CN106222273B - A specific molecular marker for identification of Gushi chicken - Google Patents
A specific molecular marker for identification of Gushi chicken Download PDFInfo
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- 241000287828 Gallus gallus Species 0.000 title claims abstract description 83
- 239000003147 molecular marker Substances 0.000 title claims abstract description 9
- 238000004519 manufacturing process Methods 0.000 claims abstract description 4
- 230000003321 amplification Effects 0.000 claims abstract 2
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract 2
- 238000000034 method Methods 0.000 claims description 5
- 239000007787 solid Substances 0.000 claims description 2
- 238000003975 animal breeding Methods 0.000 abstract description 2
- 244000144977 poultry Species 0.000 abstract description 2
- 238000012163 sequencing technique Methods 0.000 description 7
- 238000012408 PCR amplification Methods 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 5
- 238000002372 labelling Methods 0.000 description 5
- 108020004414 DNA Proteins 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- 238000012217 deletion Methods 0.000 description 4
- 230000037430 deletion Effects 0.000 description 4
- 230000000877 morphologic effect Effects 0.000 description 4
- 238000002864 sequence alignment Methods 0.000 description 4
- 210000003746 feather Anatomy 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 238000012795 verification Methods 0.000 description 3
- 240000001548 Camellia japonica Species 0.000 description 2
- 241000282994 Cervidae Species 0.000 description 2
- 241001415869 Crossoptilon crossoptilon Species 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 210000000988 bone and bone Anatomy 0.000 description 2
- 210000000349 chromosome Anatomy 0.000 description 2
- 235000018597 common camellia Nutrition 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 101150084750 1 gene Proteins 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 101000979565 Homo sapiens Protein NLRC5 Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 102100023432 Protein NLRC5 Human genes 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000010241 blood sampling Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- ZFGMDIBRIDKWMY-PASTXAENSA-N heparin Chemical compound CC(O)=N[C@@H]1[C@@H](O)[C@H](O)[C@@H](COS(O)(=O)=O)O[C@@H]1O[C@@H]1[C@@H](C(O)=O)O[C@@H](O[C@H]2[C@@H]([C@@H](OS(O)(=O)=O)[C@@H](O[C@@H]3[C@@H](OC(O)[C@H](OS(O)(=O)=O)[C@H]3O)C(O)=O)O[C@@H]2O)CS(O)(=O)=O)[C@H](O)[C@H]1O ZFGMDIBRIDKWMY-PASTXAENSA-N 0.000 description 1
- 229960001008 heparin sodium Drugs 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- LCCNCVORNKJIRZ-UHFFFAOYSA-N parathion Chemical compound CCOP(=S)(OCC)OC1=CC=C([N+]([O-])=O)C=C1 LCCNCVORNKJIRZ-UHFFFAOYSA-N 0.000 description 1
- 235000013594 poultry meat Nutrition 0.000 description 1
- 235000013613 poultry product Nutrition 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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Abstract
The present invention relates to animal breeding science, molecular genetics and molecular biology fields.More particularly to a kind of specific molecular marker for identifying gu-shi chicken.The specific molecular marker of the identification gu-shi chicken, sequence is as shown in SEQ ID NO.4, and primer sequence is as shown in SEQ ID NO.2 and 3.With the primer amplification of SEQ ID NO.2 and 3 sample DNA to be identified, it can determine that whether sample to be identified is gu-shi chicken according to the length of amplified production.For the present invention using this molecular markers for identification chicken kind, operation is relatively easy, specific height and repeated height, also adds new scientific basis with reasonable utilize for correct save of poultry variety source.
Description
Technical field
The present invention relates to animal breeding science, molecular genetics and molecular biology fields.Begin more particularly to a kind of identification is solid
The specific molecular marker of chicken.
Background technique
For a long time, animal varieties classification and authenticity mainly are carried out with routine morphological labeled analysis means.Shape
State labeled analysis is easy, economical, but since morphological feature is usually influenced by environmental factor, has phenotypical plasticity and something lost
Changeability is passed, incorrect classification and identification are easy to cause；And it is generally existing in the fubaritic many groups of morphological method
It is hidden deposit taxon, applied molecular biology technology is beneficial to cultivar identification.The limitation of Morphological Identification and constantly contracting
The systematist troop subtracted makes taxonomic development face huge challenge.
This patent is sequenced different chicken kinds using high-flux sequence means, by between the DNA sequence dna different chicken kinds
Screening comparison is carried out, finds the specific sequence occurred between different chicken kinds, and PCR amplification is carried out to this section of primers
Different chicken kinds can be carried out more with weight sequence verification to find a kind of molecular labeling by Protocols in Molecular Biology means
Accurate classification.
Summary of the invention
The purpose of the present invention is find a kind of molecular labeling for identifying gu-shi chicken specificity.
The present invention captures No. 16 chromosomes of sequencing screening difference chicken kind by target area, and is carried out using MEGA software
Sequence alignment carries out PCR amplification to different chicken kinds and is sequenced according to comparison result using software Design primers such as Oligo,
The molecular labeling of available identification gu-shi chicken specificity based on the analysis results.
The specific molecular marker of identification gu-shi chicken of the present invention, is gu-shi chicken and other chicken kinds have differences one
Duan Xulie (chr16:86,504-86,792, accession number are as follows: the total 289bp of XM_015294989.1, as shown in SEQ ID NO.1),
Gu-shi chicken in this section of sequence exist specificity 16bp base deletion (missing SEQ ID NO.1 198-213bp,
GGGAGTGGGGGGGAGG), other chicken kinds are without this feature.So the specific molecular marker of identification gu-shi chicken of the present invention,
Its sequence is as shown in SEQ ID NO.4, total 273bp.
The primer sequence of the molecular labeling are as follows:
F:AGATCCTCGCAGCCAAAGGGT(SEQ ID NO.2)
R:CAGTGTCCCCAACGCGCAGT(SEQ ID NO.3)
It is using above-mentioned primer, using the DNA of sample to be tested as mould the invention also discloses a kind of method for identifying gu-shi chicken
Plate carries out PCR amplification, determines whether sample to be identified is gu-shi chicken according to the length of amplified production.Sequence verification is carried out, if
There are the base deletions of 16bp in sequence, then are gu-shi chicken.Remaining chicken kind is then without this feature.Due to 4% Ago-Gel electricity
Swimming, it can be seen that the electrophoretic band of gu-shi chicken is located at the lower section of other chicken kind bands, but since separating effect is not accurate enough, also needs
Carry out sequence verification.Using in sequencing result, there are the base deletions of 16bp as criterion.
Using this molecular markers for identification chicken kind, operation is relatively easy, and it is also poultry product that specificity is high and repeatability is high
The correct preservation of kind resource and reasonable utilize add new scientific basis.
Detailed description of the invention
Fig. 1: gu-shi chicken and other chicken kind sequence alignment schematic diagrames.
Fig. 2: gu-shi chicken and other chicken kind agarose gel electrophoresis figures.
Specific embodiment
Embodiment:
1 materials and methods
1.1 experimental material
Choose ross chicken, black langshan chicken, cockfighting, such as hen, gu-shi chicken, hiding chicken, black-bone chicken, snow mountain hen, Red Jungle-fowl,
Xiaoshan chicken, recessive white feather chicken, camellia chicken, deer park chicken, peace card chicken, a fine breed of chicken with thick brownish feathers, large bone chicken, Wenchang Chicken, green foot fiber crops chicken, cockfighting × sieve
This hybridization 1 generation, Taihu Lake chicken, Liyang chicken, layer of green-shell egg, AA chicken, Guangxi Huang chicken, Leghorn (SPF), Xianju Chicken, white eared pheasant, totally 27
Kind different chicken kinds, wherein Xianju Chicken, camellia chicken, deer park chicken, white eared pheasant, hiding chicken, gu-shi chicken, large bone chicken, cockfighting, black langshan chicken,
12 black-bone chicken, Xiaoshan chicken, fine breed of chicken with thick brownish feathers native chicken breeds are all from country, scientia Agricultura Sinica research institute Poultry Sci research institute ground
Square fowl kind resource gene pool, Red Jungle-fowl pick up from Yunnan Province's wild animal First aid station.Other 14 chicken breeds come from this experiment
Room (Yangzhou University's animal science and technical college's genetic resources laboratory) fowl kind resource gene pool, there is note in the following documents
Carry: Guo Xiaomin, Liu Xiangping, Chang Guobin wait chicken NLRC5 promoter polymorphism to analyze [J], Chinese animal doctor's journal, and 2015,
35(11):1845-1849；Bi Yulin, Wang Hongzhi, Xu Lu wait 1 gene SNP s of difference chicken breed NFKB screening and evolutionary analysis
[J], Yangzhou University's journal, 2016,37 (1): 35-40.
1.2 target acquistion sequencing
10 samples are acquired to each chicken breed, wing venous blood sampling 0.4ml, heparin sodium is anticoagulant, and 4 DEG C of guarantors after lysate are added
It deposits spare.DNA is extracted using phenol extraction method, Huada gene company (BGI) is sent into and carries out target acquistion sequencing, choose
Gallus_gallus-4.0, which is used as, refers to genome.To the primary data that target acquistion sequencing obtains, remove in reads first
Connector pollution, then will be more than in reads 50% low quality base removal (value≤5 quality (E)), it is ensured that divide
Analyse the high quality of data.
1.3 sequence alignment
Target acquistion sequencing obtains complete sequence of the different chicken kinds on No. 16 chromosomes, using software MEGA5.1 to difference
Sequence alignment is carried out between chicken kind, finds difference condition of the different chicken kinds in sequence.
1.4 design of primers
According to sequence alignment result, chooses between different chicken kinds and Duan Xulie (chr16:86, a 504- of specific difference occur
86,792, total 289bp).Using different chicken kind DNA as template, PCR amplification is carried out using software Design primers such as Oligo, is drawn
Object sequence are as follows:
1.5 PCR amplifications and detection
PCR amplification total volume is 20 μ L:1 μ L DNA profilings (100ng/ μ L), 2 μ 10 × PCR of L Buffer, 1.5 μ L
DNTP (10m mol/L), each 1 μ L of upstream and downstream primer (10p mol/ μ L), Taq enzyme are 0.2 μ L (5U/ μ L), ddH2O 13.3μL。
PCR amplification program: 95 DEG C of initial denaturations 5min, 94 DEG C of denaturation 40s are suitable for annealing temperature 40s, and 72 DEG C of extension 40s, 35 recycle,
Last 72 DEG C of extensions 10min, 4 DEG C save backup.PCR product is sent into Shanghai Sheng Gong bioengineering limited liability company to be surveyed
Sequence simultaneously carries out electrophoresis detection, voltage 100V, electrophoresis 40min with the Ago-Gel of 4% concentration.
2 results
Pass through target acquistion sequencing, it was found that the Duan Xulie, design primer PCR that gu-shi chicken has differences with other chicken kinds
It expands and carries out resurveying sequence verifying.It was found that gu-shi chicken and other chicken kinds in this section of sequence there are specific difference, difference is
In this section of sequence there is the base deletion (GGGAGTGGGGGGGAGG) of the 16bp of specificity in gu-shi chicken, other chicken kinds are without this spy
Point (see attached drawing 1).It can also clearly be identified by agarose gel electrophoresis (see attached drawing 2A, B).Therefore, this section of sequence can
Using the molecular labeling as a kind of identification gu-shi chicken specificity.
Claims (3)
1. a kind of specific molecular marker for identifying gu-shi chicken, it is characterised in that sequence is as shown in SEQ ID NO.4, total 273bp.
2. identifying the primer of the specific molecular marker of gu-shi chicken described in claim 1, which is characterized in that its sequence such as SEQ ID
Shown in NO.2 and 3.
3. a kind of method for identifying gu-shi chicken, which is characterized in that with the primer amplification of SEQ ID NO.2 and 3 sample to be identified
DNA determines whether sample to be identified is gu-shi chicken according to the length of amplified production；The length of amplified production is 273bp's, is solid
Beginning chicken sample.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111518921A (en) * | 2020-06-29 | 2020-08-11 | 扬州大学 | Method for identifying Liancheng white duck by adopting SNP molecular marker technology |
| CN112626225A (en) * | 2020-12-04 | 2021-04-09 | 江苏省家禽科学研究所 | Method for effectively identifying Luyuan chicken species and primer combination |
| CN112481389A (en) * | 2020-12-04 | 2021-03-12 | 江苏省家禽科学研究所 | Molecular marker primer combination for identifying shou Guang chicken species and identification method |
| CN112522421A (en) * | 2020-12-04 | 2021-03-19 | 江苏省家禽科学研究所 | Method for identifying Taihu chicken by using molecular marker |
| CN112626226A (en) * | 2020-12-04 | 2021-04-09 | 江苏省家禽科学研究所 | Primer combination for detecting Langya chicken specific genetic locus and application thereof |
Citations (2)
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|---|---|---|---|---|
| CN104313129A (en) * | 2014-09-15 | 2015-01-28 | 扬州大学 | Method for obtaining game fowl specificity molecular marker |
| CN105063224A (en) * | 2015-09-06 | 2015-11-18 | 扬州大学 | Application of specific molecular marker in identifying Xueshan chicken, and kit and method for identifying Xueshan chicken |
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| US7666590B2 (en) * | 2003-12-16 | 2010-02-23 | University Of Delaware | Identification of fat and lean phenotypes in chickens using molecular markers |
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| CN104313129A (en) * | 2014-09-15 | 2015-01-28 | 扬州大学 | Method for obtaining game fowl specificity molecular marker |
| CN105063224A (en) * | 2015-09-06 | 2015-11-18 | 扬州大学 | Application of specific molecular marker in identifying Xueshan chicken, and kit and method for identifying Xueshan chicken |
Non-Patent Citations (3)
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| Identification and SNP association analysis of a novel gene in chicken;Mei X等;《Anim Genet》;20151208;第47卷(第1期);第125-127页 * |
| 固始鸡不同品系及部分外来鸡种之间遗传多样性的微卫星;邓雪娟;《中国优秀硕士学位论文全文数据库（电子期刊）农业科技辑》;20050715(第7期);D050-59 * |
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