDN3 gene
Note: LS represents Lushi chicken.
The relative copy number of table 4 chicken wings & legs with brown sauce crow skin site EDN3 gene
Note: GF represents chicken wings & legs with brown sauce.
Can find out from table 1～4: 5 Agricultural University Of He'nan's gu-shi chickens that detect are all allozygote fmfm individuality; 5 Agricultural University Of He'nan silk plumage Gallus Domesticuss are all dominant homozygote FmFm individuality; 5 Lushi of Agricultural University Of He'nan chickens are all allozygote fmfm individuality; 5 Agricultural University Of He'nan's chicken wings & legs with brown sauce are all allozygote fmfm individuality.
Because the test of sample to be tested DNA concentration calibration and quantitative fluorescent PCR all can exist certain testing error, this error control, within 20%, is not affected to the type of sentencing to black skin site.
Embodiment 2
Test kit in the present embodiment mainly comprises primer pair P, also comprises that quantitative fluorescent PCR 2 × damping fluid (mainly comprises 5U/ μ L Taq DNA Polymerase, 10 × Buffer, 25mM MgCl
2, 2mM dNTPs, SYBR Green I), sterilizing ultrapure water and fluorescent quantitation standard substance, standard substance are white skin chicken Whole Blood Genomic DNA, concentration is respectively 80,40,20,10,5,2.5ng/ μ L, for the making of fluorescent quantitation test Plays curve;
Described primer pair P is:
Upstream primer P-F:5 '-CTTGACTTTTGGGATTTACCT-3 ';
Downstream primer P-R:5 '-ACTGGTGCAGTATTTTCTGTT-3 ';
The genotypic method that detects silk plumage Gallus Domesticus, Hai Saikesi A system and filial generation crow skin site thereof in the present embodiment, comprises the following steps:
(1) preparation of sample and preservation
The brown laying hen A of 5 her Sas of Breeding Chicken Station, Henan Agricultural Univ. system, 5 silk plumage Gallus Domesticuss, the orthogonal crossover offspring of the brown laying hen A of 5 silk plumage Gallus Domesticuss and her Sa system, wing venous blood collection, add 1/3 antithrombotics, with Proteinase K method extraction blood DNA, DNA sample retention is for subsequent use in 4 DEG C of Refrigerator stores.
(2) DNA sample concentration is adjusted
Utilize micro-spectrophotometer to carry out concentration determination the DNA of testing sample, according to surveyed concentration, each testing sample DNA concentration is added to TE and be diluted to 10ng/ μ L, after dilution, measure with micro-ultraviolet spectrophotometer, 4 DEG C of Refrigerator stores are for subsequent use.
(3) fluorescent quantitative PCR
Taking primer pair P in test kit as amplimer, set up 20 μ L systems: quantitative fluorescent PCR 2 × damping fluid (mainly comprises 5U/ μ L Taq DNA Polymerase, 10 × Buffer, 25mM MgCl
2, 2mM dNTPs, SYBR Green I) and 10 μ L, ddH
2o7.0 μ L, P-F0.5 μ L, P-R0.5 μ L, DNA profiling 2 μ L; Response procedures is: 95 DEG C of denaturation 2min; 95 DEG C of sex change 30s, 57 DEG C of annealing 30s, 72 DEG C are extended 30s, 40 circulations; 72 DEG C are extended 10min; 20 DEG C of preservations.
(4) preparation of typical curve
By 6 standard substance in test kit, its concentration is respectively 80,40,20,10,5,2.5ng/ μ L, as the sample of standard curve making, utilizes absolute quantitation method to obtain the concentration of sample to be tested, i.e. relative copy number.
(5) judgement of fluorescent quantitative PCR data processing and genotype result
With the Ct value of 6 standard substance of 80,40,20,10,5,2.5ng/ μ L for ordinate zou, taking the logarithmic value of concentration as X-coordinate, Criterion curve, as shown in Figure 3.Obtain the equation y=-0.285x+7.665 of typical curve, R
2=0.9986 and the amplification efficiency 92.72% of this reaction.
Bring the Ct value of chicken to be measured into equation, obtain its corresponding logarithm concentration, then be converted into concentration, be the relative copy number of this chicken crow skin to be measured site EDN3 gene.Test-results is as shown in table 5～7.
A table 5 relative copy number of the black skin of plumage Gallus Domesticus (FmFm) site EDN3 gene
Note: WJ represents a plumage Gallus Domesticus.
The relative copy number of table 6 Hai Saikesi A system's (fmfm) black skin site EDN3 gene
Note: HA represents Hai Saikesi A system.
The relative copy number of the black skin of table 7 filial generation (Fmfm) site EDN3 gene
Note: ZJ represents that a plumage Gallus Domesticus and Hai Saikesi A are orthogonal crossover offspring.
Can find out from table 5～7: the brown laying hen A of 5 her Sas of the Agricultural University Of He'nan system of detecting is all allozygote fmfm individuality; 5 Agricultural University Of He'nan silk plumage Gallus Domesticuss are all dominant homozygote FmFm individuality; The orthogonal crossover offspring of system of the brown laying hen A of 5 silk plumage Gallus Domesticuss and her Sa system is goneoclin Fmfm individuality.
Claims (7)
1. one kind is detected the method for chicken crow skin loci gene type, it is characterized in that: comprise the following steps: taking the chicken Whole Blood Genomic DNA to be measured that comprises EDN3 gene as template, carry out fluorescent quantitative PCR taking primer pair P as primer, the DNA sequence dna of amplified fragments is as shown in SEQ ID No.3;
Described primer pair P is:
Upstream primer P-F:5 '-CTTGACTTTTGGGATTTACCT-3 ',
Downstream primer P-R:5 '-ACTGGTGCAGTATTTTCTGTT-3 '.
2. the method for detection chicken crow skin loci gene type according to claim 1, is characterized in that: described fluorescent quantitative PCR reaction system is: quantitative fluorescent PCR 2 × damping fluid 10 μ L, ddH
2o7 μ L, P-F0.5 μ L, P-R0.5 μ L, DNA profiling 2 μ L.
3. the method for detection chicken crow skin loci gene type according to claim 1, is characterized in that: described fluorescent quantitative PCR response procedures is: 95 DEG C of denaturation 2min; 40 circulations: 95 DEG C of sex change 30s, 57 DEG C of annealing 30s, 72 DEG C are extended 30s; 72 DEG C are extended 10min; 20 DEG C of preservations.
4. the method for detection chicken crow skin loci gene type according to claim 1, is characterized in that: further comprising the steps of: utilize typical curve to try to achieve the relative copy number of chicken EDN3 gene to be measured, according to the genotype in its relative copy number qualification chicken crow skin site.
5. the method for detection chicken crow skin loci gene type according to claim 4, is characterized in that: the making method of described typical curve is: using white skin chicken Whole Blood Genomic DNA as fluorescent quantitation standard substance, concentration is respectively 80,40,20,10,5,2.5ng/ μ L, carry out fluorescent quantitative PCR taking primer pair P as primer, taking the Ct value of above-mentioned six standard substance as ordinate zou, taking the logarithmic value of concentration as X-coordinate, Criterion curve, tries to achieve typical curve equation.
6. the method for detection chicken crow skin loci gene type according to claim 4, it is characterized in that: the described typical curve that utilizes asks the method for the relative copy number of chicken EDN3 gene to be measured to be: before fluorescent quantitative PCR, first the DNA of chicken to be measured is diluted to same concentration 10ng/ μ L, again by the Ct value substitution typical curve equation of chicken to be measured, obtain its corresponding logarithm concentration, be converted into again concentration, be the relative copy number of this chicken EDN3 gene to be measured.
7. the method for detection chicken crow skin loci gene type according to claim 4, it is characterized in that: described identifies that according to relative copy number the genotypic method in chicken crow skin site is: in the time that the relative copy number of chicken EDN3 gene to be measured is 2, this individuality is black skin homozygote; In the time that the relative copy number of chicken EDN3 gene to be measured is 1.5, this individuality is black skin heterozygote; In the time that the relative copy number of chicken EDN3 gene to be measured is 1, this individuality is non-black skin homozygote.
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