CN105671174A - Chicken creeping character gene and DNA molecular marker relevant to chicken creeping character - Google Patents
Chicken creeping character gene and DNA molecular marker relevant to chicken creeping character Download PDFInfo
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Abstract
The invention relates to a chicken creeping character gene and a DNA molecular marker relevant to a chicken creeping character. The molecular marker is located on a No.7 chromosome of a chicken, and a primer used for amplifying the DNA molecular marker is shown in SEQ ID NO:1-2. The invention further identifies a key gene and gene mutation of the chicken creeping character for the first time, gene mutation is caused by hemizygote deficiency of an IHH gene, the deficiency locus is located on chr7:21798705-21810600 (Ensembl, Galgal version 4), the deficiency area comprises the unique target gene IHH, and earlier-stage lethal of embryos is caused by all deficiency of the gene. The chicken creeping character gene is obtained through clone, the gene and the detecting primer of the gene have great significance on deep study on the forming mechanism of the chicken creeping character, screening of the creeping character gene in local chickens and creeping type chicken variety resource protection, study and improving work, and deep study on the limb development mechanism of human beings and other vertebrates can be promoted.
Description
Technical field
The present invention relates to genetically engineered and chicken genetic breeding field, specifically, it relates to chicken crawls character gene and the relevant DNA molecular marker of proterties of crawling to chicken.
Background technology
Dwarf-type is a kind of common abnormal limb phenotype of vertebrates, and different species have similar phenotype. Short and small phenotype, by corresponding Gene Handling, comprises sex chromosome dwarf gene and euchromosome dwarf gene. Proterties of crawling is a kind of distinctive short and small phenotype existed in chicken breed, and this phenotype is mainly characterized as that the short body of shin is short, wing is short and small. Understanding the molecule mechanism that proterties of crawling is formed, conservation and improvement work to the chicken that crawls have important meaning, also for the limb development of the mankind and other species provides reference and reference.
It is the qualitative character controlled by the dominant homogeneous lethal gene (Cp) on euchromosome that Landauer and Dunn (1930) proposes proterties of crawling, heterozygote individual shows proterties of crawling, dominant homogeneous shows as lethal type, embryonic death concentrates on the 4th day of hatching, and hatching Infant Mortality is 25%, and in offspring, the surviving rate of heterozygote is substantially not influenced, but owing to cartilage is malnutritive, cause hypoevolutism, cause shin bone to become short, show as proterties of crawling. In the world to the research research history of existing nearly 90 years of proterties of crawling, the research that proterties Embryo Gallus domesticus of crawling has been correlated with by researcher from morphology, anatomy, cytology and molecular level. Early stage research trial physical index judges the separation phenotype of embryo, and the method that this research is counted by the body segment of body early embryo 24-25h, 48-54h or morphologic method confirm that body segment number is to distinguish lethal embryo of isozygotying, assorted to close embryo and fetal tissues. Forefathers' research also shows that Cp embryo's cartilage development is abnormal, cause developmental arrest. In addition, Loewenthal (1957) is by the histology of Cp embryo and fetal tissues and histochemical analysis, sea Li Shi stationary liquid (Helly ' sfluid) is utilized to be fixed embryo, then carry out coloured differently to observe Cp embryo and the difference of normal type embryo, result show Cp isozygoty the Feulgen dyeing of embryo and normal type embryo and kytoplasm basophilia all significantly different.By nucleic acid determination research, Wallace etc. (1969) show that Cp embryo and normal type embryo also have significant difference. In addition, the cell cultures of Cp embryo and cartilage development have also been carried out correlative study by investigator. But these are distinguished the method complicated operation of embryonic phenotypes, costly, length consuming time, need special instrument, production is difficult to large-scale promotion application. Therefore, develop and a kind of utilize the molecule marker proterties detection method accurately that carries out crawling to have important meaning.
Proterties of crawling is the distinctive phenotypes of some Local chicken breeds, is distinctive limbs abnormal phenotype in chicken. Proterties of crawling is individual due to skeleton development exception, causes hypoevolutism, shin bone to become short, and wing diminishes, health is short and small. Proterties of crawling is controlled by dominant homogeneous lethal gene Cp on euchromosome, and therefore Cp gene identity is most possibly relevant to the gene and signal path gene thereof that control body skeleton development. Xingshan walnuts is the local variety originating in Guizhou Province of China, has the features such as the short body of shin is short, body body is well-balanced, meat is delicious fresh, the double use of meat egg, is the poultry genetic resources of a kind of rare preciousness. Special crawl proterties owing to this kind has and paid close attention to widely, but there is the growth problems such as hatching rate is low, shin length is inconsistent, whose body weight differs greatly, toe deformity, some Local chicken breeds such as Miyi are also owing to carrying the hatching rate that this gene seriously affects these local variety and the reguarity grown. Therefore, only clone obtain Cp gene, could fundamentally solve in breeding exist these problems, could more further investigate crawl proterties formed molecule mechanism and Local chicken breeds in this gene of examination. In addition, chicken is as an animal model, and the research of Cp gene also will provide reference for the mankind and the research of other vertebrates limb developments, also will promote the research of human cartilage development-related disorders.
Summary of the invention
It is an object of the invention to provide the relevant DNA molecular marker of a kind of proterties of crawling to chicken and application thereof.
It is a further object of the present invention to provide a kind of qualification or assistant identification is crawled the method for proterties chicken.
It is yet another object of the invention to provide chicken to crawl character gene.
In order to realize the object of the invention, the DNA molecular marker that proterties of crawling to chicken provided by the invention is relevant, it is positioned on chicken No. 7 karyomit(e)s, for the primer of the described DNA molecular marker that increases as shown in SEQIDNO:1-2.
Described DNA molecular marker is made up of the upstream and downstream sequence of long segment deletion sequence (chr7:21798705-21810600) on chicken No. 7 karyomit(e)s, and it is:
I) nucleotide sequence shown in SEQIDNO:3; Or
Ii) nucleotide sequence shown in SEQIDNO:3 is substituted, lacks and/or increases one or more Nucleotide and the nucleotide sequence of expression identical function protein; Or
Iii) under strict conditions with sequence hybridization shown in SEQIDNO:3 and express identical function protein nucleotide sequence, described stringent condition is in 0.1 × SSPE containing 0.1%SDS or the 0.1 × SSC solution containing 0.1%SDS, hybridize at 65 DEG C, and wash film with this solution; Or
Iv) and i), ii) or nucleotide sequence iii) there is more than 90% homology and express the nucleotide sequence of identical function protein.
The present invention also provides the application of described DNA molecular marker in chicken molecular mark.
The present invention also provides a kind of qualification or assistant identification to crawl the method for proterties chicken, comprises the following steps:
1) genomic dna of testing sample is extracted;
2) taking the genomic dna of testing sample as template, primer shown in SEQIDNO:1-2 is utilized to carry out pcr amplification;
3) pcr amplification product is detected; If containing described DNA molecular marker in amplified production, then judging that this individuality is as the proterties chicken that crawls; If not containing described DNA molecular marker in amplified production, then judging this individuality not as the proterties chicken that crawls.
Step 2) in PCR reaction system comprise: genomic dna 80-100ng, containing Mg2+10 × PCR damping fluid 3.0 μ l, 2.5mMdNTPs4.0 μ l, 2.5U/ μ lTaqDNA polysaccharase 1.5 μ l, 10 μm of ol/l upstream and downstream primers each 0.2 μ l, ddH2O complements to cumulative volume 25.0 μ l.
Pcr amplification program: 94 DEG C of sex change 5min; 94 DEG C of sex change 30sec, 58 DEG C of annealing 30sec, 72 DEG C extend 30sec, totally 33 circulations; 72 DEG C extend 5min; 4 DEG C of preservations.
Step 1) and 2) described in testing sample derive from chicken blood or the histoorgan from body; Step 3) in detection pcr amplification product adopt method comprise the methods such as agarose gel electrophoresis, order-checking or molecular hybridization.
The imitative method of phenol is preferably adopted to extract the wing venous blood DNA of chicken individuals to be measured.
The present invention also is provided for identifying the PCR detection kit of proterties chicken of crawling, and comprises primer shown in SEQIDNO:1-2 in described test kit.
The present invention also provides chicken to crawl character gene, described gene is positioned at the centre of long segment deletion sequence (chr7:21798705-21810600) on chicken No. 7 karyomit(e)s, this deletion sequence comprises Cp gene, its identity is IHH gene, namely causes chicken to crawl proterties by the hemizygote disappearance of IHH gene. This gene is specifically positioned at chicken genome chr7:21803719-21807290 (Ensembl database, Galgal4 version), and it is:
I) nucleotide sequence shown in SEQIDNO:6; Or
Ii) nucleotide sequence shown in SEQIDNO:6 is substituted, lacks and/or increases one or more Nucleotide and the nucleotide sequence of expression identical function protein; Or
Iii) under strict conditions with sequence hybridization shown in SEQIDNO:6 and express identical function protein nucleotide sequence, described stringent condition is in 0.1 × SSPE containing 0.1%SDS or the 0.1 × SSC solution containing 0.1%SDS, hybridize at 65 DEG C, and wash film with this solution; Or
Iv) and i), ii) or nucleotide sequence iii) there is more than 90% homology and express the nucleotide sequence of identical function protein.
The present invention described chicken that also is provided for increasing crawls the Specific PCR primers of character gene, and described primer is as shown in SEQIDNO:4-5.
The present invention further provides the PCR detection reagent containing primer shown in SEQIDNO:4-5 or test kit.
The PCR reaction system being applicable to described detection reagent or test kit comprises: genomic dna 50-500ng, containing Mg2+LAPCR damping fluid 3.0 μ l, 2.5mMdNTPs4.0 μ l, each 0.5 μ l of 10mmol/L upstream and downstream primer, LATaqDNA polysaccharase 1.5U, ddH2O complements to cumulative volume 25.0 μ l.
Pcr amplification program: 98 DEG C of sex change 10sec, 60 DEG C of annealing 30sec, 72 DEG C extend 3min, totally 30 circulations; 72 DEG C extend 10min; 4 DEG C of preservations.
The present invention utilizes bioinformatic analysis to identify chicken first and crawls the key gene of proterties and reason sudden change, namely cause by the hemizygote disappearance of IHH gene, this deletion segment is positioned at chr7:21798705-21810600, this absent region comprises unique goal gene IHH, whole disappearances of this gene, cause the early lethality of embryo. In addition, the present invention utilizes PCR method to detect the proterties chicken that crawls ingeniously, compensate for the deficiency of traditional screening method method, shortens the screening cycle.The present invention also clones and obtains chicken and crawl character gene; the examination in the further investigation of proterties formation mechenism and Local chicken breeds of crawling of this gene and detection primer pair chicken thereof crawls character gene and type chicken breeds protection of crawling, research and improvement work have important meaning, additionally it is possible to promote the further investigation of the limb development mechanism of the mankind and other vertebratess.
Accompanying drawing explanation
Fig. 1 is the DNA electrophoresis detection result adopting the imitative method of phenol to extract chicken genome in the embodiment of the present invention 1; Wherein, M is DNAMarker, numbering 1-8 is individuality to be measured.
Fig. 2 is that in the embodiment of the present invention 1, LA-PCR method amplification chicken crawls the electrophorogram of character gene; Wherein, M is 1kbDNAMarker, and swimming lane 1 and 5 amplifies gene band (3350bp) of crawling for type individuality of crawling; Swimming lane 2 is that early dead embryo does not amplify gene band of crawling; Swimming lane 3 and 4 is crawled gene band for normal individual amplification.
Fig. 3 is the PCR primer electrophoresis detection result of proterties chicken individuals of crawling in the embodiment of the present invention 2; Wherein, to be 600bpDNAMarker, numbering 1-8 be M crawls the object fragment (234bp) of proterties individuality.
Embodiment
Following examples are for illustration of the present invention, but are not used for limiting the scope of the invention. If not indicating especially, embodiment is experiment condition all conveniently, such as Sambrook equimolecular cloning experimentation handbook (SambrookJ&RussellDW, MolecularCloning:aLaboratoryManual, 2001) condition, or according to manufacturer's specification sheets advised.
Primer synthesis and order-checking complete by raw work biotechnology (Shanghai) limited-liability company.
Embodiment 1 chicken crawls the clone of character gene Cp
Intend adopting the Xingshan walnuts with complete pedigree record to be experimental subjects, set up Cp genotype to divide and leave home to be (type of crawling cock and crawl type hen hybridization), gather dead embryo, type of crawling and normal full sibs or half sibs are individual, extract genomic dna and carry out DNA quality control, each individuality builds separately library, carry out full-length genome by IlluminaHiseq2000 high-flux sequence platform to resurvey sequence, counterweight sequencing data carries out quality control and comparison, in genome range, each individuality is carried out various analysis of variance, comprise SNP, InDel (insertion and deletion), CNVs (copy number variation), gene recombination etc. the main bioinformatics software used comprises bowtie2 (sequence alignment), SAMTools (BAM processing of documents), GATK, SNPTools (SNP, little InDel analyzes), Pindel, PEMer (big InDel, restructuring), CNVnator and CNV-TV (CNV analysis). through degree of depth bioinformatic analysis and excavation, carry out Cp genome location, obtain Cp gene structure.
The crawl cloning process of character gene of chicken is as follows:
1.1 experiment material
Proterties of crawling chicken (Xingshan walnuts) is raised and sample collecting completes at China Agricultural University's poultry genetic resources and breeding test base. Blood adopts ACD anti-freezing, and the ratio of blood and ACD antithrombotics is 1:3.
1.2 extracting genome DNA and detection
Adopting the imitative method of phenol to extract poba gene group DNA, step is as follows:
(1) in 1.5mlEP pipe, add the fresh ACD anti-freezing blood sample of 30-40 μ l respectively.
(2) in each EP pipe, 700 μ l cell pyrolysis liquids are added, fully mixed even 10min.
(3) 30 μ l Proteinase Ks (final concentration is 20mg/ml) are added, vibration 10min.
(4) 55 DEG C, 220 turns/min, water-bath shaken overnight digests.
(5) blood after digestion is added the 600 saturated phenol of μ lTris, slowly put upside down centrifuge tube 10min, then 12000rpm, centrifugal 10min, carefully draw 600 μ l supernatant liquors in clean centrifuge tube with the rifle head of heavy caliber. Repeating step (5), extraction effect is better.
(6) add the phenol imitative (the saturated phenol of Tris: chloroform=1:1) of 600 μ l, slowly put upside down centrifuge tube 10min, then 12000rpm, centrifugal 10min, carefully draw 600 μ l supernatant liquors in clean centrifuge tube with the rifle head of heavy caliber.
(7) add the chloroform isoamyl alcohol (chloroform: primary isoamyl alcohol=24:1) of 600 μ l, slowly put upside down centrifuge tube 10min, then 12000rpm, centrifugal 10min, carefully draw 300 μ l supernatant liquors in clean centrifuge tube with heavy caliber rifle head.
(8) add the dehydrated alcohol of 1000 μ l, gently shake centrifuge tube, treat that adularescent flocks occurs stopping shake.
(9) carefully DNA precipitation being chosen with rifle head, be placed in the centrifuge tube washing filling 300 μ l70% ethanol, 8000rpm, centrifugal 3-5min, abandons supernatant; Or 8000rpm, centrifugal 3-5min, outwells supernatant, then adds 300 μ l70% washing with alcohol 8000rpm, and centrifugal 3-5min, abandons supernatant.
(10) ethanol is clean, room temperature makes ethanol volatilize, and dries 2-3h.
(11) 100 μ lddH are added2O dissolving DNA (or appropriate TE damping fluid), the DNA dissolved, 4 DEG C of preservations of spending the night. Then it is placed in-20 DEG C to preserve for a long time.
1.3DNA quality and Concentration Testing
DNA concentration and OD value is measured with NanoDrop2000. With 1.0% agarose gel electrophoresis detection DNA, as shown in Figure 1, the genomic dna quality of extraction is better, and master tape is single, clear for result.
1.4 design of primers
Adopt PrimerPremier6.0 software design primer, in design of primers process, avoid the appearance of the situations such as hairpin structure, primer dimer and mispairing as far as possible, make primer sequence optimization. The primer sequence used that increases is as follows:
F1:5′-TCCGTCAAGTCAGGTAAGG-3′(SEQIDNO:4)
R1:5′-CACCCACGCAACCAAGT-3′(SEQIDNO:5)
1.5 experimental technique
Taking the genomic dna of aforesaid method extraction as template, taking F1, R1 as primer, adopting LA-PCR amplification method amplification aim sequence, long segment amplification enzyme is purchased from TaKaRa company (CodeNo:RR900A).
PCR reaction system: chicken genomic dna 50-500ng, LAPCRbuffer II damping fluid is (containing Mg2+) it is the dNTPs mixed solution 4.0 μ l of 3.0 μ l, 2.5mM, each 0.5 μ l of 10mmol/L primers F 1, R1, LATaqDNA polysaccharase 1.5U, ddH2O complements to cumulative volume 25.0 μ l.
PCR reaction conditions: 98 DEG C of sex change 10sec, 60 DEG C of annealing 30sec, 72 DEG C extend 3min, totally 30 circulations; 72 DEG C extend 10min; 4 DEG C save backup.
Electrophoresis detection, purifying reclaim and sequence verification: the sepharose of compound concentration 1.5%, adopt the condition of 100V, 40min to carry out electrophoresis detection. As shown in Figure 2, individuality to be measured can amplify single band to LA-PCR amplified production. Acquisition PCR primer carrying out cut glue reclaim, the product after purifying delivers to company's sequence verification. Through order-checking, gained nucleotide sequence is as shown in SEQIDNO:6, and namely chicken crawls character gene Cp.
Embodiment 2 is crawled to chicken the acquisition of the relevant DNA molecular marker of proterties
Proterties of crawling chicken deletion sequence is positioned at chicken No. 7 karyomit(e) chr7:21798705-21810600 (Ensembl, Galgal4 version) region, therefore designing deletion sequence primer, increase deletion sequence under Standard PCR reaction conditions, detects the phenotype of individuality to be measured.
Taking the genomic dna that extracts as template (genome DNA extracting method is with embodiment 1), adopt PCR method amplification deletion sequence.Based on the upstream and downstream sequences Design primer of absent region, primer sequence used is F:5 '-TTTGGCAGCTAGAAGGGGAA-3 ' and R:5 '-GTTGCGAGAGTGAGCCATG-3 ' (SEQIDNO:1-2).
PCR reaction system (25.0 μ l): chicken genomic dna 80-100ng, 10 × PCR damping fluid is (containing Mg2+) 3.0 μ l, dNTPs (2.5mM) 4.0 μ l, Taq DNA polymerase (2.5U/ μ l) 1.5 μ l, primers F, R (10 μm of ol/l) each 0.2 μ l, ddH2O complements to cumulative volume 25.0 μ l.
Pcr amplification program: 94 DEG C of sex change 5min; 94 DEG C of sex change 30sec, 58 DEG C of annealing 30sec, 72 DEG C extend 30sec, totally 33 circulations; 72 DEG C extend 5min; 4 DEG C of preservations.
Electrophoresis detection pcr amplification product is carried out with the sepharose of concentration 1.5%, pcr amplification product is as shown in Figure 3, as can be seen from the figure proterties of crawling chicken can amplify single DNA molecular marker band (234bp), and what this band do not detected is not the proterties chicken that crawls. PCR primer glue carries out sequence verification after reclaiming, and compares with sequence in database, the DNA fragmentation amplified, and the nucleotide sequence of the DNA molecular marker that proterties of namely crawling to chicken is relevant is as shown in SEQIDNO:3.
Embodiment 3 is crawled the screening method of proterties chicken
3.1 experiment material
Xingshan walnuts father and mother are for system of family and offspring: Xingshan walnuts G0In generation, crawls type cock by 5 and 15 type hens that crawl form (planting egg to provide by livestock and poultry genetic resources management station of Guizhou Province, by the big poultry genetic resources of Chinese agriculture and the hatching of breeding test base). G0For crawling type cock and G0For crawling, the hybridization of type hen obtains G1Generation. G1Crawling in generation, the hybridization of type hen obtains G to type cock with crawling2Generation (table 1).
3.2 crawl type body detecting method
Gather the blood of all individualities, genomic dna is extracted according to the imitative method of phenol, detection DNA quality, then PCR method described in embodiment 2 is adopted to carry out genome is crawled to chicken the detection of the relevant DNA molecular marker of proterties, if occurring single DNA molecular marker band (234bp) in genomic dna sequence, then this individuality is the proterties chicken that crawls; If not occurring this band in genomic dna sequence, then this individuality to be measured is not the proterties chicken that crawls.
All individualities are carried out genotype differentiation, and experiment repeats three times, and result is as shown in table 1.
Table 1 father and mother are for system of family and offspring's idiotype detected result
As shown in Table 1, the assistant identification of the proterties chicken that crawls all can be reached 100% by the method for the present invention. The clone of this gene and detection primer pair chicken thereof the character gene of crawling of examination in proterties chicken breeding and improvement work, proterties of crawling formation mechenism and native chicken breed of crawling has important meaning.
Embodiment 4 genes of individuals somatotype and chicken crawl the association analysis of proterties
4.1 experiment material
Intend adopting the Xingshan walnuts with complete pedigree record to be experimental subjects, G0In generation, plants egg and provides by livestock and poultry genetic resources management station of Guizhou Province, hatches at the big poultry genetic resources of Chinese agriculture and breeding test base and raises. Choose healthy strong, cock that body weight is close, according to the ratio of 1:5, set up Cp genotype and divide and leave home to be (type of crawling cock and type hen hybridization of crawling). Carry out artificial insemination at 38 weeks egg-laying peaks, collect kind of an egg and carry out artificial incubation. Gathering early stage dead embryo at the embryo hatching the 4th day, the 9th day respectively, the same day of hatching gathers all individual blood.
4.2 genotype tests methods
Adopting method in embodiment 1 to extract embryo and poba gene group DNA, measure DNA concentration and OD value with NanoDrop2000, by 1.0% agarose gel electrophoresis detection DNA quality, DNA is for subsequent use in-20 DEG C of Refrigerator stores.Use embodiment 1 (SEQIDNO:4-5) and the primer described in 2, the augmentation detection of the DNA molecular marker that the proterties that undertaken crawling to chicken in genome is relevant by PCR method. According to sepharose detected result, examination is crawled the chicken of type and normal phenotype, utilize the primer (SEQIDNO:4-5) of embodiment 1, all crawl type and normal individual can amplify large fragment (3350bp) and crawls gene band (Fig. 2). Utilizing the primer (SEQIDNO:1-2) of the DNA amplification molecule marker of embodiment 2, if distinctive band (234bp) only occurs in amplification, then this individuality is type individuality of crawling; Distinctive band (234bp) do not occur, then this individuality to be measured is normal individual. Result is as shown in table 2.
The different phenotype idiotype detected result of table 2
Chi square test result shows; chicken crawls the disappearance of character gene and the phenotype close linkage of crawling (P > 0.05) of chicken; show that this DNA molecular marker can be used for crawling the marker assisted selection of build chicken and breeding; for Xingyi and other native chicken breed protection and developmental utilization have established important basis, there is important investigation and application and it is worth.
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 being made some modifications or improvements, 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, all belong to the scope of protection of present invention.
Reference
Landauer,W.,andL.C.Dunn.1930.StudiesontheCreeperfowl.I.Genetics.J.Genet.23:397-413.
Rudnick, D., andV.Hamburger.1940.Ontheidentificationofsegregatedpheno typesinprogenyfromCreeperfowlmatings.Genetics25:215-224.; Rudnick, D.1945.DifferentiationofprospectivelimbmaterialfromCreep erchickembryosincoelomicgrafts.J.Exp.Zool.100:1-17.
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Claims (10)
1. the DNA molecular marker that proterties of crawling to chicken is relevant, it is characterised in that, it is positioned on chicken No. 7 karyomit(e)s, for the primer of the described DNA molecular marker that increases as shown in SEQIDNO:1-2.
2. DNA molecular marker according to claim 1, it is characterised in that, it is:
I) nucleotide sequence shown in SEQIDNO:3; Or
Ii) nucleotide sequence shown in SEQIDNO:3 is substituted, lacks and/or increases one or more Nucleotide and the nucleotide sequence of expression identical function protein; Or
Iii) under strict conditions with sequence hybridization shown in SEQIDNO:3 and express identical function protein nucleotide sequence, described stringent condition is in 0.1 × SSPE containing 0.1%SDS or the 0.1 × SSC solution containing 0.1%SDS, hybridize at 65 DEG C, and wash film with this solution; Or
Iv) and i), ii) or nucleotide sequence iii) there is more than 90% homology and express the nucleotide sequence of identical function protein.
3. the application of DNA molecular marker described in claim 1 or 2 in chicken molecular mark.
4. a qualification or assistant identification are crawled the method for proterties chicken, it is characterised in that, comprise the following steps:
1) genomic dna of testing sample is extracted;
2) taking the genomic dna of testing sample as template, primer shown in SEQIDNO:1-2 is utilized to carry out pcr amplification;
3) pcr amplification product is detected; If containing the DNA molecular marker described in claim 1 or 2 in amplified production, then the proterties that is judged to crawl chicken.
5. method according to claim 4, it is characterised in that, step 2) in PCR reaction system comprise: genomic dna 80-100ng, containing Mg2+10 × PCR damping fluid 3.0 μ l, 2.5mMdNTPs4.0 μ l, 2.5U/ μ lTaqDNA polysaccharase 1.5 μ l, 10 μm of ol/l upstream and downstream primers each 0.2 μ l, ddH2O complements to cumulative volume 25.0 μ l;
Pcr amplification program: 94 DEG C of sex change 5min; 94 DEG C of sex change 30sec, 58 DEG C of annealing 30sec, 72 DEG C extend 30sec, totally 33 circulations; 72 DEG C extend 5min; 4 DEG C of preservations.
6. method according to claim 4 or 5, it is characterised in that, step 1) and 2) described in testing sample derive from chicken blood or the histoorgan from body; Step 3) in detection pcr amplification product adopt method comprise agarose gel electrophoresis, order-checking or molecular hybridization.
7. for the identification of the PCR detection kit of the proterties chicken that crawls, it is characterised in that, described test kit comprises primer shown in SEQIDNO:1-2.
8. chicken crawls character gene, it is characterised in that, described gene is:
I) nucleotide sequence shown in SEQIDNO:6; Or
Ii) nucleotide sequence shown in SEQIDNO:6 is substituted, lacks and/or increases one or more Nucleotide and the nucleotide sequence of expression identical function protein; Or
Iii) under strict conditions with sequence hybridization shown in SEQIDNO:6 and express identical function protein nucleotide sequence, described stringent condition is in 0.1 × SSPE containing 0.1%SDS or the 0.1 × SSC solution containing 0.1%SDS, hybridize at 65 DEG C, and wash film with this solution; Or
Iv) and i), ii) or nucleotide sequence iii) there is more than 90% homology and express the nucleotide sequence of identical function protein.
9. for the Specific PCR primers of the gene described in claim 8 that increases, it is characterised in that, described primer is as shown in SEQIDNO:4-5.
10. contain the PCR detection kit of primer described in claim 9, it is characterised in that, the PCR reaction system being applicable to described test kit comprises: genomic dna 50-500ng, containing Mg2+LAPCR damping fluid 3.0 μ l, 2.5mMdNTPs4.0 μ l, each 0.5 μ l of 10mmol/L upstream and downstream primer, LATaqDNA polysaccharase 1.5U, ddH2O complements to cumulative volume 25.0 μ l;
Pcr amplification program: 98 DEG C of sex change 10sec, 60 DEG C of annealing 30sec, 72 DEG C extend 3min, totally 30 circulations; 72 DEG C extend 10min; 4 DEG C of preservations.
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| CN113151277A (en) * | 2021-05-10 | 2021-07-23 | 安徽农业大学 | Construction method of chicken DF-1 cell IHH gene knockout stable cell strain and specific sgRNA thereof |
| CN113528521A (en) * | 2021-07-08 | 2021-10-22 | 安徽农业大学 | siRNA and application thereof |
| CN115976231A (en) * | 2022-12-29 | 2023-04-18 | 海南省崖州湾种子实验室 | Molecular marker for detecting tail-free genotype of chicken and detection primer and application thereof |
| 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 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113151277A (en) * | 2021-05-10 | 2021-07-23 | 安徽农业大学 | Construction method of chicken DF-1 cell IHH gene knockout stable cell strain and specific sgRNA thereof |
| CN113528521A (en) * | 2021-07-08 | 2021-10-22 | 安徽农业大学 | siRNA and application thereof |
| CN115976231A (en) * | 2022-12-29 | 2023-04-18 | 海南省崖州湾种子实验室 | Molecular marker for detecting tail-free genotype of chicken and detection primer and application thereof |
| 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 |
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