CN109652566A - SNP marker relevant to sheep list tire litter size, primed probe group, kit, detection method and application - Google Patents
SNP marker relevant to sheep list tire litter size, primed probe group, kit, detection method and application Download PDFInfo
- Publication number
- CN109652566A CN109652566A CN201910126727.7A CN201910126727A CN109652566A CN 109652566 A CN109652566 A CN 109652566A CN 201910126727 A CN201910126727 A CN 201910126727A CN 109652566 A CN109652566 A CN 109652566A
- Authority
- CN
- China
- Prior art keywords
- genotype
- sheep
- probe
- snp marker
- litter size
- 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.)
- Pending
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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic 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)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention provides SNP marker relevant to sheep list tire litter size, primed probe group, kit, detection method and applications, belong to sheep SNP marker technical field, the SNP marker is located at the site 40857869bp on No. 1 chromosome of sheep, the site is located at LEPR gene internal, there are C/T base mutations on the site, the genotype of the LEPR gene is CC, CT or TT, single tire litter size of the genotype CC is greater than genotype TT, and single tire litter size of the genotype TT is greater than genotype CT；The SNP marker information based on ovine genome sequence information version number be Oar_v3.1.SNP marker provided by the invention and sheep list tire litter size are significant related, and single tire litter size ability of sheep can be judged according to the SNP marker.In practical application should preferred CC type ewe as standby ewe, and rejecting heterozygous CT type ewe in the selection process.
Description
Technical field
The present invention relates to sheep SNP marker technical fields, and in particular to SNP relevant to sheep list tire litter size points
Sub- label, primed probe group, kit, detection method and application.
Background technique
In addition to a small number of sheep varieties such as Small-fat-tail sheep and sheep produce more lambs, remaining most sheep variety produces list in China
Lamb, this greatlys restrict the development of China's sheep husbandry.Sheep litter size is the complex character controlled by multiple genes, and heredity
Power is lower, is difficult to obtain larger genetic progress by traditional Phenotypic Selection.Therefore, influence sheep litter size related gene is carried out
It studies extremely important for improving sheep reproductive efficiency, quickening Sheep Breeding process, the economic benefit of raising sheep industry.With
To Fecundity Trait research gradually deeply, it is multiple control sheep litter sizes major gene resistances found successively.Researcher's hair
Gene in the TGFB superfamily accesses such as existing GDF9, BMP15 is also the major gene resistance for increasing sheep litter size.
Recently, New Zealand scientist has found in the research to Romney Marsh sheep Davisdale strain, and LEPR mutation is in addition to can
To postpone the puberty of the strain sheep, the number of eggs ovulated of sheep is had an effect on, however LEPR gene is not in TGFB superfamily access
Member.Therefore, the control methods of the gene pairs number of eggs ovulated may be different from the above major gene resistance control methods of number of eggs ovulated that influence,
The Small-fat-tail sheep in China carries this mutation.
SNP marker in LEPR gene still without finding to be closely related with sheep list tire lambing quantity at present.
Summary of the invention
The purpose of the present invention is to provide SNP markers relevant to sheep list tire litter size, primed probe group, reagent
Box, detection method and application, SNP marker provided by the invention and sheep list tire litter size are significant related, according to the SNP points
Son label can judge single tire litter size ability of sheep.
The present invention provides SNP marker relevant to sheep list tire litter size, the SNP marker is located at sheep
The site 40857869bp on No. 1 chromosome, the site are located at LEPR gene internal, and there are C/T bases on the site
Mutation, the genotype of the LEPR gene are CC, CT or TT, and single tire litter size of the genotype CC is greater than genotype TT, institute
The single tire litter size for stating genotype TT is greater than genotype CC；The SNP marker information based on ovine genome sequence letter
Breath version number is Oar_v3.1.
The present invention also provides a kind of primed probe groups for detecting LEPR genotype, including primer, the first probe and second
Probe；
The primer includes upstream primer and downstream primer, and the upstream primer has nucleosides shown in SEQ ID No.1
Acid sequence, the downstream primer have nucleotide sequence shown in SEQ ID No.2；
First probe has nucleotide sequence shown in SEQ ID No.3；
Second probe has nucleotide sequence shown in SEQ ID No.4.
The present invention also provides it is a kind of detect LEPR genotype kit, including dNTPs, Taq archaeal dna polymerase,
MgCl2, primed probe group described in PCR reaction buffer and above-mentioned technical proposal.
Preferably, the concentration of the primed probe group middle and upper reaches primer and downstream primer is independently 10 μm of ol/L.
Preferably, the concentration of first probe and the second probe is independently 10 μm of ol/L.
The present invention also provides a kind of methods for detecting LEPR genotype, comprising the following steps:
1) genomic DNA of sheep to be measured is extracted；
2) it using the step 1) genomic DNA as template, is carried out using primed probe group described in above-mentioned technical proposal real
When fluorescent quantitative PCR, LEPR genotype is determined according to the fluorescence signal detected.
Preferably, the real-time fluorescence quantitative PCR expands the system used, and every 20 μ L includes 2 μ L of genomic DNA, 2 ×
MasterMix 10 μ L, 10 μm of ol/L forward primers 1 μ L, 10 μm of 1 μ L of ol/L reverse primer；10 μm of 0.8 μ L of the first probe of ol/L,
10 μm of 0.8 μ L of the second probe of ol/L, deionized water polishing to 20 μ L.
Preferably, the program that the real-time fluorescence quantitative PCR amplification uses includes: 95 DEG C of 10min；95 DEG C of 30sec, 60 DEG C
30sec, 40 circulations.
The present invention also provides application of the SNP marker described in above-mentioned technical proposal in sheep assistant breeding.
The present invention provides SNP marker relevant to sheep list tire litter size, the SNP marker is located at sheep
The site 40857869bp on No. 1 chromosome, the site are located at LEPR gene internal, and there are C/T bases on the site
Mutation, the genotype of the LEPR gene are CC, CT or TT, and single tire litter size of the genotype CC is greater than genotype TT, institute
The single tire litter size for stating genotype TT is greater than genotype CC；The SNP marker information based on ovine genome sequence letter
Breath version number is Oar_v3.1.SNP marker provided by the invention and sheep list tire litter size are significant related, according to the SNP
Molecular labeling can judge single tire litter size ability of sheep.
The embodiment of the present invention is as the result is shown: SNP marker provided by the invention and the significant phase of sheep list tire litter size
It closes, single tire litter size ability of sheep can be judged according to the SNP marker.
Using the method for TaqmanMGB probe in detecting sheep LEPR gene pleiomorphism, the site C, T, knot high-throughput can be detected
Fruit is easy interpretation.Compared with the methods of traditional PCR-RFLP detection LEPR gene, it can detecte multiple samples every time, and be not required to
The subsequent analysis for carrying out PCR product substantially reduces detection cycle while saving testing cost, improves detection
Efficiency.
The primer and probe high specificity that the present invention uses, not will receive the interference of other genes, reduce PCR product dirt
Dye causes the risk of false positive, and result interpretation is easier.High-throughput detection can be realized to the SNP site of LEPR gene,
Sheep have potential application in large-scale molecular breeding.
Detailed description of the invention
Fig. 1 is LEPR genotype results figure of the present invention.
Specific embodiment
The present invention provides SNP markers relevant to sheep list tire litter size, which is characterized in that the SNP molecule
Label is located at the site 40857869bp on No. 1 chromosome of sheep, and the site is located at LEPR gene internal, on the site
There are C/T base mutation, the genotype of the LEPR gene is CC, CT or TT, and single tire litter size of the genotype CC is greater than
Single tire litter size of genotype TT, the genotype TT are greater than genotype CC；The SNP marker information based on sheep base
Because group sequence information version number is Oar_v3.1.
In the present invention, the accession number of the LEPR gene is preferably NM_001009763.1.
The present invention also provides a kind of primed probe groups for detecting LEPR genotype, including primer, the first probe and second
Probe；The primer includes upstream primer and downstream primer, and the upstream primer has nucleotides sequence shown in SEQ ID No.1
Column, the downstream primer have nucleotide sequence shown in SEQ ID No.2；First probe has SEQ ID No.3 institute
The nucleotide sequence shown；Second probe has nucleotide sequence shown in SEQ ID No.4.
In the present invention, the upstream primer has nucleotide sequence shown in SEQ ID No.1, and particular sequence is as follows:
5'-AGCCGAGGAGGAGCAAGG-3'；
The downstream primer has nucleotide sequence shown in SEQ ID No.2, and particular sequence is as follows:
5’-TCCCATGATCTCTTAGAGGAAGACTC-3’。
In the present invention, first probe has nucleotide sequence shown in SEQ ID No.3, the following institute of particular sequence
Show:
5'-AACCATTCTCCACCAAA-3'；The present invention preferably adds fluorescent reporter genes at 5 ' ends of the first probe,
Real-time fluorescence quantitative PCR amplification is used further to after 3 ' end addition MGB probes.
In the present invention, second probe has nucleotide sequence shown in SEQ ID No.4, the following institute of particular sequence
Show:
5'-CAACCATTCTTCACCAAA-3'；The present invention preferably adds fluorescent reporter genes at 5 ' ends of the second probe,
Real-time fluorescence quantitative PCR amplification is used further to after 3 ' end addition MGB probes.
In the present invention, the color of first probe and the fluorescent reporter gene of the second probe addition is preferably difference
, it is preferably FAM that first probe added, which is fluorescent reporter group, and the fluorescent reporter group of the second probe addition is excellent
It is selected as HEX.
The present invention also provides it is a kind of detect LEPR genotype kit, including dNTPs, Taq archaeal dna polymerase,
MgCl2, primed probe group described in PCR reaction buffer and above-mentioned technical proposal.
The present invention is to dNTPs, Taq archaeal dna polymerase, MgCl2, PCR reaction buffer and primed probe group be in reagent
Placement amount in box is not particularly limited, using the reagent placement amount of conventional kit.
In the present invention, the upstream primer of the primed probe group in the kit and the concentration of downstream primer are independent excellent
It is selected as 10 μm of ol/L；The independent preferably 10 μm of ol/L of the concentration of first probe and the second probe.
The present invention also provides a kind of methods for detecting LEPR genotype, comprising the following steps:
1) genomic DNA of sheep to be measured is extracted；
2) it using the step 1) genomic DNA as template, is carried out using primed probe group as claimed in claim 2 glimmering in real time
Fluorescent Quantitative PCR amplification, determines LEPR genotype according to the fluorescence signal detected.
The present invention is not particularly limited the method for the genomic DNA for extracting sheep to be measured, dynamic using traditional extraction
The method of object genomic DNA.
The present invention is carried out real-time using the genomic DNA as template using primed probe group described in above-mentioned technical proposal
Fluorescent quantitative PCR determines LEPR genotype according to the fluorescence signal detected.
In the present invention, the real-time fluorescence quantitative PCR expands the system used, and every 20 μ L preferably includes genomic DNA 2
μ L, 2 × MasterMix 10 μ L, 10 μm of ol/L forward primers 1 μ L, 10 μm of 1 μ L of ol/L reverse primer；10 μm of first probes of ol/L
0.8 μ L, 10 μm of 0.8 μ L of the second probe of ol/L, deionized water polishing to 20 μ L.
In the present invention, the program that the real-time fluorescence quantitative PCR amplification uses preferably includes: 95 DEG C of 10min；95℃
30sec, 60 DEG C of 30sec, 40 circulations.
The method of detection sheep LEPR genotype provided by the invention, is using ovine genome DNA as template, in system
Primer and probe described in above-mentioned technical proposal is added simultaneously.Primer is used for allele PCR amplification, joined in above-mentioned probe
2 kinds of different fluorescent markers, can match with 2 allele completely respectively.Under normal circumstances, due to probe 5 ' hold fluorophor and
Close to together, fluorescence is quenched 3 ' end quenching groups.With effective progress of PCR, the probe matched completely with template is gradually
It is cut by 5 ' → 3 ' 5 prime excision enzyme activity of Taq archaeal dna polymerase, the quenching group of the fluorophor and 3 ' ends that cause probe 5 ' to hold
Separation, quenching effect release, and reporter fluorescence group is activated；And the probe that cannot be matched completely with template, show another pair etc.
Position gene cannot effectively be cut, therefore can't detect fluorescence signal, and the variation for thus detecting fluorescent value can carry out mononucleotide
Polymorphic detection determines that sheep LEPR genotype is CC, CT or TT according to testing result；Wherein, single nucleotide polymorphism detects
It is for the nucleotide for being located at the site 40857869bp on No. 1 chromosome of sheep.
The present invention also provides application of the SNP marker described in above-mentioned technical proposal in sheep assistant breeding.
In the present invention, the application can will have the sheep individual of the high litter size character of single tire to select and remain, thus
The reproductive capacity for improving sheep has very big application value to sheep large-scale molecular breeding.
Combined with specific embodiments below to SNP marker relevant to sheep list tire litter size of the present invention, draw
Object probe groups, kit, detection method and application are further described in detail, and technical solution of the present invention includes but is not limited to
Following embodiment.
Embodiment 1
1, experimental material
Choosing 342 Small-fat-tail sheep health ewes is test object.
2, the extraction of genomic DNA
Sheep jugular vein blood collection 1ml, with EDTA anticoagulation.Erythrocyte cracked liquid cracking removal is without the red of DNA first
Cell, nucleus lysate cracking packet cell release genomic DNA, and then albumen precipitation liquid selective precipitation removes removing protein,
Last pure genomic DNA is laid equal stress on by isopropanol precipitating is dissolved in DNA lysate.
3, TaqmanMGB sonde method carries out Genotyping
For the site 40857869bp on No. 1 chromosome of sheep, (NM_001009763.1 is based on ovine genome sequence
Column information version number Oar_v3.1) design primer and probe.
The nucleotide sequence of the primer is as follows:
Forward primer: 5 '-AGCCGAGGAGGAGCAAGG-3 '；
Reverse primer: 5 '-TCCCATGATCTCTTAGAGGAAGACTC-3 '.
The nucleotide sequence of the probe is as follows:
First probe: 5 '-AACCATTCTCCACCAAA-3 ', in 5 ' end addition FAM fluorescent reporter groups, in 3 ' end additions
After MGB, probe P-G is obtained, is used for PCR amplification；
Second probe: 5 '-CAACCATTCTTCACCAAA-3 ' add in 5 ' end addition HEX fluorescent reporter groups at 3 ' ends
After adding MGB, probe P-A is obtained, is used for PCR amplification.
Primer and probe is synthesized by Zhuozhou City Ran Runji health Biotechnology Co., Ltd, using purchased from Roche company480Probes Master, PCR instrument model Roche480II real time fluorescent quantitative
PCR system.
Reaction system: using the genomic DNA of extraction as template, with the probe of above-mentioned synthesis in quantitative fluorescent PCR system
It carries out following expanding (20 μ L reaction system): genomic DNA 2 μ L, 2 × MasterMix 10 μ L, 10 μm of 1 μ of ol/L forward primer
L, 10 μm of 1 μ L of ol/L reverse primer；10 μm of ol/L probe P-G 0.8 μ L, 10 μm of ol/L probe P-A0.8 μ L, deionized water polishing
To 20 μ L.Specific step is as follows:
(1) in view of the error of pipettor and suction nozzle, general each 96 orifice plate presses 100 Response calculation systems.
(2) hole 1-24 is wild homozygote；The hole 25-46 is no mutant homozygote；The hole 47-84 is heterozygote；The hole 85-96 is yin
Property control.
(3) the continuous electric sample application device of application divides reaction system to 96 orifice plates, every 18 μ L of hole.
(4) using the volley of rifle fire in every 2 μ LDNA sample of Kong Zhongjia.
(5) it is sealed after the completion of with sealed membrane, plate centrifuge centrifugation.
(6) Roche is openedParameter is arranged in 480II real-time fluorescence quantitative PCR system, and reaction condition is such as
Under: 95 DEG C of 10min；95 DEG C of 30sec, 60 DEG C of 30sec, 40 circulations.
4, the measurement of difference LEPR genotype Small-fat-tail sheep number of eggs ovulated
Since number of eggs ovulated and litter size are positively correlated.In order to further identify the influence being mutated to Small-fat-tail sheep litter size,
12 wild type CC types are chosen from 342 Small-fat-tail sheeps according to genotyping result and 9 TT type Small-fat-tail sheeps carry out estrus synchronization,
Using the method for laparoscopic visualization corpus luteum, the number of eggs ovulated of different LEPR genotype Small-fat-tail sheeps is obtained.
5, result is analyzed
According to control amplification, parting generate three kinds of genotype and negative control there are one could not interpretation point.
See Fig. 1.
The first genotype: CC, with CC type to impinging upon on an axis, Fig. 1 Green punctuate；
Second of genotype: CT, with CT type to impinging upon on an axis, red punctuate in Fig. 1；
The third genotype: TT, with TT type to impinging upon on an axis, blue punctuate in Fig. 1；
4th kind: going out genotype without accurate interpretation, the reasons such as inadequate examination criteria of probable dna concentration cause, powder in Fig. 1
Color punctuation；
5th kind: negative control is as a result, grey punctuate in Fig. 1.
Statistical result
40857869 site different genes type analysis statistical result of No. 1 chromosome of sheep to be measured is shown in Table 1.
Table 1 detects 40857869 site different genes type analysis of No. 1 chromosome of sheep statistics
As can be drawn from Table 1, in detected Sheep Populations, wild type CC type and no mutant homozygote TT type distribution frequency phase
Seemingly, and heterozygous CT type distribution it is less.Entire Sheep Populations are based on allele C.
The site 40857869bp different genotype is associated with point with Small-fat-tail sheep litter size on No. 1 chromosome of sheep to be measured
Analysis statistical result is shown in Table 2.
2 different genotype of table and Small-fat-tail sheep litter size association analysis statistical result
Can only be examined from table 2 by t, find wild type CC type and no mutant homozygote TT type litter size it is all significant higher than miscellaneous
Zygote CT type, though the litter size of CC type is higher than TT type, difference not significant difference.
The site 40857869bp different genotype and Small-fat-tail sheep on sheep number of eggs ovulated and No. 1 chromosome is measured to ovulate
Several association analysis, statistical result are shown in Table 3.
3 difference LEPR genotype Small-fat-tail sheep number of eggs ovulated of table
Sheep reproductive capacity is reduced in order to further detect site C-T mutation, is detected by number of eggs ovulated, and examined by t
It tests, as can be drawn from Table 3, finds that the number of eggs ovulated of wild type CC type is higher than TT type difference in conspicuousness according to table 3.
By above embodiments, it can be concluded that, SNP marker provided by the invention and sheep list tire litter size are significant related,
After the site 40857869bp mutates on No. 1 chromosome of the gene, heterozygote shows significantly litter size decline,
And for no mutant homozygote TT type, the number of eggs ovulated of the genotype can be reduced significantly, may have due to the genotype preferably by
Essence and pregnant ability, finally show as little with the litter size gap of wild type.It therefore, should basis in production application
Genotype rejects all individuals of mutation T T-type in field, in later period apolegamy, should forbid CC type and TT type sheep according to genotype
Breeding, and it is phased out TT type individual.Single tire litter size ability of sheep can be judged according to the SNP marker and can be referred to
Dao Zhong sheep manufacturing enterprise is reasonably matched.
Using the method for TaqmanMGB probe in detecting sheep LEPR gene pleiomorphism, the site C, T, knot high-throughput can be detected
Fruit is easy interpretation.Compared with the methods of traditional PCR-RFLP detection LEPR gene, it can detecte multiple samples every time, and be not required to
The subsequent analysis for carrying out PCR product substantially reduces detection cycle while saving testing cost, improves detection
Efficiency.
The primer and probe high specificity that the present invention uses, not will receive the interference of other genes, reduce PCR product dirt
Dye causes the risk of false positive, and result interpretation is easier.High-throughput detection can be realized to the SNP site of LEPR gene,
Sheep have potential application in large-scale molecular breeding.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Sequence table
<110>Institute of Animal Sciences, Chinese Academy of Agricultural Sciences
<120>SNP marker relevant to sheep list tire litter size, primed probe group, kit, detection method and application
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
agccgaggag gagcaagg 18
<210> 2
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
tcccatgatc tcttagagga agactc 26
<210> 3
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
aaccattctc caccaaa 17
<210> 4
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
caaccattct tcaccaaa 18
Claims (9)
1. SNP marker relevant to sheep list tire litter size, which is characterized in that the SNP marker is located at sheep the 1st
The site 40857869bp on number chromosome, the site are located at LEPR gene internal, and it is prominent that there are C/T bases on the site
Become, the genotype of the LEPR gene is CC, CT or TT, and single tire litter size of the genotype CC is greater than genotype TT, described
Single tire litter size of genotype TT is greater than genotype CC；The SNP marker information based on ovine genome sequence information
Version number is Oar_v3.1.
2. a kind of primed probe group for detecting LEPR genotype, which is characterized in that including primer, the first probe and the second probe；
The primer includes upstream primer and downstream primer, and the upstream primer has nucleotides sequence shown in SEQ ID No.1
Column, the downstream primer have nucleotide sequence shown in SEQ ID No.2；
First probe has nucleotide sequence shown in SEQ ID No.3；
Second probe has nucleotide sequence shown in SEQ ID No.4.
3. a kind of kit for detecting LEPR genotype, including dNTPs, Taq archaeal dna polymerase, MgCl2With PCR reaction buffer,
It is characterized in that, further including primed probe group as claimed in claim 2.
4. kit according to claim 3, which is characterized in that the primed probe group middle and upper reaches primer and downstream primer
Concentration be independently 10 μm of ol/L.
5. kit according to claim 3, which is characterized in that the concentration of first probe and the second probe is independent
For 10 μm of ol/L.
6. a kind of method for detecting LEPR genotype, which comprises the following steps:
1) genomic DNA of sheep to be measured is extracted；
2) using the step 1) genomic DNA as template, it is fixed that real-time fluorescence is carried out using primed probe group as claimed in claim 2
PCR amplification is measured, LEPR genotype is determined according to the fluorescence signal detected.
7. detection method according to claim 6, which is characterized in that the real-time fluorescence quantitative PCR expands the body used
System, every 20 μ L includes 2 10 μ L, 10 μm of ol/L forward primers 1 μ L, 10 μm of ol/L of μ L, 2 × MasterMix of genomic DNA reversed
1 μ L of primer；10 μm of first probes of ol/L 0.8 μ L, 10 μm of 0.8 μ L of the second probe of ol/L, deionized water polishing to 20 μ L.
8. detection method according to claim 7, which is characterized in that the real-time fluorescence quantitative PCR expands the journey used
Sequence includes: 95 DEG C of 10min；95 DEG C of 30sec, 60 DEG C of 30sec, 40 circulations.
9. application of the SNP marker described in claim 1 in sheep assistant breeding.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910126727.7A CN109652566A (en) | 2019-02-20 | 2019-02-20 | SNP marker relevant to sheep list tire litter size, primed probe group, kit, detection method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910126727.7A CN109652566A (en) | 2019-02-20 | 2019-02-20 | SNP marker relevant to sheep list tire litter size, primed probe group, kit, detection method and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109652566A true CN109652566A (en) | 2019-04-19 |
Family
ID=66123441
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910126727.7A Pending CN109652566A (en) | 2019-02-20 | 2019-02-20 | SNP marker relevant to sheep list tire litter size, primed probe group, kit, detection method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109652566A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111440879A (en) * | 2020-04-27 | 2020-07-24 | 内蒙古大学 | Method for improving fertility of Mongolian sheep by using GDF9 gene |
| CN111500744A (en) * | 2020-04-27 | 2020-08-07 | 内蒙古大学 | Mongolian sheep fertility improvement method based on GDF9 gene coding region mutation |
-
2019
- 2019-02-20 CN CN201910126727.7A patent/CN109652566A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| JUENGEL JL等: "Mutations in the leptin receptor gene associated with delayed onset of puberty are also associated with decreased ovulation and lambing rates in prolific Davisdale sheep", 《REPROD FERTIL DEV》 * |
| NC_019458.1: "rs428867159", 《ENSEMBL GENOME BROWSER》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111440879A (en) * | 2020-04-27 | 2020-07-24 | 内蒙古大学 | Method for improving fertility of Mongolian sheep by using GDF9 gene |
| CN111500744A (en) * | 2020-04-27 | 2020-08-07 | 内蒙古大学 | Mongolian sheep fertility improvement method based on GDF9 gene coding region mutation |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110029178A (en) | SNP marker relevant to the more lamb characters of sheep list tire and its detection primer group, detection kit and application | |
| CN107619857B (en) | Method for detecting CNV (CNV) marker of beef cattle KLF8 gene and application of CNV marker | |
| CN107619870B (en) | Molecular marker capable of indicating and identifying length of sheep wool and specific primer pair and application thereof | |
| CN105176982A (en) | Method for detecting sheep FecB gene polymorphism by using Taqman MGB probes | |
| WO2023001211A1 (en) | Gene chip, molecular probe combination and kit for analyzing traits of sheep wool, and use | |
| CN110004236A (en) | SNP marker relevant to the more lamb characters of sheep and its detection ESR1 genotyping primer group, kit and application | |
| CN109234442A (en) | One kind SNP marker relevant to the more lamb characters of sheep and its detection kit and application | |
| CN106906303A (en) | One SNP marker for influenceing quality character of pork and its application | |
| CN108330201A (en) | Identify molecular labeling and its application of Tomato Mosaic Virus resistant gene | |
| CN112442542A (en) | Fetal paternity testing method | |
| CN113265476A (en) | Gene chip, molecular probe combination, kit and application for analyzing milk production performance of sheep | |
| CN108913787A (en) | SNP marker relevant to the more lambs of sheep and its application | |
| CN106939334B (en) | Method for detecting fetal DNA content in plasma of pregnant woman | |
| CN109652566A (en) | SNP marker relevant to sheep list tire litter size, primed probe group, kit, detection method and application | |
| CN107988385B (en) | Method for detecting marker of PLAG1 gene Indel of beef cattle and special kit thereof | |
| CN108866206A (en) | SNP marker relevant to the more lambs of sheep and its application | |
| CN107586857A (en) | Nucleic acid, kit and method for the red-black coat color gene of Rapid identification pig | |
| CN104726579A (en) | PCR-SSCP kit used for molecular selection of sheep meat performance | |
| CN107475414A (en) | A kind of SNP primer pairs of the method for screening the long high glycogen content parent shellfish of oyster and its correlation | |
| CN116144816A (en) | KASP molecular marker for identifying orange peel color in seedling stage and application thereof | |
| CN107937571B (en) | Nucleic acid mass spectrum paternity identification method based on information SNP set and primers thereof | |
| CN112410441A (en) | Method for identifying anti-cysticercosis trait of bee colony by using SNP marker KZ 288479.1-95621 | |
| CN112029868A (en) | Portunus trituberculatus microsatellite markers and application thereof in growth trait association analysis | |
| CN109439773A (en) | A kind of SNP marker and its detection primer sets, kit and application of the more lamb characters of sheep | |
| CN109207611A (en) | One kind SNP marker relevant to sheep heat character and its detection kit and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190419 |