CN104975097A - Kit for green-eggshell chicken feather pecking related gene detection and implementation method thereof - Google Patents

Kit for green-eggshell chicken feather pecking related gene detection and implementation method thereof Download PDF

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CN104975097A
CN104975097A CN201510420030.2A CN201510420030A CN104975097A CN 104975097 A CN104975097 A CN 104975097A CN 201510420030 A CN201510420030 A CN 201510420030A CN 104975097 A CN104975097 A CN 104975097A
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chicken
measured
digestion products
genotype
feather
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姚俊峰
王晓亮
杨长锁
王敏
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Shanghai Academy of Agricultural Sciences
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

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Abstract

The invention relates to a kit for green-eggshell chicken feather pecking related gene detection and an implementation method thereof. The method comprises the following steps: by using genome DNA (deoxyribonucleic acid) (including rs13640917C/T mutation) of a chicken to be detected as a template, carrying out PCR (polymerase chain reaction) amplification by using the primers, carrying out enzyme digestion on the obtained PCR amplification product by using restriction endonuclease AcuI, carrying out electrophoresis detection on the obtained enzyme digestion product, and judging whether the rs13640917 site deoxyribonucleotide of the chicken to be detected is wild type C or mutant T. The kit can quickly determine whether the chicken to be detected is a wild type homozygote, mutant homozygote or heterozygote.

Description

Layer of green-shell egg feather picking genes involved detection test kit and its implementation
Technical field
What the present invention relates to is a kind of technology of field of biological genes, specifically a kind of test kit and its implementation detecting layer of green-shell egg feather picking gene rs13640917C/T.
Background technology
Feather picking (feather pecking, FP) is the problem that in layer breeding, especially high-density jumpbogroup is common in raising.In bird animal husbandry, feather picking causes that the mortality ratio of chicken group rises, feed intake increases and animal welfare declines, once part, chicken group occurs that feather picking phenomenon is just easy to spread to whole chicken group, form feather picking addiction, bring huge financial loss to bird animal husbandry.The feather pecking that chicken group is serious can cause chicken feather to cover minimizing, feather damages the chicken dropped, heat loss is accelerated, in order to maintain normal body temperature, food consumption increases, increase by 27% ~ 30% (Tauson than time normal, R., Cages:how could they be improved, in The laying hen and its environment.1980, Springer.p.269 ?304.), more serious caused chicken pain, make whole chicken mine massively appetite decline, feed conversion rate reduces, cause the mortality ratio of more than 50%, huge on the economic benefit impact of layer breeding person.Mild feather picking also can make a significant impact (Buitenhuis to egg size, A., et al., Genetic and phenotypiccorrelations between feather pecking behavior, stress response, immune response, and eggquality traits in laying hens.Poultry science, 2004.83 (7): p.1077 ?1082), also can affect the price of domestic rejected layer, reduce income.
The mode of disconnected beak is mostly used to reduce chicken feather picking for a long time both at home and abroad, namely use machine to be removed at front portion 1/3 or 1/2 place of beak at chicken 7 ?10 age in days (hot cutter break beak) or 0 age in days (infrared disconnected beak), but which kind of disconnected beak mode chicken all can be made to produce stress.Along with people are to animal welfare issues growing interest, disconnected this meeting of beak to chicken cause stress behavior be subject to larger query, some countries and regions has started trial and has forbidden disconnected beak.So in the long run, along with the deep application of Molecular tools in poultry, to the feather pecking of laying hen, the laying hen strain should focusing on being produced by molecule auxiliary genetic breeding technique low feather pecking is controlled.
Find according to (2010) researchs such as Van der Poel, a SNP on HTR2C gene on No. four karyomit(e)s and chicken feather pecking have direct genetic correlation, and relevant pole is (Van der Poel significantly, et al., Across ?line SNP associationstudy for direct and associative effects on feather damage in laying hens.Behavior genetics, 2010.40 (5): p.715 ?727.).Along with the very fast development of Protocols in Molecular Biology and chicken genome research, breeding is more accurate, the molecular breeding faster that is in progress puts on the arena of history, from the mechanism of molecular biology angle research chicken feather pecking, and study candidate gene and molecular genetic marker, use the method for marker assisted selection can reach the object of breeding fast and efficiently.
Summary of the invention
The present invention is directed to prior art above shortcomings, propose a kind of layer of green-shell egg feather picking genes involved detection test kit and its implementation, can determine that chicken to be measured is wild-type homozygous body, mutant-type homozygotes or heterozygote fast.
The present invention is achieved by the following technical solutions:
The present invention relates to a kind of layer of green-shell egg feather picking genes involved detection test kit, comprising:
1) for pcr amplification to detect the primer pair of rs13640917 gene, i.e. upstream primer U and downstream primer D, wherein:
U as shown in Seq ID No.1, that is: 5 ’ ?CACAATGACCACGACAACG ?3 ';
D as shown in Seq ID No.2, that is: 5 ’ ?ATCTCACGGAACCCAAATG ?3 ';
2) restriction endonuclease AcuI, its sequence is:
5 ’ ?CTGAAG (N) 16 ↓ ?3 ' and 3 ’ ?GACTTC (N) 14 ↑ ?5 '
Wherein arrow pointed location is restriction enzyme site, and N represents any base.
The present invention relates to the implementation method of mentioned reagent box, to chicken to be measured comprise rs13640917C/T sudden change genomic dna be template, described primer is adopted to carry out pcr amplification, cut the pcr amplification product of gained again with restriction endonuclease AcuI enzyme, the digestion products that electrophoresis detection obtains also judges that the deoxyribonucleotide of the rs13640917 position of chicken to be measured is wild-type C or saltant type T.
Described judgement is specially: when electrophoresis showed is two bands, and namely digestion products is 106bp and 295bp two fragments, and representing that the genotype of described chicken to be measured is CC, is wild-type homozygous body; When electrophoresis showed is a band, namely digestion products is 401bp fragment, and representing that the genotype of described chicken to be measured is TT, is mutant-type homozygotes; When electrophoresis showed is three bands, namely digestion products is 106bp, 295bp and 401bp tri-fragments, and the genotype of described chicken to be measured is CT, is heterozygote.
Described wild-type homozygous body CC feather pecking is even more serious; Mutant-type homozygotes TT not easily produces feather pecking.
Technique effect
Utilization the invention provides test kit and its implementation detects the allelotrope whether chicken carries easily generation feather pecking, can determine chicken feather pecking, carry out corresponding feeding and management in advance to chicken, reduce the financial loss that feather picking causes.The present invention carries out marker assisted selection for feather pecking in laying hen breeding work, provide a molecular genetic marker detection method, the method and genetic marker is used to carry out marker assisted selection to the feather pecking of laying hen, greatly can reduce the cost of molecular breeding, for the molecular breeding accelerating to improve laying hen feather picking phenomenon is laid a good foundation.Method provided by the invention or test kit simple to operate, only digestion products need be carried out agarose gel electrophoresis, can determine that the rs13640917 deoxyribonucleotide of chicken to be measured is wild-type C or saltant type T.Accuracy is high, not assorted band; Consuming time short, 2 ?3 hours; Low cost; Aulomatizeted Detect can be realized.For a breed of chicken work provides convenient, will play a great role in a breed of chicken.
Accompanying drawing explanation
Fig. 1 is the agarose gel electrophoresis figure of digestion products.
Embodiment
Elaborate to embodiments of the invention below, the present embodiment is implemented under premised on technical solution of the present invention, give detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
Embodiment 1
Present embodiments provide the method for concrete detection layer of green-shell egg feather pecking: pcr amplification comprises the fragment of rs13640917 deoxyribonucleotide, the pcr amplification product of gained is cut with restriction endonuclease AcuI enzyme, electrophoresis detection digestion products, determine that the rs13640917 deoxyribonucleotide of chicken to be measured is wild-type C or saltant type T as follows, thus determine the feather picking proterties of chicken to be measured: when electrophoresis showed is two bands, the genotype of described chicken to be measured is CC, be wild-type homozygous body, easily produce feather pecking; When electrophoresis showed is a band, the genotype of described chicken to be measured is TT, is mutant-type homozygotes, not easily produces feather pecking; When electrophoresis showed is three bands, the genotype of described chicken to be measured is CT, is heterozygote.
Carry out in the pair of primers of described pcr amplification, the nucleotide sequence of a primer is specifically Seq ID No.1, and the nucleotide sequence of another primer is specifically Seq ID No.2.
The template of described pcr amplification is specially the genomic dna of described chicken to be measured.
Apply after above-mentioned primer pair carries out pcr amplification using the genomic dna of above-mentioned chicken to be measured as template, the pcr amplification product of gained is cut with restriction endonuclease AcuI enzyme, electrophoresis detection digestion products, can determine that the rs13640917 deoxyribonucleotide of chicken to be measured is wild-type C or saltant type T as follows, thus determine the feather picking proterties of chicken to be measured: if digestion products is 106bp and 295bp two fragments, the genotype of described chicken to be measured is CC, it is wild-type homozygous body, easy generation feather pecking, feather covers poor; If digestion products is 401bp fragment, the genotype of described chicken to be measured is TT, is mutant-type homozygotes, not easily produces feather picking, and feather covers better; If digestion products is 106bp, 295bp and 401bp tri-fragments, the genotype of described chicken to be measured is CT, is heterozygote.
In the following example, method therefor is ordinary method if no special instructions, raw materials usedly all can obtain from commercial channels.Primer synthesis and examining order complete by the Shanghai biological company limited of raw work.
The present embodiment specifically comprises the following steps:
One, the determination of rs13640917C/T pleomorphism site
1, pcr amplification
Select layer of green-shell egg pure lines to be experiment material, according to primers near rs13640917, the object fragment that order amplification obtains comprises rs13640917 deoxyribonucleotide, and primer sequence is as follows:
U (upstream primer): 5 ’ ?CACAATGACCACGACAACG ?3 ' (Seq ID No.1)
D (downstream primer): 5 ’ ?ATCTCACGGAACCCAAATG ?3 ' (Seq ID No.2)
With layer of green-shell egg pure lines genomic dna for template, primer U and D is used to carry out pcr amplification.
Amplification system comprises: chicken genomic dna 50ng, each 0.3 μ L of upstream and downstream primer (10pmol/L), Taq Mix7.5 μ L, and uses ddH 2o postreaction system to 15 μ L.
PCR reaction conditions is: 95 DEG C of sex change 5min; Cycling program is 95 DEG C of sex change 20s, 60 DEG C of annealing 30s, and 72 DEG C extend 30s, totally 35 circulations; Last 72 DEG C extend 10min.
2, rflp analysis
Use restriction enzyme A cuI, enzyme cuts the pcr amplification product in step 1.The cleavage sequence following (arrow pointed location is wherein restriction enzyme site, and N represents any base) of AcuI:
5’‐CTGAAG(N)16↓‐3’
3’‐GACTTC(N)14↑‐5’
Endonuclease reaction system comprises: PCR reaction mixture 4 μ L, 10 × enzyme cutting buffering liquid 1 μ L, restriction endonuclease AcuI (10U/ μ L) 0.4 μ L, ddH 2o4.6 μ L, reaction system cumulative volume 10 μ L.
Water bath with thermostatic control 37 DEG C hatches 1 hour.
Digestion products carries out 1.5% agarose gel electrophoresis (voltage 8V/cm, time 30min).Use gel imaging system is observed, and result as shown in Figure 1.
As seen from the figure, digestion products presents 3 kinds of banding patterns.Part sample shows two electrophoretic bands, and sequence length is respectively 106bp, 295bp; Part sample is shown as an electrophoretic band, and sequence length is 401bp; Remaining sample is shown as three electrophoretic bands, and sequence length is respectively 106bp, 295bp and 401bp.
3, cloning and sequencing and sequential analysis
According to PCR ?rflp analysis result, respectively the pcr amplification product sepharose of the sample of the different electrophoretic band of display is reclaimed test kit (Tian Gen biochemical technology company limited) and reclaim purifying, the DNA fragmentation connection carrier pMD reclaimed ?after 19T (precious biotechnology (Dalian) company limited), with reference to method (the Proc Natl AcadSci of Cohen etc., 69:2110), product conversion bacillus coli DH 5 alpha competent cell will be connected, according to the carboxylic Bian penicillin resistance label screening positive colony on carrier, obtain the recombinant plasmid containing reclaiming fragment.With T7 and the SP6 promoter sequence on this recombinant plasmid vector, for primer pair, it carries out nucleotide sequencing, and sequencing result shows that the expanding fragment length of different sample is 401bp, only there is the difference (C/T) of a deoxyribonucleotide.
According to sequencing result and PCR ?RFLP result, genotype is limited as follows:
If the allelotrope in this site is C, its homozygote genotype be CC, PCR ?RFLP be detected as two electrophoretic bands, sequence length is respectively 106bp, 295bp;
If the allelotrope in this site is T, its homozygote genotype be TT, PCR ?RFLP be detected as an electrophoretic band, sequence length is 401bp;
This site heterozygote genotype be CT, PCR ?RFLP be detected as three electrophoretic bands, sequence length is respectively 106bp, 295bp and 401bp.
4, the preparation of layer of green-shell egg feather pecking detection kit
Layer of green-shell egg feather pecking allelotrope detection kit comprises: pair of primers, and the nucleotide sequence of one of them primer is Seq ID No.1, and the nucleotide sequence of another primer is Seq ID No.2; Restriction endonuclease AcuI; All reagent in all reagent in step 1 in PCR reaction system and step 2 in endonuclease reaction system.
Two, the association analysis of chicken genotype and feather pecking
Layer of green-shell egg feather pecking detection kit is utilized to detect.With the genomic dna of chicken to be detected for template, according to 1 carrying out pcr amplification in step one, according to 2 carrying out rflp analysis in step one.Detect the feather scoring situation of chicken, analyze the cognation of chicken genotype and feather picking proterties.
Chicken genotype is as shown in table 1 with associating of feather picking proterties.In detected 122 chickens, CC genotype 26, CT genotype 27, TT genotype 93, has significant difference between TT type and CC type feather picking proterties.
Above results proved that rs13640917C/T is polymorphic with chicken feather picking proterties close association, can as the molecule marker of chicken feather picking character screening.
Table 1rs13640917C/T loci gene type associates with feather picking proterties, and score value is lower, and representative more easily produces feather picking.

Claims (6)

1. a layer of green-shell egg feather picking genes involved detection test kit, is characterized in that, comprising:
1) for pcr amplification to detect the primer pair of rs13640917 gene, i.e. upstream primer U and downstream primer D, wherein:
U as shown in Seq ID No.1, that is: 5 ’ ?CACAATGACCACGACAACG ?3 ';
D as shown in Seq ID No.2, that is: 5 ’ ?ATCTCACGGAACCCAAATG ?3 ';
2) restriction endonuclease AcuI, its sequence is:
5 ’ ?CTGAAG (N) 16 ↓ ?3 ' and 3 ’ ?GACTTC (N) 14 ↑ ?5 '
Wherein arrow pointed location is restriction enzyme site, and N represents any base.
2. the implementation method of a test kit according to claim 1, it is characterized in that, to chicken to be measured comprise rs13640917C/T sudden change genomic dna be template, described primer is adopted to carry out pcr amplification, cut the pcr amplification product of gained again with restriction endonuclease AcuI enzyme, the digestion products that electrophoresis detection obtains also judges that the deoxyribonucleotide of the rs13640917 position of chicken to be measured is wild-type C or saltant type T.
3. method according to claim 2, is characterized in that, described judgement is specially: when electrophoresis showed is two bands, and namely digestion products is 106bp and 295bp two fragments, and representing that the genotype of described chicken to be measured is CC, is wild-type homozygous body; When electrophoresis showed is a band, namely digestion products is 401bp fragment, and representing that the genotype of described chicken to be measured is TT, is mutant-type homozygotes; When electrophoresis showed is three bands, namely digestion products is 106bp, 295bp and 401bp tri-fragments, and the genotype of described chicken to be measured is CT, is heterozygote.
4. method according to claim 2, is characterized in that, described pcr amplification, and its amplification system comprises: chicken genomic dna 50ng, each 0.3 μ L of upstream and downstream primer 10pmol/L, Taq Mix7.5 μ L, and uses ddH 2o postreaction system to 15 μ L; PCR reaction conditions is: 95 DEG C of sex change 5min; Cycling program is 95 DEG C of sex change 20s, 60 DEG C of annealing 30s, and 72 DEG C extend 30s, totally 35 circulations; Last 72 DEG C extend 10min.
5. method according to claim 2, is characterized in that, described enzyme is cut, and its reaction system comprises: PCR reaction mixture 4 μ L, 10 × enzyme cutting buffering liquid 1 μ L, restriction endonuclease AcuI10U/ μ L0.4 μ L, ddH 2o4.6 μ L, reaction system cumulative volume 10 μ L; 1 hour is hatched by water bath with thermostatic control 37 DEG C.
6. method according to claim 2, is characterized in that, described electrophoresis detection refers to: carry out 1.5% sepharose voltage 8V/cm to digestion products, the electrophoresis of time 30min, and by gel imaging system observations.
CN201510420030.2A 2015-07-17 2015-07-17 Kit for green-eggshell chicken feather pecking related gene detection and implementation method thereof Pending CN104975097A (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106222272A (en) * 2016-08-03 2016-12-14 贵州大学 Laying ducks feather picking character important gene HTR2C molecular marker and application
CN106521008A (en) * 2016-12-28 2017-03-22 贵州大学 Laying duck traumatic feather picking susceptibility gene DEAF1 molecular marker and application
CN108707676A (en) * 2018-06-05 2018-10-26 河南农业大学 A kind of and the lethal relevant SNP marker of chicken and its application, detection primer, detection kit
CN110551832A (en) * 2019-10-18 2019-12-10 上海市农业科学院 Primer pair, kit and detection method for detecting genotype of chicken s14916609 locus

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104225029A (en) * 2014-08-29 2014-12-24 青岛蓝盈禽业科技有限公司 Chicken farming method capable of preventing chickens from pecking feather

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
CN104225029A (en) * 2014-08-29 2014-12-24 青岛蓝盈禽业科技有限公司 Chicken farming method capable of preventing chickens from pecking feather

Non-Patent Citations (2)

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Title
F. BISCARINI，H. BOVENHUIS等: ""Across-Line SNP Association Study for Direct and Associative Effects on Feather Damage in Laying Hens"，第715–727页", 《BEHAV GENET》 *
JAN J VAN DER POEL，FILIPPO BISCARINI等: ""Across-line SNP association study for (innate) immune and behavioral traits in laying hens"，Suppl 4,S18", 《BMC PROCEEDINGS》 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106222272A (en) * 2016-08-03 2016-12-14 贵州大学 Laying ducks feather picking character important gene HTR2C molecular marker and application
CN106521008A (en) * 2016-12-28 2017-03-22 贵州大学 Laying duck traumatic feather picking susceptibility gene DEAF1 molecular marker and application
CN108707676A (en) * 2018-06-05 2018-10-26 河南农业大学 A kind of and the lethal relevant SNP marker of chicken and its application, detection primer, detection kit
CN110551832A (en) * 2019-10-18 2019-12-10 上海市农业科学院 Primer pair, kit and detection method for detecting genotype of chicken s14916609 locus

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