CN105671170B - It is a kind of for identify chicken crawl character primer combination and its application - Google Patents

It is a kind of for identify chicken crawl character primer combination and its application Download PDF

Info

Publication number
CN105671170B
CN105671170B CN201610134746.0A CN201610134746A CN105671170B CN 105671170 B CN105671170 B CN 105671170B CN 201610134746 A CN201610134746 A CN 201610134746A CN 105671170 B CN105671170 B CN 105671170B
Authority
CN
China
Prior art keywords
primer
seq
chicken
character
test individual
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.)
Active
Application number
CN201610134746.0A
Other languages
Chinese (zh)
Other versions
CN105671170A (en
Inventor
侯卓成
金四华
杨宁
郑江霞
李俊英
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
China Agricultural University
Original Assignee
China Agricultural University
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by China Agricultural University filed Critical China Agricultural University
Priority to CN201610134746.0A priority Critical patent/CN105671170B/en
Publication of CN105671170A publication Critical patent/CN105671170A/en
Application granted granted Critical
Publication of CN105671170B publication Critical patent/CN105671170B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

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)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The present invention relates to molecular biology field, specifically disclose it is a kind of crawl the primer combination and its application of character for identifying chicken, the primer combination includes SEQ ID No.1, SEQ ID No.2, primer sequence shown in SEQ ID No.3 and SEQ ID No.4.Being combined using the primer can be achieved the crawl identification of character of chicken and is combined using the primer, analyzed using multiple PCR method specially using the genomic DNA of test individual as template；If multiplexed PCR amplification has a kind of product, and sequence size is 224bp, then test individual is to crawl gene dominant homozygote (Cp/Cp), shows as lethal type；If multiplexed PCR amplification, there are two types of product, sequence size is respectively 438bp and 224bp, then test individual is genetic heterozygosis (Cp/+) of crawling, character of crawling is shown as；If multiplexed PCR amplification only has a kind of product, sequence size 438bp, then test individual is stealthy homozygote (+/+), shows as wild type.

Description

It is a kind of for identify chicken crawl character primer combination and its application
Technical field
The present invention relates to molecular biology fields, specifically, being related to a kind of crawling the primer sets of character for identifying chicken It closes and its applies.
Background technique
Up to the present, it has been found that eight kinds of dwarf genes, they are located at sex chromosome and often dyeing in chicken body On body, wherein sex-kink dwarf gene dw research is more, has carried out accurate positioning and its clear mechanism of action to the gene (Agarwal,S.K.,L.A.Cogburn,and J.Burnside.1994.Dysfunctional growth hormone receptor in a strain of sex-linked dwarf chicken:evidence for a mutation in the intracellular domain.J.Endocrinol.142:427-434.).Low and small gene on autosome compared with More, wherein Cp gene (Cp:Creeper, type of crawling) is a kind of important dominant homogeneous lethal gene on autosome (Landauer,W.,and L.C.Dunn.1930.Studies on the Creeper Fowl.I.Genetics.J.Genet.23:397-413.), the character of crawling of gene control chicken.
Cutler (Cutler, I.E.1925.Reptilian fowls.J.Hered.16:352-356.) is delivered crawl first The hypothesis that crawl character is influenced by heterozygous genotypes.Landauer and Dunn (Landauer, W., and L.C.Dunn.1930.Studies on the Creeper fowl.I.Genetics.J.Genet.23:397-413.) pass through Cross experiment finds a kind of dwarf chicken for causing the abnormal development of cartilage, is named as creeper fowl (Creeper fowl), and demonstrate,prove The hereditary basis for character of crawling in fact is controlled by single Mendel's dominant gene Cp, in sub (Cp/Cp) the condition following table of dominant homogeneous It is now lethal type, heterozygote (Cp/+) performance is crawled type, allozygote (+/+) wild type is shown as, entire hatching early stage is dead Dying rate is 25%, and 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.Studies on the creeper fowl.V.The linkage of the genes for creeper and single- comb.J.Genet.26:285-290.；Landauer,W.1933.Creeper and single-comb linkage in fowl.Nature 132:606.；Taylor,L.W.1934.Creeper and single-comb linkage in the fowl.J.Hered.25:205-206.).Somes and Jr. by the Cp assignment of genes gene mapping in the Ith linkage group, with rose comb gene (R) Linkage distance is 0.4cM, with oil gland un-mixing bases because the linkage distance of (U) is 30cM (Somes, R.G., and Jr.1973.Linkage relationships in domestic fowl.J.Hered.64:217-221.).Imsland etc. By linkage analysis and high density SNP studies have shown that the inversion of 7.4Mb causes MNR2 gene location to change on No. 7 chromosomes Become, which leads to rose comb phenotype, further confirm rose comb gene (R) be located on No. 7 chromosomes (Imsland, 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,and L.Andersson.2012.The Rose-comb mutation in chickens constitutes a structural rearrangement causing both altered comb morphology and defective sperm motility.PLoS Genet.8:e1002775.).Xingshan walnuts are domestic rare ground One of square short-foot chicken breed, due to special figure of crawling, attention and concern by domestic and international expert's height.Control this The key gene of one character is Cp (Creeper) gene, which is a kind of autosomal dominant homozygosis lethal gene.In the world Have more than 80 years history (Cairns, J.M., and K.Gayer.1943.Identification of to the research of Cp gene chick embryos homozygous for the Creeper factor.J.Exp.Zool.92:229-242.； Landauer,W.1944.Length of survival of homozygous creeper fowl embryos.Science 100:553-554.；Dinner,B.1971.Chemical analysis of embryonic Creeper chicken tibia.J.Dent.Res.50:704.；Fujio,Y.,and T.Shibuya.1974.Expression of lethality Caused by Creeper gene in the chicken.Japan.J.Genet.49:87-91.), the states such as Xingyi, Miyi Interior Local chicken breeds are due to carrying the gene, and the uniformity of the hatching rate and growth and development that lead to these native chicken breeds is by serious It influences.The genetic development of character Cp gene so research control is crawled facilitates to Xingshan walnuts and China chicken elsewhere Variety source is protected, researches and develops utilization.It is mainly long according to shin to the breeding for character of crawling according to conventional breeding methods Length determine that the period is longer, expense is more.In addition, the shape of conventional detection Cp homozygous lethal embryo and natural death embryo State and cytology sampling operation are complicated, and the analysis measurement period is long, and the degree of automation is not high, detect not accurate enough.And it is sharp Carrying out molecular breeding not only with molecular marker assisted selection can be directly selected, and can be contracted significantly in Seedling selection Short breeding cycle improves the accuracy of breeding, reduces breeding expense.
Summary of the invention
In order to solve the problems in the existing technology, the object of the present invention is to provide one kind crawls character for identifying chicken Primer combination and its application.
In order to achieve the object of the present invention, technical scheme is as follows:
In a first aspect, crawl the primer combination of character the present invention provides a kind of for identifying chicken, primer combination packet Include SEQ ID No.1, SEQ ID No.2, primer sequence shown in SEQ ID No.3 and SEQ ID No.4.
The primer combination is to ensure primer specificity, sensitivity by 6.0 software design of Primer Premier Under the premise of amplification efficiency, expand to obtain required purpose band by multiplex PCR experiment, PCR product is by purifying, connection Conversion and sequencing, screening obtains after verifying in big group.
The primer combination is in exploitation molecular detection kit, poultry molecular breeding and Local Chicken Breeds development of resources and benefit There is significant advantage with aspect, the character that can crawl to chicken (Creeper) quickly, accurately and efficiently screen and identify, It is easy to operate, cheap.The primer combine high specificity, high sensitivity, it is low in cost, easy quickly, it is applied widely.It adopts It is had its unique advantages with multiple PCR method, primary first-order equation can be with three kinds of different genotype individuals of screening, and can prepare to examine Survey the presence or missing of gene order of crawling.Multi-primers combination facilitates to figure chicken and China's native chicken breed of crawling Resource is protected, researches and develops utilization.
Second aspect, the present invention provides primer combinations in the application for identifying chicken and crawling in character.
And the primer combination identifies the application in the reagent or kit that chicken crawls character in preparation.
And the described primer combination is crawled the application in character assistant breeding in chicken.
Further, the application specifically:
Using the genomic DNA of test individual as template, is combined using the primer, divided using multiple PCR method Analysis；If multiplexed PCR amplification has a kind of product, and sequence size is 224bp, then test individual is gene dominant homozygote of crawling (Cp/Cp), lethal type is shown as；If multiplexed PCR amplification, there are two types of product, sequence size is respectively 438bp and 224bp, then Test individual is genetic heterozygosis (Cp/+) of crawling, and shows as character of crawling；If multiplexed PCR amplification only has a kind of product, sequence Column size is 438bp, then test individual is stealthy homozygote (+/+), shows as wild type.
Preferably, the present invention provides the optimal multi-PRC reaction system of an expanding effect, specifically: genomic DNA 80-100ng, 10 × PCR buffer (contain Mg2+) 2.0 2.0 μ L, Taq archaeal dna polymerases (2.5U/ μ L) of μ L, dNTPs (2.5mM) 1.0 μ L, delF and delR primer (10 μm of ol/l) each 0.4 μ l of each 0.2 μ l, F and R primer (10 μm of ol/l), uses ddH2O supplement is anti- Answer system to 20.0 μ L.
Preferably, the present invention provides the optimal multi-PRC reaction condition of an expanding effect, specifically: 94 DEG C of pre- changes Property 5min；Circulation process is 94 DEG C of denaturation 30s, 57 DEG C of annealing 30s, 72 DEG C of extension 35s, and totally 35 recycle；Last 72 DEG C of extensions 10min, -20 DEG C of long-term preservations.
The third aspect, the present invention provide a kind of identification chicken and crawl the method for character.The method is to be by detecting individual No missing Cp Gene Partial sequence and No. 7 chromosome 11.896kb lack upstream and downstream sequence (chr 7:21798705- 21810600)。
The missing of the Cp gene is detected using genomic DNA as template, using multiplex PCR (multiplex-PCR Assay) method is analyzed.
Specifically: using the genomic DNA of test individual as template, combined using the primer, using multiple PCR method It is analyzed；If multiplexed PCR amplification, there are two types of product, sequence size is respectively 438bp and 224bp, then test individual is to crawl Crawl genetic heterozygosis is sub (Cp/+), shows as character of crawling.
Preferred multi-PRC reaction system and multi-PRC reaction condition are the same as described previously.
The beneficial effects of the present invention are:
The present invention provides it is a kind of utilize multiple PCR method, using chicken genomic DNA as template, using special primer pair into Row chicken crawl character mirror method for distinguishing, establish the method for normalizing of Cp genotype detection, facilitate improve China's native chicken breed Hatching rate and growth and development uniformity, be the protection, research of chicken breeds elsewhere of Xingshan walnuts and China and Development and utilization provide important references.Experiments have shown that this method can be by checking that Cp Gene Partial sequence and No. 7 chromosomes lack The upstream and downstream sequence (11.896kb, chr 7:21798705-21810600) of out-of-sequence column, to chicken, group carries out Cp genotype screen, So as to accurately and rapidly carry out Cp Genotyping to individual, and Seedling selection can be carried out to chicken group, shorten the selection time, Save breeding cost.The present invention be chicken crawl character Cp gene screening and breeding work carry out molecular marker assisted selection, mention A more accurate, quick, efficient, simple and easy, cheap molecular genetic marker detection method is supplied.Utilize the present invention The method and special primer pair of offer to chicken crawl character carry out molecular genetic marker assisted Selection, the accurate of selection can be improved Property, and Seedling selection can be carried out, breeding expense is greatly reduced, provides convenience for the breeding for character chicken breed of crawling, to place Chicken breed protection and its development and utilization have great importance.Detection method provided by the invention is easy to operate, accuracy is high, surveys The examination time is short, cheap, and can realize automation, intellectualized detection.Furthermore it is possible to establish specification according to the method for the present invention Cp genotype detection method, and detection kit can be developed, the individual of Cp gene is carried, for screening for educating for the character chicken that crawls Kind work provides convenience, protects to native chicken breed and its development and utilization have great importance.
Detailed description of the invention
Fig. 1 is Cp genotype detection design of primers schematic diagram of the present invention；Wherein, deletion sequence (chr7:21798705- 21810600), delF/delR is 224bp for expanding deletion sequence upstream and downstream sequence, amplified fragments size；F/R primer is used for Cp Gene Partial gene order is expanded, amplified fragments size is 438bp.
Fig. 2 is multiple PCR primer in the embodiment of the present invention 1 to the electrophoretogram of screening and identification；Wherein, M Marker (600bp)；Swimming lane swimming lane 1-12 is the PCR product of delF2/R2-F2/R2 primer combination；Swimming lane 13-25 is delF1/R1-F2/ The PCR product of R2 primer combination；Swimming lane 26-34 is the PCR product of delF1/R1-F1/R1 primer combination；Swimming lane 35- The target fragment of 48delF3/R3-F1/R1 primer combination；Swimming lane 49-60 is that the PCR of delF3/R3-F2/R2 primer combination is produced Object.
Fig. 3 is the electrophoretogram that multiple PCR method carries out Different Individual Cp Genotyping in the embodiment of the present invention 2；Wherein, it swims Road 1 (M) is the Marker of 600bp；Swimming lane 2-6 is that homozygous genotype is individual (Cp/Cp)；Swimming lane 7-11 is heterozygous genotypes individual (Cp/+)；Swimming lane 12-16 is recessive homozygous genotype (+/+) individual.
Specific embodiment
The preferred embodiment of the present invention is described in detail below in conjunction with embodiment.It will be appreciated that following real Providing merely to play the purpose of explanation for example is applied, is not used to limit the scope of the present invention.The skill of this field Art personnel without departing from the spirit and purpose of the present invention, can carry out various modifications and replace to the present invention.
Experimental method in following embodiments is unless otherwise specified conventional method.Examination as used in the following examples Material is tested, is what routine biochemistry reagent shop was commercially available without no specified otherwise.% in following embodiments, such as without special Illustrate, is mass percentage.Primer synthesis and sequencing are completed by Beijing Liuhe Huada Genomics Technology Co., Ltd.
The design and screening of 1 primer of embodiment combination
The design method of the primer combination, as shown in Figure 1.
1.1 design of primers
Gene of crawling for design primer is identified by high-flux sequence and by bioinformatic analysis screening, tool There are important biological significance and potential application value.The acquisition of gene order is also to be obtained by bioinformatic analysis 's.Due to the deletion sequence and multi-PRC reaction of large fragment, in addition to considering the specific, sensitive of primer during design of primers It spends, outside amplification efficiency, emphatically the position (deletion fragment intermediate sequence and deletion sequence upstream and downstream sequence) of consideration design of primers, two Kind PCR product size, two pairs of primer Tm consistency, primer proportion, parting efficiency etc..Multi-PRC reaction, can accurate area Divide different genotype individual.This primer combination streamline operation, result are accurate, efficient.
1.2 primer screening and identification
For accurate and the character individual that efficiently carries out crawling early screening and identification, according to deletion sequence and Cp gene sequence Column carry out design of primers using 6.0 software of Primer Premier, have separately designed 5 pairs of deletion sequence primers and 3 pairs of Cp bases The primer of cause, primer sequence are following (5 ' → 3 '):
DelF1:AGCCCCTCATTGTTGTCTCA (as shown in SEQ ID No.1)；
DelR1:TCGTTAAGCTGACACCTCCG (as shown in SEQ ID No.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 SEQ ID No.3)；
R1:CAGGAAGTCGCTGTAGGTG (as shown in SEQ ID No.4)；
F2:ACCTACAGCGACTTCCTG (455bp)
R2:TAGAGCAGCCCCGAGTAC
F3:AATTCCGCCCCACCTTTG (292bp)
R3:GAGTACCAGTGCACACCG
In order to obtain the good, high sensitivity of specificity, simple and efficient, easily recognizable serotype specific primer, by the above deletion sequence Primer is combined two-by-two with Cp gene sequence primer, to filter out best primer combination.
The full embryo of chicken that acquisition is hatched 4 days extracts genomic DNA with tissue extraction kit.Wing venous is taken a blood sample when 4 week old of chicken, Method, which is imitated, with traditional phenol extracts DNA, ddH2O dissolution, -20 DEG C save backup.
Using extracted genomic DNA as template, using multi-PRC reaction system (20.0 μ L): chicken genomic DNA 80- 100ng, 10 × PCR buffer (contain Mg2+) 2.0 μ L, dNTPs (2.5mM) 2.0 μ L, Taq archaeal dna polymerase (2.5U/ μ L) 1.0 μ L, delF and delR primer (10 μm of ol/l) each 0.4 μ l of each 0.2 μ l, F and R primer (10 μm of ol/l), use ddH2O supplements reactant It is to 20.0 μ L.
Multi-PRC reaction condition are as follows: 94 DEG C of initial denaturation 5min；Circulation process is 94 DEG C of denaturation 30s, 57 DEG C of annealing 30s, 72 DEG C extend 35s, totally 35 circulation；Last 72 DEG C of extensions 10min, -20 DEG C of long-term preservations.
By multi-PRC reaction and 2.0% Ago-Gel detection, and sequence verification is carried out to PCR product.As a result such as Shown in Fig. 2.As shown in Figure 2, only 5 primer combinations amplify band, respectively delF2/R2-F2/R2, delF1/R1-F2/ The primer of R2, delF1/R1-F1/R1, delF3/R3-F1/R1 and delF3/R3-F2/R2 combine, and the combination of other primers does not obtain PCR product.Wherein, M Marker, swimming lane 1-12 are delF2/R2-F2/R2 primer combination product, and the combination amplification efficiency is not Height, target fragment size is close, is not easy to carry out parting screening.DelF1/R1-F2/R2 primer combination amplification efficiency is low, it is few to have It measures primer dimer and non-specific amplification, parting band obscures (swimming lane 13-25).The combination of delF1/R1-F1/R1 primer is special Property is good, amplification efficiency is high, stripe size is suitable, is easy to parting (swimming lane 26-34).DelF3/R3-F1/R1 (swimming lane 35-48) with It is unintelligible that low primer dimer, amplification efficiency, non-specific amplification, band occurs in delF3/R3-F2/R2 (swimming lane 49-60).
Therefore, the present invention by molecule screening acquire one have the good, high sensitivity of specificity, it is simple and efficient, be easy to The advantage primer of parting combines (shown in No.1~4 SEQ ID), and advantage primer sets conjunction establishes character of quickly and accurately crawling Molecular detecting method, facilitate to figure chicken and China conservation of chicken breeds and the improvement work elsewhere of crawling.
Present invention screening, which obtains one, as a result, has specific good, high sensitivity, advantage that is simple and efficient, being easy to parting Primer combination, application are protected.
The foundation of 2 multiple PCR method of embodiment and Cp Genotyping
One, Different Individual Cp genotyping
1, experimental material.Experimental chicken group building and sample collection are in China Agricultural University's poultry genetic resources and breeding experiment It is completed in base.Xingshan walnuts are a kind of poultry genetic resources of preciousness, have special character of crawling, and character of crawling is It is controlled by single dominant gene Cp, which is dominant homogeneous lethal gene on autosome, and genetic heterozygosis leads to character of crawling, Embryonic death concentrates on the 4th day after hatching.This experiment has chosen offspring's conduct that the type cock that crawls hybridizes with the type hen of crawling Experimental subjects.
2, extracting genome DNA
The full embryo of chicken that acquisition is hatched 4 days extracts genomic DNA with tissue extraction kit.Wing venous is taken a blood sample when 4 week old of chicken, Method, which is imitated, with traditional phenol extracts DNA, ddH2O dissolution, -20 DEG C save backup.
3, multiplexed PCR amplification
The genomic DNA extracted using step 2 is template, and the specific primer obtained using embodiment 1 is to progress Cp gene Parting.
Multi-PRC reaction system (20.0 μ L): chicken genomic DNA 80-100ng, 10 × PCR buffer (contain Mg2+)2.0μ 2.0 μ L, Taq archaeal dna polymerase (2.5U/ μ L) of L, dNTPs (2.5mM), 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 supplements reaction system to 20.0 μ L.
Multi-PRC reaction condition are as follows: 94 DEG C of initial denaturation 5min；Circulation process is 94 DEG C of denaturation 30s, 57 DEG C of annealing 30s, 72 DEG C extend 35s, totally 35 circulation；Last 72 DEG C of extensions 10min, -20 DEG C of long-term preservations.
4, PCR product detects
Take 4.5 μ L PCR products to carry out 2.0% agarose gel electrophoresis detection (see Fig. 3).
All detection individuals can be divided into 3 kinds according to electrophoretic band:
1st kind: an only band, size 224bp are that dominant homogeneous is individual (Cp/Cp), and lethal type is presented in individual；
2nd kind: having two bands, it is that heterozygous genotypes are individual (Cp/+), individual is presented that size, which is respectively 224bp and 438bp, It crawls type；
3rd kind: an only band, size is respectively 438bp, and for recessive homozygous individual (+/+), wild type is presented in individual.
Two, sequence verification
The multiple PCR products of each sample of step 3 are sequenced respectively.The result shows that: Cp/Cp genotype individuals The nucleotide sequence of multiplex PCR amplification product is as shown in SEQ ID No.5；The nucleosides of the pcr amplification product of+/+genotype individuals Acid sequence is as shown in SEQ ID No.6；The multiplex PCR amplification product of Cp/+ genotype individuals is SEQ ID No.5 and SEQ ID The heterozygote of DNA shown in No.6.
Although above the present invention is described in detail with a general description of the specific embodiments, On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.

Claims (6)

1. a kind of crawl the primer combination of character for identifying chicken, which is characterized in that primer combination includes SEQ ID No.1, SEQ ID No.2, primer sequence shown in SEQ ID No.3 and SEQ ID No.4.
2. primer combination described in claim 1 identifies the application in the reagent or kit that chicken crawls character in preparation.
3. primer described in claim 1 combination is crawled the application in character assistant breeding reagent in preparation chicken.
The method of character 4. a kind of identification chicken of non-disease diagnostic purpose crawls, which is characterized in that with the genome of test individual DNA is template, is combined using primer described in claim 1, is analyzed using multiple PCR method；If multiplexed PCR amplification There are two types of product, sequence size is respectively 438bp and 224bp, then test individual is genetic heterozygosis (Cp/+) of crawling, and is shown as It crawls character；If multiplexed PCR amplification only has a kind of product, sequence size 438bp, then test individual is allozygote (+/+), show as wild type；The test individual is 4 week old chickens.
5. according to the method described in claim 4, it is characterized in that, multi-PRC reaction system are as follows: genomic DNA 80- 2.0 2.0 μ L, Taq archaeal dna polymerase of μ L, dNTPs of 100ng, 10 × PCR buffer, 1.0 μ L, SEQ ID No.1, SEQ ID Each 0.4 μ L of primer shown in each 0.2 μ L, SEQ ID No.3 of primer shown in No.2, SEQ ID No.4, uses ddH2O supplements reactant It is to 20.0 μ L.
6. method according to claim 4 or 5, which is characterized in that multi-PRC reaction condition are as follows: 94 DEG C of initial denaturation 5min； Circulation process is 94 DEG C of denaturation 30s, 57 DEG C of annealing 30s, 72 DEG C of extension 35s, and totally 35 recycle；Last 72 DEG C of extensions 10min.
CN201610134746.0A 2016-03-10 2016-03-10 It is a kind of for identify chicken crawl character primer combination and its application Active CN105671170B (en)

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 CN105671170A (en) 2016-06-15
CN105671170B true 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)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
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)

* Cited by examiner, † Cited by third party
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

Patent Citations (1)

* Cited by examiner, † Cited by third party
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)

* Cited by examiner, † Cited by third party
Title
EXPRESSION OF LETHALITY CAUSED BY CREEPER GENE IN THE CHICKEN;YOSHIHISA FUJIO等;《JAPAN.J. GENETICS》;19741231;第49卷(第2期);87-91 *
Gallus gallus DNA, including NHEJ1 and IHH gene region;Kinoshita,K.等;《Genbank Database》;20180403;Accession：LC332495.1 *
Growth of Limb Muscle is Dependent on Skeletal-Derived Indian Hedgehog;Yvette Bren-Mattison等;《Dev Biol.》;20110815;第356卷(第2期);摘要，第2页第2段，第7页第2段至第8页第1段，图3 *
STUDIES ON THE ACTION OF CREEPER GENE IN JAPANESE CHICKEN;TOHRU SHIBUYA等;《Japan. J. Genetics》;19721231;第47卷(第1期);23-32 *
The Ihh signal is essential for regulating proliferation and hypertrophy of cultured chicken chondrocytes;R.S. Ma等;《Comparative Biochemistry and Physiology, Part B》;20130806;第166卷;117-122 *

Also Published As

Publication number Publication date
CN105671170A (en) 2016-06-15

Similar Documents

Publication Publication Date Title
Sharma et al. Genetic diversity estimates point to immediate efforts for conserving the endangered Tibetan sheep of India
Karlsson et al. Leader of the pack: gene mapping in dogs and other model organisms
Kulibaba et al. Prolactin and growth hormone gene polymorphisms in chicken lines of Ukrainian selection
Porta et al. Development of a microsatellite multiplex PCR for Senegalese sole (Solea senegalensis) and its application to broodstock management
Ostroverkhova et al. Genetic diversity of the locus COI-COII of mitochondrial DNA in honeybee populations (Apis mellifera L.) from the Tomsk region
Liu et al. Genetic variability of mtDNA sequences in Chinese native chicken breeds
CN105671170B (en) It is a kind of for identify chicken crawl character primer combination and its application
CN110643716A (en) Molecular marker related to sheep tail fat weight and application thereof
Lu et al. Mapping quantitative trait loci and identifying candidate genes affecting feed conversion ratio based onto two linkage maps in common carp (Cyprinus carpio L)
Kang et al. Microsatellite loci analysis for the genetic variability and the parentage test of five dog breeds in South Korea
CN105483259A (en) SNP locus related to litter size on pig chromosome 12
CN102534006B (en) Method for detecting homozygosis or heterozygosis of silkie fiber melanin gene
CN107988385B (en) Method for detecting marker of PLAG1 gene Indel of beef cattle and special kit thereof
CN108359733B (en) Method for auxiliary detection of 100kg backfat thickness of pig
Miles et al. A genetic linkage map for the saltwater crocodile (Crocodylus porosus)
CN104131097B (en) A kind of method detecting beef cattle UCP3 gene mononucleotide polymorphism and application thereof
Yang et al. Genetic evidence for the origin of an IGF2 quantitative trait nucleotide in Chinese pigs.
CN116555442B (en) Molecular marker for identifying chicken feed utilization rate character based on ATP2A2 gene, and identification method and application thereof
CN112921101A (en) Molecular marker related to sheep remaining feed intake and application thereof
Kánainé Sipos et al. Comparative genetic analysis of natural and farmed populations of pike-perch (Sander lucioperca)
Abdulkareem The genetic variations in mitochondrial D-loop sequence for local ducks in Iraq
Zhang et al. Limited polymorphisms of two Y‐chromosomal SNPs in Chinese and Iranian sheep
Abu-Rekaiba et al. Association of novel polymorphism of esr2 gene (G40100a) with production traits of brown local Iraqi chicken
CN104131096B (en) A kind of method detecting beef cattle UCP3 gene mononucleotide polymorphism
WO2017198889A1 (en) Method for the molecular determination of the sex of birds by means of loop-mediated isothermal amplification

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