CN104611327A - Amplification and sequencing method of chicken mtDNA Cytb gene complete sequence and special primer - Google Patents
Amplification and sequencing method of chicken mtDNA Cytb gene complete sequence and special primer Download PDFInfo
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Abstract
The invention discloses an amplification and sequencing method of a chicken mtDNA Cytb gene complete sequence and a special primer. The primer for amplifying and sequencing the chicken mtDNA Cytb gene complete sequence is as shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3. The amplification and sequencing method comprises the following steps: (1) extracting DNA in feather of chicken, and detecting the quality of the DNA; (2) designing a pair of PCR primers (SEQ ID NO.1 and SEQ ID NO.2) in upstream and downstream conservative areas of the chicken mtDNA Cytb gene; (3) sequencing PCR products by using a sequencing primer (SEQ ID NO.3); (4) splicing the sequenced sequences. The detection method disclosed by the invention has the advantages of rapid speed, low cost, easiness in mastering and the like, the obtained chicken mtDNA Cytb gene complete sequence can be used in multiple fields such as chicken genetic resource diversity evaluation, maternal origin, phylogenetic analysis, and germplasm resource identification and protection.
Description
Technical field
The invention belongs to technical field of molecular biology, particularly relate to the amplification of a kind of chicken mtDNA Cytb gene complete sequence and sequence measurement thereof.
Background technology
Animal Genetic Resource diversity can provide material for breed improvement, and the change that can conform very well.China has and enriches poultry Germplasm Resources of Local, the genetic diversity of research and assessment China poultry local variety, formulate reasonably utilize and the Sustainable development of sfgd. to China's livestock industry significant.The method of genetic diversity of zoologizeing mainly contains micro-satellite and Mitochondrial DNA etc., and two kinds of methods respectively have relative merits, and Mitochondrial DNA more easily operates.Poultry (fowl) kind is mostly subject to the impact of external male animal (fowl) crossbreeding and improvement, and Population minimizing even disappears, and brings very large difficulty to research varietal salt tolerance and diversity.Mitochondrial DNA is in genetic process, and genetic material is transmitted by ooplasm, and less generation DNA recombinates, and the Mitochondrial DNA type of its wild ancestor generally can keep.This mode of inheritance, body just can represent a maternal instinct group one by one, therefore only needs a small amount of individual specimen just can reflect the genetic construction of a colony.Investigator can differentiate discontinuous matrilineage from the genetic background of complexity, even if through the cross breeding of several thousand, phylogenetic relationship is still distinct.
Chicken Mitochondrial Genome Overview, generally at about 16785bp, is closed hoop duplex structure, can self-replicating and transcribing.Comprise fgs encoder district (13 protein coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes) and 1 control region of 37 genes.Cytochrome b (Cyt b) gene is the most clearly one that in mt DNA 13 protein coding genes, structure and function is understood, and is also that coded protein is uniquely positioned at plastosome base.Cytochrome b (Cyt b) gene evolution speed is moderate, between section to kind, and even in planting, there is abundant polymorphism, be between research kind and the desirable molecule marker of intraspecific genetic differentiation degree and phyletic evolution.The some or all of sequence of Cyt b gene has been widely used in the genetic diversity of animal monoid and phyletic evolution research, the genetic information that complete sequence comprises more horn of plenty.Because Cyt b gene downstream is tRNA-Thr gene (69bp), tRNA-Pro gene (70bp), be folded into the short chain of cloverleaf pattern, the nucleotide structure order-checking difficulty of compared with normal, general backward sequencing is all difficult to successfully.Also do not use Cyt b gene complete sequence to carry out systematic research to China's poultry local variety at present, therefore set up Cyt b gene complete sequence amplification and sequence measurement be very necessary.
Summary of the invention
The object of the invention is to propose a kind of amplification of chicken mtDNA Cytb gene complete sequence and sequence measurement and primer special, speed of the present invention is fast, cost is low, be easy to grasp, effectively can detect the mtDNA Cytb gene complete sequence of chicken.
The invention provides a kind of amplification and order-checking primer of chicken mtDNA Cytb gene complete sequence, comprise PCR primer and sequencing primer, the upstream primer of described PCR primer is: 5'-TCTTACCTGGGTTCTTTCG-3', and the downstream primer of PCR primer is: 5'-TTTAGTGGAGTTGCGGTGT-3';
Described sequencing primer is Walking primer:5'-CGCCTTTGTGGGCTATGTT-3'.
Present invention also offers the amplification of a kind of chicken mtDNA Cytb gene complete sequence and sequence measurement thereof, comprise the following steps:
(1) DNA in chicken feather is extracted, and Detection job;
(2) according to the relative position of Red Jungle-fowl mtDNA Cytb gene on mtDNA whole genome sequence, in mtDNA Cytb gene upstream and downstream conservative region design one couple of PCR primers, primer sequence is:
Forward prime(SEQ ID NO.1):5'-TCTTACCTGGGTTCTTTCG-3'
Reverse primer(SEQ ID NO.2):5'-TTTAGTGGAGTTGCGGTGT-3'
(3) PCR primer order-checking and sequence assembly
Reacted by PCR, obtain object fragment, order-checking company is sent to check order after purifying, due to mtDNA Cytb downstream of gene complex structure, reverse primer cannot be measured, and designs a sequencing primer according to the mtDNA Cytb gene order that forward primer has been measured, carries out primer step and moves (Primer walking) order-checking, after allowing, forward primer and step are moved primer to record two sequences and splice, obtain mtDNACytb gene complete sequence.
Walking primer(SEQ ID NO.3)5'-CGCCTTTGTGGGCTATGTT-3'
In step 1, in chicken feather, the extraction step of DNA is: the feather that feather sample has just grown after gathering chick or becoming chicken moult, be generally 2-3 root, the down on pen feather top is cut with scissors, after clean PE gloves shred, put into 2mL centrifuge tube, each sample adds lysate 1000 μ L and Proteinase K 40 μ L shakes up, and puts 59 DEG C of water-bath digestion in shaking table.After digestion fully, add the saturated phenol of 700-800uL, shake up after 10min with the centrifugal 5min of rotating speed 10000rpm.Aspirate supernatant puts into 2mL centrifuge tube, adds the saturated phenol of 1000uL, shakes up after 5min with the centrifugal 5min of rotating speed 10000rpm.Aspirate supernatant puts into 2mL centrifuge tube, adds chloroform 1000uL, shakes up after 5min with the centrifugal 5min of rotating speed 10000rpm.Aspirate supernatant puts into 2mL centrifuge tube, adds dehydrated alcohol and is settled out DNA agglomerate, leave DNA and outwell ethanol, then DNA agglomerate with 70% alcohol wash one time, with the centrifugal 10min of rotating speed 10000rpm.Outwell ethanol, leave DNA, airing (1h), add aqua sterilisa 100uL and dissolve, put-20 DEG C of Refrigerator stores.
In step 3, PCR reaction system is: PCR Mix 25 μ L, 10 μm of each 1.5 μ L of ol/L primer, template 2 μ L, aqua sterilisa 20 μ L.
In step 3, PCR response procedures is: 95 DEG C of 5min, and (95 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 60s) 30 circulate, 72 DEG C of 4min.
The Cytb of mtDNA described in step 3 joining method is: order-checking obtains the SeqMan program splicing of sequence DNAStar 5.02 software, is compared by Red Jungle-fowl reference sequences NC_007235 in splicing sequence and GenBank, cuts primer and unnecessary sequence.
Above-mentioned method, described pcr amplification specific band is 1490bp, comprise mtDNA Cytb gene complete sequence and upstream and downstream partial sequence, its nucleotides sequence is classified as: (lowercase represents Cytb full length gene sequence, capitalization represents upstream and downstream sequence, black underscore represents upstream and downstream primer location, and dash area represents that step moves primer location):
TCTTACCTGG GTTCTTTCGC CCTCACAATC CTTACAACGA TCCTACTTAT CCAAAAATAA 60
ACTAatggca cccaacattc gaaaatccca ccccctacta aaaataatta acaactccct 120
aatcgacctc ccagccccat ccaacatctc tgcttgatga aatttcggct ccctattagc 180
agtctgcctc atgacccaaa tcctcaccgg cctactacta gccatgcact acacagcaga 240
cacatcccta gccttctcct ccgtagccca cacttgccgg aacgtacaat acggctgact 300
catccggaat ctccacgcaa acggcgcctc attcttcttc atctgtatct tccttcacat 360
cggacgaggc ctatactacg gctcctacct ctacaaagaa acctgaaaca caggagtaat 420
cctcctcctc acactcatag ccac
ctcccat ggggccaaat 480
atcattctga ggggccaccg ttatcacaaa cctattctca gcaattccct acattggaca 540
caccctagta gagtgagcct gaggaggatt ctcagtcgac aacccaaccc ttacccgatt 600
cttcgcctta cacttcctcc tcccctttgc aatcgcaggt attactatca tccacctcac 660
cttcctacac gaatcaggct caaacaaccc cctaggcatc tcatccgact ctgacaaaat 720
tccatttcac ccatactact ccttcaaaga cattctgggc ttaactctca tactcacccc 780
attcctaaca ctagccctat tctcccccaa cctcctagga gacccagaaa acttcacccc 840
agcaaaccca ctagtaaccc ccccacatat caaaccagaa tgatattttc tattcgccta 900
tgccatccta cgctccatcc ccaacaaact tggaggtgta ctagccctag cagcctcagt 960
cctcatcctc ttcctaatcc ccttcctcca caaatctaaa caacgaacaa taaccttccg 1020
accactctcc caaaccctat tctgacttct agtagccaac cttcttatcc taacctgaat 1080
cggaagccaa ccagtagaac accccttcat catcattggc caaatagcat ccctctctta 1140
cttcaccatc ctacttatcc tcttccccac aatcggaaca ctagaaaaca aaatactcaa 1200
ctactaaAAT ACTCTAATAG TTTATGAAAA ACATTGGTCT TGTAAACCAA AAACTGAAGA 1260
CTCCACCCTT CTTAGAGTAT CAGAAAAGGA GGACTCAAAC CTCCATCTCC AGCTCCCAAA 1320
GCTGGTATTT TCAAATAAAC TACTCTCTGA AACCCTTAAA CCGCCCGAAT TGCCCCCCGA 1380
GACAACCCAC GCACAAGCTC TAGTACAACA AACAAAGCTA ACAACAAACC TCACCCAGCC 1440
ACCAAAAACA ACCCAACCCC ACATGAATAA A
ACACCGCAA CTCCACTAAA1490
。
Compared with prior art, the present invention has following beneficial effect:
1, detection method of the present invention has the advantages such as speed is fast, cost is low, easy grasp, effectively can detect the mtDNACytb gene complete sequence of chicken, thus provides effective research platform for chicken molecular genetics and Molecular Phylogeny and Evolution research.2 pairs of primers total length that could increase is needed to compare with (HEILONGJIANG ANIMAL SCIENCE AND VETERINARY MEDICINE, the 2nd phase, 51st ~ 53 pages in 2013) such as Wu cicada, more easy easy operation;
2, the invention provides a kind of method of cost-saving, simple and easy to do amplification chicken mtDNA Cytb gene complete sequence, chicken mtDNA Cytb gene complete sequence provided by the invention can be applicable to chicken genetic resources diversity evaluation, maternal origin, phylogenetic analysis and multiple field such as germplasm identification and protection;
3, the present invention adopts feather collection, substitutes traditional blood sampling, reduces the injury to laboratory animal, and can obtain high-quality DNA profiling.
Embodiment
The amplification of chicken mtDNA Cytb gene complete sequence and an order-checking primer, comprise PCR primer and sequencing primer.
The upstream primer of PCR primer is: 5'-TCTTACCTGGGTTCTTTCG-3',
The downstream primer of PCR primer is: 5'-TTTAGTGGAGTTGCGGTGT-3'.
Sequencing primer is Walking primer:5'-CGCCTTTGTGGGCTATGTT-3'.
The complete sequence amplification of a kind of chicken mtDNA Cytb gene and sequence measurement, comprise the following steps:
(1) DNA in chicken feather is extracted, and Detection job;
(2) in chicken mtDNA Cyt b gene upstream and downstream conservative region design one couple of PCR primers, the fragment of Cytb gene is comprised by PCR reaction amplification, through agarose gel electrophoresis; The upstream primer of described PCR primer is: 5'-TCTTACCTGGGTTCTTTCG-3', and the downstream primer of PCR primer is: 5'-TTTAGTGGAGTTGCGGTGT-3',
(3) design sequencing primer to check order to pcr amplification product, described sequencing primer is Walking primer:5'-CGCCTTTGTGGGCTATGTT-3';
(4) obtain sequence to order-checking to splice, and cut the unnecessary sequence of upstream and downstream, obtain chicken mtDNA Cyt b gene complete sequence, chicken mtDNA Cyt b gene total length is 1143bp.
The described PCR reaction system for the chicken mtDNA Cyt b gene complete sequence that increases is: PCR Mix 25 μ L, 10 μm of each 1.5 μ L of ol/LPCR primer upstream and downstream primer, template 2 μ L, aqua sterilisa 20 μ L.
Described PCR reaction conditions is: 95 DEG C of 5min; 95 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 60s, 30 circulations; 72 DEG C of 4min.
DNA step in described extraction chicken feather is: the feather 2-3 root that described feather sample has just grown after gathering chick or becoming chicken moult, cut the down on pen feather top, 2mL centrifuge tube is put into after feather shreds, each sample adds lysate 1000 μ L and Proteinase K 40 μ L shakes up, and puts 59 DEG C of water-bath digestion in shaking table; After digestion fully, add the saturated phenol of 700-800uL, shake up after 10min with the centrifugal 5min of rotating speed 10000rpm;
Aspirate supernatant puts into 2mL centrifuge tube, adds the saturated phenol of 1000uL, shakes up after 5min with the centrifugal 5min of rotating speed 10000rpm;
Aspirate supernatant puts into 2mL centrifuge tube, adds chloroform 1000uL, shakes up after 5min with the centrifugal 5min of rotating speed 10000rpm;
Aspirate supernatant puts into 2mL centrifuge tube, adds dehydrated alcohol and is settled out DNA agglomerate, leave DNA and outwell ethanol, then DNA agglomerate with 70% alcohol wash one time, with the centrifugal 10min of rotating speed 10000rpm; Outwell ethanol, leave DNA, airing 1h, add aqua sterilisa 100uL and dissolve, put-20 DEG C of Refrigerator stores.
Described to order-checking obtain sequence carry out splicing step for: by described in step 3 order-checking obtain sequence splice through the SeqMan program of DNAStar 5.02 software, Red Jungle-fowl reference sequences NC_007235 in splicing sequence and GenBank is compared, cut primer and unnecessary sequence, obtain chicken mtDNA Cytb full length gene.
The research of embodiment 1 Silky fowl phyletic evolution and genetic diversity
1.1 DNA sample preparations
The feather just grown after gathering 30 Silky fowl neck moult, male and female half and half, random sampling.Cut the down on pen feather top with scissors, after clean PE gloves shred, put into 2mL centrifuge tube, each sample adds lysate 1000 μ L and Proteinase K 40 μ L shakes up, and puts 59 DEG C of water-bath digestion in shaking table.After digestion fully, add the saturated phenol of 700-800uL, shake up after 10min with the centrifugal 5min of rotating speed 10000rpm.Aspirate supernatant puts into 2mL centrifuge tube, adds the saturated phenol of 1000uL, shakes up after 5min with the centrifugal 5min of rotating speed 10000rpm.Aspirate supernatant puts into 2mL centrifuge tube, adds chloroform 1000uL, shakes up after 5min with the centrifugal 5min of rotating speed 10000rpm.Aspirate supernatant puts into 2mL centrifuge tube, adds dehydrated alcohol and is settled out DNA agglomerate, leave DNA and outwell ethanol, then DNA agglomerate with 70% alcohol wash one time, with the centrifugal 10min of rotating speed 10000rpm.Outwell ethanol, leave DNA, airing (1h), add aqua sterilisa 100uL and dissolve, put-20 DEG C of Refrigerator stores.
1.2 5 subspecies Red Jungle-fowl Cytb gene complete sequences are downloaded
From GenBank database, download the sequence that Red Jungle-fowl 5 subspecies Gallus gallus bankiva, Gallus gallus gallus, Gallus gallus murghi, Gallus gallus jabouillei, Gallus gallus spadiceus etc. have measured mtDNA Cytb gene complete sequence, carry out Phylogenetic Analysis with Silky fowl.
1.3 pcr amplification
PCR amplification system (50 μ L): PCRMix (Nanjing Bo Erdi company) 25 μ L, 10 μm of each 1.5 μ L of ol/L primer, template 2 μ L, aqua sterilisa 20 μ L.Response procedures is: 95 DEG C of 5min, and (95 DEG C of 30s, 56 DEG C of 60s, 72 DEG C of 60s) 30 circulate, 72 DEG C of 4min.1.5% LMP agarose (Promega company) detected through gel electrophoresis PCR primer, reclaims purifying with test kit (Promega company).Moving primer together with forward primer and step after purifying send order-checking company to check order.
1.4 sequence assembling and analysis
Order-checking obtains the sequence SeqMan program of DNAStar 5.02 software and splices, and is compared by Red Jungle-fowl reference sequences NC_007235 in splicing sequence and GenBank, cuts primer and unnecessary sequence, obtains chicken mtDNA Cytb full length gene.。With DnaSP version 5.10 software statistics polymorphic site number and mutational site sum, haplotype number, the various degree of haplotype, diverse oligonucleotide degree etc.Carry out changing and the statistics of transversion number and based composition analysis with MEGA 4.0, and adopt adjacent method phylogenetic tree construction with it, grow tree and select Kimura 2 model, repeat for 1000 times.
The present invention is described in conjunction with most preferred embodiment, but after having read foregoing of the present invention, those skilled in the art can make various changes or modifications the present invention, and these equivalent form of values fall within the application's appended claims limited range equally.
Claims (7)
1. the amplification of chicken mtDNA Cytb gene complete sequence and order-checking primer, it is characterized in that, comprise PCR primer and sequencing primer, the upstream primer of described PCR primer is: 5'-TCTTACCTGGGTTCTTTCG-3', and the downstream primer of PCR primer is: 5'-TTTAGTGGAGTTGCGGTGT-3';
Described sequencing primer is Walking primer:5'-CGCCTTTGTGGGCTATGTT-3'.
2. the amplification of chicken mtDNA Cytb gene complete sequence and a sequence measurement, is characterized in that, comprise the following steps:
(1) DNA in chicken feather is extracted, and Detection job;
(2) design a pair PCR primer according to claim 1 at chicken mtDNA Cyt b gene upstream and downstream conservative region, comprised the fragment of Cytb gene by PCR reaction amplification, detect through agarose gel electrophoresis;
(3) sequencing primer described in claim 1 is utilized to check order to pcr amplification product;
(4) obtain sequence to order-checking to splice, and cut the unnecessary sequence of upstream and downstream, obtain chicken mtDNA Cyt b gene complete sequence, chicken mtDNA Cyt b gene total length is 1143bp.
3. the amplification of chicken mtDNA Cytb gene complete sequence according to claim 2 and sequence measurement thereof, it is characterized in that, the described PCR reaction system for the chicken mtDNA Cyt b gene complete sequence that increases is: PCR Mix 25 μ L, 10 μm of each 1.5 μ L of ol/LPCR primer upstream and downstream primer, template 2 μ L, aqua sterilisa 20 μ L.
4. the amplification of the chicken mtDNA Cytb gene complete sequence according to Claims 2 or 3 and sequence measurement thereof, it is characterized in that, described PCR reaction conditions is: 95 DEG C of 5min; 95 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 60s, 30 circulations; 72 DEG C of 4min.
5. the amplification of chicken mtDNA Cytb gene complete sequence according to claim 2 and sequence measurement thereof, it is characterized in that, DNA step in described extraction chicken feather is: the feather 2-3 root that described feather sample has just grown after gathering chick or becoming chicken moult, cut the down on pen feather top, 2mL centrifuge tube is put into after feather shreds, each sample adds lysate 1000 μ L and Proteinase K 40 μ L shakes up, and puts 59 DEG C of water-bath digestion in shaking table; After digestion fully, add the saturated phenol of 700-800uL, shake up after 10min with the centrifugal 5min of rotating speed 10000rpm;
Aspirate supernatant puts into 2mL centrifuge tube, adds the saturated phenol of 1000uL, shakes up after 5min with the centrifugal 5min of rotating speed 10000rpm;
Aspirate supernatant puts into 2mL centrifuge tube, adds chloroform 1000uL, shakes up after 5min with the centrifugal 5min of rotating speed 10000rpm;
Aspirate supernatant puts into 2mL centrifuge tube, adds dehydrated alcohol and is settled out DNA agglomerate, leave DNA and outwell ethanol, then DNA agglomerate with 70% alcohol wash one time, with the centrifugal 10min of rotating speed 10000rpm; Outwell ethanol, leave DNA, airing 1h, add aqua sterilisa 100uL and dissolve, put-20 DEG C of Refrigerator stores.
6. the amplification of chicken mtDNA Cytb gene complete sequence according to claim 2 and sequence measurement thereof, it is characterized in that, described in step (4), sequence is obtained to order-checking and carry out splicing step for SeqMan program splicing order-checking described in step 3 being obtained sequence DNAStar5.02 software, Red Jungle-fowl reference sequences NC_007235 in splicing sequence and GenBank is compared, cut primer and unnecessary sequence, obtain chicken mtDNA Cytb full length gene.
7. the amplification of chicken mtDNA Cytb gene complete sequence according to claim 2 and sequence measurement thereof, it is characterized in that, order-checking obtains the nucleotides sequence comprising the specific amplification band of the 1490bp of mtDNA Cytb full length gene and upstream and downstream partial sequence and is classified as:
TCTTACCTGG GTTCTTTCGC CCTCACAATC CTTACAACGA TCCTACTTAT CCAAAAATAA 60ACTAatggca cccaacattc gaaaatccca ccccctacta aaaataatta acaactccct 120aatcgacctc ccagccccat ccaacatctc tgcttgatga aatttcggct ccctattagc 180agtctgcctc atgacccaaa tcctcaccgg cctactacta gccatgcact acacagcaga 240cacatcccta gccttctcct ccgtagccca cacttgccgg aacgtacaat acggctgact 300catccggaat ctccacgcaa acggcgcctc attcttcttc atctgtatct tccttcacat 360cggacgaggc ctatactacg gctcctacct ctacaaagaa acctgaaaca caggagtaat 420cctcctcctc acactcatag ccaccgcctt tgtgggctat gttctcccat ggggccaaat 480atcattctga ggggccaccg ttatcacaaa cctattctca gcaattccct acattggaca 540caccctagta gagtgagcct gaggaggatt ctcagtcgac aacccaaccc ttacccgatt 600cttcgcctta cacttcctcc tcccctttgc aatcgcaggt attactatca tccacctcac 660cttcctacac gaatcaggct caaacaaccc cctaggcatc tcatccgact ctgacaaaat 720tccatttcac ccatactact ccttcaaaga cattctgggc ttaactctca tactcacccc 780attcctaaca ctagccctat tctcccccaa cctcctagga gacccagaaa acttcacccc 840agcaaaccca ctagtaaccc ccccacatat caaaccagaa tgatattttc tattcgccta 900tgccatccta cgctccatcc ccaacaaact tggaggtgta ctagccctag cagcctcagt 960cctcatcctc ttcctaatcc ccttcctcca caaatctaaa caacgaacaa taaccttccg 1020accactctcc caaaccctat tctgacttct agtagccaac cttcttatcc taacctgaat 1080cggaagccaa ccagtagaac accccttcat catcattggc caaatagcat ccctctctta 1140cttcaccatc ctacttatcc tcttccccac aatcggaaca ctagaaaaca aaatactcaa 1200ctactaaAAT ACTCTAATAG TTTATGAAAA ACATTGGTCT TGTAAACCAA AAACTGAAGA 1260CTCCACCCTT CTTAGAGTAT CAGAAAAGGA GGACTCAAAC CTCCATCTCC AGCTCCCAAA 1320GCTGGTATTT TCAAATAAAC TACTCTCTGA AACCCTTAAA CCGCCCGAAT TGCCCCCCGA 1380GACAACCCAC GCACAAGCTC TAGTACAACA AACAAAGCTA ACAACAAACC TCACCCAGCC 1440ACCAAAAACA ACCCAACCCC ACATGAATAA AACACCGCAA CTCCACTAAA 1490。
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| CN105385699A (en) * | 2015-12-09 | 2016-03-09 | 重庆国际旅行卫生保健中心 | cytB specific sequences of four rats in river ports of three gorges area and molecular identification method |
| CN108060234A (en) * | 2017-12-13 | 2018-05-22 | 安徽师范大学 | The amplimer and amplification method of a kind of Tiger Shrike and Lanius schach Linnaeus cytochrome b gene |
| CN115058522A (en) * | 2022-07-21 | 2022-09-16 | 江苏省家禽科学研究所 | Molecular marker for identifying broiler varieties containing white rocco blood sources and application |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105385699A (en) * | 2015-12-09 | 2016-03-09 | 重庆国际旅行卫生保健中心 | cytB specific sequences of four rats in river ports of three gorges area and molecular identification method |
| CN108060234A (en) * | 2017-12-13 | 2018-05-22 | 安徽师范大学 | The amplimer and amplification method of a kind of Tiger Shrike and Lanius schach Linnaeus cytochrome b gene |
| CN115058522A (en) * | 2022-07-21 | 2022-09-16 | 江苏省家禽科学研究所 | Molecular marker for identifying broiler varieties containing white rocco blood sources and application |
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