CN105671170A - Primer combination for identifying creeping character of chicken and application thereof - Google Patents
Primer combination for identifying creeping character of chicken and application thereof Download PDFInfo
- Publication number
- CN105671170A CN105671170A CN201610134746.0A CN201610134746A CN105671170A CN 105671170 A CN105671170 A CN 105671170A CN 201610134746 A CN201610134746 A CN 201610134746A CN 105671170 A CN105671170 A CN 105671170A
- Authority
- CN
- China
- Prior art keywords
- character
- chicken
- combination
- primer
- primers
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
- 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
- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- 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
- C12Q2600/158—Expression markers
-
- 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
- C12Q2600/16—Primer sets for multiplex assays
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to the field of molecular biology, in particular to a primer combination for identifying the creeping character of chicken and the application thereof.The primer combination comprises primer sequences as shown in the SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4.The creeping character of chicken can be identified by means of the primer combination.Specifically, the genomic DNA of an individual to be tested is taken as the template, and analysis is conducted with the multiple PCR method by means of the primer combination; if one product is obtained through multiple PCR amplification and the sequence size of the product is 224 bp, the individual to be tested is a creep gene dominant homozygote (Cp/Cp), and the individual is of the lethal type; if two products are obtained through multiple PCR amplification and the sequence sizes of the products are 438 bp and 224 bp respectively, the individual to be tested is a creep gene heterozygote (Cp/+), and the individual has the creeping character; if only one product is obtained through multiple PCR amplification and the sequence size of the product is 438 bp, the individual to be tested is a recessive homozygote (+/+), and the individual is of the wild type.
Description
Technical field
The present invention relates to biology field, specifically, relate to a kind of for differentiating that chicken crawls the combination of primers of character and application thereof.
Background technology
Up to the present, chicken body has been found that eight kinds of dwarf genes, they lay respectively on sex chromosome and autosome, wherein Sex linkage dwarf gene dw research is more, carry out this gene being accurately positioned and clear and definite its mechanism of action (Agarwal, S.K., L.A.Cogburn, andJ.Burnside.1994.Dysfunctionalgrowthhormonereceptorina strainofsex-linkeddwarfchicken:evidenceforamutationinthe intracellulardomain.J.Endocrinol.142:427-434.). Low and small gene on autosome is more, wherein Cp gene (Cp:Creeper, crawl type) it is a kind of important dominant homogeneous lethal gene (Landauer on autosome, W., andL.C.Dunn.1930.StudiesontheCreeperfowl.I.Genetics.J.Ge net.23:397-413.), the character of crawling of this Gene Handling chicken.
Cutler (Cutler, I.E.1925.Reptilianfowls.J.Hered.16:352-356.) first delivers character of crawling and is subject to the hypothesis of heterozygous genotypes impact. Landauer and Dunn (Landauer, W., andL.C.Dunn.1930.StudiesontheCreeperfowl.I.Genetics.J.Ge net.23:397-413.) pass through cross experiment, find a kind of dwarf chicken causing the abnormal growth of cartilage, called after creeper fowl (Creeperfowl), and the hereditary basis of character of confirming to crawl is to be controlled by single Mendel dominant gene Cp, lethal type is shown as under dominant homogeneous (Cp/Cp) condition, heterozygote (Cp/+) shows type of crawling, allozygote (+/+) show as wild type, whole hatching Infant Mortality is 25%, the embryonic death time concentrates on the 4th day of hatching.
Genetics research shows, Cp gene and leaf-comb or rose comb chain (Landauer, W.1932.Studiesonthecreeperfowl.V.Thelinkageofthegenesfor creeperandsingle-comb.J.Genet.26:285-290.;Landauer, W.1933.Creeperandsingle-comblinkageinfowl.Nature132:606., Taylor, L.W.1934.Creeperandsingle-comblinkageinthefowl.J.Hered.2 5:205-206.). Somes and Jr. by Cp gene mapping in the Ith linkage group, it is 0.4cM with the linkage distance of rose comb gene (R), it is 30cM (Somes with the linkage distance of oil gland isolated genes (U), R.G., andJr.1973.Linkagerelationshipsindomesticfowl.J.Hered.64: 217-221.). Imsland etc. are shown by linkage analysis and high density SNP research, on No. 7 chromosomes, the inversion of 7.4Mb causes MNR2 gene location to change, this gene inversion causes rose comb phenotype, it is further characterized by rose comb gene (R) and is positioned at (Imsland on No. 7 chromosomes, F., C.Feng, H.Boije, B.Bed'hom, V.Fillon, B.Dorshorst, C.J.Rubin, R.Liu, Y.Gao, X.Gu, Y.Wang, D.Gourichon, M.C.Zody, W.Zecchin, A.Vieaud, M.Tixier-Boichard, X.Hu, F.Hallbook, N.Li, andL.Andersson.2012.TheRose-combmutationinchickensconsti tutesastructuralrearrangementcausingbothalteredcombmorph ologyanddefectivespermmotility.PLoSGenet.8:e1002775.). Xingshan walnuts is one of domestic rare some places Bantam kind, owing to having special build of crawling, receives attention and the concern of domestic and international expert height. the key gene controlling this character is Cp (Creeper) gene, and this gene is that a kind of autosomal dominant isozygotys lethal gene. in the world to existing more than 80 year history (Cairns, J.M., the andK.Gayer.1943.Identificationofchickembryoshomozygousfo rtheCreeperfactor.J.Exp.Zool.92:229-242. of the research of Cp gene, Landauer, W.1944.Lengthofsurvivalofhomozygouscreeperfowlembryos.Sc ience100:553-554., Dinner, B.1971.ChemicalanalysisofembryonicCreeperchickentibia.J. Dent.Res.50:704., Fujio, Y., andT.Shibuya.1974.ExpressionoflethalitycausedbyCreeperge neinthechicken.Japan.J.Genet.49:87-91.), the domestic Local chicken breeds such as Xingyi, Miyi, owing to carrying this gene, causes that the incubation rate of these native chicken breeds and the regularity of growth promoter are severely impacted. so, research controls to crawl the genetic development of character Cp gene, contributes to Xingshan walnuts and China's chicken breeds elsewhere are protected, researched and developed utilization. conventionally breeding method, to the selection-breeding of character of crawling, mainly length according to shin length determines, the cycle is longer, and expense is many. it addition, the detection Cp of routine isozygotys, lethal embryo is complicated with the morphology of natural death embryo and cytology sampling operation, analyzes the mensuration cycle long, and automaticity is not high, detects not accurate enough. and utilize molecular marker assisted selection to carry out molecular breeding and not only can directly select, and can select in early days, it is greatly shortened breeding cycle, improves the accuracy of breeding, reduce breeding expense.
Summary of the invention
In order to solve problems of the prior art, it is an object of the invention to provide a kind of for differentiating that chicken crawls the combination of primers of character and application thereof.
In order to realize the object of the invention, technical scheme is as follows:
First aspect, the invention provides a kind of for differentiating that chicken crawls the combination of primers of character, and described combination of primers includes the primer sequence shown in SEQIDNo.1, SEQIDNo.2, SEQIDNo.3 and SEQIDNo.4.
Described combination of primers is through PrimerPremier6.0 software design, under the premise guaranteeing primer specificity, sensitivity and amplification efficiency, required purpose band is obtained through multiplex PCR experiment amplification, PCR primer converts and order-checking through purification, connection, and after verifying in big colony, screening obtains.
Described combination of primers has significant advantage in exploitation molecular detection kit, poultry molecular breeding and Local Chicken Breeds Resources and utilization, character (Creeper) of chicken can being crawled carries out quickly, screens accurately and efficiently and identify, simple to operate, cheap. This combination of primers high specificity, highly sensitive, with low cost, easy quickly, applied widely. Adopt multiple PCR method to have the advantage of its uniqueness, primary first-order equation can three kinds of different genotype individualities of examination, and can prepare to detect existence or the disappearance of gene order of crawling. This multi-primers is combined with helping to crawling build chicken and China's native chicken breed resource is protected, researched and developed utilization.
Second aspect, the invention provides described combination of primers in the application differentiating that chicken crawls in character.
And in preparation, described combination of primers differentiates that chicken crawls the application in the reagent of character or test kit.
And described combination of primers crawls the application in character assistant breeding chicken.
Further, described application particularly as follows:
With the genomic DNA of test individual for template, utilize described combination of primers, adopt multiple PCR method to be analyzed; If multiplexed PCR amplification has a kind of product, and sequence size is 224bp, then test individual is gene dominant homozygote (Cp/Cp) of crawling, and shows as lethal type; If multiplexed PCR amplification has two kinds of products, sequence size is 438bp and 224bp respectively, then test individual is genetic heterozygosis (Cp/+) of crawling, and shows as character of crawling; If only a kind of product of multiplexed PCR amplification, sequence size is 438bp, then test individual be stealth homozygote (+/+), shows as wild type.
As preferably, the present invention provides the multi-PRC reaction system that an expanding effect is best, particularly as follows: genomic DNA 80-100ng, 10 × PCR buffer is (containing Mg2+) 2.0 μ L, dNTPs (2.5mM) 2.0 μ L, Taq DNA polymerase (2.5U/ μ L) 1.0 μ L, delF and delR primer (10 μm of ol/l) each 0.2 μ l, F and R primer (10 μm of ol/l) each 0.4 μ l, uses ddH2O postreaction system is to 20.0 μ L.
As preferably, the present invention provides the multi-PRC reaction condition that an expanding effect is best, particularly as follows: 94 DEG C of denaturation 5min; Circulation process is 94 DEG C of degeneration 30s, 57 DEG C of annealing 30s, and 72 DEG C extend 35s, totally 35 circulations; Last 72 DEG C extend 10min ,-20 DEG C long-term preservations.
The third aspect, the present invention provides a kind of and differentiates that chicken crawls the method for character. Described method is by detecting whether individuality lacks Cp Gene Partial sequence and No. 7 chromosome 11.896kb disappearance upstream and downstream sequence (chr7:21798705-21810600).
The disappearance of described Cp gene detects with genomic DNA for template, adopts multiplex PCR (multiplex-PCRassay) method to be analyzed.
Particularly as follows: with the genomic DNA of test individual for template, utilize described combination of primers, multiple PCR method is adopted to be analyzed; If multiplexed PCR amplification has two kinds of products, sequence size is 438bp and 224bp respectively, then test individual is genetic heterozygosis (Cp/+) of crawling, and shows as character of crawling.
Preferred multi-PRC reaction system and multi-PRC reaction condition are with described previously.
The beneficial effects of the present invention is:
The invention provides one and utilize multiple PCR method; with chicken genomic DNA for template; primer special is adopted to crawl the method for character identification to carrying out chicken; establish the method for normalizing of Cp genotype detection; contribute to the regularity of incubation rate and the growth promoter improving China's native chicken breed, provide important references for the protection of Xingshan walnuts and China's chicken breeds elsewhere, research and development utilization. Experiment proves that this method can pass through to check the upstream and downstream sequence (11.896kb of Cp Gene Partial sequence and No. 7 chromosome deficiency sequences, chr7:21798705-21810600), chicken group is carried out Cp genotype screen, such that it is able to accurately and rapidly individuality is carried out Cp gene type, and chicken group can be carried out Seedling selection, shorten the selection time, save breeding cost. The present invention is that chicken crawls the examination of character Cp gene and breeding work carries out molecular marker assisted selection, it is provided that more accurate, quick, efficient, simple, a cheap molecular genetic marker detection method. Utilize method provided by the invention and primer special that character that chicken is crawled is carried out molecular genetic marker assisted Selection; the accuracy of selection can be improved; and Seedling selection can be carried out; greatly reduce breeding expense; breeding for character chicken breed of crawling provides facility, and native chicken breed protection and exploitation thereof are had great importance. Detection method provided by the invention is simple to operate, accuracy is high, and the testing time is short, low price, and can realize automatization, intellectualized detection. Additionally; the Cp genotype detection method of specification can be set up according to the inventive method, and detection kit can be developed, carry the individuality of Cp gene for examination; in order to crawl, character a breed of chicken work provides facility, and native chicken breed protection and exploitation thereof are had great importance.
Accompanying drawing explanation
Fig. 1 is Cp genotype detection design of primers schematic diagram of the present invention; Wherein, deletion sequence (chr7:21798705-21810600), delF/delR is used for expanding deletion sequence upstream and downstream sequence, and amplified fragments is sized to 224bp; F/R primer is used for expanding Cp Gene Partial gene order, and amplified fragments is sized to 438bp.
Fig. 2 is the multiple PCR primer electrophoretogram to screening with qualification in the embodiment of the present invention 1; Wherein, M is Marker (600bp); Swimming lane swimming lane 1-12 is the PCR primer of delF2/R2-F2/R2 combination of primers; Swimming lane 13-25 is the PCR primer of delF1/R1-F2/R2 combination of primers; Swimming lane 26-34 is the PCR primer of delF1/R1-F1/R1 combination of primers; The purpose fragment of swimming lane 35-48delF3/R3-F1/R1 combination of primers; Swimming lane 49-60 is the PCR primer of delF3/R3-F2/R2 combination of primers.
Fig. 3 is the electrophoretogram that in the embodiment of the present invention 2, multiple PCR method carries out Different Individual Cp gene type; Wherein, the Marker that swimming lane 1 (M) is 600bp; Swimming lane 2-6 is homozygous genotype individual (Cp/Cp); Swimming lane 7-11 is heterozygous genotypes individual (Cp/+);Swimming lane 12-16 is for individual for recessive homozygous genotype (+/+).
Detailed description of the invention
Below in conjunction with embodiment, the preferred embodiment of the present invention is described in detail. It will be appreciated that providing merely to play descriptive purpose of following example, be not used to the scope of the present invention is limited. Those skilled in the art is when without departing substantially from the objective of the present invention and spirit, it is possible to the present invention carries out various amendment and replacement.
Experimental technique in following embodiment, if no special instructions, is conventional method. Test material used in following embodiment, without without specified otherwise, being what routine biochemistry reagent shop was commercially available. % in following embodiment, if no special instructions, is weight/mass percentage composition. Primer synthesis and order-checking complete by Beijing Liuhe Huada Genomics Technology Co., Ltd.
The design of embodiment 1 combination of primers and screening
The method for designing of described combination of primers, as shown in Figure 1.
1.1 design of primers
It is through high-flux sequence and through bioinformatic analysis examination qualification for designing the gene of crawling of primer, there is important biological significance and potential using value. The acquisition of gene order is also obtained by bioinformatic analysis. Deletion sequence and multi-PRC reaction due to large fragment, in design of primers process except considering the specificity of primer, sensitivity, amplification efficiency, consider emphatically the position (deletion fragment intermediate sequence and deletion sequence upstream and downstream sequence) of design of primers, two kinds of PCR primer sizes, two pairs of primer Tm concordance, primer proportioning, typing efficiency etc. Multi-PRC reaction, it is possible to accurately distinguish different genotype individuality. This combination of primers streamline operation, result are accurately, efficiently.
1.2 primer screenings and qualification
In order to accurately and the individual early screening of the character that efficiently carries out crawling and qualification, according to deletion sequence and Cp gene order, PrimerPremier6.0 software is adopted to carry out design of primers, separately design the primer of 5 pairs of deletion sequence primers and 3 pairs of Cp genes, its primer sequence following (5 ' → 3 '):
DelF1:AGCCCCTCATTGTTGTCTCA (as shown in SEQIDNo.1);
DelR1:TCGTTAAGCTGACACCTCCG (as shown in SEQIDNo.2);
delF2:GAACCCTCGCTCCTGAAA(355bp)
delF2:CTCGGTGGCTCCCTATTA
delF3:CCAGCCCCTCATTGTTGTCT(182bp)
delR3:CGCAAGGTGCCACTTATTTATT
delF4:GAACCCTCGCTCCTGAAA(255bp)
delF4:CACGCAAGGTGCCACTTAT
delF5:GAACCCTCGCTCCTGAAA(356bp)
delR5:GCTCGGTGGCTCCCTATT
F1:CTGCCTTGTGCGTTCTCA (as shown in SEQIDNo.3);
R1:CAGGAAGTCGCTGTAGGTG (as shown in SEQIDNo.4);
F2:ACCTACAGCGACTTCCTG(455bp)
R2:TAGAGCAGCCCCGAGTAC
F3:AATTCCGCCCCACCTTTG(292bp)
R3:GAGTACCAGTGCACACCG
In order to obtain good, highly sensitive, simple and efficient, the easily recognizable serotype specific primer of specificity, above deletion sequence primer and Cp gene order primer be combined between two, to filter out best combination of primers.
Gather the hatching full embryo tissue extraction test kit of the chicken of 4 days and extract genomic DNA. Wing venous blood collection during chicken 4 week old, extracts DNA, ddH by the imitative method of tradition phenol2O dissolves, and-20 DEG C save backup.
With the genomic DNA that extracts for template, adopt multi-PRC reaction system (20.0 μ L): chicken genomic DNA 80-100ng, 10 × PCR buffer is (containing Mg2+) 2.0 μ L, dNTPs (2.5mM) 2.0 μ L, Taq DNA polymerase (2.5U/ μ L) 1.0 μ L, delF and delR primer (10 μm of ol/l) each 0.2 μ l, F and R primer (10 μm of ol/l) each 0.4 μ l, uses ddH2O postreaction system is to 20.0 μ L.
Multi-PRC reaction condition is: 94 DEG C of denaturation 5min; Circulation process is 94 DEG C of degeneration 30s, 57 DEG C of annealing 30s, and 72 DEG C extend 35s, totally 35 circulations; Last 72 DEG C extend 10min ,-20 DEG C long-term preservations.
Through the agarose gel detection of multi-PRC reaction and 2.0%, and PCR primer is carried out sequence verification. Result is as shown in Figure 2. As shown in Figure 2, only 5 combination of primers amplify band, respectively the combination of primers of delF2/R2-F2/R2, delF1/R1-F2/R2, delF1/R1-F1/R1, delF3/R3-F1/R1 and delF3/R3-F2/R2, and other combination of primers do not obtain PCR primer. Wherein, M is Marker, and swimming lane 1-12 is delF2/R2-F2/R2 combination of primers product, and this combination amplification efficiency is not high, purpose clip size is close, not easily carry out typing examination. DelF1/R1-F2/R2 combination of primers amplification efficiency is low, have a small amount of primer dimer and non-specific amplification, typing band fuzzy (swimming lane 13-25). DelF1/R1-F1/R1 combination of primers specificity is good, amplification efficiency is high, stripe size is suitable, be prone to typing (swimming lane 26-34). DelF3/R3-F1/R1 (swimming lane 35-48) and delF3/R3-F2/R2 (swimming lane 49-60) occurs that primer dimer, amplification efficiency are low, non-specific amplification, band are unintelligible.
Therefore, the present invention acquires one by molecular screening and has the advantage combination of primers (shown in SEQIDNo.1～4) that specificity is good, highly sensitive, simple and efficient, be prone to typing, this advantage combination of primers establishes the molecular detecting method of character of crawling quickly and accurately, contributes to the conservation to crawl build chicken and China's chicken breeds elsewhere and improvement work.
Thus, present invention screening obtains one and has the advantage combination of primers that specificity is good, highly sensitive, simple and efficient, be prone to typing, and application is protected.
The foundation of embodiment 2 multiple PCR method and Cp gene type
One, Different Individual Cp gene type assay
1, experiment material. Experimental chicken group builds and sample collection completes in China Agricultural University's poultry genetic resources with breeding experiment base. Xingshan walnuts is the poultry genetic resources of a kind of preciousness, having special character of crawling, its character of crawling is to be controlled by single dominant gene Cp, and this gene is dominant homogeneous lethal gene on autosome, genetic heterozygosis cause character of crawling, embryonic death to concentrate on hatching after the 4th day. This experiment have chosen crawls type cock with the offspring of type hen hybridization of crawling as experimental subject.
2, extracting genome DNA
Gather the hatching full embryo tissue extraction test kit of the chicken of 4 days and extract genomic DNA. Wing venous blood collection during chicken 4 week old, extracts DNA, ddH by the imitative method of tradition phenol2O dissolves, and-20 DEG C save backup.
3, multiplexed PCR amplification
With the genomic DNA of step 2 extraction for template, utilize the specific primer that embodiment 1 obtains to carrying out Cp gene type.
Multi-PRC reaction system (20.0 μ L): chicken genomic DNA 80-100ng, 10 × PCR buffer is (containing Mg2+) 2.0 μ L, dNTPs (2.5mM) 2.0 μ L, Taq DNA polymerase (2.5U/ μ L) 1.0 μ L, delF and delR primer (10 μm of ol/l) each 0.2 μ l, F and R primer (10 μm of ol/l) each 0.4 μ l, uses ddH2O postreaction system is to 20.0 μ L.
Multi-PRC reaction condition is: 94 DEG C of denaturation 5min; Circulation process is 94 DEG C of degeneration 30s, 57 DEG C of annealing 30s, and 72 DEG C extend 35s, totally 35 circulations; Last 72 DEG C extend 10min ,-20 DEG C long-term preservations.
4, PCR primer detection
Take 4.5 μ LPCR products and carry out 2.0% agarose gel electrophoresis detection (see Fig. 3).
All detection individualities can be divided into 3 kinds according to electrophoretic band:
1st kind: an only band, being sized to 224bp, for dominant homogeneous individual (Cp/Cp), individuality presents lethal type;
2nd kind: having two bands, size is 224bp and 438bp respectively, for heterozygous genotypes individual (Cp/+), individuality presents the type of crawling;
3rd kind: an only band, size is 438bp respectively, and for recessive homozygous individual (+/+), individuality presents wild type.
Two, sequence verification
Respectively the multiple PCR products of each sample of step 3 is checked order. Result shows: the nucleotide sequence of the multiplexed PCR amplification product of Cp/Cp genotype individuals is such as shown in SEQIDNo.5; The nucleotide sequence of the pcr amplification product of+/+genotype individuals is such as shown in SEQIDNo.6; The multiplexed PCR amplification product of Cp/+ genotype individuals is the heterozygote of DNA shown in SEQIDNo.5 and SEQIDNo.6.
Although, above the present invention is described in detail with a general description of the specific embodiments, but on basis of the present invention, it is possible to it is made some modifications or improvements, and this will be apparent to those skilled in the art. Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to the scope of protection of present invention.
Claims (10)
1. one kind for differentiating that chicken crawls the combination of primers of character, it is characterised in that described combination of primers includes the primer sequence shown in SEQIDNo.1, SEQIDNo.2, SEQIDNo.3 and SEQIDNo.4.
2. the combination of primers described in claim 1 is differentiating that chicken crawls the application in character.
3. in preparation, the combination of primers described in claim 1 differentiates that chicken crawls the application in the reagent of character or test kit.
4. the combination of primers described in claim 1 is crawled the application in character assistant breeding chicken.
5. application according to claim 4, it is characterised in that with the genomic DNA of test individual for template, utilizes the combination of primers described in claim 1, adopts multiple PCR method to be analyzed; If multiplexed PCR amplification has a kind of product, and sequence size is 224bp, then test individual is gene dominant homozygote (Cp/Cp) of crawling, and shows as lethal type; If multiplexed PCR amplification has two kinds of products, sequence size is 438bp and 224bp respectively, then test individual is genetic heterozygosis (Cp/+) of crawling, and shows as character of crawling; If only a kind of product of multiplexed PCR amplification, sequence size is 438bp, then test individual be stealth homozygote (+/+), shows as wild type.
6. application according to claim 5, it is characterised in that multi-PRC reaction system is: genomic DNA 80-100ng, 10 × PCR buffer 2.0 μ L, dNTPs2.0 μ L, Taq DNA polymerase 1.0 μ L, the each 0.2 each 0.4 μ L of μ L, F and R primer of delF and delR primer, uses ddH2O postreaction system is to 20.0 μ L.
7. the application according to claim 5 or 6, it is characterised in that multi-PRC reaction condition is: 94 DEG C of denaturation 5min; Circulation process is 94 DEG C of degeneration 30s, 57 DEG C of annealing 30s, and 72 DEG C extend 35s, totally 35 circulations; Last 72 DEG C extend 10min.
8. one kind differentiates that chicken crawls the method for character, it is characterised in that with the genomic DNA of test individual for template, utilizes the combination of primers described in claim 1, adopts multiple PCR method to be analyzed; If multiplexed PCR amplification has two kinds of products, sequence size is 438bp and 224bp respectively, then test individual is genetic heterozygosis (Cp/+) of crawling, and shows as character of crawling.
9. method according to claim 8, it is characterised in that multi-PRC reaction system is: genomic DNA 80-100ng, 10 × PCR buffer 2.0 μ L, dNTPs2.0 μ L, Taq DNA polymerase 1.0 μ L, the each 0.2 each 0.4 μ L of μ L, F and R primer of delF and delR primer, uses ddH2O postreaction system is to 20.0 μ L.
10. method according to claim 8 or claim 9, it is characterised in that multi-PRC reaction condition is: 94 DEG C of denaturation 5min; Circulation process is 94 DEG C of degeneration 30s, 57 DEG C of annealing 30s, and 72 DEG C extend 35s, totally 35 circulations; Last 72 DEG C extend 10min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610134746.0A CN105671170B (en) | 2016-03-10 | 2016-03-10 | It is a kind of for identify chicken crawl character primer combination and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610134746.0A CN105671170B (en) | 2016-03-10 | 2016-03-10 | It is a kind of for identify chicken crawl character primer combination and its application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105671170A true CN105671170A (en) | 2016-06-15 |
| CN105671170B CN105671170B (en) | 2018-12-14 |
Family
ID=56308087
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610134746.0A Active CN105671170B (en) | 2016-03-10 | 2016-03-10 | It is a kind of for identify chicken crawl character primer combination and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105671170B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108342502A (en) * | 2018-02-05 | 2018-07-31 | 西南大学 | It identifies the PCR primer pair of wheat ms1b recessiveness sterile genes and primer pair combines and its detection method |
| CN110169391A (en) * | 2019-06-24 | 2019-08-27 | 四川省畜牧科学研究院 | Dwarf-type Local chicken breeds breed protection and selection method |
| CN116411090A (en) * | 2023-05-24 | 2023-07-11 | 江苏省家禽科学研究所 | SNP locus primer combination for identifying pettitoes and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102618649A (en) * | 2012-04-01 | 2012-08-01 | 广州市权诚生物科技有限公司 | Chicken dwarf foot related molecular detection method |
-
2016
- 2016-03-10 CN CN201610134746.0A patent/CN105671170B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102618649A (en) * | 2012-04-01 | 2012-08-01 | 广州市权诚生物科技有限公司 | Chicken dwarf foot related molecular detection method |
Non-Patent Citations (5)
| Title |
|---|
| KINOSHITA,K.等: "Gallus gallus DNA, including NHEJ1 and IHH gene region", 《GENBANK DATABASE》 * |
| R.S. MA等: "The Ihh signal is essential for regulating proliferation and hypertrophy of cultured chicken chondrocytes", 《COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY, PART B》 * |
| TOHRU SHIBUYA等: "STUDIES ON THE ACTION OF CREEPER GENE IN JAPANESE CHICKEN", 《JAPAN. J. GENETICS》 * |
| YOSHIHISA FUJIO等: "EXPRESSION OF LETHALITY CAUSED BY CREEPER GENE IN THE CHICKEN", 《JAPAN.J. GENETICS》 * |
| YVETTE BREN-MATTISON等: "Growth of Limb Muscle is Dependent on Skeletal-Derived Indian Hedgehog", 《DEV BIOL.》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108342502A (en) * | 2018-02-05 | 2018-07-31 | 西南大学 | It identifies the PCR primer pair of wheat ms1b recessiveness sterile genes and primer pair combines and its detection method |
| CN110169391A (en) * | 2019-06-24 | 2019-08-27 | 四川省畜牧科学研究院 | Dwarf-type Local chicken breeds breed protection and selection method |
| CN116411090A (en) * | 2023-05-24 | 2023-07-11 | 江苏省家禽科学研究所 | SNP locus primer combination for identifying pettitoes and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105671170B (en) | 2018-12-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Sorenson et al. | Numts: a challenge for avian systematics and population biology | |
| CN103667429B (en) | Method of screening silky character of chicken by SNP (Single Nucleotide Polymorphism) detection | |
| Douglas et al. | Complete mitochondrial DNA sequence analysis of Bison bison and bison–cattle hybrids: Function and phylogeny | |
| CN113046444A (en) | SNP marker combination for tracing and identifying beef cattle individual and meat product and application thereof | |
| CN108410994A (en) | It is a kind of influence sheep Fecundity Trait SNP marker and its application | |
| OLANO‐MARIN et al. | A genome‐wide set of 106 microsatellite markers for the blue tit (Cyanistes caeruleus) | |
| CN106906303A (en) | One SNP marker for influenceing quality character of pork and its application | |
| CN110760599B (en) | Cannabis harfish microsatellite molecular marker locus, polymorphism primer and application | |
| Mikulíček et al. | Hybridization between three crested newt species (Triturus cristatus superspecies) in the Czech Republic and Slovakia: comparison of nuclear markers and mitochondrial DNA | |
| Kang et al. | Microsatellite loci analysis for the genetic variability and the parentage test of five dog breeds in South Korea | |
| CN105671170A (en) | Primer combination for identifying creeping character of chicken and application thereof | |
| CN102534006B (en) | Method for detecting homozygosis or heterozygosis of silkie fiber melanin gene | |
| CN102352410A (en) | Method for rapidly improving number of eggs produced by Bian chicken through DNA (deoxyribonucleic acid) labeling | |
| CN104131097B (en) | A kind of method detecting beef cattle UCP3 gene mononucleotide polymorphism and application thereof | |
| Miles et al. | A genetic linkage map for the saltwater crocodile (Crocodylus porosus) | |
| CN104694538B (en) | The SNP marker related to the more toe characters of chicken and its application | |
| CN102220410B (en) | Method for auxiliary identification of chickens with different weight properties | |
| CN103602744B (en) | Primers, kit and detection method for detecting gene type of dominant white feather site of chicken | |
| CN112176073B (en) | PROS1 gene molecular marker related to chicken carcass traits and application | |
| CN103789445B (en) | Molecular genetic marker for weight of jinghai yellow chicken at 12 weeks and application thereof | |
| Mustafa et al. | Comparative analysis of genome wide difference in Red Sindhi and Holstein cattle breeds using dense SNP marker | |
| Melka et al. | Identification of genomic differences between Hanwoo and Holstein breeds using the Illumina Bovine SNP50 BeadChip | |
| Igawa et al. | Development and validation of PCR-RFLP assay to identify three Japanese brown frogs of the true frog genus Rana | |
| CN110835651B (en) | Primer and kit for detecting indel multiple allele markers of chicken CDKN3 gene promoter region and application of primer and kit | |
| Komarova et al. | Genetic structure of natural populations of sterlet (Acipenser ruthenus L.) in the catchment basins of the Kama and Ob rivers based on polymorphic ISSR markers |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |