CN101974647B - Molecular biological variety identification method for Huangjiaji chicken - Google Patents
Molecular biological variety identification method for Huangjiaji chicken Download PDFInfo
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Abstract
The invention discloses a molecular biological variety identification method for a Huangjiaji chicken, which comprises the following steps: accurately acquiring COI gene sequences of mitochondrion DNAs of a Huangjiaji chicken and a tested chicken based on gene cloning and DNA sequencing technologies, calculating variable sites, haplotypes and average nucleotide differences, nucleotide degrees of ramification and net genetic distances of the Huangjiaji chicken and the tested chicken by means of DnaSP4.10.7 software, constructing colony clustering charts and haplotype phylogenetic trees of the Huangjiaji chicken and the tested chicken, and determining whether the Huangjiaji chicken and the tested chicken are of the same variety according to the results of the constructed colony clustering charts and haplotype phylogenetic trees. By identifying whether the Huangjiaji chicken is true or false based on the precise software calculation and analysis results of the differences between varieties in genetic information, the invention has the characteristics of high accuracy, short identification time, low cost and the like.
Description
Technical field
The invention belongs to biological variety authenticate technology field, be specifically related to the molecular biology identification method of the good lucky chicken breed of a kind of emperor.
Background technology
The good lucky chicken of emperor is the novel fryer kind of high-quality of the up-to-date cultivation of modern new agriculture limited-liability company, and because of bright fragrant, the flavor alcohol of its meat, juice are many, the extensive parent who has received the human consumer looks at.But some illegal enterprises on the market, raiser's illegal production and to sell the phenomenon of personation, the good lucky chicken of counterfeit emperor of common occurrence, heavy damage the reputation of the good lucky chicken brand of emperor, influenced the development of modern new agriculture limited-liability company.Evaluation to the poultry kind mainly is to be labeled as according to examining breeding archives and performance test with morphology at present.But along with the continuous maturation of breeding technique, the macroscopic features of parent and filial generation has bigger similarity, is difficult to distinguish from macroscopic features.Identify just not enough science, accurate so utilize the morphology mark to carry out poultry kind (strain).If carry out performance test; The not only manpower of labor, time and high performance measurement expense; And the result of performance measurement also raised the influence of each link factor, so performance test also is difficult to accurately reflect the real production performance and the germplasm characteristic of each kind (strain).Dna molecular marker based on gene clone and dna sequencing technical development can disclose hereditary difference the most basic between the kind (base difference) accurately.The Mitochondrial DNA molecule marker just belongs to particularly COI (cytochrome C oxidase subunit base I) gene of this type mark; It has characteristics such as genetic independence, strict matrilinear inheritance, copy number height, inorganization specificity, rate of evolution be fast, has been widely used in colonies' evaluations such as whale, the mankind, birds.
Summary of the invention
Technical problem to be solved by this invention is the deficiency to above-mentioned prior art, provides a kind of accuracy high, and qualification time is short, the molecular biology identification method of the good lucky chicken breed of emperor that cost is low.
For solving the problems of the technologies described above, the technical scheme that the present invention adopts is: the molecular biology identification method of the good lucky chicken breed of a kind of emperor is characterized in that this method may further comprise the steps:
(1) extracts total DNA that the good lucky chicken of emperor and tested chicken comprise Mitochondrial DNA respectively through the benzene phenol-chloroform method;
(2) according to the COI gene order (AP003322) of the Red Jungle-fowl Mitochondrial DNA of delivering on the GeneBank and COI gene order (AB086102) the design three couples of primer PF1 (SEQ ID NO.3) and the PR1 (SEQ ID NO.4) of silk plumage Gallus Domesticus Mitochondrial DNA; PF2 (SEQ IDNO.5) and PR2 (SEQ ID NO.6), PF3 (SEQ ID NO.7) and PR3 (SEQ ID NO.8); The sequence of said primer is respectively:
Upstream primer PF1:5 '-TACAGCCTAACGCTTCAACA-3 ';
Downstream primer PR1:5 '-GGTTGCGGTCGGTAAGTAGT-3 '.
Upstream primer PF2:5 '-GCCCATGCTTTCGTCATAA-3 ';
Downstream primer PR2:5 '-TGGGATGGCGATGATTATTG-3 '.
Upstream primer PF3:5 '-CGCCATCCCAACTGGTAT-3 ';
Downstream primer PR3:5 '-GATGAGGCGTCTTGAAAG-3 '.
(3) utilize primer described in the step (2) that the COI gene of the good lucky chicken Mitochondrial DNA of emperor is carried out pcr amplification, with the product cloning of amplification and the complete sequence (SEQ ID NO.1) of the COI gene that obtains the good lucky chicken Mitochondrial DNA of emperor of on the dna sequencing appearance, checking order;
(4) according to the complete sequence design primer PF (SEQ ID NO.9) and the PR (SEQ ID NO.10) of the COI gene of the good lucky chicken Mitochondrial DNA of emperor that records in the step (3); The sequence of said primer is:
Upstream primer PF:5 '-GCACAGGATGGACAGTTTAC-3 ';
Downstream primer PR:5 '-ATAGCATAGGGGGGTCTCAT-3 '.
Utilize said primer to obtain the fragment gene sequence (SEQ ID NO.2) in the good lucky chicken COI gene of the emperor who can be used for cultivar identification, check order, and accurately read sequencing result through Chromas1.49 software;
(5) utilize primer described in the step (4) to obtain the corresponding gene order of a fragment gene sequence (SEQ ID NO.2) in the good lucky chicken COI gene of the emperor who records in tested chicken and the step (4), check order, and accurately read sequencing result through Chromas1.49 software;
(6) sequencing result that the sequencing result that step (4) is obtained and step (5) obtain carries out manual work and pursues base and read and check;
(7) variant sites, haplotype, average nucleotide difference, Nucleotide degree of ramification and the clean genetic distance through the good lucky chicken of DnaSP4.10.7 software statistics emperor and tested chicken; And according to the variant sites of good lucky chicken of emperor and tested chicken, haplotype, average nucleotide difference, Nucleotide degree of ramification and clean genetic distance; Confirm the average degree of variation of good lucky chicken of emperor and tested chicken gene order; Average degree of variation is not more than 2% and is same kind, and average degree of variation is a different varieties greater than 2%;
(8) the utilization biological software makes up colony's dendrogram and the haplotype phylogenetic tree of good lucky chicken of emperor and tested chicken; Confirm according to the colony's dendrogram that makes up and the result of haplotype phylogenetic tree whether good lucky chicken of emperor and tested chicken are same kind; To be one type be same kind then if the haplotype of the good lucky chicken of emperor and tested chicken gathers, otherwise be different varieties.
Making up the good lucky chicken of emperor and colony's dendrogram of tested chicken and the method for haplotype phylogenetic tree described in the above-mentioned steps (8) is: utilization MEGA4.0 software is estimated the two-parameter genetic distance of Kimura between good lucky chicken of emperor and the tested chicken; And, use NETWORK4.5.0.1 software building colony's dendrogram and haplotype phylogenetic tree simultaneously based on Kimura two-parameter model application UPGMA/NJ method structure colony's dendrogram and haplotype phylogenetic tree.
The present invention compared with prior art has the following advantages: the present invention utilizes interracial genetic information difference to identify the good lucky chicken breed of true and false emperor through the accurate result who calculates and analyze of software, has accuracy height, qualification time weak point and low cost and other advantages.
Through embodiment technical scheme of the present invention is done further detailed description below.
Description of drawings
Fig. 1 is colony's dendrogram of good lucky chicken of the embodiment of the invention 2 emperors and tested chicken.
Fig. 2 is the haplotype phylogenetic tree of good lucky chicken of the embodiment of the invention 2 emperors and tested chicken.
Fig. 3 is colony's dendrogram of good lucky chicken of the embodiment of the invention 3 emperors and tested chicken.
Fig. 4 is the haplotype phylogenetic tree of good lucky chicken of the embodiment of the invention 3 emperors and tested chicken.
Embodiment
Embodiment 1
Tested chicken is the good lucky chicken of emperor:
(1) extracts total DNA that the good lucky chicken of emperor (1, the kind code name is HJJ1) and tested chicken (2 of the good lucky chickens of emperor, the kind code name is SHJJ1 and SHJJ2) comprise Mitochondrial DNA respectively through the benzene phenol-chloroform method;
(2) according to the COI gene order AP003322 of the Red Jungle-fowl Mitochondrial DNA of delivering on the GeneBank and COI gene order AB086102 design three couples of primer PF1 (SEQ ID NO.3), the PR1 (SEQ ID NO.4) of silk plumage Gallus Domesticus Mitochondrial DNA, PF2 (SEQ ID NO.5), PR2 (SEQ ID NO.6) and PF3 (SEQ ID NO.7), PR3 (SEQ ID NO.8); The sequence of said primer is respectively:
Upstream primer PF1:5 '-TACAGCCTAACGCTTCAACA-3 ';
Downstream primer PR1:5 '-GGTTGCGGTCGGTAAGTAGT-3 '.
Upstream primer PF2:5 '-GCCCATGCTTTCGTCATAA-3 ';
Downstream primer PR2:5 '-TGGGATGGCGATGATTATTG-3 '.
Upstream primer PF3:5 '-CGCCATCCCAACTGGTAT-3 ';
Downstream primer PR3:5 '-GATGAGGCGTCTTGAAAG-3 '.
(3) utilize primer described in the step (2) that the COI gene of the good lucky chicken Mitochondrial DNA of emperor is carried out pcr amplification, with the product cloning of amplification and the complete sequence (SEQ ID NO.1) of the COI gene that obtains the good lucky chicken Mitochondrial DNA of emperor of on the dna sequencing appearance, checking order;
Utilize primer PF1, PR2 carries out pcr amplification: dna profiling 100ng, 10 * buffer2.5 μ L; 25mM mg ion 2 μ L, 10mM dNTP 0.5 μ L, mix primer (upstream primer PF1 and downstream primer PR1 respectively are 10pmol/ μ L) 0.5 μ L; TaqDNA polysaccharase 1U adds water to 25 μ L.PCR response procedures: 94 ℃ of preparatory sex change 5min; 94 ℃ of sex change 45s; 58 ℃ of annealing 45s; 72 ℃ are extended 1min; Carry out 35 circulations; Last 72 ℃ are extended 10min;
Utilize primer PF2, PR2 carries out pcr amplification: dna profiling 100ng, 10 * buffer2.5 μ L; 25mM mg ion 2 μ L, 10mM dNTP 0.5 μ L, mix primer (upstream primer PF2 and downstream primer PR2 respectively are 10pmol/ μ L) 0.5 μ L; TaqDNA polysaccharase 1U adds water to 25 μ L.PCR response procedures: 94 ℃ of preparatory sex change 5min; 94 ℃ of sex change 45s; 56 ℃ of annealing 45s; 72 ℃ are extended 1min; Carry out 35 circulations; Last 72 ℃ are extended 10min;
Utilize primer PF3, PR3 carries out pcr amplification: dna profiling 100ng, 10 * buffer2.5 μ L; 25mM mg ion 2 μ L, 10mM dNTP 0.5 μ L, mix primer (upstream primer PF3 and downstream primer PR3 respectively are 10pmol/ μ L) 0.5 μ L; TaqDNA polysaccharase 1U adds water to 25 μ L.PCR response procedures: 94 ℃ of preparatory sex change 5min; 94 ℃ of sex change 45s; 59 ℃ of annealing 45s; 72 ℃ are extended 1min; Carry out 35 circulations; Last 72 ℃ are extended 10min;
(4) according to the complete sequence design primer PF (SEQ ID NO.9) and the PR (SEQ ID NO.10) of the COI gene of the good lucky chicken Mitochondrial DNA of emperor that records in the step (3):
Upstream primer PF:5 '-GCACAGGATGGACAGTTTAC-3 ';
Downstream primer PR:5 '-ATAGCATAGGGGGGTCTCAT-3 '.
Utilize said primer to obtain the fragment gene sequence (SEQ ID NO.2) in the good lucky chicken COI gene of the emperor who can be used for cultivar identification, check order, and accurately read sequencing result through Chromas1.49 software;
Utilize primer PF, PR carries out pcr amplification: dna profiling 100ng, 10 * buffer2.5 μ L; 25mM mg ion 2 μ L, 10mM dNTP 0.5 μ L, mix primer (upstream primer and downstream primer respectively are 10pmol/ μ L) 0.5 μ L; TaqDNA polysaccharase 1U adds water to 25 μ L.PCR response procedures: 94 ℃ of preparatory sex change 5min; 94 ℃ of sex change 45s; 55 ℃ of annealing 45s; 72 ℃ are extended 1min; Carry out 38 circulations; Last 72 ℃ are extended 10min;
(5) utilize primer described in the step (4) to obtain the corresponding gene order of a fragment gene sequence (SEQ ID NO.2) in the good lucky chicken COI gene of the emperor who records in tested chicken and the step (4), check order, and accurately read sequencing result through Chromas1.49 software; The same step of pcr amplification condition and response procedures (4);
(6) sequencing result that the sequencing result that step (4) is obtained and step (5) obtain carries out manual work and pursues base and read and check;
(7) variant sites, haplotype, average nucleotide difference, Nucleotide degree of ramification and the clean genetic distance through the good lucky chicken of DnaSP4.10.7 software statistics emperor and tested chicken; And, confirm the average degree of variation of good lucky chicken of emperor and tested chicken gene order according to the variant sites of good lucky chicken of emperor and tested chicken, haplotype, average nucleotide difference, Nucleotide degree of ramification and clean genetic distance;
(8) utilization MEGA4.0 software is estimated the two-parameter genetic distance of Kimura between good lucky chicken of emperor and the tested chicken; And use colony's dendrogram and the haplotype phylogenetic tree that the UPGMA/NJ method makes up good lucky chicken of emperor and tested chicken based on the Kimura two-parameter model, use colony's dendrogram and the haplotype phylogenetic tree of good lucky chicken of NETWORK4.5.0.1 software building emperor and tested chicken simultaneously.
Qualification result: the upstream and downstream primer that adopts design; Obtained the COI gene one segment length 651bp sequence that the good lucky chicken of emperor and tested chicken amount to 3 individuals through sequencing technologies; Variant sites is not found in the comparison of utilization Chromas1.49 software; Utilization DnaSP4.10.7 software discovery all sequences has a kind of haplotype (Hap1:CAG), and the various degree of haplotype is 0.000 between good lucky chicken of emperor and the tested chicken sequence, and the diverse oligonucleotide degree is 0.000; Average nucleotide difference is 0.000, and the average degree of variation of gene order is 0%.
Through the gained result, drawing kind to be measured is the good lucky chicken of emperor.
Embodiment 2
Tested chicken is native chicken:
(1) passes through total DNA that benzene phenol-chloroform method extraction good lucky chicken of emperor (1, the kind code name is HJJ1) and tested chicken (3 of native chickens, kind code name are TJ1, TJ2 and TJ3) comprise Mitochondrial DNA;
(2) according to the COI gene order AP003322 of the Red Jungle-fowl Mitochondrial DNA of delivering on the GeneBank and COI gene order AB086102 design three couples of primer PF1 (SEQ ID NO.3), the PR1 (SEQ ID NO.4) of silk plumage Gallus Domesticus Mitochondrial DNA, PF2 (SEQ ID NO.5), PR2 (SEQ ID NO.6) and PF3 (SEQ ID NO.7), PR3 (SEQ ID NO.8); The sequence of said primer is respectively:
Upstream primer PF1:5 '-TACAGCCTAACGCTTCAACA-3 ';
Downstream primer PR1:5 '-GGTTGCGGTCGGTAAGTAGT-3 '.
Upstream primer PF2:5 '-GCCCATGCTTTCGTCATAA-3 ';
Downstream primer PR2:5 '-TGGGATGGCGATGATTATTG-3 '.
Upstream primer PF3:5 '-CGCCATCCCAACTGGTAT-3 ';
Downstream primer PR3:5 '-GATGAGGCGTCTTGAAAG-3 '.
(3) utilize primer described in the step (2) that the COI gene of the good lucky chicken Mitochondrial DNA of emperor is carried out pcr amplification, with the product cloning of amplification and the complete sequence (SEQ ID NO.1) of the COI gene that obtains the good lucky chicken Mitochondrial DNA of emperor of on the dna sequencing appearance, checking order;
(4) according to the complete sequence design primer PF (SEQ ID NO.9) and the PR (SEQ ID NO.10) of the COI gene of the good lucky chicken Mitochondrial DNA of emperor that records in the step (3):
Upstream primer PF:5 '-GCACAGGATGGACAGTTTAC-3 ';
Downstream primer PR:5 '-ATAGCATAGGGGGGTCTCAT-3 '.
Utilize said primer to obtain the fragment gene sequence (SEQ ID NO.2) in the good lucky chicken COI gene of the emperor who can be used for cultivar identification, check order, and accurately read sequencing result through Chromas1.49 software;
(5) utilize primer described in the step (4) to obtain the corresponding gene order of a fragment gene sequence (SEQ ID NO.2) in the good lucky chicken COI gene of emperor that records in tested chicken (native chicken) and the step (4); Check order, and accurately read sequencing result through Chromas1.49 software;
(6) sequencing result that the sequencing result that step (4) is obtained and step (5) obtain carries out manual work and pursues base and read and check;
(7) variant sites, haplotype, average nucleotide difference, Nucleotide degree of ramification and the clean genetic distance through the good lucky chicken of DnaSP4.10.7 software statistics emperor and tested chicken (native chicken); And, confirm the average degree of variation of good lucky chicken of emperor and tested chicken (native chicken) gene order according to the variant sites of good lucky chicken of emperor and tested chicken (native chicken), haplotype, average nucleotide difference, Nucleotide degree of ramification and clean genetic distance;
(8) utilization MEGA4.0 software is estimated the two-parameter genetic distance of Kimura between good lucky chicken of emperor and the tested chicken (native chicken); And use colony's dendrogram and the haplotype phylogenetic tree that the UPGMA/NJ method makes up good lucky chicken of emperor and tested chicken based on the Kimura two-parameter model; Use colony's dendrogram of good lucky chicken of NETWORK4.5.0.1 software building emperor and tested chicken simultaneously, and the haplotype phylogenetic tree.
Pcr amplification condition in the present embodiment and response procedures are with embodiment 1.
Qualification result: adopt the upstream and downstream primer of design, obtained the COI gene one segment length 651bp sequence that the good lucky chicken of emperor and tested chicken (native chicken) amount to 4 individuals through sequencing technologies, 3 variant sites (C-G/53bp places are found in the comparison of utilization Chromas1.49 software; The A-T/282bp place, the C-G/606bp place), utilization DnaSP4.10.7 software discovery has 4 kinds of haplotypes; 1 good lucky chicken individuals of emperor is enjoyed a kind of haplotype (Hap1:CAG), and 3 tested chickens (native chicken) are shared 3 kinds of haplotypes (Hap2, Hap3; Hap4:GAC, CTC, CAC); The various degree of haplotype is 0.800 between good lucky chicken of emperor and tested chicken (native chicken) sequence; The diverse oligonucleotide degree is 0.00195, and the average degree of variation that average nucleotide difference is a gene order between 1.26667,2 kinds is greater than 2%.Utilization MEGA4.0 computed in software goes out that the two-parameter genetic distance of Kimura is 0.00257 between Huang Jiaji chicken and the tested chicken (native chicken); Use the UPGMA method based on the Kimura two-parameter model and make up colony's dendrogram (like Fig. 1), the good lucky chicken of emperor gathers in different branches with tested chicken (native chicken).Utilization NETWORK4.5.0.1 software building haplotype phylogenetic tree (like Fig. 2), (Hap2, Hap3 Hap4) gather in different branches the haplotype 1 (Hap1) that the good lucky chicken individuals of emperor is enjoyed with 3 kinds of shared haplotypes of 3 tested chickens (native chicken).
Through the gained result, drawing kind to be measured is not the good lucky chicken of emperor.
Embodiment 3
Tested chicken is a black-bone chicken:
(1) passes through total DNA that benzene phenol-chloroform method extraction good lucky chicken of emperor (1, the kind code name is HJJ1) and tested chicken (3 of black-bone chickens, kind code name are WJ1, WJ2 and WJ3) comprise Mitochondrial DNA;
(2) according to the COI gene order AP003322 of the Red Jungle-fowl Mitochondrial DNA of delivering on the GeneBank and COI gene order AB086102 design three couples of primer PF1 (SEQ ID NO.3), the PR1 (SEQ ID NO.4) of silk plumage Gallus Domesticus Mitochondrial DNA, PF2 (SEQ ID NO.5), PR2 (SEQ ID NO.6) and PF3 (SEQ ID NO.7), PR3 (SEQ ID NO.8); The sequence of said primer is respectively:
Upstream primer PF1:5 '-TACAGCCTAACGCTTCAACA-3 ';
Downstream primer PR1:5 '-GGTTGCGGTCGGTAAGTAGT-3 '.
Upstream primer PF2:5 '-GCCCATGCTTTCGTCATAA-3 ';
Downstream primer PR2:5 '-TGGGATGGCGATGATTATTG-3 '.
Upstream primer PF3:5 '-CGCCATCCCAACTGGTAT-3 ';
Downstream primer PR3:5 '-GATGAGGCGTCTTGAAAG-3 '.
(3) utilize primer described in the step (2) that the COI gene of the good lucky chicken Mitochondrial DNA of emperor is carried out pcr amplification, with the product cloning of amplification and the complete sequence (SEQ ID NO.1) of the COI gene that obtains the good lucky chicken Mitochondrial DNA of emperor of on the dna sequencing appearance, checking order;
(4) according to the complete sequence design primer PF (SEQ ID NO.9) and the PR (SEQ ID NO.10) of the COI gene of the good lucky chicken Mitochondrial DNA of emperor that records in the step (3):
Upstream primer PF:5 '-GCACAGGATGGACAGTTTAC-3 ';
Downstream primer PR:5 '-ATAGCATAGGGGGGTCTCAT-3 '.
Utilize said primer to obtain the fragment gene sequence (SEQ ID NO.2) in the good lucky chicken COI gene of the emperor who can be used for cultivar identification, check order, and accurately read sequencing result through Chromas1.49 software;
(5) utilize primer described in the step (4) to obtain the corresponding gene order of a fragment gene sequence (SEQ ID NO.2) in the good lucky chicken COI gene of emperor that records in tested chicken (black-bone chicken) and the step (4); Check order, and accurately read sequencing result through Chromas1.49 software;
(6) sequencing result that the sequencing result that step (4) is obtained and step (5) obtain carries out manual work and pursues base and read and check;
(7) variant sites, haplotype, average nucleotide difference, Nucleotide degree of ramification and the clean genetic distance through the good lucky chicken of DnaSP4.10.7 software statistics emperor and tested chicken (black-bone chicken); And, confirm the average degree of variation of good lucky chicken of emperor and tested chicken (black-bone chicken) gene order according to the variant sites of good lucky chicken of emperor and tested chicken (black-bone chicken), haplotype, average nucleotide difference, Nucleotide degree of ramification and clean genetic distance;
(8) utilization MEGA4.0 software is estimated the two-parameter genetic distance of Kimura between good lucky chicken of emperor and the tested chicken (black-bone chicken); And use colony's dendrogram and the haplotype phylogenetic tree that the UPGMA/NJ method makes up good lucky chicken of emperor and tested chicken based on the Kimura two-parameter model; Use colony's dendrogram and the haplotype phylogenetic tree of good lucky chicken of NETWORK4.5.0.1 software building emperor and tested chicken simultaneously, according to the result of the colony's dendrogram and the haplotype phylogenetic tree of two kinds of softwares.
Pcr amplification condition in the present embodiment and response procedures are with embodiment 1.
Qualification result: adopt the upstream and downstream primer of design, obtained the COI gene one segment length 651bp sequence that the good lucky chicken of emperor and tested chicken (black-bone chicken) amount to 4 individuals through sequencing technologies, 3 variant sites (T-A/9bp places are found in the comparison of utilization Chromas1.49 software; The A-C/46bp place, the C-G/386bp place), utilization DnaSP4.10.7 software discovery has 3 kinds of haplotypes; 1 good lucky chicken individuals of emperor is enjoyed a kind of haplotype (Hap1:TAC); The shared 2 kinds of haplotypes of 3 tested chickens (black-bone chicken) (Hap2, Hap3:TCG, ACG); The various degree of haplotype is 0.733 between good lucky chicken of emperor and tested chicken (black-bone chicken) sequence; The diverse oligonucleotide degree is 0.00236, and the average degree of variation that average nucleotide difference is a gene order between 1.53333,2 kinds is greater than 2%.Utilization MEGA4.0 computed in software goes out that the two-parameter genetic distance of Kimura is 0.0036 between Huang Jiaji chicken and the tested chicken (black-bone chicken); Use the UPGMA/NJ method based on the Kimura two-parameter model and make up colony's dendrogram (like Fig. 3), the good lucky chicken of emperor gathers in different branches with tested black-bone chicken.Utilization NETWORK4.5.0.1 software building haplotype phylogenetic tree (like Fig. 4), 1 haplotype 1 (Hap1) that the good lucky chicken individuals of emperor is enjoyed, (Hap2 Hap3) gathers in different branches 32 kinds of shared haplotypes of tested black-bone chicken.
Through the gained result, drawing kind to be measured is not the good lucky chicken of emperor.
The above; It only is preferred embodiment of the present invention; Be not that the present invention is done any restriction, every technical spirit all still belongs in the protection domain of technical scheme of the present invention any simple modification, change and equivalent structure transformation that above embodiment did according to the present invention.
Claims (2)
1. the molecular biology identification method of the good lucky chicken breed of emperor is characterized in that, this method may further comprise the steps:
(1) extracts total DNA that the good lucky chicken of emperor and tested chicken comprise Mitochondrial DNA respectively through the benzene phenol-chloroform method;
(2) according to the COI gene order AP003322 of Red Jungle-fowl Mitochondrial DNA and three pairs of primers of COI gene order AB086102 design of silk plumage Gallus Domesticus Mitochondrial DNA; The sequence of said primer is respectively:
Upstream primer PF1:5 '-TACAGCCTAACGCTTCAACA-3 ';
Downstream primer PR1:5 '-GGTTGCGGTCGGTAAGTAGT-3 '.
Upstream primer PF2:5 '-GCCCATGCTTTCGTCATAA-3 ';
Downstream primer PR2:5 '-TGGGATGGCGATGATTATTG-3 '.
Upstream primer PF3:5 '-CGCCATCCCAACTGGTAT-3 ';
Downstream primer PR3:5 '-GATGAGGCGTCTTGAAAG-3 '.
(3) utilize primer described in the step (2) that the COI gene of the good lucky chicken Mitochondrial DNA of emperor is carried out pcr amplification, with the product cloning of amplification and the complete sequence of the COI gene that obtains the good lucky chicken Mitochondrial DNA of emperor of on the dna sequencing appearance, checking order;
(4) according to the complete sequence design primer of the COI gene of the good lucky chicken Mitochondrial DNA of emperor that records in the step (3); The sequence of said primer is:
Upstream primer PF:5 '-GCACAGGATGGACAGTTTAC-3 ';
Downstream primer PR:5 '-ATAGCATAGGGGGGTCTCAT-3 '.
Utilize said primer to obtain the fragment gene sequence in the good lucky chicken COI gene of the emperor who can be used for cultivar identification, check order, and accurately read sequencing result through Chromas1.49 software;
(5) utilize primer described in the step (4) to obtain the corresponding gene order of a fragment gene sequence in the good lucky chicken COI gene of the emperor who records in tested chicken and the step (4), check order, and accurately read sequencing result through Chromas1.49 software;
(6) sequencing result that the sequencing result that step (4) is obtained and step (5) obtain carries out manual work and pursues base and read and check;
(7) variant sites, haplotype, average nucleotide difference, Nucleotide degree of ramification and the clean genetic distance through the good lucky chicken of DnaSP4.10.7 software statistics emperor and tested chicken; And confirm the average degree of variation of good lucky chicken of emperor and tested chicken gene order according to the variant sites of good lucky chicken of emperor and tested chicken, haplotype, average nucleotide difference, Nucleotide degree of ramification and clean genetic distance; Be same kind if average degree of variation is not more than 2%, average degree of variation is a different varieties greater than 2%;
(8) the utilization biological software makes up colony's dendrogram and the haplotype phylogenetic tree of good lucky chicken of emperor and tested chicken; Confirm according to the colony's dendrogram that makes up and the result of haplotype phylogenetic tree whether good lucky chicken of emperor and tested chicken are same kind; To be one type be same kind then if the haplotype of the good lucky chicken of emperor and tested chicken gathers, otherwise be different varieties.
2. the molecular biology identification method of the good lucky chicken breed of a kind of emperor according to claim 1; It is characterized in that; Making up the good lucky chicken of emperor and colony's dendrogram of tested chicken and the method for haplotype phylogenetic tree described in the step (8) is: utilization MEGA4.0 software is estimated the two-parameter genetic distance of Kimura between good lucky chicken of emperor and the tested chicken; And, use NETWORK4.5.0.1 software building colony's dendrogram and haplotype phylogenetic tree simultaneously based on Kimura two-parameter model application UPGMA/NJ method structure colony's dendrogram and haplotype phylogenetic tree.
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| CN112513298B (en) * | 2018-12-12 | 2023-12-01 | 忠南大学校产学协力团 | Mononucleotide polymorphism marker set for distinguishing genetic background or variety of local chicken and application thereof |
| CN113005207B (en) * | 2021-04-23 | 2021-10-22 | 江苏省家禽科学研究所 | Haploid marker for identifying camellia chicken variety based on sex chromosome and application thereof |
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| CN101016565A (en) * | 2007-02-09 | 2007-08-15 | 江苏省家禽科学研究所 | Method of identifying chicken variety by DNA strip shape coding |
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| CN101016565A (en) * | 2007-02-09 | 2007-08-15 | 江苏省家禽科学研究所 | Method of identifying chicken variety by DNA strip shape coding |
Non-Patent Citations (2)
| Title |
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| 包文斌，等.中国家鸡和红色原鸡mtDNA 控制区遗传多态性及系统进化分析.《畜牧兽医学报》.2008,第39卷(第11期),1449-1459. * |
| 薛茂云,等.基于线粒体COⅠ基因狼山鸡和鹿苑鸡DNA条形编码分析.《扬州大学学报(农业与生命科学版)》.2009,第30卷(第4期),26-29. * |
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