CN105316424B - One kind molecular labeling relevant to Gaoyou duck weight, chest leg flesh weight and chest muscle muscle fiber trait, its acquisition methods and application - Google Patents
One kind molecular labeling relevant to Gaoyou duck weight, chest leg flesh weight and chest muscle muscle fiber trait, its acquisition methods and application Download PDFInfo
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Abstract
The invention discloses a kind of molecular labelings relevant to Gaoyou duck weight, chest leg flesh weight and chest muscle muscle fiber trait and its acquisition methods and application, the molecular labeling to be located at the 190th of nucleotide shown in SEQ ID NO:1, and the nucleotide in the site is G or T.PCR amplification Gaoyou duck genomic DNA is sequenced after the detection reaction of amplified production linked enzyme, obtains molecular labeling relevant to Gaoyou duck weight, chest leg flesh weight and chest muscle muscle fiber trait by sequence analysis.The molecular labeling is applied in screening Gaoyou duck weight, chest leg flesh weight and chest muscle muscle fiber trait, can be used for the Seedling selection in family selective breeding, molecule foundation is provided in apolegamy, to improve weight when Gaoyou duck listing, increases economic benefit.
Description
Technical field
The invention belongs to the technical fields of poultry genetic engineering, more particularly, to a kind of and Gaoyou duck weight, chest leg flesh
Weight molecular labeling relevant with chest muscle muscle fiber trait, its acquisition methods and application.
Background technique
Insulin growth factor -1 (IGF-1) belongs to insulin family member, is one of the important gene of growth of animal axis, tool
Have and promotes cell Proliferation and ontogenetic function.IGF-1 can promote albumen synthesis, flesh by the IGFs receptor on target organ
Jerky cell Proliferation, myoblast differentiation and myotube fusion etc., to promote the development of skeletal muscle.Test discovery, in duck embryonic
Phase, the interior injection external source IGF-1 of egg can significantly improve the weight and chest muscle weight of duck after hatching, illustrate growth of the IGF-I to duckling
With facilitation.
Single nucleotide polymorphism (single nucleotide polymorphism, SNP), is primarily referred to as in genome water
Flat DNA sequence polymorphism caused by a single nucleotide variation.SNP as third generation molecular labeling, have quantity it is more,
The features such as distribution is wide, representativeness is strong and genetic stability is good.Make at present in the analysis of genetic diversity of animals and plants, gene
It is widely used in the research of figure, molecular mark and functional gene.In conjunction with round pcr, electrophoresis, fluorescence
The SNP detection method of lamp method plays an important role in animals and plants genetic breeding.Gaoyou duck is one of national three big ducks,
Growth and development is fast, with good meat quality, belongs to meat egg dual-purpose type local varieties.It is (artificial with individual inheritance characteristic is changed in breeding work
Mutagenesis) it compares, job change genetic structure is relatively easy.And in the prior art not yet discovery with Gaoyou duck weight, chest leg
Flesh weight and the relevant molecular labeling of chest muscle muscle fiber trait and application.
Summary of the invention
In order to solve the shortcomings of the prior art, the present invention provides a kind of and Gaoyou duck weight, chest leg flesh weight and chests
The relevant molecular labeling of flesh muscle fiber trait, its preparation method and application.
It is weighed with Gaoyou duck weight, chest leg flesh and the relevant molecular labeling of chest muscle muscle fiber trait the present invention provides a kind of,
The molecular labeling is located at the 190th of nucleotide shown in SEQ ID NO:1, and the nucleotide in the site is G or T.
The present invention also provides a kind of acquisition molecules relevant to Gaoyou duck weight, chest leg flesh weight and chest muscle muscle fiber trait
The method of label, PCR amplification Gaoyou duck genomic DNA are sequenced after the detection reaction of amplified production linked enzyme, are analyzed by sequence
Obtain molecular labeling relevant to Gaoyou duck weight, chest leg flesh weight and chest muscle muscle fiber trait.
Wherein, PCR amplification the primer is to sequence as shown in SEQ ID NO:2 and SEQ ID NO:3.
Wherein, such as SEQ ID NO:4 of probe sequence used in Ligase detection reaction, SEQ ID NO:5 and SEQ ID NO:6
It is shown.
Wherein, Ligase detection reaction product length 170bp is genotype GG, Ligase detection reaction product length
172bp is genotype TT, and Ligase detection reaction product length 170bp and 172bp are genotype GT.
The present invention also provides a kind of molecular labelings relevant to Gaoyou duck weight, chest leg flesh weight and chest muscle muscle fiber trait
Application in screening Gaoyou duck weight, chest leg flesh weight and chest muscle muscle fiber trait.
Beneficial effects of the present invention: the present invention provides a kind of and Gaoyou duck weight, chest leg flesh weight and chest muscle myofibrous
The relevant molecular labeling of shape, for the single nucleotide polymorphism in the molecular labeling site, designing specific primer amplification includes SNP
Then the genetic fragment in site is attached enzyme detection reaction, sequencing analysis being capable of simple, quick, at low cost, accurate inspection
Survey the polymorphism of its mononucleotide.Genotype and 70 age in days weight of Gaoyou duck, chest leg flesh weight and chest muscle to above-mentioned molecular locus
Muscle fiber trait is associated analysis, can be used for the Seedling selection in family selective breeding, provides molecule foundation, in apolegamy to mention
Weight when high Gaoyou duck lists increases economic benefit.
Detailed description of the invention
Fig. 1 is the detection of ABI3730 type Genetic Analyser and Genemapper analysis result.
Specific embodiment
The present invention is carried out below with reference to embodiment explanation is explained in detail.
Molecular labeling relevant to Gaoyou duck weight, chest leg flesh weight and chest muscle muscle fiber trait of the invention, the molecule
Label is located at the 190th of nucleotide shown in SEQ ID NO:1, and the nucleotide in the site is G or T, leads to the gene loci
Single nucleotide polymorphism.PCR amplification obtains molecule mark relevant to Gaoyou duck weight, chest leg flesh weight and chest muscle muscle fiber trait
Note, nucleotides sequence are classified as the partial genome sequence of duck IGF-1, and sequence is as shown in SEQ ID NO:1.In the sequence
There is the mutation of a G190-T190 at 190bp, leads to the single nucleotide polymorphism in the site.
For the single nucleotide polymorphism in above-mentioned site, the invention also discloses the methods that it is obtained, wherein for expanding
Forward and reverse primer pair sequence of the IGF-1 gene and the detection gene point mutation is as shown in SEQ ID NO:2-3.
Wherein for being attached enzyme detection reaction (LDR), required LDR probe sequence such as SEQ to above-mentioned amplified production
Shown in ID NO:4-6.
Molecular labeling relevant to Gaoyou duck weight, chest leg flesh weight and chest muscle muscle fiber trait of the invention is for screening height
Postal duck weight, chest leg flesh weight and chest muscle muscle fiber trait.
The side of acquisition of the invention molecular labeling relevant to Gaoyou duck weight, chest leg flesh weight and chest muscle muscle fiber trait
Method, comprising the following steps:
1, the acquisition of duck blood sample
Test duck of the invention comes from Jiangsu Province's Gaoyou duck group, the same age in days Gaoyou fed in the case where identical daily ration is horizontal
Duck and Dual Gold kind duck group.Using disposable syringe from 1ml blood is extracted under duck wing in vein, injection is through high pressure sterilization and fills
It in the 1.5ml centrifuge tube of ethylenediamine tetra-acetic acid (EDTA) anti-coagulants for having 200 μ l 2% sterile, gently shakes up, records wing number ,-
80 DEG C save backup.
2, the extraction of DNA
The extracting of genomic DNA uses phenol-chloroform extraction method
(1) it takes above-mentioned 30 μ l of whole blood to be placed in 1.5ml centrifuge tube, is separately added into 470 μ 1 × SET of l buffers, 12.5 μ l
The 10mg/ml Proteinase K of 20%SDS (lauryl sodium sulfate) and 6 μ l are put in 55 DEG C of water-baths after mixing and stay overnight；
(2) sample is taken out in 1.5ml centrifuge tube, and 500 μ l are added and are saturated phenol, jog 20min, 10000rpm centrifugation
10min；
(3) supernatant is taken, 500 μ l saturation phenol is added again, jog 20min, 10000rpm are centrifuged 10min；
(4) supernatant is taken, 500 μ l chloroform-isoamyl alcohol (23:1) jog 20min, 10000rpm are added and are centrifuged 10min；
(5) supernatant is taken, is added 1ml ice dehydrated alcohol (- 20 DEG C), is oscillated to precipitate DNA, 10000rpm centrifugation
Ethyl alcohol is poured out after 10min；
(6) to clean DNA with 75% ethyl alcohol of 1ml primary, outwells ethyl alcohol, is placed in drying in 50 DEG C of drying boxes；
(7) distilled water after 300 μ l sterilizing is added after DNA is completely dried dissolves, overnight with dissolution in 50 DEG C of water-baths
DNA；DNA stoste 1:100 is diluted and is carried out spectrophotometer detectable concentration, OD value is 1.85-1.94.
(8) DNA concentration is diluted to 50ng/ μ l, deposits in -20 DEG C of refrigerators and saves backup.
3, PCR amplification
Amplification includes the segment in the site G190T: according to duck IGF-1 gene (NW_004677293) complete sequence of GenBank
Design includes forward and reverse primer in the site, is synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.With above-mentioned Gaoyou
Duck genome is template, is deposited in forward and reverse primer comprising site sequence to be measured, Taq archaeal dna polymerase, buffer environment, dNTPs
In case, it is expanded under PCR reaction condition, PCR product size is 232bp, is reacted for LDR.
The primer pair sequence is as follows:
Upstream primer sequence are as follows: 5 '-TTGCATGGAGTGGATGTGAT-3 ' (SEQ ID NO:2)；
Downstream primer sequence are as follows: 5 '-GCAGGAATAAGAGTGCCATGT-3 ' (SEQ ID NO:3)
The specific steps of pcr amplification reaction are as follows:
1) pcr amplification reaction system prepares: in the PCR thin-wall tube of 200 μ l, 1 μ l DNA profiling (50ng/ μ l), 2 is added
μl Bufer、0.6μl Mg2+(3mmol/L), 2 μ l dNTP (2mmol/L), 0.2 μ l Taq archaeal dna polymerase (5U/ μ l), 2 μ l
Primer mix、12.2μl ddH2O, totally 20 μ l PCR reaction system, mixes well rear of short duration centrifugation, is eventually adding 20 μ l stones
Wax oil.
2) pcr amplification reaction program is arranged: being expanded on Perkin-Elmer Gene Amp PCR Systems9600
Increase reaction, pcr amplification reaction program are as follows: 95 DEG C of denaturation 2min, 40 circulations (94 DEG C of 90s, 50 DEG C of 1min 30s, 65 DEG C of 30s)
65 DEG C of extension 10min, obtain pcr amplification product.
3) 2 μ l reaction products electrophoresis in 3.0% agarose gel, 0.5 × TBE after reaction, is taken, whether is detection reaction
Success, remaining reaction product save under conditions of -20 DEG C.
4, Ligase detection reaction (LDR)
Three LDR probes are designed, LDR reaction is then carried out；The sequence difference of three probes is as follows:
G190T_modify:
P-ATTTCCCAGAATACAGCTGAGAATATTTTTTTTTTTTTTTTTTTTTTTTTTT
TTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM(SEQ ID NO:4)
G190T_G:
TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTTTGTCATT
TATTATTTGCTTTC(SEQ ID NO:5)
G190T_T:
TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
TTTTTTTTTTTTTTGTTTGTCATTTATTATTTGCTTTA(SEQ ID NO:6)
Wherein, the 3 ' ends of G190T-modify with FAM modification (blue-fluorescence), 5 ' end phosphorylations, so as to it is other
Two specific probe connections；3 ' the terminal bases of probe G190T-G are corresponding with G, the 3 ' terminal bases of probe G190T-T with
T is corresponding.
The specific steps of LDR reaction are as follows:
1) isometric ddH is added in pcr amplification product2O dilution, the template as Ligase detection reaction；
2) in the PCR thin-wall tube of 200 μ l, 4 μ l templates, 11 × Buffer of μ l, 1 μ l Probe mix (2pmol/ is added
μl)、0.05μl Taq DNA ligase(2U/μl)、4μlddH2O mixes well rear of short duration centrifugation, is eventually adding 10 μ l paraffin
Oil；
3) PCR reaction tube is put into PCR instrument and is attached enzyme detection reaction, react amplification program are as follows: 95 DEG C of denaturation 2min,
40 circulations (94 DEG C of 15s, 50 DEG C of 25s)；
4) 10 μ l Loading Buffer, 0.25 μ l ABI GS-500ROX fluorescent marker are added in each loading hole
Molecular weight standard is eventually adding the LDR product of 0.5 μ l, mixes well rear loading, carries out sequencing gel with ABI3730 sequenator
Capillary Electrophoresis；
5) data collection, the correction of swimming lane line, the measurement of migration clip size and correction are carried out using GENESCANTM672 software
Inherent molecular weight standard.
5, genotyping and judgement
Above-mentioned LDR reaction product is detected by ABI3730 type Genetic Analyser, according to the length of LDR product and each genotype
It is associated analysis by Genemapper, parting figure is shown in that attached drawing 1, the LDR product length of genotype GG are 170bp, genotype
The LDR product length of TT is 172bp, and the LDR product length of genotype GT is the mixing of 170bp and 172bp.
6, the association analysis of the SNP marker of IGF-1 gene and 70 age in days Gaoyou duck weight and chest leg flesh weight
The association analysis of SNP marker and 70 age in days Gaoyou duck weight and chest leg the flesh weight of 1 IGF-1 gene of table
Note: () interior digital representation number of individuals；Colleague's data, different lowercase letter indication differences are significant (P < 0.05), identical
Lowercase letter indication difference is not significant.
Different genotype and 70 age in days Gaoyou duck weight, chest muscle weight and gastrocnemius to the SNP marker of IGF-1 gene
Carried out the detection application of association analysis again, the results are shown in Table 1 for association analysis, the SNP marker site of IGF-1 gene with
Gaoyou duck weight, chest muscle are in extremely significant related (P < 0.01) again；It is again in significant related (P < 0.05) to gastrocnemius；Wherein genotype
GG is all remarkably higher than genotype GT and TT in 70 age in days Gaoyou duck weight, chest muscle weight and gastrocnemius again, compares GT and TT, GT is 70
Age in days Gaoyou duck weight and chest muscle are significantly higher than TT again, and the gastrocnemius method of double differences is not different significant.This illustrates GG genotype for 70 ages in days
The weight of Gaoyou duck, chest leg flesh are beneficial gene type again, can be as the molecular labeling of raising weight, chest leg flesh again breeding speed.
In breeding screening, GG type is selected, the genotype can be fixed in group, weight when Gaoyou duck listing can be effectively improved.
7, the association analysis of the SNP marker of IGF-1 gene and 70 age in days Gaoyou duck chest muscle muscle fibre characteristics
The association analysis of the SNP marker of 2 IGF-1 gene of table and 70 age in days Gaoyou duck chest muscle muscle fibre characteristics
Note: () interior digital representation number of individuals；Colleague's data, different lowercase letter indication differences are significant (P < 0.05), identical
Lowercase letter indication difference is not significant.
Different genotype and 70 age in days Gaoyou duck chest muscle muscle fibre characteristics to the SNP marker of IGF-1 gene carry out
The detection application of association analysis, the results are shown in Table 2 for association analysis, the SNP marker site of IGF-1 gene and 70 ages in days
Gaoyou duck chest muscle area of muscle fiber, muscle fibre average diameter are significant related (P < 0.01)；Wherein genotype GG is in 70 age in days Gaoyou
Duck area of muscle fiber and diameter of muscle fiber are all remarkably higher than genotype GT and TT, area of muscle fiber be substantially less than genotype GT and
TT；Compare GT and TT, difference is not significant between area of muscle fiber, diameter of muscle fiber and muscle fibre density.This show the site with
70 age in days Gaoyou duck muscle fiber traits are expected to become to the progress of 70 age in days Gaoyou duck muscle fiber traits there is significant correlation
The molecular labeling of selection.
Presently preferred embodiments of the present invention is not intended to limit the invention, all made in substantive content of the present invention
Any modification, equivalent replacement and simple modifications etc., should all be included in the protection scope of the present invention.
Claims (5)
1. a kind of molecular labeling relevant to Gaoyou duck weight, chest leg flesh weight and chest muscle muscle fiber trait, which is characterized in that described
The nucleotide sequence of molecular labeling is as shown in SEQ ID NO:1, and the 190th of SEQ ID NO:1 is G or T.
2. a kind of obtain molecule relevant to Gaoyou duck weight, chest leg flesh weight and chest muscle muscle fiber trait described in claim 1
The method of label, which is characterized in that PCR amplification Gaoyou duck genomic DNA is sequenced after the detection reaction of amplified production linked enzyme,
Molecular labeling relevant to Gaoyou duck weight, chest leg flesh weight and chest muscle muscle fiber trait is obtained by sequence analysis；Ligase inspection
Surveying reaction product length 170bp is genotype GG, and Ligase detection reaction product length 172bp is genotype TT, connection
It is genotype GT that enzyme, which detects reaction product length 170bp and 172bp,.
3. acquisition according to claim 2 molecule relevant to Gaoyou duck weight, chest leg flesh weight and chest muscle muscle fiber trait
The method of label, which is characterized in that PCR amplification the primer is to sequence as shown in SEQ ID NO:2 and SEQ ID NO:3.
4. acquisition according to claim 2 molecule relevant to Gaoyou duck weight, chest leg flesh weight and chest muscle muscle fiber trait
The method of label, which is characterized in that probe sequence used in Ligase detection reaction such as SEQ ID NO:4, SEQ ID NO:5 and
Shown in SEQ ID NO:6.
5. molecular labeling relevant to Gaoyou duck weight, chest leg flesh weight and chest muscle muscle fiber trait described in claim 1 is sieving
Select the application in Gaoyou duck weight, chest leg flesh weight and chest muscle muscle fiber trait.
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| CN112852979B (en) * | 2021-03-25 | 2022-05-10 | 中国农业科学院北京畜牧兽医研究所 | Molecular marker related to duck breast muscle fiber diameter character and application thereof |
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| Title |
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| IGF-1对鸭胚肌肉发育影响及其分子机理研究;刘贺贺;《中国博士学位论文全文数据库（电子期刊）》;20130715;第D050-13页 |
| 淮南麻鸭IGF-1基因单核苷酸多态性及其与生长性能关系研究;赵聘等;《信阳农业高等专科学校学报》;20140630;第24卷(第2期);第99-104页 |
| 鸭肝脏IGF1基因早期发育表达;宋迟等;《江苏农业科学》;20150831;第43卷(第8期);第42-44页 |
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