CN104313129A - Method for obtaining game fowl specificity molecular marker - Google Patents
Method for obtaining game fowl specificity molecular marker Download PDFInfo
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Abstract
The invention discloses a method for obtaining a game fowl specificity molecular marker. Chromosomes 16 of different chicken breeds are screened through target region capture sequencing; sequence comparison is carried out through the adoption of MEGA software; primers are designed through software such as Oligo according to comparison results; and PCR amplification is carried out on the different chicken breeds, and sequencing verification is carried out. Sequencing results show that specificity differences exist between game fowl and other chicken breeds on a section of sequence, the differences are that Gushi chicken have the specific 18-bp deficiency (CATTCCCGTTCCCGTTCG) on the section of sequence, and other chicken breeds do not have the characteristic, and clear identification can be achieved through agarose gel electrophoresis. In this way, the section of sequence can serve as the molecular marker for identifying the game fowl specificity. When the molecular marker is used for identifying the chicken breeds, operation is relatively simple, the specificity is high, and the repeated performance is high; a new scientific basis is added for correctly saving and reasonably using fowl breed resources
Description
Technical field:
The present invention relates to animal breeding science, molecular genetics and biology field.
Technical background:
Cockfighting is the amusement tradition nearly all had all over the world, utilizes the bird of Galliformes (also having other objects) to play in the feature that rutting sedson is bellicose.Cockfighting is the chicken breed for contest and amusement, is apt to struggle against and famous rare bird, has another name called dozen chicken, sting chicken, army chicken with kind beating.Henan cockfighting in cockfighting is one of Chinese Sweet and sour chicken kind.In Chinese native game chicken breeds kind, build is maximum, and quantity is maximum, and its chest, leg flesh are flourishing, and can be used as the raw data of broiler chicken breeding, main product is in cities such as Kaifeng, Henan Province, Zhengzhou, Luoyang, Zhoukou City.Henan cockfighting somatotypes has 4 kinds, coarse loose type, fine type, compact type and careful compact type.
For a long time, mainly animal varieties classification and authenticity is carried out with routine morphological labeled analysis means.Morphology labeled analysis is easy, economical, but because morphological feature is usually by the impact of environmental factors, has phenotypical plasticity and genetic variability, easily cause incorrect classification and identification; And ubiquitously in the fubaritic many colonies of morphological method hiddenly deposit taxon, applied molecular biology technology will be conducive to cultivar identification.The limitation of Morphological Identification and the taxonomist troop of constantly reduction, make taxonomic development face huge challenge.
The present invention utilizes high-flux sequence means to check order to different chicken kind, by carrying out examination comparison to the DNA sequence dna between different chicken kind, find the specific sequence occurred between different chicken kind, and pcr amplification and heavy sequence verification are carried out to this section of primers, to passing through Protocols in Molecular Biology means, find a kind of molecule marker, can classify more accurately to different chicken kind.
Summary of the invention:
The object of the invention is to obtain the specific molecule marker of cockfighting.
Technical scheme of the present invention is:
A kind of method obtaining cockfighting specific molecular marker, it is characterized in that: No. 16 karyomit(e)s of being caught order-checking examination 27 kinds of chicken kinds by target area, and utilize MEGA software to carry out sequence alignment, Oligo software design primer is utilized according to comparison result, pcr amplification is carried out to different chicken kind and carries out sequence verification, can obtain the specific molecule marker of cockfighting according to analytical results, i.e. CATTCCCGTTCCCGTTCG, concrete steps are as follows:
Materials and methods
1.1 experiment material
Choose Luo Si chicken, black langshan chicken, cockfighting, as hen, gu-shi chicken, Tibetan chicken, Gallus Domesticus, snow mountain hen, Red Jungle-fowl, Xiaoshan chicken, recessive white feather chicken, camellia chicken, deer park chicken, pacify card chicken, a fine breed of chicken with thick brownish feathers, large bone chicken, Wenchang Chicken, blue or green pin fiber crops chicken, Taihu Lake chicken, Liyang chicken, layer of green-shell egg, AA chicken, the yellow chicken in Guangxi, Leghorn, Xianju Chicken, white eared pheasant, the different chicken kind of Guangxi Huang chicken 27 kinds;
Order-checking is caught in 1.2 target areas
Gather 10 samples to each chicken breed, wing venous blood collection 0.4ml, adds 0.5mol/LNa
2eDTA antithrombotics 2 μ L, saves backup in 4 DEG C after adding lysate 4ml; Utilize phenol extraction method to extract DNA, send into Hua Da genome company and carry out target acquistion order-checking, choose Gallus_gallus-4.0 as reference genome;
1.3 sequence alignment
Target area is caught order-checking and is obtained the complete sequence of different chicken kind on No. 16 karyomit(e)s, utilizes software MEGA5.1 to carry out sequence alignment between different chicken kind, finds the difference condition of different chicken kinds in sequence;
1.4 design of primers
According to sequence alignment result, choose the one section of sequence occurring specificity difference between different chicken kind, utilize different chicken kind DNA as template, and utilize Oligo software design primer to carry out pcr amplification, Primer is: DOUJI; Seat: chromosome16; 5 '-3 ' primer sequence be: F:CCGCGCTGACCTCGTCTCGCTGTGT, R:CTCGGCGGGGCAGCAGCTAATTCAG; Annealing temperature: 66.3 DEG C;
1.5PCR amplification and detection
Pcr amplification cumulative volume is 20 μ L:1 μ LDNA templates, and its concentration is 100ng/ μ L, 2 μ L10 × PCRBuffer, 1.5 μ LdNTP, concentration is 10mmol/L, each 1 μ L of upstream and downstream primer, and concentration is 10pmol/ μ L, Taq enzyme is 0.2 μ L, and concentration is 5U/ μ L, ddH2O13.3 μ L; Pcr amplification program: 95 DEG C of denaturation 5min, 94 DEG C of sex change 40s, suitable annealing temperature 40s, 72 DEG C extend 40s, 35 circulations, and last 72 DEG C extend 10min, and 4 DEG C save backup; PCR primer is carried out checking order and uses the sepharose of 2% concentration to carry out electrophoresis detection, voltage is 100V, electrophoresis 40min;
Result
Order-checking is caught by target area, find one section of sequence that cockfighting and other chicken kinds there are differences, the amplification of design primer PCR is carried out resurveying sequence verifying with agarose gel electrophoresis, find that cockfighting and other chicken kinds exist specificity difference in this section of sequence, difference is: in this section of sequence, cockfighting exists the disappearance of specific 18 bp, i.e. CATTCCCGTTCCCGTTCG.
Of the present invention is a little this molecular markers for identification chicken kind of application, and operation is relatively simple, and specificity is high and repeated high, is also that correct preservation and the Appropriate application of poultry variety source adds new scientific basis.
Accompanying drawing explanation
Accompanying drawing 1 is cockfighting and other chicken kind sequence diagram;
Accompanying drawing 2 is cockfighting and other chicken kind agarose gel electrophoresis figure.
Embodiment
Below in conjunction with accompanying drawing, the present invention is described in further detail:
Embodiment
Technical scheme of the present invention is: No. 16 karyomit(e)s of being caught the different chicken kind of order-checking examination by target area, and utilize MEGA software to carry out sequence alignment, the software design primers such as Oligo are utilized according to comparison result, different chicken kind is carried out pcr amplification and checked order, the specific molecule marker of qualification cockfighting can be obtained according to analytical results.The materials and methods adopted and the result obtained as follows:
1 materials and methods
1.1 experiment material
Choose Luo Si chicken, black langshan chicken, cockfighting, as hen, gu-shi chicken, hide chicken, Gallus Domesticus, 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, blue or green pin fiber crops chicken, Taihu Lake chicken, Liyang chicken, layer of green-shell egg, AA chicken, the yellow chicken in Guangxi, Leghorn, Xianju Chicken, white eared pheasant, the different chicken kind of the yellow chicken 27 kinds in Guangxi, wherein Xianju Chicken, camellia chicken, deer park chicken, white eared pheasant, hide chicken, gu-shi chicken, large bone chicken, Henan cockfighting, langshan chicken, calm and peaceful Gallus Domesticus, Xiaoshan chicken, Beijing Fatty Chicken 12 native chicken breeds are all from the local fowl kind resource gene pool of country of Poultry Sci institute of Scientia Agricultura Sinica research institute, Red Jungle-fowl picks up from wildlife First aid station of Yunnan Province, other 14 chicken breeds are from Yangzhou University's animal science and technical college's genetic resources laboratory fowl kind resource gene pool,
1.2 target acquistion order-checkings
Gather 10 samples to each chicken breed, wing venous blood collection 0.4ml, adds 0.5mol/LNa
2eDTA antithrombotics 2 μ L, saves backup in 4 DEG C after adding lysate 4ml; Utilize phenol extraction method to extract DNA, send into Hua Da genome company and carry out target acquistion order-checking, choose Gallus_gallus-4.0 as reference genome.
1.3 sequence alignment
Target acquistion order-checking obtains the complete sequence of different chicken kind on No. 16 karyomit(e)s, utilizes software MEGA5.1 to carry out sequence alignment between different chicken kind, finds the difference condition of different chicken kinds in sequence.
1.4 design of primers
According to sequence alignment result, choose the one section of sequence occurring specificity difference between different chicken kind.Utilize different chicken kind DNA as template, utilize the software design primers such as Oligo to carry out pcr amplification, primer sequence is:
1.5PCR amplification and detection
Pcr amplification cumulative volume is 20 μ L:1 μ LDNA templates, and its concentration is 100ng/ μ L, 2 μ L10 × PCRBuffer, 1.5 μ LdNTP, concentration is 10mmol/L, each 1 μ L of upstream and downstream primer, and concentration is 10pmol/ μ L, Taq enzyme is 0.2 μ L, and concentration is 5U/ μ L, ddH2O13.3 μ L; Pcr amplification program: 95 DEG C of denaturation 5min, 94 DEG C of sex change 40s, suitable annealing temperature 40s, 72 DEG C extend 40s, 35 circulations, and last 72 DEG C extend 10min, and 4 DEG C save backup; PCR primer is sent into Shanghai Sheng Gong biotechnology limited-liability company carry out checking order and use the sepharose of 2% concentration to carry out electrophoresis detection, voltage is 100V, electrophoresis 40min.
2 results
Checked order by target acquistion, found one section of sequence that cockfighting and other chicken kinds there are differences, design primer PCR increases and carries out sequence checking of resurveying.Find that cockfighting and other chicken kinds exist specificity difference in this section of sequence, difference is that cockfighting exists the disappearance (CATTCCCGTTCCCGTTCG) of specific 18 bp in this section of sequence, and other chicken kinds are without this feature (see accompanying drawing 1).Also can be identified clearly (see accompanying drawing 2) by agarose gel electrophoresis.Therefore, this section of sequence can as the specific molecule marker of a kind of cockfighting.Apply this molecular markers for identification chicken kind, operation is relatively simple, and specificity is high and repeated high, is also that correct preservation and the Appropriate application of poultry variety source adds new scientific basis.
Claims (1)
1. one kind obtains the method for cockfighting specific molecular marker, it is characterized in that: No. 16 karyomit(e)s of being caught order-checking examination 27 kinds of chicken kinds by target area, and utilize MEGA software to carry out sequence alignment, Oligo software design primer is utilized according to comparison result, pcr amplification is carried out to different chicken kind and carries out sequence verification, can obtain the specific molecule marker of cockfighting according to analytical results, i.e. CATTCCCGTTCCCGTTCG, concrete steps are as follows:
Materials and methods
1.1 experiment material
Choose Luo Si chicken, black langshan chicken, cockfighting, as hen, gu-shi chicken, Tibetan chicken, Gallus Domesticus, snow mountain hen, Red Jungle-fowl, Xiaoshan chicken, recessive white feather chicken, camellia chicken, deer park chicken, pacify card chicken, a fine breed of chicken with thick brownish feathers, large bone chicken, Wenchang Chicken, blue or green pin fiber crops chicken, Taihu Lake chicken, Liyang chicken, layer of green-shell egg, AA chicken, the yellow chicken in Guangxi, Leghorn, Xianju Chicken, white eared pheasant, the different chicken kind of Guangxi Huang chicken 27 kinds;
Order-checking is caught in 1.2 target areas
Gather 10 samples to each chicken breed, wing venous blood collection 0.4ml, adds 0.5mol/LNa
2eDTA antithrombotics 2 μ L, saves backup in 4 DEG C after adding lysate 4ml; Utilize phenol extraction method to extract DNA, send into Hua Da genome company and carry out target acquistion order-checking, choose Gallus_gallus-4.0 as reference genome;
1.3 sequence alignment
Target area is caught order-checking and is obtained the complete sequence of different chicken kind on No. 16 karyomit(e)s, utilizes software MEGA5.1 to carry out sequence alignment between different chicken kind, finds the difference condition of different chicken kinds in sequence;
1.4 design of primers
According to sequence alignment result, choose the one section of sequence occurring specificity difference between different chicken kind, utilize different chicken kind DNA as template, and utilize Oligo software design primer to carry out pcr amplification, Primer is: DOUJI; Seat: chromosome16; 5 '-3 ' primer sequence be: F:CCGCGCTGACCTCGTCTCGCTGTGT, R:CTCGGCGGGGCAGCAGCTAATTCAG; Annealing temperature: 66.3 DEG C;
1.5PCR amplification and detection
Pcr amplification cumulative volume is 20 μ L:1 μ LDNA templates, and its concentration is 100ng/ μ L, 2 μ L10 × PCRBuffer, 1.5 μ LdNTP, concentration is 10mmol/L, each 1 μ L of upstream and downstream primer, and concentration is 10pmol/ μ L, Taq enzyme is 0.2 μ L, and concentration is 5U/ μ L, ddH2O13.3 μ L; Pcr amplification program: 95 DEG C of denaturation 5min, 94 DEG C of sex change 40s, suitable annealing temperature 40s, 72 DEG C extend 40s, 35 circulations, and last 72 DEG C extend 10min, and 4 DEG C save backup; PCR primer is carried out checking order and uses the sepharose of 2% concentration to carry out electrophoresis detection, voltage is 100V, electrophoresis 40min;
Result
Order-checking is caught by target area, find one section of sequence that cockfighting and other chicken kinds there are differences, the amplification of design primer PCR is carried out resurveying sequence verifying with agarose gel electrophoresis, find that cockfighting and other chicken kinds exist specificity difference in this section of sequence, difference is: in this section of sequence, cockfighting exists the disappearance of specific 18 bp, i.e. CATTCCCGTTCCCGTTCG.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104823919A (en) * | 2015-05-18 | 2015-08-12 | 贵州省畜牧兽医研究所 | Cultivation method and application of recessive white feather new strain of Xiaoxiang chicken in southeast of Guizhou province |
| CN105907867A (en) * | 2016-05-26 | 2016-08-31 | 江苏省家禽科学研究所 | SNP marker used for identifying Langshan chicken breed specificity and method for determining SNP site |
| CN105950733A (en) * | 2016-05-26 | 2016-09-21 | 江苏省家禽科学研究所 | SNP (Single Nucleotide Polymorphism) marker for identifying specificity of Luyuan chicken breed and method for determining SNP sites |
| CN106222273A (en) * | 2016-08-03 | 2016-12-14 | 扬州大学 | A kind of specific molecular marker identifying gu-shi chicken |
| CN105063224B (en) * | 2015-09-06 | 2018-08-21 | 扬州大学 | The method of the kit and identification of application and identification snow mountain hen of the one species specific molecular labeling in identifying snow mountain hen |
| 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 |
| CN112522420A (en) * | 2020-12-04 | 2021-03-19 | 江苏省家禽科学研究所 | Specific genetic site, primer combination and method for Liyang chicken species identification |
| 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 |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104823919A (en) * | 2015-05-18 | 2015-08-12 | 贵州省畜牧兽医研究所 | Cultivation method and application of recessive white feather new strain of Xiaoxiang chicken in southeast of Guizhou province |
| CN104823919B (en) * | 2015-05-18 | 2017-03-15 | 贵州省畜牧兽医研究所 | The breeding method of southeast of Guizhou Province Fructus Foeniculi chicken recessive white feather new lines and application |
| CN105063224B (en) * | 2015-09-06 | 2018-08-21 | 扬州大学 | The method of the kit and identification of application and identification snow mountain hen of the one species specific molecular labeling in identifying snow mountain hen |
| CN105907867A (en) * | 2016-05-26 | 2016-08-31 | 江苏省家禽科学研究所 | SNP marker used for identifying Langshan chicken breed specificity and method for determining SNP site |
| CN105950733A (en) * | 2016-05-26 | 2016-09-21 | 江苏省家禽科学研究所 | SNP (Single Nucleotide Polymorphism) marker for identifying specificity of Luyuan chicken breed and method for determining SNP sites |
| CN105950733B (en) * | 2016-05-26 | 2020-04-21 | 江苏省家禽科学研究所 | SNP marker for identifying specificity of Luyuan chicken species and method for determining SNP locus |
| CN106222273A (en) * | 2016-08-03 | 2016-12-14 | 扬州大学 | A kind of specific molecular marker identifying gu-shi chicken |
| CN106222273B (en) * | 2016-08-03 | 2019-03-15 | 扬州大学 | A kind of specific molecular marker for identifying gu-shi chicken |
| 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 |
| CN112522420A (en) * | 2020-12-04 | 2021-03-19 | 江苏省家禽科学研究所 | Specific genetic site, primer combination and method for Liyang chicken species identification |
| 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 |
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