CN101016565A - Method of identifying chicken variety by DNA strip shape coding - Google Patents
Method of identifying chicken variety by DNA strip shape coding Download PDFInfo
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- CN101016565A CN101016565A CN 200710020054 CN200710020054A CN101016565A CN 101016565 A CN101016565 A CN 101016565A CN 200710020054 CN200710020054 CN 200710020054 CN 200710020054 A CN200710020054 A CN 200710020054A CN 101016565 A CN101016565 A CN 101016565A
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Abstract
The invention discloses a method to appraise chicken species with DNA encoding strip in molecular biology technical domain, which comprises the following steps: utilizing DNA test-sequence technology; checking gene sequence of the chicken group and standard control sample mitochondria cell coloring matter C oxidase I(cytochrome C oxidase I COI); proceeding chicken specie checking according to point of discontinuity of COI sequence, haploid type, haploid type multiple degree (Hd), nucleic acid multiple degree (Pi), genetic distance of double parametric model among species, nucleic acid branch degree among species, specific site of each specie and DNA classified image. This invention can reflect the difference among species compared to the other methods, which result can be scientific proof to appraise genuine-fake of chicken species.
Description
Technical field
The present invention relates to the method for identifying chicken variety by DNA strip shape coding, use dna sequencing technical measurement mitochondrial cytochrome C oxydase I (cytochrome C oxidase I COI) gene order specifically, utilize the particular section of this specific gene of COI to identify chicken breed, belong to technical field of molecular biology as the DNA barcode.
Background technology
Because standard is also owed in China poultry kind market, the kind of cultivating is very different, fake and forged, shoddy phenomenon happens occasionally, evaluation to the poultry kind mainly is to be labeled as according to examining the breeding archives and carrying out performance test with morphology at present, along with improving constantly of breeding level, the macroscopic features of parent and filial generation has bigger similarity, such as being male parent with the indigenous chicken kind, hybridizing with the Recessive Chicken female parent, its hybridization first filial generation is just similar especially to the indigenous chicken kind, is difficult to distinguish from macroscopic features.Modern specialized strain is mainly paid attention to production performance, the macroscopic features of strain might not be in full accord, such as the external high-yield egg chicken of introducing, commodity godmother chicken back striped by the plumage colour identification has multiple phenotype, therefore the evaluation between its strain can not be distinguished by appearance simply, identifies just not enough science so utilize the morphology mark to carry out poultry kind (strain).If carry out performance test, not only to take a long time (laying hen and kind about 72 weeks of chicken, about 66 weeks of meat kind chicken, fryer commodity about 14 weeks in generation), and cost is also very high, and the result of mensuration also will be raised the influence of each link factor, is difficult to accurately reflect the germplasm characteristic of each strain.And identify the polymorphism that can only reflect few part DNA of kind indirectly with isozyme and protein labeling.Therefore, above-mentioned these methods can not science, carry out the evaluation of poultry kind exactly, and qualification time is long, and cost is also very high.
Summary of the invention
Purpose of the present invention is exactly the defective that will overcome aspects such as poor accuracy, cost height and qualification time that traditional cultivar identification method exists be long, invents a kind of science, chicken breed authentication method accurately.
Technical scheme of the present invention is achieved in that the method for identifying chicken variety by DNA strip shape coding, it is characterized in that utilizing mitochondrial cytochrome C oxydase I (the cytochrome C oxidase I COI) gene order of dna sequencing technical measurement tested chicken colony and standard control sample, according to the mutational site in the COI sequence of order-checking, haplotype, the various degree of haplotype (Hd), diverse oligonucleotide degree (Pi), two-parameter model genetic distance between kind, Nucleotide degree of ramification between kind, the DNA classification chart of the specific site of each kind and kind carries out chicken breed to be identified, the steps include:
(1) tested chicken breed and standard control sample are randomly drawed blood sample, extract genomic dna with phenol-chloroform method;
(2) according to the conserved regions design primer among the Chinese Red Jungle-fowl mt DNA COI;
(3) primer of utilization design carries out pcr amplification to evaluation chicken group's mt DNACOI, pcr amplification product is reclaimed, and carry out two-way order-checking on ABI 3730 sequenators;
(4) sequencing result being carried out sequence reads and compares;
(5) statistics haplotype, average nucleotide difference and diverse oligonucleotide degree, Nucleotide degree of ramification, clean genetic distance and nucleotide diversity site determine that inter-variety variance still is variation in the kind;
(6) carry out cluster according to mtDNA COI sequence according to the UPGMA method, determine whether to belong to same kind or same colony.
Sample size is 12 individualities in the described tested chicken kind of randomly drawing blood sample and the standard control sample chicken kind, and the ratio of hen and cock is 1: 1.
Because Mitochondrial DNA Terminal oxidase COI gene has relative conservative property in same species, there are enough variations can distinguish different kinds again, what it disclosed is the difference of hereditary material DNA, compare with methods such as using phenotypic character, production performance, biochemical marker and other genetic markers, more can reflect interracial difference really.Utilize the DNA bar coding to identify the method for chicken breed, its result can be used as the scientific basis of identifying the chicken breed true and false.
Embodiment
(1) to randomly drawing 6 cocks in tested chicken breed and the standard control sample, 6 hens, the wing venous blood collection, blood sample extracts genomic dna with phenol-chloroform method;
(2) according to the 3 pairs of primers of conserved regions design among the Chinese Red Jungle-fowl mt DNA COI (sequence number) that delivers on the Gen Bank;
(3) utilize 3 pairs of primers that evaluation chicken group's mt DNA COI is carried out pcr amplification, pcr amplification product is reclaimed and on ABI 3730 sequenators, carry out two-way order-checking;
(4) order-checking is read by Chromas software, and each sequencing result carried out manually one by one base reads and check and correction repeatedly, and by two-way repeat the order-checking proofread and correct, the sequence that obtains is compared with CLUSTALW software, and belong to the mitochondrial COI gene sequence with GenBank chicken (Gallus Gallus) and compare, calculate genetic distance with the Kimura two parameter method in the MEGA3.0 software.Use adjacent method (NJ) and make up phylogenetic tree.Use DNAsp 4.10 statistics haplotypes, average nucleotide difference and diverse oligonucleotide degree, Nucleotide degree of ramification, clean genetic distance and nucleotide diversity site etc.;
(5), determine in the chicken breed and COI gene order degree of variation between kind according to the haplotype of tested chicken kind and standard control chicken breed COI gene order, average nucleotide difference and diverse oligonucleotide degree, Nucleotide degree of ramification and clean genetic distance.If on average make a variation less than 2%, then be same kind, be different varieties greater than 2%;
(6) according to both difference of specific site of COI gene order, take a decision as to whether same chicken kind;
(7) use Mega3.0 software, carry out cluster according to the UPGMA method, be that a class then is same kind if measure poly-in the chicken, otherwise be different varieties with standard control chicken kind according to mtDNA COI sequence.
Claims (2)
1, the method for identifying chicken variety by DNA strip shape coding, it is characterized in that utilizing mitochondrial cytochrome C oxydase I (the cytochrome C oxidase I COI) gene order of dna sequencing technical measurement tested chicken colony and standard control sample, according between the various degree of mutational site, haplotype, haplotype (Hd), diverse oligonucleotide degree (Pi), kind in the COI sequence of order-checking between two-parameter model genetic distance, kind the DNA classification chart of the specific site of Nucleotide degree of ramification, each kind and kind carry out chicken breed and identify, the steps include:
(1) tested chicken breed and standard control sample are randomly drawed blood sample, extract genomic dna with phenol-chloroform method;
(2) according to the conserved regions design primer among the Chinese Red Jungle-fowl mt DNA COI;
(3) primer of utilization design carries out pcr amplification to evaluation chicken group's mt DNACOI, pcr amplification product is reclaimed, and carry out two-way order-checking on ABI 3730 sequenators;
(4) sequencing result being carried out sequence reads and compares;
(5) statistics haplotype, average nucleotide difference and diverse oligonucleotide degree, Nucleotide degree of ramification, clean genetic distance and nucleotide diversity site determine that inter-variety variance still is variation in the kind;
(6) carry out cluster according to mtDNA COI sequence according to the UPGMA method, determine whether to belong to same kind or same colony.
2, the method for identifying chicken variety by DNA strip shape coding according to claim 1 is characterized in that sample size is 12 individualities in the described tested chicken kind of randomly drawing blood sample and the standard control sample chicken kind, and the ratio of hen and cock is 1: 1.
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| CN 200710020054 CN101016565A (en) | 2007-02-09 | 2007-02-09 | Method of identifying chicken variety by DNA strip shape coding |
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| CN 200710020054 CN101016565A (en) | 2007-02-09 | 2007-02-09 | Method of identifying chicken variety by DNA strip shape coding |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101974647A (en) * | 2010-11-26 | 2011-02-16 | 现代新农业（集团）股份有限公司 | Molecular biological variety identification method for Huangjiaji chicken |
| CN103266174A (en) * | 2013-05-22 | 2013-08-28 | 青岛农业大学 | PCR (Polymerase Chain Reaction) primer and identification method of athetis lepigone and sibling species thereof |
| CN112513298A (en) * | 2018-12-12 | 2021-03-16 | 忠南大学校产学协力团 | Single nucleotide polymorphism marker set for distinguishing genetic background or variety of local chicken and application thereof |
| CN113005207A (en) * | 2021-04-23 | 2021-06-22 | 江苏省家禽科学研究所 | Haploid marker for identifying camellia chicken variety based on sex chromosome and application thereof |
-
2007
- 2007-02-09 CN CN 200710020054 patent/CN101016565A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101974647A (en) * | 2010-11-26 | 2011-02-16 | 现代新农业（集团）股份有限公司 | Molecular biological variety identification method for Huangjiaji chicken |
| CN101974647B (en) * | 2010-11-26 | 2012-03-21 | 现代新农业（集团）股份有限公司 | Molecular biological variety identification method for Huangjiaji chicken |
| CN103266174A (en) * | 2013-05-22 | 2013-08-28 | 青岛农业大学 | PCR (Polymerase Chain Reaction) primer and identification method of athetis lepigone and sibling species thereof |
| CN103266174B (en) * | 2013-05-22 | 2014-12-10 | 青岛农业大学 | PCR (Polymerase Chain Reaction) primer and identification method of athetis lepigone and sibling species thereof |
| CN112513298A (en) * | 2018-12-12 | 2021-03-16 | 忠南大学校产学协力团 | Single nucleotide polymorphism marker set for distinguishing genetic background or variety of local chicken and application thereof |
| 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 |
| CN113005207A (en) * | 2021-04-23 | 2021-06-22 | 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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