CN107766698A - A kind of domestic sheep of identification whether the method containing argali blood relationship - Google Patents
A kind of domestic sheep of identification whether the method containing argali blood relationship Download PDFInfo
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- 239000008280 blood Substances 0.000 title claims abstract description 30
- 210000004369 blood Anatomy 0.000 title claims abstract description 30
- 238000000034 method Methods 0.000 title claims abstract description 21
- 241000283903 Ovis aries Species 0.000 claims abstract description 44
- 108020005196 Mitochondrial DNA Proteins 0.000 claims abstract description 38
- 102000054766 genetic haplotypes Human genes 0.000 claims abstract description 31
- 241000283901 Ovis aries musimon Species 0.000 claims abstract description 19
- 241000611732 Ovis vignei Species 0.000 claims abstract description 17
- 108020004414 DNA Proteins 0.000 claims abstract description 12
- 238000012163 sequencing technique Methods 0.000 claims abstract description 11
- 230000003321 amplification Effects 0.000 claims abstract description 7
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 7
- 238000007400 DNA extraction Methods 0.000 claims abstract description 5
- 238000012408 PCR amplification Methods 0.000 claims abstract description 4
- 241000283898 Ovis Species 0.000 claims abstract 13
- 229920000669 heparin Polymers 0.000 claims description 6
- 238000002887 multiple sequence alignment Methods 0.000 claims description 5
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- 210000003484 anatomy Anatomy 0.000 description 1
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- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B10/00—ICT specially adapted for evolutionary bioinformatics, e.g. phylogenetic tree construction or analysis
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- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B30/00—ICT specially adapted for sequence analysis involving nucleotides or amino acids
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Abstract
The invention belongs to gene engineering technology field, disclose a kind of domestic sheep of identification whether the method containing argali blood relationship.Including：Individual DNA extractions；The Software for Design of Primer 5.0 includes the primer of sheep mtDNA D Loop sequences, using the DNA of extraction as template, enters performing PCR amplification；The PCR primer sequencing of amplification, DNAstar softwares are arranged to sequencing result, spliced, assembled；Download domestic sheep mtDNA D loop haplotypes A and B sequence from ncbi database and mouflon (Ovis musimon), urial, Yuan sheep, argali dimension formula subspecies, argali Kazakhstan subspecies, argali Mongolia subspecies, ovis ammon hodgsonii and argali are named the sequences of subspecies；ClustalW softwares carry out multiple alignment, Mega 6.06 Neighbor Joining program construction evolutionary relationship trees, with this precise Identification to above sequence.
Description
Technical field
Whether the invention belongs to gene engineering technology field, more particularly to a kind of domestic sheep of identification contains argali blood relationship
Method.
Background technology
Domestic sheep (Ovis aries) is one of animal tamed earliest, and substantial amounts of meat, hair, skin are provided for the mankind
Deng the means of production.On the origin of domestic sheep, in general viewpoint thinks that domestic sheep possesses common ancestors with wild sheep.
Paleontologist, Animal genetics man by archaeology, individual morphology compare with the features such as anatomy think domestic sheep with it is wild
There is common ancestor in sheep, or genetic connection be present；Cytogenetics discloses domestic sheep (2n=54) and Siberia mountain goat
(2n=52), mouflon (Ovis musimon) (European mouflon (Ovis musimon) and Asia mouflon (Ovis musimon), 2n=54), argali (2n=56), urial (2n
=58) etc. had differences in the chromosome number of wild sheep, but in areas such as Xinjiang of China, Tibet, Qinghai, domestic sheep with
Wild argali can raise up seed.In the local monoid of the Tibet sheep of western China, some local monoids (Jia Luoyang)
There is similarity in the characteristic aspects such as build, appearance, angle-style and wild argali, by the features such as build, appearance and angle-style come
Judge the blood relationship or directly tamed by argali that such as Jia Luo sheep monoids contain wild argali.
Sry genes are a paternal inheritance genes on the mammal Y chromosome, using its CDS sequence analysis Ah
Strangle safe sheep, to hide be Jia Luoyang, hides the affiliation for being OUra sheep with domestic sheep, argali and mouflon (Ovis musimon), it is believed that Altai Sheep,
Tibetan is Jia Luoyang, to hide be OUra sheep and common sheep, argali and mouflon have identical Sry gene orders, originating from altogether
Same ancestors or Jia Luoyang are tamed by argali.
In summary, the problem of prior art is present be：1) west is judged by visual properties such as build, appearance, angle-style
Hide domestication or the origin of the local monoid of sheep, it is impossible to objectively solve whether these local monoids originate from argali in itself from species
Or the blood relationship containing argali；2) the CDS sequences of Sry genes are highly conserved, and evolutionary rate is slower, are unsuitable for solving with the gene
The local monoid of Tibet sheep is originating from argali or contains argali blood relationship.
The content of the invention
The problem of existing for prior art, whether the invention provides a kind of domestic sheep of identification contains argali blood relationship
Method.
The present invention be achieved in that a kind of domestic sheep of identification whether the method containing argali blood relationship, the connoisseur
Whether the method containing argali blood relationship comprises the following steps foster sheep：
Step 1, the collection of Jia Luo sheep blood samples or tissue sample, individual DNA extractions；
Step 2, according to the domestic sheep mtDNA complete sequences that accession number in ncbi database is NC001941, use
Primer 5.0 designs primer, using Jia Luoyang DNA sample as template, carries out the PCR amplifications of mtDNA D-loop complete sequences；
Step 3, the mtDNA D-loop of amplification PCR primer, sequencing company is sent to carry out the survey of mtDNA D-loop sequences
Sequence, the sequencing result that DNAstar softwares enter itinerant trader Lip river sheep mtDNA D-loop are arranged, spliced, assembled；
Step 4, downloaded from ncbi database domestic sheep mtDNA D-loop haplotypes A and haplotype B sequence and
European wild sheep-mouflon (Ovis musimon), urial, Yuan sheep, argali dimension formula subspecies, argali Kazakhstan subspecies, argali Mongolia are sub-
Kind, ovis ammon hodgsonii and argali name subspecies sequence；
Step 5, mitochondria D-Loop Multiple Sequence Alignment is carried out using ClustalW softwares, Mega's 6.06
The evolutionary relationship tree of Neighbor-Joining program constructions mouflon (Ovis musimon), urial, Yuan sheep and each subspecies of argali, judges to rub
Not whether logical sequence sheep, urial, Yuan sheep and each subspecies of argali etc. are classified as with Jia Luoyang haplotype A, haplotype B and haplotype C
One kind, determine that Jia Luoyang has common ancestors or shared blood relationship with mouflon (Ovis musimon), urial, Yuan sheep and each subspecies of argali
Relation.
Further, the step 1 specifically includes：Using jugular vein blood collection 5-8mL, inject added with sodium heparin anticoagulant
In disposal vacuum heparin tube, 180 ° gently overturn and mix 5-6 times, acquire 62 individuals altogether, transport laboratory back in time, and -20
DEG C save backup.
Further, the step 2 designs primer using Primer 5.0 and included：
Sense primer SEQ ID NO：1 and anti-sense primer SEQ ID NO：2.
Further, the PCR reaction systems cumulative volume of the PCR amplifications is the μ of 50 μ L, 2 × Taq PCR MasterMix 25
L, each 1.0 μ L of upstream and downstream primer, DNA profiling 2.0 μ L, ultra-pure water H2O 21.0μL；PCR response procedures are 94 DEG C of pre-degeneration
5min, 94 DEG C of 30s are denatured, annealed 56~63 DEG C, extend 72 DEG C of 90s, totally 34 circulations, 72 DEG C of extensions 5min, 16 DEG C of hold.
Further, the step 5 includes：It is each that itinerant trader Lip river sheep, Mo Folun sheep, Yuan sheep, argali are entered using ClustalW softwares
The mtDNA D-Loop complete sequences of the colonies such as subspecies and domestic sheep mtDNA D-Loop haplotype A and haplotype B carry out more
Sequence alignment, the sequence after comparison is recycled to Mega 6.06 Neighbor-Joining program constructions mouflon (Ovis musimon), east
Argali, Yuan sheep and the evolutionary relationship tree of each subspecies of argali.
Advantages of the present invention and good effect are：The method that evolutionary relationship tree is built by mtDNA D-Loop complete sequences can
To identify whether domestic sheep contains argali blood relationship exactly.Mitochondrial DNA is extranuclear heredity material, have molecular weight it is small,
Evolutionary rate is fast, inorganization is specific and the characteristic such as strict matrilinear inheritance, compared to build, appearance, angle-style and conservative Sry
Gene, the evolutionary relationship tree of mtDNA D-Loop structures more can identify whether domestic sheep contains disk exactly from molecular level
The blood relationship of sheep.
Brief description of the drawings
Fig. 1 be the domestic sheep of identification provided in an embodiment of the present invention whether the method flow diagram containing argali blood relationship.
Embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
The application principle of the present invention is explained in detail below in conjunction with the accompanying drawings.
As shown in figure 1, the domestic sheep of identification provided in an embodiment of the present invention whether the method containing argali blood relationship include with
Lower step：
S101：The collection of Jia Luo sheep blood samples or tissue sample, individual DNA extractions；
S102：According to the domestic sheep mtDNA complete sequences that accession number in ncbi database is NC001941, using Primer
5.0 design primers, using Jia Luoyang DNA sample as template, carry out the amplification of mtDNA D-loop complete sequences；
S103：The mtDNA D-loop of amplification PCR primer, sequencing company is sent to carry out the survey of mtDNA D-loop sequences
Sequence, the sequencing result that DNAstar softwares enter itinerant trader Lip river sheep mtDNA D-loop are arranged, spliced, assembled；
S104：Domestic sheep mtDNA D-loop haplotypes A and haplotype B sequence and Europe is downloaded from ncbi database
Continent wild sheep-mouflon (Ovis musimon) (Ovis aries musimon), urial (Ovis ammon nigrimontana), Yuan sheep
(Ovis ammon argnli), argali dimension formula subspecies (Ovis vignei bochariensis), argali Kazakhstan subspecies
(Ovis ammon collium), argali Mongolia subspecies (Ovis ammon darwini), ovis ammon hodgsonii (Ovis ammon
Hodgsoni) named the sequences such as subspecies (Ovis ammon ammon) with argali；
S105：Mitochondria D-Loop Multiple Sequence Alignment is carried out using ClustalW softwares, Mega's 6.06
The evolutionary relationship tree of Neighbor-Joining program constructions mouflon (Ovis musimon), urial, Yuan sheep and each subspecies of argali, so as to sentence
Disconnected mouflon (Ovis musimon), urial, Yuan sheep and each subspecies of argali etc. whether the haplotype A, haplotype B and haplotype C with Jia Luoyang
Be classified as one kind, so that it is determined that Jia Luoyang and mouflon (Ovis musimon), urial, Yuan sheep and each subspecies of argali exist common ancestors or
Shared genetic connection.
The application principle of the present invention is further described with reference to specific embodiment.
Whether the method containing argali blood relationship comprises the following steps the domestic sheep of identification provided in an embodiment of the present invention：
(1) sample collection
Meet Jia Luo sheep standards in Jia Luo sheep original producton location-Sichuan Province Abazangzuqiangzu Autonomous Prefecture Aba County Jia Luo towns selection
Individual, using jugular vein blood collection 5-8mL, inject in the disposal vacuum heparin tube added with sodium heparin anticoagulant, 180 ° are gently
It is reverse to mix 5-6 times, 62 individuals are acquired altogether, transport laboratory back in time, and -20 DEG C save backup.
(2) DNA is extracted
DNA extractions are carried out using TIANamp Genomic DNA Kit kits.1) the μ L of blood sampling 200, are added to
In 1.5mL EP pipes, 20 μ L Proteinase K Solution is added, mixes, adds 200 μ L buffer solution GB, it is fully reverse to mix, 70
10min is placed in DEG C water-bath；2) EP pipes are taken out, add 200 μ L absolute ethyl alcohols, fully vibration mixes 15s, by resulting solution
It is transferred in adsorption column CB3,12000rpm centrifugation 30s, outwells waste liquid in collecting pipe, 500 μ L GD is added to adsorption column CB3,
12000rpm centrifuges 30s, outwells waste liquid in collecting pipe；3) to adsorption column CB3 add 600 μ L rinsing liquid PW, 12000rpm from
Heart 30s, waste liquid in collecting pipe is outwelled, be repeated once and add 600 μ L rinsing liquid PW into adsorption column CB3, centrifuge and outwell waste liquid；
4) adsorption column CB3 is put back in collecting pipe, 12000rpm centrifugation 2min outwell waste liquid, and adsorption column CB3 is placed in into room temperature number point
Clock, then be transferred in a new EP pipe, elution buffer TE being vacantly added dropwise to the middle part of adsorption column, room temperature places 2~
5min, 12000rpm centrifuge 2min, and the liquid in EP pipes is the DNA solution extracted, carry out detected through gel electrophoresis, and -20 DEG C
Save backup.
(3) design of primers
With reference to domestic sheep mtDNA complete sequences (accession number NC001941) in NCBI data, set using Primer 5.0
Count primer, sense primer SEQ ID NO：1 is 5 '-CTCCCTAAGACTCAAGGAAGAAGC-3 ', anti-sense primer SEQ ID NO：
2 are, 5 '-GAGGGATTCTGAGTTCCTTCTTCG-3 ', primer is synthesized by Sangon Biotech (Shanghai) Co., Ltd..
(4) PCR is expanded
PCR reaction systems cumulative volume is 50 μ L, 2 × Taq PCR MasterMix 25 μ L, each 1.0 μ L of upstream and downstream primer,
DNA profiling 2.0 μ L, ultra-pure water H2O 21.0μL；PCR response procedures are 94 DEG C of 5min of pre-degeneration, are denatured 94 DEG C of 30s, annealing 56
~63 DEG C, extend 72 DEG C of 90s, totally 34 circulations, 72 DEG C of extensions 5min, 16 DEG C of hold.The PCR primer of amplification send raw work biology work
Journey (Shanghai) limited company carries out mtDNA D-Loop sequencings.Using DNAstar softwares to Jia Luo sheep mtDNA D-loop
Sequencing result arranged, spliced and assembled.
(5) wild sheep mtDNA D-loop sequences
Domestic sheep mtDNA D-Loop haplotype A (NCBI accession number is downloaded from ncbi database：
) and haplotype B (AF039577.1), Mo Folun sheep (AY091487.1 and AY091488.1), urial AF039578.1
(AY091493.1 and AY091494.1), Yuan sheep (AJ238292.1, AJ238296.1, AJ238297.1, AJ238300.1,
AJ238301.1, AJ238302.1, AJ238303.1, AJ238304.1, AJ238305.1, AJ238293.1 and
AJ238299.1), argali dimension formula subspecies (AY091490.1, AY091491.1 and AF039580.1), argali Kazakhstan are sub-
Kind of (AY091492.1), argali Mongolia subspecies (AF242348.1), ovis ammon hodgsonii (JX673912.1) and argali are named Asia
Kind (AF242347.1).
(6) evolutionary relationship tree is built
Enter the mtDNA D- of the colonies such as itinerant trader Lip river sheep, Mo Folun sheep, Yuan sheep, each subspecies of argali using ClustalW softwares
Loop complete sequences and domestic sheep mtDNA D-Loop haplotype A and haplotype B carry out Multiple Sequence Alignment, by the sequence after comparison
Row recycle Mega 6.06 Neighbor-Joining program constructions mouflon (Ovis musimon), urial, Yuan sheep and each subspecies of argali
Evolutionary relationship tree.
(7) Relationship iden- tification
Jia Luo sheep mtDNA D-Loop complete sequence analysis shows that Jia Luo sheep has 3 kinds of haplotypes, i.e. haplotype A, list
Times type B and haplotype C, is accounted for leading with haplotype A, and therefore, Jia Luoyang has 3 kinds of maternal origins.European Mo Folun sheep and Jia Luo
Together, evidence shows that European mountain sheep-Mo Folun sheep has to the maternal origin of Jia Luo sheep to sheep haplotype B individual cluster
Contribution, and urial, Yuan sheep, the subspecies of argali (are tieed up formula subspecies, Kazakhstan's subspecies, Mongolian subspecies, Tibet subspecies and referred to
Name subspecies) etc. cluster outside Jia Luoyang 3 kinds of haplotypes, to the time of Jia Luoyang maternal origin without contribution or differentiation compared with
It is early.Therefore, in Jia Luo sheeps, the blood relationship or domestic sheep that do not contain wild argali have just been broken up before by domestication.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
All any modification, equivalent and improvement made within refreshing and principle etc., should be included in the scope of the protection.
Sequence table
<110>Sichuan Province Grassland research institute
<120>A kind of domestic sheep of identification whether the method containing argali blood relationship
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
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<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
Claims (5)
1. a kind of domestic sheep of identification whether the method containing argali blood relationship, it is characterised in that whether the domestic sheep of identification
Method containing argali blood relationship comprises the following steps：
Step 1, the collection of Jia Luo sheep blood samples or tissue sample, individual DNA extractions；
Step 2, according to the domestic sheep mtDNA complete sequences that accession number in ncbi database is NC001941, using Primer
5.0 design primers, using Jia Luoyang DNA sample as template, carry out the PCR amplifications of mtDNA D-loop complete sequences；
Step 3, the mtDNA D-loop of amplification PCR primer, sequencing company is sent to carry out the sequencing of mtDNA D-loop sequences,
The sequencing result that DNAstar softwares enter itinerant trader Lip river sheep mtDNA D-loop is arranged, spliced, assembled；
Step 4, domestic sheep mtDNA D-loop haplotypes A and haplotype B sequence and Europe are downloaded from ncbi database
Wild sheep-mouflon (Ovis musimon), urial, Yuan sheep, argali dimension formula subspecies, argali Kazakhstan subspecies, argali Mongolia subspecies, disk
Sheep Tibet subspecies and argali are named subspecies sequence；
Step 5, using ClustalW softwares to being downloaded in domestic sheep mtDNA D-Loop haplotypes A and B, ncbi database
MtDNA D-Loop sequences and Jia Luoyang mtDNA D-Loop sequences carry out Multiple sequence alignments, the sequence after comparison uses again
The Neighbor-Joining program construction evolutionary relationship trees of the softwares of Mega 6.06, judge mouflon (Ovis musimon), urial, Yuan sheep
And whether each subspecies of argali with Jia Luoyang haplotype A, haplotype B and haplotype C are classified as one kind, determine Jia Luoyang and Mo Fulun
Common ancestors or shared genetic connection be present in sheep, urial, Yuan sheep and each subspecies of argali.
2. identify as claimed in claim 1 domestic sheep whether the method containing argali blood relationship, it is characterised in that the step
One specifically includes：Using jugular vein blood collection 5-8mL, inject in the disposal vacuum heparin tube added with sodium heparin anticoagulant, 180 °
Gently overturn and mix 5-6 times, acquire 62 individuals altogether, transport laboratory back in time, -20 DEG C save backup.
3. identify as claimed in claim 1 domestic sheep whether the method containing argali blood relationship, it is characterised in that the step
Two design primer using Primer 5.0 includes：
Sense primer SEQ ID NO：1 and anti-sense primer SEQ ID NO：2.
4. identify as claimed in claim 1 domestic sheep whether the method containing argali blood relationship, it is characterised in that the PCR
The PCR reaction systems cumulative volume of amplification is 50 μ L, 2 × Taq PCR MasterMix 25 μ L, each 1.0 μ L of upstream and downstream primer,
DNA profiling 2.0 μ L, ultra-pure water H2O 21.0μL；PCR response procedures are 94 DEG C of 5min of pre-degeneration, are denatured 94 DEG C of 30s, annealing 58
DEG C, extend 72 DEG C of 90s, totally 34 circulations, 72 DEG C of extensions 5min, 16 DEG C of hold.
5. identify as claimed in claim 1 domestic sheep whether the method containing argali blood relationship, it is characterised in that the step
Five include：Enter the mtDNA D-Loop of each subspecies colony of itinerant trader Lip river sheep, Mo Folun sheep, Yuan sheep, argali using ClustalW softwares
Complete sequence and domestic sheep mtDNA D-Loop haplotype A and haplotype B carry out Multiple Sequence Alignment, by the sequence after comparison again
Utilize entering for Mega 6.06 Neighbor-Joining program constructions mouflon (Ovis musimon), urial, Yuan sheep and each subspecies of argali
Change relational tree.
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| CN201710847511.0A CN107766698B (en) | 2017-09-19 | 2017-09-19 | Method for identifying whether domestic sheep contains sheep cutlet |
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| CN201710847511.0A CN107766698B (en) | 2017-09-19 | 2017-09-19 | Method for identifying whether domestic sheep contains sheep cutlet |
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| CN107766698A true CN107766698A (en) | 2018-03-06 |
| CN107766698B CN107766698B (en) | 2021-10-01 |
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| CN108611425A (en) * | 2018-04-23 | 2018-10-02 | 四川省草原科学研究院 | The Genetic diversity evaluation method of sheep sources group |
Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1320627A2 (en) * | 2000-03-15 | 2003-06-25 | The Johns Hopkins University | Mitochondrial dosimeter |
| CN1428439A (en) * | 2001-12-27 | 2003-07-09 | 赵兴波 | Method for making animal genetic information identification by utilizing mt DNA fingerprint |
| US20040029133A1 (en) * | 2001-11-26 | 2004-02-12 | Mitokor, Inc. | Mitochondrial DNA polymorphisms |
| CN101260439A (en) * | 2008-03-03 | 2008-09-10 | 深圳出入境检验检疫局动植物检验检疫技术中心 | Amplification primer for detecting pigeon source component by PCR-mtDNA, detecting kit and using method thereof |
| CN102226216A (en) * | 2011-05-30 | 2011-10-26 | 河北省农林科学院植物保护研究所 | Universal primers for identifying bacillus and classification method using same |
| US8158772B2 (en) * | 2004-04-16 | 2012-04-17 | National Institute Of Agrobiological Sciences | Oligonucleotide sequences that identify species of animal |
| CN103343161A (en) * | 2013-05-10 | 2013-10-09 | 赵兴波 | Analysis method for genetic variation of sheep mitochondrial genome |
| CN103409519A (en) * | 2013-07-30 | 2013-11-27 | 新疆维吾尔自治区种羊与羊毛羊绒质量安全监督检验中心 | Gene probe for identifying ingredients of sheep origin and goat origin |
| CN103865924A (en) * | 2014-02-20 | 2014-06-18 | 中国农业科学院兰州畜牧与兽药研究所 | MtDNA D-loop complete sequence of tibetan sheep in tibet plateau and detection method thereof |
| CN103952421A (en) * | 2014-05-06 | 2014-07-30 | 中国农业科学院兰州畜牧与兽药研究所 | MtDNA (mitochondrial Deoxyribonucleic Acid) Cytb (Cytochrome b) complete sequence synthesis method for Tibetan plateau sheep |
| CN106148518A (en) * | 2016-07-01 | 2016-11-23 | 中国农业科学院兰州畜牧与兽药研究所 | A kind of Tibetan plateau sheep mitochondrion complete genome DNA and detection method thereof and application |
-
2017
- 2017-09-19 CN CN201710847511.0A patent/CN107766698B/en not_active Expired - Fee Related
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1320627A2 (en) * | 2000-03-15 | 2003-06-25 | The Johns Hopkins University | Mitochondrial dosimeter |
| US20040029133A1 (en) * | 2001-11-26 | 2004-02-12 | Mitokor, Inc. | Mitochondrial DNA polymorphisms |
| CN1428439A (en) * | 2001-12-27 | 2003-07-09 | 赵兴波 | Method for making animal genetic information identification by utilizing mt DNA fingerprint |
| US8158772B2 (en) * | 2004-04-16 | 2012-04-17 | National Institute Of Agrobiological Sciences | Oligonucleotide sequences that identify species of animal |
| CN101260439A (en) * | 2008-03-03 | 2008-09-10 | 深圳出入境检验检疫局动植物检验检疫技术中心 | Amplification primer for detecting pigeon source component by PCR-mtDNA, detecting kit and using method thereof |
| CN102226216A (en) * | 2011-05-30 | 2011-10-26 | 河北省农林科学院植物保护研究所 | Universal primers for identifying bacillus and classification method using same |
| CN103343161A (en) * | 2013-05-10 | 2013-10-09 | 赵兴波 | Analysis method for genetic variation of sheep mitochondrial genome |
| CN103409519A (en) * | 2013-07-30 | 2013-11-27 | 新疆维吾尔自治区种羊与羊毛羊绒质量安全监督检验中心 | Gene probe for identifying ingredients of sheep origin and goat origin |
| CN103865924A (en) * | 2014-02-20 | 2014-06-18 | 中国农业科学院兰州畜牧与兽药研究所 | MtDNA D-loop complete sequence of tibetan sheep in tibet plateau and detection method thereof |
| CN103952421A (en) * | 2014-05-06 | 2014-07-30 | 中国农业科学院兰州畜牧与兽药研究所 | MtDNA (mitochondrial Deoxyribonucleic Acid) Cytb (Cytochrome b) complete sequence synthesis method for Tibetan plateau sheep |
| CN106148518A (en) * | 2016-07-01 | 2016-11-23 | 中国农业科学院兰州畜牧与兽药研究所 | A kind of Tibetan plateau sheep mitochondrion complete genome DNA and detection method thereof and application |
Non-Patent Citations (5)
| Title |
|---|
| XIN CAI 等: "Bioinformatic Analysis of Phylogenetic Evolution of Chinese Cattle", 《2011 5TH INTERNATIONAL CONFERENCE ON BIOINFORMATICS AND BIOMEDICAL ENGINEERING》 * |
| XIN CAI 等: "Median-Joining Network Analysis of Phylogeny of Goat Breeds from South China", 《2011 5TH INTERNATIONAL CONFERENCE ON BIOINFORMATICS AND BIOMEDICAL ENGINEERING》 * |
| 包鹏甲 等: "中国五个绵羊群体mtDNA D-环遗传多样性研究", 《家畜生态学报》 * |
| 郭彦斌 等: "利用mtDNA D-loop区研究中国10个绵羊品种的遗传多样性与起源", 《农业生物技术学报》 * |
| 韩旭 等: "中国地方绵羊线粒体DNA D-loop区遗传多样性研究", 《遗传育种》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108611425A (en) * | 2018-04-23 | 2018-10-02 | 四川省草原科学研究院 | The Genetic diversity evaluation method of sheep sources group |
| CN114181934A (en) * | 2018-04-23 | 2022-03-15 | 四川省草原科学研究院 | Extraction method of sheep blood genome DNA (deoxyribonucleic acid), application of extraction method and genetic diversity evaluation method of sheep resource population |
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