CN107988376A - A kind of application of relevant SNP marker of the colour of skin in black-bone chicken Genotyping - Google Patents
A kind of application of relevant SNP marker of the colour of skin in black-bone chicken Genotyping Download PDFInfo
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Abstract
The present invention provides a kind of application of relevant SNP marker of the colour of skin in black-bone chicken Genotyping, and the SNP marker is located at the transcription initiation site upstream 2228bp of TYR gene promoter areas, and it is adenine A or thymidine T to have polymorphic base.The SNP genotype detections primer has very strong specificity, and test strip is single, and genotyping result is accurate.Make it that whole operation process is easier, efficient, cost is lower with reference to the detection method of offer, realize fast and accurately individual screening, foundation is provided for the development of black-bone chicken breed breeding and kind protection.
Description
Technical field
The invention belongs to molecular Biological Detection field, and in particular to a kind of relevant SNP marker of the colour of skin is in black-bone chicken base
Because of the application in parting.
Background technology
The skin color of chicken is a kind of very important varietal characteristic as qualitative character.Since different market comsuptons needs
Ask, the skin color of chicken has important economic use.The skin color of poultry is broadly divided into white, yellow, black three kinds, wherein black
Skin is using black-bone chicken as representative.
It is to contain a large amount of melanin in vivo that black-bone chicken, which has specific nourishing and medical value, its most significant feature,.Crow
As a kind of important active material, it has to remove free radical, anti-oxidant, anti-aging and improve body exempts from bone chicken dark pigment
Epidemic disease power and other effects.It is micro- that research shows that black-bone chicken melanin can be enriched with Cu, Mn, Fe, Co and Ni etc., and above-mentioned element can be with
The cellular immunity of body is directly participated in, and participates in three big metabolism approach, while suppresses the gene caused by mutagens
Mutation.Xu Xinglian etc. show that black-bone chicken melanin can effectively remove ultra-oxygen anion free radical by experiment, has super oxygen disproportionation
The effect of enzyme.Prove that black-bone chicken melanin can extend the average life span of drosophila at the same time, improve the Sexual potency of drosophila, significantly reduce fruit
The lipofuscin content of fly（P<0.05）, show that black-bone chicken melanin has the function that to slow down aging.Described in summary, in black-bone chicken body
Melanocyte is that it plays the material base of medicinal nourishing effects, in black-bone chicken body the increase of melanocyte deposition it is medicinal can to effectively improve its
Value.In addition, in the long-term phenomenon for occurring some individuals colour of skin during numerous and shoaling of black-bone chicken, and albino is sold
Price is far below black individual, has seriously affected the breeding income of black boneblack chicken.Therefore, the choosing of black-bone chicken black character is carried out
Educate, improve melanin content in black-bone chicken body, have great importance to current black-bone chicken breeding production.
Tyrosinase（tyrosinae, TYR）It is rate-limiting enzyme important in melanin synthesis process, it can catalytic tyrosine
Oxidation produces DOPA and DOPA quinone.The missing of tyrosinase normal function will cause B16 cell path to block, and make animal table
Reveal albefaction trait phenotypes.TYR genes are located on No. 1 chromosome of chicken, are made of 5 extrons and 4 intrones, correlation is ground
It is related to the plumage color and the colour of skin of chicken to study carefully the hereditary variation for showing TYR genes.The researchs such as Zhang point out that two heredity of TYR genes become
Ectopic sites have significant correlation with chicken muscle yellowish pink（zhanget al, 2010）.Lu Xiaoping etc. have studied him and stay black-bone chicken
TYR gene pleiomorphisms, detect 20 SNP sites altogether, and association analysis shows that the genetic mutation of TYR and plumage color character are notable（Land
Dawn screen etc., 2015）.Zhang etc. obtains 649 and the relevant gene of the black-bone chicken colour of skin, wherein TYR by RNA-seq technologies
Gene differential expression in black and white skin histology, TYR are up-regulation gene（zhang et al, 2015）.The studies above
Show, TYR genes can be as the candidate gene of the research black-bone chicken colour of skin.
SNP is the third generation molecular labeling after RFLP, microsatellite marker, very wide in animals and plants genetics research
General, genetic stability is high.At present, SNP detections are applied in animals and plants trait associations extensively, carry out objective trait molecular genetic
Marker development.The research of genome SNP provides important clue for fields such as species genetic analysis, disease diagnosis and therapies.Pass
The SNP detection method of system includes single-strand conformation polymorphism（SSCP）, direct sequencing, TaqMan probe typing, restricted
Segment length polymorphism（RFLP）Deng.But for these methods there are certain shortcoming, wherein SSCP is complicated, time-consuming, is not suitable for big
Measure sample detection.PCR sequencing PCR and TaqMan probe Genotype testing cost are too high, and RFLP is not suitable for a large amount of sample detections.
SNP is located at the 26S Proteasome Structure and Function that gene coding region may directly affect protein, and then influences interaction between albumen, causes correspondence
The change of shape phenotype.And the functional genetic variation of promoter region may control the opening and closing that the mRNA of gene is expressed, sieve
The promoter region SNP of a certain character major gene resistance is selected to can be used for the different variant phenotypes for studying same character, elaboration causes a
The hereditary basis of phenotypic difference between body.Therefore, the polymorphism of TYR gene promoter areas is studied, analyzes itself and the black-bone chicken colour of skin
Correlation, filters out the molecular labeling that can be used for black-bone chicken colour of skin selection and breeding, will greatly accelerate black-bone chicken breed breeding, improves black bone
Chicken individuals quality, increases breeding income.
The content of the invention
The present invention is intended to provide different from the prior art and the relevant mutational site of the black-bone chicken colour of skin, and should by the site
For detecting the relevant SNP genotype of the black-bone chicken colour of skin, foundation is provided for the development of black-bone chicken breed breeding and kind protection.
In order to solve the above technical problems, the present invention is achieved through the following technical solutions：
A kind of application of relevant SNP marker of the colour of skin in black-bone chicken Genotyping, the SNP marker are located at TYR gene promoters
At the transcription initiation site upstream 2228bp of sub-district, it is adenine A or thymidine T to have polymorphic base.
The application be based on SNP site design PCR amplification primer, using the amplimer to sample to be tested DNA into
Row PCR amplification, amplified production are imaged into row agarose gel electrophoresis, finally according to the presence or absence of purpose band in amplified production
Determine the genotype of the SNP marker of the sample to be tested.Show TYR promoter regions SNP according to test result analysis（c.-2228A
>T）There is significant correlation with the black-bone chicken colour of skin, AA genotype black-bone chicken black individual numbers pole is significantly higher than TT types.The SNP can
Carry out black-bone chicken individual choice and breeding work as molecular labeling.
The design method of the reverse amplimer of the PCR amplification primer is as follows：By SNP site reverse primer 3 ＇
End, the 3rd base that will be close to SNP site are changed into base mismatch C, its sequence is
R1：5’-AACTCTAACAGAACCATCATTTCTT-3’
R2：5’-AACTCTAACAGAACCATCATTTCTA-3’
Positive universal primer F：5-’CTGAAAGGAGGTTGTAGTGAGC-3’.
The pcr amplification reaction condition：94 DEG C of pre-degeneration 5min, cyclic amplification stage：94 DEG C of 30s denaturation, 55 DEG C
30s anneals, 72 DEG C of 30s extensions, circulates 30 times, 10min end extensions eventually are finally kept the temperature at 72 DEG C.After amplified reaction, PCR
Product carries out electrophoresis detection, and detection genotypic results are respectively AA, AT, TT.
During the agarose gel electrophoresis, Ago-Gel concentration is 2.0%-2.5%, voltage 130V, electrophoresis 30 minutes.
A kind of method for detecting above-mentioned SNP marker：Choose dark skin black-bone chicken, the white skin black-bone chicken of same individual
It is some, genomic DNA is extracted, each individual DNA carries out mixed pond by equivalent, and design primer P1, P2, P3 carry out PCR amplification, obtain
Amplified fragments, are analyzed by DNA sequence chromatogram after sequencing and carry out SNP site detection, wherein institute in candidate gene TYR promoter regions
It is as follows to state primer sequence：
P1：F：5-’GCTCAGGGGAGACCTTATTGC-3’
R：5-’GCTGGTTAACAGAGGAATCTCCT-3’
P2：F：5-’CATCAGTAGGTCAAAAAGGGTG-3’
R：5-’GAAATTACCCCTTGAAGGAGG-3’
P3：F：5-’CTGCAACAATGACAACTAAACTC-3’
R：5-’GACTCTGCTACTGTGAACCCTC-3’.
Primer P1, P2, P3 pcr amplification reaction program is：In 94 DEG C of pre-degeneration 5min, cyclic amplification stage：94℃ 30s
Denaturation, 30s annealing, 72 DEG C of 1min extensions, circulate 32 times, 10 min end extensions eventually are finally kept the temperature at 72 DEG C, wherein, wherein P1
The annealing temperature of primer is 60 DEG C, and wherein the annealing temperature of P2 primers is 56 DEG C, and wherein the annealing temperature of P3 primers is 58 DEG C.
The invention has the advantages that：
SNP genotype detections primer provided by the invention has very strong specificity, and test strip is single, and genotyping result is accurate.
Make it that whole operation process is easier, efficient, cost is lower with reference to the detection method of offer, according to existing test operation journey
Sequence, everyone detection that can complete 200-300 sample in one day.SNP marker provided by the invention can be used for the black-bone chicken colour of skin
Molecular mark, realizes fast and accurately individual screening, is provided for the development of black-bone chicken breed breeding and kind protection
Foundation.
Figure of description
Fig. 1 detects PCR product sequencer map for SNP site of the present invention；
Fig. 2 is SNP genotypings electrophoresis result figure of the present invention；
Fig. 3 is to use PCR sequencing PCR to carry out result figure of the obtained genotype of Genotyping for AA；
Fig. 4 is to use PCR sequencing PCR to carry out result figure of the obtained genotype of Genotyping for AT；
Fig. 5 is to use PCR sequencing PCR to carry out result figure of the obtained genotype of Genotyping for TT；
Fig. 6 is primer P1, P2 and P3 amplified production electrophoretogram.
Embodiment
Materials and methods
Experimental animal and sample collection：
This experiment selects 188 adult healthy Muchuan crow boneblack chickens altogether, and experimental subjects is in the black phoenix black-bone chicken industry in Muchuan County
Under the unified feeding and management condition of Co., Ltd.Blood collection：Blood is gathered by wing venous using disposable blood collection pin
In the vacuum blood collection tube containing sodium citrate anticoagulant, and fully mix.Record the black-bone chicken individual colour of skin at the same time（It is black, shallow
It is black, white）.
Instrument and equipment：
Low-temperature and high-speed centrifuge, ultramicrospectrophotometer, adjustable micropipettor, gel imaging system, electronic balance, height
Press sterilizing pan, thermostat water bath, electrophoresis apparatus, grads PCR instrument.
Extracting genome DNA
1st, take 20ul anticoagulated whole bloods to be put into a 1.5ml centrifuge tube, add 500ul DNA extracting solutions（0.1M Tris-Hcl、
0.5M Nacl, 0.05M EDTA-2Na, 2% SDS, 0.1% beta -mercaptoethanol, 0.5% polyvinylpyrrolidone）, fully mix,
65 DEG C of water-bath 30min, during which concussion are vortexed for several times.
2nd, 10min is centrifuged with 12000r/min, supernatant is transferred to new 1.5ml centrifuge tubes by careful take out, in supernatant
Add the 3M NaAC of 1/10 volume（pH=5.2）, 4 DEG C of standing 20min, add 300ul chloroform-isoamyl alcohols（24:1）, acutely
Concussion, 4 DEG C of standing 20min, 12000r/min centrifugation 10min.
3rd, supernatant is shifted to new 1.5ml centrifuge tubes, adds isometric isopropanol（- 20 DEG C of precoolings）, gently overturn mixed
Even, room temperature places 30min, and 12000r/min centrifugation 5min, abandon supernatant.
4th, 75% ethanol wash of 500ul precipitation is added, 12000r/min centrifugation 5min, abandon supernatant.
5th, the 4th step is repeated, 75% alcohol unnecessary in centrifuge tube is suctioned out, 100-200ul ddH2O are added after air-drying
Dissolved dilution, into concentration and purity testing, A260/280 and A260/230 ratios are respectively interposed in 1.87 ~ 1.95,2.3 ~ 2.5, -20
DEG C preserve.
The examination of TYR gene promoter areas SNP
Each 8 of black-bone chicken black and white skin subjects are chosen, each individual equivalent DNA is subjected to mixed pond.By designing primer
P1, P2 and P3 complete the PCR amplification of candidate gene TYR promoter region DNA sequence dnas, and amplification electrophoretogram is as shown in fig. 6, amplified production
Sequencing technologies analysis is carried out, TYR gene promoter area SNP examinations are completed by the general analysis of sequencer map.
Design of primers is according to Genbank database chicken TYR gene orders Chromosome 1, NC_006088.4
（187920666..187970514）.Primer is synthesized by Chengdu Qing Ke Zi Xi Bioisystech Co., Ltd, and primer information is shown in Table 1.
1 primer information table of table
It is sequenced using sanger and carries out PCR product sequencing, sequencing result is as shown in Figure 1, in promoter region upstream from start codon
A mutational site is identified at 2228bp（SNP）.
SNP genotyping primers design：
5 softwares (PREMIER Biosoft, USA) of Genotyping special primer design sampling Primer Premier.By SNP
Site is placed in 3 ＇ ends of reverse primer, and the 3rd base that will be close to SNP site is changed into base mismatch C, and designs positive general draw
Thing F, primer sequence are as follows：
Positive universal primer F：5-’CTGAAAGGAGGTTGTAGTGAGC-3’
R1：5’- AACTCTAACAGAACCATCATTTCTT-3’
R2：5’-AACTCTAACAGAACCATCATTTCTA-3’
Expanding fragment length：349bp, optimal 55 DEG C of TM values.
Individual specimen PCR amplification and electrophoresis
Each sample need to design two groups of PCR reaction systems（25ul）, A groups primer be by positive universal primer F and reverse primer R1,
B groups are forward direction universal primer F and reverse primer R2, its PCR reaction systems according to the form below 2 is prepared：
Table 2：PCR reaction systems（25ul）
PCR reaction conditions：Above-mentioned mixed liquor is vortexed and mixes and centrifuges, PCR instrument is immediately placed on, is expanded.In 94 DEG C of pre- changes
Property 5min, cyclic amplification stage：94 DEG C of 30s denaturation, 55 DEG C of 30s annealing, 72 DEG C of 30s extensions, are circulated 30 times, finally 72
DEG C insulation 10min eventually end extension.After amplified reaction, PCR product is positioned over 4 DEG C and treats electrophoresis detection.Delayed with 1 × TBE electrophoresis
The Ago-Gel of fliud flushing configuration 2.0%, voltage 130V, electrophoresis 30 minutes, gel imaging system detection Genotyping.
The present invention is by taking 8 sample electrophoresis figures as an example, if as shown in Fig. 2, A groups have a target stripe, B groups are without band, then its SNP
Genotype is AA, if A groups have target stripe without target stripe, B groups, then its SNP genotype is TT, if A B groups have band,
Its SNP genotype is AT.
The present invention will be contrasted according to the genotyping result of the method for the present invention and by sequencing result at the same time, randomly select electrophoresis
Numbering is 1 on figure（AT types）、4（TT）Type, 7（AA types）Each one of sample is using the sequencing of Sanger PCR sequencing PCRs, sequencing such as Fig. 3, figure
4th, it is as a result consistent with the method for the present invention genotyping result shown in Fig. 5.
SNP c.-2228A>T and Muchuan crow boneblack chicken colour of skin characters correlation
The present invention utilizes SNP c.-2228A>T specific gene serotype specific primers, complete 188 black-bone chicken genes of individuals type analysis,
With reference to individual colour of skin characteristics record as a result, being blocked genotypic results and black-bone chicken colour of skin character with SPSS statistical softwares
Square independence test, statistical result is as shown in table 3, SNP c.-2228A>T has significant phase with the Muchuan crow boneblack chicken colour of skin
Guan Xing.A is dominant in black-bone chicken dark skin subjects, and albino T is dominant.
3 SNP c.-2228A of table>T combines frequency and Chi-square Test with the colour of skin
Claims (6)
- A kind of 1. application of relevant SNP marker of the colour of skin in black-bone chicken Genotyping, it is characterised in that：The SNP marker position At the transcription initiation site upstream 2228bp of TYR gene promoter areas, it is adenine A or thymidine to have polymorphic base T。
- 2. a kind of application of the relevant SNP marker of colour of skin according to claim 1 in black-bone chicken Genotyping, its feature It is：The application is based on SNP site design PCR amplification primer, and PCR expansions are carried out to sample to be tested DNA using amplimer Increase, amplified production is imaged into row agarose gel electrophoresis, finally according to determining the presence or absence of purpose band in amplified production The genotype of the SNP marker of sample to be tested.
- 3. a kind of application of the relevant SNP marker of colour of skin according to claim 1 in black-bone chicken Genotyping, its feature It is：The design method of the reverse amplimer of the amplimer is as follows：SNP site is set to be located at 3 ＇ ends of reverse primer, will The 3rd base close to SNP site is changed into base mismatch C, its sequence isR1：5’-AACTCTAACAGAACCATCATTTCTT-3’R2：5’-AACTCTAACAGAACCATCATTTCTA-3’Positive universal primer F：5’-CTGAAAGGAGGTTGTAGTGAGC-3’.
- 4. a kind of application of the relevant SNP marker of colour of skin according to claim 2 in black-bone chicken Genotyping, its feature It is：The PCR amplification program is as follows：94 DEG C of pre-degeneration 5min, cyclic amplification stage：94 DEG C of 30s denaturation, 55 DEG C of 30s are moved back Fire, 72 DEG C of 30s extension, is circulated 30 times, finally keeps the temperature 10min end extensions eventually at 72 DEG C, after amplified reaction, PCR product into Row electrophoresis detection.
- 5. a kind of application of the relevant SNP marker of colour of skin according to claim 2 in black-bone chicken Genotyping, its feature It is：During the agarose gel electrophoresis, with the Ago-Gel of 1 × TBE electrophoretic buffers configuration 2.0%-2.5%, electrophoresis electricity Press as 130V, electrophoresis 30 minutes.
- A kind of 6. method for detecting the relevant SNP marker of the colour of skin as claimed in claim 1, it is characterised in that：The method is as follows： It is some to choose dark skin black-bone chicken, the white skin black-bone chicken of same individual, extracts DNA, equivalent DNA is subjected to mixed pond, if Count primer P1, P2, P3 and carry out PCR amplification, obtain candidate gene TYR promoter region amplified fragments, SNP site inspection is carried out after sequencing Survey, wherein the primer sequence is as follows：P1：F：5-’GCTCAGGGGAGACCTTATTGC-3’R：5-’GCTGGTTAACAGAGGAATCTCCT-3’P2：F：5-’CATCAGTAGGTCAAAAAGGGTG-3’R：5-’GAAATTACCCCTTGAAGGAGG-3’P3：F：5-’CTGCAACAATGACAACTAAACTC-3’R：5-’GACTCTGCTACTGTGAACCCTC-3’The method of the detection relevant SNP marker of the colour of skin according to claim 6, it is characterised in that：The pcr amplification reaction Program is：In 94 DEG C of pre-degeneration 5min, cyclic amplification stage：94 DEG C of 30s denaturation, 30s annealing, 72 DEG C of 1min extensions, circulation 32 times, 10 min end extensions eventually are finally kept the temperature at 72 DEG C, the wherein annealing temperature of P1 primers is 60 DEG C, the wherein annealing of P2 primers Temperature is 56 DEG C, and wherein the annealing temperature of P3 primers is 58 DEG C.
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Cited By (9)
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| CN110135703A (en) * | 2019-04-24 | 2019-08-16 | 江苏兴牧农业科技有限公司 | A kind of black-bone chicken selection |
| CN111657226A (en) * | 2020-05-29 | 2020-09-15 | 湖北省农业科学院畜牧兽医研究所 | Purification breeding method of black-bone goats |
| CN114480669A (en) * | 2022-01-28 | 2022-05-13 | 中国农业科学院北京畜牧兽医研究所 | Molecular marker related to chicken breast muscle color character and application thereof |
| CN115181740A (en) * | 2022-07-05 | 2022-10-14 | 北京擎科生物科技有限公司 | Lysate, PCR reaction kit and method |
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| CN110135703A (en) * | 2019-04-24 | 2019-08-16 | 江苏兴牧农业科技有限公司 | A kind of black-bone chicken selection |
| CN110135703B (en) * | 2019-04-24 | 2021-12-14 | 江苏兴牧农业科技有限公司 | Breeding method of black-bone chicken |
| CN111657226A (en) * | 2020-05-29 | 2020-09-15 | 湖北省农业科学院畜牧兽医研究所 | Purification breeding method of black-bone goats |
| CN111657226B (en) * | 2020-05-29 | 2021-12-07 | 湖北省农业科学院畜牧兽医研究所 | Purification breeding method of black-bone goats |
| CN114480669A (en) * | 2022-01-28 | 2022-05-13 | 中国农业科学院北京畜牧兽医研究所 | Molecular marker related to chicken breast muscle color character and application thereof |
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| CN115181805B (en) * | 2022-02-23 | 2023-08-11 | 华南农业大学 | Molecular marker related to yellow-feather broiler leg skin yellowness and application thereof |
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