CN103667489A - DNA (Deoxyribonucleic Acid) marking method for identifying domestic duck, muscovy duck and mule duck - Google Patents
DNA (Deoxyribonucleic Acid) marking method for identifying domestic duck, muscovy duck and mule duck Download PDFInfo
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Abstract
The invention provides a DNA (Deoxyribonucleic Acid) marking method for identifying domestic duck, muscovy duck and mule duck, and mainly relates to the technical field of analytical biochemistry. The DNA marking method is characterized in that a pair of primers P1 is mainly designed and has the sequence shown as that an upstream primer F is in a sequence of 5'-CGTGCCCAACGAACTCTT-3', and a downstream primer R is in the sequence of 5'-CAGGGCGAACATGTGGA-3'; a PCR (Polymerase Chain Reaction) strip of about 523bp can be specifically amplified from duck DNA by PCR amplification and agarose gel electrophoresis; then the PCR-RFLP (Restriction Fragment Length Polymorphism) technology is performed, and the digestion result can be observed. The marketing method provided by the invention has the advantages of being fast, flexible, specific and the like, and brings a reliable method for identifying domestic duck, muscovy duck and mule duck groups.
Description
Technical field
A kind of DNA marker method that the present invention relates to effective connoisseur duck, kind duck and half kind of duck, belongs to technical field of analytical biochemistry.
Background technology
DNA molecular marker is that to take nucleotide diversity between individuality be basic genetic marker, the direct difference between detection of biological individuality on DNA level, it is the direct reflection of biont heritable variation on DNA level, compare with methods such as applying phenotypic character, production performance, biochemical marker, more can reflect really genetic background and the sibship of fowl kind.Utilize poultry kind DNA Genetic Markers, its result can be used as the scientific basis of identifying the poultry kind true and false.
The molecule marker that DNA tracing technology is used has AFLP(amplified fragment length polymorphism) mark, the micro-satellite of SSR() mark and SNP(single nucleotide polymorphism) mark.SNP mark is the third generation DNA molecular marker of American scholar proposition in 1996.SNP refers to the variation of single core thuja acid on the same site of genome, comprises conversion, transversion, deletion and insertion.SNP is modal a kind of in heritable variation, has the high feature of the wide density of distribution, and average every 1000 bases just have 1 SNP to exist.SNP mark is because of widely distributed, and inheritance stability, and suitable high throughput automated analysis, identify molecule marker of greatest concern so day by day become animal identification, and progressively replaces AFLP mark and SSR mark.
SNP mark is that gene order single core thuja acid morphs and produces the DNA molecular marker of polymorphism, and it is two equipotential gene polymorphics, and therefore a SNP can distinguish three animal individuals in theory, comprises two homozygotes and a heterozygote.The present invention utilizes the PCR-RFLP technology in SNP mark to distinguish a duck, kind duck, half kind of duck three class animal population.The method is simple and efficient, safe and reliable, expense is cheap, from effectively identifying in essence and distinguish family duck, kind duck and half Fan Yasanlei duck colony.
At present, on young seedling market, impure, the shoddy phenomenon of cultivar origin happens occasionally, the invention of the method, for family duck, kind duck and half kind of duck varieties confusion on effective solution market, effectively the loss of a protection man duck, kind duck and half kind of duck varieties pure and excellent genes is all significant; Meanwhile, can replace to a certain extent a kind duck live population, for kind duck varieties resource conservation provides the safest a kind of, the store method that the most reliable, standing charges are minimum.
Summary of the invention
The object of the present invention is to provide a kind of DNA marker method of effective connoisseur duck, kind duck and half kind of duck, the present invention mainly utilizes the PCR-RFLP technology in SNP mark effectively to distinguish a duck, kind duck, half kind of duck three class animal population.
For achieving the above object, the present invention adopts following technical scheme:
In the duck MC1R gene coding region containing SNP restriction enzyme site, design a pair of P1 primer, primer sequence is:
Upstream primer F:5'-CGTGCCCAACGA ACTCTT-3';
Downstream primer R:5'-CAGGGCGAACATGTGGA-3'.
A DNA marker method for connoisseur duck, kind duck and half kind of duck, its concrete steps comprise:
(1) venous blood collection, EDTA anti-freezing ,-20 ℃ of preservations; Genomic dna test kit extracts total DNA, and Telomerase (TE) dissolves, and-20 ℃ save backup;
(2) the primer upstream primer having designed: 5'-GCCAGTGAGGGCAACCAGAG-3'
Downstream primer: 5'-CTACCAGGAGCACAGCACCA-3' increases to the DNA of family duck, kind duck and half kind of duck respectively; Product reclaims, also forward and reverse order-checking of clone; SeqMan software in application DNAStar is compared to sequencing result, search SNP;
(3) design of primer
Utilize software Oligo 6.0 and Primer Primer5.0, at the duck MC1R gene coding region design pair of primers P1 containing SNP restriction enzyme site, upstream primer F and downstream primer R; Expection amplifies the PCR band of about 523bp;
(4) pcr amplification
Utilize primer P1 to carry out pcr amplification to a kind duck, half kind of duck and the Duck genome DNA of family, PCR reaction system is: 2 * Taq MasterMix, 12.5 μ L, upstream primer F 1 μ L, downstream primer R 1 μ L, template DNA 2 μ L, supplement ddH2O to final volume 25 μ L; PCR reaction conditions: 94 ℃ of 5 min, 94 ℃ of 50 s, 55.5 ℃ of 35 s, 72 ℃ of 40 s, totally 35 circulations; 72 ℃ of 10 min, 4 ℃ of preservations; The agarose gel electrophoresis that PCR product is 2% through massfraction detects expanding effect;
(5) PCR-RFLP detects
Get amplified production 5-6 μ L, add restriction enzyme Hin6
i1 μ L, 10 * Buffer, 1 μ L, adds ddH
2o is to final volume 10 μ L, 37 ℃ of endonuclease reactions spend the night, enzyme is cut the agarose gel electrophoresis that product is 3% with massfraction and is detected, adopt DL1000 DNA Marker as the standard control of molecular weight, observe enzyme and cut result, Bio-Rad gel imaging system is observed the preservation of taking pictures, by banding pattern judgement genotype;
(6) result
Utilize primer P1 to 3 Lei Ya colonies increase, cloning and sequencing comparison, find that A399G is positioned at restriction enzyme digestion Hin6
irecognition site (G*CGC), 1 available PCR-PFLP method detects the duck MC1R genetic marker of SNP, is the DNA marker method of connoisseur duck, kind duck and half kind of duck.
Duck genes of individuals group DNA, after primer P1 amplification electrophoresis, obtains that a brightness is strong, front and back are without the object band (Fig. 1) of the identical 523bp of non-specific band and size and desired value, so can continue RFLP restriction analysis.Amplified production has a Hin6
irestriction enzyme site, is cut to 334bp and two fragments of 189bp by product, and wherein AA genotype shows 1 band (523bp), and GG genotype shows 2 bands (334bp+189bp), and heterozygote AG genotype shows 3 bands (523bp+334bp+189bp).Range gene type as shown in Figure 2.
Family's duck varieties of measuring comprises that Beijing duck, the white duck of Fujian agriculture, Putian coot, mountain sheldrake, Taiwan White change duck, khaki Kang Beier duck.
The invention has the advantages that: the inventive method can directly be distinguished a duck, kind duck, half kind of duck three class animal population by this DNA molecular marker effectively, can be in duck, kind Ya Hebanfan duck colony a kind of reliable authentication method is provided, for long-term preservation and the utilization of duck DNA gene pool provides certain technical support.
Accompanying drawing explanation
Fig. 1 primer P
1amplified production electrophoresis result.
Fig. 2 PCR-RFLP restriction enzyme mapping.
Embodiment
Different duck colony's genotype distributions and gene frequency are in Table 1.As shown in Table 1, MC1R Gene A 399G be in mutational site distribute in duck, Fan Ya colony very single, wherein to change the family such as duck, a Ka Jikang Bel duck duck idiotype be all GG for the white duck of Fujian agriculture, Putian coot, Beijing duck, mountain sheldrake, Taiwan White, only has a kind of allelotrope G; The genotype of white kind of duck is all AA, only has a kind of allelotrope A; In the Ban Fan duck colony of this site, be low polymorphic, genotype be take AG as main (0.981), and minority is GG (0.019), and A, G allelotrope are suitable.
This research and utilization PCR-RFLP technology for detection the polymorphism in 8 MC1R of kind/colony Gene A 399G sites, result shows, Beijing duck, the white duck of Fujian agriculture, Putian coot, Taiwan White change 6 kinds such as duck, khaki Kang Beier duck, kind duck and all show as extreme singlet distribution, and wherein the genotype of 5 family's duck varieties is GG type; The genotype of kind duck is all AA type.This shows that A399G site is in quite conservative in duck, kind duck; But completely different between the two, being in duck only appears in this site G allelotrope, A/G catastrophic event does not occur in kind duck, this does not belong to for family duck and kind duck originate from the evidence that provides new together, illustrate that family duck and certain one-phase of kind duck evolutionary process have produced differentiation on this site, and then occurred different homozygous genotypes.In this research, though half kind of duck tool polymorphism belongs to extremely low polymorphic, genotype is almost heterozygote AG type entirely, and its frequency is up to 0.981; GG type only accounts for 0.019(and can ignore); G gene frequency is no better than A allelotrope, and this may be that family duck is relevant with kind duck intergeneric hybridization product with half kind of duck, and the genotype in this site is between two parents.
the different duck of table 1 colony's genotype and gene frequency
embodiment 1
(1) blood sampling 30 μ L genomic dna test kits extract total DNA, and TE dissolves, and-20 ℃ save backup.
(2) utilize primer P2 to carry out pcr amplification to the genomic dna of blood sample to be measured.PCR reaction system is: 2 * Taq MasterMix, 12.5 μ L, and upstream primer (F) 1 μ L, downstream primer (R) 1 μ L, template DNA 2 μ L, supplement ddH
2o is to final volume 25 μ L.PCR reaction conditions:: 94 ℃ of 5 min, 94 ℃ of 50 s, 55.5 ℃ of 35 s, 72 ℃ of 40 s, totally 35 circulations; 72 ℃ of 10 min, 4 ℃ of preservations.
(3) get amplified production 6 μ L, add restriction enzyme Hin6 I 1 μ L, 10 * Buffer, 1 μ L, adds ddH
2o is to final volume 10 μ L.37 ℃ of endonuclease reactions spend the night.Enzyme is cut product and is detected with 3% agarose gel electrophoresis, adopt the DNA Marker of DL1000 as the standard control of molecular weight, observe enzyme and cut result, Bio-Rad gel imaging system is observed the preservation of taking pictures, by banding pattern judgement genotype, and then the duck varieties of detected blood sample is judged.
(4) survey after blood sample PCR product enzyme is cut and only occur 1 band (523bp), illustrate that this duck kind is for kind duck.
embodiment 2
(1) blood sampling 30 μ L genomic dna test kits extract total DNA, and TE dissolves, and-20 ℃ save backup.
(2) utilize primer P2 to carry out pcr amplification to the genomic dna of blood sample to be measured.PCR reaction system is: 2 * Taq MasterMix, 12.5 μ L, and upstream primer (F) 1 μ L, downstream primer (R) 1 μ L, template DNA 2 μ L, supplement ddH
2o is to final volume 25 μ L.PCR reaction conditions:: 94 ℃ of 5 min, 94 ℃ of 50 s, 55.5 ℃ of 35 s, 72 ℃ of 40 s, totally 35 circulations; 72 ℃ of 10 min, 4 ℃ of preservations.
(3) get amplified production 6 μ L, add restriction enzyme Hin6 I 1 μ L, 10 * Buffer, 1 μ L, adds ddH2O to final volume 10 μ L.37 ℃ of endonuclease reactions spend the night.Enzyme is cut product and is detected with 3% agarose gel electrophoresis, adopt the DNA Marker of DL1000 as the standard control of molecular weight, observe enzyme and cut result, Bio-Rad gel imaging system is observed the preservation of taking pictures, by banding pattern judgement genotype, and then the duck varieties of detected blood sample is judged.
(4) there are 2 bands (334bp+189bp), illustrate that this duck kind is for family duck.
embodiment 3
(1) blood sampling 30 μ L genomic dna test kits extract total DNA, and TE dissolves, and-20 ℃ save backup.
(2) utilize primer P2 to carry out pcr amplification to the genomic dna of blood sample to be measured.PCR reaction system is: 2 * Taq MasterMix, 12.5 μ L, and upstream primer (F) 1 μ L, downstream primer (R) 1 μ L, template DNA 2 μ L, supplement ddH
2o is to final volume 25 μ L.PCR reaction conditions:: 94 ℃ of 5 min, 94 ℃ of 50 s, 55.5 ℃ of 35 s, 72 ℃ of 40 s, totally 35 circulations; 72 ℃ of 10 min, 4 ℃ of preservations.
(3) get amplified production 5 μ L, add restriction enzyme Hin6 I 1 μ L, 10 * Buffer, 1 μ L, adds ddH
2o is to final volume 10 μ L.37 ℃ of endonuclease reactions spend the night.Enzyme is cut product and is detected with 3% agarose gel electrophoresis, adopt the DNA Marker of DL1000 as the standard control of molecular weight, observe enzyme and cut result, Bio-Rad gel imaging system is observed the preservation of taking pictures, by banding pattern judgement genotype, and then the duck varieties of detected blood sample is judged.
(4) there are 3 bands (523bp+334bp+189bp), illustrate that this duck kind is half kind of duck.
The foregoing is only preferred embodiment of the present invention, all equalizations of doing according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.
SEQUENCE LISTING
<110> Fujian Province Academy Of Agricultural Sciences Animal Husbandry And Veterinary Medicine Institute
The DNA marker method of <120> connoisseur duck, kind duck and half kind of duck
<130>4
<160>4
<170>PatentIn version 3.3
<210>1
<211>18
<212>DNA
<213> artificial sequence
<400>1
cgtgcccaac gaactctt 18
<210>2
<211>17
<212>DNA
<213> artificial sequence
<400>2
cagggcgaac atgtgga 17
<210>3
<211>20
<212>DNA
<213> artificial sequence
<400>3
gccagtgagg gcaaccagag 20
<210>4
<211>20
<212>DNA
<213> artificial sequence
<400>4
ctaccaggag cacagcacca 20
Claims (3)
1. a DNA marker method for connoisseur duck, kind duck and half kind of duck, is characterized in that:
In the duck MC1R gene coding region containing SNP restriction enzyme site, design a pair of P1 primer, primer sequence is:
Upstream primer F:5'-CGTGCCCAACGA ACTCTT-3';
Downstream primer R:5'-CAGGGCGAA CATGTGGA-3'.
2. a DNA marker method for connoisseur duck, kind duck and half kind of duck, is characterized in that: its concrete steps comprise:
(1) duck is adopted to jugular vein blood sampling, EDTA anti-freezing ,-20 ℃ of preservations; Genomic dna test kit extracts total DNA, and Telomerase TE dissolves, and-20 ℃ save backup;
(2) utilize the primer having designed
Upstream primer: 5'-GCCAGTGAGGGCAACCAG AG-3';
Downstream primer: 5'-CTACCAGGAGCACAGCACCA-3', increases to the DNA of family duck, kind duck and half kind of duck respectively; Product reclaims, also forward and reverse order-checking of clone; SeqMan software in application DNAStar is compared to sequencing result, search SNP;
(3) design of primer
At the duck MC1R gene coding region design pair of primers P1 containing SNP restriction enzyme site, upstream primer F and downstream primer R;
(4) pcr amplification
Utilize primer P1 to carry out pcr amplification to a kind duck, half kind of duck and the Duck genome DNA of family, PCR reaction system is: 2 * Taq MasterMix, 12.5 μ L, upstream primer F 1 μ L, downstream primer R 1 μ L, template DNA 2 μ L, supplement ddH2O to final volume 25 μ L; PCR reaction conditions: 94 ℃ of 5 min, 94 ℃ of 50 s, 55.5 ℃ of 35 s, 72 ℃ of 40 s, totally 35 circulations; 72 ℃ of 10 min, 4 ℃ of preservations; The agarose gel electrophoresis that PCR product is 2% through massfraction detects expanding effect;
(5) PCR-RFLP detects
Get amplified production 5-6 μ L, add restriction enzyme Hin6
i1 μ L, 10 * Buffer, 1 μ L, adds ddH
2o is to final volume 10 μ L, 37 ℃ of endonuclease reactions spend the night, enzyme is cut the agarose gel electrophoresis that product is 3% with massfraction and is detected, adopt DL1000 DNA Marker as the standard control of molecular weight, observe enzyme and cut result, Bio-Rad gel imaging system is observed the preservation of taking pictures, by banding pattern judgement genotype;
(6) result
Utilize primer P1 to 3 Lei Ya colonies increase, cloning and sequencing comparison, find that A399G is positioned at restriction enzyme digestion Hin6
irecognition site, 1 available PCR-PFLP method detects the duck MC1R genetic marker of SNP, is the DNA marker method of connoisseur duck, kind duck and half kind of duck.
3. the DNA marker method of a kind of connoisseur duck according to claim 2, kind duck and half kind of duck, is characterized in that: family duck comprises that Beijing duck, the white duck of Fujian agriculture, Putian coot, mountain sheldrake, Taiwan White change duck, khaki Kang Beier duck.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105349689A (en) * | 2015-12-15 | 2016-02-24 | 西北农林科技大学 | Molecular marker for identifying muscovy duck eggs and applications thereof |
| CN105969887A (en) * | 2016-06-30 | 2016-09-28 | 华南农业大学 | Cairina moschata laying-related SNP (single nucleotide polymorphism) site and application thereof |
| CN108004330A (en) * | 2017-12-15 | 2018-05-08 | 中国农业大学 | A kind of molecular labeling and its application for being used to identify maple leaf duck |
| CN108004331A (en) * | 2017-12-15 | 2018-05-08 | 中国农业大学 | A kind of molecular labeling and its application for being used to identify Beijing duck |
| CN108018359A (en) * | 2017-12-15 | 2018-05-11 | 中国农业大学 | A kind of molecular labeling and its application for being used to identify cherry valley duck |
| CN111334588A (en) * | 2020-04-20 | 2020-06-26 | 四川农业大学 | Molecular marker related to black and brown feather characters of duck and application thereof |
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| CN103409562A (en) * | 2013-09-02 | 2013-11-27 | 福建省农业科学院畜牧兽医研究所 | PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) method for distinguishing goose parvovirus from muscovy duck parvovirus |
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| CN103409562A (en) * | 2013-09-02 | 2013-11-27 | 福建省农业科学院畜牧兽医研究所 | PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) method for distinguishing goose parvovirus from muscovy duck parvovirus |
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105349689A (en) * | 2015-12-15 | 2016-02-24 | 西北农林科技大学 | Molecular marker for identifying muscovy duck eggs and applications thereof |
| CN105349689B (en) * | 2015-12-15 | 2018-08-24 | 西北农林科技大学 | Differentiate molecular labeling and its application of kind duck's egg |
| CN105969887A (en) * | 2016-06-30 | 2016-09-28 | 华南农业大学 | Cairina moschata laying-related SNP (single nucleotide polymorphism) site and application thereof |
| CN105969887B (en) * | 2016-06-30 | 2019-10-22 | 华南农业大学 | A kind of relevant SNP site and its application of laying eggs to a kind duck |
| CN108004330A (en) * | 2017-12-15 | 2018-05-08 | 中国农业大学 | A kind of molecular labeling and its application for being used to identify maple leaf duck |
| CN108004331A (en) * | 2017-12-15 | 2018-05-08 | 中国农业大学 | A kind of molecular labeling and its application for being used to identify Beijing duck |
| CN108018359A (en) * | 2017-12-15 | 2018-05-11 | 中国农业大学 | A kind of molecular labeling and its application for being used to identify cherry valley duck |
| CN108004331B (en) * | 2017-12-15 | 2020-09-18 | 中国农业大学 | Molecular marker for identifying Beijing duck and application thereof |
| CN108004330B (en) * | 2017-12-15 | 2020-09-22 | 中国农业大学 | Molecular marker for identifying maple leaf ducks and application thereof |
| CN108018359B (en) * | 2017-12-15 | 2020-11-03 | 中国农业大学 | Molecular marker for identifying cherry valley duck and application thereof |
| CN111334588A (en) * | 2020-04-20 | 2020-06-26 | 四川农业大学 | Molecular marker related to black and brown feather characters of duck and application thereof |
| CN111334588B (en) * | 2020-04-20 | 2022-07-22 | 四川农业大学 | Molecular marker related to black and brown feather characters of duck and application thereof |
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